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DIFFERENTIAL EFFECT OF IMPROVED CITRUS ROOTSTOCKS AND NUTRITION ON THE GENE EXPRESSION IN Candidatus Liberibacter asiaticus (CaLas) -

INFECTED ‘VALENCIA’ SWEET ORANGE TREES

By

ADITI DILIP SATPUTE

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2017

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To the Florida citrus growers and researchers

4

ACKNOWLEDGMENTS

I would like to express my thanks to my outstanding advisor Dr. Jude Grosser for

giving me the opportunity to work on my doctoral degree and his guidance throughout

my studies. Dr. Grosser encouraged my research and allowed me to grow as a

research scientist. His dedication and perseverance towards the betterment of the citrus

industry inspire me to support the agricultural community. I would like to extend my

thanks to my committee members Drs. Christine Chase, Fred Gmitter and Matias Kirst

for their suggestions, brilliant comments, and input to achieve the goal of my Ph.D.

Project. Thanks to the Kirst lab members Christopher Dervinis and Isabela Sant’Anna

for their help in sequencing preparation and data analysis. Thanks to Quibin Yu in the

Gmitter lab for introducing me to different software which I used for functional analysis

of the genes. would like to recognize Dr. Manjul Dutt for his guidance in my greenhouse

study.

Thanks to my laboratory colleagues and greenhouse team who helped me in

conducting my laboratory and greenhouse work smoothly and efficiently. I would like to

acknowledge the Horticultural Sciences Department chair Dr. Kevin Folta, the Citrus

Research and Development Foundation, and the New Varieties Development and

Management Corporation for their financial support of this project.

My spiritual sustenance thrives through a trust and faith in the Almighty. I would

not be able to come to the USA and pursue a doctoral degree without the support of my

family and friends back in India. Thanks to my parents and sister for supporting me

financially and emotionally in my endeavors. I am grateful to my dear friends, Jian Wu,

Utpal Handique, Kiran Dixit, Flavia Zambon, and Mathew Mattia for their unconditional

support, brainstorming discussions, and making my life more enjoyable throughout

5

hardships of my Ph.D. program. There are also many people who contributed pleasant

memories throughout my Ph.D. program. Thanks to all of them.

6

TABLE OF CONTENTS page

ACKNOWLEDGMENTS .................................................................................................. 4

LIST OF TABLES .......................................................................................................... 10

LIST OF FIGURES ........................................................................................................ 13

ABSTRACT ................................................................................................................... 15

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ..................................................... 18

Introduction ............................................................................................................. 18 Field Study ....................................................................................................... 21

Greenhouse Study ........................................................................................... 21 Literature Review .................................................................................................... 21

Citrus Genomics ............................................................................................... 21

Breeding for citrus improvement ................................................................ 21 Effect of citrus rootstock on scion in scion/rootstock combinations ............ 28

Citrus Bacterial Disease: Huanglongbing (HLB) ............................................... 29 Geographic distribution of HLB .................................................................. 30

HLB complex, pathogen-host-vector interaction ........................................ 30 Status of HLB in Florida and recent developments in HLB research ......... 33

Effect of HLB infection on scion performance; nutritional deficiencies and low quality fruit production ............................................................... 35

Interaction of citrus roots with HLB ............................................................ 37 Breeding of citrus scion and rootstock for HLB tolerance and resistance .. 38

Use of Sequencing Technologies in Plants ...................................................... 39 Sequencing technologies in non-model plants ........................................... 39 Transcriptome analysis using RNA sequencing ......................................... 41 Citrus-HLB interaction: omics studies ........................................................ 42 Transcriptomic analysis of CaLas-infected leaves of rough lemon and

sweet orange .......................................................................................... 43

Transcriptomic analysis between CaLas-infected and healthy roots and stem sampled from ‘Valencia’/Swingle combination ............................... 43

Transcriptomic analysis of DEGs in CaLas-infected leaves between non-grafted US-897 and Cleopatra mandarin rootstocks ........................ 44

Genes associated with HLB susceptibility .................................................. 45 Genes related to HLB tolerance ................................................................. 47

Comprehensive transcriptome and gene expression network analysis of HLB-citrus interactions ............................................................................ 48

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Transcriptomic and proteomic analysis of CaLas-infected and non-infected roots of ‘Sanhu’ red tangerine ................................................... 50

Proteomic analysis between healthy and CaLas-infected ‘Madam Vinous’ sweet orange ............................................................................. 50

Comparative proteomic analysis of symptomatic and pre-symptomatic CaLas-infected grapefruit leaves ............................................................ 51

Research Overview ................................................................................................ 51

2 COMPARATIVE ANALYSIS OF TRANSCRIPTOME AND PLANT PHENOTYPES OF CaLas-INFECTED SCION/ROOTSTOCK COMBINATIONS .. 54

Introduction ............................................................................................................. 54 Materials and Methods............................................................................................ 59

Plant Materials .................................................................................................. 59

Sampling .......................................................................................................... 59

Fruit Juice Quality Analysis .............................................................................. 60 CaLas and Citrus Tristeza Virus (CTV) Detection ............................................ 60 RNA Extraction and Quantification ................................................................... 61

RNA Library Preparation and RNA Sequencing ............................................... 62 Raw Data Processing ....................................................................................... 63

Functional Analysis and Gene Ontology of DEGs ............................................ 64 Validation of the RNA-seq Data ........................................................................ 64

Results .................................................................................................................... 67

Fruit Juice Quality Analysis .............................................................................. 67 PCR Detection of HLB and ELISA Detection of CTV ....................................... 67

RNA Extraction, RNA Library Preparation, and RNA Sequencing .................... 68

Raw Data Processing ....................................................................................... 68

Functional Analysis of Differentially Expressed Genes .................................... 69 Validation of Differentially Expressed Genes using qRT-PCR .......................... 70

Discussion .............................................................................................................. 71

3 DIFFERENTIAL EXPRESSION ANALYSIS OF HORMONAL METABOLISM-ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE ........................................................ 90

Introduction ............................................................................................................. 90 Materials and Methods............................................................................................ 95

Plant Material ................................................................................................... 95

Sampling, RNA extraction, and RNA sequencing ............................................. 95 Results .................................................................................................................... 96

HLB Detection and RNA Sequencing Output ................................................... 96

Differential Expression of Hormonal Regulation-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW Combinations . 96

Leaf samples .............................................................................................. 96 Root samples ............................................................................................. 98

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Differential Expression of Hormonal Regulation-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW Combinations ... 99

Leaf samples .............................................................................................. 99 Root samples ........................................................................................... 100

Discussion ............................................................................................................ 102

4 DIFFERENTIAL EXPRESSION OF PLANT IMMUNITY AND DEFENSE-ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE ...................................................... 130

Introduction ........................................................................................................... 130 Materials and Methods.......................................................................................... 136

Plant Material ................................................................................................. 136

Sampling, RNA extraction, and RNA sequencing ........................................... 136

Results .................................................................................................................. 137 HLB Detection and RNA Sequencing Output ................................................. 137 Differential Expressed of Defense-Associated Genes in Leaves and Roots

of Asymptomatic VAL/CAN and VAL/SW combinations .............................. 137 Leaf samples ............................................................................................ 137

Root samples ........................................................................................... 140 Differentially Expression of Defense-Associated Genes in Leaves and

Roots of Symptomatic VAL/CAN and VAL/SW combinations ..................... 140

Leaf Samples ........................................................................................... 140 Root Samples .......................................................................................... 142

Discussion ............................................................................................................ 144

5 DIFFERENTIAL EXPRESSION OF PLANT GROWTH AND DEVELOPMENT-ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE ...................................................... 182


Plant Material ................................................................................................. 188

Sampling, RNA Extraction, and RNA Sequencing .......................................... 189 Results .................................................................................................................. 189

HLB Detection and RNA Sequencing Output ................................................. 189 Differentially Expressed Plant Growth and Development-Associated Genes

in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW combinations ............................................................................................... 190

Leaf samples ............................................................................................ 190

Root samples ........................................................................................... 193 Differentially Expressed Plant Growth and Development-Associated Genes

in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW Combinations .............................................................................................. 193

Leaf samples ............................................................................................ 193

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Root samples ........................................................................................... 196

Discussion ............................................................................................................ 197

6 EFFECTS OF IMPROVED CITRUS ROOTSTOCK AND ENHANCED NUTRIENT FORMULATION ON HLB-DISEASE SEVERITY IN ‘VALENCIA’ SWEET ORANGE SCION .................................................................................... 240


Plant Material and Nutrition Treatment ........................................................... 244 CaLas-Inoculation and Detection ................................................................... 246 Sampling ........................................................................................................ 247 RNA Extraction and Gene Expression Quantification ..................................... 248 Plant Phenotype Analysis ............................................................................... 249

Results .................................................................................................................. 249

CaLas-Detection ............................................................................................. 249 DGE Analysis of The Defense and Transporter Genes in Leaves .................. 251

Differential expression analysis of the NPR1 gene .................................. 251

Differential expression analysis of the NPR3 gene .................................. 251 Differential expression analysis of the PP2B gene ................................... 252

Differential expression analysis of the ZRT2 gene ................................... 252 Differential expression analysis of the NRAMP2 gene ............................. 253 Differential expression analysis of the NIP6 gene .................................... 253

DGE Analysis of Defense and Transporter Genes in Roots .................... 254 Plant Phenotype Analysis ............................................................................... 254

Discussion ............................................................................................................ 255

7 SUMMARY AND CONCLUSIONS ........................................................................ 272

APPENDIX

A SUPPLEMENTAL DATA FOR CHAPTER 2 ......................................................... 279

B SUPPLEMENTAL DATA FOR CHAPTER 5 ......................................................... 287

LIST OF REFERENCES ............................................................................................. 313

BIOGRAPHICAL SKETCH .......................................................................................... 336

10

LIST OF TABLES

Table page 2-1 Experimental treatments and combinations ........................................................ 75

2-2 Comparison pairs used for differential gene expression analysis in leaves ........ 75

2-3 Analysis of HLB and CTV detection in the experimental samples ...................... 75

2-4 Reads obtained from the sequencing run ........................................................... 76

2-5 Functional analysis of significant DEGs in leaves of the asymptomatic treatment between VAL/CAN and ....................................................................... 77

2-6 Functional analysis of significant DEGs in leaves of the symptomatic treatment between .............................................................................................. 78

2-7 Functional analysis of significant DEGs in roots of the symptomatic treatment between VAL/CAN and VAL/SW ........................................................................ 79

2-8 Genes used for validation of RNA-sequencing results ....................................... 80

3-1 Experimental treatments and scion/rootstock combinations ............................. 110

3-2 Comparison pairs used for DGE analysis in leaves and roots of the ...................... experimental scion/rootstock combinations ...................................................... 110 3-3 Differentially expressed hormonal metabolism-associated genes

significantly upregulated in leaves of ................................................................ 111

3-4 Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of ................................................................................... 114

3-5 Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of ................................................................................... 119

3-6 Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of the ............................................................................. 121

3-7 Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the ................................................................................ 125


4-1 Experimental treatments and combinations ...................................................... 151

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4-2 Comparison pairs used for differential gene expression analysis in leaves ............ and roots of the experimental scion/rootstock combinations ............................ 151 4-3 Differentially expressed immunity and defense-associated genes significantly

upregulated in leaves of ................................................................................... 152

4-4 Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of ................................................................................... 153

4-5 Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of ................................................................................... 156

4-6 Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of the ............................................................................. 158

4-7 Differentially expressed immunity and defense-associated genes significantly upregulated in roots of the ................................................................................ 163


5-1 Experimental treatments and scion/rootstock combinations ............................. 206

5-2 Comparison pairs used for differential gene expression analysis in leaves ...... 206 and roots of the experimental scion/rootstock combinations ............................ 206 5-3 Differentially expressed growth and development -associated genes

significantly upregulated in leaves of ................................................................ 207

5-4 Differentially expressed growth and development-associated genes significantly upregulated in leaves of ................................................................ 210

5-5 Differentially expressed growth and development -associated genes significantly upregulated in leaves of ................................................................ 214

5-6 Differentially expressed growth and development-associated genes significantly upregulated in leaves of the .......................................................... 219

5-7 Differentially expressed growth and development -associated genes significantly upregulated in roots of the ............................................................ 224

5-8 Differentially expressed growth and development -associated genes significantly upregulated in roots of the ............................................................ 228

6-1 Rootstock treatments. ....................................................................................... 263

6-2 Controlled release fertilizer formulations. ......................................................... 263

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6-3 Primer sequences ............................................................................................. 264

6-4 Gene expression analyses in the roots ............................................................. 265

6-5 Phenotypic measurements including tree diameters below and above the graft union, and no.of ........................................................................................ 266

A-1 Identification of samples for RNA-seq run ........................................................ 279

A-2 Individual RNA-seq library pooling calculations for the sequencing.................. 280

A-3 Shell Script used to identify DGE between the comparisons mentioned in ...... 282

B-1 Differentially expressed and significantly upregulated growth associated genes in leaves of the ....................................................................................... 287

B-2 Differentially expressed and significantly upregulated growth associated genes in the leaves of ....................................................................................... 289

B-3 Differentially expressed and significantly upregulated growth associated genes in leaves of the symptomatic .................................................................. 290

B-4 Differentially expressed and significantly upregulated growth associated genes in the leaves of the ................................................................................. 294

B-5 Differentially expressed and significantly upregulated growth associated genes in the roots of ......................................................................................... 297

B-6 Differentially expressed and significantly upregulated growth associated genes in roots of the ......................................................................................... 305

13

LIST OF FIGURES

Figure page 2-1 VAL/CAN combination field trees. ...................................................................... 81

2-2 VAL/SW combination field trees. ........................................................................ 82

2-3 Juice qualityt analysis. A) Year 2015, B) Year 2016. .......................................... 83

2-4 RNA quality and quantity analysis. A) Leaf RNA B) Root RNA........................... 84

2-5 Read mapping statistics. .................................................................................... 85

2-6 Display of Volcano plot of using CummeRbund. ................................................. 86

2-7 Blast2GO functional analysis of DEGs in leaves (A and B) and roots (C and D) of the asymptomatic VAL/CAN and VAL/SW combinations………………………….87

2-8 Blast2GO functional analysis of DEGs in leaves (A and B) and roots (C and D) of the symptomatic VAL/CAN and VAL/SW combintions ……………………………87

2-9 qRT-PCR based DEGs validation. ...................................................................... 89

3-1 Graphical presentation of Hormonal regulation in plants. ................................. 128

3-2 Graphical presentation of DEGs involved in hormonal metabolism. ................. 129

4-1 Graphical presentation of DEGs involved in environmental biotic and ............. 179

4-2 Graphical presentation of DEGs involved in biotic and abiotic stress. .............. 180

4-3 Display of HLB-induced biotic stress responses in the leaves of the HLB .............. asymptomatic VAL/CAN and VAL/SW leaves. ................................................. 180 4-4 Display of HLB-induced biotic stress responses in leaves of the HLB- ................... symptomatic VAL/CAN and VAL/SW leaves. ................................................... 181 4-5 Display of HLB-induced biotic stress responses in roots of the HLB- ..................... symptomatic VAL/CAN and VAL/SW leaves. ................................................... 181 5-1 Graphical presentation of secondary metabolite biosynthesis pathway ............ 235

5-2 Graphical presentation of cell wall modification-associated pathways .............. 236

5-3 Graphical presentation of nutrient transportation-associated genes found in DGE analysis of asymptomatic and symptomatic VAL/CAN ........................ 237

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5-4 Graphical presentation of Nitrogen metabolism-associated ................................... genes found DGE analysis of asymptomatic and symptomatic ....................... 237 5-5 Graphical presentation of carbohydrate metabolism-associate genes ........ 238

5-6 Graphical presentation of genes encoding transcription regulators .................... in plant growth and development, and found in DGE analysis of...................... 239 6-1 CaLas-infected Valencia sweet orange (VAL) stick grafts. ............................... 267

6-2 CaLas detection from leaves and roots of different combinations. ................... 268

6-3 Gene expression analysis of the selected genes in leaves. A) NPR1, B) ......... 269

6-4 Phenotypic differences in rootstock-nutrient formulation combinations. ........... 271

6-5 Plant phenotype at 37 WAB. A) VAL grafted onto Sw rootstock. B) VAL ......... 271

7-1 Graphic presenting summary of CaLas-infected VAL/SW combination. ........... 278

7-2 Graphic presenting summary of CaLas-infected VAL/CAN combination. ......... 278

A-1 Tuxedo pipeline components. (Adapted from Trapnell et al., 2012) ................. 281

15

Abstract of Dissertation Presented to the Graduate School

of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

DIFFERENTIAL EFFECT OF IMPROVED CITRUS ROOTSTOCKS AND NUTRITION

ON THE GENE EXPRESSION IN Candidatus Liberibacter asiaticus (CaLas) -INFECTED ‘VALENCIA’ SWEET ORANGE TREES

By

Aditi Dilip Satpute

August 2017

Chair: Jude Grosser Major: Horticultural Sciences

Rootstocks are a key component of commercial citrus production. Therefore,

rootstock improvement is a major breeding objective of citrus breeding programs.

Improved citrus rootstocks are a potential solution to combat Huanglongbing (HLB), a

bacterial disease which is caused by putative causal agent CaLas. The citrus breeding

program at the University of Florida, Citrus Research and Education Center (UF-CREC)

has developed many putative HLB-tolerant rootstocks that can enhance ‘Valencia’

sweet orange scion sustainability and fruit quality under endemic HLB condition.

Differential transcriptomic analysis of HLB -asymptomatic and -symptomatic ‘Valencia’

(VAL) scion grafted onto UF-CREC improved candidate (CAN) rootstock (a putatively

HLB tolerant rootstock, hybrid of Hirado Buntan pummelo and Cleopatra mandarin) and

commercially used Swingle (SW) rootstock, showed significant differential expression

regulation of transcripts involved in the jasmonic acid (JA), ethylene (ET), abscisic acid

(ABA), auxin (AU) and brassinosteroid (BR) hormonal metabolism. In asymptomatic

(leaves) and symptomatic (leaves and roots) VAL/SW leaves showed significant

16

upregulation of genes encoding in ABA response regulation, AU biosynthesis, and ET

biosynthesis and receptors as compared to the respective treatments of VAL/CAN

indicating possible activation of response to abiotic stress, and strong involvement of

AU and ET mediated responses in CaLas-infected VAL/SW. In VAL/CAN, significant

upregulation of AU response factors and BR response genes suggests that the

enhanced plant sustainability might be the outcome of AU-BR interactions. VAL/SW

also showed upregulation of different JA biosynthesis genes suggesting a defense

activation, possibly against the psyllid phloem feeding. The transcriptome comparison

results also showed a greater number of defense-associated genes upregulated in

leaves and roots of VAL/SW combination which seem to exhibit a high energy

requirement condition that compromises plant growth. Therefore, strong upregulation of

defense genes in VAL/SW seems to be a reason for poor plant health in the advanced

stage of CaLas-infection. Whereas significant upregulation of nutrient transporters, cell

wall modification genes, phloem regeneration associated genes, growth factors and AU-

BR interactions suggest a better energy distribution balance between defense and

growth in VAL/CAN plants. In a greenhouse study, VAL grafted onto a UF-CREC

created improved complex tetraploid (4x) rootstock and SW showed significant

differences in the plant phenotype and nutrient transporter genes expression. CaLas-

infected-VAL/4x plants had a superior phenotype and lower HLB bacterial titer as

compared to VAL/SW under traditional and enhanced controlled release fertilizer

(ECRF). Also, CaLas infected -VAL/SW phenotype improved under ECRF. Our findings

in the field and greenhouse experiments support the hypothesis that rootstock can

differentially reprogram CaLas-infected scion to improve plant performance. Moreover,

17

there appears to be a significant rootstock-nutrition interaction that plays a role in the

defense response.

18

CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW

Introduction

Orange juice is a popular beverage consumed all around the world. Florida ranks

as the 2nd largest producer of the orange juice in the world and the leading supplier of

orange juice in the USA. However, the endemic spread of Huanglongbing (HLB) or

“citrus greening” disease started a rapid decline of the Florida citrus industry. The major

concerns associated with HLB to Florida citrus industry are decreasing fruit and juice

production. HLB is caused by a putative Candidatus species, Candidatus Liberibacter

asiaticus (CaLas) in the USA. CaLas is a gram-negative (Garnier et al., 1984),

biotrophic and phloem residing bacteria belonging to the alpha subdivision of the

proteobacteria (Jagoueix et al., 1994). The dissemination of CaLas is possible because

of its vector the Asian citrus psyllid (Diaphrina citri Kuwayama) which was found first in

1998 in Florida (Halbert and Manjunath, 2004). Currently, there is no definite solution to

control HLB. HLB control practices in Florida mostly relied on vector suppression and

nutrition management that increases production costs. HLB preventive practices are not

cost-effective, rather in some cases, they are a great financial burden to the citrus

farmers. In Florida, an increasing number of HLB-affected citrus trees in the past few

years have been leading to many abandoned citrus orchards. Declining fruit production

is also decreasing the juice production and, also, has caused business losses to

farmers. Long term-effective and affordable solutions are required to achieve

sustainable citrus production that will make farmers less dependent on chemical control

strategies. Citrus breeding has the potential to offer a solution to HLB through the

creation of tolerant or resistant cultivars that can mitigate HLB symptoms or even

19

challenge the CaLas survival. Hence, breeding of HLB tolerant cultivars is important to

lessen the disease severity and improve sustainable high quality fruit and juice

production. In Florida, declining juice quality of HLB affected fruit is a matter of grave

concern, as domestic and international juice markets are highly competitive.

Most commercial citrus rootstock and scion cultivars that are used in Florida are

highly susceptible to HLB. It has also been reported that once a plant is CaLas-infected,

bacteria can move quickly into the root system, and lead to significant loss of feeder

roots, especially in the Swingle citrumelo (Citrus paradisi [Macf.] x Poncirus trifoliata [L.]

Raf) rootstock (Graham et al., 2013). Thus, feeder root loss leads to overall poor tree

health and significantly reduced productivity. Also, HLB susceptible scion/rootstock

combinations showed callose and starch deposition, and vascular system blockage

which are the primary reasons for CaLas-infected plant health deterioration (Etxeberria

et al., 2009; Achor et al., 2010). Improved HLB-tolerant citrus cultivars are badly needed

with mechanisms that reduce HLB-affected plant phloem damage and root loss. Scion

and rootstock are equally important for plant performance. Woody fruit-tree rootstocks

impart many beneficial traits such as disease resistance, soil pH tolerance, better fruit

quality, and improved root systems (Martines-Ballista et al., 2012; Car, 1973; Webster,

1995). Citrus rootstocks are also crucial to improve plant horticultural performance

(Ribeiro et al., 2014). The citrus breeding program at the University of Florida- Citrus

Research and Education Center, Lake Alfred, FL (UF-CREC) has a major citrus

rootstock improvement program. The rootstock improvement program includes

conventional breeding at the diploid level, somatic hybridization via protoplast fusion to

produce allotetraploid rootstock hybrids (Grosser and Gmitter, 2011), and more recently

20

conventional breeding at the tetraploid level (Grosser et al., 2015). The program also is

heavily involved in the evaluation of UF-CREC created rootstock germplasm and

material obtained from rootstock breeding programs around the world. Growers are

actively involved in the evaluation of newly developed citrus varieties in field trials for

release in a recently employed fast-track release program. One of the priorities of the

CREC-rootstock breeding program is to develop HLB-tolerant rootstocks for high quality

fruit and juice production. Putative HLB-tolerant candidate rootstocks (CANs) in

greenhouse pre-testing and field trials showed that CaLas-infected sweet orange (Citrus

sinensis [L.] Osbeck) and grapefruit (Citrus paradisi [Macf.]) scion varieties grafted onto

CAN rootstocks had better plant performance as compared to commercial rootstocks;

this was attributed to regulatory mechanisms that are altered due to differential

rootstock genetics (Castle et al., 2015). Therefore, differential regulatory process

analysis should shed light on factors contributing to the improved plant phenotype in

Valencia/CAN combinations. Improved plant performances and increased sustainability

of CaLas-infected Valencia/CANs combinations as compared to Valencia/Swingle

combination led to our hypothesis that CAN rootstocks can alter the scion gene

expression by triggering changes in the plant defenses, hormonal signaling pathways,

plant growth and development regulations, and nutrient transportation processes, when

the plant is CaLas-infected.

Considering the promising results of UF-CREC developed CAN rootstock in

reducing HLB disease severity in the grafted scion, the aim of this project is to gain a

better understanding of the molecular mechanisms that are responsible for the

tolerance of the Valencia/CAN combinations using a transcriptomic approach. The

21

comparative differential gene expression (DGE) analysis between Valencia/Swingle and

Valencia/CAN combinations will also be helpful to study the HLB-citrus interactions at

asymptomatic and symptomatic stages of CaLas-infection.

Field Study

• Comparative DGE analysis of leaves and roots of CaLas-infected and the asymptomatic Valencia/CAN and Valencia/Swingle combinations

• Comparative DGE analysis of leaves and roots of CaLas-infected and the symptomatic Valencia/CAN and Valencia/Swingle combinations

Greenhouse Study

• Study of phenotypic differences in CaLas-infected plants response to interaction between citrus rootstock and nutrient formulations’ combinations

• DGE analysis of transporter and defense associated genes in CaLas-infected plants in response to citrus rootstocks and nutrient formulations’ combination

Literature Review

Citrus Genomics

Breeding for citrus improvement

Citrus fruits; sweet orange, mandarin (Citrus reticulata Blanco.), lemon (Citrus

limon L. [Burm.] f.), grapefruit varieties are among the highly consumed horticultural

crops globally (Burke, 1967). The origin of citrus is claimed by some to be in the Indian

subcontinent (Scora, 1975). It is also reported by others that Yunnan province in China

a region where citrus might have originated (Gmitter and Hu, 1990) Tropical and

subtropical climatic conditions favor citrus production (Burke, 1967). The commercially

produced citrus cultivars are mostly hybrids that originated from four citrus progenitors,

namely mandarin, pummelo (Citrus maxima Merr.), citron (Citrus medica L.) and

papeda species (Scora, 1975; Li et al., 2010). Citrus species are primarily diploid (2n=

2x) and constituted of 18 chromosomes (x=9). A few naturally occurring citrus cultivars

22

are triploid (3x) and occasionally tetraploid (4x). Genomic studies of progenitor citrus

species showed that the smallest and largest haploid genome size of citrus species,

respectively, are 360 MB in mandarins and 390 MB in citron (Ollitrault et al., 1994). The

haploid genome size of secondary species of citrus such as sweet oranges, sour

oranges (Citrus aurantium L.), grapefruit, and lemons is between 360 and 390

MB/haploid(Gmitter et al., 2012). The joint venture of international citrus genetics

researchers and sequencing centers worldwide initiated the International Citrus

Genome Consortium (ICGC). ICGC is a platform for citrus genetics and breeding

researchers worldwide to share and exchange the knowledge of contemporary citrus

genetics research (ICGC, 2004). ICGS facilitates free citrus genome-based databases

and tools through a portal called Phytozome (Michael and Jackson, 2013). Citrus

genome databases are widely used to study citrus population genetics and

domestication events. The citrus genome databases also used to study targeted

genome editing of sweet orange using CRISPR technology (Jia and Wang, 2014),

genetic transformation (Dutt et al., 2015), gene predictions that are responsible for plant

phenotypes (Dornelas et al., 2007), and a genome wide association study (Minamikawa

et al., 2017). The study of diverse mandarins, pummelos, and orange genome

sequencing showed that cultivated mandarins are the results of admixture of pummelo

into the mandarin, and also, found that a Chinese wild mandarin is significantly diverse

from ancestral mandarin species Citrus reticulata (Wu et al., 2014). Information

available in the sequenced citrus genomes’ databases has opened many opportunities

for citrus breeders and molecular biologists to perform advanced citrus research.

23

Major human edible or non-edible crops in the world evolved through adaptation,

domestication and manual breeding processes. Human civilization stimulated

intervention in the natural breeding process to develop cultivars that are better suited for

climatic, social, and economic conditions of human society at a given space and time.

Hence, breeding is an inseparable part of any agricultural and horticulture based

industry, and so it is with citrus cultivar development. Citrus species origine is proposed

to be in the Asia continent, in parts of China and India (Scora, 1975; Gmitter and Hu,

1990) . Over the years, trade between the different geographical locations led to the

transport of citrus species from eastern countries to the rest of the world. Also, citrus

hybrids are being developed in all major citrus breeding programs through conventional

and modern breeding techniques. The conventional citrus breeding method includes

hybridization followed by a selection of superior performing individuals. Marker-assisted

selection (MAS) enables efficient screening of superior individuals. Somatic

hybridization via protoplast fusion is also the most popular and widely used

biotechnique in citrus breeding (Gmitter, 1990; Grosser and Gmitter, 2011). Protoplast

fusion techniques have overcome some limitations of conventional breeding, bypassing

the barriers including sexual incompatibles between certain breeding parents, and male

or female sterility issues. Protoplast fusion allows ploidy manipulation, inter- or intra-

species organelle exchange and gene transfer to create somatic hybrids and cybrids

(Grosser et al., 1992; Guo et al., 2013). Citrus improvement programs also use natural

or induced mutations and somaclonal variation for variety development. An Individual

with a desired mutation or somaclonal variation can be used as a potential parent in the

hybridization/selection program. Omics-assisted, molecular marker-assisted, and

24

genetic-engineering techniques are gaining popularity in modern plant improvement

research. The Agrobacterium-based transformation has also been successfully used in

citrus breeding to transfer target genes (Dutt and Grosser, 2011). However,

controversies over genetically modified organisms (GMO) and environmental

regulations limit their commercial use. Newly emerging non-GMO genome editing

‘CRISPR Cas9’ technique has been gaining the attention of citrus breeders for potential

to achieve certain citrus breeding objectives (Jia and Wang, 2014)

Commercial citrus plants are grown by grafting a scion onto a rootstock. Unlike

non-grafted plants, both the scion and rootstock are equally important for plant

performance and survival. Therefore, scion and rootstock genetic improvements are

equally important objectives of citrus breeding programs. One primary objective of citrus

scion breeding is focused on manipulating ploidy level to create seedless triploids and

high-quality fruits. Somatic hybridization is key to creating allotetraploid breeding

parents that can be used in interploid crosses to generate seedless triploids (Grosser

and Gmitter 2011). In citrus improvement, embryo rescue technique is used for efficient

triploid embryo recovery (Viloria and Grosser, 2005; Shen et al., 2011). Polyembryony is

associated with nucellar embryony which produces multiple embryos in a single one

seed, all clones of the mother. Polyembryonic selections are difficult to use as a female

parent in conventional crosses due to a lack of zygotic embryo production. Therefore, in

scion breeding, efficient production of zygotic embryos (monoembryony) is preferred to

the nucellar embryony (polyembryony) in diverse female parents (Grosser and Gmitter,

2011). Other objectives of scion breeding programs are developing cultivars that

combine cold hardiss, easy peeling, disease resistance, improved juice flavor and color,

25

and expanded periods of harvesting. Among disease resistant cultivars, ‘Lakeland

limequat’ is a well known cultivar that is a canker resistant hybrid of kumquat

(Fortunella [Swing.]) and Key lime (Citrus aurantifolia Swing.) (Viloria and Grosser,

2005). ‘LB8-9’ SugarBelle® has proven to be the most HLB-tolerant commercially

available scion (Stover et al., 2016), and it is being used heavily in the UF-CREC

breeding program. Allotetraploid and autotetraploid zipperskin mandarins can be used

as elite parents to create easy peel and triploid seedless fruits (Grosser et al., 2010).

UF-CREC scion breeding program has, also, developed various mandarin, orange and

grapefruit cultivars which are accepted for commercial citrus production (Grosser et al.,

2015). Protoplast fusion also generates cybrids that allow the exchange of parental

organelles (mitochondria and/or chloroplast DNA) with or without mixing of the nuclear

genomes. Cybridization also has good potential to improve certain traits. Diploid cybrids

generated from the fusion of embryogenic callus of ‘Dancy’ mandarin with ‘Ruby red’

grapefruit showed increased brix of the juice and extended fruit harvesting period

(Satpute et al., 2015). The mechanism for this change has not been determined. Many

commercially used citrus scions are developed at United States Department of

Agriculture (USDA)- citrus breeding program. Some of the imporatnt commercially

accepted are ‘Orlando’ and ‘Minneola’ Tangelos (Citrus paradisi x Citrus reticulata),

‘Osceola’, ‘Lee’, ‘Nova’ and ‘Robinson’ which are hybrids of ‘Clementine’ mandarion

with ‘Orlando’ tangelo, ‘Page’ a hybrid of ‘Minneola’ Tangelos with ‘Clementine’,

‘Sunburst’ (‘Robinson’ x ‘Osceola’) (McCollum, 2007).

Another important component of citrus improvement programs is rootstock

breeding. Rootstock breeding history goes back to approximately 1900 AC and can be

26

defined as early-, and modern-era based on international trade of citrus fruits,

development of budwood protection programs, and commercialization of the citrus

industry (Castle, 2010). Citrus rootstock characteristics evaluations are different from

those of scion breeding. Therefore, sources of genetic variation and selection methods

are also different than for scion breeding. Sexual and somatic hybridization are primary

approaches for rootstock development. Genetic engineering may also play a role in

citrus rootstock improvement programs. In a citrus rootstock development program,

plant material from any source or developed by any technique can be entered into the

system at any time point, that can then be screened based on the potential of the plant

material to improve a desirable trait or multiple traits (Gmitter et al., 2007).

Today, different citrus rootstocks such as sour orange (Citrus aurantium L.),

Cleopatra mandarin, rough lemon (Citrus jambhiri Lush.), Volkamer lemon (Citrus

volkmeriana Ten. & pesq.), citranges (P. trifoliata x C. sinensis), and citrumelos (C.

Paradisi x P. trifoliata) are used in commercial citrus production. Citrus rootstock

breeding is primarily aimed to develop cultivars with improved disease resistance and

tree size-controlling attributes. In addition, there are many other rootstock-related citrus

tree attributes that can be achieved through citrus rootstock improvement (Castle,

2010). These attributes include yield, precocity of bearing, fruit quality, adaptation to

high soil pH, scion compatibility, and ease of propagation. Louzada et al. (1992)

reported the creation of potential disease resistant 4x somatic hybrids created via

protoplast fusion. Somatic hybrids created between ‘Caipira’ sweet orange embryogenic

parent fused with Volkamer lemon, Rangpur lime (Citrus limonia L. osbeck), and sour

orange as the non-embryogenic parents showed potential resistance to citrus blight and

27

citrus tristeza virus CTV (Mendes et al., 2001). The soil borne disease complex

Diaprepes/Phytophthora is a serious problem for citrus production in Florida. The

rootstock program at UF-CREC has developed several improved rootstock cultivars.

Among these, a somatic hybrid combining ‘Nova’ mandarin + ‘Hirado Buntan Pink’ HBP

pummelo and its progeny exhibited tolerance to the Diaprepes/Phytophthora complex

(Grosser and Gmitter, 2011). Somatic hybrids created at the UF-CREC rootstock

breeding program have shown many improved horticultural traits, including dwarf plant

stature and higher fruit production in field trials with commercial scions. Somatic hybrids

created between sour orange + Rangpur lime and sour orange + Palestine sweet lime

yielded 20,000 kg fruit per acre with 3-4 m tree height (Grosser and Gmitter, 2011).

Traditional rootstock propagation in nurseries requires polyembryonic rootstock seed as

necessary to generate true-to-type uniform seedlings. Therefore, embryo genetics is

highly important in rootstock breeding. The citrus breeding program at the UF-CREC

has also pioneered the creation of superior 4x allotetraploid “tetrazyg” cultivars.

Tetrazyg rootstocks are a combination of three or four cultivars that bring desirable traits

from different citrus germplasm into one cultivar via conventional breeding using

somatic hybrids or other tetrazygs as parents. Some of these tetrazygs were derived

from the following crosses: [‘Nova’ mandarin + ‘HBP’ pummelo x ‘Cleopatra’ mandarin +

argentine trifoliate orange], [‘Nova’ mandarin + ‘HBP’ pummelo x ‘Succari’ + argentine

trifoliate orange], [‘Nova’ mandarin + ‘HBP’ pummelo x Cleopatra mandarin + sour

orange], [Nova + ‘HBP’ pummelo x sour orange + Palestine sweet lime] (Grosser and

Gmitter, 2011). In addition to the improved horticultural traits, UF-CREC developed 2X

and 4x rootstocks have also shown a lower incidence of HLB as compared to the

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commercially used rootstocks in the grafted scions after 5 years in the field under heavy

HLB pressure (Grosser, 2013; Castle et al., 2016). Many USDA developed rootstocks

are also commercially used to grow citrus. Some of USDA-developed rootstocks: US-

812 US-802, US-897, and US-942, have potential to combat against CTV,

Diaprepes/Phytophthora complex, and also, act as candidate rootstock for HLB

tolerance (Bowman et al., 2016). In Florida, the epidemic of HLB has brough many

challenges to the use of traditional scion and rootstock cultivars. The newly developed

scions and rootstocks are the potential cultivars that may establish in the citrus industry

because of their their ability to fight against HLB.

Effect of citrus rootstock on scion in scion/rootstock combinations

A majority of commercially important horticultural crops are grown using

scion/rootstock combinations. In horticultural crops, fruits are commercially important

plant parts. In citrus, fruit and juice are equally important commodities. Citrus rootstocks

have a great impact on regulating fruit and juice quality (Castle, 1995; Ghnaim et al.,

2006). Rootstocks have been used for crop production for over 2000 years ( Webster,

1995). Rootstocks significantly contribute to increasing or decreasing tree vigor, higher

plant productivity, increasing precocity, increasing plant immunity, changes in plant

biomass, plant growth pattern, and the biochemical and physiological status of the plant.

The roles of rootstocks in fruit crops like mango, apple, citrus, and stone fruits have

been thoroughly studied. In apple, the ‘Malling’ (M) series of clonal rootstocks is well

known for dwarfing and use in high density plantings. The ‘Polish’ (P) series is used for

collar rot resistance. The ‘Budagovasky’ (Bud) series is used worldwide for winter-

hardiness. In pear, the famous Quince rootstock is widely used to obtain higher quality

fruits from the grafted scion (Webster, 1995). In citrus: rough lemon, sour orange,

29

Rangpur lime, Cleopatra mandarin, Swingle citrumelo, and Carrizo citrange (Poncirus

trifoliata X ‘Washington’ sweet orange) rootstocks are popular (Castle, 2010). Although

rootstocks are critical in regulating scion performance, the mechanisms responsible for

changes in scion performance are still unclear. It is hypothesized that the rootstock-

scion union leads to internal changes in the quantity and ratio of endogenous

hormones, movement of plant assimilates such as sugar, amino acids, mineral

nutrients, and the amount of water uptake (Webster, 1995). Biotechnological research

tools have facilitated the understanding of molecular mechanisms involved in rootstock-

scion interactions. A new discovery in this direction is the mRNA, small RNA, peptides,

or amino acids long distance transport through phloem in grafted vegetables and woody

plants. A heterografting experiment between tomato rootstock and potato scion showed

the physical transfer of mRNA between rootstock and scion that led to changes in scion

leaf morphology (Kudo and Harada, 2007). In woody plants, long distance transport of

mRNA between rootstock and scion was reported in apple (Stoeckli et al., 2011). Also,

gibberellic acid-insensitive (GAI) mRNA transcript (Zhang et al., 2012), and transcript

encoding NAC domain protein exchanges were found across graft unions in pear scion

and rootstock (Zhang et al., 2013). Considering the importance of the rootstock in

regulating scion performance, improved citrus rootstocks can be a potential antidote to

reduce the detrimental effects of HLB in CaLas-infected commercial citrus trees.

Citrus Bacterial Disease: Huanglongbing (HLB)

Huanglongbing is the mandarin language-originated word meaning “yellow

dragon disease”. Worldwide, HLB is known by different names such as greening in

South Africa, mottle leaf in Philippines, and dieback in India. In South Africa, greening-

affected sweet oranges were found to have disintegrated and necrotic phloem present

30

in their vascular system. In Indonesia, HLB is recognized as ‘Vein phloem

degeneration.' The different names of HLB in the disease affected areas were

commonly derived from typical symptoms of HLB on the host plant. HLB affected areas

were first found to be reported in India. In 1919, Reinking reported “yellow shoot” of

citrus in the study of diseases of economic plants in southern China and later it was

known as HLB. In the 21st century, HLB has become a highly destructive epidemic citrus

disease (Bové, 2006; Bové, 2014).

Geographic distribution of HLB

The origin of HLB is found to be in the Asian continent. In the 18th century, the

die back symptoms were first observed in citrus orchards in India (Capoor, 1963), and

yellow shoot symptoms were found in southern China citrus orchards in the late 1800’s

(Zhao, 1981). This suggests the origin of HLB to be in the Asian continent, either in

India or China. In Africa, HLB was first observed in the late 1920s (da Graca et al.,

2016). In 2004, HLB was first confirmed in the western world in the state of Sao Paulo in

Brazil, and later, in 2005, it was confirmed in the state of Florida in the USA (Halbert,

2005). In the USA, HLB is also detected in other citrus producing states; Texas,

Arizona, California, South Carolina, Georgia and Louisiana (da Graca et al., 2016). The

expanding HLB infection is becoming a threat to other citrus producing countries too.

These countries include Australia, Mediterranean basin countries, Middle Eastern

countries: Iran, Turkey, and other Caribbean countries: Cuba, Belize, and Mexico.

HLB complex, pathogen-host-vector interaction

The presumed causal agent of HLB is Candidatus Liberibacter spp. bacteria. In

1970, the causal organism of HLB was confused with a virus or Mycoplasma like

organism (MLO) because of its presence in the plant's phloem and yellow shoot

31

symptoms (Doi et al., 1967). However, in 1970, Laflache and Bove proved that the HLB

associated pathogen is a gram-negative bacterium that has a triple layer of outer

peptidoglycan membrane, and it belongs to the alpha-proteobacteria in the family of

Rhizobiaceae. Presumably, three species of the HLB causing Candidatus Liberibacter

genus have been identified: americanus, asiaticus, and africanus. The most destructive

species of putative HLB causing bacteria is Candidatus Liberibacter asiaticus which is

prominently found in the American and Asian continents. The other two species, namely

africanus, and americanus have been found in African countries and in Brazil,

respectively (Bové, 2006). The current research shows that the putative HLB causing

bacteria is pleomorphic during growth, and is not limited to the shoots. Rather, bacteria

colonize the roots even before the appearance of visible symptoms on leaves, and

cause stunted root growth (Graham et al., 2013). CaLas bacteria are thus far non-

culturable which continues to be a major constraint in understanding the host-pathogen

interactions (Jagoueix et al., 1997). In earlier days, CaLas diagnosis was conducted

using biological index or antibodies against HLB (Garnier et al., 1991). However, in

recent years, the HLB diagnosis has been routinely performed using polymerase chain

reaction (PCR) technique (Li et al., 2006; Ananthakrishnan et al., 2013). The complete

genome sequencing of CaLas obtained from an infected psyllid revealed that the

bacterium has a small genome size of 1.23 MB, and lacks type III and IV secretion

systems. Gene annotation showed that CaLas genome contains a high number of

genes, particularly for low a small genome, involved in cell motility (4.5%) and transport

activity (8%) (Duan et al., 2009). Li et al. (2012) showed several ATI-binding cassette

32

(ABC) transporters are also present in the CaLas that are involved in importing

metabolites, co-enzymes, and heavy metals from host plants.

Another important component of the HLB disease complex is the insect vector;

namely Asian citrus psyllid (ACP) Diaphorina citri Kuwayama and Trioza erytreae that

cause transmission of CaLas. ACP is a vector of CaLas, whereas, T. erytreae is a

vector of Candidatus Liberibacter africanus. The interaction of scion-rootstock grafts

and its effect on the HLB complex is crucial since CaLas is a phloem-limited- bacteria

and can also be transmitted by grafting. The third key component of the HLB disease

complex is the host. Most Citrus species are HLB-susceptible (Folimonova et al.,

2009). Murraya paniculata belongs to the family Rutaceae and is also highly susceptible

to HLB (Damsteegt et al., 2010). Whereas, trifoliate orange and rough lemon have

shown moderate tolerance to HLB (Albrecht and Bowman, 2012a; Fan et al., 2012).

Interactions of HLB and citrus cultivars are discussed in the following sections.

HLB symptom development depends on the geographic location, temperature,

and citrus types. CaLas is observed to be heat-tolerant. Therefore, CaLas pathogen can

survive and develop symptoms in the temperatures above 30 degrees in hot tropical

and subtropical regions of the world, whereas africanus species is heat-sensitive and

cannot survive hot temperatures. The African psyllid vector T. erytreae can survive in

cool temperatures. Hence, Candidatus Liberibacter africanus-caused HLB is

predominantly found in African countries or in cool and dry places. HLB affected plants

are found to have non-systemic or localized presence of CaLas in different plant organs

such as leaves, roots, and fruit. Hilf (2011) reported that CaLas DNA could not be

detected from seedlings grown from CaLas-infected ‘Sanguenelli’ and ‘Conner’ sweet

33

orange fruit. A similar study investigating potential seed mediated CaLas transmission

from sweet orange fruits also demonstrated that seedlings grown from infected fruits

were not HLB positive over a period of up three years. The authors did not detect any of

the typical HLB symptoms such as blotchy mottle or chlorosis from the regenerated

seedlings and these plants remained qPCR negative for the duration of the study

(Hartung et al., 2010). HLB-affected trees exhibit leaves with mottled, blotchy

appearance and corky mid-veins, as well as stunted root growth and significant loss of

lateral roots biomass (Graham et al., 2013). HLB-affected fruits are lopsided with small

sizes (Vashisth et al., 2016). The internal fruit quality of HLB-affected fruit is highly

compromised. Juice extracted from HLB-affected fruits possesses off flavor and low

total soluble content (Baldwin et al., 2010; Dagulo et al., 2010; Plotto et al., 2010). An

HLB-affected tree also shows the presence of small pointed and erect leaves known as

“rabbit ears,” twin dieback, fruit drop, and off-season flowering.

Status of HLB in Florida and recent developments in HLB research

Florida is the 2nd largest orange juice supplier in the world. ‘Valencia’ and

‘Hamlin’ sweet oranges are the most popular juice producing scion varieties in Florida,

and they are highly susceptible to HLB (Castle, 2013). In Florida, HLB was first found in

Miami Dade county, in 2005 (Bové, 2006). Since then, in the last 13 years, the HLB

infection rate in Florida has approached almost 80% (Singerman and Useche, 2016).

The endemic spread of HLB is causing severe economic losses to the citrus growers

and the citrus industry. However, the combination of UF-CREC and USDA citrus

research team efforts, financial support from the government, and growers’

determination may help to achieve sustainable citrus production under the pressure of

HLB.

34

Prevalent HLB, in Florida, has not only affected the citrus production but also

affected the livelihood of people depending on the citrus industry. Statistical analysis of

economic impacts of citrus greening (HLB) in Florida reported that during 2006-07

through 2010-11, the state of Florida had severe income losses because of reduction in

in citrus industry generated revenues, and about 48% total jobs were lost in the citrus

industry associated employment. Each year, Florida orange production is declining in

comparison to the previous year. Efforts are being taken to improve the production of

HLB affected trees. The citrus research teams at the UF-CREC and USDA are involved

in developing improved HLB tolerant and resistant citrus cultivars, efficient nutrition and

HLB management practices, control of psyllids, and pathological identification of early

disease detection, etc. The citrus breeding program at the UF-CREC has developed

new improved HLB tolerant scion and rootstock selections using conventional and

modern biotechnological approaches. These selections are being tested under

controlled greenhouse conditions and at different trial locations to analyze their

response under HLB. Nutrition is a key factor in HLB disease development. Macro- and

micro-nutrient deficiencies such as zinc (Zn), phosphorous (P), boron (B), calcium (Ca)

and magnesium (Mg) are prominent in the HLB-affected trees. Advanced foliar nutrition

programs are found to be expensive and less effective (Gottwald et al., 2012). The

recent studies of soil application of controlled release fertilizers supplemented with

enhanced micro-nutrient concentrations have shown improved health of HLB-affected

commercial trees (Spyke et al., 2017).

Psyllid control is crucial to minimize the spread of HLB in the citrus orchard.

Insecticidal foliar sprays and scouting are being implemented to identify the presence of

35

the vector and control its population in HLB-affected orchards. Neonicotinoid-containing

pesticides are systemic and have long-lasting residual effects which are used in

controlling psyllids (Boina and Bloomquist, 2015). In addition, engineering-inspired

automated drone surveys and aerial imaging techniques have been used to identify the

spacial patterns of HLB infection in the citrus orchards (Garcia-Ruiz et al., 2013).

Thermotherapy, chemotherapy, and antibiotics have also shown promising but mixed

results. However, these may not be curable solutions but a temporary resort for HLB-

affected plants. The successful development of a viable HLB solution also depends on

the awareness of growers about the dynamics of the disease, information on available

options to manage infected orchards, and sharing HLB success stories among growers.

The citrus extension specialists are the mediators that reach out to the growers and can

effectively convey the research from the laboratory to the field. Programs like the Citrus

Health Management program (CHMA) are being implemented to bring citrus growers

together to combat HLB and become involved in the area-wide HLB management

programs. Also, the UF-implemented fast-track release program provides growers the

opportunity to test newly developed putative HLB-tolerant rootstocks or scions to

accelerate their commercialization.

Effect of HLB infection on scion performance; nutritional deficiencies and low quality fruit production

Mineral nutrients are invaluable for plant growth, development and defense.

Plants encounter environmental and biotic stresses which may cause nutrient

deficiencies in the plants. The nutritional deficiencies in the plants are the biomarkers of

the plant stress and are mostly seen as symptoms on the plants (McCauley et al.,

2011). Balanced nutrition is a key for healthy and productive life for all living beings on

36

the earth, and plants are not an exception. Plant physiology and defense are highly

interrelated to the nutrition level of plants (Dordas, 2008). Imbalance in the plant

nutritional status affects plant growth and thus, reduces immunity to fight against pest

and diseases, resulting in declining yields.

HLB-affected plants exhibit Zn deficiency like-blotchy mottled symptoms on the

leaves. In addition, B deficiency is prominent in the HLB-affected plants, which show

vein-corking symptoms on the leaves. HLB-affected plants are found to be deficient in

Mn, Fe, Ca, Mg and P, whereas, potassium (K) level is found to be increased (Spann

and Schuman, 2009). Therefore, HLB-affected plants seem to be severely depleted in

the pool of essential secondary and micronutrients.

Nutrient uptake depends on the plant’s demand for nutrition, the activity of nutrient

transporters, and assimilation and movement of the nutrients to the sink tissue. Uptake

and movement of nutrients in the plants may be inhibited by many constraints such as

inadequate nutrient supply, dysfunctional transporters, or impaired transporting

passages. Of these reasons, reduced nutrient supply and impaired vascular (phloem)

system are responsible for nutrient deficiencies and source-sink imbalance in the HLB

affected and the symptomatic citrus plants. Callose depositions, starch accumulation,

and phloem degeneration also hinder translocation of nutrients in HLB-affected plants,

causing premature fruit-drop and low-quality fruits. Small and lopsided fruits produced

from HLB-affected plants are non-marketable in the fresh fruit market and produce

mediocre quality juice (Bessanezi et al., 2009). Fruits harvested from HLB- symptomatic

and asymptomatic trees have been found to contain higher concentrations of limonin

and nominin, and undesirable juice flavor (Baldwin et al., 2010). Consistently declining

37

fruit yields and fruit juice quality, and escalating expenditures on nutrition programs due

to HLB, affect the grower’s interest in investing in citrus orchard management or in new

citrus plantings.

Interaction of citrus roots with HLB

The citrus rootstock is invaluable for the citrus tree health, enhanced immunity,

fruit quality, production, and tree hardiness (Ribeiro et al., 2014). The performances of

citrus scions grafted on commercial rootstocks such as Swingle, Cleopatra, Sour orange

have been observed to be highly compromised under HLB pressure in Florida. Fibrous

roots are important for nutrient and water uptake. HLB-affected plants have been

observed to show a decline of 30%-50% capacity of water and nutrient uptake due to

the stunted root growth and loss of fibrous root density (Johnson and Graham, 2015).

Also, presymptomatic CaLas-infected root systems have been found to lose about 30%

root density (Graham et al., 2013). Johnson et al. (2014), reported presymptomatic

colonization of CaLas in infected plants. A greenhouse study on the movement of

CaLas in HLB-affected plants showed that CaLas colonizes roots first, uses root-starch

to multiply, and then moves to the scion. HLB-affected plants exhibit substantial dieback

and low starch content (Etxeberria et al., 2009). A similar observation was reported by

Li et al. (2003) showing a rapid decline in the carbohydrate reserve in the roots of

phloem disrupted and girdled plants. The decrease in the CaLas-infected roots starch

suggests that CaLas multiplies at the cost of root-starch and thus, reduces the supply of

carbohydrates for root growth. Root starvation leads to stunted root growth and

depletion of nutrients and water uptake (Johnson et al., 2014). Increasing awareness

about root damage in HLB-citrus interactions is drawing the attention of growers to the

importance of rootstocks in HLB disease control and maintaining root health.

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Breeding of citrus scion and rootstock for HLB tolerance and resistance

HLB is prevalent in Florida and now affects more than 80% of citrus trees

(Singerman and Useche, 2016), and has reduced Florida citrus fruit and juice

marketability. Commercial sweet orange and grapefruit varieties account for 95% of

citrus fruit production in Florida, all which are highly susceptible to HLB. The popular

rootstocks: Swingle citrumelo, Cleopatra mandarin, and Carrizo citrange, and scions:

‘Valencia,' and ‘Hamlin’ sweet oranges, and ‘Ruby Red’ and ‘Marsh’ grapefruits are

intolerant to HLB. New rootstock varieties in Florida have been developed for

Diprepes/Phytophthora complex resistance, citrus tristeza virus (CTV) resistance, cold

hardiness, higher vigor, etc. Florida-grown sweet orange and grapefruit juice quality is

favored in the domestic and international market. However, the epidemic of HLB in

citrus orchards has been decreasing tree survival and sustainable fruit production.

Understanding the urgent need for HLB tolerant or resistant cultivars, the citrus

breeders at UF-CREC and USDA have been developing improved cultivars that can

survive and produce sustainable fruit yield under the high impact of HLB. In the case of

HLB, sometimes CaLas-infected plants show biological tolerance. However, it is short

lived. There are no truly HLB-resistant cultivars available. Realistic expectations and

knowledge of scion/rootstock combination field performance are required to produce

biologically HLB-tolerant and economically profitable citrus rootstock or scion cultivars

(Castle, 2016). In addition, the knowledge of CaLas strains, vector and nutrition

dynamics are important in finding true tolerance to HLB.

Field evaluations of UF-CREC developed rootstocks at St. Helena, Dundee, FL,

showed fruits harvested from seven-year old CaLas-infected ‘Valquarius’ and ‘Vernia’

scions grafted onto different tetraploid somatic hybrid and ‘tetrazyg’ rootstocks had

39

higher amounts of soluble solids/per box than commercial rootstocks (Grosser et al.,

2013). Scions grafted onto UF-CREC developed tetraploid and diploid rootstock showed

a lower percentage of CaLas-infection as compared to the scions grafted onto

commercial rootstocks after 5 years (Grosser 2013). The UF-CREC developed UFR

series rootstock hybrids have shown tolerance and lower incidence of HLB with

increased yield/tree (Castle et al., 2016). Also, UF-CREC developed scions ‘LB8-9’

(Sugar Belle®), and Orie Lee Late (OLL) sweet orange varieties have shown promising

tolerance to HLB when grafted onto conventional rootstocks (Grosser et al.; Stover et

al., 2016). The USDA citrus breeding program has also developed improved HLB-

tolerant rootstock cultivars, of which mandarin and Poncirus trifoliata hybrid rootstocks

are showing promising HLB tolerance, such as US-942 (Vicky, 2014). Conventional

scion/rootstock combinations ‘LB8-9’ (Sugar Belle®) /sour orange and Tango/Kurhakse

have been reported to show high growth rate and increased production in the presence

of HLB (Stover et al., 2016)

Use of Sequencing Technologies in Plants

Sequencing technologies in non-model plants

Model plant species have many biological and physiological characteristics that

make them an appropriate choice for genomic research. The complete genome

sequence of Arabidopsis has opened the door for genomic studies of other plants with

more complex genomes. Plant genomic information is useful for breeding and

understanding the evolutionary development of the species. However, not all plant

species genomes are amenable to sequencing using available technologies. The four

major constraints in building high quality genome assembly in non-model plants are

discussed by Hirsch and Buell (2013). These constraints are genome duplication, ploidy

40

level, heterozygosity, and repetitive sequences. In non-model plants, sequencing

guided genomic studies may produce inadequate information. Hence, model plants

such as Arabidopsis, rice, and maize genomes are commonly used as a reference

genome for alignment and annotation of newly studied plant species. In recent years,

the next generation sequencing (NGS) platforms have enabled fast and reliable

extraction of genomic information from non-model species (Strickler et al., 2012;

Unamba et al., 2015). Today, many plant genomes have been sequenced including

Citrus species such as ‘Clementine’ mandarin (Citrus clementine) and Citrus sinensis

(Wu et al., 2014). NGS platforms are less time-consuming, and comparatively

inexpensive. NGS-high throughput sequencing (HTS) platforms are dominated by

companies such as Illumina (sequencing by synthesis), and ABI/SOLiD (bridge

amplification), and new innovative technologies providers are emerging as competitors.

Bioinformatics are important to analyze the sequencing information to identify genes

and their functional characterization (Thimm et al., 2004; Conesa et al., 2005; Magi et

al., 2010).

The sequencing technologies have facilitated genome sequencing, transcriptome

profiling, development of single nucleotide polymorphism (SNP) and microsatellite

markers, genome wide association (GWAS) of markers with traits of interest,

identification of copy number variation, co-expression analysis of genes, and

methylation pattern profiling (Lister et al., 2009; Voelkerding et al., 2009). In horticultural

crops, NGS platforms have been used to sequence genomes de novo and to study

regulatory mechanisms (Abdurakhmonov, 2016). NGS platforms facilitate analysis of

regulatory genes, metabolic pathways, protein networking, and identifying molecular

41

markers associated with horticultural traits. The information obtained from NGS

platforms can be used to improve the marketable traits of the plant produce through

breeding.

Transcriptome analysis using RNA sequencing

RNA sequencing (RNA-seq) technology is a revolutionary tool of NGS that can

perform transcriptome profiling, gene expression analysis, and re-sequencing.

According to the central dogma of molecular biology, sequential conversion of DNA to

proteins goes through two steps. The first one is a transcription of biopolymer DNA into

RNA, and the second is a translation of RNA into proteins. The aim of RNA-seq

technology is to catalogue the transcriptome of an organism. Plants contain messenger

RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). In addition, small

RNAs, micro RNAs, non-coding RNAs have been found to play a key role in plant

responses (Chu and Rana, 2006; Wolfswinkel and Ketting, 2010). Transcriptomic

studies use different approaches to profile the quantitative or qualitative differential RNA

changes in the given condition of an organism (Wang et al., 2009). One of the

approaches in studying transcriptomics is the use of microarray analysis that allows

identifying gene expression changes using a hybridization technique. However, its

application is limited to quantify known genes of the species (Wang et al., 2009). In

contrast to the microarray method, RNA-seq is a sequence-based approach, and more

reliable because of short reads and deep sequencing (Sims et al., 2014). RNA-seq

produces raw data in the form of short strings of nucleotides that are called reads. The

reads can be reproducible for any biological and technical replicates. However,

technical difficulties may arise in laboratory preparations of cDNA that may need to be

optimized for different species. Analysis of RNA-seq generated data requires a highly

42

precise computational method for mapping and quantifying the genes or transcripts. The

computational techniques perform reads cleaning, reads alignment to the reference

genome, expression quantification, normalization of gene expression counts, differential

gene/isoform expression, and in some cases transcriptome reconstruction (Garber et

al., 2011).

Citrus-HLB interaction: omics studies

Citrus cultivars have shown a various range of reactions to HLB in infected

plants. Among these, most of commercially used mandarin, sweet orange and grapefruit

varieties are sensitive to HLB, showed strong symptoms of HLB.

Whereas,’Volkameriana’ and ‘Eureka’ lemons, ‘Mexican,' ‘Persian’ and ‘Palestine

Sweet’ limes, ‘Meiwa’ Kumquat, Citron, Citrus micrantha, Poncirus trifoliata, and

Severnia buxifolia are moderately tolerant or tolerant to HLB. Pummelo varieties; ‘HBP,'

‘Siamese Sweet’, ‘Ling Ping Yau’, and other citrus such as C. amblycarpa, C. indica,

and Cleopatra mandarin showed variable interaction with HLB, exhibiting mild HLB

symptoms and less reduction in growth as compared to the sensitive cultivars

(Folimonova et al., 2009). The UF-CREC developed the UFR series rootstocks and

‘LB8-9’ SugarBelle® scion, and the USDA created the US series rootstocks and some

scion showing improved tolerance to HLB. The interactions between citrus and HLB are

also studied using transcriptomic and proteomic analyses which showed that HLB

infection could significantly alter the expression of the genes that are involved in plant

defense, hormonal regulations, sugar metabolism, nutrient metabolism, and vascular

tissue development. The gene expression and protein profile changes differ with the

stage of symptoms, plant tissue, and the cultivars. Some of these interactions are

discussed in the following sections.

43

Transcriptomic analysis of CaLas-infected leaves of rough lemon and sweet orange

Time-course dependent comparative differential gene expression analysis was

conducted between CaLas-infected and non-grafted leaves sampled from Rough lemon

and ‘Madam Vinous’ sweet orange using microarray technology (Fan et al., 2012). The

results of microarray analysis showed that rough lemon upregulated callose hydrolyzing

1–3 β glucanase transcripts at the late stage (27 weeks after infection) of the infection.

Rough lemon also overexpressed group of genes encoding cell wall modifying proteins

xyloglucan endotransglycosylases (XET) at the late stage of infection compared to the

sweet orange. In addition, CaLas-infected rough lemon leaves showed activation of

genes encoding defense associated WRKY transcription factors and signaling kinases

at the early stage of infection (5 weeks after infection). In contrast, CaLas-infected

sweet orange leaves upregulated defense related genes at the late stage of infection.

The results of the comparative transcriptome analysis between CaLas-infected leaves

of rough lemon and ‘Madam Vinous’ sweet orange showed the variety dependent

response of citrus to HLB. This study showed that the delayed defense in the sweet

orange leaves might contribute to the disease development. Whereas, early stage

defense activation and cell wall modifying enzymes in rough lemon might inhibit the

further spread of CaLas, and create a barrier to the spread of HLB in the non-infected

plant parts.

Transcriptomic analysis between CaLas-infected and healthy roots and stem sampled from ‘Valencia’/Swingle combination

Microarray based comparative transcriptome analysis between HLB-affected and

healthy ‘Valencia’/Swingle combinations showed that a total of 885 genes in stems and

111 genes in roots were differentially expressed (Aritua et al., 2013). The

44

downregulated genes in the HLB-affected plant tissue belonged to carbohydrate

metabolism, cell wall biogenesis, biotic and abiotic stress response, transportation, cell

wall organization, hormone signaling, metal binding and redox functional categories. In

the same study, microscopy analysis of HLB-affected roots was also conducted.

Microscopy of the HLB affected stem tissue showed the collapse and thickening of the

cell wall, and also showed the depletion of starch in the roots of CaLas-infected plants

as compared to the healthy plants.

Transcriptomic analysis of DEGs in CaLas-infected leaves between non-grafted US-897 and Cleopatra mandarin rootstocks

Microarray assisted comparative transcriptomic analysis between CaLas-infected

USDA developed US-897, and commercial Cleopatra mandarin rootstocks (both non-

grafted) showed 3412 genes were significant differently expressed in response to

CaLas infection (Albrecht and Bowman, 2012b). A total of 326 genes were considerably

overexpressed in Cleopatra compared to only 17 genes in the US-897 rootstock. Genes

belonging to different biological functional categories were upregulated in CaLas-

infected Cleopatra leaves. These include genes encoding expansin proteins (6 fold),

pathogenesis related (PR) proteins, enzymes of carbohydrate metabolism, proteins

involved in oxidation-reduction processes and other unknown proteins. Genes involved

in plant defense were also upregulated (10-40 fold) in Cleopatra leaves. In CaLas-

infected US-897 leaves, genes encoding for biotic stress related protein

CONSTITUTIVE DISEASE RESISTANCE (CDR1), COPPER/ZINC SUPEROXIDE

DISMUTASE 1 (CDS1), and VITAMIN C DEFECTIVE 2 (VTC 2) were upregulated.

CDR1 is associated with salicylic acid mediated plant defense. CDS1 is a reactive

oxygen species (ROS) scavenger that downregulates the hormful effects of ROS. The

45

results of this study suggested that upregulation of antioxidants such as CSD1 and

defense related gene CDR1 in US-897 might be a possible mechanisms that counters

the attack the HLB pathogen. Overall, the DGE analysis in leaves of CaLas-infected

tolerant US-897 and susceptible Cleopatra mandarin suggested that susceptibility or

tolerance to HLB is correlated to the magnitude and speed of the plant defense

mechanism to counter attack the pathogen infection.

Genes associated with HLB susceptibility

Symptoms are the physical manifestation of the disease. The disease symptoms

are the results of stress generated in the plant because of unfavorable pathogen

colonization and/or plant defense response at the different stages of disease

development. Comparative DGE analysis of CaLas-infected leaves of susceptible

Cleopatra mandarin and tolerant US-897 showed upregulation of genes that were

overexpressed only in Cleopatra but not in US-897 (Albrecht and Bowman, 2012b).

Among these, expression of the gene encoding Myb-like HTH transcriptional regulator

family protein was found to be increased by 200-fold in Cleopatra but not in US-897. A

Myb-like transcriptional regulator was also expressed in Bos Noir phytoplasma infection

of grapevine and related to the resistance to the phytoplasma infection (Albertazzi et al.,

2009). In the case of HLB, higher expression of the gene encoding for Myb-like HTH

transcriptional regulator was found in susceptible Cleopatra cultivar. Therefore, this

study suggested that the Myb-like HTH transcription regulator, unlike in grapevine

disease, is associated with HLB susceptibility (Albrecht and Bowman, 2012b). HLB-

affected plants exhibit symptoms similar to that of Zn deficiency. Therefore, susceptible

CaLas-infected Cleopatra showed upregulation of ZINC TRANSPORTER 5

PRECURSOR (ZIP5) to compensate for disease-induced Zn deficiency. Upregulation of

46

ZIP5 was also observed in HLB-affected ‘Valencia’ fruit (Martinelli et al., 2013). CaLas-

infected plants show excessive accumulation of starch (Etxeberria et al., 2009). CaLas-

infected Cleopatra mandarin and other sweet oranges such as ‘Valencia’ and ‘Madam

Vinous’ showed the significantly elevated level of genes encoding enzymes involved in

the starch biosynthesis (Fan et al., 2012; Albrecht and Bowman, 2012b). The higher

accumulation of starch in HLB-affected plants is supported by a phenomenon called

microbial volatile-induced starch accumulation process (MIVOISAP) which is a microbial

strategy for the survival inside the host tissue where pathogen-induced volatiles affect

the carbohydrate metabolism and accumulation of starch in the plant organs (Ezquer et

al., 2010). Also, downregulation of plastidial thioredoxin level is associated with plant

pathogen activated volatiles which cause starch accumulation (Ezquer et al., 2010).

Thioredoxin family protein level was also downregulated in CaLas-infected Cleopatra

suggesting its connection to the starch accumulation and therefore, susceptibility to

HLB. Transcripts encoding PHLOEM PROTEIN 2-B15 (PP2-B15) was strongly

upregulated in Cleopatra compared to the US-897. At the initial stage of HLB

development, upregulation of transcripts encoding PP2 is beneficial in being involved in

differentiation of vascular tissue and plugging of sieve plates. In the advanced stage of

HLB, upregulated expression of transcripts encoding PP2 results in a barrier for the

translocation of nutrients through phloem which ultimately creates local or systemic

nutrient deficiencies. A homologue of GAST1 (GASA1) was found to be highly

upregulated (>15 fold change) in the CaLas-infected ‘Madam Vinous’ sweet orange

leaves compared to the CaLas-infected rough lemon leaves, suggesting its potential

role as an HLB-susceptibility associated gene (Fan et al., 2012).

47

Genes related to HLB tolerance

In the absence of HLB resistance, long-lasting HLB tolerance is vital to keep

citrus production sustainable. Citrus types such as certain pummelos, rough lemon,

Poncirus trifoliata, and some other citrus hybrids have been found to exhibit tolerance to

HLB. However, the endurance and production capacity of the HLB-tolerant cultivars are

not verified. Transcriptome comparison of non-grafted tolerant US-897, and commercial

and HLB-susceptible Cleopatra rootstock was reported (Albrecht and Bowman, 2012b).

The genes that were upregulated in CaLas-infected US-897 and healthy Cleopatra

identify HLB-tolerance candidate genes. Among these, oxido-reductase family 2-

OXOGLUTARATE (2OG) AND FE (II) DEPENDENT OXYGENASE, VEIN

PATTERNING 1 (VEP1), GLUCOSE TRANSPORTER 1 (GLT1), that are involved in

plant defense, herbivore defense, and in export of photoassimilates from chloroplasts

respectively, were upregulated over 30-fold (Albrecht and Bowman, 2012b). UDP-

glycosyl transferase (UGT) superfamily proteins are crucial to impart tolerance to the

virus infection in tobacco. Transcripts encoding UGT superfamily proteins were

expressed in abundance in non-infected US-897. Transcripts encoding PR proteins;

OSMOTIN LIKE PROTEINS (OSM 34) and PLANT DEFENSIN (PDF2.2) were also

overexpressed in CaLas-infected US-897 (Albrecht and Bowman, 2012b). Proteomic

analysis conducted by Martinelli et al., (2016) reported the significant role of

detoxification enzyme; S-glutathione transferases in the moderate HLB tolerant

‘Volkamer’ lemon leaves. Transcriptomic comparison between putative tolerant

‘Jackson’ grapefruit and susceptible ‘Marsh’ grapefruit showed the upregulated

expression of auxin-negative regulators SAUR-like genes and upregulated basal

defense genes in the ‘Jackson’ grapefruit (Wang et al., 2016). UF-CREC developed

48

UFR series rootstocks and ‘SugarBelle®’ scion have also shown sustainable tolerance

to HLB in different scion/rootstock combinations (Stover et al., 2016). However, genetic

regulators that contribute the potential tolerance in the UFR rootstocks and ‘LB8-9’

SugarBelle® scion need to be analyzed.

Comprehensive transcriptome and gene expression network analysis of HLB-citrus interactions

A comprehensive view of HLB response networks was presented using different

HLB-citrus transcriptomic studies (Zheng and Zhao, 2013). The results of the previously

studied HLB-citrus interaction transcriptome datasets were used to build a system view

of the HLB response network using Pearson correlation coefficient-based gene

coexpression analysis. The gene coexpression networks were presented by the core

network, subnetworks, and hubs that showed the interrelation of the genes with each

other. Among these, carbohydrate metabolism, transport, and hormone response

networks had large hubs that were then further subdivided into subnetworks. The

hormonal network showed that plant hormones such as ethylene (ET), abscisic acid

(ABA), salicylic acid (SA), jasmonic acid (JA), and gibberellin (GA) response network

were overrepresented in the HLB-citrus interaction. Subnetwork analysis of SA showed

overrepresentation of protein degradation component UBIQUTINATION 10-LIKE

(UBQ10), transcriptional regulator WRKY40, ASYMMETRIC LEAVES 1 (AS1), MYB,

and carbohydrate metabolism enzyme GSTU7. At the early stage of CaLas infection, it

is difficult to find any visible HLB symptoms, but transcriptome analysis of early stage

HLB infection showed that the plant is already responding to the HLB infection by

overexpressing associated defense genes. In the early stage, the HLB response

subnetwork showed upregulation of ENHANCED DISEASE SUSCEPTIBILITY (EDS-1)

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like gene, transcripts encoding Tetratricopeptide repeat (TPR)- like superfamily protein

and NAC domain containing NAC096 transcription factor. Further subnetwork analysis

revealed that transport is a key process that overexpressed in the HLB response.

Overexpression of the PP2-LIKE gene was reported in resistance to phloem feeding

aphids in Arabidopsis (Zhang et al., 2011a). Upregulation of closest homolog of

Arabidopsis PP2 in citrus at the late and very late stage of infection suggests its role in

defense response to CaLas infection. The subnetwork of PP2 showed the presence of

genes encoding Zn transporter proteins. The presence of Zn family transporter

transcripts in the PP2 subnetwork suggests that Zn binding proteins are important in

arresting the development of disease at an early stage of infection. The results of this

work also showed the overlapping and interconnection of different biological processes

that were activated or suppressed in response to HLB and will be helpful in

understanding the key genes and mechanisms that are potentially involved in regulation

of CaLas-citrus interaction.

Citrus co-expression networks built from microarray data also showed significant

presence of HLB network (Du et al., 2015). The results of this study showed 28 nodes,

75 edges, and 0.198 density of the HLB network using random matrix theory. The

highest rank gene ontology (GO) terms represented in HLB module was programmed

cell death (PCD). The PCD network showed upregulation of transcripts encoding

POLYUBIQUITIN 10, SPHINGOID BASE HYDROXYLASE, Glutaredoxin family

proteins, RING finger E3 ubiquitin ligases, and BCL-2-ASSOCIATED ANTHOGENE.

50

Transcriptomic and proteomic analysis of CaLas-infected and non-infected roots of ‘Sanhu’ red tangerine

Comparative analysis of transcriptome and proteome of CaLas-infected and non-

infected roots of ‘Sanhu’ red tangerine showed a total of 3956 genes, and 70 proteins

were differentially regulated (Zhong et al., 2015). CaLas-infected ‘Sanhu’ tangerine

roots showed downregulation of genes involved in ubiquitin dependent protein

degradation pathway, secondary metabolism, cytochrome P450s, UDP-glucosyl

transferase, and pentatricopeptide repeat containing proteins. Upregulation of SPERM

SPECIFIC PROTEIN 411, COPPER ION BINDING protein, GERMIN-LIKE proteins,

SUBTILIN-LIKE proteins and SERINE CARBOXYPEPTIDASE-LIKE 40 protein were

observed in the proteomic comparisons, and the transcriptomic study showed that the

transcripts encoding same genes were also uregulated in the same comparison

suggesting CaLas has evolved a counter attack to the host through proteolysis. CaLas-

infected ‘Sanhu’ roots showed upregulation of a gene encoding PP2, and

downregulation of GLS7, a callose synthase gene. A gene encoding Beta-glucosidase,

a callose hydrolyzing enzyme, was upregulated in the CaLas-infected roots as

compared to the non-infected roots in ‘Sanhu.' Based on these results, authors of this

study concluded that the roots of ‘Sanhu’ tangerine are trying to achieve a balance

between callose synthesis and degradation enzymes as a part of the defense against

HLB.

Proteomic analysis between healthy and CaLas-infected ‘Madam Vinous’ sweet orange

iTRAQ based proteomic analysis between healthy and CaLas-infected sweet

orange leaves showed differential proteomic changes in the asymptomatic and

symptomatic leaves (Fan et al., 2011). A total of 686 proteins were expressed uniquely

51

in the differential comparison between asymptomatic and symptomatic leaves. Among

these, MIRACULIN LIKE proteins, CU/ZN SUPEROXIDE DISMUTASE, and

CHITINASE were significantly accumulated in the CaLas-infected asymptomatic leaves.

Comparative proteomic analysis of symptomatic and pre-symptomatic CaLas-infected grapefruit leaves

‘Duncan’ grapefruit leaves were analyzed for comparative protein profiles

changes in response to the CaLas infection using 2D protein gels (Nwugo et al., 2013b).

This study identified 191 significantly altered protein spots in response to CaLas

infection. Of these, 56 proteins were downregulated in CaLas-infected and symptomatic

‘Duncan’ leaves as compared to healthy leaves. These proteins were associated with

photosynthesis, protein synthesis, and metabolism. Whereas, an OXYGEN-EVOLVING

ENHANCER (OEE) and RNA polymerase B TRANSCRIPTIONAL FACTOR 3 (BTF3)

proteins were downregulated in pre-symptomatic and symptomatic ‘Duncan’ leaves.

This study also found CU/ZN SUPEROXIDE DISMUTASE, CHITINASE, lectin-related

proteins, MIRACULIN-LIKE proteins, and peroxiredoxin proteins were upregulated in

CaLas infected ‘Duncan’ leaves. The significance of this study was to establish the

correlation between protein and nutritional profiles in CaLas-infected and symptomatic

‘Duncan’ leaves, where increased concentrations of potassium in the CaLas-infected

‘Duncan’ leaves were correlated to the upregulated granule-bound starch synthase

proteins.

Research Overview

Since the first incidence of HLB in Florida, there are a considerable number of

studies conducted that have generated a lot of information about the HLB disease

complex. The lack of enough information about CaLas biology limits the research

52

opportunities to find solutions for HLB control. HLB is a high impact disease that is

causing vast economic losses to the Florida citrus industry. The contemporary HLB

research is focused on developing CaLas and psyllid control strategies including

thermotherapy, antibiotics, and chemical control. However, to control the endemic

spread of HLB, economically viable solutions are required. The vulnerability of existing

commercial citrus cultivars to HLB has been studied under both laboratory and field

conditions. The improved citrus scions or rootstocks may bring a practical solution that

can reverse the negative impact of HLB in CaLas-infected plants. Therefore,

economically enduring-HLB tolerant citrus cultivars can be a long-term solution to

combat the existing sustainability issues associated with HLB-affected plants. There are

a few rootstocks and scion cultivars showing some enhanced level of HLB tolerance

that have been released for commercial citrus production, and several additional

promising selections in the breeding pipeline. The overall goal of this study is to identify

the molecular mechanisms that potentially contribute to the sustainability of ‘Valencia’

sweet orange scion grafted onto two selected putative HLB-tolerant CAN rootstocks

currently in the breeding pipeline: 42x20-04-48 (‘HBP’ x ‘Cleopatra’ mandarin) and 4x

hybrid (‘Amblycarpa’ mandarin + ‘Volkamer’ lemon cybrid) x [‘Nova’ + ‘HBP’ pummelo x

Argentine trifoliate orange]. In addition, the effect of root applied enhanced controlled-

release micronutrient formulations was also analyzed to test the performance of the

HLB susceptible Valencia/Swingle as well as the latter Valencia/CAN combination

above. The results of this study will be a useful contribution in developing HLB tolerant

cultivars by providing insights into the mechanisms responsible for the tolerance

53

response. Finally, a better understanding of rootstock genetics/nutrition interactions

should lead to improved nutrition practices for HLB management.

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CHAPTER 2 COMPARATIVE ANALYSIS OF TRANSCRIPTOME AND PLANT PHENOTYPES OF

CaLas-INFECTED SCION/ROOTSTOCK COMBINATIONS

Introduction

In Florida, commercial citrus production is focused on growing sweet orange

(Citrus sinensis [L.] Osbeck) and grapefruit (Citrus paradisi [Macf.]) varieties. Sweet

orange cultivars are grown for homeland and premium export quality juice production.

However, HLB-affected citrus trees make it more difficult to fulfill the export quality juice

standards because of off-flavors and color, and lowered Brix-acid ratio. The endemic

spread of HLB has resulted in the consistently declining net productivity of HLB-affected

citrus orchards. In 2014-15, Florida commercial citrus production was the lowest

recorded citrus production with increased gross loss as compared to previous years

since 2009 (USDA, 2014). Increasing economic losses caused by HLB in Florida, and

the lack of true resistance to HLB in the citrus germplasm demand economically

enduring HLB-tolerant cultivars that have potential to reduce HLB severity and increase

sustainability of HLB-affected plants. Field- and greenhouse-based studies of the

interaction between various Citrus species and closely related genera with HLB have

been conducted. The interactions between citrus cultivars and HLB are classified as

sensitive (chlorosis, vein corking and reduced growth, death), tolerant (little or no HLB

symptoms), moderately tolerant (mild HLB symptoms on older leaves or scattered

chlorosis on groups of leaves), or inconsistent presence or absence of bacteria based

on symptomatology and Candidatus Liberibacter asiaticus (CaLas) bacteria titer in the

plants (Folimonova et al., 2009). Of these responses, tolerant or moderately tolerant

responses were found in some closely related species and genera of citrus. Trifoliate

orange (Poncirus trifoliata [L.] Raf), rough lemon (Citrus jambhiri Lush.), pummelos

55

(Citrus maxima Merr.), lemons (Citrus limon L. [Burm.] f.), and their hybrids have shown

tolerance to HLB (Fan et al., 2012; Albrecht and Bowman, 2012b; Aritua et al., 2013;

Fan et al., 2013; Martinelli et al., 2016). However, the tolerance of these citrus species

and their hybrids is not consistent. The performance consistency of HLB-tolerant

cultivars is highly dependent on the psyllid control, interaction with other diseases,

environmental challenges, nutritional program, and farm management practices

(Gottwald et al., 2012; Haapalainen, 2014; Castle, 2015). Against all the odds of HLB,

citrus breeders have engaged in developing improved citrus cultivars for HLB tolerance,

and perhaps even resistance using different breeding approaches.

The University of Florida, Citrus Research and Education Center (UF-CREC) at

Lake Alfred is the world renowned citrus research facility. UF-CREC research teams

have contributed to the Florida citrus industry over the past 100 years through their

research, extension, and teaching programs (www.crec.ifas.ufl.edu). The breeding

program at UF-CREC has always been engaged in developing the improved citrus

cultivars that can fulfill the demand of growers and citrus-associated food industries

(Grosser et al., 2015). In the UF-CREC citrus breeding program, putative HLB-tolerant

rootstock and scion hybrids have been developed by crossing non-commercial HLB-

tolerant and commercially accepted citrus cultivars. There is no citrus cultivar available

to defend against HLB and improved plant performance when the plant is CaLas-

infected. However, HLB tolerance can be improved by enhancing regeneration ability of

the phloem, regrowth of the affected root system, and reducing CaLas titer. Differential

gene expression (DGE) analysis of CaLas-infected rough lemon and sweet orange

showed the higher phloem regeneration ability of rough lemon (Fan et al., 2012). Rough

56

lemon tolerance to HLB is also reported in India (Cheema et al., 1982). Some of the

pummelo cultivars and trifoliate orange are found to be less affected by HLB (Shokrollah

et al., 2011). However, these cultivars cannot produce commercially accepted fruit and

juice as compared to the commercially used rootstock/scion combinations. In Florida,

commercially used rootstock cultivars: Swingle citrumelo (Citrus paradisi x Poncirus

trifoliata), Cleopatra mandarin (Citrus reticulata Blanco), Carrizo citrange (Poncirus

trifoliata X ‘Washington’ sweet orange), and scions ‘Valencia’ and ‘Hamlin’ sweet

oranges, and grapefruit cultivars are HLB susceptible. Therefore, to create HLB-tolerant

and commercially viable cultivars, hybrids between HLB-tolerant Citrus species and

high quality fruit producing cultivars are required.

The UF-CREC citrus breeding program has developed scion and rootstock

hybrids which exhibit HLB-tolerance with improved plant performance. The newly

developed putative-HLB tolerant rootstocks have been showing promising results in

mitigating HLB severity in the CaLas-infected plants. Some of these putative HLB-

tolerant rootstocks, which have been commercially available since 2009, yield higher

fruit production and increased juice quality in grafted CaLas-infected scion (Castle et al.,

2016). Also, some of the scion hybrids have shown reduced HLB symptoms. However,

there is no published data available on commercial scion tolerance except ‘LB8-9’

SugarBelle® variety (Stover et al., 2016). The tolerance of the newly developed cultivars

might not be economically viable because of possible scion-rootstock compatibility

issues, the impact of rootstock on the scion, or undesirable changes in the fruit quality.

Field evaluations of the CaLas-infected scion/newly developed rootstock combinations

are important to test the economic viability of the new rootstock hybrids. A field trial of

57

one of these newly developed and putative HLB-tolerant candidate rootstocks (CAN)

exhibited superior plant phenotype of CaLas-infected ‘Valencia’ scion grafted onto it as

compared to the Valencia/Swingle control combination after 7 years in the field under

heavy HLB pressure. The performances of these combinations were measured in terms

of visual inspection of HLB symptoms on leaves and fruit quality parameters (Brix, acid,

Brix-acid ratio, and total sugars.). The consistency of HLB-tolerance and plant

sustainability of CaLas-infected ‘Valencia’/CAN suggested a more enduring

performance of the rootstock when the plant is CaLas-infected. Therefore,

‘Valencia’/CAN trees were selected to analyze transcriptomic differences in the leaves

and roots of trees identified in the field exhibiting asymptomatic and symptomatic stages

as compared to the commercial standard ‘Valencia’/Swingle combination.

In the contemporary biological sciences, omics-assisted methods of research are

very popular and widely used to reveal the regulatory mechanisms in the different living

organisms. The omics studies require precision instrumentation and high throughput

computing technology to give quicker and highly accurate results. The demand and

competition between technological developments are out competing the old techniques

of omics-assisted research, and transcriptome studies are no exception. The next

generation sequencing-based RNA sequencing (RNA-seq) techniques are rapidly

evolving. RNA-seq not only identifies the known transcripts/genes but also discloses the

unidentified genes, identifies alternate splicing events, quantifies the isoforms,

measures differential allelic expression, and detects non-coding RNAs. High throughput,

improved resolution, and less background noise have increased the popularity of NGS

techniques in the global gene expression studies of plants, animals, microbes, and

58

humans. The International Citrus Genomics Consortium (ICGC) is a collaboration of a

worldwide group of scientists, students, and industry leaders associated with citrus. The

goal of ICGC is to understand the underlying genetic structure of citrus. Many diverse

pummelo, mandarin and sweet orange citrus genomes have sequenced (Xu et al.,

2013; Wu et al., 2014). The Clementine mandarin (Citrus clementina) and sweet orange

genomes have also sequenced, and data are available on the Phytozome portal.

Phytozome is the plant comparative genomics portal of the Department of Energy’s

Joint Genome Institute (Goodstein et al., 2012). The NGS or microarray technologies

have been used to perform DGE analysis in many citrus studies. These studies are

physiological development (Terol et al., 2016), nutrient deficiency (Licciardello et al.,

2013), disease and pest interactions (Gandía et al., 2007; Boava et al., 2011; Wang et

al., 2016), genotypic differences, and gene expression network (Du et al., 2015). HLB-

citrus interactions are also studied using global gene expression transcriptomic

approach (Albrecht and Bowman, 2008; Fan et al., 2011; Febres et al., 2012; Martinelli

et al., 2012). RNA-seq technology has been used successfully in understanding the

molecular mechanisms that cause phenotypic changes. The aim of this study is to

determine potential molecular mechanisms that enhance sustainability in ‘Valencia’

grafted onto the CAN rootstock with RNA-seq. The results of this study are important to

the understanding of possible molecular mechanisms which are involved in reducing

HLB damage in citrus and developing HLB-tolerant or resistant citrus cultivars using

conventional breeding as well as advanced biotechnological approaches such as gene

transfer and genome editing.

59

Materials and Methods

Plant Materials

Two scion/rootstock combinations were used in this experiment. Field grown

seven year-old experimental plants were identified in the Lee Family's Alligator Grove,

east of St. Cloud, Florida. The first combination of trees was ‘Valencia' (VAL) sweet

orange grafted onto a putative HLB-tolerant candidate (CAN) rootstock. The CAN

rootstock (46x20-04-48) is a hybrid of ‘Hirado Buntan Pink' (HBP) pummelo and

Cleopatra mandarin. The 2nd combination was ‘Valencia' (VAL) scion grafted onto

Swingle citrumelo (SW), a standard commercial rootstock. Swingle is a hybrid of

grapefruit and trifoliate orange. In each combination of VAL/CAN and VAL/SW, plants

were divided into two treatments based on the visible presence of HLB-like symptoms

(Table 2-1). Highly infected and symptomatic trees in each combination grouped into

the symptomatic treatment (Figure 2-1A and B; Figure 2-2A and B). Whereas, trees with

very few or no visible symptoms were grouped into the asymptomatic treatment (Figure

2-1C and D; Figure 2-2C and D). All biological replicates in each treatment and

combination were tested using quantitative PCR (qPCR) based CaLas detection, which

validated that all trees in the study were indeed infected. Enzyme-linked immunosorbent

assay (ELISA) was performed to detect Citrus Tristeza Virus (CTV) in the samples.

Sampling

Sampling of different plant organs (leaves, roots, and fruits) was conducted to

study DGE analysis and fruit juice quality of the plants. In each treatment of both

scion/rootstock combinations, leaves and fibrous roots from all quadrants of the tree

were collected for CaLas detection, DGE analysis and real time PCR based gene

expression validation studies, in April 2015. Leaves (December 2014, April 2015,

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December 2015) and fruits (April 2015 and April 2016) were sampled in two consecutive

years to analyze changes in HLB status and fruit juice quality. Three separate biological

replicates of asymptomatic and symptomatic treatments were selected in each

VAL/CAN and VAL/SW combination for DGE analysis and juice quality testing. Leaf and

root tissues collected for the RNA sequencing, were saved at -80º Celsius until RNA

extraction.

Fruit Juice Quality Analysis

Juice quality of fruit collected from each biological replicate in each treatment

was analyzed. In each biological replicate, approximately 30–35 fruit were collected to

analyze the juice quality. The commercial juice quality is judged by its Brix, acid

percentage, and Brix-acid ratio. The same analyses were conducted for fruit harvested

in years 2015 and 2016 from the both treatments of VAL/SW and VAL/CAN

combinations. Fruit Juice analyses were conducted at the Tropicana facility located at

Bradenton, FL.

CaLas and Citrus Tristeza Virus (CTV) Detection

Approximately 10 fully expanded leaves and enough root tips were collected from

both the treatments in VAL/CAN and VAL/SW combinations to extract DNA for CaLas-

detection. GenElute™ Plant genomic DNA Miniprep kit was used to extract DNA from

the petiole and leaf midrib (Sigma Aldrich, Woodlands, TX). Root DNA was isolated

using PowerMax® Soil DNA Isolation kit (MOBIO Laboratories, Inc., Carlsbad, CA). The

extracted DNA was then quantified in a NanoDrop ND-100 spectrophotometer (Thermo

Scientific, Wilmington, DE). The qPCR assays were performed using CaLas specific

probe and primer pairs which are developed by Ananthakrishnan et al. (2013). The

cytochrome c oxidase (cox) subunit Vc gene forward and reverse primer pair, and

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corresponding probe were used as internal control. Amplification was performed for 40

cycles using ABI7500 real-time PCR machine (Applied Biosystems, Foster City, CA)

and the TaqMan® Gene Expression Master Mix (Applied Biosystems). All reactions

were carried out in a 25 µl reaction volume containing 50 ng DNA and 0.3 µM probe and

primer pair concentrations. The CTV detection was performed using the ELISA plates

that are pre-coated with CTV specific antibody (Agdia Co, Elkhart, IN). A total of 0.25

gram of leaf midrib was used to diagnose the CTV from each leaf sample. The

procedure for CTV detection was performed according to the manufacturer’s protocol

(SRA78900, Agdia). CTV-36 and healthy plant tissue were used as the positive and

negative controls, respectively.

RNA Extraction and Quantification

PureLink® Plant RNA Reagent-small scale procedure was used to extract total

RNA from 0.1 gram samples of leaves or roots (ThermoFisher Scientific, Waltham, MA).

PureLink® Plant RNA reagent contains Trizol reagent which uses the guanidium

isothiocyanate RNase inhibitor and phenol-chloroform protein extraction method. It is a

non-column based RNA extraction method that may cause DNA contamination in the

eluted product. Hence, total extracted RNA was treated with DNase. Approximately 100

µl of eluted total RNA was treated with DNA-free™ kit (ThermoFisher Scientific). For

each biological replicate of leaf and root samples for both treatments, RNA was isolated

twice and then pooled together as one sample for downstream processes. RNA

extracted from leaves was tagged A and B followed by the experiment ID, and the roots

RNA samples named as C and D followed by the experiment ID (Table A-1). The quality

and quantity of DNase treated total RNA were determined using the 0.8% agarose gel

and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). RNA integrity (RIN)

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number generated by Bioanalyzer was used to determine the quality of RNA and its

acceptability for RNA-seq library preparations.

RNA Library Preparation and RNA Sequencing

DNase treated total RNA with acceptable RIN value was used to prepare RNA

libraries. Poly-A mRNA magnetic isolation kit (NEW ENGLAND Biolabs® Inc, Ipswich,

MA) and NEBNext Ultra Directional RNA Library Kit for Illumina (NEW ENGLAND

Biolabs® Inc) were used to prepare an RNA library for each sample. NEBNext Ultra

Directional RNA Library Kit for Illumina contains Illumina-specific adapters and

multiplexing index that can be used in Illumina sequencing platform. The NEBNext-

developed protocol is optimized for 200 base pair (bp) RNA insert. For RNA library

preparation, one microgram RNA was used as the starting amount. The manufacturer’s

protocol was followed to prepare RNA libraries of each sample. The protocol contains

steps for preparation of first strand reaction buffer and random primer mix, mRNA

isolation, fragmentation and priming starting with total RNA, first strand cDNA synthesis,

second strand cDNA synthesis, purification of the double-stranded cDNA using 1.8X

Agencourt AMPure XP Beads (Beckman Coulter Inc., Indianapolis, IN), end preparation

of cDNA library, adaptor ligation, purification of the ligation reaction using AMPure XP

Beads, PCR enrichment of adaptor-ligated DNA, and purification of the PCR reaction

using AMPure XP Beads. A total 24 RNA libraries was prepared and sent to the UF

Interdisciplinary Center for Biotechnology Research (ICBR) gene expression core for

sequencing. All RNA libraries were diluted and then pooled together to test the quality

parameters suitable for sequencing (Table A-2). Two sets of pooled libraries were used

for RNA sequencing. Paired-end sequencing, 2 x 150, was carried on Illumina

Nextseq500 high throughput platform using the sequencing by synthesis (SBS) method.

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Raw Data Processing

Nextseq500-generated raw sequencing data were processed using the Tuxedo

bioinformatics pipeline to get the comparative DGE results (Trapnell et al., 2012) (Figure

A-1). The UF High Performance Computing (HPC) developed HiPerGator.1 server was

used to compute and store data processing results. Illumina Nextseq500 platform

generates raw data in the form of fastq files. Raw data processing was executed using a

Unix-based shell script command line (Table A-3). The raw data obtained from each

biological replicate were organized into directories to perform the pairwise comparison

between the treatments, tissues, and the scion/rootstock combinations (Table 2-2). The

Trimmomatic program was used to remove the low-quality reads and trim the Illumina

adapters by assigning leading (20), trailing (20), slidedown (4:20), minlen (36), and

phredscore (33) parameters. Trimmomatic algorithm-generated output files contained

the data which were ready to use for the alignment. The Bowtie 2 program was used to

index the Trimmomatic generated clean sequencing reads and C. clementina, a

reference genome (Phytozome.org.net). The indexed RNA seq reads were then

mapped to the indexed C. clementine genomic data using a TopHat2 algorithm

(Trapnell et al., 2012). Cufflinks and Cuffmerge algorithms were used to quantify the

abundance of the transcripts in each biological replicate and then consolidated into a

master transcriptome file (Trapnell et al., 2012). The differential gene expression

changes between the treatments (Table 2-2) were analyzed using the Cuffdiff program.

Cuffdiff normalizes the expression level of differentially expressed genes (DEGs) based

on fragments per kilobase per million (FPKM) as explained by Trapnell et al., 2010. The

DGE changes were expressed in the log2 fold change (log2FC). CummeRbund, an R

64

statistical package based program, was used to overview and visualize the results of

Cufffdiff output (Goff et al., 2012).

Functional Analysis and Gene Ontology of DEGs

DEGs were chosen based on significance determined by a Cuffdiff algorithm,

and used for gene ontology (GO) and functional analysis studies. DEGs obtained in the

comparative analysis between VAL/CAN and VAL/SW treatments were annotated to the

Arabidopsis thaliana genes, which are available in the Phytozome database. Functional

analysis of the differentially expressed and A. thaliana annotated genes was performed

using MapMan tool (Usadel et al., 2009).The signaling pathways and regulatory

components of the significant differentially expressed genes were analyzed using

Pathway Studio (Nikitin et al., 2003) and MapMan. DEGs were assigned into gene

ontology (GO) biological processes using a Blast2GO algorithm (Conesa et al., 2005).

The biological importance of the significant differentially expressed genes was

determined by combining the results from MapMan, Blast2GO and Pathway Studio.

Validation of the RNA-seq Data

Quantitative real-time reverse transcriptase (qRT-RT) PCR technique was used

to validate the genes that were differentially expressed in the RNA-seq analysis. Seven

genes were selected to validate gene expression from RNA-seq results (Figure 2-9).

These genes were selected based on their significant changes in the expression of

levels in the leaves and roots of symptomatic and asymptomatic treatments between

VAL/CAN and VAL/SW combinations. These genes encode BON ASSOCIATED

PROTEIN 2 (BAP2), BR (Brassinosteroid) LEUCINE RICH RECEPTOR BRI1, NPR1-

like 3 (NPR3), MYO-INOSITOL PHOSPHATE SYNTHASE 2 (MIPS2), Zim domain

containing JAZ8, Pectin methyl esterase inhibitor (PMEI) and LIPID TRANSFER

65

PROTEIN 1 (LTP1). The gene encoding BRI1 (Ciclev10022388) is a BR receptor kinase

and found to be significantly upregulated in the asymptomatic VAL/CAN leaves as

compared to the asymptomatic VAL/SW leaves. BAP2 is identified as a negative

regulator of programmed cell death (PCD) in Arabidopsis (Yang et al., 2007). The loss

of BAP2 function was found to induce a hypersensitive response in Arabidopsis. BAP2

was significantly upregulated in the symptomatic leaves and roots, and downregulated

in the asymptomatic leaves as compared to respective treatments and organs in the

VAL/SW, Therefore, BAP2 was selected to validate the (Ciclev10022388) expression.

NPR3 is a SA signaling receptor, however, it is a negative regulator of defense

response through its interaction with NPR1 (Shi et al., 2013). Significant downregulation

of NPR3 (Ciclev10017873) in the asymptomatic and symptomatic VAL/CAN leaves

indicates the possibility of upregulating NPR1 expression and, therefore NPR1- induced

defense in the VAL/CAN leaves as compared to VAL/SW. Considering the significant

role of NPR3 in regulating NPR1 expression, NPR3 was selected to validate its

expression pattern. MIPS is involved in a rate limiting step in the synthesis of myo-

inositol which is a critical component in the developmental stages (Loewus, 1970;

Donahue et al., 2010). The gene encoding (Ciclev10025400). was upregulated in the

asymptomatic and symptomatic VAL/CAN leaves as compared to the respective

VAL/SW leaves. Therefore, MIPS2 was selected to validate its expression pattern. JAZ8

acts as JA repressor (Wasternack and Hause, 2013). Significant changes in the

expression of JAZ8 may be a clue to understand a JA-SA antagonism in CaLas-infected

plants. Therefore JAZ8 (Ciclev1001798) was selected to validate its expression pattern.

PMEIs are involved in plant development and defense. The overexpression PMEI in

66

Arabidopsis is found to influence the Botrytis cinerea induced susceptibility, and

thereby, confers resistance to Botrytis cinereal (Lionetti et al., 2007). Therefore, PMEI

(Ciclev10007964) was selected to validate RNA-seq expression results. Lipid transfer

proteins (LTPs) is a small class of proteinsthat is involved in various plant functions

such as cutin formation, embryogenesis, defense against phytopathogens, and plant

adaptations to various environmental conditions (Finkina et al., 2016). LTP1

(Ciclev1003524) expression was significantly downregulated in asymptomatic and

symptomatic VAL/CAN leaves as compared to respective VAL/SW leaves. Considering

the critical role of LTP in plant growth and defense, and significant downregulation in

VAL/CAN leaves, LTP1 was also selected for validation.

Forward and reverse primers were designed for the selected genes using

Integrated DNA Technologies (IDT) technologies Primer Quest Tool (IDT, Coralville, IA),

and then amplified using a SYBR green based gene expression protocol. For

normalization, the GLYCERADEHYDE-3-PHOSPHATE DEHYDROGENASE C2

(GAPC) gene was used as the reference gene control. Power SYBR® green RNA-to-

Ct™ 1-Step RT-PCR (Applied Biosystems) master mix was used to convert total RNA

into cDNA, and then cDNA fragments were amplified with the primer pair. A total of 50

ng DNase free RNA and 500 nm of each primer concentration were used to amplify and

quantify the qRT-RT gene expression. Amplification was performed over 40 cycles on a

StepOnePlus™ real-time PCR machine (Applied Biosystems).

Relative gene expression quantification was calculated using the ΔΔCt method

(Livak and Schmittgen, 2001). For each sample, the Ct value of GAPC was subtracted

from the Ct value for the gene of interest (ΔCt). ΔΔCt was calculated by subtracting ΔCt

67

of VAL/SW from ΔCt of VAL/CAN, and the results are presented as log2FC scale. The

visual presentation of the data was created using Microsoft Office Excel10.

Results

Fruit Juice Quality Analysis

In 2015, the Brix values of fruit collected from all the treatments and

combinations were not significantly different. The average Brix value of fruit collected

from the asymptomatic VAL/SW was higher followed by asymptomatic VAL/CAN,

symptomatic VAL/SW and symptomatic VAL/CAN (Figure 2-3A). In 2015, although the

Brix value remained nonsignificantly different in fruit collected from all scion/rootstock

combinations, the acidity percentage was significantly higher in fruit collected from the

symptomatic VAL/SW plants (Figure 2-3A). In 2016, fruit collected from the symptomatic

VAL/CAN combination followed the similar pattern of Brix, acidity percentage, and BAR

ratio as the symptomatic and asymptomatic VAL/SW combination (Figure 2-3B).

PCR Detection of HLB and ELISA Detection of CTV

All 24 samples were tested to identify the CaLas presence in leaf and root

tissues collected from the symptomatic and asymptomatic treatments of VAL/CAN and

VAL/SW combinations (Table 2-3). In April 2015, leaves collected from asymptomatic

VAL/SW biological replicates were CaLas negative; whereas roots collected from

asymptomatic VAL/SW were CaLas positive (Table 2-3). All biological replicates in

symptomatic and asymptomatic VAL/SW combination were CTV positive except one

biological replicate (sample #21) in the asymptomatic treatment. In the same year,

symptomatic leaves and roots collected in VAL/SW combination were all CaLas

positive. In asymptomatic VAL/CAN combination, leaves collected from sample #22 was

CaLas and CTV negative, and sample #23 leaves were CaLas and CTV positive. In

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asymptomatic VAL/CAN combination, sample #24 was CTV-infected with the absence

of CaLas in the leaves, but roots were CaLas-infected. All biological replicates in the

symptomatic VAL/CAN combination were CTV negative; however, VAL leaves were

CaLas positive (Table 2-3). Roots sampled from symptomatic VAL/CAN combination

were either negative (sample #5) or found to have higher Ct value in the range of 34–

36. In December 2015, all biological replicates in symptomatic and asymptomatic

treatments of VAL/CAN and VAL/SW combinations were PCR CaLas positive (Table 2-

3).

RNA Extraction, RNA Library Preparation, and RNA Sequencing

RNA extracted from leaves showed a 260/280 ratio of ≥ 2 and clear separation of

18s and 26s ribosomal bands on the 0.8% agarose gel (Figure 2-4A). Whereas, it was

difficult to get high quality RNA from root tissue (Figure 2-4B). Multiple RNA extractions

were conducted to get high quality RNA from the roots. The average size of libraries

prepared for the sequencing was 390 bp as quantified by qPCR results at the ICBR

facility (Table A-2). The leaf samples tested in this study obtained about 28-53 million

reads/sample, and the root samples obtained 10–40 million reads/sample (Table 2-4).

Raw Data Processing

Trimmomatic trimming, Bowtie indexing, and Tophat alignment generated ≥ 60%

of mapped and paired reads of total raw reads in leaf samples of both the treatments.

except two leaf samples in the asymptomatic VAL/CAN combination that generated a

lower percentage of alignment to the reference genome. In root samples of both the

treatments, a total 56 to 97% of the raw reads were mapped to the C. clementine

genome (Figure 2-5). The leaf and root samples that generated a lower percentage of

alignment to the reference genome were re-sequenced to increase the reliability of the

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DEG analysis. The Cuffdiff significance test showed that comparative analysis between

the asymptomatic treatment of both scion/rootstock combinations had 1163 genes that

were significant differentially expressed in leaves with a false discovery rate (FDR) q ≤

0.04 (Figure 2-6A). Whereas roots of the asymptomatic treatment showed very few,

only 49 genes were significant (q ≤ 0.04) differentially regulated. Comparative

transcriptomic analysis of leaves of the symptomatic VAL/CAN and VAL/SW

combination showed 1437 significant (q ≤ 0.04) differentially expressed genes (Figure

2-6B). Whereas, roots in the same treatment showed 2025 significantly regulated (q ≤

0.04) genes (Figure 2-6C).

Functional Analysis of Differentially Expressed Genes

Functional analysis software: MapMan, Blast2Go, and Pathway Studio were

used to find functional annotation of the differentially regulated genes. Comparative

gene expression analysis between leaves of the asymptomatic VAL grafted onto CAN

and SW rootstock showed differentially regulated genes belonged to signaling,

transport, biotic stress, hormone metabolism, jasmonic acid (JA) and ethylene (ET)

stimulus, salicylic acid (SA) signaling pathway, response to chitin and wounding,

intracellular signal transduction, starch and sucrose metabolism, response to water

deprivation functional, and transport categories (Table 2-5, and Figure 2-7A and B).

However, the comparative DGE analysis in the roots of the asymptomatic treatment of

VAL/SW and VAL/CAN did not show the involvement of functional categories except

photosynthesis processes, glutamine metabolism, response to light, and microtubule

polymerization (Figure 2-7C and D).

In the symptomatic treatment, DGE analysis between VAL grafted onto the CAN,

and SW rootstocks showed significant differences in the genes belonging to following

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functional categories: flavonoid metabolism, cell wall pectin esterases metabolism, lipid

metabolism, response to chitin and wounding, lignin biosynthesis, water deprivation,

defense response to bacterium, respiratory burst involved in the defense, response to

JA stimulus, and response to water deprivation (Table 2-6, and Figure 2-8A and B).

Whereas, comparative transcriptomic analysis between CAN and SW roots in the

symptomatic VAL/CAN, and VAL/SW combinations showed that genes belonging to

RNA regulation of transcription factors (TF), wounding response, response to water

deprivation, flavonoid biosynthesis, nitrate transport, response to auxin (AU) stimulus,

JA-mediated signaling pathway, ET stimulus, starch and sucrose metabolism,

brassinosteroid (BR) metabolism, phenylpropanoid biosynthesis, and negative

regulation of programmed cell death (PCD) categories were differentially expressed.

(Table 2-7, and Figure 2-8C and D).

Validation of Differentially Expressed Genes using qRT-PCR

To validate the results of RNA-seq data, seven genes that had a distinct

expression pattern in the RNA seq study in both the scion/rootstock combinations were

selected (Table 2-8). These genes are encoding BAP2, BRI1, NPR3, MIPS2, JAZ8,

PMEI and LTP1. All seven genes validated RNA seq results of with respect to leaves of

the asymptomatic treatment. Also, the expression level changes were not significantly

different between RNA-seq and qRT-PCR Log2 FC (Figure 2-9A to F). In leaves of the

symptomatic treatment, qRT -PCR amplified BAP2, BRI1, NPR3, MIPS2, JAZ8, LTP1,

and PMEI transcripts expression levels validated RNA-seq generated expression

pattern (Figure 2-9A to G). The genes selected to validate the DGE analysis in roots of

the symptomatic treatments were BAP2 and JAZ8. All three genes showed a similar

pattern of gene expression using qRT-PCR as that of RNA-seq (Figure 2-9A, and E).

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Discussion

HLB is the most serious disease ever affecting Florida’s citriculture. In the

absence of a definite cure for the disease, HLB-tolerant cultivars can potentially be a

solution to increase tree survival and plant productivity under endemic HLB spread. The

VAL/CAN plants selected in this study showed an enhanced plant sustainability and

better fruit quality under pressure of HLB. In Florida, ‘Valencia’ and ‘Hamlin’ sweet

oranges are important scion varieties for juice production. Higher Brix and BAR, and

lower acidity percentage fruits fetch a higher price in the market. In year 2015, fruit

harvested from asymptomatic VAL/SW combination had the highest Brix value followed

by symptomatic VAL/CAN, asymptomatic VAL/CAN, and symptomatic VAL/SW Brix

values. Although there was no significant difference in the Brix of VAL fruit harvested

from the different combinations and treatments, fruit harvested from the symptomatic

VAL/SW had significantly higher acidity as compared to fruit harvested from the

symptomatic VAL/CAN combination. Higher acidity percentage of symptomatic VAL/SW

fruit seems to be an effect of HLB infection. Both the treatments in VAL/CAN

combination had higher Brix and BAR values of fruit suggesting that CAN rootstock can

possibly maintain the optimum juice quality when the plant is CaLas-infected. Higher Ct

values and high Brix of VAL/CAN also indicate that VAL/CAN combination is probably

less affected by CaLas-infection. In contrast, lower Ct and low Brix in symptomatic

VAL/SW indicate that at the advanced stage of CaLas-infection, VAL/SW combination

had low fruit juice quality as compared to VAL/CAN.

The use of NGS technologies is not limited to the model organisms; rather its use

is expanding in studies of non-model organisms too. The transcriptomic analyses of

HLB-citrus interactions are mostly studied using the microarray technique (Albrecht and

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Bowman, 2008; Febres et al., 2012; Aritua et al., 2013). In recent years, a few studies of

RNA-seq based transcriptome analysis of HLB-affected leaves, roots and fruit have also

been published (Martinelli et al., 2013; Wang et al., 2016; Zhong et al., 2016). The use

of RNA-seq technology in this study explored the differential gene regulation between

combinations and treatments.

Results obtained from all three functional analysis software: MapMan, Pathway

Studio, and Blast2GO showed similarity in their output. In the comparison between

leaves of the asymptomatic VAL/SW and VAL/CAN, response to wounding and chitin,

JA and ET metabolism, negative regulators of PCD, response to water deprivation, and

secondary metabolism functional categories were overrepresented by the GO analysis.

The overrepresentation of wounding and chitin response and JA-ET signaling pathways

suggests the CaLas-infected plants were responding to the herbivore attack.

Involvement of JA-ET pathways and chitin responses in the herbivore attacks was also

reported in Arabidopsis (Foyer et al., 2015). CaLas, HLB putative causal agent, is

vectored by the psyllid insect Diaphorina citri Kuwayama. Psyllids feed on the plant

vascular system and transmit CaLas into the plant cells. Therefore, the GO term

enrichment showing wounding response, chitin response, and JA-ET signaling

pathways suggest that many DGE changes were in response to the phloem-feeding of

psyllids.

DGE analysis of leaves and roots of symptomatic VAL/SW and VAL/CAN

combinations overrepresented the flavonoid biosynthesis pathway, lignin metabolism,

cell wall degrading enzymes, and pectin methyl esterases pathways suggesting that

genes associated with cell wall modification were significantly altered in their expression

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level at an advanced stage of HLB disease development. Blast2GO biological functional

analysis showed an overrepresentation of protein phosphorylation and proteolysis

functional categories in leaves of symptomatic VAL/CAN. Protein phosphorylation action

triggers the conformational changes in proteins that alter biological properties (Ubersax

and Ferrell, 2007). Proteolysis plays a vital role in cleansing the plant system during

stress responses (Vierstra, 1996). Taken together, GO enrichment of the

phosphorylation and proteolysis term in the symptomatic leaves of VAL/CAN suggests

that at symptomatic stage CaLas-infected VAL leaves had differential changes at the

post-transcriptional level.

MapMan analysis of functional categories showed significant differential

regulation of BR metabolism-related genes in roots of the symptomatic treatment

comparison between the VAL/CAN and VAL/SW combinations. BR hormone is involved

in plant growth and defense response (Nakashita et al., 2003; Haubrick and Assmann,

2006). External application of BR has been shown to induce defense in the CaLas-

infected greenhouse and field plants (Canales et al., 2016). Significant reprogramming

of BR metabolism associated genes in roots of the symptomatic treatment between the

VAL/CAN and VAL/SW combinations suggests the potential involvement of BR in

response to HLB.

SA-mediated systemic acquired resistance (SAR) signaling pathway, leucine rich

receptor kinase, MAPK cascade, and plant oxidative burst associated genes were also

significantly reprogrammed in the transcriptomic comparisons between VAL/SW and

VAL/CAN combinations in both the treatments. The overrepresentation of SAR

mediated signaling and plant hypersensitive reaction functional categories indicates the

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activation of plant defense. Functional analysis of DEGs also overrepresented water

deprivation and abscisic acid (ABA) response pathways in leaves and roots of the

symptomatic and asymptomatic treatments in both scion/rootstock combinations. ABA is

associated with drought response (Fernando and Schroeder, 2016). The GO analysis

showed that ABA signaling functional category was overrepresented which Indicates

CaLas-infected plants show a response to water deprivation and salt stress.

Overall analysis of functional categories showed that plant defense to HLB was

activated by activating genes related to SA-mediated signaling, JA and ET-dependent

signaling, protein kinases, and secondary metabolite pathways. Whereas, genes

involved in nitrate, metal, and sugar transporters suggest development-associated

genes were also differentially regulated in response to rootstock differences. The

functional analysis of DGE also highlighted the involvement of ABA and BR signaling

pathway genes in response to rootstock differences in CaLas-infected plants. Functional

analysis of genes differentially expressed between VAL/SW and VAL/CAN plants

showed DGE in response to asymptomatic and symptomatic stages of HLB

development. The genes that are contributing to the differential reprogramming are not

discussed in this Chapter. The detailed analysis of upregulation and downregulation of

genes involved in the functional categories is discussed in Chapter 3, Chapter 4, and

Chapter 5.

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Table 2-1. Experimental treatments and combinations

Rootstock Rootstock Parents Scion Treatments based on visual

observations of HLB symptoms

Swingle; 2n (SW)

Grapefruit X Trifoliate orange

Valencia sweet orange (VAL)

Symptomatic VAL/SW

Asymptomatic VAL/SW

Putative HLB-tolerant

candidate; 2n (CAN)

‘HBP’ Pummelo X Cleopatra Mandarin


Slightly Symptomatic

VAL/CAN

Asymptomatic

VAL/CAN

Approximately 7- year old trees planted in the Lee Family’s Alligator Grove east of St. Cloud, FL.

Table 2-2. Comparison pairs used for differential gene expression analysis in leaves and roots of the experimental scion/rootstock combinations

Leaves Roots

Asymptomatic VAL/SW vs. Asymptomatic VAL/CAN


Symptomatic VAL/SW vs. Symptomatic VAL/CAN


Table 2-3. Analysis of HLB and CTV detection in the experimental samples

Experiment Sample ID

Rootstock

Scion

Treatment

HLB detection (2014 April)

HLB detection (2015 December)

CTV detection (2015 December)

Leaves Roots Leaves Leaves

13 SW VAL Symptomatic Pos Pos Pos Pos



19 SW VAL Asymptomatic Neg Pos Pos Pos

20 SW VAL Asymptomatic Neg Pos Pos Pos

21 SW VAL Asymptomatic Neg Pos Pos Neg

5 CAN VAL Symptomatic Pos Neg Pos Neg

6 CAN VAL Symptomatic Pos Pos Pos Neg

7 CAN VAL Asymptomatic Pos Pos Pos Neg

22 CAN VAL Asymptomatic Neg Neg Pos Neg

23 CAN VAL Asymptomatic Pos Pos Pos Pos

24 CAN VAL Asymptomatic Neg Pos Pos Pos

Pos, PCR positive; Neg, PCR negative

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Table 2-4. Reads obtained from the sequencing run Experimental Sample ID

Plant tissue Rootstock Scion Treatment NEB index* No. of reads

13B Leaves SW VAL Symptomatic i504-i702 45,474,082

13D Roots SW VAL Symptomatic i508-i703 38,740,620

14A Leaves SW VAL Symptomatic i505-i702 41,910,010


15B Leaves SW VAL Symptomatic I506-i702 41,476,846


19B Leaves SW VAL Asymptomatic i507-i702 43,050,176

19D Roots SW VAL Asymptomatic i506-i701 36,758,134

20B Leaves SW VAL Asymptomatic i508-i702 30,822,930


21A Leaves SW VAL Asymptomatic i501-i703 28,336,160


5A Leaves CAN VAL Symptomatic i501-i701 38,546,288

5C Roots CAN VAL Symptomatic i502-i703 10,798,204

6A Leaves CAN VAL Symptomatic i502-i701 35,288,378

6D Roots CAN VAL Symptomatic i503-i703 12,792,946

7B Leaves CAN VAL Symptomatic i503-701 9,952,680

7C Roots CAN VAL Asymptomatic i504-i703 13,831,332

22B Leaves CAN VAL Asymptomatic i501-i702 49,335,730





24D Roots CAN VAL Asymptomatic i507-i703 116,236

* New England Biolabs® Inc developed Illumina index used for multiplexing the samples for the sequencing run

77

Table 2-5. Functional analysis of significant DEGs in leaves of the asymptomatic treatment between VAL/CAN and VAL/SW

Pathway studio MapMan

Biological functional category p-value Bin Name† elements* p-value

response to chitin 1.07E-76 transport 55 2.98E-04

response to wounding 3.15E-49 RNA.regulation of transcription.WRKY domain transcription factor family

9 9.47E-04

respiratory burst involved in defense response

3.72E-48 RNA.regulation of transcription. PHOR1 4 0.0036

intracellular signal transduction 2.41E-34 misc.GDSL-motif lipase 6 0.0040

response to jasmonic acid stimulus 8.09E-34 signalling 72 0.0055

protein targeting to membrane 1.58E-33 secondary metabolism.flavonoids 11 0.0082

response to ethylene stimulus 2.48E-33 signalling.calcium 15 0.0094

regulation of plant-type hypersensitive response

2.72E-33 misc 80 0.0101

jasmonic acid mediated signaling pathway 3.42E-30 stress 56 0.0193

ethylene biosynthetic process 5.21E-30 hormone metabolism. ethylene 15 0.0212

negative regulation of programmed cell death 8.53E-28 development. storage proteins 2 0.0267

defense response to fungus 2.05E-27 secondary metabolism.flavonoids.dihydroflavonols

4 0.0268

response to water deprivation 8.61E-27 AP2/EREBP, APETALA2/Ethylene-responsive element binding protein family

9 0.0310

jasmonic acid biosynthetic process 8.40E-23 signalling.receptor kinases.S-locus glycoprotein like

2 0.0381

negative regulation of defense response 1.15E-22 hormone metabolism.ethylene.signal transduction

9 0.0389

response to fungus 4.16E-22 protein.postranslational modification.kinase 9 0.0411

response to high light intensity 6.01E-22 transport.ABC transporters and multidrugresistance systems

8 0.0427

abscisic acid mediated signaling pathway 1.03E-21 DNA.unspecified 2 0.0438

salicylic acid mediated signaling pathway 3.85E-21 signalling.receptor kinases 34 0.0494

systemic acquired resistance, salicylic acid mediated signaling pathway

2.02E-20 signalling.in sugar and nutrient physiology 4 0.0529

†Classification of the measured parameter into a set a functional category in the MapMan analysis tool. Top 20 (based on p value) biological functional categories are presented that were identified in Pathway studio and MapMan (Wilcoxon rank sum test), *No. of DGE present

78

Table 2-6. Functional analysis of significant DEGs in leaves of the symptomatic treatment between VAL/CAN and VAL/SW

Pathway studio MapMan

Biological functional category p-value Bin Name†

elements* p-value

response to heat 3.91E-53 stress.abiotic.heat 32 9.4E-09

response to high light intensity 2.14E-52 stress 71 1.9E-07

response to hydrogen peroxide 2.33E-41 stress.abiotic 43 7.6E-07

protein folding 3.02E-31 secondary metabolism.flavonoids 12 6.9E-05

response to wounding 1.60E-29 cell wall.pectin*esterases 7 2.2E-03

response to water deprivation 1.98E-25 cell 37 2.5E-03

response to chitin 2.23E-23 secondary metabolism 51 3.7E-03

lignin biosynthetic process 3.53E-21 lipid metabolism.lipid degradation. lysophospholipases

5 5.3E-03

response to cold 1.74E-19 cell wall 38 5.9E-03

response to karrikin 2.77E-19 signalling. receptor kinases 30 6.0E-03

response to salt stress 3.47E-18 secondary metabolism.flavonoids.chalcones

4 9.5E-03

response to red light 8.74E-18 secondary metabolism. flavonoids.dihydroflavonols

4 9.5E-03

chloroplast 9.98E-18 misc.protease inhibitor/seed storage/lipid transfer protein (LTP) family protein

6 9.7E-03

response to endoplasmic reticulum stress

1.19E-17 transport.amino acids 11 1.1E-02

jasmonic acid mediated signaling pathway

6.79E-17 cell wall.degradation 8 1.4E-02

response to sucrose stimulus 8.73E-17 signalling 64 1.8E-02

defense response to bacterium 1.89E-16 misc.glutathione S transferases 4 2.1E-02

response to jasmonic acid stimulus 3.76E-16 signalling.receptor kinases.leucine rich repeat XII

2 2.2E-02

plasma membrane 6.54E-16 RNA.regulation of transcriptionMYB-related transcription factor family

5 2.8E-02

†Classification of the measured parameter into a set a functional category in the MapMan analysis tool. Top 20 (based on p value) biological functional categories are presented that were identified in Pathway Studio and MapMan (Wilcoxon rank sum test), *No. of DGE present.

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Table 2-7. Functional analysis of significant DEGs in roots of the symptomatic treatment between VAL/CAN and VAL/SW Pathway studio MapMan

Biological functional category p-value Bin Name†

elements* p-value

response to chitin 2.25E-52 metal handling.binding, chelation and storage 4 0.00462

negative regulation of programmed cell death 5.12E-40 RNA.regulation of transcription 109 0.00743

response to ethylene stimulus 7.53E-38 hormone metabolism.brassinosteroid 5 0.01214

protein targeting to membrane 2.60E-37 RNA.regulation of transcription.WRKY domain transcription factor family

6 0.01672

regulation of plant-type hypersensitive response

4.27E-37 transport. nitrate 5 0.01802

response to wounding 9.30E-37 misc 123 0.02027

jasmonic acid mediated signaling pathway 1.34E-35 DNA.unspecified 3 0.02214

defense response to fungus 5.25E-34 transport.metal 10 0.02372

response to jasmonic acid stimulus 1.52E-31 transporter.sugars.sucrose 2 0.02514

endoplasmic reticulum unfolded protein response

1.93E-31 RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive element binding protein family

5 0.02973

respiratory burst involved in defense response 1.53E-29 RNA.regulation of transcription.HB, Homeobox transcription factor family

5 0.02999

MAPK cascade 8.57E-28 Gluconeogenese/ Glyoxylate cycle 3 0.03244

systemic acquired resistance, salicylic acid mediated signaling pathway

1.77E-27 hormone metabolism.brassinosteroid.synthesis-degradation

4 0.03314

negative regulation of defense response 3.87E-27 hormone metabolism.gibberelin 9 0.03667

abscisic acid mediated signaling pathway 2.31E-26 Hormone metabolism. brassinosteroid. synthesis-degradation.sterols.other

2 0.03689

salicylic acid biosynthetic process 8.79E-26 RNA.regulation of transcription. Aux/IAA family 5 0.04019

salicylic acid mediated signaling pathway 5.59E-24 protein. degradation. serine protease 6 0.04252

response to water deprivation 2.71E-23 misc. myrosinases-lectin-jacalin 2 0.04442

intracellular signal transduction 1.24E-21 RNA 124 0.04917

regulation of hydrogen peroxide metabolic process

2.07E-19 protein. degradation.aspartate protease 4 0.05295

ethylene biosynthetic process 2.68E-19 metal handling. acquisition 3 0.05327 †Classification of the measured parameter into a set a functional category in the MapMan analysis tool. Top 20 (based on p value) biological functional categories are presented that were identified in Pathway Studio and MapMan (Wilcoxon rank sum test), *No. of DGE present.

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Table 2-8. Genes used for validation of RNA-sequencing results

Gene Identification Abbr∞. Primer sequence*

Ciclev10005926m.g/ Pectin methyl esterase inhibitor

PMEI F- 5’-GCTTCTCTTCGCGGTTAGAT -3’

R- 5’-CAGTTGTGGTTTGGTTGATAG-3’

Ciclev10022388m.g/BON associated protein 2

BON F- 5’-TTCGTCACCATCACCACATAC-3’

R- 5’-TCCGGGACTACAGGTTCTAAT-3’

Ciclev10003524m.g/Lipid transfer protein 1

LTP1 F- 5’-CATGTGAGCAAGTGACAATCTG-3’

R- 5’-CTAGGATAAGAATTAAGGGCGTACT-3’

Ciclev10024737m.g/BR leucine rich receptor

BRI F- 5’-CTCCGGGTCCTTTGTCTGATATT-3’

R- 5’-ACCGAATCTACTGGGAACTTTAC-3’

Ciclev10017873m.g/LOC102621158/Non-expressor of protein 3

NPR3 F- 5’-AGGTTCTCAGCCTCCGGATTA-3’

R- 5’-CCATCGGATTCCTCATTTC-3’

Ciclev10017198m.g/Zim domain JAZ 8 JAZ8 F- 5’-GCCTTCTTCCTTATTCCGACTC-3’

R- 5’-GCTAAGCTCCTGTGCTTTCT-3’

Ciclev10025400m/Myo-inositol phosphate synthase 2

MIPS2 F- 5’-TGTTTGGAGGATGGGACATTAG-3’

R- 5’-GCATACAAGGTGGAAGGAGAA-3’

Glyceradehyde-3-phosphate dehydrogenase C2

GAPC F- 5’-GGAAGGTCAAGATCGGAATCAA-3’ R- 5’-CGTCCCTCTGCACfGATGACTCT-3’

*F; forward primer sequence, R; reverse primer sequence, ∞ Abbreviations used in the texts

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A B

C D Figure 2-1. VAL/CAN combination field trees.

A) symptomatic (Year 2015), B) symptomatic (Year 2016). C) asymptomatic (Year 2015), D) asymptomatic (Year 2016)

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A B

C D Figure 2-2. VAL/SW combination field trees.

A) symptomatic (Year 2015), B) symptomatic (Year 2016), C) asymptomatic (Year 2015), D) asymptomatic (Year 2016)

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Figure 2-3. Juice qualityt analysis. A) Year 2015, B) Year 2016. Sympt: symptomatic; Asympt: asymptomatic, BAR: Brix-acid ratio.

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Figure 2-4.RNA quality and quantity analysis. A) Leaf RNA B) Root RNA.

Electrophoresis gel based quality/quantity analysis of leaf and root RNA tissue. The quality of RNA is based on 260/280 ratio obtained from NanoDrop technique, and RNA quantity is expressed in ng/µl. Each lane contains 50 ng RNA. Leaf and root samples have different identifications. Leaves are designated either A or B, and roots are designated either C or D followed by sample ID.

B

A

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Figure 2-5. Read mapping statistics. Raw reads were trimmed and indexed, and then mapped to the C. clementina genome for downstream analysis.

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Figure 2-6. Display of Volcano plot of using CummeRbund.

A) leaves of asymptomatic VAL/SW (X19B) and VAL/CAN (X22B), B) leaves of symptomatic treatment VAL/SW (X5C) and VAL/CAN (X19C), C) roots of symptomatic treatment VAL/SW (X5C) and VAL/CAN (X19)

A

B

C

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Figure 2-7. Blast2GO functional analysis of DEGs in leaves (A and B) and roots (C and D) of the asymptomatic VAL/CAN and VAL/SW combinations. Biological (bp) functional categories identified (Top 10 and p value < 10-6) in upregulated (up) and downregulated (down) genes in the VAL/CAN as compared to the VAL/SW. Graphics are adapted from Blast2GO statistics results. L, leaves; R, roots

A

B

C

D

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Figure 2-8. Blast2GO functional analysis of DEGs in leaves (A and B) and roots (C and D) of the symptomatic VAL/CAN and VAL/SW combinations.

Biological (bp) functional categories identified (Top 10 and p value < 10-6) in upregulated (up) and downregulated (down) genes in the VAL/CAN as compared to the VAL/SW. Graphics are adapted from Blast2GO statistics results. L, leaves; R, root

A C

B D

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Figure 2-9. qRT-PCR based DEGs validation.

A) BAP2, B) BRI1, C) JAZ8, D) LTP1, E) NPR3, F) MIPS2, G) PMEI

A

B

C

D

E

F

G

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CHAPTER 3 DIFFERENTIAL EXPRESSION ANALYSIS OF HORMONAL METABOLISM-

ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB

DISEASE

Introduction

As sessile organisms, plants constantly need to cope up with their surrounding

environment for growth and survival. Evolutionary, plants have developed the sensory

system to sense the environmental changes through plant growth regulators (PGRs).

PGRs or plant hormones are vital sensing chemicals that are important in plant growth

and defense. Plant hormones have a significant role in stress signal perception,

transduction, and response. Hence, hormones are crucial for plant growth and

sustainability. PGRs are low molecular weight chemicals that are secreted by

specialized cells and that affect the metabolism or behavior of other cells possessing

functional receptors. The well-known PGRs are auxins (AU), gibberellins (GA),

cytokinins (CT), abscisic acid (ABA), ethylene (ET), salicylic acid (SA), jasmonic acid

(JA), brassinosteroids (BR), and strigolactone (ST) (McCourt, 1999). Some signaling

peptides are also acknowledged as PGRs (Wang and Irving, 2011). AU, CT, GA, and

BR are highly regulated in plant growth (Werner et al., 2001; Liscum and Reed, 2002;

Birnbaum and Benfey, 2004). Whereas, SA, JA, and ET are plant defense associated

hormones (Pieterse et al., 2009). BRs and ABA are implicated in the plant development

as well as plant defenses (Haubrick and Assmann, 2006; Cao et al., 2011). The studies

on plant functional biology and genetics revealed that the PGRs are multitasking

chemicals, either working individually or in combination with other hormones, which

supports the concept of hormonal crosstalk (McCourt, 1999; Swarup et al., 2002;

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Pieterse et al., 2012; Vanstraelen and Benková, 2012). The crosstalk between PGRs is

limited not only to the plant growth and development. Hormonal signaling is also

involved in plant immunity, nutrient uptake and transport, and metal homeostasis (Rubio

et al., 2009; Krouk et al., 2011). Interdependence of AU and BR showed the regulatory

network and negative feedback mechanism between AU and BR response pathways in

seedling growth of Arabidopsis thaliana (Nemhauser et al., 2004). The interplay

between GA and BR in development and defense signaling is discussed by Lieselotte et

al., 2014. JA and SA -induced antagonism and its molecular differences in the

necrotrophic and biotrophic pathogen infection is evident in many plant species (Kunkel

and Brooks, 2002; Pieterse et al., 2009; Leon-Reyes et al., 2010). The developmental

role of AU, CT, GA, ABA, and ET is well known. However, these PGRs are also

involved in the plant defense signaling (Cao et al., 2011; Giron et al., 2013).

Hormonal homeostasis is a key to performing plant functions at an optimum

level. Environmental imbalances, physiological changes, and plant growth initiate the

hormonal changes in the plant. Hormonal regulation hierarchy starts with signal

perception followed by hormone synthesis, signal transduction and the response

activation (Figure 3-1). Hormonal concentration is subsequently regulated by negative

feedback mechanisms that include negative regulation of hormone synthesis,

catabolism or sequestration (Woodward and Bartel, 2005; Brenner et al., 2012). The

hormonal activation input signal is perceived by a binding receptor which initiates the

signal transduction. Each PGR has its specific receptor kinase (RK), signal transduction

ligands, protein-coupled receptors (G-coupled proteins) to bring a hormone into action

(Wang and Irving, 2011). Examples of RKs are CT specific cytokinin regulated kinase 1

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(CRK1) (Brenner et al., 2012), ET specific ethylene regulated 1 (ETR1) (Gallie, 2015),

GA specific gibberellin insensitive dwarf 1 (GID1), ABA specific -regulatory components

of ABA receptor/pyrabactin resistance protein1/PYR-like proteins (RCARs/PYR1/PYLs)

(Raghavendra et al., 2010), and BR specific brassinosteroid insensitive 1 (BRI1)

(Haubrick and Assmann, 2006). Once the hormone is perceived by RKs, the next step

is signal transduction. Signaling transduction activates the functional transcription

factors (TFs) such as AU activated auxin response factors (ARFs), JA-dependent MYC,

GA-dependent phytochrome interacting factor (PIF) and ET activated ethylene response

factor 1 (ERF1). The hormone specific TFs activate downstream hormonal responses.

Regulation of hormonal turnover is controlled by repressor proteins which downregulate

the hormonal response. Hence, the repression proteins are either repressed or

degraded by S-phase kinase associated protein1 (SKP1), cullin and F-box protein

complexes (SCF) upon hormonal reception (Woodward and Bartel, 2005; Raghavendra

et al., 2010; Brenner et al., 2012). In different hormonal signaling pathways, SCF-

mediated degradation of repressor proteins is characterized. In AU signaling, the

SCFTIR1 complex represses the auxin/indole-3-acetic acid (AUX/IAA) hormonal receptor

repressor. TIR1 is an auxin specific F-box protein which co-represses AUX/IAA

repression and thereby activates AU response element (AuxRE) and ARFs (Woodward

and Bartel, 2005; Grones and Friml, 2015; Li et al., 2016). In GA signaling, DELLA

proteins are the receptor repressors that downregulate the GA response. GID1 is an F-

box protein and a GA receptor. In the presence of GA, GID binds to DELLA and

degrades it. Once the DELLA is deactivated, GA-responsive PIF genes induce the GA-

activated growth response (Frgerioi et al., 2006). Jasmonate zim-domain (JAZ) proteins

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are negative regulators of MYC transcription factors. JAZ repression is removed by

coronitine-insensitive 1 (COI1) and SCF complex which then activates JA-responsive

genes (Wasternack and Hause, 2013). Similarly, the role of other hormone-specific

receptors, SCF complex and responsive genes are well characterized by screening

mutants in A. thaliana (Wang and Irving, 2011). The activity of hormonal receptor

suppressor is crosslinked or controlled by other hormonal receptors, repressors, TFs,

and responsive genes. One of the impacts of hormone crosstalk is a plant growth and

defense trade-off (Wei Wang et al., 2012; Denancé et al., 2013; Huot et al., 2014).

Hormonal regulations in plants are a highly coordinated and synchronized process to

maintain a balance between plant growth and defense.

Transcriptomic studies of CaLas-infected susceptible and HLB-tolerant citrus

cultivars underscored the role of PGRs in the HLB-citrus interaction (Canales et al.,

2016; Fan et al., 2012; Martinelli et al., 2013; Albretch and; Bowman, 2011). The

regulatory network analysis of diverse CaLas-infected scion/rootstock combination at

various stages of infection showed the altered expression levels of AU, GA, ABA, ET,

JA, and SA-regulated genes. In HLB-citrus interaction studies, AU, GA, JA, and SA-

regulated functional categories were overrepresented by gene ontology (GO) algorithms

(Zheng and Zhao, 2013). In CaLas-infected plants, the JA and ET-response activated

ENDOCHITINASE gene expression was found to be repressed at the early stage (5

weeks after infection) of infection in ‘Valencia’ sweet orange (Citrus sinensis [L.]

Osbeck) scion grafted onto Cleopatra rootstock (Albrecht and Bowman, 2012b).

Whereas, CaLas-B232 (Thailand strain) and Citrus Tristeza Virus (CTV) infected

‘Valencia’ overexpressed the JA-responsive LOX2 encoding gene (Fu et al., 2016). SA-

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induced CONSTITUTIVE DISEASE RESISTANCE (CDR1) was upregulated in the

leaves of non-grafted HLB-tolerant US-897 rootstock (Albrecht and Bowman, 2012b).

GA-induced cell organization and biogenesis related gene encoding GA-responsive

GAS1 protein homolog GASA was overexpressed in rootstock US-897 leaves (Albrecht

and Bowman, 2012b). Transcriptomic analysis of fruit harvested from CaLas-infected

‘Valencia’/Swingle combination was reported by Martinelli et al., 2012. HLB-

symptomatic fruit showed overexpression of AU biosynthesis and signal transduction

related transcripts. Also, transcripts encoding ET biosynthesis, receptors, and

responsive genes such as ETR1, ERF1, ERF2 and ethylene forming enzyme (EFE)

were induced in the HLB-symptomatic ‘Valencia’ fruit. However, GA and CT metabolism

related ENT-KAURENOIC ACID HYDROXYLASE 2 (KAO2) and GASA1 genes were

downregulated in the CaLas-infected ‘Valencia’ fruit. In addition, transcript encoding

LOX2 was also suppressed. Overall, transcriptomic studies revealing HLB-citrus

interaction showed that hormonal changes in the CaLas-infected plants vary with the

scion and rootstock cultivars (Albrecht and Bowman, 2012b; Nwugo et al., 2013a;

Zhong et al., 2016). Differential gene expression (DGE) changes in the HLB-affected

plants are tissue and stage of infection specific (Fan et al., 2012; Martinelli et al., 2012;

Zheng and Zhao, 2013). Field and laboratory -based experiments provide evidence that

exogenous hormonal treatments can manipulate transcriptome of CaLas-infected plants

to promote growth and induce defense (Shen et al., 2013; Canales et al., 2016).

Knowledge of hormonal changes is a key to understanding the HLB-citrus

interaction. The goal of comparative transcriptomic analysis between ‘Valencia’ grafted

onto the putative HLB-tolerant candidate rootstock 42x20-04-48 (CAN) or the HLB-

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susceptible commercial rootstock Swingle citrumelo is to emphasize the vital role of

citrus rootstocks in modulating the scion hormonal network in response to CaLas-

infection.


Plant Material

Two combinations of scion and rootstocks were used in this experiment (Table 3-

1). Field grown seven-year-old experimental plants were planted in the Lee Family's

Alligator Grove, east of St. Cloud, Florida. The one combination of trees was ‘Valencia'

(VAL) sweet orange grafted onto a putative HLB-tolerant candidate (CAN) rootstock.

The CAN rootstock (46x20-04-48) is a hybrid of ‘Hirado Buntan Pink' pummelo (HBP)

(Citrus maxima Merr.) and Cleopatra mandarin (Citrus reticulata Blanco.). The 2nd

combination was ‘Valencia' (VAL) scion grafted onto standard commercial Swingle

citrumelo (SW) rootstock. Swingle is a hybrid of grapefruit (Citrus paradisi [Macf.]) and

trifoliate orange (Poncirus trifoliata [L.] Raf). Each combination of VAL/CAN and

VAL/SW, plants were divided into two treatments based on the visible presence of HLB-

like symptoms (Table 3-2). Highly infected and symptomatic trees in each combination

grouped into the symptomatic treatment, whereas, trees with very few or no visible

symptoms were grouped into the asymptomatic treatment. All biological replicates in

each treatment and combination were tested using quantitative PCR (qPCR) based

CaLas detection, and enzyme-linked immunosorbent assay (ELISA) assisted CTV

detection.

Sampling, RNA extraction, and RNA sequencing

A detailed protocol of sampling, RNA extraction, and RNA sequencing is

explained in Chapter 2. In brief, differentially expressed genes (DEGs) in the pairwise

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comparison between asymptomatic VAL/CAN and VAL/SW combinations, and

symptomatic VAL/CAN, and VAL/SW combinations were obtained using RNA-seq

Tuxedo pipeline (Chapter 2). The significant DEGs in leaves and roots were annotated

to the C. clementina genome database in the Phytozome V1.0 (Goodstein et al., 2012).

Functional categories of the significantly expressed genes were identified using A.

thaliana annotation in the Phytozome server and MapMan (Thimm et al., 2004) software

(Chapter 2.). Blast2GO algorithm (Conesa et al., 2005) also used to identify molecular,

cellular, and biological functional categories. An overview of DEGs was developed using

PageMan analysis tool in the MapMan software (Usadel et al., 2009).

Results

HLB Detection and RNA Sequencing Output

The results of qRT PCR-based HLB detection and RNA-seq output in all

combinations and treatments are discussed in Chapter 2.

Differential Expression of Hormonal Regulation-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW Combinations

Leaf samples

The results of DGE studies combined with gene functional analysis tools showed

a significantly different regulation of genes involved in all the hormonal pathways in the

comparison of asymptomatic treatment between VAL/CAN and VAL/SW combinations

in leaves. In the asymptomatic treatment, CaLas-infected leaves of VAL/CAN had a

greater number of AU and BR metabolism related genes that were significantly

upregulated as compared to the asymptomatic leaves of VAL/SW combination (Figure

3-2), whereas, SA, JA, ET, AU and ABA hormonal signaling genes were strongly

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upregulated in the leaves of asymptomatic of VAL/SW as compared to leaves of

asymptomatic VAL/CAN combination.

Leaves of asymptomatic VAL/CAN combination significantly upregulated genes

encoding BR biosynthesis STEROL METHYL ESTERASE (SMT1), BRI1, HERCULES

RECEPTOR KINASE (HERK1), and BR KINASE INHIBITOR (BKI1) as compared to

leaves of the asymptomatic VAL/SW combination (Table 3-3). Genes involved in

different steps of AU signaling and homeostasis were also upregulated in the

asymptomatic VAL/CAN combination. These include genes encoding AU regulators

such IAA14, AUX/IAA, MYO-INOSITOL-1-PHOSPAHTE SYNTHASE-2 (MIPS2), UDP-

GLUCOSYL TRANSFERASE 74B1 (UGT74B1) and AU responsive genes such as

SAUR-like, ARF8, and ARF16 (Table 3-3). Leaves collected from asymptomatic

VAL/CAN also significantly upregulated genes encoding JA biosynthesis LOX2 and SA

biosynthesis phenylalanine ammonia lyase 1 (PAL1). Genes encoding ET-responsive

SHN1 TF also significantly overexpressed in leaves of the asymptomatic VAL/CAN

combination. In the asymptomatic treatment, leaves collected from VAL/CAN also

showed significant upregulation of genes encoding cytokinin-dependent response

regulators ARR9, Che-Y like two components responsive regulator, GIBBERELLIN

DEGRADING GA 2-OXIDASE (GA2OX), and the ABA biosynthesis gene NINE-CIS-

EPOXYCAROTENOID DIOXYGENASE 5 (NCED5) (Table 3-3).

In the asymptomatic VAL/SW leaves, AU dependent IAA-LEUCINE RESISTANT

(ILR)-like 6 (ILL6), IAA-ALANINE RESISTANT 3 (IAR3), and genes encoding

glucosinolate biosynthesis CYP83B1 and R2R3-Myb77 transcription factor were

overexpressed. JA and ET response genes were highly upregulated in leaves of the

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asymptomatic VAL/SW combination (Table 3-4). ET biosynthesis EFE, and genes

encoding ET receptors such as ERT2, ERF5, ERF12, ERF13, ET signal transduction

apetala 2 (AP2) domain containing, REDOX RESPONSIVE TRANSCRIPTION

FACTORS (RRTF1), and ET-responsive ELEMENT BINDING PROTEIN (EBP) were

significantly upregulated in leaves of the asymptomatic VAL/SW as compared to leaves

of the asymptomatic VAL/CAN. JA biosynthesis gene ALLENE OXIDE CYCLASE 3

(AOC3), and genes encoding LOX3 and JA response transcription factors WRKY30 and

WRKY 33, ZIM domain containing JA repressors such as JAZ1, JAZ8 and JAZ10 and

JA catabolic turnover CYP94C1 were upregulated in the VAL leaves of asymptomatic

VAL/SW combination as compared to VAL leaves of asymptomatic VAL/CAN in the

range of 2-4 log2 fold change (log2 FC) (Table 3-4). Asymptomatic VAL/SW leaves also

showed significant upregulation of ABA signaling genes at higher expression levels

compared to the asymptomatic VAL/CAN combination (Table 3-4). These include genes

encoding ABA-responsive GRAM domain containing TFs, F-box EMPFINDLICHER IM

DUNKELROTEN LICHT 1-LIKE PROTEIN 3 (EDL-3), abscisic acid 8'-hydroxylase

(CYP707A1) and type 2C protein phosphatases (PP2Cs). Genes encoding transcription

factors involved in SA activated resistance (SAR) pathway, WRKY70 and thioredoxin

family proteins GRX480, were overexpressed in the asymptomatic VAL grafted onto the

SW rootstock as compared to the asymptomatic VAL grafted onto CAN rootstock.

Root samples

The comparative gene expression analysis of roots collected from the

asymptomatic treatment of VAL/CAN and VAL/SW did not show differential expression

of many hormonal metabolism associated genes.

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Differential Expression of Hormonal Regulation-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW Combinations

Leaf samples

Leaves sampled from symptomatic CaLas-infected VAL grafted onto the CAN

rootstock significantly upregulated BR and CT -metabolism and -response related

genes. Whereas CaLas-infected VAL grafted onto SW rootstock showed significant

upregulation of ET, JA and AU signaling genes (Figure 3-2).

In leaves of the symptomatic VAL/CAN combination, genes encoding sterol

biosynthesis- CYCLOARTENOL SYNTHASE 1 (CAS1), STEROL

METHYLTRANSFERASE1 (SMT1) and DWF5 were overexpressed as compared to

leaves of symptomatic VAL/SW combination (Figure 3-2). In addition, genes encoding

BR SIGNALING KINASE-2 (BSK2) and BR ENHANCED EXPRESSION 3 (BEE3) were

upregulated in leaves of VAL/CAN as compared to the VAL/SW in the symptomatic

treatment (Table 3-5). CT and GA metabolism related genes increased in their

expression level in leaves of the symptomatic VAL/CAN as compared to symptomatic

VAL/SW. These included CYTOKININ OXIDASE 7 (CKX7) gene, and genes encoding

chase domain containing histidine kinase; WOODEN LEG (WOL), and CT-two

component system associated ARR4, APRR2, and APRR10 (Table 3-4). Genes

involved in SA, JA, and ET -hormones activated plant defense responses did not

upregulate at their higher expression level in the symptomatic VAL/CAN combination

(Figure 3-2). However, transcripts encoding PAL1 which is a key enzyme in the SAR

signaling pathway were upregulated. Genes involved in the ET or JA biosynthesis did

not overexpress significantly in the symptomatic VAL/CAN combination; whereas,

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genes encoding in the AU response regulators were significantly upregulated (Table 3-

5).

In the symptomatic treatment, VAL/SW overexpressed a greater number of JA,

ET, AU, and ABA metabolism associated genes (Table 3-6). In the same treatment,

VAL/SW leaves did not show a significant increase in expression level of genes

involved in SA metabolism related genes except WRKY70 TF. However, SA-responsive

SIGMA FACTOR BINDING PROTEIN1 (SIB1) upregulated by 2 log2 FC. The gene

encoding negative regulator of NON-EXPRESSOR OF PATHOGENESIS-RELATED 1

(NPR1), NPR3, was also overexpressed in the leaves collected from the symptomatic

VAL/SW combination. Genes involved in the JA, ET, AU, and ABA -biosynthesis,

regulation, and negative regulation were substantially increased in their expression level

in the symptomatic VAL/SW combination (Table 3-6). BASIC CHITINASE (HCHIB) is a

pathogenicity related (PR) protein which the expression level upregulates in response to

higher expression of JA and ET. Expression of HCHIB significantly upregulated in the

leaves of the VAL/SW as compared to leaves of VAL/CAN in the symptomatic

treatment. Notably, transcripts encoding JA repressors JAZ1 and JAZ8 were also

strongly induced in the leaves of the symptomatic VAL/SW combination (Table 3-6).

Root samples

In the symptomatic treatment, CaLas-infected roots collected from the CAN and

SW rootstock showed significant differences in the BR, CT, GA, SA, JA, and ABA -

regulated genes. Fibrous roots collected from the VAL/CAN combination showed

significant upregulation of a higher number of genes involved in BR, GA, and ABA

metabolism (Table 3-7). Roots collected from VAL/CAN combination, also, showed

significant overexpression of defense associated SA, JA and ET hormonal signaling and

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responsive genes as compared to roots collected from VAL/SW, in the symptomatic

treatment. In the same treatment, CAN roots showed significant upregulation of genes

encoding negative regulators of JA, SA, and GA hormonal response (Table 3-7). These

negative regulators are JAZ1, JAZ8, JAZ10, NPR3, GIBBERELLIC ACID INSENSITIVE

(GAI), and RGA like-3 (RGL-3) proteins. Roots collected from symptomatic VAL/CAN

significantly overexpressed genes encoding transcriptional activators of SA, JA, and

ABA as compared to roots collected from symptomatic VAL/SW combination. These

transcription activators are THIOREDOXIN PROTEINS (GRX480), MYC and PYL4

(Table 3-7). Roots collected from the symptomatic VAL/CAN combination showed

significant overexpression of the genes involved in the BR-mediated hormonal signaling

pathway as compared to the roots collected from the symptomatic VAL/SW combination

(Figure 3-2). These genes are encoding BR biosynthesis SQUALENE

MONOOXYGENASE (XF1), BAS1, CAS1 and BRI like kinase receptors (Table 3-7).

In the symptomatic treatment, CaLas-infected SW roots overexpressed JA, ET,

AU, and ABA metabolism associated genes as compared to CAN roots. In the

symptomatic treatment, transcripts of JA biosynthesis AOC4 and JA responsive PR4

were significantly upregulated in roots collected from VAL/SW as compared to

VAL/CAN roots (Table 3-8). Genes encoding AU transporter such as EFFLUX

PINFORMED 4 (PIN4) and LIKE-AUXIN RESISTANT-1 (LAX1), AU regulator GH3.1

and IAA29 significantly upregulated in the roots of symptomatic VAL/SW as compared

to symptomatic VAL/CAN (Table 3-8). The ABA transcription activator ABSCISIC ACID

-DEFICIENT 4 (ABA4) transcripts were also significantly overexpressed in roots of the

symptomatic VAL/SW combination. In the symptomatic treatment, VAL/SW roots

102

significantly upregulated ET RESPONSE SENSOR-1 (ERS1) and ET degradation ACC

OXIDASE (ACO) transcripts.

Discussion

The comparative gene expression analysis in leaves of the HLB-asymptomatic

treatment between VAL grafted onto putative tolerant CAN and the susceptible SW

rootstock showed an abundance of significant differentially regulated genes in the

category of hormonal metabolism. However, roots collected from CAN and SW

rootstocks in the same treatment did not show significant expression of genes involved

in the hormonal regulations. To confirm the results, the RNA-seq protocol and data

analysis of comparison between asymptomatic -VAL/CAN and -VAL/SW roots were

performed twice. , However, results again showed very few DEGs. CAN and SW

rootstocks have different genotypes; therefore, it is expected that the DGE analysis

should have generated more numbers of differentially regulated genes in response to

CaLas infection. However, the consistent results of DGE analysis of the asymptomatic

VAL/CAN and VAL/SW roots suggests that the lower number of DEGs may not be a

result of genetic differences between two rootstocks, rather it is probably a result of the

encountered technical difficulty to obtain good quality RNA from the roots. The read

mapping data also showed that roots collected from samples 24D and 19D which

represent biological replicate in asymptomatic VAL/CAN and VAL/SW combinations,

respectively, had very low number of reads paired and mapped. Also, sample 24D also

showed the lowest number of reads (0.1 million).

Transcriptome analysis of VAL leaves in the asymptomatic VAL/CAN

combination showed significant upregulation of genes encoding AU conjugators such as

MIPS, AU transporter, AU responsive ARFs, BR biosynthesis, BR kinase receptors as

103

compared to asymptomatic VAL/SW leaves. Upregulation of BR and AU metabolism

associated genes in the asymptomatic VAL/CAN suggests that CAN rootstock

reprograms the AU and BR metabolism in VAL scion at the asymptomatic stage of

CaLas-infection. Pathogen-infected plants are marked with enhanced AU metabolism

(Ludwig-Müller, 2015). Also, pathogens manipulate AU and GA pathways to facilitate

cell wall expansion to spread the pathogen infection (Ma and Ma, 2016). Induction of

genes encoding AU response repressors, AUX/IAA regulators and negative regulators

of GA biosynthesis in asymptomatic VAL/CAN combination suggests the possible

mechanism of CAN rootstock in inhibiting AU and GA -induced cell expansion, and

CaLas infection spread in the scion. Whereas, SW rootstock significantly upregulated

AU biosynthesis genes such as ILL6, IAR3, and transcripts encoding CYP83B1 which

increase the AU pool in the plant. Overexpression of these genes in asymptomatic

VAL/SW combination supports the previous studies that showed the potential

involvement of pathogen-induced AU production (Mutka et al., 2013; Castillo-Lizardo et

al., 2015). Cytokinin is an important hormone that regulates shoot and root

morphogenesis (Werner et al., 2001). VAL leaves collected from asymptomatic

VAL/CAN combination showed significant upregulation of genes encoding positive and

negative CT response regulators suggesting CT-mediated growth or defense responses

were induced in the asymptomatic VAL/CAN combination. Whereas, genes encoding

ABA TFs such as C-REPEAT BINDING FACTOR 4 (CBF4) and ABA-responsive

drought tolerance PP2C were significantly upregulated in leaves of the asymptomatic

treatment of VAL/SW. Upregulation of CBF4 and PP2C genes was observed in plant

drought adaptation (Singh et al., 2015; Haake et al., 2016). CaLas-infected plants have

104

shown reduced root growth (Graham et al., 2013) which may reduce the uptake of

nutrients and water from the rhizosphere. The highly upregulated CBF4 and PP2C

transcripts in the asymptomatic VAL/SW suggest that reduction in the root growth might

activate drought tolerance signaling in plants.

Plant defense hormone genes encoding SA-, and ET- response regulator, and

JA biosynthesis and response regulators were upregulated in leaves of the

asymptomatic VAL/SW combination as compared to leaves of the asymptomatic

VAL/CAN, in which ET and SA responses were strongly indicated through upregulation

of genes encoding transcription regulators such as ERF1, ERF13, AP2, RRTF1, EBP,

GRX480 and WRKY 70. JA biosynthesis LOX3 gene was significantly upregulated in

leaves of the VAL/SW symptomatic treatment when compared to VAL/CAN leaves.

However, the JAZ8 gene (encoding a Jasmonate Zim domain JAZ protein) was

significantly upregulated in the asymptomatic VAL/SW leaves when compared to

asymptomatic VAL/CAN leaves. Jasmonate Zim domain JAZ proteins play an important

role in repressing JA activated signaling cascade through interaction between JAZ and

COI1 (Wasternack and Hause, 2013). The JA and ET signaling pathways induce upon

necrotrophic and herbivore infection (Simms and Rausher, 1987). The upregulated

JAZs encoding genes in the VAL/SW combination suggest the suppression of the JA

pathway and activation of the ET signaling cascade. However, significant upregulation

of gene encoding LOX3 is still need to invistage to understand its potential role in

asymptomatic VAL/SW leaves. A significant upregulation of many ET-regulated genes

supports the HLB-associated leaf senescence events in the plants. Ethylene production

is found to be increased in the biotrophic and necrotrophic pathogen virulence

105

(Weingart et al., 2001). The strongly upregulated transcripts of ET regulators suggest

that CaLas-infection overproduces or promotes the plant to overexpress the ET

signaling pathway in the VAL/SW combination. NPR3 gene, a negative regulator of

NPR1, was also significantly overexpressed in leaves of the asymptomatic VAL/SW

combination. The SAR exhibited by upregulating SA-mediated defense pathway was

potentially suppressed by its negative regulator known as NPR3. Altogether,

comparison of DGE analysis between leaves of asymptomatic VAL/CAN and VAL/SW

suggests that in leaves of asymptomatic VAL/SW, there is a critical reprogramming

between SA-JA-ET hormone metabolism to suppress the CaLas infection but, CaLas

might be targeting the ET signaling pathway to manipulate plant induced defense and

facilitates the pathogen infection. Also, CaLas infection induced AU and ABA

metabolism genes in the HLB presymptomatic VAL/SW combination. Defense

associated SA, JA and ET hormonal response related genes were not significantly

upregulated in asymptomatic VAL/CAN leaves as compared to leaves collected from

asymptomatic VAL/SW. However, CAN rootstock promoted the BR-dependent signaling

pathway and downregulated AU production in the VAL scion grafted onto it at the

asymptomatic stage of the CaLas-infection, suggesting that BR-AU interactions might

play an important role that is potentially altering the VAL gene expression in VAL/CAN

combination at the asymptomatic stage of HLB.

Transcriptomic analysis of CaLas-infected leaves collected from the symptomatic

treatment of VAL/CAN and VAL/SW combinations showed genes involved in the

hormonal metabolism were significantly upregulated in the VAL leaves as well as roots

collected from CaLas-infected CAN and SW rootstocks. The abundance of DEGs in the

106

leaves and roots shows that trees at the symptomatic stage of CaLas-infection respond

differentially compared to those at the asymptomatic stage.

DGE analysis of hormone metabolism and regulation related genes at the

advanced stage of CaLas-infection showed that CAN and SW rootstock could

differentially reprogram the hormone regulation in the leaves and roots of the grafted

plants. Putative HLB-tolerant CAN rootstock differentially upregulated BR, CT, and GA

metabolism related genes in roots, and reprogrammed the BR, CT, and AU metabolic

signaling related genes in the VAL scion. The symptomatic VAL/CAN combination

showed upregulation of AU response regulators ARFs as well as AU repressor IAAs.

Whereas, the CaLas-infected symptomatic VAL/SW combination showed

overexpression AU biosynthesis genes that can increase AU pool in the plants, and

therefore, can facilitate spread of bacteria via cell expansion. Altogether, this suggests

that the CAN rootstock probably attains AU homeostasis in HLB-symptomatic leaves

and roots by upregulating AU response regulators and repressors as compared to

upregulated AU biosynthesis genes in the VAL/SW combination. Genes encoding BR

and CT receptor kinases were overexpressed in leaves and roots of the symptomatic

VAL/CAN as compared to symptomatic VAL/SW, suggesting that CAN rootstock

activates the BR and CT -dependent immunity and growth responses in leaves and

roots at the symptomatic stage of HLB-affected plants. Jasmonic acid hormone

metabolism related genes were not significantly upregulated in the leaves of the

symptomatic VAL/CAN as compared to symptomatic VAL/SW. However, the JA-

induced MYC2, a gene involved in the JA defense signaling branch, overexpressed in

the symptomatic VAL/CAN roots. In addition, roots collected from symptomatic

107

VAL/CAN combination showed the upregulation of genes encoding the negative

regulators of JA, SA, and GA. The activation of the MYC associated response in the

roots is attributed to the interaction between JA repressor JAZ and GA repressor

DELLA proteins as reviewed in the hormones crosstalk literature (De Bruyne et al.,

2014). In the absence of GA, DELLA interacts with JAZ which released the JAZ induced

JA repression. The suppression of JAZ leads to the activation of MYC signaling

pathway (Navarro et al., 2008). MYC singling branch activates the JA signaling and

suppression of SA-induced responses (Pieterse et al., 2009). Also, JA and ABA-induced

MYC and PYL4 TFs have a synergistic effect on the herbivore resistance (Lackman et

al., 2011). Therefore, upregulation of transcripts encoding MYC and PYL4 TFs, and

overexpression of DELLA indicate that VAL/CAN plants had defense activated for

herbivore (psyllid) attack.

The symptomatic treatment of VAL/SW combination modulated JA, ET, AU, and

ABA hormonal metabolism associated genes in the VAL leaves and SW roots. SA-

mediated signaling pathway genes were not upregulated at higher expression levels in

either of the tissues suggesting that JA-ET dependent SA antagonism suppressed the

SA pathway (Pieterse et al., 2012). Moreover, overexpression of AU and ABA

metabolism pathway genes supported the downregulation of SA-dependent defenses

as studied by De Torres Zabala et al. (2009). Upregulation of genes involved in the

ABA metabolism and signaling pathway in the symptomatic treatment of the VAL/SW

roots as compared to symptomatic VAL/CAN roots supports the HLB induced root

growth reduction, and possible water deficiency associated abiotic stress in the plant.

The water deficiency associated abiotic stress might activate ABA signaling in the

108

symptomatic VAL/SW roots. Antagonism between ABA and SA has also been reported

when overexpression of NCED5, an ABA biosynthesis gene, accumulated JA and

lowered SA content in Arabidopsis (Fan et al., 2009). In symptomatic VAL/SW roots, the

significant upregulation of NCED5 suggests that ABA might play an important role in

downregulating SAR response. Auxin-responsive GH3 is an important modulator in AU

homeostasis that inactivates AU by conjugating excess AU with amino acids (Staswick,

2005). Higher expression of transcripts encoding GH3.1 in the roots of the symptomatic

treatment of the VAL/SW as compared to symptomatic VAL/CAN roots suggests that

VAL/SW is possibly controlling excess AU accumulation.

Overall, the DGE analysis of the genes involved in hormonal metabolism

regulation suggests that susceptibility of the SW rootstock is significantly attributed to

the AU accumulation, water deficiency induced ABA responses and ET metabolism

overexpression. Although ET is associated with the plant defenses to the pathogen, the

overwhelming ET induces response imbalances between JA-SA interaction. Also, ABA

and AU -induced responses strongly downregulate SA-regulated defense in the

advanced stage of symptom development. The JA activated pathway seems to be a

part of SA-JA hormonal crosstalk and response to psyllid attack. The improved

tolerance induced by the CAN rootstock can be attributed to the substantial upregulation

of BR metabolism and AU homeostasis in the presymptomatic and symptomatic stages

of CaLas infection. Crosstalk between DELLA and JAZ proteins significantly bypass the

JA suppression and activates MYC branch. The upregulation of JA-induced MYC also

explains the suppression ET and SA signaling pathway in the symptomatic VAL/CAN

roots. Overall, the data showed that CAN rootstock reprograms the VAL scion by

109

attaining AU homeostasis, and BR-CT-JA regulated growth and defense responses

under CaLas infection. Whereas SW is unable to defy AU and ET -induced reactions in

the CaLas-infected plant, at least in the infection phases studied. In conclusion, DGE

analysis of two different scion-rootstock combinations study showed that rootstock can

differentially regulate the scion transcriptome in response to the HLB infection, affecting

overall tree tolerance potentially through hormonal regulations.

110

Table 3-1. Experimental treatments and scion/rootstock combinations



Swingle; 2n (SW)



Symptomatic VAL/SW

Asymptomatic VAL/SW

putative HLB-tolerant

candidate; 2n (CAN)




VAL/CAN

Asymptomatic

VAL/CAN


Table 3-2. Comparison pairs used for DGE analysis in leaves and roots of the experimental scion/rootstock combinations

Leaves Roots

Asymptomatic VAL/CAN vs. Asymptomatic VAL/SW


Symptomatic VAL/CAN vs. Symptomatic VAL/SW


.

111

Table 3-3. Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of asymptomatic VAL/CAN combination as compared to asymptomatic VAL/SW leaves Arabidopsis Gene

C.clementina_ID log2 FC Arabidopsis-define Associated hormone

Functional description

AT3G45140 Ciclev10014207m.g 3.07029 lipoxygenase 2 JA JA Biosynthesis

LHY Ciclev10018964m.g 2.81304 Homeodomain-like superfamily protein

AU Circadian clock regulation

TT4 Ciclev10001405m.g 2.03082 Chalcone and stilbene synthase family protein

AU negative regulator of AU transport

AT3G09610 Ciclev10012263m.g 2.00758 Homeodomain-like superfamily protein

AU Circadian clock

MIPS2 Ciclev10025400m.g 1.94841 myo-inositol-1-phosphate synthase 2

AU Auxin storage and transport

MYB3 Ciclev10009286m.g 1.86113 myb domain protein 3 AU Myb domain transcription factor

SHN1 Ciclev10009447m.g 1.69017 Integrase-type DNA-binding superfamily protein

ET Cutin Biosynthesis

TINY2 Ciclev10027587m.g 1.66184 Integrase-type DNA-binding superfamily protein

ET putative transcription factor

F20P5.26 Ciclev10032167m.g 1.58935 myb-like transcription factor family protein

AU Myb transcription factor

TT7 Ciclev10019637m.g 1.56945 Cytochrome P450 superfamily protein

AU Flavonoid synthesis, Convert naringenin to eriodictyol

ARR9 Ciclev10021937m.g 1.56679 response regulator 9 CT Type-A response regulators seem to act as negative regulators of the cytokinin signaling

AT1G08810 Ciclev10031946m.g 1.55202 myb domain protein 60 JA Stomatal regulation

T12P18.14 Ciclev10032900m.g 1.40527 RING/U-box superfamily protein

AU Proteasome/ protein degradation

ARF16 Ciclev10030860m.g 1.34252 auxin response factor 16 AU Auxin responsive gen

IAA14 Ciclev10026522m.g 1.29996 indole-3-acetic acid inducible 14

AU IAA/AUX gene family

NRT1.1 Ciclev10019412m.g 1.29906 nitrate transporter 1.1 AU Nitrate transporter

ATAUX2-11 Ciclev10022370m.g 1.26724 AUX/IAA transcriptional regulator family protein

AU Auxin response repressor

F10A5.21 Ciclev10029633m.g 1.26191 SAUR-like auxin-responsive protein family

AU Auxin responsive gene

112

Table 3-3. Continued Arabidopsis Gene

C.clementina_ID log2 FC Arabidopsis-define Associated hormone


GLIP1 Ciclev10025893m.g 1.2467 GDSL lipase 1 ET Ethylene signaling, antimicrobial resistance

GPRI1 Ciclev10015266m.g 1.24409 GBF\'s pro-rich region-interacting factor 1

SA Chloroplast development/regulation of chlorophyll biosynthesis

ZFP8 Ciclev10012347m.g 1.1678 zinc finger protein 8 SA transcription factor required for the initiation of inflorescence trichomes to GA and CT

BRI1 Ciclev10024737m.g 1.11348 Leucine-rich receptor-like protein kinase family protein

BR BR Signaling

AT1G68530 Ciclev10031329m.g 1.05234 3-ketoacyl-CoA synthase 6 JA long chain fatty acid biosynthesis, Wax biosynthesis

BKI1 Ciclev10001740m.g 1.04793 BRI1 kinase inhibitor 1 BR Negative regulator of brassinosteroid signaling

F27J15.20 Ciclev10021342m.g 1.03707 Duplicated homeodomain-like superfamily protein

AU Transcription factors


AU IAA/AUX gene family, repressor of AU responsive gene

MBG8.12 Ciclev10008106m.g 1.01028 Major facilitator superfamily protein

SA folate transport

GA2OX8 Ciclev10015702m.g 0.995038 gibberellin 2-oxidase 8 GA inactivates GA

SMT1 Ciclev10031748m.g 0.966705 sterol methyltransferase 1 BR Sterol Biosynthesis (cycloartenol to 24-methylene cycloarteno)

MGH6.18 Ciclev10008343m.g 0.955683 Major facilitator superfamily protein

ET affinity carnitine transporter involved in the active cellular uptake of carnitine

HB-2 Ciclev10021374m.g 0.952059 homeobox protein 2 AU Transcription factor

EXPA1 Ciclev10032548m.g 0.904924 expansin A1 GA loosening and extension of cell wall

TCP14 Ciclev10020275m.g 0.868728 TEOSINTE BRANCHED, cycloidea and PCF (TCP) 14

CT affect internode length

COP1 Ciclev10014491m.g 0.813941 Transducin/WD40 repeat-like superfamily protein

GA repressor of photomorphogenisis and activates etiolation in dark

ARF8 Ciclev10014198m.g 0.805212 auxin response factor 8 AU Promote JA production and flower maturation

HERK1 Ciclev10027823m.g 0.777615 hercules receptor kinase 1 BR required for cell elongation during vegetative growth BR independent manner

113


C.clementina_ID log2 FC Arabidopsis-define Associated hormones



ET Cutin Biosyntehsis

AT1G06620 Ciclev10005208m.g 1.18259 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

JA Oxido-reductase

APRR2 Ciclev10031120m.g 1.23171 CheY-like two-component responsive regulator family protein

CT Ripening and chlorophyll development

BKI1 Ciclev10001740m.g 1.04793 BRI1 kinase inhibitor 1 BR Negative regulator of brassinosteroid signaling

F27J15.20 Ciclev10021342m.g 1.03707 Duplicated homeodomain-like superfamily protein

AU Transcription factors


AU IAA/AUX gene family, repressor of AU responsive gene

MBG8.12 Ciclev10008106m.g 1.01028 Major facilitator superfamily protein

SA folate transport

GA2OX8 Ciclev10015702m.g 0.995038 gibberellin 2-oxidase 8 GA inactivates GA

SMT1 Ciclev10031748m.g 0.966705 sterol methyltransferase 1 BR Sterol Biosynthesis (cycloartenol to 24-methylene cycloarteno)

MGH6.18 Ciclev10008343m.g 0.955683 Major facilitator superfamily protein

ET affinity carnitine transporter involved in the active cellular uptake of carnitine

HB-2 Ciclev10021374m.g 0.952059 homeobox protein 2 AU Transcription factor

EXPA1 Ciclev10032548m.g 0.904924 expansin A1 GA loosening and extension of cell wall

TCP14 Ciclev10020275m.g 0.868728 TEOSINTE BRANCHED, cycloidea and PCF (TCP) 14

CT affect internode length

COP1 Ciclev10014491m.g 0.813941 Transducin/WD40 repeat-like superfamily protein

GA repressor of photomorphogenisis and activates etiolation in dark


HERK1 Ciclev10027823m.g 0.777615 hercules receptor kinase 1 BR

required for cell elongation during vegetative growth BR independent manner

114

Table 3-4. Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of asymptomatic VAL/SW combination as compared to asymptomatic VAL/CAN leaves Arabidopsis Gene

C.clementina_ID log2 FC* Arabidopsis-define Associated hormones


CBF4 Ciclev10007068m.g -5.4123 C-repeat-binding factor 4 ABA ABA induce abiotic stress related TF

F12K2.11 Ciclev10028740m.g -4.8287 F-box family protein ABA

WRKY33 Ciclev10011386m.g -4.71604 WRKY DNA-binding protein 33 SA JA regulated transcription factor

AT2G31945 Ciclev10033110m.g -4.63576 hypothetical protein ET hypothetical protein

CBF4 Ciclev10007068m.g -4.4286 C-repeat-binding factor 4 ABA ABA induce abiotic stress related TF

CYP83B1 Ciclev10025429m.g -4.36975 cytochrome P450, family 83, subfamily B, polypeptide 1

ET glucosinolate biosynthesis, elavated auxins, JA suppressor

EDL3 Ciclev10020962m.g -4.2594 EID1-like 3 ABA

F3H9.15 Ciclev10029923m.g -4.2588 hypothetical protein ABA hypothetical protein

SIB1 Ciclev10002803m.g -4.2317 sigma factor binding protein 1 SA Hyperactive defense _SA accumulation

F22D22.22 Ciclev10005971m.g -4.16158 Acyl-CoA N-acyltransferases (NAT) superfamily protein

ET GCN5-related N-acetyltransferase-like protein

T5J17.9 Ciclev10025235m.g -4.08285 Cupredoxin superfamily protein SA L-ascorbate oxidase/oxidation-reduction

DREB2C Ciclev10032029m.g -3.8805 Integrase-type DNA-binding superfamily protein

ABA abiotic tolerance

JAZ8 Ciclev10017198m.g -3.76589 jasmonate-zim-domain protein 8 ET JA biosynthesis suppressor

MYB15 Ciclev10005629m.g -3.71609 myb domain protein 15 AU ABA induced drought/ stress tolerance

ERF1 Ciclev10005820m.g -3.60745 ethylene response factor 1 ET Ethylene transcription factor

F22D22.22 Ciclev10005998m.g -3.5945 Acyl-CoA N-acyltransferases (NAT) superfamily protein

ET GCN5-related N-acetyltransferase-like protein

CYP81D8 Ciclev10027560m.g -3.47832 cytochrome P450, family 81, subfamily D, polypeptide 8

ET response to karrikin

ERF-1 Ciclev10021652m.g -3.47224 ethylene responsive element binding factor 1

ET/ABA Involved in the regulation of gene expression

T19D16.6 Ciclev10000618m.g -3.37243 Protein kinase superfamily protein ET/ABA putative receptor-like protein kinase

WRKY40 Ciclev10026105m.g -3.29586 WRKY DNA-binding protein 40 SA/JA Suppress PAMP induce ROS and seedling growth inhibition/suppress PTI

JAZ1 Ciclev10026376m.g -3.25918 jasmonate-zim-domain protein 1 SA/JA/ABA JA repressor gene

AT3G57450 Ciclev10006321m.g -3.13197 JA/ABA uncharacterized protein

RRTF1 Ciclev10028930m.g -3.10646 redox-responsive transcription factor 1

ET involved in the regulation of gene expression by stress factors

115




K18I23.10 Ciclev10033100m.g -3.06587 ET uncharacterized protein

ATAF1 Ciclev10001976m.g -3.0475 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein

ABA

CCR3 Ciclev10030732m.g -3.0404 CRINKLY4 related 3 ET Serine/threonine-protein kinase

CYP94C1 Ciclev10028253m.g -3.01833 cytochrome P450, family 94, subfamily C, polypeptide 1

AU JA Lle catabolic turn over

ERF1 Ciclev10021622m.g -3.00672 ethylene response factor 1 ET key integrator of ethylene and jasmonate signals in the regulation defenses.

JAZ1 Ciclev10026527m.g -2.89212 jasmonate-zim-domain protein 1 SA JA respressor gene

T1F9.17 Ciclev10016832m.g -2.87894 F-box family protein ET F-box protein

AT5G57150 Ciclev10009285m.g -2.84995 basic helix-loop-helix (bHLH) DNA-binding superfamily protein

JA transcription factor bHLH35

WRKY40 Ciclev10008930m.g -2.8097 WRKY DNA-binding protein 40 SA/JA/ABA Suppress PAMP induce ROS and seedling growth inhibition/suppress PTI

F18A17.4 Ciclev10007383m.g -2.78663 Protein kinase family protein with leucine-rich repeat domain

ET/ABA Protein kinase family protein with leucine-rich repeat domain

ERF13 Ciclev10021995m.g -2.77454 ethylene-responsive element binding factor 13

ET/AU components of stress signal transduction pathways

WRKY33 Ciclev10000654m.g -2.7374 WRKY DNA-binding protein 33 SA JA regualted transcription factor

MPA22.3 Ciclev10015253m.g -2.70883 ARM repeat superfamily protein ET U-box domain-containing protein 21; Functions as an E3 ubiquitin ligase

TT7 Ciclev10025396m.g -2.65351 Cytochrome P450 superfamily protein

AU Flavonoid synthesis, Convert naringenin to eriodictyol

HERK1 Ciclev10030714m.g -2.64783 hercules receptor kinase 1 BR Receptor-like protein kinase required for cell elongation during vegetative growth

PLA-2 Ciclev10019666m.g -2.6299 alpha/beta-Hydrolases superfamily protein

ET

JAZ1 Ciclev10008653m.g -2.60165 jasmonate-zim-domain protein 1 SA JA siganling respressor

T32G9.25 Ciclev10002675m.g -2.52729 ET uncharacterized protein

F13A10.15 Ciclev10023517m.g -2.43694 P-loop containing nucleoside triphosphate hydrolases superfamily protein

ET The enzyme involved in the ethylene biosynthesis.

CYP707A1 Ciclev10011655m.g -2.4304 cytochrome P450, family 707, subfamily A, polypeptide 1

ABA ABA Biosynthesis

116




T32G9.25 Ciclev10002675m.g -2.52729 ET uncharacterized protein

F13A10.15 Ciclev10023517m.g -2.43694 P-loop containing nucleoside triphosphate hydrolases superfamily protein

ET Enzyme involved in the ethylene biosynthesis.

CYP707A1 Ciclev10011655m.g -2.4304 cytochrome P450, family 707, subfamily A, polypeptide 1

ABA ABA Biosynthesis


JA/ABA/AU JA Lle catabolic turn over

EFE Ciclev10015962m.g -2.39971 ethylene-forming enzyme ET/SA activating ethylene biosynthesis

HAI2 Ciclev10028495m.g -2.3323 highly ABA-induced PP2C gene 2 ABA negative regulator of PCD, SA, JA, and ET

LOX3 Ciclev10024819m.g -2.25448 lipoxygenase 3 SA Linolenic acid

MAPKKK19 Ciclev10025910m.g -2.0696 mitogen-activated protein kinase kinase kinase 19

ET mitogen-activated protein kinase kinase kinase 19

F3G5.22 Ciclev10032889m.g -2.05007 C2H2 and C2HC zinc fingers superfamily protein

ET zinc finger of Arabidopsis thaliana 11

AP2C1 Ciclev10028590m.g -2.0242 Protein phosphatase 2C family protein

ABA

DIC2 Ciclev10001822m.g -2.01391 dicarboxylate carrier 2 ET May be involved in protecting plant cells against oxidative stress damage

AT5G26170 Ciclev10009761m.g -2.00255 WRKY DNA-binding protein 50 JA Mediate SA and repress JA

EBP Ciclev10021059m.g -1.99823 ethylene-responsive element binding protein

CT involved in the regulation of gene expression by stress factors


ABA


AU involved in the regulation of gene expression by stress factors

BT1 Ciclev10020762m.g -1.93758 BTB and TAZ domain protein 1 AU substrate-specific adapter of an E3 ubiquitin-protein ligase complex

MYBR1 Ciclev10001979m.g -1.91955 myb domain protein r1 AU/ABA Confers resistance to abiotic stresses dependent of ABA

STZ Ciclev10002297m.g -1.91779 salt tolerance zinc finger AU involved in jasmonate (JA) early signaling response.

ERF12 Ciclev10016982m.g -1.88941 ERF domain protein 12 ET Ehtylene resposnive gene

GRX480 Ciclev10017106m.g -1.82247 Thioredoxin superfamily protein SA SA-responsive gene

117




MQL5.25 Ciclev10021067m.g -1.81767 myb-like transcription factor family protein

GA Transcription factor

AT1G32640 Ciclev10011214m.g -1.80925 Basic helix-loop-helix (bHLH) DNA-binding family protein

JA Transcription factor

ERF5 Ciclev10021285m.g -1.7995 ethylene responsive element binding factor 5

ET components of stress signal transduction pathways

ILL6 Ciclev10000964m.g -1.77065 IAA-leucine resistant (ILR)-like gene 6

AU IAA storage

KCS11 Ciclev10007916m.g -1.76019 3-ketoacyl-CoA synthase 11 BR Cutin/wax synthesis to prevent water loss and pathogen atatck

F14D16.17 Ciclev10023101m.g -1.72698 SA uncharacterized protein

T6G21.2 Ciclev10028965m.g -1.66046 Polynucleotidyl transferase, ribonuclease H-like superfamily protein

ET/ABA It is a component of the CCR4 complex involved in the control of gene expression

AT4G16760 Ciclev10019196m.g -1.63443 acyl-CoA oxidase 1 JA May be involved in the biosynthesis of jasmonic acid

AATP1 Ciclev10011449m.g -1.62721 AAA-ATPase 1 SA Proteosome

T23A1.8 Ciclev10028539m.g -1.54257 Protein kinase superfamily protein SA kinase 3

AATP1 Ciclev10011474m.g -1.54166 AAA-ATPase 1 SA Preoteosome

MWD22.13 Ciclev10005570m.g -1.51981 Integrase-type DNA-binding superfamily protein

ET ethylene-responsive transcription factor ERF105

AOC3 Ciclev10032510m.g -1.46679 allene oxide cyclase 3 ET/ABA production of 12-oxo-phytodienoic acid (OPDA), a precursor of jasmonic acid

F14G6.20 Ciclev10002430m.g -1.42173 ET uncharacterized protein

F12L6.31 Ciclev10032209m.g -1.36132 Protein of unknown function (DUF506)

ET uncharacterized protein

NPR3 Ciclev10017873m.g -1.33014 NPR1-like protein 3 SA SA modulator

AT5G35735 Ciclev10015492m.g -1.32348 Auxin-responsive family protein SA Auxine responsive gene

PKT3 Ciclev10020068m.g -1.2937 peroxisomal 3-ketoacyl-CoA thiolase 3

ABA

WRKY70 Ciclev10032192m.g -1.23958 WRKY DNA-binding protein 70 SA SA responsive gene

ATERDJ3A Ciclev10011366m.g -1.1066 DNAJ heat shock N-terminal domain-containing protein

ABA

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F2O15.22 Ciclev10012000m.g -1.09079 zinc finger (C3HC4-type RING finger) family protein

ET C3H4 type zinc finger protein

AOS Ciclev10000825m.g -0.989301 allene oxide synthase SA JA biosyntehsis * The negative sign in the column of log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination.

119

Table 3-5. Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW leaves Arabidopsis Gene



CKX7 Ciclev10007999m.g 1.86652 cytokinin oxidase 7 CT degradation of cytokinin

WRKY75 Ciclev10032943m.g 1.57081 WRKY DNA-binding protein 75 ET Interacts specifically with the W box a frequently occurring elicitor- responsive cis-acting element

IAA19 Ciclev10026400m.g 1.327 indole-3-acetic acid inducible 19 AU Auxin response repressor

IAA14 Ciclev10005789m.g 1.21806 indole-3-acetic acid inducible 14 AU Auxin response repressor

HCT Ciclev10026558m.g 1.18827 hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase

AU lignin bisynthesis, flavonoid accumulation/auxin tranport

ATAUX2-11

Ciclev10022370m.g 1.16366 AUX/IAA transcriptional regulator family protein


KCS6 Ciclev10031329m.g 1.10481 3-ketoacyl-CoA synthase 6 JA an essential step of the cuticular wax production

F24J1.30 Ciclev10031937m.g 1.06029 Homeodomain-like superfamily protein

CT

ARR4 Ciclev10032281m.g 1.01137 response regulator 4 SA Modulates red light signaling through its interaction with the phytochrome B photoreceptor

AIR3 Ciclev10014351m.g 0.995049 Subtilisin-like serine endopeptidase family protein

AU Au resonsive root gene, activated by NAC transcription factor

T12H17.10 Ciclev10022581m.g 0.971691 SAUR-like auxin-responsive protein family

AU Auxin response gene

KAO2 Ciclev10031341m.g 0.87059 ent-kaurenoic acid hydroxylase 2 GA a key step in gibberellins (GAs) biosynthesis

IAA8 Ciclev10001479m.g 0.841252 indoleacetic acid-induced protein 8


ABF2 Ciclev10001159m.g 0.840412 abscisic acid responsive elements-binding factor 2

ABA nvolved in ABA and stress responses and acts as a positive component of glucose signal transduction.

BSK2 Ciclev10019840m.g 0.83354 BR-signaling kinase 2 BR BR-signaling kinase 2

F12K2.11 Ciclev10012130m.g 0.811637 F-box family protein ABA F-box family protein

ATAUX2-11

Ciclev10006002m.g 0.808084 AUX/IAA transcriptional regulator family protein


NDL1 Ciclev10008801m.g 0.767112 N-MYC downregulated-like 1 AU Auxine transport and hence root architcture

RPN10 Ciclev10025759m.g 0.706887 regulatory particle non-ATPase 10

AU growth develop via proteosome of ABA signaling protein ABI5/DPBF1

120


C.clementina_ID log2 FC Arabidopsis-definition Associated hormones


APRR2 Ciclev10031120m.g 0.697944 CheY-like two-component responsive regulator family protein

CT


ARR11 Ciclev10031052m.g 0.675231 response regulator 11 CT

CYP707A2 Ciclev10028346m.g 0.643668 cytochrome P450, family 707, subfamily A, polypeptide 2

ABA ABA hydroxylation, Control ABA during mid-Maturation

121

Table 3-6. Differentially expressed hormonal metabolism-associated genes significantly upregulated in leaves of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN leaves Arabidopsis Gene

C.clementina_ID log2 FC * Arabidopsis-definition Hormones associated


LOX2 Ciclev10014199m.g -2.98016 lipoxygenase 2 JA JA biosynthesis

JAZ1 Ciclev10026376m.g -2.83654 jasmonate-zim-domain protein 1 SA JA repressor

LOX2 Ciclev10014204m.g -2.83407 lipoxygenase 2 JA JA biosynthesis

EXPA1 Ciclev10021897m.g -2.79461 expansin A1 GA loosening and extension of cell wall

JAZ8 Ciclev10017198m.g -2.73427 jasmonate-zim-domain protein 8 JA Repressor of jasmonate responses

ERF1 Ciclev10021622m.g -2.66907 ethylene response factor 1 JA Binds to the GCC- box pathogenesis-related promoter element

AT4G08850 Ciclev10024069m.g -2.51968 Leucine-rich repeat receptor-like protein kinase family protein

ET

WRKY40 Ciclev10008930m.g -2.3154 WRKY DNA-binding protein 40 SA Supress PAMP induce ROS and seedling growth inhibition/supress PTI

WRKY33 Ciclev10011386m.g -2.24588 WRKY DNA-binding protein 33 SA JA response postive regulator.

SIB1 Ciclev10002803m.g -2.20916 sigma factor binding protein 1 SA Hyperactive defense _SA accumulation

DREB2C Ciclev10032029m.g -1.9199 Integrase-type DNA-binding superfamily protein

ABA Binding to the C-repeat/DRE element mediates high salinity- and abscisic acid-inducible transcription


ERF1 Ciclev10016995m.g -1.87617 ethylene response factor 1 JA Binds to the GCC- box pathogenesis-related promoter element

TCTP Ciclev10002699m.g -1.79532 translationally controlled tumor protein

AU Involved in calcium binding and microtubule stabilization

ACS6 Ciclev10019920m.g -1.7656 1-aminocyclopropane-1-carboxylic acid (acc) synthase 6

AU Ethylene synthesis, local gene mediated and basal resistance

ATERDJ3A Ciclev10011366m.g -1.74509 DNAJ heat shock N-terminal domain-containing protein

ABA DNA J domain-containing protein

AP2C1 Ciclev10028590m.g -1.67441 Protein phosphatase 2C family protein

ABA Protein phosphatase that negatively regulates defense respones. I

OPCL1 Ciclev10000764m.g -1.6669 OPC-8:0 CoA ligase1 JA Contributes to jasmonic acid biosynthesis by initiating the beta-oxidative chain shortening of its precursors.

AT3G60690 Ciclev10022560m.g -1.60776 SAUR-like auxin-responsive protein family

SA Auxin response gene

Rap2.6L Ciclev10005787m.g -1.59504 related to AP2 6l ET

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C.clementina_ID log2 FC Arabidopsis-definition Hormones associated


EDL3 Ciclev10020962m.g -1.58192 EID1-like 3 ABA F box protein, ABA regulation

CYP81D8 Ciclev10025420m.g -1.50646 cytochrome P450, family 81, subfamily D, polypeptide 8

ET

PYL4 Ciclev10032781m.g -1.49605 PYR1-like 4 ABA required for ABA- mediated responses such as stomatal closure and germination inhibition

F5H14.15 Ciclev10015144m.g -1.47575 Integrase-type DNA-binding superfamily protein

ET


JA JA Lle catabolic turn over

MFB16.16 Ciclev10032901m.g -1.45981 SAUR-like auxin-responsive protein family

AU Auxin response gene

ATAF1 Ciclev10001976m.g -1.45707 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein

JA

T29M8.8 Ciclev10009540m.g -1.45152 Integrase-type DNA-binding superfamily protein

ET

T1F9.17 Ciclev10016832m.g -1.43897 F-box family protein ET

F5E6.17 Ciclev10004474m.g -1.35904 Plant neutral invertase family protein

ET

CBF4 Ciclev10007068m.g -1.35588 C-repeat-binding factor 4 ABA freezing tolerance and cold acclimation, drought adaptations

PYR1 Ciclev10012769m.g -1.33539 Polyketide cyclase/dehydrase and lipid transport superfamily protein

ABA Inhibits the activity of group-A protein phosphatases type 2C (PP2Cs) when activated by ABA

T1J8.15 Ciclev10011571m.g -1.31988 UDP-Glycosyltransferase superfamily protein

ET

TT4 Ciclev10025807m.g -1.30409 Chalcone and stilbene synthase family protein

AU negative regulator of AU transport/ flavonoid accumulation/chalcone synthase_Naringin


ABA ; Acts as a transcriptional activator


ABA


WRKY33 Ciclev10000654m.g -1.31137 WRKY DNA-binding protein 33 SA JA regulate transcription factor

123





JA glucasinolate biosynthesis, elavated auxins, JA suppressor

PR4 Ciclev10029528m.g -1.23902 pathogenesis-related 4 JA JA responsive gene

WRKY30 Ciclev10020744m.g -1.23552 WRKY DNA-binding protein 30 ET

MEE59 Ciclev10029307m.g -1.22607 maternal effect embryo arrest 59 ET

EBF1 Ciclev10007739m.g -1.21117 EIN3-binding F box protein 1 ET

AP22.63 Ciclev10000389m.g -1.187 ARM repeat superfamily protein SA

EBP Ciclev10005701m.g -1.16377 ethylene-responsive element binding protein

CT

ABP1 Ciclev10022400m.g -1.15672 endoplasmic reticulum auxin binding protein 1

AU Auxin binding protein involved in cell elongation and cell division/receptor of AU

LOX3 Ciclev10024819m.g -1.11808 lipoxygenase 3 SA JA responsive genes

LECRKA4.2 Ciclev10013896m.g -1.06804 lectin receptor kinase a4.1 ABA involved in negative regulation of abscisic acid response in seed germination


ABA

T11A7.19 Ciclev10005012m.g -1.04622 Integrase-type DNA-binding superfamily protein

ET

F3F20.16 Ciclev10022741m.g -1.00246 basic helix-loop-helix (bHLH) DNA-binding superfamily protein

ET

MWD22.13 Ciclev10005570m.g -1.00067 Integrase-type DNA-binding superfamily protein

ET

ABI2 Ciclev10008610m.g -0.934027 Protein phosphatase 2C family protein

ABA Repressor of the abscisic acid (ABA) signaling pathway that regulates numerous ABA responses

MKD10.10 Ciclev10011584m.g -0.932938 Methylenetetrahydrofolate reductase family protein

JA proline dehydrogenase 2; Converts proline to delta-1-pyrroline-5-carboxylate

PYL11 Ciclev10006096m.g -0.879625 PYR1-like 11 ABA Receptor for ABA, required for ABA- mediated responses

WRKY70 Ciclev10012055m.g -0.807275 WRKY DNA-binding protein 70 SA SA acid transcription factor

EIN4 Ciclev10018972m.g -0.806884 Signal transduction histidine kinase, hybrid-type, ethylene sensor

ET Acts as a redundant negative regulator of ethylene signaling

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DHS2 Ciclev10028255m.g -0.764296 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase

CT

LOX5 Ciclev10004281m.g -0.746513 PLAT/LH2 domain-containing lipoxygenase family protein

JA regulate root architecture, leniolate biosynthesis

WRKY70 Ciclev10032192m.g -0.718461 WRKY DNA-binding protein 70 SA SA acid transcription factor

NPR3

Ciclev10017873m.g -0.682176 NPR1-like protein 3 SA SA modulator

* The negative sign in the column of log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination.

125

Table 3-7. Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW roots

Arabidopsis Gene



JAZ8 Ciclev10017198m.g 4.77853 jasmonate-zim-domain protein 8 JA

CYP94C1 Ciclev10028254m.g 3.63521 cytochrome P450, family 94, subfamily C, polypeptide 1

JA

NPR3 Ciclev10031749m.g 3.61027 NPR1-like protein 3 SA SA modulator

JAZ1 Ciclev10026376m.g 3.45108 jasmonate-zim-domain protein 1 JA/SA JA repressor

JAZ1 Ciclev10026527m.g 3.18591 jasmonate-zim-domain protein 1 JA/SA JA repressor

CYP83B1 Ciclev10004006m.g 3.1829 cytochrome P450, family 83, subfamily B, polypeptide 1

AU

MYC2 Ciclev10019749m.g 2.61256 Basic helix-loop-helix (bHLH) DNA-binding family protein

JA JA, ABA transcriptional activator

ERF13 Ciclev10024187m.g 2.52228 ethylene-responsive element binding factor 13

SA

BAS1 Ciclev10007914m.g 2.33409 Cytochrome P450 superfamily protein BR

GAI Ciclev10031305m.g 2.20437 GRAS family transcription factor family protein

GA/SA Della protein

AOS Ciclev10000825m.g 1.99711 allene oxide synthase JA JA biosynthesis gene

F1M20.4 Ciclev10024165m.g 1.92242 Leucine-rich repeat protein kinase family protein

JA

RGL1 Ciclev10004586m.g 1.90692 RGA-like 1 GA/SA Della protein

DHS1 Ciclev10025342m.g 1.78471 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 1

JA

JAZ10 Ciclev10032858m.g 1.7251 jasmonate-zim-domain protein 10 JA

HERK2 Ciclev10014250m.g 1.69952 hercules receptor kinase 2 BR

NCED5 Ciclev10014639m.g 1.60548 nine-cis-epoxycarotenoid dioxygenase 5 ABA

GRX480 Ciclev10017106m.g 1.55861 Thioredoxin superfamily protein SA

BKI1 Ciclev10001740m.g 1.52823 BRI1 kinase inhibitor 1 BR

AT5G59845 Ciclev10029695m.g 1.4799 Gibberellin-regulated family protein GA

F14G6.12 Ciclev10031658m.g 1.37629 Auxin efflux carrier family protein AU

PYL4 Ciclev10032781m.g 1.2223 PYR1-like 4 ABA

RR5 Ciclev10022334m.g 1.2182 response regulator 5 GA

GA20OX1 Ciclev10005157m.g 1.2112 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

GA

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F12K2.11 Ciclev10012130m.g 1.20727 F-box family protein ABA

T11I11.4 Ciclev10001889m.g 1.2061 F-box family protein BR

AIR12 Ciclev10032615m.g 1.19412 auxin-responsive family protein AU

DREB2C Ciclev10032029m.g 1.17466 Integrase-type DNA-binding superfamily protein

ABA


AU

RGA1 Ciclev10010825m.g 1.14157 GRAS family transcription factor family protein

GA Della protein

GRX480 Ciclev10017057m.g 1.09409 Thioredoxin superfamily protein SA

MYC2 Ciclev10011214m.g 1.08555 Basic helix-loop-helix (bHLH) DNA-binding family protein

SA JA, ABA transcriptional activator

GA2OX6 Ciclev10024433m.g 1.08346 gibberellin 2-oxidase 6 GA

PSBO2 Ciclev10026034m.g 1.0783 photosystem II subunit O-2 JA

RGA1 Ciclev10003213m.g 1.06425 GRAS family transcription factor family protein

GA Della protein

ERF1 Ciclev10016995m.g 1.03474 ethylene response factor 1 JA

ATAF1 Ciclev10001976m.g 1.00984 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein

JA

LTA2 Ciclev10031397m.g 1.00336 2-oxoacid dehydrogenases acyltransferase family protein

JA

BAS1 Ciclev10033321m.g 0.995538 Cytochrome P450 superfamily protein BR

ARF16 Ciclev10030860m.g 0.984785 auxin response factor 16 AU

GH3.1 Ciclev10025211m.g 0.965343 Auxin-responsive GH3 family protein BR

ARF2 Ciclev10019257m.g 0.948293 auxin response factor 2 AU

GAI Ciclev10011367m.g 0.935485 GRAS family transcription factor family protein

GA/SA Della protein

ERF13 Ciclev10022734m.g 0.890543 ethylene-responsive element binding factor 13

JA

JAR1 Ciclev10019459m.g 0.887257 Auxin-responsive GH3 family protein JA

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Table 3-8. Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN roots

Arabidopsis Gene

C.clementina_ID log2 FC* Arabidopsis-definition Hormones associated

NCED4 Ciclev10031003m.g

-1.73638

nine-cis-epoxycarotenoid dioxygenase 4

ABA

PIN4 Ciclev10012938m.g -1.50665 Auxin efflux carrier family protein SA

ABA4 Ciclev10012575m.g -1.36332 abscisic acid (aba)-deficient 4 ABA

F24J13.5 Ciclev10032036m.g -1.34888 Domain of unknown function (DUF220) SA

AOC4 Ciclev10012528m.g -1.31543 allene oxide cyclase 4 AU

ARF19 Ciclev10007286m.g -1.27446 auxin response factor 19 AU

ASA1 Ciclev10031010m.g -1.25436 anthranilate synthase alpha subunit 1 AU

SERK1 Ciclev10000597m.g -1.20804 somatic embryogenesis receptor-like kinase 1 AU

PAP1 Ciclev10026076m.g -1.16013 phytochrome-associated protein 1 ET

HK5 Ciclev10010162m.g -1.16251 histidine kinase 5 CT

DFL1 Ciclev10007746m.g -1.15876 Auxin-responsive GH3 family protein AU

F24J1.30 Ciclev10031937m.g -1.14421 Homeodomain-like superfamily protein AU

OEP16-1 Ciclev10029517m.g -1.13309 outer plastid envelope protein 16-1 AU

MGH6.18 Ciclev10008092m.g -1.12944 Major facilitator superfamily protein JA

K19P17.6 Ciclev10008806m.g -1.1056 Serine/threonine-protein kinase WNK (With No Lysine)-related

JA

F17H15.18 Ciclev10007358m.g -1.05918 Leucine-rich receptor-like protein kinase family protein SA

CYP83B1 Ciclev10000924m.g -1.03948 cytochrome P450, family 83, subfamily B, polypeptide 1 AU

F12K2.11 Ciclev10023799m.g -1.01711 F-box family protein GA

LAX1 Ciclev10031413m.g -1.01 like AUXIN RESISTANT 1 SA

KAO2 Ciclev10031341m.g -0.958024 ent-kaurenoic acid hydroxylase 2 GA

IAA8 Ciclev10025988m.g -0.933771 indoleacetic acid-induced protein 8 AU

F5A9.17 Ciclev10016176m.g -0.909571 anthranilate synthase beta subunit 1 AU

ARF4 Ciclev10027839m.g -0.869553 auxin response factor 4 AU

CYP714A1 Ciclev10031065m.g -0.857293 cytochrome P450, family 714, subfamily A, polypeptide 1 CT * The negative sign in the column of log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination.

128

Figure 3-1. Graphical presentation of Hormonal regulation in plants. (adapted from Plant physiology and development, sixth edition,2015)

129

Figure 3-2. Graphical presentation of DEGs involved in hormonal metabolism. Hormonal metabolism overview in the PageMan depicting DGE in leaves

and roots of HLB-asymptomatic and -symptomatic treatment in VAL/CAN and VAL/SW combinations. Log2 FC are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated

and VAL/SW combination. Asymp; Asymptomatic, Sympt; symptomatic

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CHAPTER 4

DIFFERENTIAL EXPRESSION OF PLANT IMMUNITY AND DEFENSE-ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS

AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB DISEASE

Introduction

Plants live in a complex environment where they interact with a variety of

organisms. Some plant associations with organisms are beneficial, whereas some are

detrimental. The compatible or incompatible interaction between plants and pathogens

leads to the activation of plant defense responses. Immunity responses to plant

pathogens categorize into non-host specific and host-specific interactions. Non-host

specific defense involves modulating plant structures and/or secreting chemical signals

to stop the entry of the pathogen or to alert the neighboring plant tissue to prepare itself

for the impending pathogen infection. The structural modifications include thickening of

the leaf surface, the closing of stomata to stop a pathogen entry, and secretion of

volatiles or phenolics to deter the plant pathogens.

The interaction of a plant innate immunity system with a host-specific pathogen is

a two-branched system. The first component of the plant immunity is called PAMP-

triggered immunity (PTI). PTI is the interaction between the pattern recognition

receptors (PRR) that are located the plant membrane and pathogen-associated

molecular patterns (PAMP). A classic example to understand PRR induced PAMP

recognition is elucidated in the model plants. The interaction between Pseudomonas

syriangae induced flagellin 2 (FLG2) and the flagellin sensitive 2 (FLS2) transmembrane

receptor kinase was reported in Arabidopsis thaliana (Chinchilla, 2006). Failure or

weakened PTI induces the second component of the plant innate immunity which is

known as effector-triggered immunity (ETI) (Jones and Dangl, 2006). Effectors and

131

elicitors are host-specific pathogen virulence factors that target the plant's immunity.

Interactions between resistance (R) genes of the plants and avirulence (AVR or elicitor)

genes of the pathogens determine the compatibility of the pathogen with its host. The

successful interaction of R-AVR gene products results in resistance, whereas failure of

this interaction leads to susceptibility to the disease. The ETI responses activate nuclear

R genes that result in a hypersensitive reaction (HR). Within few hours of the infection,

the HR results in programmed cell death (PCD) that stops the spread of pathogen within

the plant. Genomics studies of R genes have identified the R gene-encoded signature

protein domains (Jones and Dangl, 2006; Wu and Zhou, 2013). These include a nuclear

binding site (NBS), leucine-rich repeats (LRR) and a Toll-interleukin 1 receptor (TIR).

The interaction between ETI and the effector-triggered susceptibility (ETS) determines

plant survival.

Plant defense strategies also involve hormonal crosstalk (Pieterse et al., 2012).

Modulation of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) hormones

through a network of transcription factors (TFs), secondary messengers, and regulatory

factors contribute to plant immunity (Pieterse et al., 2009). The antagonism between SA

and JA responses has been reviewed extensively (Reymond and Farmer, 1998;

Pieterse et al., 2009; Robert-Seilaniantz et al., 2011). The SA-induced defense

signaling pathway results in systemic acquired resistance (SAR) which is effective

mostly against biotrophs and hemibiotrophs. Whereas, JA and ET activate induced

systemic resistance (ISR) is effective against necrotrophs and herbivores (Simms and

Rausher, 1987; Dam and Oomen, 2008). The role of SA, JA, and ET is not restricted to

biotrophs or necrotrophs, but SAR and ISR signaling components can interchange

132

between the SA, JA, and ET-induced immunity responses through hormonal crosstalk

(Pieterse et al., 2009; Pieterse et al., 2012). The functional genomics of the plant growth

hormones: auxins (AU), cytokinins (CT), and gibberellins (GA) showed that these

hormones are key in the plant defense responses too (Navarro et al., 2008; Bari and

Jones, 2009; Giron et al., 2013). The critical role of hormone-associated receptor

kinases such as the BR RECEPTOR BR INSENSITIVE 1 (BRI1), ET RECEPTOR ET

REGULATED 1 (ETR1), and CT RECEPTOR CT RESPONSE 1 (CRE1) in the plant

defense and growth was studied using the A. thaliana model plant (Huot et al., 2014).

Abscisic acid (ABA) was also found to be involved in defense response (Cao et al.,

2011). Nakashita et al. (2003) reported brassinosteroid (BR)-induced disease resistance

to tobacco mosaic virus (TMV) in tobacco (Nicotiana tabcum cv. Xanthi) and to

P.syringae in rice (Oryza Sativa L). JA and ET-regulated plant defenses were also

found in response to herbivore attack (Vos et al., 2005; Pieterse et al., 2012; Foyer et

al., 2015). Studies of phloem-feeding insects (PFI)-plant interactions showed that PFI

infestation can modulate SA, JA, and ET hormone regulated genes expression, and

thereby influence the different defense signaling pathways (Thompson and Goggin,

2006; Foyer et al., 2015). Transcriptomic reprogramming PFI-infected host plants are

reviewed in A. thaliana, Sorghum bicolor, Nicotiana attenuate, Oryza sativa, Malus

domestica, Lycopersium esculantum (Vos et al., 2005; Foyer et al., 2015).

Hormonal or innate plant immune responses are perceived, amplified, and

transduced to activate defense via various signaling molecules. The category of signal

receptor molecules is comprised of receptor-like kinases (RLKs), receptor-like proteins

(RLPs), PRRs, mitogen-activated protein kinase (MAPK) cascades, secondary

133

messenger molecules such as calcium (Ca2+), cyclic nucleotide gated channels

(CNGC), reactive oxygen species (ROS), and ROS scavengers and, lipids. RLKs do

not function only as receptors but also act as a surveillance system to detect potential

threats to plants (Zhou et al., 2017). RLKs are phosphorylated by an enzyme kinase

upon recognition of PAMP, MAMP (Microbe-associated molecular pattern) or herbivore-

associated molecular patterns (HAMP). Phosphorylation initiates signal transduction.

RLKs are also critical in defense-induced hormonal signaling (Wu and Zhou, 2013).

WALL-ASSOCIATED KINASE 1 (WAK1) proteins are the receptors activated by

herbivores or by PFI induced wounding (Ferrari et al., 2013). Wounding in plants leads

to damage associated molecular pattern (DAMP) alarming signals. During pathogen

induced wounding or PFI interaction with plants, cell wall degrading enzymes are

secreted that act as threat signal for the host plant. In this scenario, plants activate

DAMP signals. DAMP signals are exhibited as secretion of oligogalacturonide (OGs)

such as chitinase, glucanase, callose deposition enzymes, and production of ROS.

WAK1 recognized the OG induced DAMP signals to activate downstream defense

signaling (Ferrari et al., 2013). The signals received through receptors are then

amplified through a MAPK cascade, Ca2+ signaling pathways, changes in the cell wall

pH, ROS production and lipid signaling. Calcium is the most ubiquitous second

messenger in the plants. Calcium-dependent protein kinase (CDPK), Calmodulin (CaM),

CaM-like protein (CML), glutamate-like receptors (GLRs), CNGCs are tightly

interdependent in the Ca2+ mediated signal propagation in the pathogen infected plants

(Ma et al., 2009; Schulz et al., 2013). The plasma membranes are primarily made up of

lipids. Activation of hydrolytic enzymes that break the bonds between lipids are

134

implicated as messenger molecules in a variety of cellular processes. Among these

enzymes, acyl hydrolyzes and phospholipases (PLAs) A, C, and D lead to the

conformational changes in plasma membrane upon pathogen infection, and confer non-

host specific defense responses (Canonne et al., 2011).

The different strategies of plant defense discussed above have been reported in

various Citrus diseases as well. However, the defense mechanisms involved in citrus -

huanglongbing (HLB) interaction are still unclear because of limitations in culturing HLB

causing putative bacteria Candidatus Liberibacter asiaticus (CaLas). The epidemic of

HLB in the Florida-citrus groves is destructive. The current HLB control practices are

only partially successful, and a temporary solution. Some citrus and related genera such

as certain pummelos (Citrus maxima Merr.), lemons (Citrus limon L. [Burm.] f.), trifoliate

orange (Poncirus trifoliata [L.] Raf), and finger lime (Microcitrus australasica F. Muell.)

show tolerance to HLB. However, these species might have limited commercial viability

(Folimonova et al., 2009). In contrast, the commercially viable scions ‘Valencia,' and

‘Hamlin' sweet oranges (Citrus sinensis [L.] Osbeck), and rootstocks such as Cleopatra

mandarin (Citrus reticulata Blanco), Swingle citrumelo (Citrus paradisi x Poncirus

trifoliata) are not HLB-tolerant or resistant. Therefore, for a long term HLB-solution, new

combinations of two or more of the tolerant citrus species and/or cultivars will be

required to produce HLB-tolerant/resistant and commercially profitable citrus varieties.

Some of such improved hybrids have been created in the University of Florida (UF) and

U.S Department of Agriculture (USDA) sponsored citrus breeding programs and are

being analyzed for their performance under CaLas-infection. The comparative

transcriptomic and proteomic analyses between CaLas-infected and healthy plant

135

tissues among different citrus varieties, including newly improved citrus hybrids, showed

the reprogramming of defense-associated genes (Albretch and Bowman, 2008; Fan et

al., 2011; Fan et al., 2012; Wang et al., 2016). Comparative analysis between CaLas-

infected susceptible and tolerant cultivars showed modulation in the expression levels of

genes encoding scavengers of superoxide, PHLOEM PROTEIN 2B (PP2B),

DEFECTIVE INDUCED RESISTANCE 1(DIR1), RECEPTOR-LIKE PROTEIN KINASE

33 (RLP33), PLANT DEFENSIN, OSMOTIN-like protein (OSM), CHITINASE, HEAT

SHOCK PROTEINS (HSPs), THAUMATIN-like proteins, MIRACULIN-like proteins,

glutathione transferases, secondary metabolites involved in the glycosylation and cell

wall modification, and SA-JA-ET dependent signaling regulators (Albretch and Bowman,

2008; Fan et al., 2011; Martinelli et al., 2013; Zheng and Zhao, 2013; Wang et al., 2016;

Zhong et al., 2016). Altogether, this suggests that CaLas infection does not induce

pathogen-specific plant immunity in these citrus cultivars but induces nonspecific plant

defenses. Biological and economic tolerance to HLB is an urgent need to reduce HLB

severity as necessary for sustainable and profitable citrus fruit production.

The improved candidate (CAN) hybrid rootstock (46x20-04-48; ‘Hirado Buntan

Pink’ (HBP) pummelo x Cleopatra mandarin) used in this study was selected based on

the better juice quality and reduced HLB symptoms of the ‘Valencia' scion grafted onto

it. This suggests that improved CAN rootstock has potential to address the need of

biological and economic viability of HLB-tolerance. The goal of the comparative

transcriptomic study is to analyze the differential reprogramming of defense related

genes that were induced in the CaLas-infected ‘Valencia' scion due to the rootstock

differences, comparing the CAN to the susceptible commercial Swingle citrumelo (SW).

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Plant Material

Two combinations of scion and rootstocks were used in this experiment (Table 4-

1). Field grown seven-year-old experimental plants were planted in the Lee Family's

Alligator Grove, east of St. Cloud, Florida. The one combination of trees was ‘Valencia'

(VAL) sweet orange grafted onto putative HLB-tolerant candidate (CAN) rootstock. The

CAN rootstock is a hybrid between ‘HBP’ pummelo and ‘Cleopatra' mandarin. The 2nd

combination was ‘Valencia' (VAL) scion grafted onto Swingle citrumelo (SW) rootstock.

Swingle is a hybrid of grapefruit (Citrus paradisi [Macf.]) and trifoliate orange. Each

combination of VAL/CAN and VAL/SW plants was divided into two treatments based on

the visible presence of HLB-like symptoms (Table 4-2). Highly infected and symptomatic

trees in each combination grouped into the symptomatic treatment, whereas trees with

fewer symptoms or no visible symptoms were grouped into the asymptomatic treatment.

All biological replicates in each treatment and combination were tested using

quantitative PCR (qPCR) based CaLas detection and enzyme-linked immunosorbent

assay (ELISA) -assisted citrus tristeza virus (CTV) detection.

Sampling, RNA extraction, and RNA sequencing


explained in Chapter 2. In brief, differentially expressed genes (DEGs) in the pairwise


symptomatic VAL/CAN, and VAL/SW combinations were obtained using RNA-Seq

Tuxedo pipeline (Table 4-2). The significant differentially regulated genes in leaves and

roots tissue were annotated to the C. clementina genome database in Phytozome V1.0

(Goodstein et al., 2012). Functional categories of the DEGs were identified using A.

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thaliana annotation in the Phytozome server and MapMan sottware (Thimm et al., 2004)

(Chapter 2). The Blast2GO algorithm (Conesa et al., 2005) was also used to identify

molecular, cellular, and biological functional categories. An overview of the DEGs was

developed using PageMan analysis tool in the MapMan software (Usadel et al., 2009).

Results


The results of qRT PCR-based HLB detection and RNA-Seq output in all

combinations and treatments are presented in Chapter 2.

Differential Expressed of Defense-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW combinations

Leaf samples

The results of transcriptome studies combined with Mapman-Pageman analysis

tools showed that asymptomatic treatment VAL/CAN leaves did not show a significant

increase in the expression levels of genes associated with defense signaling and innate

immunity as compared to VAL/SW leaves. Leaves collected from CaLas-infected

asymptomatic VAL/CAN showed upregulation of a few genes related to non-host

resistance and PFI induced defense. Among these, genes encoding ET-induced-GDSL

LIPASE-LIKE 1 (GLIP1), BRI1 receptor kinase, LIPID TRANSFER PROTEIN (LTP),

MILDEW RESISTANCE LOCUS O-like (MLO-like), CIPK 20, and PP2-B15 were

significantly upregulated more than 1 log2 fold change (log2 FC) (Table 4-3). In the

asymptomatic treatment, genes involved in the secondary metabolite flavonoid and

terpenoid biosynthesis pathway were significantly overexpressed in leaves of the

VAL/CAN combination as compared to leaves of VAL/SW (Figure 4-3). These genes

are encoding TRANSPARENT TESTA 4 (TT4), TRANSPARENT TESTA 7 (TT7),

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TERPENE SYNTHASE 14 (TPS14), FLAVANONE-3 HYDROXYLASE (F3H) and

UGT72B1. In the asymptomatic VAL/CAN combination, genes encoding PECTIN

METHYLESTERASE 44 (PME 44), glucosinolate UGT74B1 and cytochrome P450

superfamily CYP98A3 were also significantly upregulated (Table 4-3).

Leaves collected from the asymptomatic VAL/SW combination showed a greater

number of significantly upregulated defense signaling and innate immunity-related

genes as compared to leaves collected from asymptomatic VAL/SW (Figure 4-1 and 4-

3). These genes are encoding proteins involved in secondary metabolite biosynthesis

pathways, PAMP-induced receptor kinases, WRKY TFs, PATHOGENESIS RELATED

(PR) proteins, Ca2+ mediated signaling components, MAPK cascade, glutamate

receptor-like GRLs, HSPs, and PHOSPHOLIPASE A2. Transcripts encoding the ETI

triggered NUCLEOTIDE BINDING-adaptor shared by APAF-1, R proteins and CED-4

(NB-ARC) domain containing disease resistance, and leucine-rich repeat (LRR) family

proteins were, also, significantly overexpressed in asymptomatic VAL/SW leaves. Gene

encoding TÓXICOS EN LEVADURA (ATL2), a RING-H2 family protein that responds to

elicitors, was also significantly upregulated in the asymptomatic VAL/SW leaves as

compared to leaves collected from asymptomatic VAL/CAN (Table 4-4). Asymptomatic

VAL/SW leaves showed a significant overexpression of genes encoding SA-induced

RECEPTOR LECTIN KINASE (RLK), RLP33, RLK1 and GLR2.7 in the range of 2 to 4

log2 FC (Table 4-4). Genes encoding of defense signal amplifier MAPKs such as MPK3,

MKK4, MAPKK5, MAPKKK13, MAPKKK18, and MAPKKK19 were also significantly

overexpressed in the asymptomatic treatment of VAL/SW leaves. In the Ca2+ mediated

signaling branch of defense, genes encoding CPK32, CML37, and calmodulin binding

139

family protein EDA39 were significantly upregulated in the range of 1 to 3 log2 FC in the

leaves of asymptomatic VAL/SW combination as compared to leaves collected from

asymptomatic VAL/CAN (Table 4-4). Genes involved in the SA, JA, and ET -mediated

defense were significantly overexpressed in the asymptomatic VAL/SW leaves as

compared to asymptomatic VAL/CAN leaves (Figure 4-3). Asymptomatic VAL/SW

leaves showed significant upregulation of genes encoding SA-induced WRKY70,

WRKY50, thioredoxin superfamily GRX480, SIGMA FACTOR BINDING PROTEIN 1

(SIB1), a positive regulator of JA-dependent defense WRKY33, and JA-responsive PR4

and BASIC CHITINASE as compared to the asymptomatic VAL/CAN leaves. The non-

host resistance induced Ca2+ dependent PENETRATION 3 (PEN3) upregulated in the

asymptomatic VAL/SW leaves. In the category of enzymes, transcripts encoding PLA

2A, cytochrome 450 family monooxygenase CYP83B1, beta-glucosidase, UDP

glucosyltransferase UGT73B4, glutathione transferases and beta 1,3 glucan hydrolyses

were significantly overexpressed in the asymptomatic VAL/SW leaves (Figure 4-3).

Asymptomatic VAL/SW leaves, also, significantly upregulated transcripts encoding

negative regulator of SA-induced plant defense NPR3 gene. Negative regulators of HR-

induced programmed cell death genes were also strongly overexpressed in the

asymptomatic VAL/SW combination. These included genes encoding for MAC/Perforins

domain-containing protein CAD and NSL1 proteins, BON ASSOCIATED PROTEIN 2

(BAP2), and ABA-induced PP2C (Table 4-4). In addition, genes encoding mitogen-

activated MKK4, WRKY 40, WRKY 48 and WRKY 60 were co-expressed strongly in

asymptomatic VAL/SW leaves as compared to asymptomatic VAL/CAN leaves (Table

4-4).

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Root samples

Roots collected from asymptomatic VAL/CAN and VAL/SW combination did not

show many DEGs in the defense category (Figure 4-1). However, roots collected from

the CAN rootstock overexpressed transcripts encoding cell wall degrading enzymes

BETA-GALACTOSIDASE 12 and BETA-XYLOSIDASE 1.

Differentially Expression of Defense-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW combinations

Leaf Samples

Rootstock genetic differences showed differential regulation of defense-

associated genes in symptomatic VAL scion. Leaves collected from the symptomatic

VAL/CAN combination showed a low number of defense-associated genes those were

significantly upregulated as compared to symptomatic VAL/SW leaves. These genes

are encoding LTP, PHENYLALANINE AMMONIA LYASE (PAL), ZINC FINGER

PROTEIN 7, a protein from pectin methylesterase inhibitor (PMEI) family, and

COPPER/ZINC SUPEROXIDE DISMUTASE 2 (CSD2). Symptomatic VAL/CAN leaves

also showed significant upregulation of multiple transcripts encoding aspartyl proteases,

the SA-positive regulator MYB-like HTH transcriptional regulator family protein

ASYMMETRIC LEAVES 1(AS1), NB-ARC DOMAIN CONTAINING DISEASE

RESISTANCE, and CHITINASE A. (Table 4-5). Genes involved in the lignin

biosynthesis and flavonoid networking were also significantly upregulated in

symptomatic VAL/CAN leaves as compared to symptomatic VAL/SW leaves (Table 4-

5).

Leaves collected from the symptomatic VAL/SW combination showed strong

overexpression of plant immunity and defense-induced genes as compared to

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symptomatic VAL/CAN leaves (Figure 4-1 and 4-4). Symptomatic VAL/SW leaves

showed upregulation of genes encoding pathogen signaling and ETI-induced numerous

NB-ARC domain, TIR-containing disease resistance proteins, and LRR receptors (Table

4-6). Symptomatic VAL/SW leaves significantly overexpressed transcripts encoding

signaling kinases such as RECEPTOR KINASE 3 (RPK3), RECEPTOR-LIKE PROTEIN

56 (RLP 56) RECEPTOR LECTIN KINASE (RLK), MALECTIN-like LRR, RLP1, RLP 13,

CYSTEINE-RICH RLK 3 , GRL 2.7, GRL 3.6 , WAK, and RLP 33 (Table 4-6). Leaves

collected from symptomatic VAL/SW tees also showed overexpression of the transcripts

encoding biotic stress induced mitogen-activated and secondary defense messengers

(Table 4-6). In the category of secondary defense messengers, transcripts of Ca2+

dependent lipid binding CaLB domain containing protein, and LIPID-DEPENDENT

PHOSPHOLIPASE 2A, PHOSPHOLIPASE C2, and non-specific PHOSPHOLIPASE C3

were upregulated in symptomatic VAL/SW leaves (Table 4-6). Leaves collected from

symptomatic VAL/SW combination also upregulated genes encoding enzyme in the

defense responses. These include oxidoreductase family proteins, peroxidase

superfamily proteins ascorbate peroxidase and 2-oxoglutarate (2OG), Fe(ii)-dependent

oxygenase superfamily proteins, serine protease inhibitors, aspartyl proteases, beta-

glucosidases, glutathione transferases, callose forming cytochrome P450

monooxygenase CYP83B1, LTP1, and ET-induced GDSL-like lipase superfamily

protein (Table 4-6 and Figure 4-4). Hormone activated defense and signaling genes

were also significantly upregulated in HLB-symptomatic VAL/SW combination (Figure 4-

4). Transcripts encoding hormonal activated WRKY 70, SIB1, WRKY 33 and BASIC

CHITINASE were significantly upregulated in symptomatic VAL/SW leaves as

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compared to symptomatic VAL/CAN leaves (Table 4-6). In the symptomatic treatment,

VAL/SW leaves showed significant upregulation of multiple genes encoding enzymes or

proteins in the phenylpropanoid activated lignin biosynthesis pathway as compared to

VAL/CAN leaves (Table 4-6). Genes encoding HSPs were overexpressed in the

symptomatic VAL/SW leaves. Highly upregulated HSPs were found in the symptomatic

VAL/SW leaves treatment were HSP20, HSP70T.2, HSP90.1, HSP17.6A, and HSP22.

Leaves collected from symptomatic VAL/SW also showed significant overexpression of

genes encoding negative regulators of hormones response, HR induced cell death, JA

signaling, and MAPK regulators. These negative regulators are NPR3, BAP2, JAZ

proteins, WRKY40, and histidine kinase PP2C.

Root Samples

Comparative transcriptomic analysis in the symptomatic treatment between

VAL/CAN and VAL/SW roots showed significant upregulation of transcripts encoding

ETI-induced TIR-NBS-LRR (Ciclev10018458m.g), NB-ARC (Ciclev10018492m.g) and

LRR (Ciclev10010475m.g) domain containing disease resistance genes in VAL/CAN

roots. Symptomatic VAL/CAN roots also significantly overexpressed transcripts

encoding protein kinases, MAPKKK19, MAPKKK18, MAPKKK15, members of lectin

protein kinase family and cysteine-rich RLK10 (Table 4-7). However, symptomatic

VAL/CAN roots did not show upregulation of many hormone activated defense-

associated genes except the thioredoxin GRX480 gene. Symptomatic VAL/CAN roots

also showed significant overexpression of transcripts encoding enzymes that are

involved in cell wall modification, flavonoid biosynthesis, and ROS scavenging, as well

as glutathione transferases, cytochrome P450 monooxygenases, phospholipases and

protease inhibitors as compared to symptomatic VAL/SW roots (Figure 4-5). Among

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these, transcripts encoding BETA-XYLOSIDASE 1, an enzyme involved in defense

response by callose deposition CYP83B1, PMEI protein, GLUTATHIONE S-

TRANSFERASE TAU 8, and PHOSPHOLIPASE A 2A were strongly upregulated (Table

4-7). Roots collected from the symptomatic of VAL/CAN combination showed significant

overexpression of genes encoding negative regulators of defense as compared to

symptomatic VAL/SW roots. These genes are encoding NPR3, JAZ proteins (JAZ1 and

JAZ10), RPM INTERACTING PROTEIN 4 (RIN4), and BAP2 (Table 4-7)

In the symptomatic treatment, VAL/SW roots showed significant upregulation of

genes encoding various NB-ARC domain containing (Ciclev10001996m.g,

Ciclev10023911m.g, Ciclev10014222m.g, Ciclev10027635m.g), and TIR-NBS-LRR

class (Ciclev10004174m.g) disease resistance proteins as compared to the VAL/CAN

roots (Table 4-8). Transcripts encoding kinase superfamily proteins such as cysteine-

rich RLK (CSRLK29 and CSRLK10), lectin proteins, and LRR signaling receptor kinase

family proteins (II, III, IV, V, VI, VIII-1, X, XII, XII, and XIV) were also significantly

overexpressed in the symptomatic VAL/SW roots as compared to the symptomatic

VAL/CAN roots (Figure 4-1). Genes involved in the plant basal defense were also highly

upregulated in the symptomatic VAL/SW roots as compared to the symptomatic

VAL/CAN roots. Among these, genes encoding STRICTOSIDINE SYNTHASE-LIKE-4

(SSL-4), PATHOGENESIS-RELATED 4 (PR4), OSMOTIN 34 (OSM34), and a homolog

of EP3-CHITINASE strongly overexpressed in the symptomatic VAL/SW roots (Table 4-

8). In the category of enzyme-induced defense responses, many genes encoding

glutathione S-transferase Tau and Phi family, and NAD(P)-linked oxidoreductase family

were strongly overexpressed in roots of the symptomatic VAL/SW combination. Many

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genes encoding pleiotropic drug resistance and multidrug resistance-associated

proteins (PDR4, PDR9, MDR3, and MDR11) also upregulated in the symptomatic

VAL/SW roots. In response to the presence of CaLas, defense mechanisms were

strongly expressed in the symptomatic SW roots (Figure 4-1 and 4-2). However, genes

encoding negative response of the defense carbonic anhydrase (CA2) and RIN4 were

also significantly overexpressed in symptomatic VAL/SW roots (Table 4-8).

Discussion

Differential gene expression (DGE) analysis of VAL leaves, and roots sampled

from both VAL/SW and VAL/CAN combinations showed differential transcriptomic

reprogramming in response to the asymptomatic and symptomatic stages of HLB

disease development. In the asymptomatic treatment, VAL/SW showed a greater

number plant defense and immunity response genes upregulated in leaves as

compared to the VAL/CAN leaves (Figure 5-1 and 5-2). The DGE analysis of the

asymptomatic root tissue did not show significant expression differences of defense-

associated genes. As discussed in Chapter 3, in the symptomatic treatment, VAL/CAN

leaves did not show upregulation of hormone-induced genes that are involved in the

biotic responses. However, ET-induced GLIP1, and BR-induced receptor kinase

transcripts were overexpressed in the asymptomatic VAL/CAN leaves (Table 4-3). The

Role of GLIP is discussed in activating systemic signaling in interaction with fungal and

bacterial pathogens (Kwon et al., 2009). The role of BR in plant growth has been

reviewed in many crops, and BRI-induced defense responses are discussed in many

studies (Nakashita et al., 2003; Albrecht et al., 2012; Choudhary et al., 2012).

Therefore, upregulation of GLIP and BRI1 in the asymptomatic VAL/CAN tissues

suggests plant defense activation. The interaction between BR and SA showed that BR

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antagonizes the SA-induced NPR1 gene expression (De Vleesschauwer et al., 2012).

Significant downregulation of SA-induced defense genes expression levels, and

increased expression level of BRI1 in the asymptomatic VAL/CAN leaves indicates that

BR may play an important role to improve HLB tolerance in VAL/CAN plants. CaLas

bacterial infection in the plants is vectored by psyllids. Psyllids feed on the citrus trees

by puncturing the phloem tissue which helps the bacterial spread. The wound created

by psyllid-feeding in the vascular tissue is sensed as a potential threat through HAMP or

DAMP signaling. HAMP- or DAMP-induced defense strategies involve cell wall

modifications such as secretion of PMEs and secondary metabolite-induced defenses

that fight against the pathogen attack (Redovnikovic et al., 2008; Foyer et al., 2015).

Overexpression of PME 44 and glucosinolate UGT74B1 in the asymptomatic VAL/CAN

leaves as compared to asymptomatic VAL/SW leaves suggests that the CAN rootstock

induces wounding defense in response to psyllid feeding in the VAL. Plant flavonoids

are important for physiological processes. In addition, flavonoids also contribute to the

plant defense responses to herbivore infestation or pathogen infection (Gould, 2004).

Herbivore attack also triggers Ca2+ dependent defenses (Schulz et al., 2013; Foyer et

al., 2015). Upregulation of transcripts encoding the proteins in flavonoid biosynthesis

pathway, cell wall modification enzymes, and Ca2+ dependent protein kinase suggests

that the CAN rootstock is strongly reacting to the psyllid attack at the asymptomatic

stage of HLB.

Leaves collected from the asymptomatic treatment of VAL/SW showed

upregulation of PTI-induced genes. Receptor kinases which are part of PTI system can

recognize potential threats to the plants through their signaling components (Zhou et al.,

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2017). Upregulation of genes encoding RLK in the asymptomatic VAL/SW leaves as

compared to asymptomatic VAL/CAN leaves suggests that SW has a robust threat

recognition system which strongly activated defense mechanisms in the asymptomatic

VAL/SW combination. In addition, overexpression of genes involved in the defense

signal cascade such as Ca2+ (Schulz et al., 2013), MAP kinase (Pedley and Martin,

2005), phospholipase (Canonne et al., 2011) and HSPs (Martinelli et al., 2012) supports

the strong defense of SW rootstock towards HLB. Glutamate receptors are key players

in the disease priming (Manzoor et al., 2013; Mousavi et al., 2013). Overexpression of

transcripts encoding glutamate receptors in the asymptomatic VAL/SW combination

suggests that SW has an active signaling system which is priming the non-infected

portion of the tree. In the asymptomatic treatment, SA-JA-ET regulated defense

response genes were upregulated in VAL/SW leaves as compared VAL/CAN leaves.

Among these, SAR signaling thioredoxin, WRKY TFs and JA positive regulator

WRKY33 genes were overexpressed. SIB1 is a SA-regulated protein that confers

resistance to P. syriangae (Chalovich and Eisenberg, 2005). Herbivore-induced

CHITINASE and PR4 transcript upregulation is reported in HLB transcriptomics

analyses (Fan et al., 2011; Sharma et al., 2011). Genes encoding SIB1, CHITINASE,

and PR4 were, also, found to be upregulated in the asymptomatic VAL/SW leaves as

compared to asymptomatic VAL/CAN leaves suggesting SA and JA induced defenses

in the VAL/SW leaves. Genes encoding cell modifying beta-glucosidase (Morant et al.,

2008), and 1,3 glucan hydrolyse (Levy et al., 2007) have a crucial role in activating

chemical defense against herbivores. Significant upregulation of transcripts encoding

beta-glucosidase and 1,3 glucan hydrolyse in asymptomatic VAL/SW leaves indicated

147

that in the asymptomatic VAL/SW trees defense was also exhibited by modifying cell

walls structures. In the asymptomatic treatment, the ETI signature NB-ARC and LRR

domain-encoding genes were also overexpressed in VAL/SW leaves as compared to

VAL/CAN leaves. ETI- induced defense is mostly R-gene mediated defense. There is

no R-AVR interaction yet identified in the HLB-citrus interaction, although asymptomatic

VAL/SW showed overexpression of ETI and PTI-associated genes as well as

upregulation of negative regulators of HR induced cell death. The activation of genes

involving in PTI-, ETI-induced defense suggests that at an early stage of CaLas

infection, VAL/SW trees initiate a strong defense response. In asymptomatic VAL/SW

leaves, the gene encoding NPR3, a negative regulator NPR1, was also significantly

upregulated, indicating the possible downregulation of NPR1 dependent defense.

However, more analyses are required to conclude the role of NPR3 upregulation in

defense downregulation in CaLas infected VAL/SW leaves.

Transcriptomic analysis of HLB symptomatic VAL/CAN and VAL/SW leaves

showed similar results as the asymptomatic treatment, but with the stronger

overexpression of the genes. Symptomatic VAL grafted onto the putative HLB-tolerant

CAN rootstock showed increased expression of genes encoding redox enzymes.

Reactive oxygen species (ROS) are a byproduct of oxidation burst reaction in the basal

defense mechanism (Glazebrook, 2005). However, ROS are harmful to the plant.

Therefore, to neutralize ROS effect, plant activates oxidoreductase enzymes. In the

symptomatic treatment, VAL/CAN leaves showed upregulation of genes encoding

thioredoxins, dismutases, glutathione transferases and glutaredoxins as compared to

VAL/SW leaves. PAL is a key enzyme in phenylpropanoid pathway which is important in

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secondary metabolite biosynthesis pathways such as lignin, flavonoids, and

phytoalexins (Hahlbrock and Scheel, 1989). These secondary metabolites have a

significant role in plant defenses (Besseau et al., 2007). Significant upregulation of PAL

and PMEI in the symptomatic VAL/CAN leaves suggests that CAN rootstock triggered

the secondary metabolite-induced plant defense via cell wall modification in the HLB-

symptomatic VAL. In the symptomatic VAL/CAN combination, roots showed

upregulation of transcripts encoding NB-ARC and LRR protein domains, and MAPK

cascade suggesting that at the symptomatic stage VAL/CAN may potentially activate

ETI-induced defense.

Comparative transcriptomic analysis of leaves and roots sampled from the

symptomatic VAL/SW combination showed significant upregulation of PTI-induced

receptors in response to HLB. In the symptomatic VAL/SW combination, nonhost-

specific defense responses exhibited by upregulating transcripts encoding detoxification

enzymes, activation of secondary messengers such as MAPK, Ca2+-Calmodium

dependent stress response, HSPs, secondary metabolite activated cell wall modification

enzymes, multidrug associated antibiotic resistance proteins, and many ETI-induced

genes. The overall transcriptomic data of symptomatic leaves and roots collected from

the VAL/SW combination showed that SW was overreacting to the CaLas-infection in

the scion by activated PTI- and ETI-activated defense responses.

CaLas-infected plants trigger callose deposition. Callose deposition is a strategy

to stop pathogen spread in the plants (Koh et al., 2012). However, in HLB-citrus

interaction, overwhelming callose depositions not only blocks the spread of CaLas but

also causes significant blockage of the phloem, leading to an inadequate food supply to

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the sink tissue (Achor et al., 2010; Fan et al., 2012). The monooxygenase p 450

CYP83B1 gene is involved in the callose synthesis. VAL/SW combination strongly

upregulated CYP83B1 in asymptomatic as well as symptomatic VAL, and callose

degrading 1,3- glucan hydrolase transcripts were upregulated only in the asymptomatic

VAL grafted onto SW. Whereas, the CAN rootstock triggered CYP83B1 expression only

in the symptomatic roots. The overexpression of transcripts encoding CYP83B1 and

1,3-glucan hydrolase in the asymptomatic VAL/SW treatment suggests that SW can

regulate the callose production and turnover at the asymptomatic stage only. At an

advanced stage of CaLas-infection, SW is unable to repair plants from uncontrolled

callose depositions and leads to the physical damage to the phloem. Whereas, the CAN

rootstock promotes callose synthesis at an advanced stage of CaLas infection only.

Also, expression of the PP2 gene at the asymptomatic stage supports that the CAN

rootstock is not hypersensitive to the CaLas infection, rather it is gradually deploying its

defense strategies based on the stage of HLB disease development in the plant.

Altogether, combined data suggests that SW is highly sensitive to the CaLas infection,

and VAL scion grafted on the SW overact to the disease causing metabolic imbalances

detrimental to the tree. Upregulation transcripts encoding negative regulators of HR

reaction such as CAD, BON, WRKY60, WRKY40, and NPR3 in the asymptomatic

VAL/SW combination suggests that SW was controlling the overwhelming plant defense

response in VAL by activating the negative controller of the defense.

In this study, VAL grafted onto the CAN and SW rootstocks showed many

commonalities in HLB-defense responses. These commonalities were activation of

genes encoding PTI receptors-induced signaling, detoxification enzymes, LTP

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superfamily genes, callose depositions, chitinase, and secondary metabolite-triggered

pathways. However, the levels of gene expression were substantially higher in the

VAL/SW combination in the asymptomatic stage of CaLas-infection and enhanced

further in the symptomatic stage of infection. Moreover, VAL grafted onto SW strongly

upregulated R genes encoding NB-ARC and LRR domain. Whereas, the putative HLB-

tolerant CAN rootstock upregulated a low level of ETI involved defense genes and

defense triggered by secondary metabolite pathways such as cell wall modifying pectin

methylesterases, lignin biosynthesis and detoxification processes at an optimum level.

In summary, transcriptomic analysis of VAL grafted onto CAN and SW rootstock

at two stages of HLB-infection showed that in the absence of disease-specific

resistance, SW activates many branches of non-host specific resistance at a higher

intensity. Whereas, the CAN rootstock exhibits a more balanced level of induction of

defense-associated genes, which may help the plant to invest adequate energy

resources needed for routine growth and sustainability.

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Table 4-1. Experimental treatments and combinations



Swingle; 2n (SW)



Symptomatic VAL/SW

Asymptomatic VAL/SW

putative HLB-tolerant

candidate; 2n (CAN)




VAL/CAN

Asymptomatic

VAL/CAN

Approximately 7-year old citrus trees in the Lee Family’s Alligator Grove east of St. Cloud, FL.


Leaves Roots





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Table 4-3. Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of asymptomatic VAL/CAN combination as compared to asymptomatic VAL/SW leaves Arabidopsis Gene

C. Clementina_ID Log2 FC Arabidopsis-define Functional description


Essential for lignin biosysntheis

CIPK20 Ciclev10015231m.g 2.594 CBL-interacting protein kinase 20 Calcium signaling

TT4 Ciclev10001405m.g 2.03082 Chalcone and stilbene synthase family protein flavonoid accumulation/chalcone synthase

PP2-B15 Ciclev10003574m.g 1.95793 Phloem protein 2-B15

MJE7.13 Ciclev10017274m.g 1.88599 Lipid transfer protein

SHN1 Ciclev10009447m.g 1.69017 Integrase-type DNA-binding superfamily protein cutin Biosyntehsis

TINY2 Ciclev10027587m.g 1.66184 Integrase-type DNA-binding superfamily protein putative transcription factor

F20P5.26 Ciclev10032167m.g 1.58935 myb-like transcription factor family protein

TPS14 Ciclev10014707m.g 1.5717 terpene synthase 14

TT7 Ciclev10019637m.g 1.56945 Cytochrome P450 superfamily protein Flavonoid syntehsis, Convert naringenin to eriodictyol

AT1G08810 Ciclev10031946m.g 1.55202 myb domain protein 60

T12P18.14 Ciclev10032900m.g 1.40527 RING/U-box superfamily protein

GLIP1 Ciclev10025893m.g 1.2467 GDSL lipase 1

MLO-12 Ciclev10031130m.g

1.2383 Seven membrane MLO family protein


PAL1 Ciclev10011175m.g 1.1827 PHE ammonia lyase 1

AT1G06620 Ciclev10005208m.g 1.18259 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

UGT74B1 Ciclev10031201m.g 1.6869 UDP-glucosyl transferase 74B1

ZFP8 Ciclev10012347m.g 1.1678 zinc finger protein 8

BRI1 Ciclev10024737m.g 1.11348 Leucine-rich receptor-like protein kinase family protein

AT1G68530 Ciclev10031329m.g 1.05234 3-ketoacyl-CoA synthase 6 Wax metabolism

PME44 Ciclev10007993m.g 1.0514 pectin methylesterase 44

BKI1 Ciclev10001740m.g 1.04793 BRI1 kinase inhibitor 1

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Table 4-4. Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of asymptomatic VAL/SW combination as compared to asymptomatic VAL/CAN leaves

Arabidopsis Gene

C. Clementina_ID Log2 FC* Arabidopsis-define

PLA2A Ciclev10001337m.g -6.06084 phospholipase A 2A

PUB23 Ciclev10028564m.g -5.67279 plant U-box 23

BAP2 Ciclev10022337m.g -5.14962 BON association protein 2

WRKY33 Ciclev10011386m.g -4.71604 WRKY DNA-binding protein 33


GLR2.7 Ciclev10014227m.g -4.29173 glutamate receptor 2.7

SIB1 Ciclev10002803m.g -4.2317 sigma factor binding protein 1

T5J17.9 Ciclev10025235m.g -4.08285 Cupredoxin superfamily protein

MYB15 Ciclev10005629m.g -3.71609 myb domain protein 15

CML37 Ciclev10002523m.g -3.55957 calmodulin like 37


JAZ10 Ciclev10032858m.g -3.31943 jasmonate-zim-domain protein 10


AT3G14470 Ciclev10030540m.g -3.27492 NB-ARC domain-containing disease resistance protein


EDA39 Ciclev10008000m.g -3.24025 calmodulin-binding family protein

CML37 Ciclev10022753m.g -2.91453 calmodulin like 37




MPK3 Ciclev10028667m.g -2.7404 mitogen-activated protein kinase 3


K14A3.2 Ciclev10020507m.g -2.69615 Protein kinase superfamily protein



MLO6 Ciclev10028092m.g -2.49702 Seven transmembrane MLO family protein

HSP90.1 Ciclev10004456m.g -2.37564 heat shock protein 90.1

SYP121 Ciclev10012164m.g -2.30668 syntaxin of plants 121

154


C. Clementina_ID Log2 FC Arabidopsis-define


F13E7.22 Ciclev10015599m.g -2.20645 ARM repeat superfamily protein

PTR3 Ciclev10004600m.g -2.20386 peptide transporter 3

F19K19.4 Ciclev10020517m.g -2.13696 Protein kinase superfamily protein




T19F6.150 Ciclev10001334m.g -1.95347 alpha/beta-Hydrolases superfamily protein


MYBR1 Ciclev10001979m.g -1.91955 myb domain protein r1

CPK32 Ciclev10004707m.g -1.89183 calcium-dependent protein kinase 32

AT1G73805 Ciclev10019990m.g -1.87666 Calmodulin binding protein-like


GRX480 Ciclev10017106m.g -1.82247 Thioredoxin superfamily protein

YLS9 Ciclev10012678m.g -1.80769 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family



PR4 Ciclev10029528m.g -1.75558 pathogenesis-related 4

ATL6 Ciclev10001478m.g -1.71906 RING/U-box superfamily protein

HSPRO2 Ciclev10031447m.g -1.71163 ortholog of sugar beet HS1 PRO-1 2

T6G21.2 Ciclev10028965m.g -1.66046 Polynucleotidyl transferase, ribonuclease H-like superfamily protein

CLH1 Ciclev10021103m.g -1.64284 chlorophyllase 1

RLK Ciclev10011218m.g -1.63894 receptor lectin kinase

NSL1 Ciclev10014498m.g -1.62922 MAC/Perforin domain-containing protein

SNAP33 Ciclev10021320m.g -1.56942 soluble N-ethylmaleimide-sensitive factor adaptor protein 33

T23A1.8 Ciclev10028539m.g -1.54257 Protein kinase superfamily protein

RHD2 Ciclev10030649m.g -1.48973 NADPH/respiratory burst oxidase protein D


AOC3 Ciclev10032510m.g -1.46679 allene oxide cyclase 3

155


C. Clementina_ID Log2 FC Arabidopsis-define


MES1 Ciclev10026284m.g -1.40122 methyl esterase 1


F12L6.31 Ciclev10032209m.g -1.36132 Protein of unknown function (DUF506)

NPR3 Ciclev10017873m.g -1.33014 NPR1-like protein 3


AT3G09830 Ciclev10011770m.g -1.21939 Protein kinase superfamily protein

THI1 Ciclev10008765m.g -1.20972 thiazole biosynthetic enzyme, chloroplast (ARA6) (THI1) (THI4)

F14N23.22 Ciclev10007796m.g -1.16039 Ankyrin repeat family protein


PEN3 Ciclev10024701m.g -1.13472 ABC-2 and Plant PDR ABC-type transporter family protein

AT3G07720 Ciclev10015897m.g -1.13279 Galactose oxidase/kelch repeat superfamily protein

CAD1 Ciclev10017858m.g -1.10825 MAC/Perforin domain-containing protein

PLDBETA1 Ciclev10018726m.g -1.08624 phospholipase D beta 1

MEE62 Ciclev10014476m.g -1.05602 Leucine-rich repeat protein kinase family protein

ATL2 Ciclev10005780m.g -1.049 TOXICOS EN LEVADURA 2

OBP2 Ciclev10016017m.g -1.0399 Dof-type zinc finger DNA-binding family protein

AOS Ciclev10000825m.g -0.989301 allene oxide synthase



MKK4 Ciclev10025845m.g -0.926951 mitogen-activated protein kinase kinase 4

NDR1 Ciclev10009510m.g -0.889822 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

FC1 Ciclev10008171m.g -0.852234 ferrochelatase 1 * The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination.

156

Table 4-5. Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW leaves

C. Clementina_ID Arabi ID Gene name Bin Name† Log2 FC

Arabidopsis_define

Ciclev10013132m.g AT3G53980 LTP 2.89136 Bifunctional inhibitor/ lipid-transfer protein

Ciclev10019637m.g AT5G07990 TT7 Secondary metabolism. flavonoids. dihydroflavonols. flavonoid 3''-monooxy genase

2.4112 Cytochrome P450 superfamily protein

Ciclev10009896m.g AT1G11530 CXXS1 redox.thioredoxin 1.7951 C-terminal cysteine residue is changed to a serine 1

Ciclev10032749m.g AT5G05270 K18I23.7 secondary metabolism. flavonoids.chalcones

1.735 Chalcone-flavanone isomerase family protein

Ciclev10011175m.g AT2G37040 PAL1 secondary metabolism. phenylpropanoids.lignin biosynthesis.PAL

1.7321 PHE ammonia lyase 1

Ciclev10029196m.g AT2G28190 CSD2 redox. dismutases and catalases 1.7118 copper/zinc superoxide dismutase 2

Ciclev10014888m.g AT1G23200 PMEI 1.68029 Plant invertase/pectin methylesterase inhibitor

Ciclev10014887m.g AT1G61820 Bglu 46 misc.gluco-, galacto- and mannosidases 1.663 beta glucosidase 46

Ciclev10011175m.g AT2G37040 PAL1 phenylpropanoids.lignin biosynthesis.PAL 1.6364 PHE ammonia lyase 1

Ciclev10012523m.g AT4G39230 T22F8.130 secondary metabolism. flavonoids.isoflavonols

1.5868 NmrA-like negative transcriptional regulator family protein

Ciclev10029158m.g AT4G34050 CCoAOMT1 phenylpropanoids. lignin biosynthesis.CCoAOMT

1.5718 S-adenosyl-L-methionine-dependent methyltransferases superfamily protein

Ciclev10017119m.g AT2G44310 F4I1.12 signalling.calcium 1.5296 Calcium-binding EF-hand family protein

Ciclev10019532m.g AT1G65060 4CL3 phenylpropanoids.lignin biosynthesis.4CL 1.5252 4-coumarate:CoA ligase 3

Ciclev10032697m.g AT3G55120 TT5 secondary metabolism.flavonoids. chalcones.chalcone isomerase

1.518 Chalcone-flavanone isomerase family protein

Ciclev10017223m.g AT4G27280 M4I22.90 signalling.calcium 1.5084 Calcium-binding EF-hand family protein

Ciclev10025931m.g AT3G51240 F3H flavonoids. dihydroflavonols. 1.4821 flavanone 3-hydroxylase

Ciclev10011175m.g AT2G37040 PAL1 phenylpropanoids.lignin biosynthesis.PAL 1.4486 PHE ammonia lyase 1

Ciclev10023013m.g AT1G03020 F10O3.16 redox.glutaredoxins 1.4283 Thioredoxin superfamily protein



157

Table 4-5. Continued

C. Clementina_ID Arabi ID Gene name

Bin Name Log2 FC

Arabidopsis_define

Ciclev10020737m.g AT1G64500 F1N19.7 redox.glutaredoxins 1.3653 glutaredoxin family protein

Ciclev10025280m.g AT3G21240 4CL2 lignin biosynthesis.4CL 1.3604 4-coumarate:CoA ligase 2

Ciclev10001524m.g AT5G28840 GME redox.ascorbate and glutathione.ascorbate.gME

1.3447 gDP-D-mannose 3\',5\'-epimerase

Ciclev10027805m.g AT2G28470 BGAL8 misc.gluco-, galacto- and mannosidases.beta-galactosidase

1.274 beta-galactosidase 8

Ciclev10025174m.g AT5G10250 DOT3 signalling.light 1.2462 Phototropic-responsive NPH3 family protein

Ciclev10009963m.g AT5G54490 PBP1 signalling.calcium 1.2104 pinoid-binding protein 1

Ciclev10014664m.g AT5G23400 K19M13.1 stress.biotic.PR-proteins 1.2098 Leucine-rich repeat (LRR) family protein

Ciclev10001618m.g AT4G39330 CAD9 phenylpropanoids.lignin biosynthesis.CAD 1.202 cinnamyl alcohol dehydrogenase 9

Ciclev10027450m.g AT2G14820 NPY2 signalling.light 1.2004 Phototropic-responsive NPH3 family protein

Ciclev10026558m.g AT5G48930 HCT phenylpropanoids.lignin biosynthesis.HCT 1.1883 hydroxycinnamoyl-CoA shikimate/

Ciclev10008184m.g AT4G27570 T29A15.60 secondary metabolism.flavonoids.anthocyanins

1.1563 UDP-glycosyltransferase superfamily protein

Ciclev10032084m.g AT1G59960 F23H11.27 secondary metabolism.flavonoids.chalcones

1.1562 NAD(P)-linked oxidoreductase superfamily protein

Ciclev10004868m.g AT5G54010 K19P17.18 secondary metabolism.flavonoids.dihydroflavonols


Ciclev10011191m.g AT5G02010 ROPgEF7 signalling.g-proteins 1.1358 RHO guanyl-nucleotide exchange factor 7

Ciclev10008010m.g AT3G18080 BGLU44 misc.gluco-, galacto- and mannosidases 1.1328 B-S glucosidase 44

Ciclev10011238m.g AT2G36570 F1O11.20 signalling.receptor kinases.leucine rich repeat III

1.1275 Leucine-rich repeat protein kinase family protein

Ciclev10009291m.g AT1G18250 ATLP-1 stress.biotic 1.102 Pathogenesis-related thaumatin superfamily protein

Ciclev10029208m.g AT2G15220 F15A23.4 stress.biotic 1.0977 Plant basic secretory protein (BSP)

Ciclev10019937m.g AT4G03100 F4C21.2 signalling.g-proteins 1.0688 Rho gTPase activating protein with PAK-box/P21-Rho-binding domain

Ciclev10028153m.g AT3G45290 MLO3 stress.biotic.signalling.MLO-like 1.056 Seven transmembrane MLO family protein

158

Ciclev10005522m.g AT5G24090 CHIA stress.biotic.PR-proteins 1.0559 chitinase A



Bin Name Log2 FC Arabidopsis_define

Ciclev10019884m.g AT3G49260 iqd21 signalling.calcium 1.0464 IQ-domain 21

Ciclev10005421m.g AT3G16920 CTL2 stress.biotic 1.0294 chitinase-like protein 2

Ciclev10025807m.g AT5G13930 TT4 secondary metabolism.flavonoids.chalcones. naringenin-chalcone synthase

1.0259 Chalcone and stilbene synthase family protein

Ciclev10012089m.g AT2G37630.1 AS1 1.01105 myb-like HTH transcriptional regulator family protein

Ciclev10018681m.g AT1G50180 F14I3.19 stress.biotic.PR-proteins 1.0162 NB-ARC domain-containing disease resistance protein

Ciclev10019927m.g AT4G01070 gT72B1 secondary metabolism.flavonoids.dihydroflavonols


Ciclev10004254m.g AT5G61480 PXY signalling.receptor kinases.leucine rich repeat XI


†Classification of the measured parameter into a set a functional category in the MapMan analysis tool .

159

Table 4-6. Differentially expressed immunity and defense-associated genes significantly upregulated in leaves of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN leaves C. Clementina_ID Arabi ID Gene name Bin Name† Log2 FC* Arabidopsis_define

Ciclev10022906m.g AT2G38870 T7F6.4 stress.biotic -4.7945 Serine protease inhibitor, potato inhibitor I-type family protein

Ciclev10019788m.g AT1G02850 BGLU11 misc.gluco-, galacto- and mannosidases -4.0365 beta glucosidase 11

Ciclev10012028m.g AT1G17020 SRG1 secondary metabolism.flavonoids. -3.9855 senescence-related gene 1

Ciclev10022906m.g AT2G38870 T7F6.4 stress.biotic -3.8684 Serine protease inhibitor, potato inhibitor I-type family protein

Ciclev10012554m.g AT3G09640 APX2 redox.ascorbATe and glutathione.ascorbate -3.59 ascorbate peroxidase 2

Ciclev10028831m.g AT3G12500 HCHIB stress.biotic -3.4308 basic chitinase

Ciclev10022211m.g AT1G17860 F2H15.9 stress.biotic.PR-proteins.proteinase inhibitors.trypsin inhibitor

-3.3336 Kunitz family trypsin and protease inhibitor protein


Ciclev10010312m.g AT2G34930 F19I3.16 stress.biotic.PR-proteins -3.0454 disease resistance family protein / LRR family protein

Ciclev10012028m.g AT1G17020 SRG1 secondary metabolism. flavonoids.flavonols -3.0234 senescence-related gene 1

Ciclev10012033m.g AT4G35160 T12J5.30 secondary metabolism.phenylpropanoids -3.0045 O-methyltransferase family protein

Ciclev10020640 AT5G33370 -2.8938 GDSL-like lipas superfamily

Ciclev10010312m.g AT2G34930 F19I3.16 stress.biotic.PR-proteins -2.8648 disease resistance family protein / LRR family protein

Ciclev10002523m.g AT5G42380 CML37 signalling.calcium -2.7983 calmodulin like 37

Ciclev10014185m.g AT3G05660 RLP33 stress.biotic.kinases -2.7599 receptor like protein 33

Ciclev10004174m.g AT5G17680 MVA3.30 stress.biotic.PR-proteins -2.6676 disease resistance protein (TIR-NBS-LRR class), putative

Ciclev10005251m.g AT5G54160 OMT1 secondary metabolism.phenylpropanoids. lignin biosynthesis.COMT

-2.5961 O-methyltransferase 1

Ciclev10004431m.g AT1G69730 T6C23.7 signalling.receptor kinases. wall associated kinase

-2.589 Wall-associated kinase family protein

Ciclev10030526m.g AT3G47570 AT3G47570 signalling.receptor kinases. leucine rich repeat XII

-2.5361 Leucine-rich repeat protein kinase family protein

Ciclev10024069m.g AT4G08850 AT4G08850 signalling.receptor kinases. leucine rich repeat XII

-2.5197 Leucine-rich repeat receptor-like protein kinase family protein


Ciclev10030795m.g AT3G54800 AT3g54800 signalling.lipids -2.4475 lipid-binding START dpmain

160




Ciclev10012859m.g AT5G06690 WCRKC1 redox.thioredoxin -2.4015 WCRKC thioredoxin 1



Ciclev10030965m.g AT1G70530 CRK3 signalling.receptor kinases.DUF 26

-2.272 cysteine-rich RLK (RECEPTOR-like protein kinase) 3

Ciclev10005259m.g AT3G16510 -2.268 Ca dependent lipid binding protein

Ciclev10007443m.g AT4G21380 RK3 signalling.receptor kinases.S-locus glycoprotein like

-2.262 receptor kinase 3

Ciclev10021170m.g AT1G73500 MKK9 signalling.MAP kinases -2.2613 MAP kinase kinase 9

Ciclev10020496m.g AT5G50120 MPF21.14 signalling.g-proteins -2.0453 Transducin/WD40 repeat-like superfamily protein


Ciclev10006362m.g AT2G02100 LCR69 stress.biotic -1.9539 low-molecular-weight cysteine-rich 69

Ciclev10025139m.g AT1G07390 RLP1 signalling.receptor kinases.misc -1.9471 receptor like protein 1

Ciclev10021347m.g AT2G20142 AT2g20142 stress.biotic.receptors -1.9366 Toll-Interleukin-Resistance (TIR) domain family protein

Ciclev10000733m.g AT2G18750 MSF3.13 signalling.calcium -1.8842 Calmodulin-binding protein

Ciclev10024830m.g AT5G39000 MXF12.10 signalling.receptor kinases.CATharanthus roseus-like RLK1

-1.8597 Malectin/receptor-like protein kinase family protein

Ciclev10011218m.g AT2G37710 RLK signalling.receptor kinases.misc -1.8338 receptor lectin kinase

Ciclev10008261m.g AT5G20050 AT5g20050 signalling.receptor kinases.misc -1.8089 Protein kinase superfamily protein

Ciclev10007779m.g AT4G27290 M4I22.100 signalling.receptor kinases.S-locus glycoprotein like

-1.7824 S-locus lectin protein kinase family protein

Ciclev10014227m.g AT2G29120 gLR2.7 signalling.in sugar and nutrient physiology

-1.7203 glutamate receptor 2.7

Ciclev10000764m.g AT1G20510 OPCL1 secondary metabolism.phenylpropanoids

-1.6669 OPC-8:0 CoA ligase1

Ciclev10019897m.g AT2G31880 SOBIR1 signalling. receptor kinases.leucine rich repeat XI


Ciclev10029391m.g AT5G49290 RLP56 stress. biotic -1.5727 receptor like protein 56

161

Table 4-6. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_define

Ciclev10030540m.g AT3G14470 AT3g14470 stress.biotic.PR-proteins -1.553 NB-ARC domain-containing disease resistance protein

Ciclev10025139m.g AT1G07390 RLP1 signalling.receptor kinases.misc -1.5475 receptor like protein 1

Ciclev10007687m.g AT1G56130 T6H22.8 signalling. receptor kinases. leucine rich repeAT VIII-2

-1.5418 Leucine-rich repeat transmembrane protein kinase

Ciclev10030935m.g AT3G08510 PLC2 signalling.phosphinositides. phosphoinositide phospholipase C

-1.5174 phospholipase C 2

Ciclev10029158m.g AT4G34050 CCoAOMT1 secondary metabolism. phenylpropanoids. lignin biosynthesis.CCoAOMT

-1.5099

S-adenosyl-L-methionine-dependent methyltransferases superfamily protein

Ciclev10022063m.g AT3G54420 EP3 stress.biotic -1.4639 homolog of carrot EP3-3 chitinase

Ciclev10002523m.g AT5G42380 CML37 signalling.calcium -1.4481 calmodulin like 37

Ciclev10017113m.g AT3G26740 CCL signalling.light -1.4356 CCR-like

Ciclev10006796m.g AT3G14460 AT3g14460 stress.biotic.PR-proteins -1.3837 LRR and NB-ARC domains-containing disease resistance protein

Ciclev10007443m.g AT4G21380 RK3 signalling.receptor kinases. S-locus glycoprotein like


Ciclev10012028m.g AT1G17020 SRg1 secondary metabolism. flavonoids.flavonols

-1.358 senescence-related gene 1

Ciclev10009715m.g AT4G32690 gLB3 redox.heme -1.3562 hemoglobin 3

Ciclev10014185m.g AT3G05660 RLP33 stress.biotic.kinases -1.343 receptor like protein 33

Ciclev10019637m.g AT5G07990 TT7 secondary metabolism.flavonoids. dihydroflavonols.flavonoid 3''-monooxygenase

-1.3278 Cytochrome P450 superfamily protein

Ciclev10007383m.g AT5G25930 F18A17.4 signalling.receptor kinases.leucine rich repeAT XI

-1.2912 Protein kinase family protein with leucine-rich repeat domain

Ciclev10018721m.g AT1G53440 T3F20.24 signalling.receptor kinases. leucine rich repeAT VIII-2

-1.2889 Leucine-rich repeat transmembrane protein kinase

Ciclev10029528m.g AT3G04720 PR4 stress.biotic -1.239 pathogenesis-related 4

Ciclev10000515m.g AT2G33580 F4P9.35 signalling.receptor kinases.lysine motif -1.2066 Protein kinase superfamily protein

162


Ciclev10004551m.g AT2G23770 F27L4.5 signalling.receptor kinases.lysine motif

-1.2018 protein kinase family protein / peptidoglycan-binding LysM domain-containing protein

Ciclev10026139m.g AT4G38660 T9A14.6 stress.biotic -1.127 Pathogenesis-related thaumatin superfamily protein


Ciclev10007843m.g AT5G48150 PAT1 signalling.light -1.1042 gRAS family transcription factor


Ciclev10025933m.g AT4G37990 ELI3-2 secondary metabolism.phenylpropanoids. lignin biosynthesis.CAD

-1.0697 elicitor-activated gene 3-2


Ciclev10013896m.g AT5G01550 LECRKA4.2 signalling.receptor kinases.misc

-1.068 lectin receptor kinase a4.1

Ciclev10028839m.g AT5G58490 MQJ2.6 secondary metabolism. phenylpropanoids.lignin biosynthesis.CCR1

-1.0506 NAD(P)-binding Rossmann-fold superfamily protein

Ciclev10008000m.g AT4G33050 EDA39 signalling.calcium -1.0224 calmodulin-binding family protein * The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. †Classification of the measured parameter into a set a functional category in the MapMan analysis tool.

163

Table 4-7. Differentially expressed immunity and defense-associated genes significantly upregulated in roots of the symptomatic VAL/CAN combination as compared to symptoaatic VAL/SW roots

C. Clementina_ID Arabi ID Gene name Bin Name† log2 FC Arabidopsis_define

Ciclev10005808m.g AT3G09270 ATGSTU8, misc.glutathione S transferases 7.69171 glutathione S-transferase TAU 8

Ciclev10004369m.g AT5G49360 5.4693

beta-xylosidase 1

Ciclev10010871m.g AT1G45616 RLP6 stress.biotic 4.67947 receptor like protein 6

Ciclev10023289m.g AT3G03305 misc.calcineurin-like phosphoesterase family protein

4.26751 Calcineurin-like metallo-phosphoesterase superfamily protein

Ciclev10010227m.g AT4G19810 stress.biotic 4.08565 Glycosyl hydrolase family protein with chitinase insertion domain

Ciclev10015868m.g AT1G59960 secondary metabolism.flavonoids.chalcones



3.83467 O-methyltransferase 1

Ciclev10018458m.g AT4G12010 stress.biotic.PR-proteins 3.76207 Disease resistance protein (TIR-NBS-LRR class) family

Ciclev10002326m.g AT1G78780 stress.biotic 3.73548 pathogenesis-related family protein

Ciclev10019989m.g AT1G12740 CYP87A2 misc.cytochrome P450 3.66014 cytochrome P450, family 87, subfamily A, polypeptide 2

Ciclev10028254m.g AT2G27690 CYP94C1 misc.cytochrome P450 3.63521 cytochrome P450, family 94, subfamily C, polypeptide 1

Ciclev10000579m.g AT4G19010 secondary metabolism. phenylpropanoids

3.61678 AMP-dependent synthetase and ligase family protein

Ciclev10013402m.g AT3G52780 PAP20 misc. acid and other phosphatases

3.43161 Purple acid phosphatases superfamily protein

Ciclev10031811m.g AT5G05600 secondary metabolism. flavonoids. anthocyanins

3.22194 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

Ciclev10022123m.g AT3G54420 CHIV,EP3 stress.biotic 3.22026 homolog of carrot EP3-3 chitinase Ciclev10004006m.g AT4G31500 CYP83B1, SUR2 secondary metabolism.CYP83B1

phenylacetaldoxime monooxygenase

3.1829 cytochrome P450, family 83, subfamily B, polypeptide 1

Ciclev10025910m.g AT5G67080 MAPKKK19 3.10097 mitogen-activated protein kinase kinase kinase 19

Ciclev10006212m.g AT2G38870

stress.biotic 3.08222 Serine protease inhibitor, potato inhibitor I-type family protein

164


C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define

Ciclev10020505m.g AT3G29590 AT5MAT secondary metabolism. flavonoids. anthocyanins.anthocyanin 5-aromatic acyltransferase

3.07757 HXXXD-type acyl-transferase family protein

Ciclev10026130m.g AT1G49570 misc.peroxidases 3.0196 Peroxidase superfamily protein

Ciclev10024663m.g AT3G03080 misc.oxidases - copper, flavone etc. 2.87948 Zinc-binding dehydrogenase family protein

Ciclev10030404m.g AT1G22380 AtUGT85A3, misc.UDP glucosyl and glucoronyl transferases 2.85423 UDP-glucosyl transferase

Ciclev10022392m.g AT1G24620 signalling.calcium 2.83459 EF hand calcium-binding protein family


Ciclev10024825m.g AT3G51480 GLR3.6 signalling.in sugar and nutrient physiology 2.81218 glutamate receptor 3.6


Ciclev10014733m.g AT5G21105 redox.ascorbate and glutathione.ascorbate 2.76489 Plant L-ascorbate oxidase

Ciclev10032971m.g AT1G53540 stress.abiotic.heat 2.75707 HSP20-like chaperones superfamily protein

Ciclev10016441m.g AT5G45920 misc.GDSL-motif lipase 2.71557 SGNH hydrolase-type esterase superfamily protein

Ciclev10009896m.g AT1G11530 ATCXXS1, CXXS1

redox.thioredoxin 2.67416 C-terminal cysteine residue is changed to a serine 1

Ciclev10027566m.g AT4G25150 misc.acid and other phosphatases 2.66793 HAD superfamily, subfamily IIIB acid phosphatase

Ciclev10012795m.g AT3G02100 misc.UDP glucosyl and glucoronyl transferases 2.58069 UDP-Glycosyltransferase superfamily protein


Ciclev10015723m.g AT4G35150 misc.O-methyl transferases 2.36635 O-methyltransferase protein Ciclev10005279m.g AT4G35160 secondary metabolism.phenylpropanoids 2.36441 O-methyltransferase protein

Ciclev10014726m.g AT4G20820 misc.nitrilases, *nitrile lyases, berberine bridge enzymes, reticuline oxidases, troponine reductases

2.35686 FAD-binding Berberine family protein


misc.nitrilases, *nitrile lyases, berberine bridge enzymes, reticuline oxidases, troponine reductases


Ciclev10028206m.g AT1G19250 FMO1 misc.oxidases - copper, flavone etc. 2.33429 flavin-dependent monooxygenase 1

165

Table 4.7 Continued C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define

Ciclev10012170m.g AT2G37130 misc.peroxidases 2.24626 Peroxidase superfamily protein Ciclev10006099m.g AT1G24020 MLP423 stress.abiotic.unspecified 2.24602 MLP-like protein 423 Ciclev10025245m.g AT1G23010 LPR1 misc.oxidases - copper, flavone etc. 2.23901 Cupredoxin superfamily protein

Ciclev10026237m.g AT5G10050 misc.short chain dehydrogenase/reductase (SDR)

2.16514 NAD(P)-binding Rossmann superfamily protein

Ciclev10015130m.g AT5G09590 HSC70-5 stress.abiotic.heat 2.13309 mitochondrial HSO70 2

Ciclev10012221m.g AT2G29290 secondary metabolism.N misc.alkaloid-like 2.11522 NAD(P)-binding Rossmann-superfamily protein

Ciclev10015460m.g AT1G05100 MAPKKK18 signalling.MAP kinases 2.09831 mitogen-activated protein kinase kinase kinase 18

Ciclev10023652m.g AT5G46940 misc.invertase/pectin methylesterase inhibitor family protein

2.09779 Plant invertase/pectin methylesterase inhibitor superfamily protein

Ciclev10012087m.g AT5G54160 ATOMT1,OMT1 secondary metabolism.phenylpropanoids .lignin biosynthesis.COMT


Ciclev10005971m.g AT2G32030 misc.GCN5-related N-acetyltransferase 2.0696 Acyl-CoA N-acyltransferases (NAT) superfamily protein

Ciclev10028550m.g AT5G05320 misc.oxidases - copper, flavone etc. 1.97786 FAD/NAD(P)-binding oxidoreductase family protein

Ciclev10020092m.g AT2G43820 SGT1,UGT74F2 misc.UDP glucosyl and glucoronyl transferases 1.94311 UDP-glucosyltransferase 74F2 Ciclev10020006m.g AT1G73880 UGT89B1 misc.UDP glucosyl and glucoronyl transferases 1.88065 UDP-glucosyl transferase 89B1




misc.UDP glucosyl and glucoronyl transferases

1.85576 UDP-Glycosyltransferase superfamily protein

Ciclev10014991m.g AT4G15440 CYP74B2,HPL1 misc.cytochrome P450 1.84067 hydroperoxide lyase 1 Ciclev10033585m.g AT5G12080 ATMSL10,

MSL10 signalling.unspecified 1.78826 mechanosensitive channel of

small conductance-like 10 Ciclev10017553m.g AT5G39160

stress.abiotic.unspecified 1.7776 RmlC-like cupins superfamily

protein Ciclev10009083m.g AT2G26660 ATSPX2,SPX2 stress.abiotic 1.75478 SPX domain gene 2 Ciclev10021721m.g AT4G01700

stress.biotic 1.73295 Chitinase family protein

Ciclev10027770m.g AT4G35290 GLR3.2,GLUR2 signalling.in sugar and nutrient physiology 1.72276 glutamate receptor 2 Ciclev10028742m.g AT2G15760

signalling.calcium 1.72214 Protein of unknown function

(DUF1645)

166

Table 4-7. Continued C. Clementina_ID Arabi ID Gene name Bin Name log2 FC Arabidopsis_define

Ciclev10010475m.g AT2G34930 stress.biotic.PR-proteins 1.70967 disease resistance family protein / LRR family protein

Ciclev10014250m.g AT1G30570 HERK2 signalling.receptor kinases. Catharanthus roseus-like RLK1

1.69952 hercules receptor kinase 2

Ciclev10005251m.g AT5G54160 ATOMT1 phenylpropanoids.lignin biosynthesis.COMT 1.68109 O-methyltransferase 1

Ciclev10018492m.g AT3G14460 stress.biotic.PR-proteins 1.67065 LRR and NB-ARC domains-containing disease resistance protein

Ciclev10014400m.g AT5G55090.1 MAPKKK15 1.64914 mitogen-activated protein kinase kinase kinase 15



Ciclev10004033m.g AT4G14640 CAM8 signalling.calcium 1.63822 calmodulin 8

Ciclev10022063m.g AT3G54420 ATCHITIV, ATEP3

stress.biotic 1.63251 homolog of carrot EP3-3 chitinase

Ciclev10002897m.g AT4G15800 RALFL33 signalling.misc 1.6218 ralf-like 33 Ciclev10017106m.g AT1G28480 GRX480 redox.glutaredoxins 1.55861 Thioredoxin superfamily protein Ciclev10001555m.g AT5G54160 ATOMT1 phenylpropanoids.lignin biosynthesis.COMT 1.55643 O-methyltransferase 1

Ciclev10025794m.g AT4G37560 misc.misc2 1.54191 Acetamidase/Formamidase family protein



Ciclev10007392m.g AT3G15354 SPA3 signalling.light 1.51088 SPA1-related 3

Ciclev10029995m.g AT3G47570 signalling. receptor kinases. leucine rich repeat XII


Ciclev10033625m.g AT3G21420 secondary metabolism.flavonoids.flavonols 1.4951 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

Ciclev10014879m.g AT1G09970 LRR XI-23, RLK7

signalling. receptor kinases. leucine rich repeat XI

1.48134 Leucine-rich receptor-like protein kinase family protein

Ciclev10012494m.g AT2G29290 secondary metabolism. N misc. alkaloid-like

1.48086 NAD(P)-binding Rossmann-fold superfamily protein

Ciclev10015452m.g AT5G48230 ACAT2, EMB1276

secondary metabolism.isoprenoids.mevalonate Pathway.acetyl-CoA C-acyltransferase

1.46867 acetoacetyl-CoA thiolase 2

Ciclev10008832m.g AT4G17190 FPS2 secondary metabolism.isoprenoids.mevalonate Pathway.farnesyl pyrophosphate synthetase

1.46627 farnesyl diphosphate synthase 2

167


Ciclev10014386m.g AT4G21390 B120 signalling.receptor kinases. S-locus glycoprotein like

1.45309 S-locus lectin protein kinase family protein

Ciclev10017621m.g AT3G25070 RIN4 stress. biotic 1.44031 RPM1 interacting protein 4 Ciclev10021781m.g AT2G45130 ATSPX3,SPX3 stress. abiotic 1.43376 SPX domain gene 3

Ciclev10031440m.g AT3G07320 misc.beta 1,3 glucan hydrolases.glucan endo-1,3-beta-glucosidase

1.4059 O-Glycosyl hydrolases family 17 protein




Ciclev10010546m.g AT2G18950 ATHPT,HPT1

secondary metabolism.isoprenoids.tocopherol biosynthesis.homogentisate phytyltransferase

1.38739 homogentisate phytyltransferase 1

Ciclev10013555m.g AT5G15720 GLIP7 misc.GDSL-motif lipase 1.36034 GDSL-motif lipase 7

Ciclev10001970m.g AT5G64260 EXL2 signalling.in sugar and nutrient physiology

1.34893 EXORDIUM like 2



Ciclev10009394m.g AT2G43290 MSS3 signalling.calcium 1.33131 Calcium-binding EF-hand family protein

Ciclev10013430m.g AT1G22380 AtUGT85A3, UGT85A3

misc.UDP glucosyl and glucoronyl transferases

1.29935 UDP-glucosyl transferase 85A3

Ciclev10030627m.g AT5G13980 misc.gluco-, galacto- and mannosidases.alpha-mannosidase

1.29804 Glycosyl hydrolase family 38 protein

Ciclev10004353m.g AT3G57630 misc.UDP glucosyl and glucoronyl transferases

1.29128 exostosin family protein

Ciclev10031948m.g AT1G17020 ATSRG1,SRG1 secondary metabolism.flavonoids.flavonols

1.28935 senescence-related gene 1

Ciclev10003438m.g AT5G56790 signalling.receptor kinases.proline extensin like

1.28125 Protein kinase superfamily protein


Ciclev10028092m.g AT1G61560 ATMLO6,MLO6 stress.biotic.signalling.MLO-like 1.24667 Seven transmembrane MLO family protein

168



1.24368 Protein kinase protein with adenine nucleotide alpha hydrolases-like domain

Ciclev10025531m.g AT1G51880 RHS6 signalling.receptor kinases.misc 1.23064 root hair specific 6




Ciclev10010546m.g AT2G18950 ATHPT,VTE2 isoprenoids.tocopherol biosynthesis. homogentisate phytyltransferase


Ciclev10013555m.g AT5G15720 GLIP7 misc.GDSL-motif lipase 1.36034 GDSL-motif lipase 7






Ciclev10009394m.g AT2G43290 MSS3 signalling.calcium 1.33131 Calcium-binding EF-hand family protein

Ciclev10013430m.g AT1G22380 AtUGT85A3 misc.UDP glucosyl and glucoronyl transferases


Ciclev10030627m.g AT5G13980 misc.gluco-, galacto- and mannosidases.alpha-mannosidase

1.29804 Glycosyl hydrolase family 38 protein


1.29128 exostosin family protein

Ciclev10031948m.g AT1G17020 ATSRG1, SRG1

secondary metabolism.flavonoids.flavonols

1.28935 senescence-related gene 1




Ciclev10028092m.g AT1G61560 ATMLO6, MLO6

stress.biotic.signalling.MLO-like 1.24667 Seven transmembrane MLO family protein


1.24368 Protein kinase protein with adenine nucleotide alpha hydrolases-like domain

Ciclev10025531m.g AT1G51880 RHS6 signalling.receptor kinases.misc 1.23064 root hair specific 6

Ciclev10017610m.g AT5G11720 misc.gluco-, galacto- and mannosidases.alpha-galactosidase

1.22991 Glycosyl hydrolases family 31 protein

169


Ciclev10026072m.g AT4G33420 misc.peroxidases 1.22299 Peroxidase superfamily protein Ciclev10007081m.g AT1G24020 MLP423 stress.abiotic.unspecified 1.21946 MLP-like protein 423


Ciclev10007347m.g AT2G25140 CLPB-M,HSP98.7 stress.abiotic.heat 1.20902 casein lytic proteinase B4

Ciclev10002683m.g AT4G36040 stress.abiotic.heat 1.20273 Chaperone DnaJ-domain superfamily protein




Ciclev10033587m.g AT1G78380 ATGSTU19,GSTU19 misc.glutathione S transferases 1.19974 glutathione S-transferase TAU 19

Ciclev10015249m.g AT5G17540 stress.biotic 1.19939 HXXXD-type acyl-transferase family protein

Ciclev10011715m.g AT5G67150 secondary metabolism.phenylpropanoids


Ciclev10011540m.g AT1G22380 AtUGT85A3,UGT85A3 misc.UDP glucosyl and glucoronyl transferases


Ciclev10011544m.g AT3G21760 HYR1 misc.UDP glucosyl and glucoronyl transferases


Ciclev10027371m.g AT2G18950 ATHPT,HPT1VTE2

secondary metabolism.isoprenoids.tocopherol biosynthesis.homogentisate phytyltransferase


Ciclev10022154m.g AT1G78380 ATGSTU19,GSTU19 misc.glutathione S transferases 1.17975 glutathione S-transferase TAU 19

Ciclev10008330m.g AT5G16970 AER, AT-AER misc.oxidases - copper, flavone etc.

1.17411 alkenal reductase

Ciclev10027393m.g AT5G10530 signalling.receptor kinases.legume-lectin

1.17294 Concanavalin A-like lectin protein kinase family protein



Ciclev10004432m.g AT4G15560 CLA1,DEF,DXPS2,DXS secondary metabolism.isoprenoids.non-mevalonate Pathway.DXS

1.16461 Deoxyxylulose-5-phosphate synthase

Ciclev10000878m.g AT1G28390 signalling.receptor kinases.crinkly like


170


Ciclev10028274m.g AT2G36800 DOGT1,UGT73C5 stress.biotic 1.16225 don-glucosyltransferase 1




Ciclev10028419m.g AT5G59580 UGT76E1 misc.UDP glucosyl and glucoronyl transferases

1.13938 UDP-glucosyl transferase 76E1

Ciclev10028637m.g AT2G27500 misc.beta 1,3 glucan hydrolases.glucan endo-1,3-beta-glucosidase

1.13502 Glycosyl hydrolase superfamily protein


Ciclev10017726m.g AT3G26300 CYP71B34 misc.cytochrome P450 1.13121 cytochrome P450, family 71, subfamily B, polypeptide 34

Ciclev10002752m.g AT1G24620 signalling.calcium 1.13112 EF hand calcium-binding protein family

Ciclev10005491m.g AT5G24090 ATCHIA,CHIA stress.biotic.PR-proteins 1.12166 chitinase A

Ciclev10005201m.g AT1G06620 redox.ascorbate and glutathione 1.12095

2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

Ciclev10015883m.g AT5G46080 signalling.receptor kinases.misc 1.11658 Protein kinase superfamily protein



†Classification of the measured parameter into a set a functional category in the MapMan analysis tool

171

Table 4-8. Differentially expressed hormonal metabolism-associated genes significantly upregulated in roots of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN roots

C. Clementina_ID Arabi ID Gene Name Bin Name† Log2 FC* Arabidospsis_define

Ciclev10029536m.g AT3G04720 HEL, PR-4,PR4 stress.biotic -5.93811 pathogenesis-related 4


-4.98469 FAD-binding Berberine family protein

Ciclev10015735m.g AT3G51420 ATSSL4, SSL4

secondary metabolism. N misc.alkaloid-like

-4.85918 strictosidine synthase-like 4

Ciclev10002362m.g AT3G54420 ATCHITIV, ATEP3

stress.biotic -4.81539 homolog of carrot EP3-3 chitinase

Ciclev10021347m.g AT2G20142 stress.biotic.receptors -4.47026 Toll-Interleukin-Resistance (TIR) domain family protein

Ciclev10021130m.g AT1G59960 secondary metabolism. flavonoids.chalcones

-3.65164 NAD(P)-linked oxidoreductase superfamily protein

Ciclev10005925m.g AT5G51440 stress.abiotic.heat -3.64355 FAD-binding Berberine family protein

Ciclev10027635m.g AT1G12280 stress.biotic.PR-proteins -3.62651 LRR and NB-ARC domains-containing disease resistance protein

Ciclev10023568m.g AT5G54160 ATOMT1,OMT1 secondary metabolism. phenylpropanoids.lignin biosynthesis.COMT


Ciclev10006671m.g AT3G09270 ATGSTU8, GSTU8

misc.glutathione S transferases -3.55462 glutathione S-transferase TAU 8

Ciclev10020838m.g AT1G17020 ATSRG1,SRG1 secondary metabolism.flavonoids.flavonols -3.52534 senescence-related gene 1

Ciclev10030418m.g AT2G29420 ATGSTU7, GSTU7

misc.glutathione S transferases -3.23252 glutathione S-transferase tau 7

Ciclev10026235m.g AT4G17080 signalling.phosphinositides. phosphatidylinositol-4-phosphate 5-kinase

-3.21452 Histone H3 K4-specific methyltransferase SET7/9 family protein

Ciclev10027185m.g AT4G16660 stress.abiotic.heat -3.20734 heat shock protein 70 (Hsp 70) family protein

Ciclev10030281m.g AT1G35710 signalling.receptor kinases.l eucine rich repeat XII

-3.1971 Protein kinase family protein with leucine-rich repeat domain

Ciclev10007645m.g AT4G27300 signalling.receptor kinases.S-locus glycoprotein like


172

Table 4-8. Continued C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC Arabidospsis_define

Ciclev10022118m.g AT5G39120 stress.abiotic.unspecified -3. 00553 RmlC-like cupins superfamily protein

Ciclev10024032m.g AT4G23180 CRK10 signalling.receptor kinases.DUF 26 -2.99508 cysteine-rich RLK (RECEPTOR-like protein kinase) 10

Ciclev10026966m.g AT3G26300 CYP71B34 misc.cytochrome P450 -2.9662 cytochrome P450, family 71, subfamily B, polypeptide 34

Ciclev10020212m.g AT1G78860 misc.myrosinases-lectin-jacalin -2.77135 D-mannose binding lectin protein with Apple-like carbohydrate-binding domain

Ciclev10021499m.g AT3G22600 misc.protease inhibitor/seed storage/ lipid transfer protein (LTP) family protein

-2.72343 Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily protein



Ciclev10023259m.g AT5G37940 misc.oxidases - copper, flavone etc. -2.68329 Zinc-binding dehydrogenase family protein

Ciclev10028275m.g AT2G45550 CYP76C4 misc.cytochrome P450 -2.68182 cytochrome P450, family 76, subfamily C, polypeptide 4

Ciclev10024525m.g AT2G20142 stress.biotic.receptors -2.64764 Toll-Interleukin-Resistance (TIR) domain family protein

Ciclev10014222m.g AT3G46530 RPP13 stress.biotic -2.63199 NB-ARC domain-containing disease resistance protein




-2.61685 UDP-Glycosyltransferase superfamily protein

Ciclev10009711m.g AT3G18430 signalling.calcium -2.58112 Calcium-binding EF-hand family protein

Ciclev10021079m.g AT2G45400 BEN1 secondary metabolism.flavonoids. flavonols.dihydrokaempferol 4-reductase


Ciclev10015853m.g AT1G17020 SRG1 secondary metabolism.flavonoids.flavonols



misc.short chain dehydrogenase/reductase (SDR)


Ciclev10010519m.g AT4G27290 signalling.receptor kinases. S-locus glycoprotein like


173




Ciclev10026415m.g AT4G37990 ATCAD8 phenylpropanoids.lignin biosynthesis -2.43536 elicitor-activated gene 3-2

Ciclev10031740m.g AT2G18950 ,VTE2 isoprenoids.tocopherol biosynthesis. homogentisate phytyltransferase

-2.41184 homogentisate phytyltransferase 1

Ciclev10001726m.g AT1G71695 misc.peroxidases -2.38363 Peroxidase superfamily protein

Ciclev10012710m.g AT3G09270 GSTU8 misc.glutathione S transferases -2.35917 glutathione S-transferase TAU 8

Ciclev10023911m.g AT4G27190 stress.biotic.PR-proteins -2.34945 NB-ARC domain-containing disease resistance protein

Ciclev10014618m.g AT1G66910 signalling. wheat LRK10 like -2.3204 Protein kinase superfamily protein

Ciclev10005336m.g AT2G36690 misc.oxidases - copper, flavone etc. -2.3067 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

Ciclev10023959m.g AT2G29420 GSTU7 misc.glutathione S transferases -2.30659 glutathione S-transferase tau 7

Ciclev10005186m.g AT4G02340 misc.misc2 -2.29788 alpha/beta-Hydrolases superfamily protein

Ciclev10002119m.g AT2G47730 GSTF8 misc.glutathione S transferases -2.25253 glutathione S-transferase phi 8

Ciclev10027191m.g AT1G18980 stress.abiotic.unspecified -2.2015 RmlC-like cupins superfamily protein

Ciclev10007070m.g AT2G38870 stress.biotic -2.19967 Serine protease inhibitor, potato inhibitor I-type family protein

Ciclev10032737m.g AT2G30860 GSTF9 misc.glutathione S transferases -2.17214 glutathione S-transferase PHI 9




Ciclev10012590m.g AT1G20030 stress.biotic -2.1235 Pathogenesis-related thaumatin superfamily protein

Ciclev10007471m.g AT1G65800 ARK2,RK2 signalling.receptor kinases.S-locus glycoprotein like


Ciclev10004174m.g AT5G17680 stress.biotic.PR-proteins -2.06446 disease resistance protein (TIR-NBS-LRR class), putative

Ciclev10024069m.g AT4G08850 signalling.receptor kinases. leucine rich repeat XII


Ciclev10027249m.g AT3G50740 UGT72E1 phenylpropanoids.lignin biosynthesis -1.98165 UDP-glucosyl transferase 72E1

Ciclev10026825m.g AT2G17880 stress.abiotic.heat -1.96993 Chaperone DnaJ-domain superfamily protein

174


Ciclev10008287m.g AT3G16490 IQD26 signalling.calcium -1.96815 IQ-domain 26

Ciclev10025863m.g AT2G23540 misc.GDSL-motif lipase -1.93432 GDSL-like Lipase/Acylhydrolase superfamily protein

Ciclev10014789m.g AT4G21410 CRK29 signalling.receptor kinases.DUF 26 -1.91438 cysteine-rich RLK (RECEPTOR-like protein kinase) 29

Ciclev10004282m.g AT3G57330 ACA11 signalling.calcium -1.87353 autoinhibited Ca2+-ATPase 11

Ciclev10024795m.g AT4G37640 ACA2 signalling.calcium -1.86552 calcium ATPase 2



Ciclev10002259m.g AT4G36810 GGPS1 secondary metabolism.isoprenoids.non-mevalonate Pathway.geranylgeranyl pyrophosphate synthase

-1.83556 geranylgeranyl pyrophosphate synthase 1

Ciclev10006554m.g AT1G31690 misc.oxidases - copper, flavone etc. -1.80585 Copper amine oxidase family protein

Ciclev10022349m.g AT3G14630 CYP72A9 misc.cytochrome P450 -1.77144 cytochrome P450, family 72, subfamily A, polypeptide 9



Ciclev10020864m.g AT5G22300 AtNIT4,NIT4 secondary metabolism. glucosinolates. degradation. nitrilase

-1.74216 nitrilase 4

Ciclev10028457m.g AT4G14210 PDS3 secondary metabolism. isoprenoids.carotenoids. phytoene dehydrogenase

-1.72224 phytoene desaturase 3

Ciclev10022230m.g AT1G18650 PDCB3 misc.beta 1,3 glucan hydrolases -1.71515 callose-binding protein 3

Ciclev10026248m.g AT4G37000 ACD2, ATRCCR

stress.biotic -1.70408 accelerated cell death 2 (ACD2)

Ciclev10023412m.g AT3G03080 misc.oxidases - copper, flavone etc. -1.69884 Zinc-binding dehydrogenase protein

Ciclev10005988m.g AT5G52390 signalling.in sugar and nutrient physiology

-1.69616 PAR1 protein


stress.abiotic -1.69162 Pathogenesis-related thaumatin superfamily protein

Ciclev10022143m.g AT5G26990 stress.abiotic.drought/salt -1.67829 Drought-responsive family protein

Ciclev10001524m.g AT5G28840 GME redox.ascorbate and glutathione.ascorbate.GME

-1.66612 GDP-D-mannose 3\',5\'-epimerase

Ciclev10014803m.g AT5G44400 misc.nitrilases, *nitrile lyases, berberine reticuline oxidases, troponine reductases


175


Ciclev10029467m.g AT1G53540 stress.abiotic.heat -1.65675 HSP20-like chaperones superfamily protein

Ciclev10007723m.g AT1G68400 signalling.receptor kinases. leucine rich repeat III

-1.62931 leucine-rich repeat transmembrane protein kinase family protein

Ciclev10021886m.g AT3G07500 signalling.light -1.60937 Far-red impaired responsive (FAR1) family protein



Ciclev10010023m.g AT1G52240 PIRF1,ROPGEF11 signalling.G-proteins -1.60309 RHO guanyl-nucleotide exchange factor 11

Ciclev10021946m.g AT4G27700 misc.rhodanese -1.58722 Rhodanese/Cell cycle control phosphatase superfamily protein

Ciclev10025807m.g AT5G13930 ATCHS,CHS,TT4 secondary metabolism.flavonoids.chalcones. naringenin-chalcone synthase

-1.57816 Chalcone and stilbene synthase family protein

Ciclev10022100m.g AT4G11650 ATOSM34,OSM34 stress.abiotic -1.5733 osmotin 34


-1.56011 alpha 1,4-glycosyltransferase family protein

Ciclev10001996m.g AT4G27220 stress.biotic.PR-protei Ciclev10032517m.g ns

-1.55872 NB-ARC domain-containing disease resistance protein

Ciclev10026553m.g AT1G73040 misc.myrosinases-lectin-jacalin -1.55512 Mannose-binding lectin superfamily protein





Ciclev10024896m.g AT2G23200 signalling. receptor kinases. Catharanthus roseus-like RLK1

-1.49825 Protein kinase superfamily protein

Ciclev10021994m.g AT1G01200 ATRAB-A3, signalling.G-proteins -1.49313 RAB GTPase homolog A3

Ciclev10022104m.g AT4G11650 ATOSM34,OSM34 stress.abiotic -1.47446 osmotin 34

Ciclev10029528m.g AT3G04720 HEL, PR-4,PR4 stress.biotic -1.46394 pathogenesis-related 4

Ciclev10019699m.g AT3G48310 CYP71A22 misc.cytochrome P450 -1.44637 cytochrome P450, family 71, subfamily A, polypeptide 22



176

Table 4-8. continued C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC Arabidospsis_define

Ciclev10030258m.g AT2G32520 misc.misc2 -1.4157 alpha/beta-Hydrolases superfamily protein

Ciclev10006703m.g AT2G34790 EDA28,MEE23 misc.nitrilases, *nitrile lyases, berberine bridge enzymes, reticuline oxidases, troponine reductases


Ciclev10021726m.g AT3G25070 RIN4 stress.biotic -1.3748 RPM1 interacting protein 4

Ciclev10025102m.g AT1G16030 Hsp70b stress.abiotic.heat -1.36969 heat shock protein 70B

Ciclev10026139m.g AT4G38660 stress.biotic -1.35471 Pathogenesis-related thaumatin superfamily protein



Ciclev10018645m.g AT3G63380 signalling.calcium -1.34747 ATPase E1-E2 type family protein / haloacid dehalogenase-like hydrolase family protein


Ciclev10033694m.g AT2G44290 misc.protease inhibitor/seed storage/lipid transfer protein (LTP) family protein

-1.30822 Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin superfamily protein

Ciclev10026558m.g AT5G48930 HCT secondary metabolism.phenylpropanoids.lignin biosynthesis.HCT

-1.30046 hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase

Ciclev10000846m.g AT4G31940 CYP82C4 misc.cytochrome P450 -1.29287 cytochrome P450, family 82, subfamily C, polypeptide 4



Ciclev10019883m.g AT1G71380 CEL3 misc.gluco-, galacto- and mannosidases.endoglucanase

-1.28489 cellulase 3

Ciclev10006106m.g AT1G24020 MLP423 stress.abiotic.unspecified -1.27801 MLP-like protein 423

Ciclev10004621m.g AT4G13700 PAP23 misc.acid and other phosphatases -1.26566 purple acid phosphatase 23

Ciclev10008059m.g AT5G39090 secondary metabolism.flavonoids.anthocyanins. anthocyanin 5-aromatic acyltransferase

-1.26197 HXXXD-type acyl-transferase family protein

Ciclev10005812m.g AT3G09270 GSTU8 misc.glutathione S transferases -1.23756 glutathione S-transferase TAU 8


-1.23187 Chalcone-flavanone isomerase family protein

177


Ciclev10002693m.g AT1G21550 signalling. calcium -1.22067 Calcium-binding EF-hand family protein

Ciclev10000597m.g AT1G71830 SERK1 Signaling.leucine rich repeat II -1.20804 somatic embryogenesis receptor-like kinase 1

Ciclev10014418m.g AT4G22130 SRF8 signalling.leucine rich repeat V -1.19878 STRUBBELIG-receptor family 8

Ciclev10002706m.g AT1G21550 signalling. calcium -1.18526 Calcium-binding EF-hand family protein

Ciclev10018210m.g AT5G17770 ATCBR,CBR, redox.misc -1.16839 NADH:cytochrome B5 reductase 1

Ciclev10000895m.g AT5G10610 CYP81K1 misc. cytochrome P450 -1.16162 cytochrome P450, family 81, subfamily K, polypeptide 1

Ciclev10025325m.g AT1G11000 MLO4 stress.biotic.signalling.MLO-like -1.15979 Seven transmembrane MLO family protein

Ciclev10019330m.g AT5G48800 signalling. light -1.15247 Phototropic-responsive NPH3 family protein

Ciclev10032706m.g AT3G07410 AtRABA5b signalling.G-proteins -1.12896 RAB GTPase homolog A5B

Ciclev10011520m.g AT2G40890 CYP98A3 misc. cytochrome P450 -1.12441 cytochrome P450, family 98, subfamily A, polypeptide 3

Ciclev10025254m.g AT5G63710 signalling.receptor kinases. leucine rich repeat II


Ciclev10007314m.g AT5G53890 PSKR2 signalling..leucine rich repeat X -1.09988 phytosylfokine-alpha receptor 2

Ciclev10026012m.g AT1G15950 CCR1, IRX4

secondary metabolism. phenylpropanoids.lignin biosynthesis.CCR1

-1.09732 cinnamoyl coa reductase 1

Ciclev10008835m.g AT3G47860 CHL stress.abiotic -1.09088 chloroplastic lipocalin


Ciclev10007366m.g AT5G51350 signalling.receptor kinases. leucine rich repeat XIV

-1.0812 Leucine-rich repeat transmembrane protein kinase family protein

Ciclev10007358m.g AT2G25790 signalling.receptor kinases. leucine rich repeat IV

-1.05918 Leucine-rich receptor-like protein kinase family protein

Ciclev10026349m.g AT2G22570 NIC1 secondary metabolism.phenylpropanoids -1.05823 nicotinamidase 1

Ciclev10008865m.g AT1G35190 secondary metabolism. N misc.alkaloid-like

-1.04569 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

Ciclev10000174m.g AT1G06840 signalling.receptor kinases. leucine rich repeat VIII-1


178


C. Clementina_ID Arabi ID Gene Name

Bin Name Log2 FC Arabidospsis_define

Ciclev10000924m.g AT4G31500 CYP83B1, SUR2

secondary metabolism. sulfur-containing.glucosinolates.synthesis. shared.CYP83B1 phenylacetaldoxime monooxygenase

-1.03948 cytochrome P450, family 83, subfamily B, polypeptide 1

Ciclev10028758m.g AT3G19000 redox.ascorbate and glutathione -1.03174

2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

Ciclev10006362m.g AT2G02100 LCR69, PDF2.2

stress.biotic -1.02554 low-molecular-weight cysteine-rich 69

Ciclev10005795m.g AT4G25570 ACYB-2 redox.ascorbate and glutathione -1.00919 Cytochrome b561/ferric reductase transmembrane protein family

Ciclev10025368m.g AT1G53710 misc.calcineurin-like phosphoesterase family protein

-1.00463 Calcineurin-like metallo-phosphoesterase superfamily protein

* The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. †Classification of the measured parameter into a set a functional category in the MapMan analysis tool.

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Figure 4-1. Graphical presentation of DEGs involved in environmental biotic and abiotic signaling.Biotic and abiotic signaling overview in the PageMan depicting DGE in leaves

and roots of HLB-asymptomatic and -symptomatic treatment in VAL/CAN and VAL/SW combinations. Log2 fold changes are indicated as a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated and VAL/SW combination. Asymp; Asymptomatic, Sympt; symptomatic

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Figure 4-2. Graphical presentation of DEGs involved in biotic and abiotic stress. Stress overview in the PageMan depicting DGE in leaves and roots of the HLB-asymptomatic and -symptomatic treatments in VAL/CAN and VAL/SW combinations. Log2 fold changes are indicated as a gradient between blue (upregulated in VAL/CAN combination) and red (upregulated in VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic

Figure 4-3. Display of HLB-induced biotic stress responses in the leaves of the HLB asymptomatic VAL/CAN and VAL/SW leaves. The log2 fold change of DGE was

analyzed using MapMan. Log2 fold changes are indicated as a gradient between blue (upregulated in the asymptomatic VAL/CAN) and red (upregulated in asymptomatic VAL/SW combination).

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Figure 4-4 Display of HLB-induced biotic stress responses in leaves of the HLB- symptomatic VAL/CAN and VAL/SW leaves. The log2 fold change of DGE was

analyzed using MapMan. Log2 fold changes are indicated as a gradient blue (upregulated in the symptomatic VAL/CAN combination) and red (upregulated in symptomatic VAL/SW combination).

Figure 4-5. Display of HLB-induced biotic stress responses in roots of the HLB- symptomatic VAL/CAN and VAL/SW leaves. The log2 fold change of DGE was

analyzed using MapMan. Log2 fold changes are indicated as a gradient between blue (upregulated in the symptomatic VAL/CAN combination) and red (upregulated in symptomatic VAL/SW combination).

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CHAPTER 5 DIFFERENTIAL EXPRESSION OF PLANT GROWTH AND DEVELOPMENT-

ASSOCIATED GENES IN LEAVES AND ROOTS OF TWO SCION/ROOTSTOCK COMBINATIONS AT ASYMPTOMATIC AND SYMPTOMATIC STAGES OF HLB

DISEASE

Introduction

Physical growth and development are essential for the plant life cycle. Various

components of plant growth are embryogenesis, development of seed and flower, fruit

set and ripening, development of structural components and their expansion, growth of

reproductive and vegetative organs, plant senescence and plant death, and interaction

of plant with biotic and abiotic stress. Plant growth is a highly coordinated process which

mostly depends on energy production (photosynthesis) and consumption (respiration),

supply and demand of photosynthates flow between source-sink organs, regeneration

of secondary plant growth, reproduction, and external and/or internal signaling

mechanism. Plant growth may be compromised severely under biotic and abiotic

stresses. When plants are impacted by biotic and abiotic challenges, defense is

inevitable for survival, but plant sustainability is indeed dependent on continued

photosynthesis and respiration for plant growth. In citrus, (huanglongbing) HLB-affected

plants have shown the decline in plant production and sustainability. Interactions

between HLB and citrus are still unclear. Anatomical and molecular analysis of HLB

affected citrus trees suggests that a disparity between plant growth and defense affects

the plant sustainability. Anatomical studies of Candidatus Liberibacter asiaticus

(CaLas) -infected citrus cultivars have shown a severely affected plant photosynthesis

machinery, interrupted phloem channels, reduced root growth, and disproportionate

distribution of plant photosynthates between sink-source tissue, leading to the

appearance of HLB symptoms on the leaves (Etxeberria et al., 2009; Folimonova and

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Achor, 2010; Koh et al., 2012). Molecular analysis of the CaLas-infected leaves, roots

and fruits samples supported the findings from the anatomical studies (Fan et al., 2012;

Albrecht and Bowman, 2012b; Aritua et al., 2013). The anatomical, transcriptomic,

proteomic, and microscopic -assisted studies of HLB-citrus interaction in various

cultivars suggest that activation of many non-host specific defenses, inability to repair

damaged phloem system, and limited available photosynthate resources hamper plant

growth. Improved plant growth can, therefore, help to enhance the plant sustainability

and performance of HLB-affected plants. This Chapter discusses the possible role of a

putative HLB-tolerant candidate rootstock (CAN) to promote the plant growth in

asymptomatic and symptomatic CaLas-infected ‘Valencia’ scion.

Plant tissues and organs are often injured because of insect feeding, wounding,

disease attack and abiotic stresses. Damaged tissues are either repaired, regenerated

or removed by selective cell death to keep essential plant biological functions working.

Regeneration is a common strategy for plants to repair the damage caused by biotic

and abiotic stress. The plant vascular system is a crucial system for plant survival and

has a capacity to repair or regenerate damaged phloem, xylem and cambium tissues.

Plant tissue culture techniques and microscopy have revealed a hierarchy of plant

growth and development from totipotent meristematic tissue (Allan, 2015). Anatomical

studies on plant regeneration of the model plant Arabidopsis thaliana showed that

induction of the phloem regeneration is a three-step process: maintenance of stem cell

microenvironment, proliferation and growth, and differentiation of dividing cells into

tissues. Studies in Arabidopsis showed that Auxins (AU), gibberellins (GA), cytokinins

(CT) and ethylene (ET) regulate vascular cambium activity and differentiation of

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secondary vascular tissue (Ursache et al., 2013). The genes encoding vascular tissue

regeneration transcription factors (TFs) such as KNOX1, AP-2 , HD ZIPIII, KANADI

(KAN), LATERAL ORGAN BOUNDARIES (LBD), and Vascular related NAC domain

protein-1 (VND1) (Pang et al., 2008; Chen et al., 2014; Zhou et al., 2014). However,

there are not enough studies on the genetic aspect of how the totipotent cells

differentiate into different primary and secondary vascular structures. Microarray

analysis of secondary vascular tissue regeneration in girdled Populus tissue showed

changes in gene expression at each step of xylem, phloem and cambium regeneration

(Zhang et al., 2011b). The study of girdled Populus reported differential changes in the

expression level of genes encoding transcription factors (TFs) belong to R2R3-MYB,

WRKY, DNA- binding with one zinc finger (DOFs), Knotted1-like homeobox (KNOX) and

other phloem specific TFs. Also, the comparative gene expression analysis between

xylem, phloem, and cambium regeneration stages showed effects of AU, CT and GA

responsive genes in the vascular tissue development (Zhang et al., 2011b). A review of

the genetic and hormonal regulation of cambial development reported that AU, CT, and

GA act synergistically to regulate cambial development (Ursache et al., 2013). HLB-

affected plants are a classic example of vascular tissue (phloem) damage caused by

psyllid (Diaphrina citri Kuwayama) feeding (piercing) and callose/starch depositions in

the phloem, which block the source-sink transport in the plants (Koh et al., 2012).

Weakening strength of HLB-affected plants is the indirect effect of CaLas-infection and

the direct impact of photosynthate supply shortage for plant growth, development, and

defense (Cimò et al., 2013). Considering the significant impact of phloem damage in

developing HLB symptoms and reducing plant sustainability, developing strategies that

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can increase phloem regeneration ability in the CaLas-infected plants is a priority for

citrus researchers.

Plant secondary metabolites are involved in plant development and growth. The

secondary metabolites are divided into 3 major categories: terpenes, phenolics, and

nitrogen containing compounds. The groups of plant hormones such as GA and

brassinosteroids (BR) are considered as diterpenes and tetraterpenes (Haubrick and

Assmann, 2006; Urbanov et al., 2011). Abscisic acid (ABA) is a C15 terpene produced

by degradation of a carotenoid precursor (Raghavendra et al., 2010). The carotenoids

(red, orange, yellow colored) are tetraterpenes which are important pigments in the

plant photosystem (Singh and Sharma, 2015). Phenylalanine (PHE) is a key

intermediate in the secondary metabolite biosynthesis which belongs to the phenolics

group. Many phenolic compounds in the plants are derived by PHE catabolic enzyme

phenylalanine ammonia lyase (PAL). PAL is also a branching point which leads to

different biosynthetic pathways (Hahlbrock and Scheel, 1989). PAL acts as a signal in

response to nutrient level changes, light response, and potential plant danger signaling.

Phenylpropanoid, a derivative of PHE, is involved in lignin biosynthesis (Besseau et al.,

2007). Lignin is the component of plant cell wall cytoskeleton which supports cell

integrity and strengthens the plant vascular system (Ruprecht and Persson, 2012).

Phenolic compounds are divided into major 4 subgroups: flavonoids, flavones,

isoflavonoids, and tannins. These phenolic subgroups are involved in developing plant

pigmentation, cell wall modification, protection from the oxidative burst, and plant

defense, (Gould, 2004; Besseau et al., 2007; Hussey et al., 2013).

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Plants are autotrophs that produce their own energy using photosystems coupled

with organelles that metabolize the energy into the form of adenosine triphosphate

(ATP). Energy harvesting chloroplasts and energy production mitochondria are

inevitable organelles in the plant cells that balance the energy demand and supply

throughout the plant life cycle. Photosynthesis and plant respiration synthesized

metabolites, and ATPs are the life driving force in plants. Plant metabolites are the

complex molecules that are synthesized from simple sugar monomers or amino acids

and other inorganic building blocks. Carbohydrates (CHOs) or sugars are invaluable for

plant growth and defense (Sturm and Tang, 1999; Koch, 2004). Roles of monomers or

polymers of CHOs are well known, and they are involved in the diverse plant biological

functions. Polymers such as starch and fructans are the nocturnal energy reserve when

photosynthesis is inactive (Orzechowski, 2008) whereas, sucrose is the primary

provider of energy to the growing plant tissue. CHOs are not only the source of energy

but also provide the plant cytoskeleton. The polysaccharides such as

homogalacturonan (HGA), rhamnogalacturonan I (RGI), rhamnogalacturonan II (RGII)

and xylogalacturonan (XGA) are components of the cell wall cytoskeleton. HGA is a

component of pectins in the plant cell wall. Pectinmethylesterases (PMEs) are a

carbohydrate esterase that catalyzes dimethyl esterification of HGA, and it remodels

the plant cell wall during plant growth and defense (Pelloux et al., 2007).

Nutrient availability controls plant development (Krouk et al., 2011). Plant

minerals are essentials to produce plant energy and required metabolites. Plant

minerals are divided into macronutrients: nitrogen (N), phosphorus (P), potassium (K)

secondary nutrients: magnesium (Mg), calcium (Ca), Sulfur (S) and micronutrients: iron

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(Fe), manganese (Mn), zinc (Zn), copper (Cu), and boron (B), nickel (Ni) and

molybdenum (Mo). Macro, secondary and micronutrients are important plant regulatory

components are essential for plant growth, energy production, maintenance plant

structures, cellular redox environment, and resource supply to the source organs. Active

plant growth, reproduction, and defense demand more energy and, therefore, nutrient

requirement is more in the plant. Hence, nutrient homeostasis is critical to perform plant

functions at optimum levels. Nutrient homeostasis is regulated through plant internal or

cellular signaling system which creates hormonal changes, enhanced or reduced metal

transporter activity, and expansion of the root system. The different components of

nutrient homeostasis are highly interdependent (Krouk et al., 2011). Genomics studies

in dicot and monocot plants revealed the differential expressional changes in the

transporter genes to maintain the metal homeostasis in response to mineral

deficiencies (Wintz et al., 2003; Grotz and Guerinot, 2006).

Resource limitations or pathogen infection can lead to nutrient deficiencies or

heavy metal toxicity in plants. It is not unusual that disease affected plants are deficient

in various mineral nutrients. The depleted pool of nutrients in the disease affected plants

can be enhanced by nutrient rich fertilizer applications. However, the time and space

interaction is highly correlated to revive the plant’s health under nutrient deficit and lack

of immunity conditions. The similar scenario of space-time-disease complex induced

nutrient deficiency is seen in the CaLas-infected citrus plants. HLB-affected citrus plants

are found to exhibit Ca, Mg, Zn, Mn and B deficiencies in leaves, whereas K

concentration is found to be increased (Gottwald et al., 2007). The same deficiencies

were found to be even much greater in roots from both greenhouse and field trees

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infected with CaLas (J.W. Grosser, personal communication). The dysfunctionality of

vascular systems in HLB-affected plants not only imbalances the source-sink

compartmentalization but also creates localized or systemic nutrient deficiencies which

inhibit plant growth (Nwugo et al., 2013b). There is no comprehensive study on the

nutrition challenges in the CaLas infected citrus plants. Another factor that strongly

impacts the nutrient translocation and transport in the CaLas-infected citrus plants is the

scion/rootstock combination. Effect of citrus rootstocks on mineral nutrition was

discussed by Wutscher (1973). The purpose of studying differential transcriptomic

changes between a putatively HLB-tolerant rootstock and the HLB-susceptible Swingle

is to analyze the role of genes encoding nutrient transporters and transmembrane

proteins to reprogram CaLas-infected scion growth and development.


Plant Material

Two combinations of scion and rootstock were used in this experiment. Field

grown seven-year-old experimental plants were planted in the Lee Family's Alligator

Grove, east of St. Cloud, Florida. The first combination of trees was ‘Valencia' (VAL)

sweet orange (Citrus sinensis [L.] Osbeck) grafted onto a putative HLB-tolerant

candidate (CAN) rootstock. The CAN rootstock is a hybrid of ‘Hirado Buntan Pink'

pummelo (HBP) (Citrus maxima Merr.) and ‘Cleopatra' mandarin (Citrus reticulata

Blanco.). The 2nd combination was ‘Valencia' (VAL) scion grafted onto commercially

important Swingle citrumelo (SW) rootstock. Swingle is a hybrid of grapefruit (Citrus

paradisi [Macf.]) and trifoliate orange (Poncirus trifoliata [L.] Raf). Each combination of

VAL/CAN and VAL/SW, plants were divided into two treatments based on the visible

presence of HLB-like symptoms (Table 5-2). Highly infected and symptomatic trees in

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each combination were grouped into the symptomatic treatment. Whereas, trees with

fewer or no symptoms were grouped into the asymptomatic treatment (Table 5-1). All

biological replicates in each treatment and combination were tested using quantitative

PCR (qPCR) based CaLas detection, and ELISA assisted citrus tristeza virus (CTV)

detection.

Sampling, RNA Extraction, and RNA Sequencing


explained in chapter#2. In brief, differentially expressed genes (DEGs) in the pairwise


symptomatic VAL/CAN, and VAL/SW combinations were identified with RNA-Seq and

the Tuxedo pipeline (Chapter 2). The significant differentially regulated genes in leaves

and roots tissue were annotated to C. clementina genome database in the Phytozome

V1.0 (Goodstein et al., 2012). Functional categories of the significantly expressed genes

were identified using A. thaliana annotation in the Phytozome server and MapMan

(Thimm et al., 2004) software (Chapter 2.). Blast2GO algorithm (Conesa et al., 2005)

was also used to identify molecular, cellular and biological functional categories.

Overview of DEG developed using PageMan analysis tool in the MapMan software

(Usadel et al., 2009).

Results


The results of qRT PCR-based HLB detection and RNA-Seq output in all

combinations and treatments are discussed in Chapter 2.

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Differentially Expressed Plant Growth and Development-Associated Genes in Leaves and Roots of Asymptomatic VAL/CAN and VAL/SW combinations

Leaf samples

The pairwise comparison between the leaf samples of the asymptomatic

treatment of VAL/CAN and VAL/SW combinations showed differential expression of

genes encoding the phloem regeneration specific transcription factors (TFs). From this

list, genes encoding TFs belonging to G2 like transcription factor family KANADI, LBD2,

late elongation hypocotyl (LHY), and homeobox domain (HB2 and HB16) were

upregulated in the VAL/CAN leaves as compared to VAL/SW leaves (Table 5-3).

Growth regulating factor 7 (GRF7) is a positive regulator of growth that suppresses the

growth inhibiting osmotic stress responsive genes, was also overexpressed in the

leaves of the asymptomatic VAL/CAN combination. In asymptomatic treatment, VEIN

PATTERNING 1 (VEP1) gene was upregulated in the asymptomatic VAL/CAN leaves

as compared to VAL/SW leaves (Table 5-3). Leaves collected from asymptomatic

VAL/CAN overexpressed genes encoding AUXIN RESPONSIVE FACTOR 8 and 16

(ARF8 and 16), CT- associated response regulator ARR9 and CT-two components

responsive regulator APRR2. In asymptomatic treatment, DGE analysis showed

overexpression of genes involved in the secondary metabolite pathways in VAL/CAN

leaves as compared to VAL/SW leaves. These genes are encoding PMEs,

XYLOGLUCAN ENDOTRANSGLYCOSYLASE/HYDROLASE 33 (XTH33), Expansin

family protein (EXP), anthocyanin and carotenoid biosynthesis TRANSPARENT TESTA

4 and 7 (TT4 and TT7), FLAVANONE 3-DIOXYGENASE (F3H), PAL, Wax biosynthesis

and 3-KETOACYL-COA SYNTHASE 6 (KCS6) (Figure 5-1 and 5-2). In the category of

carbohydrate metabolism, genes encoding starch degrading Vacuolar invertases and

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MYO-INOSITOL PHOSPHATE SYNTHASE 2 (MIPS2) were increased in their

expression in the asymptomatic VAL/CAN leaves, whereas, starch biosynthesis, starch

degradation and storage, synthesis genes were upregulated in the asymptomatic

VAL/SW combination. Multiple transcripts encoding trehalose synthesis gene were

strongly increased in their expression levels in the asymptomatic VAL/CAN leaves

(Table 5-3).

In the asymptomatic treatment, genes encoding nutrient transporters NODULIN-

LIKE PROTEINS (NIP), MAJOR INTRINSIC PROTEIN (MIP), oligopeptide transporters

and ATO BINDING CASSETTE (ABC) superfamily proteins, were also significantly

upregulated in VAL/CAN leaves as compared to VAL/SW leaves (Figure 5-3). Nutrient

transporter genes were AMMONIUM TRANSPORTER 1;2 (AMT1;2), and carbohydrate

transmembrane INOSITOL TRANSPORTER (INT1) (Table 5-2). Nutrient transporters

such as ZINC TRANSPORTER PRECURSOR 1 (ZIP1), potassium inward rectifier K

TRANSPORTER 1(AKT), nitrate transporter NRT1.1, low affinity phosphate transporter

PHT2;1, 2 oxoglutarate and malate valve transporter DECARBOXYLATE

TRANSPORTER 1 (DiT1) and genes encoding water channels NOD26-like intrinsic

protein NIP 4;2 and plasma membrane intrinsic protein PIP 2;4 and PIP 2;5 were also

upregulated in the asymptomatic VAL/CAN leaves as compared to asymptomatic

VAL/SW leaves (Table 5-3).

VAL leaves collected from the asymptomatic VAL/SW combination were

significantly upregulated in the ABA and ET hormone-induced responsive genes. In this

category, gene encoding ABA induced GRAM domain containing protein, ET signal

transduction ERF1 and ET forming enzyme (EFE) were overexpressed in the range of

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2-4 log2 FC. Genes encoding TFs involved in plant development were also

overexpressed in the asymptomatic VAL/SW combination. These include LOB domain

containing protein LBD 41, homeobox domain containing HB7 and many NAC domain

containing (NAC002, NAC084, NAC084, NAC090, and NAC061), and SCARECROW-

like SCR13 and SCR14 gene (Table 5-4). Asymptomatic VAL/SW also showed

significant upregulation of genes encoding proteins and enzymes involved in the

secondary metabolite pathways (Figure 5-1). These genes are encoding cell wall

metabolism and lignin biosynthesis associated; UDP-D-XYLOSE SYNTHASE 2,

ARABINOGALACTAN-PROTEIN 3 (AGP3), XYLOGLUCAN

ENDOTRANSGLYCOSYLASE 6 (XTR6), ARABINOGALACTAN PROTEIN 16 (AGP16)

and CELLULOSE SYNTHASE- like D3 (CSLD3). In the asymptomatic treatment, genes

encoding proteins those are involved in dehydration response: LATE

EMBRYOGENESIS ABUNDANT 14 (LEA14), EARLY RESPONSE DEHYDRATION

(EDR7), and RESPONSIVE to DESICCATION 26 (RD26) were significantly upregulated

in VAL/SW leaves as compared to VAL/CAN leaves (Table 5-4 and Figure 5-6). In the

category of CHO metabolism, genes encoding starch forming and starch catabolic

invertases (neutral and cell wall) were significantly overexpressed in the asymptomatic

VAL/SW leaves (Figure 5-5). The chloroplast GERANYLGERANYL REDUCTASE

(GGR) gene was also upregulated in the asymptomatic VAL/SW leaves as compared to

VAL/CAN leaves. In the category of transporter functional category, not many nutrient

transporter genes were overexpressed in the asymptomatic VAL/SW leaves. However,

phosphate transporters PHT1;4 and PHT 1;9 were significantly upregulated. DGE

analysis of asymptomatic VAL/SW leaves showed significant overexpression of

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PEPTIDE TRANSPORTER 3 (PTR3), GLUCOSE 6-PHOSPHATE TRANSPORTER

(GPT2), METAL TOLERANCE PROTEIN B1 (MTPB1) and genes encoding heavy

metal transport/detoxification superfamily proteins (Table 5-4).

Root samples

The DGE analysis between roots collected from the asymptomatic VAL/CAN,

and VAL/SW combinations showed significantly upregulated CHO metabolism related

genes in the VAL/SW roots.

Differentially Expressed Plant Growth and Development-Associated Genes in Leaves and Roots of Symptomatic VAL/CAN and VAL/SW Combinations

Leaf samples

In the symptomatic treatment, leaves collected from VAL/CAN combination

showed significant upregulation of genes encoding hormone-dependent plant growth

regulators such as auxin responsive ARF8, REVEILLEA1 (REV1), subtilisin like

protease known as AIR3, and LIKE-AUXIN RESISTANT 2 (LAX2). In the symptomatic

treatment, leaves collected from VAL/CAN showed significant upregulation of genes

encoding BR ENHANCED EXPRESSION 3 (BEE3), BR SIGNALING KINASE (BSK2),

GA biosynthesis KAO2, and CT-induced response regulators ARR4 and chase domain

containing histidine kinase WOL as compared to VAL/SW leaves (Table 5-3 and Table

B-3). In the symptomatic VAL/CAN combination, many genes involved in the

phenylpropanoid pathway induced lignin biosynthesis and flavonoids biosynthesis were

also significantly upregulated (Figure 5-1 and 5-2). Among these, CAFFEOYL-COA 3-

O-METHYLTRANSFERASE (CCoAOMT), 4-COUMARATE-COA LIGASE-3 (4CL3),

CINNAMYL-ALCOHOL-DEHYDROGENASE CAD4 and CAD9,

HYDROXYLCINNAMOYL-COA-SHIKIMATE TRANSFER (HCT), TRANSPARENT

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TESTA (TT7, TT5, TT4), CINNAMOYL-COA REDUCTASE 1 (CCR1), and genes

involved anthocyanin biosynthesis were also overexpressed (Table 5-5 and Table B-3).

Genes encoding proteins involved in the chloroplast development and photosynthesis

reactions: photosystem II polypeptide subunit, DEOXYLULOSE-5-PHOSPHATE

SYNTHASE (DXS), NON-PHOTOCHEMICAL QUENCHING 1(NPQ1),

MONOGALACTOSYL DIACYLGLYCEROL SYNTHASE1 (MGD1), CYCLOPHILIN 38,

and ATP SYNTHASE DELTA SUBUNIT (ATPD) were significantly upregulated in the

symptomatic VAL/CAN leaves as compared to symptomatic VAL/SW leaves (Table 5-

5). In the CHO metabolism category, starch, sucrose, raffinose and Myo-inositol

phosphate biosynthesis related genes were overexpressed in symptomatic VAL/CAN

combination (Table 5-5). Leaves collected from the symptomatic VAL/CAN combination

overexpressed genes encoding developing vessel and xylem differentiation related NAC

domain protein1 VND1, BSK2, LBD11, MADS box SEPALLATA 4 (SEP4), VEP1, and

NAC transcription factors-like (NTL9) (Table 5-4).

Symptomatic VAL/CAN leaves showed significant upregulation of many nutrient

transporter genes that generally respond to nutrient deficiency or increased nutrient

transportation (Figure 5-3). These include high affinity nitrate transporter NRT2;7,

phosphate transporter PHT2;1, potassium transporter AKT1 and AKT2, sulfate

transmembrane transporter SULTR4;1 and SULTR4;2, CALCIUM ANION EXCHANGE

CAX1 and CAX7, and CAX interacting protein CXIP4. Among water channel and sugar

transporters, NIP4;2, PIP1;4, INT1, and inorganic phosphate transmembrane

transporter encoding genes were upregulated in leaves of the symptomatic VAL/CAN

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combination. In the symptomatic treatment, transcripts encoding chloroplast ATPase

subunits and mitochondrial carrier A BOUT DE SOUFFLE (BOU) genes were

also upregulated in VAL/CAN leaves as compared to VAL/SW leaves (Table 5-5).

In the symptomatic VAL/SW combination, leaves showed significant upregulation

of a number of genes involved in the starch biosynthesis. Among these STARCH

BRANCHING ENZYME SBE2.1 and SBE2.2, ADP-GLUCOSE

PYROPHOSPHORYLASE (AGPase), and ALPHA GLUCAN PHOSPHORYLASE 2

(PHS2) were upregulated (Figure 5-5). The transcriptomic data also showed the

upregulation of starch biosynthesis regulator GLUCAN WATER DIKINASE or STARCH

EXCESS 1 (SEX1) gene in the symptomatic VAL/SW leaves. In the symptomatic

treatment, VAL/SW leaves showed significantly upregulated starch degrading neutral

SUCROSE INVERTASE gene transcripts (Table 5-6). Transcripts encoding the

photosystem I and II polypeptide subunits, RUBISCO ACTIVASE (RCA), ferredoxin

oxidoreductase family HY2, CHLORORESPIRATORY REDUCTION 3 (CCR3) and ATP

SYNTHASE were upregulated VAL/SW leaves as compared to VAL/CAN, in the

symptomatic treatment. There was no significant overexpression of secondary vascular

growth associated genes in leaves of the symptomatic VAL/SW combination, except a

few genes involved in cell wall modification, lignin biosynthesis, and flavonoid

metabolism pathway. Among transportation category, transcripts encoding phosphate

transporter, zinc efflux, amino acid carriers, mitochondrial membrane carriers,

phosphate transporter, and Zn transporter precursor 1; ZIP1 were significantly

upregulated in leaves of the symptomatic VAL/SW combination. Transcripts encoding

transition metal ion transporters such as NATURAL RESISTANCE ASSOCIATED

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MACROPHAGE PROTEIN (NRAMP), HEAVY METAL ATPase (HMA1), a copper

transporter COP1, CATION EXCHANGERS (CAXs), and sugar transporters were also

significantly upregulated in leaves of the symptomatic VAL/SW combination (Table 5-6).

Root samples

RNA extracted from roots of the symptomatic VAL/CAN combination showed

significant overexpression of the genes encoding auxin dependent Au efflux carrier

family protein F14G6.12, Au responsive AIR12, ARF16 and ARF2, and BR-induced

stem elongation promoting HERCULES RECEPTOR KINASE 2 (HERK2) (Table 5-7). In

addition, PHYTOSULFOKININE precursor 3 (PSK3) growth factor transcripts

upregulated by 3.34 log2 FC in CAN roots as compared to the SW roots in the

symptomatic treatment. Roots collected from the symptomatic VAL/CAN combination

showed significantly upregulated expression of genes encoding TFs of GARP family

KAN4, and NAC domain containing NAC047, which are involved in the secondary

vascular tissue development (Table 5-7 and Table B-5). In the symptomatic treatment,

roots collected from VAL/CAN combination did not show significant upregulation of any

starch synthesis precursor genes (Figure 5-5). However, glucogenesis pathway genes,

trehalose metabolism, and sucrose synthesis genes were significantly overexpressed

(Table 5-7). Roots collected from the symptomatic VAL/CAN combination showed an

abundant number of transcripts encoding cell wall modification enzymes such as PMEs,

BETA XYLOSIDASE 1 (BXL1) (Figure 5-2). Roots from symptomatic VAL/CAN

combination showed upregulation of nitrate, iron and phosphate transporters: NRT1.1,

NRT2.7, NRT1:2, Fe carriers, IRON-REGULATED 2 (IREG2), FER-like regulated iron

uptake, FERRIC OXIDASE 7 (FRO7), and Phosphate transporters: PHT1;3, PHT1;7;

PHT2;1 (Table 5-7). In symptomatic VAL/CAN, transition metal carriers NRAMP6,

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HMA5, organic cation/carnitine transporter OCT1, METAL TOLERANCE PROTEIN

(MTP), and CATION EFFLUX EXCHANGER (CAX) transcripts were also upregulated.

Other transporter genes upregulated in the symptomatic VAL/CAN roots encoded heavy

metal detoxification superfamily proteins, nodulin MtN21-like intrinsic protein,

TONOPLAST INTRINSIC PROTEIN (TIP), oligopeptide sugar and mitochondrial

substrate carriers (Table 5-7).

Roots collected from the symptomatic treatment of VAL/SW showed

upregulation of a few genes associated with root and shoot growth, including Au efflux

carrier PIN4, Au response factor ARF19 and LAX1. Genes encoding homeobox like

transcription factors that impact plant development and lateral root initiation such as

HB13, HB7, HB8 and SHORT ROOTS (SHR) were significantly overexpressed in the

symptomatic VAL/SW roots. In the symptomatic treatment, VAL/SW roots showed

significant overexpression of transcripts encoding NAC domain containing XND1,

NAC036, NAC042, NAC096, AINTGUMENTA-LIKE 5 (AIL5), SCR, and HAT22 TFs

(Table 5-8). Roots collected from symptomatic VAL/SW combination, also, showed

upregulation of amino acid and mitochondrial transmembrane carrier genes, and Nod-

like 26 and plasma membrane major intrinsic protein (MIP) genes that are involved in

water transport.

Discussion

Routine plant growth and defense trade-off is a matter of energy allocation.

Plants generally do well in distributing energy under stress conditions. However, the

burden of stress may disrupt the energy distribution balance between routine growth

and defense. The complexity of CaLas-infection in citrus possibly leads to the

imbalanced distribution of the energy resources in susceptible citrus scion/rootstock

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combinations. In previous chapters, HLB-induced differentially regulated hormonal

response genes and defense associated genes were discussed. This chapter discusses

the qualitative and quantitative differential changes growth and development -

associated genes in HLB asymptomatic and symptomatic VAL/CAN and VAL/SW

combinations.

HLB affected plants exhibit damaged vascular system which is caused by callose

depositions and starch accumulation. The asymptomatic VAL/CAN leaves showed

upregulated genes encoding TFs that are involved in phloem regeneration. They were

KANADI and LBD2. Upregulation of transcripts encoding KANADI TF also reported in

girdled Populus in the regenerating sieve elements (Zhang et al., 2011b; Hussey et al.,

2013; Chen et al., 2014). Therefore, a significant increase in the KANADI encoded

transcripts suggests that VAL/CAN plants possibly has increased phloem regeneration

capacity in the asymptomatic CaLas-infected plants. Circadian rhythms maintain the

plant biological clock. Transcription factors LHY, and REV1 are involved in regulating

circadian clock regulation (Schaffer et al., 1998; Rawat et al., 2009). Transcriptomics

studies of CaLas-infected citrus varieties reported a reduction in the LHY transcripts

expression at the late stage of infection in sweet orange (Fu et al., 2016), Ponkan

(Zhong et al., 2016), and Rough lemon (Fan et al., 2012). The growth regulating factor

(GRF) and growth interacting factor (GIF) duo have a key role in plant organogenesis

(Kim and Tsukaya, 2015). Of these, GRF7 is a negative regulator of osmotic stress-

responsive genes that generally inhibit plant growth. The role of GRF7 has been studied

in ABA-induced osmotic stress. ABA-induced drought stress upregulates dehydration-

responsive element binding protein 2A (DREB2A), which increases tolerance to osmotic

199

stress and retards plant growth. GRF7 binds to the DREB2A promoter region and

therby, downregulates DREB2A induced growth and reproduction inhibition under

normal environmental conditions (Kim et al., 2012). Upregulation of GRF7 transcripts in

the leaves of asymptomatic VAL/CAN suggests the stimulation of growth in the

asymptomatic VAL/CAN leaves. In asymptomatic treatment, VAL/SW leaves

upregulated ABA biosynthesis genes, as discussed in the results presented in Chapter

4. This indicates the drought-like or abiotic stress conditions created in the CaLas-

infected plants that increase expression of dehydration related and dehydration

adaptation associated genes. Upregulation of transcripts encoding KANADI, LHY,

GRF7 in the asymptomatic and REV1 in the asymptomatic leaves of VAL/CAN

combinations highlights the CAN and SW rootstock induced growth changes in the

HLB-affected VAL scion.

In both symptomatic and asymptomatic treatments, VAL/CAN leaves showed

significant upregulation of flavonoid biosynthesis, anthocyanin production, PMEs, and

XTH33 transcripts. The role of PME (Pelloux et al., 2007) and XTH (Divol et al., 2007)

has been well studied in plant-herbivore interactions. PMEs are, also, actively involved

in the cell wall modification and expansions during plant growth (Pelletier et al., 2014).

Vascular-related NAC domain 1 VND1 is involved in cell wall biosynthesis (Zhou et al.,

2014), and this gene was upregulated only in the symptomatic VAL/CAN combination.

Upregulation of cell wall modification related genes in both treatments of VAL/CAN

leaves as compare to VAL/SW leaves, suggests the potential involvement of a cell wall

modification mechanism to defend the CaLas-infection and Psyllid-induced wounding in

the VAL/CAN combination.

200

Ethylene induced-AP2 TFs and NAC domain containing proteins are implicated

in plant growth and stress response, and genes encoding these proteins were

upregulated in the asymptomatic VAL/SW leaf combination. NAC domain proteins are

implicated in plant senescence, stress, and cell wall biosynthesis (Wang et al., 2011;

Nuruzzaman et al., 2013; Podzimska-Sroka et al., 2015). NAC090 transcripts were

highly upregulated in the asymptomatic VAL/SW leaves. Although the NAC090 specific

biological function has not yet been studied, overexpression of transcripts encoding ET-

activated TFs and NAC genes supports the activation of the biotic stress response, leaf

senescence and cell wall modifications in both the asymptomatic and symptomatic

VAL/SW leaves.

Hormone-induced, growth-associated genes were differentially expressed in

VAL/CAN, and VAL/SW leaves. Plant growth hormones BR, CT and AU signaling

pathways were strongly stimulated in the asymptomatic VAL by CAN rootstock in CaLas

infected plants. AU and BR have been reported to work synergistically in plant

developmental processes such as stem elongation and expansion (Nemhauser et al.,

2004). Although AUs are involved in the plant susceptibility to the disease, they are

indispensable plant growth hormones which promote plant development through their

responsive genes. AU promotes plant growth through their ARF responsive genes

(Liscum and Reed, 2002; Li et al., 2016). Also, interdependency of BR and AU signaling

operates via ARFs, as reported in A. thaliana (Nemhauser et al., 2004). The

upregulated ARF genes identified in this study, ARF8 and ARF16, are implied in plant

parthenocarpy (Goetz et al., 2007) and control of seed germination (Liu et al., 2013).

Upregulation of ARF8 was also reported in the regenerating phloem in girdled Populus

201

(Zhang et al., 2011b). Therefore, despite the involvement of ARF8 in the fruit set, a

significant overexpression of the ARF8 in the symptomatic and asymptomatic VAL/CAN

leaves suggests a possible role in phloem regeneration specific to the CAN rootstock. In

HLB -asymptomatic and -symptomatic VAL/SW combinations, SW rootstock strongly

upregulated ABA, AU, and ET-regulated genes expression. However, the

overexpression of ET and ABA along with AU biosynthesis genes appeared to be the

negative effect of CaLas infection interaction rather than a benefit to plant development.

A key factor that severely reduces the sustainability of CaLas-infected plants is

the deficiency of essential micronutrients (Schumann and Spann, 2009), and

imbalanced sink-source partitioning (Fan et al., 2010). Leaves of HLB-affected citrus

plants have shown an increase in the K while Ca, B, Fe, Zn, Mn, and Mg are reduced

significantly (Schumann and Spann, 2009). The same deficiencies are even greater in

roots of infected trees (J.W. Grosser, personal communication). Zn deficiency-like

symptoms are typical of the HLB-affected plants. Plants generally increase the uptake

and the transporter’s activity of deficient nutrients. The overexpression of ZIP5

precursor was reported in the HLB-susceptible Cleopatra rootstock (Albrecht and

Bowman, 2012b). The increased expression of Zn efflux transporter ZIP1 and ZIP4

genes in the symptomatic VAL/SW combination suggests that plant experienced Zn

deficiency. Therefore, to obtain Zn either from soil or storage organelles, ZIP transporter

genes expression was enhanced. The role of ZIP family proteins in the Zn homeostasis

is explained in many plant nutrients studies (Sinclair and Krämer, 2012; Assunção et al.,

2013; Bashir et al., 2016). Cu transporter genes such as HMA1 and COPT1 were also

overexpressed in the leaves collected from the symptomatic VAL/SW. The Cu

202

transporters are present on the chloroplast envelope and play a significant role in

photosynthetic electron transport (Grotz and Guerinot, 2006). Increased expression of

chloroplast localized metal transporter transcripts suggests enhanced chloroplast

activity in the symptomatic VAL/SW leaves. Phosphorus deficiency is also prominent in

HLB-affected plants, and occurs because of the interference of small RNAs (Zhao et al.,

2013). Phosphorus starvation-induced P transporters PHT 2;1 (asymptomatic and

symptomatic leaves) and PHT1;3 and PHT1;7 (symptomatic roots) transcripts were

upregulated in the VAL/CAN as compared to VAL/SW in the respective treatments and

tissues. Whereas, VAL/SW combination showed upregulation of PHT1;4 and PHT1;9 in

asymptomatic leaves as compared to asymptomatic VAL/CAN leaves.

Excessive starch biosynthesis is a signature of HLB-affected plants (Etxeberria et

al., 2009). Enhanced production of transcripts encoding starch biosynthesis AGPase,

starch branching SBE2.1 and SBE2.2, starch excess 1 (SEX1) was seen in the

symptomatic VAL/SW leaves as compared to symptomatic VAL/CAN leaves. However,

starch degrading enzyme and invertase genes upregulation was observed only in the

asymptomatic VAL/SW leaves. The pattern of starch biosynthesis and catabolism gene

transcripts in the VAL/SW combination supports the typical behavior of the HLB-

susceptible cultivars. Trehalose is a common sugar in lower organisms such as

bacteria, fungi, and invertebrates, but also accumulates in plants when they are infected

with micro-organisms. The role of trehalose in the plant is still uncertain. However, it is

predicted that trehalose may interfere with sugar sensing and plant development (Müller

et al., 1999). Overexpression of trehalose synthesis genes in the asymptomatic

VAL/SW combination suggests a higher CaLas activity in the infected rootstock. While

203

the asymptomatic VAL/CAN combination showed upregulation of genes encoding

starch degrading enzymes, vacuolar invertases, and raffinose synthesis and MIPS

biosynthesis proteins highlighted the genetic response of CAN rootstock to avoid starch

accumulations and utilization of other sugar resources.

Roots obtained from the asymptomatic treatment of VAL/CAN and VAL/SW

showed very few differentially expressed genes. Among these, strong upregulation of

genes associated with the light reaction system of chloroplasts was observed in the

asymptomatic VAL/SW roots. In contrast, many genes were differentially expressed in

the roots collected from the symptomatic treatment of the VAL/CAN and VAL/SW. At

the advanced stage of infection, CAN roots showed significant upregulation of BR and

AU response genes. Whereas, SW roots were found to enhance the expression of

genes that increase lateral root spread. These genes were AU efflux carrier PIN4,

SHORT ROOTS (SHR), SCR, and WUSCHEL related WOX4. HLB-affected SW

rootstock show decreased root growth. Increased expression of lateral root growth

associated genes suggests the SW is promoting root growth to reach out to the nutrient

resources available in the soil. Higher expression of the HD-zip II family HAT22 gene in

the SW roots supports the theory of reduced root growth and the ABA-dependent

drought response in the HLB affected plants (Huang et al., 2008).

Roots collected from the symptomatic treatment of VAL/CAN combination

showed upregulated genes encoding phloem regenerating KANADI TF that was also

upregulated in the leaves of the VAL/CAN. Upregulation of KANADI in the roots and

leaves of the VAL/CAN combination indicates phloem regeneration ability is specific to

the putative HLB-tolerant CAN rootstock. Phytosulfokine-α is a signaling peptide and

204

acts as a root growth factor in Arabidopsis suspension cell culture (Kutschmar et al.,

2009). In the absence of any other significant root promoting factors, phytosulfokines

precursor 3 and 4 may support enhanced root growth in the CaLas-infected CAN

rootstock. Nitrate and sulfur transporters, major intrinsic protein, and nodulin-like NIP

transporter transcripts were downregulated in the CaLas-infected roots of SW.

However, the transcripts encoding nitrate transporters, sulfate transmembrane

transporters, calcium exchanger (CAX), water and sugar transporter activities were

upregulated in roots of the symptomatic VAL/CAN combination. In addition, transcripts

for a mitochondrial carrier BOU that is involved in plant meristem growth and a

mitochondrial substrate carrier were also upregulated in the roots of the symptomatic

VAL/CAN combination, suggesting upregulated mitochondrial activity in the CaLas-

infected VAL/CAN plants. The upregulation of N, S, Ca, sugar, and water transporter

transcripts in the CAN rootstock as compared to the SW rootstock suggests a possible

increased root growth of the CAN rootstock under CaLas-infection. Upregulation of

transition metal transporter genes in the symptomatic VAL/CAN roots suggests that the

infected plant is trying to achieve homeostasis by effluxing nutrients from the storage

organelles (Hall and Williams, 2003; Krämer et al., 2007).

Plants under stress often exhibit compromised growth. Optimizing the balance

and allocation of energy between routine growth and defense is critical when the plant is

consistently under stress. CaLas-infected citrus plants exhibit a similar scenario, and it

appears that the susceptible SW rootstock partitions too much energy to defense at the

expense of energy needed for adequate nutrition management. Use of HLB-tolerant

rootstock cultivars and optimal nutrition management may be the key to prolonging the

205

economic viability of CaLas-infected plants. The study of comparative transcriptomic

analysis between VAL/CAN and VAL/SW in symptomatic and asymptomatic treatments

showed that rootstock has potential to reprogram the scion transcriptome at different

stages of HLB disease development. The changes in the scion are not only manipulated

based on rootstock specific genetics, but also the stage of HLB disease development in

the scion. The results of this study suggest that enhanced VAL performance in the

infected plants is probably due to a combination of phloem regeneration ability, nutrient

homeostasis, and BR-AU dependent plant development induced by the putative HLB-

tolerant CAN rootstock.

206

Table 5-1. Experimental treatments and scion/rootstock combinations

Rootstock Rootstock Parents Scion Treatments based on visual observations of HLB symptoms

Swingle; 2n (SW)



Symptomatic VAL/SW

Asymptomatic VAL/SW

putative HLB-tolerant candidate; 2n (CAN)



Slightly Symptomatic VAL/CAN

Asymptomatic VAL/CAN



Leaves Roots





207

Table 5-3. Differentially expressed growth and development -associated genes significantly upregulated in leaves of asymptomatic VAL/CAN combination as compared to asymptomatic VAL/SW leaves C. Clementina_ID Arabi ID Gene name Bin Name† Log2 FC Arabidopsis_define

Ciclev10000557m.g AT2G13610 T10F5.15 transport.ABC transporters and multidrugresistance systems

3.62462 ABC-2 type transporter family protein

Ciclev10031051m.g AT2G40460 T2P4.19 transport.peptides and oligopeptides 3.25629 Major facilitator superfamily protein

Ciclev10018964m.g AT1G01060 LHY RNA. regulation of transcription. MYB-related transcription factor family

2.81304 Homeodomain-like superfamily protein

Ciclev10005222m.g AT5G23730 MRO11.23 development.unspecified 2.4234 Transducin/WD40 repeat-like superfamily protein

Ciclev10033853m.g AT5G06710 HAT14 development. unspecified 2.30573 homeobox from Arabidopsis thaliana

Ciclev10019808m.g AT1G64780 AMT1;2 transport. ammonium 2.24333 ammonium transporter 1;2

Ciclev10016510m.g AT3G14770 AT3G14770 development. unspecified 2.08371 Nodulin MtN3 family protein

Ciclev10001405m.g AT5G13930 TT4 secondary metabolism. flavonoids. chalcones.naringenin-chalcone synthase

2.03082 Chalcone and stilbene synthase family protein

Ciclev10025400m.g AT2G22240 MIPS2 minor CHO metabolism.myo-inositol.InsP Synthases

1.94841 myo-inositol-1-phosphate synthase 2

Ciclev10001292m.g AT1G32240 KAN2 RNA. regulation of transcription. G2-like transcription factor family, GARP


Ciclev10020116m.g AT2G43330 INT1 transporter. sugars 1.78643 inositol transporter 1

Ciclev10002276m.g AT1G21460 F24J8.9 development. unspecified 1.74987 Nodulin MtN3 family protein

Ciclev10021834m.g AT2G45190 AFO development. unspecified 1.74896 Plant-specific transcription factor YABBY family protein

Ciclev10000639m.g AT1G59740 F23H11.6 transport. peptides and oligopeptides 1.74264 Major facilitator superfamily protein

Ciclev10007412m.g AT2G26650 AKT1 transport. potassium 1.7288 K+ transporter 1

Ciclev10031484m.g AT3G29670 AT3G29670

secondary metabolism.flavonoids. anthocyanins. anthocyanin 5-aromatic acyltransferase


Ciclev10031201m.g AT1G24100 UGT74B1 secondary metabolism.sulfur-containing.glucosinolates.synthesis.

1.6869 UDP-glucosyl transferase 74B1

Ciclev10014707m.g AT1G61680 TPS14 secondary metabolism. isoprenoids.terpenoids

1.57168 terpene synthase 14

Ciclev10008549m.g AT1G02630 T14P4.9 transport.unspecified cations 1.54003 Nucleoside transporter family protein

Ciclev10025197m.g AT4G36710 AP22.56 development. unspecified 1.50117 GRAS family transcription factor

Ciclev10002627m.g AT1G78020 F28K19.24 development. 1.50002 Protein of unknown function (DUF581)

208



Bin Name Log2 FC

Arabidopsis_definition

Ciclev10015713m.g AT3G12750 ZIP1 transport.metal 1.44897 zinc transporter 1 precursor

Ciclev10012022m.g AT3G02380 COL2 development.unspecified 1.41699 CONSTANS-like 2

Ciclev10011184m.g AT5G15410 DND1 transport.cyclic nucleotide or calcium regulated channels

1.41698 Cyclic nucleotide-regulated ion channel family protein

Ciclev10001346m.g AT2G02450 NAC035 development.unspecified 1.39801 NAC domain containing protein 35

Ciclev10000054m.g AT1G27940 PGP13 transport.ABC transporters and multidrugresistance systems

1.34906 P-glycoprotein 13

Ciclev10025364m.g AT4G36920 AP2 development.unspecified 1.33959 Integrase-type DNA-binding superfamily protein

Ciclev10019134m.g AT1G12240 BETA-FRUCT4

major CHO metabolism. degradation.sucrose.invertases.vacuolar


Ciclev10000788m.g AT2G30300 T9D9.11 development.unspecified 1.31886 Major facilitator superfamily protein

Ciclev10023499m.g AT4G01840 KCO5 transport.potassium 1.30922 Ca2+ activated outward rectifying K+ channel 5

Ciclev10019412m.g AT1G12110 NRT1.1 transport.nitrate 1.29906 nitrate transporter 1.1

Ciclev10011188m.g AT2G28260 CNGC15 transport.cyclic nucleotide or calcium regulated channels

1.29503 cyclic nucleotide-gated channel 15

Ciclev10020894m.g AT1G18590 SOT17 secondary metabolism. sulfur-containing. glucosinolates.

1.25659 sulfotransferase 17

Ciclev10012382m.g AT5G37820 NIP4;2 transport.Major Intrinsic Proteins.unspecified 1.24622 NOD26-like intrinsic protein 4;2

Ciclev10029283m.g AT2G28500 LBD11 RNA.regulation of transcription. AS2, Lateral Organ Boundaries Gene Family

1.23564 LOB domain-containing protein 11

Ciclev10019450m.g AT1G12480 OZS1 transport. metabolite transporters at the mitochondrial membrane

1.20934 C4-dicarboxylate transporter/malic acid transport protein

Ciclev10006697m.g AT1G79360 OCT transport.misc 1.2087 organic cation/carnitine transporter 2

Ciclev10011167m.g AT2G37360 F3G5.15 transport. ABC transporters and multidrug resistance systems


Ciclev10008176m.g AT5G55180 MCO15.13 misc.beta 1,3 glucan hydrolases 1.19447 O-Glycosyl hydrolases family 17 protein

Ciclev10011175m.g AT2G37040 PAL1 secondary metabolism. phenylpropanoids. lignin biosynthesis.PAL


Ciclev10003356m.g AT1G10960 FD1 PS.lightreaction.other electron carrier (ox/red).ferredoxin

1.162 ferredoxin 1

Ciclev10032524m.g AT2G40610 EXPA8 cell wall. modification 1.14952 expansin A8

209

Table 5-3. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition

Ciclev10014769m.g AT5G12860 DiT1 transport.metabolite transporters at the mitochondrial membrane

1.14632 dicarboxylate transporter 1

Ciclev10010626m.g AT3G29590 AT5MAT secondary metabolism.flavonoids.anthocyanins. anthocyanin 5-aromatic acyltransferase


Ciclev10011857m.g AT3G58060 AT3G58060 transport.metal 1.13998 Cation efflux family protein

Ciclev10018412m.g AT3G13620 AT3G13620 transport.amino acids 1.12853 Amino acid permease family protein

Ciclev10028975m.g AT5G60660 PIP2;4 transport.Major Intrinsic Proteins.PIP 1.11673 plasma membrane intrinsic protein 2;4

Ciclev10025931m.g AT3G51240 F3H secondary metabolism.flavonoids. dihydroflavonols. flavanone 3-dioxygenase

1.11237 flavanone 3-hydroxylase

Ciclev10019927m.g AT4G01070 GT72B1 secondary metabolism.flavonoids.dihydroflavonols


Ciclev10032164m.g AT1G10550 XTH33 cell wall.modification 1.0937 xyloglucan:xyloglucosyl transferase 33

Ciclev10030988m.g AT2G39210 T16B24.15 development. unspecified 1.08131 Major facilitator superfamily protein


Ciclev10026013m.g AT4G40060 HB16 RNA.regulation of transcription.HB,Homeobox transcription factor family

1.06148 homeobox protein 16

Ciclev10001422m.g AT4G24220 VEP1 development.unspecified 1.0546 NAD(P)-binding Rossmann-fold superfamily protein

Ciclev10031329m.g AT1G68530 KCS6 secondary metabolism.wax 1.05234 3-ketoacyl-CoA synthase 6

Ciclev10007993m.g AT4G33220 PME44 cell wall.pectin*esterases.misc 1.05144 pectin methylesterase 44

Ciclev10008106m.g AT5G54860 MBG8.12 transport. misc 1.01028 Major facilitator superfamily protein

†Classification of the measured parameter into a set a functional category in the MapMan analysis tool.

210

Table 5-4. Differentially expressed growth and development-associated genes significantly upregulated in leaves of asymptomatic VAL/SW combination as compared to asymptomatic VAL/CAN leaves C. Clementina_ID Arabi ID Gene name Bin Name† Log2 FC* Arabidopsis_define

Ciclev10001337m.g AT2G26560 PLA2A development. storage proteins -6.06084 phospholipase A 2A

Ciclev10007068m.g AT5G51990 CBF4 RNA. regulation of transcription. AP2/EREBP/Ethylene-responsive element

-4.42861 C-repeat-binding factor 4

Ciclev10031823m.g AT1G12060 BAG5 development.unspecified -4.38215 BCL-2-associated athanogene 5

Ciclev10025429m.g AT4G31500 CYP83B1 secondary metabolism. glucosinolates. synthesis. shared. CYP83B1


Ciclev10008936m.g AT4G38460 GGR secondary metabolism. geranylgeranyl pyrophosphate synthase

-3.97354 geranylgeranyl reductase

Ciclev10015210m.g AT1G61800 GPT2 transport. metabolite transporters at the envelope membrane

-3.77132 glucose-6-phosphate/phosphate translocator 2

Ciclev10029032m.g AT5G22380 NAC090 development. unspecified -3.57421 NAC domain containing protein 90

Ciclev10026710m.g AT1G53885 AT1G53903 development. unspecified -3.54895 Protein of unknown function (DUF581)

Ciclev10004474m.g AT3G06500 F5E6.17 major CHO metabolism degradation.sucrose.invertases.neutral

-3.51543 Plant neutral invertase family protein

Ciclev10020033m.g AT5G41330 MYC6.4 transport. potassium -3.29693 BTB/POZ domain with WD40/YVTN repeat-like protein

Ciclev10010340m.g AT2G46330 AGP16 cell wall.cell wall proteins.AGPs -3.194 arabinogalactan protein 16

Ciclev10025259m.g AT3G13790 ATBFRUCT1 major CHO metabolism.degradation. sucrose.invertases.cell wall

-2.93558 Glycosyl hydrolases family 32 protein

Ciclev10032304m.g AT1G69490 NAP development. unspecified -2.9254 NAC-like, activated by AP3/PI

Ciclev10029146m.g AT1G07050 F10K1.24 development. unspecified -2.90762 CCT motif family protein

Ciclev10016832m.g AT1G61340 T1F9.17 development. unspecified -2.87894 F-box family protein

Ciclev10016434m.g AT5G14000 NAC084 Development. unspecified -2.82346 NAC domain containing protein 84

Ciclev10005127m.g AT5G19120 T24G5.20 development. storage proteins -2.7325 Eukaryotic aspartyl protease family protein

Ciclev10012978m.g AT5G06760 LEA4-5 development. late embryogenesis abundant

-2.65074 Late Embryogenesis Abundant 4-5

Ciclev10011765m.g AT3G28960 AT3G28960 transport. amino acids -2.51719 Transmembrane amino acid transporter family protein

Ciclev10030691m.g AT1G23870 TPS9 minor CHO metabolism. trehalose. potential TPS/TPP

-2.3844 trehalose-phosphatase/synthase 9

211



Bin Name Log2 FC Arabidopsis_definition

Ciclev10001976m.g AT1G01720 ATAF1 development.unspecified -2.29197 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein

Ciclev10022068m.g AT3G02550 LBD41 RNA.regulation of transcription.AS2,Lateral Organ Boundaries Gene Family

-2.26832 LOB domain-containing protein 41

Ciclev10008778m.g AT4G25810 XTR6 cell wall.modification -2.19406 xyloglucan endotransglycosylase 6

Ciclev10008812m.g AT4G27410 RD26 development.unspecified -2.18013 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein

Ciclev10011715m.g AT5G67150 K21H1.11 secondary metabolism -2.15822 HXXXD-type acyl-transferase family protein

Ciclev10021027m.g AT2G47180 GolS1 minor CHO metabolism.raffinose family.galactinol synthases.known

-2.08193 galactinol synthase 1


Ciclev10025170m.g AT1G80300 NTT1 transport.misc -1.97371 nucleotide transporter 1

Ciclev10005375m.g AT5G24520 TTG1 development.unspecified -1.92709 Transducin/WD40 repeat-like protein

Ciclev10015213m.g AT5G40780 LHT1 transport.amino acids -1.92289 lysine histidine transporter 1

Ciclev10010901m.g AT2G36380 PDR6 transport.ABC transporters and multidrugresistance systems

-1.91774 pleiotropic drug resistance 6

Ciclev10024949m.g AT4G15560 CLA1 secondary metabolism.isoprenoids. non-mevalonate Pathway.DXS

-1.91766 Deoxyxylulose-5-phosphate synthase

Ciclev10001513m.g AT1G35910 F10O5.8 minor CHO metabolism.trehalose. -1.88035 Haloacid dehalogenase-like hydrolase (HAD) superfamily protein

Ciclev10031846m.g AT1G68840 RAV2

RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive element binding protein family

-1.86528 related to ABI3/VP1 2

Ciclev10009681m.g AT4G31290 F8F16.110 transport.unspecified cations -1.86316 ChaC-like family protein

Ciclev10000764m.g AT1G20510 OPCL1 secondary metabolism.phenylpropanoids


Ciclev10012678m.g AT2G35980 YLS9 development.unspecified -1.80769 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Ciclev10000098m.g AT3G03050 CSLD3 cell wall.cellulose synthesis.cellulose synthase

-1.8039 cellulose synthase-like D3

Ciclev10025933m.g AT4G37990 ELI3-2 secondary metabolism.phenylpropanoids.lignin biosynthesis.CAD


212


Ciclev10001398m.g AT2G18950 HPT1 secondary metabolism.isoprenoids. tocopherol biosynthesis.


Ciclev10000437m.g AT4G07960 CSLC12 cell wall.cellulose synthesis -1.69397 Cellulose-synthase-like C12

Ciclev10004839m.g AT5G18840 F17K4.90 transporter.sugars -1.66238 Major facilitator superfamily protein


Ciclev10008865m.g AT1G35190 T32G9.27 secondary metabolism.N misc.alkaloid-like -1.58563 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily protein

Ciclev10001869m.g AT3G05290 PNC1 transport.metabolite transporters at the mitochondrial membrane

-1.58107 peroxisomal adenine nucleotide carrier 1

Ciclev10012227m.g AT2G36950 T1J8.13 transport.misc -1.52815 Heavy metal transport/detoxification superfamily protein

Ciclev10027848m.g AT1G07530 SCL14 RNA.regulation of transcription. GRAS transcription factor family

-1.5275 SCARECROW-like 14

Ciclev10032519m.g AT5G13790 AGL15 development.unspecified -1.42152 AGAMOUS-like 15

Ciclev10028637m.g AT2G27500 F10A12.18 misc.beta 1,3 glucan hydrolases.glucan endo-1,3-beta-glucosidase

-1.4192 Glycosyl hydrolase superfamily protein

Ciclev10004592m.g AT5G46050 PTR3 transport.peptides and oligopeptides -1.41478 peptide transporter 3

Ciclev10028125m.g AT4G34950 AT4G34950 development.unspecified -1.39279 Major facilitator superfamily protein

Ciclev10026128m.g AT2G22500 UCP5 transport.metabolite transporters at the mitochondrial membrane

-1.38581 uncoupling protein 5

Ciclev10014248m.g AT1G06410 TPS7 minor CHO metabolism.trehalose.potential TPS/TPP

-1.34744 trehalose-phosphatase/synthase 7

Ciclev10010508m.g AT1G01470 LEA14 development.late embryogenesis abundant -1.33518 Late embryogenesis abundant protein

Ciclev10000819m.g AT1G76430 PHT1;9 transport.phosphate -1.32555 phosphate transporter 1;9

Ciclev10000991m.g AT1G77380 AAP3 transport.amino acids -1.31739 amino acid permease 3

Ciclev10009180m.g AT2G23810 TET8 development. unspecified -1.29853 tetraspanin8

Ciclev10005151m.g AT1G64760 F13O11.7 Misc. beta 1,3 glucan hydrolases. glucan endo-1,3-beta-glucosidase

-1.27931 O-Glycosyl hydrolases family 17 protein

Ciclev10011989m.g AT5G61430 NAC100 development. unspecified -1.27176 NAC domain containing protein 100

Ciclev10011458m.g AT4G08250 T12G13.90 development. unspecified -1.27045 GRAS family transcription factor

Ciclev10033125m.g AT1G13980 GN development.unspecified -1.23772 sec7 domain-containing protein

Ciclev10015168m.g AT1G03940 F21M11.13 secondary metabolism.flavonoids. anthocyanins.


213

Table 5-4. Continued C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition

Ciclev10015356m.g AT5G36160 MAB16.11 secondary metabolism.isoprenoids.tocopherol biosynthesis

-1.19921 Tyrosine transaminase family protein

Ciclev10014811m.g AT4G17230 SCL13 development.unspecified -1.19414 SCARECROW-like 13

Ciclev10005251m.g AT5G54160 OMT1 secondary metabolism.phenylpropanoids.lignin biosynthesis.COMT


Ciclev10019368m.g AT3G49530 NAC062 development.unspecified -1.18215 NAC domain containing protein 62

Ciclev10004729m.g AT5G26340 MSS1 transporter.sugars -1.18122 Major facilitator superfamily protein

Ciclev10008010m.g AT3G18080 BGLU44 misc.gluco-, galacto- and mannosidases -1.15326 B-S glucosidase 44

Ciclev10030771m.g AT1G78950 AT1G78950 secondary metabolism.isoprenoids.terpenoids -1.13938 Terpenoid cyclases family protein

Ciclev10024701m.g AT1G59870 PEN3 transport.ABC transporters and multidrugresistance systems

-1.13472 ABC-2 and Plant PDR ABC-type transporter family protein

Ciclev10007531m.g AT4G26140 BGAL12 misc.gluco-, galacto- and mannosidases.beta-galactosidase

-1.1213 beta-galactosidase 12

Ciclev10017858m.g AT1G29690 CAD1 development.unspecified -1.10825 MAC/Perforin domain-containing protein

Ciclev10030499m.g AT1G10760 SEX1 major CHO metabolism.degradation.starch.glucan water dikinase

-1.06235 Pyruvate phosphate dikinase, PEP/pyruvate binding domain

Ciclev10024781m.g AT3G21250 MRP6 transport.ABC transporters and multidrug resistance systems

-1.04104 multidrug resistance-associated protein 6

Ciclev10016017m.g AT1G07640 OBP2 secondary metabolism. sulfur-containing.glucosinolates.regulation.indole

-1.0399 Dof-type zinc finger DNA-binding family protein

Ciclev10028621m.g AT1G08200 AXS2 cell wall. precursor synthesis.AXS -1.03176 UDP-D-apiose/UDP-D-xylose synthase 2

Ciclev10007428m.g AT2G18700 TPS11 minor CHO metabolism. trehalose.potential TPS/TPP

-1.01948 trehalose phosphatase/synthase 11


214

Table 5-5. Differentially expressed growth and development -associated genes significantly upregulated in leaves of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW leaves

C. Clementina_ID Arabi ID Name Bin Name† Log2 FC


Ciclev10018964m.g AT1G01060 LHY RNA. regulation of transcription. MYB-related transcription factor family


Ciclev10025400m.g AT2G22240 MIPS2 minor CHO metabolism.myo-inositol.Insp Synthases

2.32245 myo-inositol-1-phosphate synthase 2

Ciclev10000557m.g AT2G13610 T10F5.15 transport.ABC transporters and multidrugresistance systems


Ciclev10032927m.g AT2G03710 SEP-4 RNA.regulation of transcription.MADS box transcription factor family

2.11262 K-box region and MADS-box transcription factor family protein

Ciclev10012453m.g AT1G73830 BEE3 RNA.regulation of transcription. bHLH,Basic Helix-Loop-Helix family

2.10314 BR enhanced expression 3

Ciclev10022564m.g AT4G35580 NTL9 development. 1.94511 NAC transcription factor-like 9

Ciclev10012022m.g AT3G02380 COL2 development. 1.9157 CONSTANS-like 2

Ciclev10011175m.g AT2G37040 PAL1 secondary metabolism.phenylpropanoids.liGnin biosynthesis.PAL


Ciclev10023918m.g AT4G11080 T22B4.60 RNA.regulation of transcription.HiGh mobility Group (HMG) family

1.82442 HMG (high mobility group) box protein

Ciclev10029351m.g AT1G24625 ZFP7 RNA.regulation of transcription.C2H2 zinc finger family

1.82312 zinc finger protein 7

Ciclev10031051m.g AT2G40460 T2P4.19 transport.peptides and oligopeptides 1.80227 Major facilitator superfamily protein

Ciclev10002208m.g AT2G22800 HAT9 RNA.regulation of transcription.HB,Homeobox transcription factor family

1.72417 Homeobox-leucine zipper protein family

Ciclev10005365m.g AT1G04770 F13M7.24 development. 1.68141 Tetratricopeptide repeat (TPR)-like superfamily protein

Ciclev10014888m.g AT1G23200 F26F24.2 cell wall.pectin*esterases.misc 1.68029 Plant invertase/pectin methylesterase inhibitor superfamily

Ciclev10012332m.g AT5G46800 BOU transport.metabolite transporters AT the mitochondrial membrane

1.67303 Mitochondrial substrate carrier family protein

Ciclev10014887m.g AT1G61820 BGLU46 misc.Gluco-, Galacto- and mannosidases 1.66298 beta glucosidase 46

Ciclev10026114m.g AT2G18060 VND1 development. 1.65703 vascular related NAC-domain protein 1

Ciclev10021740m.g AT2G44940 T13E15.25 1.57875 Integrase-type DNA-binding superfamily protein

215


C. Clementina_ID Arabi ID Name Bin Name Log2 FC


Ciclev10018481m.g AT3G60160 MRP9 transport. ABC transporters and multidrug resistance systems

1.57287 multidrug resistance-associated protein 9

Ciclev10029158m.g AT4G34050 CCoAOMT1 secondary metabolism.Phenylpropanoids lignin biosynthesis. CCoAOMT

1.57178 S-adenosyl-L-methionine-dependent methyltransferases superfamily

Ciclev10032943m.g AT5G13080 WRKY75 RNA.regulation of transcription. WRKY domain transcription factorfamily

1.57081 WRKY DNA-binding protein 75

Ciclev10026193m.g AT1G16070 TLP8 RNA.regulation of transcription.TUB transcription factor family

1.52709 tubby like protein 8

Ciclev10019532m.g AT1G65060 4CL3 secondary metabolism.lignin biosynthesis.4CL

1.52516 4-coumarate:CoA ligase 3

Ciclev10016123m.g AT5G13870 XTH5 cell wall.modification 1.5178 xyloglucan endotransglucosylase/hydrolase 5

Ciclev10007850m.g AT2G26690 F18A8.6 transport. nitrate 1.5111 Major facilitator superfamily protein

Ciclev10020573m.g AT1G17140 ICR1 development. 1.5097 interactor of constitutive active rops 1

Ciclev10018574m.g AT1G02730 CSLD5 cell wall.cellulose synthesis. 1.48285 cellulose synthase-like D5

Ciclev10001467m.g AT4G19420 T5K18.200 cell wall.pectin*esterases.acetyl esterase 1.46834 Pectinacetylesterase family protein

Ciclev10007412m.g AT2G26650 AKT1 transport.potassium 1.4549 K+ transporter 1

Ciclev10029283m.g AT2G28500 LBD11 RNA.regulation of transcription. AS2,Lateral Organ Boundaries Gene Family

1.41069 LOB domain-containing protein 11

Ciclev10019828m.g

AT5G53660

GRF7

1.38033

growth-regulating factor 7

Ciclev10029663m.g AT2G21650 MEE3 RNA.regulation of transcription. MYB-related transcription factor family


Ciclev10028651m.g AT3G19450 CAD4 secondary metabolism. phenylpropanoids. lignin biosynthesis.CAD

1.37762 GroES-like zinc-binding alcohol dehydrogenase family protein

Ciclev10025280m.g AT3G21240 4CL2 secondary metabolism .phenylpropanoids. lignin biosynthesis.4CL

1.3604 4-coumarate: CoA ligase 2

Ciclev10016587m.g AT5G23420 HMGB6 RNA.regulation of transcription Nucleosome/chromatin assembly group

1.35371 high-mobility group box 6


1.34562 pleiotropic drug resistance 4

Ciclev10028106m.g AT3G61430 PIP1A transport.Major Intrinsic Proteins. 1.3356 plasma membrane intrinsic protein 1A

Ciclev10026400m.g AT3G15540 IAA19 RNA.regulation Aux/IAA family 1.327 indole-3-acetic acid inducible 19

216




Ciclev10001335m.g AT5G07050 MOJ9.22 development. 1.30628 nodulin MtN21 /EamA-like transporter family protein

Ciclev10002351m.g AT4G09650 ATPD PS.lightreaction. ATP synthase. delta chain

1.30371 ATP synthase delta-subunit gene

Ciclev10020116m.g AT2G43330 INT1 transporter. sugars 1.29893 inositol transporter 1

Ciclev10014902m.g AT2G05160 F5G3.6 RNA.regulation of transcription 1.28597 CCCH-type zinc finger family protein with RNA-binding domain

Ciclev10027805m.g AT2G28470 BGAL8 misc.Gluco-, Galacto- and mannosidases. beta-Galactosidase



Ciclev10031284m.g AT5G03760 CSLA09 cell wall. cellulose synthesis 1.25163 Nucleotide-diphospho-sugar transferases superfamily protein

Ciclev10024243m.g AT4G00370 ANTR2 transporter.sugars 1.24987 Major facilitator superfamily protein

Ciclev10027856m.g AT3G19620 AT3G19620 cell wall. degradation. mannan-xylose-arabinose-fucose

1.22686 Glycosyl hydrolase family protein

Ciclev10002793m.g AT1G60950 FED A PS.lightreaction. other electron carrier (ox/red).ferredoxin

1.22329 2Fe-2S ferredoxin-like superfamily protein

Ciclev10005789m.g AT4G14550 IAA14 RNA.regulation of transcription. Aux/IAA family

1.21806 indole-3-acetic acid inducible 14

Ciclev10028271m.g AT2G21050 LAX2 transport. amino acids 1.21711 like AUXIN RESISTANT 2

Ciclev10014586m.g AT2G32540 CSLB04 cell wall. cellulose synthesis. cellulose synthase

1.20872 cellulose synthase-like B4

Ciclev10000171m.g AT1G58370 RXF12 cell wall. degradation. mannan-xylose-arabinose-fucose

1.20747 glycosyl hydrolase family10 protein / carbohydrate-binding domain-containing protein

Ciclev10001618m.g AT4G39330 CAD9 secondary metabolism. phenylpropanoids. lignin biosynthesis. CAD

1.20204 cinnamyl alcohol dehydrogenase 9

Ciclev10015053m.g AT1G30690 T5I8.14 transport. misc 1.19847 Sec14p-like phosphatidylinositol transfer family protein

Ciclev10026558m.g HCT hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase

1.18827 lignin bisynthesis, flavonoid accumulation/auxin tranport

Ciclev10015129m.g AT5G14570 NRT2.7 transport. nitrate 1.16621 high affinity nitrate transporter 2.7

Ciclev10006002m.g AT5G43700 AtAUX2-11

217




Ciclev10019376m.g AT5G53370 PME cell wall.pectin*esterases.misc 1.1444 pectin methylesterase PCR fragment F

Ciclev10033132m.g AT1G51400 F5D21.10 PS. Light reaction. photosystem II.PSII polypeptide subunits

1.14218 Photosystem II 5 kD protein

Ciclev10029551m.g AT2G30570 PSBW PS. Light reaction. photosystem II.PSII polypeptide subunits

1.14013 photosystem II reaction center W

Ciclev10012263m.g AT3G09600 AT3G09610 RNA.regulation of transcription.MYB-related transcription factor family


Ciclev10008010m.g AT3G18080 BGLU44 misc.Gluco-, Galacto- and mannosidases

1.13277 B-S glucosidase 44

Ciclev10020360m.g AT3G61950 AT3G61950 RNA.regulation of transcription.bHLH,Basic Helix-Loop-

1.1307 basic helix-loop-helix (bHLH) DNA-binding superfamily protein

Ciclev10002276m.g AT1G21460 F24J8.9 development. 1.12259 Nodulin MtN3 family protein

Ciclev10014787m.g AT1G11580 PMEPCRA cell wall.pectin*esterases.PME 1.11503 methylesterase PCR A

Ciclev10001578m.g AT1G75880 T4O12.12 development. 1.10425 SGNH hydrolase-type esterase superfamily

Ciclev10025272m.g AT3G18660 PGSIP1 major CHO metabolism.synthesis.starch

1.09889 plant glycogenin-like starch initiation protein 1

Ciclev10000352m.g AT1G78060 F28K19.27 cell wall.degradation. mannan-xylose-arabinose-fucose

1.09628 Glycosyl hydrolase family protein

Ciclev10030439m.g AT2G26500 T9J22.17 PS.lightreaction.cytochrome b6/f 1.09046 cytochrome b6f complex subunit (petM), putative

Ciclev10025455m.g AT5G65380 MNA5.11 development. 1.08679 MATE efflux family protein

Ciclev10029284m.g AT4G38960 F19H22.60 RNA. regulation of transcription.C2C2(Zn) CO-like, Constans-like zinc finger family

1.07817 B-box type zinc finger family protein

Ciclev10005222m.g AT5G23730 MRO11.23 development. 1.07461 Transducin/WD40 repeat-like superfamily protein

Ciclev10011587m.g AT1G66330 T27F4.8 development. 1.07222 senescence-associated family protein

Ciclev10012382m.g AT5G37820 NIP4;2 transport. Major Intrinsic Proteins. 1.06595 NOD26-like intrinsic protein 4;2

Ciclev10015976m.g AT4G28500 NAC073 development. 1.06452 NAC domain containing protein 73

Ciclev10011060m.g AT3G53720 CHX20 transport. metal 1.06451 cation/H+ exchanger 20

Ciclev10031937m.g AT1G69580 F24J1.30 RNA. regulation of transcription. G2-like transcription factor family, GARP


Ciclev10005376m.g AT5G49330 MYB111 RNA.regulation of transcription.MYB domain transcription factor family

1.04245 myb domain protein 111

218


Ciclev10008143m.g AT5G10770 T30N20.40 RNA.regulation of transcription. 1.03541 Eukaryotic aspartyl protease family protein

Ciclev10016510m.g AT3G14770 AT3G14770 development. 1.03147 Nodulin MtN3 family protein

Ciclev10022322m.g AT4G28660 PSB28 PS.liGhtreaction.photosystem II.PSII polypeptide subunits

1.02672 photosystem II reaction center PSB28 protein

Ciclev10014611m.g AT5G14880 T9L3.180 transport.potassium 1.0197 Potassium transporter family protein

Ciclev10005737m.g AT4G15920 DL4000C development. 1.0178 Nodulin MtN3 family protein

Ciclev10032281m.g AT1G10470 ARR4 RNA. regulation of transcription. ARR

1.01137 response regulator 4

Ciclev10012089m.g AT2G37630 AS1 RNA.regulation of transcription.MYB domain transcription factor family

1.01105 myb-like HTH transcriptional regulator family protein

. †Classification of the measured parameter into a set a functional category in the MapMan analysis tool.

219

Table 5-6. Differentially expressed growth and development-associated genes significantly upregulated in leaves of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN leaves

C. Clementina_ID Arabi ID Gene name Bin Name† Log2 FC* Arabidopsis_define

Ciclev10019788m.g AT1G02850 BGLU11 misc.Gluco-, Galacto- and mannosidases

-4.03647 beta glucosidase 11

Ciclev10005306m.g AT3G12750 ZIP1 transport. metal -3.52287 zinc transporter 1 precursor

Ciclev10030504m.g AT3G13080 MRP3 transport. ABC transporters and multidrug resistance systems


Ciclev10004563m.g AT5G53130 CNGC1 transport. cyclic nucleotide or calcium regulated channels

-3.23314 cyclic nucleotide gated channel 1

Ciclev10012033m.g AT4G35160 T12J5.30 secondary metabolism. phenylpropanoids -3.00446 O-methyltransferase family protein

Ciclev10021897m.g AT1G69530 EXPA1 cell wall.modification -2.79461 expansin A1

Ciclev10015210m.g AT1G61800 GPT2 transport.metabolite transporters at the envelope membrane

-2.58556 glucose-6-phosphate/phosphate translocator 2

Ciclev10028125m.g AT4G34950 AT4G34950 development. -2.53515 Major facilitator superfamily protein

Ciclev10011844m.g AT3G52450 PUB22 RNA. regulation of transcription. PHOR1 -2.47552 plant U-box 22

Ciclev10022651m.g AT4G02620 VHA -F transport.p- and v-ATPases. H+-transportinG two-sector ATPase

-2.40948 vacuolar ATPase subunit F family protein

Ciclev10015253m.g AT5G37490 MPA22.3 RNA. regulation of transcription. PHOR1 -2.38096 ARM repeat superfamily protein

Ciclev10026710m.g AT1G53885 AT1G53903 development. -2.37761 Protein of unknown function (DUF581)

Ciclev10008930m.g AT1G80840 WRKY40 RNA.regulation of transcription. WRKY domain transcription factor family

-2.3154 WRKY DNA-binding protein 40



Ciclev10029146m.g AT1G07050 F10K1.24 development. -2.2876 CCT motif family protein

Ciclev10012920m.g AT2G37060 NF-YB8 RNA.regulation of transcription. CCAAT box binding factor family, HAP3

-2.25882 nuclear factor Y, subunit B8

Ciclev10029464m.g AT5G59820 RHL41 RNA.regulation C2H2 zinc finger family -2.24303 C2H2-type zinc finger family protein

Ciclev10019000m.g AT4G23990 CSLG3 cell wall.cellulose synthesis. cellulose synthase

-2.24166 cellulose synthase like G3

Ciclev10028195m.g AT4G39210 APL3 major CHO metabolism. synthesis.starch.AGPase

-2.2362 Glucose-1-phosphate adenylyltransferase family protein

Ciclev10008031m.g AT3G18830 PMT5 transporter.sugars -2.16781 polyol/monosaccharide transporter 5

Ciclev10031308m.g AT5G19500 T20D1.20 transport.amino acids -2.08241 Tryptophan/tyrosine permease

Ciclev10012180m.g AT1G07030 F10K1.26 transport.metabolite transporters at the mitochondrial membrane

-2.06237 Mitochondrial substrate carrier family protein

220


Ciclev10014642m.g AT4G04450 WRKY42 RNA. regulation of transcription. WRKY domain transcription factor family

-2.02977 WRKY family transcription factor

Ciclev10000819m.g AT1G76430 PHT1;9 transport. phosphate -2.00302 phosphate transporter 1;9

Ciclev10008812m.g AT4G27410 RD26 development. -1.99503 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein

Ciclev10019426m.g AT5G62680 MRG21.10 transport. peptides and oligopeptides -1.97926 Major facilitator superfamily protein

Ciclev10006432m.g AT4G18780 IRX1 cell wall.cellulose synthesis. cellulose synthase

-1.92039 cellulose synthase family protein

Ciclev10032029m.g AT2G40340 DREB2C RNA. regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive

-1.9199 Integrase-type DNA-binding superfamily protein

Ciclev10000444m.g AT5G45380 DUR3 transport. cations -1.87776 solute:sodium symporters;urea transmembrane transporters

Ciclev10004729m.g AT5G26340 MSS1 transporter. sugars -1.85513 Major facilitator superfamily protein

Ciclev10015364m.g AT3G07650 COL9 RNA. regulation.C2C2(Zn) CO-like, Constans-like zinc finger family

-1.80653 CONSTANS-like 9

Ciclev10002699m.g AT3G16640 TCTP development. -1.79532 translationally controlled tumor protein

Ciclev10013735m.g AT2G39730 RCA PS.calvin cycle.rubisco interacting -1.75115 rubisco activase

Ciclev10007550m.g AT5G55930 OPT1 transport. peptides and oligopeptides -1.75075 oligopeptide transporter 1

Ciclev10021699m.g AT2G31180 MYB14 RNA.regulation of transcription. MYB domain transcription factor family

-1.74308 myb domain protein 14


Ciclev10008419m.g AT1G10970 ZIP4 transport. metal -1.72394 zinc transporter 4 precursor

Ciclev10005629m.g AT3G23250 MYB15 RNA. regulation of transcription. MYB domain transcription factor family


Ciclev10000991m.g AT1G77380 AAP3 transport. amino acids -1.69006 amino acid permease 3

Ciclev10000764m.g AT1G20510 OPCL1 secondary metabolism. phenylpropanoids


Ciclev10011765m.g AT3G28960 AT3G28960 transport. amino acids -1.61969 Transmembrane amino acid transporter family protein

Ciclev10008106m.g AT5G54860 MBG8.12 transport.misc -1.6124 Major facilitator superfamily protein

Ciclev10005787m.g AT5G13330 Rap2.6L RNA.regulation of transcription. AP2/EREBP, APETALA2/Ethylene-

-1.59504 related to AP2 6l

221


Ciclev10012678m.g AT2G35980 YLS9 development. -1.59396 Late embryogenesis abundant (LEA) hydroxyproline-rich glycoprotein family

Ciclev10025170m.g AT1G80300 NTT1 transport.misc -1.58246 nucleotide transporter 1


Ciclev10016943m.g AT2G01590 CRR3 PS.lightreaction.NADH DH -1.5454 chlororespiratory reduction 3

Ciclev10015841m.g AT4G32551 LUG development. -1.54317 LisH dimerisation motif;WD40/YVTN repeat-like-containing domain

Ciclev10020717m.g AT3G04070 NAC047 development. -1.52212 NAC domain containing protein 47

Ciclev10015488m.g AT3G20810 AT3G20810 RNA.regulation of transcription. JUMONJI family


Ciclev10015144m.g AT2G20880 F5H14.15 RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive


Ciclev10025169m.g AT5G66770 MUD21.1 development. -1.46315 GRAS family transcription factor

Ciclev10001956m.g AT1G01720 ATAF1 development. -1.45707 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein

Ciclev10018971m.g AT4G10770 OPT7 transport.peptides and oligopeptides -1.45605 oligopeptide transporter 7

Ciclev10009540m.g AT1G19210 T29M8.8 RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive


Ciclev10024781m.g AT3G21250 MRP6 transport.ABC transporters and multidrugresistance systems


Ciclev10016832m.g AT1G61340 T1F9.17 development. -1.43897 F-box family protein

Ciclev10000256m.g AT5G03650 SBE2.2 major CHO metabolism.synthesis. starch.starch branching

-1.43493 starch branching enzyme 2.2



Ciclev10007831m.g AT1G52190 F9I5.4 transport.peptides and oligopeptides -1.37681 Major facilitator superfamily protein

Ciclev10004474m.g AT3G06500 F5E6.17 major CHO metabolism.degradation.sucrose. invertases.neutral

-1.35904 Plant neutral invertase family protein

222


Ciclev10007068m.g AT5G51990 CBF4 RNA.regulation.AP2/EREBP, APETALA2/Ethylene-responsive

-1.35588 C-repeat-binding factor 4

Ciclev10024715m.g AT1G15520 PDR12 transport. ABC transporters and multidrug resistance systems



Ciclev10030544m.g AT1G68710 F24J5.6 RNA. regulation of transcription. bZIP transcription factor family

-1.31858 ATPase E1-E2 type family protein / haloacid dehalogenase-like hydrolase family protein

Ciclev10020150m.g AT2G39890 PROT1 transport.amino acids -1.3185 proline transporter 1





Ciclev10008617m.g AT2G26150 HSFA2 RNA.regulation of transcription.HSF,Heat -shock transcription factor family

-1.29278 heat shock transcription factor A2

Ciclev10016017m.g AT1G07640 OBP2 RNA.regulation of transcription. C2C2(Zn) DOF zinc finger family


Ciclev10020744m.g AT5G24110 WRKY30 RNA. regulation of transcription. WRKY domain transcription factor family


Ciclev10029506m.g AT5G59030 COPT1 transport. metal -1.22809 copper transporter 1


Ciclev10021112m.g AT3G17611 RBL14 RNA. regulation of transcription. -1.20945 RHOMBOID-like protein 14

Ciclev10010930m.g AT3G28345 AT3G28345 transport. ABC transporters and multidrug resistance systems

-1.19386 ABC transporter family protein

Ciclev10014849m.g AT5G14940 F2G14.60 transport. peptides and oligopeptides -1.16618 Major facilitator superfamily protein

Ciclev10016655m.g AT1G78600 LZF1 RNA.regulation of transcription. C2C2(Zn) CO-like, Constans-like zinc finger family

-1.16328 light-regulated zinc finger protein 1

Ciclev10014968m.g AT3G21690 AT3G21690 transport.misc -1.12647 MATE efflux family protein

Ciclev10025318m.g AT1G15960 NRAMP6 transport. metal -1.12107 NRAMP metal ion transporter 6

Ciclev10024910m.g AT4G37270 HMA1 transport.metal -1.11554 heavy metal atpase 1

Ciclev10027815m.g AT3G46970 PHS2 major CHO metabolism. degradtion. starch.starch phosphorylase

-1.10124 alpha-glucan phosphorylase 2

Ciclev10025933m.g AT4G37990 ELI3-2 secondary metabolism. phenylpropanoids. lignin biosynthesis. CAD


223


Ciclev10005403m.g AT5G62020 HSFB2A RNA.regulation of transcription.HSF,Heat-shock transcription factor family

-1.05955 heat shock transcription factor B2A

Ciclev10011551m.g AT3G54090 FLN1 major CHO metabolism.degradation.sucrose.fructokinase

-1.05271 fructokinase-like 1

Ciclev10028839m.g AT5G58490 MQJ2.6 secondary metabolism.phenylpropanoids.lignin biosynthesis.CCR1


Ciclev10005012m.g AT2G41710 T11A7.19 RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive element binding protein family


Ciclev10032043m.g AT1G60470 GolS4 minor CHO metabolism.raffinose family.Galactinol synthases.putative


Ciclev10012712m.g AT4G17900 T6K21.80 RNA.regulation of transcription.unclassified -1.04179 PLATZ transcription factor family protein

Ciclev10026222m.g AT3G15840 PIFI PS.lightreaction.cyclic electron flow-chlororespiration

-1.03408 post-illumination chlorophyll fluorescence increase

Ciclev10013238m.g AT3G51860 CAX3 transport.metal -1.00947 cation exchanger 3

Ciclev10032205m.g AT1G14140 F7A19.22 transport.metabolite transporters AT the mitochondrial membrane


Ciclev10018376m.g AT5G40240 MSN9.140 development. -1.00119 nodulin MtN21 /EamA-like transporter family protein


224

Table 5-7. Differentially expressed growth and development -associated genes significantly upregulated in roots of the symptomatic VAL/CAN combination as compared to symptomatic VAL/SW roots C. Clementina_ID Arabi ID Gene name Bin Name† log2 FC Arabidopsis_define

Ciclev10006459m.g AT2G46320 transport. metabolite transporters at the mitochondrial membrane


Ciclev10004563m.g AT5G53130 CNGC1 transport. cyclic nucleotide or calcium regulated channels

6.83261 cyclic nucleotide gated channel 1

Ciclev10030461m.g AT1G74840 RNA. regulation of transcription. MYB-related transcription factor family


Ciclev10004369m.g AT5G49360 BXL1 cell wall.degradation. mannan-xylose-arabinose-fucose

5.4693 beta-xylosidase 1

Ciclev10006044m.g 5.29825

Ciclev10028730m.g AT2G22780 PMDH1 Gluconeogenesis. Malate DH 5.19677 peroxisomal NAD-malate dehydrogenase 1

Ciclev10002178m.g AT1G45474 LHCA5 PS.lightreaction. photosystem I.LHC-I 4.61703 photosystem I light harvesting complex gene 5





Ciclev10000579m.g AT4G19010 secondary metabolism.phenylpropanoids 3.61678 AMP-dependent synthetase and ligase family protein

Ciclev10020067m.g AT1G09230 RNA.RNA binding 3.35782 RNA-binding (RRM/RBD/RNP motifs) family protein

Ciclev10001337m.g AT2G26560 PLA2A,PLP2 development. storage proteins 3.34391 phospholipase A 2A

Ciclev10029683m.g AT3G44735 AtPSK3,PSK1 development. 3.23871 PHYTOSULFOKINE 3 PRECURSOR

Ciclev10031811m.g AT5G05600 secondary metabolism. flavonoids. anthocyanins


Ciclev10004006m.g AT4G31500 ATR4,CYP83B1 secondary metabolism. sulfur-containing. Glucosinolates. synthesis. shared. CYP83B1

3.1829 cytochrome P450, family 83, subfamily B, polypeptide 1

Ciclev10001683m.g AT5G65640 bHLH093 RNA. regulation of transcription. bHLH, Basic Helix-Loop-Helix family

3.16781 beta HLH protein 93

Ciclev10023271m.g AT4G24040 AtTRE1, TRE1 minor CHO metabolism.trehalose.trehalase 3.12288 trehalase 1

225


Ciclev10020505m.g AT3G29590 At5MAT secondary metabolism.flavonoids. anthocyanins. anthocyanin 5-aromatic acyltransferase


Ciclev10022611m.g 2.84401

Ciclev10031846m.g AT1G68840 RAV2,TEM2 RNA.regulation of transcription. AP2/EREBP, APETALA2 /Ethylene-responsive

2.8375 related to ABI3/VP1 2

Ciclev10016395m.g 2.69999

Ciclev10003789m.g AT5G10830 development. 2.69943 S-adenosyl-L-methionine-dependent methyltransferases superfamily protein

Ciclev10017240m.g AT4G18372 RNA.processing 2.63955 Small nuclear ribonucleoprotein family protein

Ciclev10015129m.g AT5G14570 NRT2.7 transport.nitrate 2.62048 high affinity nitrate transporter 2.7

Ciclev10003278m.g 2.59092

Ciclev10028639m.g AT1G68010 HPR PS.photorespiration. hydroxypyruvate reductase

2.55923 hydroxypyruvate reductase

Ciclev10027618m.g 2.54474

Ciclev10005315m.g AT2G39510 development. 2.39411 nodulin MtN21 /EamA-like transporter family protein


2.36441 O-methyltransferase family protein

Ciclev10016953m.g 2.35172

Ciclev10019412m.g AT1G12110 CHL11, NRT1, NRT1.1

transport.nitrate 2.3453 nitrate transporter 1.1


Ciclev10020053m.g AT4G00050 UNE10 RNA.regulation of transcription. bHLH,Basic Helix-Loop-Helix family



development. 2.11625 nodulin MtN21 /EamA-like transporter family protein


Ciclev10012221m.g AT2G29290 secondary metabolism.N misc.alkaloid-like


226



Ciclev10012221m.g AT2G29290 secondary metabolism.N misc.alkaloid-like


Ciclev10014396m.g AT4G16370 OPT3 transport.peptides and oligopeptides

2.10862 oligopeptide transporter

Ciclev10012087m.g AT5G54160 OMT1 secondary metabolism.phenylpropanoids. lignin biosynthesis. COMT


Ciclev10022680m.g AT4G25470 CBF2, DREB1C


2.07929 C-repeat/DRE binding factor 2

Ciclev10004816m.g AT1G51340 transport.misc 2.06909 MATE efflux family protein

Ciclev10011092m.g AT3G53720 CHX20 transport.metal 2.02928 cation/H+ exchanger 20

Ciclev10014463m.g AT3G21090 transport.ABC transporters and multidrug resistance systems


Ciclev10006265m.g 2.01839


minor CHO metabolism. others 2.01374 Galactose mutarotase-like superfamily protein

Ciclev10013246m.g

2.00591

Ciclev10009186m.g AT1G67940 AtNAP3,AtSTAR1 transport. ABC transporters and multidrugresistance systems

2.00073 non-intrinsic ABC protein 3

Ciclev10007552m.g AT5G55930 OPT1 transport.peptides and oligopeptides

1.96663 oligopeptide transporter 1

Ciclev10006706m.g AT1G51310 RNA.processing 1.95365

transferases;tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferases

Ciclev10013694m.g AT1G61110 NAC025 development. 1.91859 NAC domain containing protein 25

Ciclev10030170m.g AT1G58030 CAT2 transport.amino acids 1.90109 cationic amino acid transporter 2


development. 1.87951 nodulin MtN21 /EamA-like transporter family protein

Ciclev10004641m.g AT3G16910 AAE7, ACN1 Gluconeogenese/ Glyoxylate cycle

1.91768 acyl-activating enzyme 7

227


Ciclev10004728m.g AT3G47420 ,PS3 transporter.membrane system unknown

1.76366 phosphate starvation-induced gene 3

Ciclev10011350m.g AT3G10960 AZG1 transport.misc 1.76324 AZA-guanine resistant1

Ciclev10032377m.g AT2G40100 LHCB4.3 PS.lightreaction. photosystem II. LHC-II

1.74389 light harvesting complex photosystem II

Ciclev10032508m.g AT4G17030 EXLB1,EXPR1 cell wall. modification 1.72426 expansin-like B1

Ciclev10030073m.g AT5G58460 CHX25 transport.metal 1.71876 cation/H+ exchanger 25

Ciclev10011458m.g AT4G08250 development. 1.71687 GRAS family transcription factor

Ciclev10005251m.g AT5G54160 OMT1 secondary metabolism. phenylpropanoids. lignin biosynthesis.COMT


Ciclev10020033m.g AT5G41330 transport.potassium 1.6809 BTB/POZ domain with WD40/YVTN repeat-like protein

Ciclev10026128m.g AT2G22500 PUMP5, UCP5 transport. metabolite transporters at the mitochondrial membrane

1.67363 uncoupling protein 5

Ciclev10029134m.g AT4G01470 TIP1.3, GAMMA-TIP3

transport.Major Intrinsic Proteins.TIP

1.67336 tonoplast intrinsic protein 1;3

Ciclev10020693m.g AT1G08650 PPCK1

Glycolysis.cytosolic branch.phospho-enol-pyruvATe carboxylase kinase (PPCK)

1.652 phosphoenolpyruvate carboxylase kinase 1

Ciclev10011292m.g AT1G68570 transport.peptides and oligopeptides

1.64396 Major facilitator superfamily protein

Ciclev10003716m.g AT1G62280 SLAH1 transport.metabolite transporters AT the mitochondrial membrane

1.64275 SLAC1 homologue 1

Ciclev10017962m.g AT1G20480 secondary metabolism.phenylpropanoids


Ciclev10000235m.g AT5G49740 FRO7 metal handling.acquisition 1.63453 ferric reduction oxidase 7

Ciclev10032534m.g AT4G01470 ATTIP1.3,GAMMA-TIP3

transport.Major Intrinsic Proteins.TIP

1.61344 tonoplast intrinsic protein 1;3

228


Ciclev10013636m.g AT2G38100 transport. peptides and oligopeptides

1.59611 proton-dependent oligopeptide transport (POT) family protein

Ciclev10030345m.g AT1G58030 CAT2 transport. amino acids 1.5896 cationic amino acid transporter 2

Ciclev10010291m.g AT4G30380 cell wall. modification 1.58202 Barwin-related endoglucanase

Ciclev10027932m.g AT2G28260 CNGC15, CNGC15

transport. cyclic nucleotide or calcium regulated channels

1.58065 cyclic nucleotide-gated channel 15

Ciclev10001555m.g AT5G54160 ATOMT1, OMT1

secondary metabolism. phenylpropanoids.lignin biosynthesis.COMT


Ciclev10004694m.g AT3G54700 PHT1;7 transport. phosphate 1.53939 phosphate transporter 1;7

Ciclev10025117m.g AT1G17840 ABCG11, WBC11,

transport. ABC transporters and multidrug resistance systems

1.52843 white-brown complex homolog protein 11

Ciclev10033117m.g AT3G25882 NIMIN-2 RNA. regulation of transcription. unclassified

1.52719 NIM1-interacting 2

Ciclev10004580m.g AT5G53130 CNGC1, CNGC1


1.51247 cyclic nucleotide gated channel 1


minor CHO metabolism. others 1.50309 Galactose mutarotase-like superfamily protein


transport. metal 1.50292 Cation efflux family protein

†Classification of the measured parameter into a set a functional category in the MapMan analysis tool.

229

Table 5-8. Differentially expressed growth and development -associated genes significantly upregulated in roots of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN roots

C. Clementina_ID Arabi ID Gene name Bin Name† Log2 FC* Arabidopsis_definition

Ciclev10030369m.g AT4G12240 RNA. regulation of transcription.C2H2 zinc finger family

-5.57768 zinc finger (C2H2 type) family protein

Ciclev10033320m.g -5.33999

Ciclev10026846m.g -5.04682

Ciclev10015735m.g AT3G51420 SSL4 secondary metabolism. N misc.alkaloid-like

-4.85918 strictosidine synthase-like 4


-3.65164 NAD(P)-linked oxidoreductase superfamily protein

Ciclev10021785m.g AT5G15090 VDAC3 transport. porins -3.5579 voltage dependent anion channel 3

Ciclev10023568m.g AT5G54160 OMT1 secondary metabolism. phenylpropanoids. lignin biosynthesis. COMT


Ciclev10009879m.g AT3G15270 SPL5 development. squamosa promoter binding like (SPL)

-3.55512 squamosa promoter binding protein-like 5

Ciclev10013967m.g -3.54226

Ciclev10020838m.g AT1G17020 SRG1 secondary metabolism. flavonoids.flavonols


Ciclev10012978m.g AT5G06760 LEA4-5 development.late embryogenesis abundant

-3.48679 Late Embryogenesis Abundant 4-5

Ciclev10031060m.g AT1G68570 transport.peptides and oligopeptides -3.04482 Major facilitator superfamily protein

Ciclev10009855m.g AT3G24450 metal handling.binding, chelation, and storage

-3.0277 Heavy metal transport/detoxification superfamily protein

Ciclev10010905m.g AT3G53480 ABCG37, ATPDR9,

transport.ABC transporters and multidrugresistance systems



-2.82074 Major facilitator superfamily protein

Ciclev10032964m.g -2.79867

Ciclev10029192m.g AT2G28660 metal handling. binding, chelation, and storage

-2.77507 Chloroplast-targeted copper chaperone protein

Ciclev10004454m.g AT4G24000 ATCSLG2, CSLG2

cell wall. cellulose synthesis.cellulose synthase

-2.72604 cellulose synthase like G2

Ciclev10032043m.g AT1G60470 AtGolS4, GolS4

minor CHO metabolism raffinose family.galactinol synthases.putative


230



transport. metabolite transporters at the mitochondrial membrane





Ciclev10028859m.g AT5G59800 MBD7 RNA.regulation of transcription. Methyl binding domain proteins

-2.56108 methyl-CPG-binding domain 7

Ciclev10020692m.g AT3G14420 PS. photorespiration.glycolate oxidase -2.52311 Aldolase-type TIM barrel family protein

Ciclev10002410m.g AT3G54340 AP3, ATAP3 development. -2.50372 K-box region and MADS-box transcription factor family protein

Ciclev10025594m.g AT2G17840 ERD7 development. -2.43618 Senescence/dehydration-associated protein-related

Ciclev10026415m.g AT4G37990 ATCAD8, ELI3 secondary metabolism.phenylpropanoids.lignin biosynthesis.CAD


Ciclev10031740m.g AT2G18950 TPT1, VTE2 secondary metabolism.isoprenoids.tocopherol biosynthesis.homogentisate phytyltransferase


Ciclev10021733m.g AT2G45050 GATA2 RNA. regulation of transcription.C2C2(Zn) gatA transcription factor family

-2.40858 GATA transcription factor 2

Ciclev10024556m.g AT1G02460 cell wall. degradation. pectate lyases and polygalacturonases

-2.39309 Pectin lyase-like superfamily protein

Ciclev10010063m.g -2.3711


-2.33995 cyclic nucleotide-gated channels

Ciclev10000828m.g AT1G22710 SUC2, SUT1 transporter. sugars.sucrose -2.33944 sucrose-proton symporter 2

Ciclev10009098m.g AT5G11450 PS.lightreaction.photosystem II.PSII polypeptide subunits

-2.31656 Mog1/PsbP/DUF1795-like photosystem II reaction center PsbP family protein

Ciclev10005117m.g AT5G23870 cell wall.pectin*esterases.acetyl esterase -2.26826 Pectinacetylesterase family protein

Ciclev10018145m.g AT2G38300 RNA.regulation of transcription. G2-like transcription factor family, GARP

-2.23641 myb-like HTH transcriptional regulator family protein

Ciclev10011184m.g AT5G15410 CNGC2, DND1


-2.20161 Cyclic nucleotide-regulated ion channel family protein

231


Ciclev10004151m.g AT3G53480 ATPDR9, PDR9 transport. ABC transporters and multidrug resistance systems


Ciclev10005189m.g AT3G57670 NTT,WIP2 RNA. regulation of transcription. C2H2 zinc finger family

-2.18112 C2H2-type zinc finger family protein

Ciclev10016525m.g AT4G05180 PSBQ-2,PSII-Q PS. lightreaction. photosystem II. PSII polypeptide subunits

-2.15586 photosystem II subunit Q-2

Ciclev10031067m.g AT1G69850 NRT1:2, NTL1 transport. nitrate -2.14956 nitrate transporter 1:2

Ciclev10000981m.g AT5G49630 AAP6 transport. amino acids -2.13237 amino acid permease 6


-2.12847 Major facilitator superfamily protein

Ciclev10018956m.g AT1G63680 ATMURE, MURE, PDE316

development. -2.09081 acid-amino acid ligases;ligases;ATP binding;ATP binding;ligases

Ciclev10018229m.g AT3G13080 ATMRP3, MRP3 transport. ABC transporters and multidrug resistance systems


Ciclev10012939m.g -2.06516

Ciclev10014333m.g AT5G40390 SIP1 minor CHO metabolism. raffinose family.raffinose synthases.known

-2.03661 Raffinose synthase family protein

Ciclev10015613m.g AT3G19330 RNA.processing -2.03177 Protein of unknown function (DUF677)

Ciclev10029560m.g -2.02771

Ciclev10000639m.g AT1G59740 transport. peptides and oligopeptides -2.00619 Major facilitator superfamily protein

Ciclev10010139m.g -2.00448

Ciclev10009761m.g AT5G26170 WRKY50 RNA.regulation of transcription.WRKY domain transcription factor family


Ciclev10014531m.g AT3G28180 ATCSLC04, ATCSLC4,

cell wall.cellulose synthesis -1.98624 Cellulose-synthase-like C4

Ciclev10027249m.g AT3G50740 UGT72E1 secondary metabolism. phenylpropanoids.lignin biosynthesis

-1.98165 UDP-glucosyl transferase 72E1

Ciclev10011381m.g AT3G54140 ATPTR1,PTR1 transport. peptides and oligopeptides -1.97959 peptide transporter 1

Ciclev10012384m.g AT4G00430 PIP1;4, PIP1E,TMP-C

transport.Major Intrinsic Proteins.PIP -1.96847 plasma membrane intrinsic protein 1;4

Ciclev10006914m.g AT3G24310 ATMYB71,MYB305 RNA.regulation of transcription.MYB domain transcription factor family


Ciclev10023956m.g AT2G44745 RNA.regulation of transcription -1.94641 WRKY family

232


Ciclev10033373m.g AT5G65550 secondary metabolism.flavonoids.anthocyanins.


Ciclev10015364m.g AT3G07650 COL9 RNA.regulation of transcription. C2C2(Zn) CO-like, Constans-like zinc finger family

-1.89385 CONSTANS-like 9

Ciclev10022631m.g AT1G03650 RNA.regulation of transcription.Histone acetyltransferases

-1.86557 Acyl-CoA N-acyltransferases (NAT) superfamily protein

Ciclev10000950m.g AT5G65380 development. -1.86012 MATE efflux family protein

Ciclev10022568m.g AT2G45660 AGL20, SOC1

RNA.regulation of transcription.MADS box transcription factor family

-1.83583 AGAMOUS-like 20

Ciclev10002259m.g AT4G36810 GGPS1 secondary metabolism.isoprenoids.non-mevalonate Pathway.geranylgeranyl pyrophosphate synthase

-1.83556 geranylgeranyl pyrophosphate synthase 1

Ciclev10010448m.g AT5G10970 RNA.regulation of transcription. C2H2 zinc finger family

-1.83409 C2H2 and C2HC zinc fingers superfamily protein

Ciclev10014984m.g AT1G30650 ATWRKY14, WRKY14

RNA. regulation of transcription. WRKY domain transcription factor family


Ciclev10031966m.g AT5G46590 anac096, NAC096

development. -1.81485 NAC domain containing protein 96

Ciclev10017079m.g AT3G43660 development. -1.81291 Vacuolar iron transporter (VIT) family protein

Ciclev10033420m.g AT5G65060 AGL70, FCL3, MAF3

RNA.regulation of transcription. MADS box transcription factor family

-1.80234 K-box region and MADS-box transcription factor family protein

Ciclev10014919m.g AT2G05760 transport.misc -1.79557 Xanthine/uracil permease family protein

Ciclev10003119m.g AT1G75900 development. -1.76406 GDSL-like Lipase/Acylhydrolase superfamily protein

Ciclev10018903m.g AT5G41610 CHX18 transport.metal -1.75524 cation/H+ exchanger 18

Ciclev10026061m.g AT3G50410 OBP1 RNA.regulation of transcription. C2C2(Zn) DOF zinc finger family

-1.75489 OBF binding protein 1

Ciclev10013028m.g AT5G07050 development. -1.74759 nodulin MtN21 /EamA-like transporter family protein

Ciclev10020864m.g AT5G22300 AtNIT4,NIT4 secondary metabolism. sulfur-containing. glucosinolates.degradation.nitrilase

-1.74216 nitrilase 4

Ciclev10006176m.g AT5G49120 development. -1.73729 Protein of unknown function (DUF581)

233


Ciclev10009942m.g -1.72917

Ciclev10028757m.g AT1G08290 WIP3 RNA. regulation of transcription.C2H2 zinc finger family

-1.72753 WIP domain protein 3

Ciclev10028457m.g AT4G14210 PDS, PDS3 secondary metabolism. isoprenoids. carotenoids. phytoene dehydrogenase

-1.72224 phytoene desaturase 3

Ciclev10033966m.g AT5G20420 CHR42 RNA.regulation of transcription. Chromatin Remodeling Factors

-1.7158 chromatin remodeling 42



Ciclev10028385m.g AT1G08230 GAT1 transport.amino acids -1.70612 Transmembrane amino acid transporter family protein

Ciclev10022854m.g -1.69008

Ciclev10012089m.g AT2G37630 AS1, MYB91 RNA.regulation of transcription.MYB domain transcription factor family

-1.68872 myb-like HTH transcriptional regulator family protein

Ciclev10007316m.g AT4G32730 MYB3R-1, MYB3R1 RNA.regulation of transcription. MYB domain transcription factor family

-1.68528 Homeodomain-like protein

Ciclev10009245m.g AT1G04250 AXR3, IAA17 RNA.regulation of transcription.Aux/IAA family

-1.67523 AUX/IAA transcriptional regulator family protein

Ciclev10013296m.g

-1.66375

Ciclev10021650m.g AT4G00950 MEE47 RNA.regulation of transcription.C2C2(Zn) DOF zinc finger family

-1.66157 Protein of unknown function (DUF688)




metal handling.binding, chelation, and storage


Ciclev10028494m.g AT4G34530 CIB1 RNA.regulation of transcription.bHLH,Basic Helix-Loop-Helix family

-1.61697 cryptochrome-interacting basic-helix-loop-helix 1

Ciclev10015729m.g AT4G21440 ATM4, MYB102

RNA.regulation of transcription.MYB domain transcription factor family

-1.61341 MYB-like 102

Ciclev10014775m.g AT2G32950 COP1 development. -1.60543 Transducin/WD40 repeat-like superfamily protein

234






Ciclev10015474m.g AT3G27400 cell wall. degradation.pectate lyases and polygalacturonases


Ciclev10009128m.g AT5G56840 RNA.regulation of transcription. MYB-related transcription factor family

-1.54732 myb-like transcription factor family protein

Ciclev10007249m.g AT2G26910 ATPDR4, PDR4 transport.ABC transporters and multidrug resistance systems


Ciclev10000054m.g AT1G27940 PGP13 transport.ABC transporters and multidrug resistance systems

-1.53159 P-glycoprotein 13

Ciclev10014713m.g AT3G10600 CAT7 transport.amino acids -1.5313 cationic amino acid transporter 7

Ciclev10025631m.g AT5G66460 cell wall.degradation.mannan-xylose-arabinose-fucose

-1.52491 Glycosyl hydrolase superfamily protein

Ciclev10003379m.g AT5G64530 ANAC104, XND1 development. -1.50295 xylem NAC domain 1 * The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. †Classification of the measured parameter into a set a functional category in the MapMan analysis tool.

235

Figure 5-1. Graphical presentation of secondary metabolite biosynthesis pathway involved genes found in DGE analysis of asymptomatic and symptomatic

VAL/CAN and VAL/SW -leaves and -roots. Log2 fold changes are indicated as

a gradient between blue (up-regulated in VAL/CAN combination) and red (up-regulated VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic

236

Figure 5-2. Graphical presentation of cell wall modification-associated pathways involved genes found in DGE analysis of asymptomatic and symptomatic



237

Figure 5-3. Graphical presentation of nutrient transportation-associated Genes found in DGE analysis of asymptomatic and symptomatic VAL/CAN



Figure 5-4. Graphical presentation of Nitrogen metabolism-associated genes found DGE analysis of asymptomatic and symptomatic VAL/CAN



238

Figure 5-5. Graphical presentation of carbohydrate metabolism-associate genes found

in DGE analysis of asymptomatic and symptomatic VAL/CAN and VAL/SW -leaves and -roots. Log2 fold changes are indicated as


239

Figure 5-6. Graphical presentation of genes encoding transcription regulators involved in plant growth and development, and found in DGE analysis of asymptomatic and symptomatic VAL/CAN and VAL/SW leaves and roots. Log2 fold changes are indicated as a gradient between blue (up-regulated in VAL/CAN

combination) and red (up-regulated and VAL/SW combination). Asymp; Asymptomatic, Sympt; symptomatic

240

CHAPTER 6 EFFECTS OF IMPROVED CITRUS ROOTSTOCK AND ENHANCED NUTRIENT FORMULATION ON HLB-DISEASE SEVERITY IN ‘VALENCIA’ SWEET ORANGE

SCION

Introduction

Plants are autotrophic organisms which synthesis their own food by the process

of photosynthesis. The products of photosynthesis are called photosynthates, and they

perform various plant functions. Plant photosynthates are translocated from area of

supply called sources, to areas of metabolism, storage, and growth, called sinks. Biotic

or abiotic stress may cause imbalance between source and sink tissue photosynthates

allocation/distribution. Plants face environmental challenges in their natural habitat.

Hence, plant’s surveillance and defense systems are activated to defend the

environmental adversaries. Pathogen detection and resistance are energy consuming

processes that can create an imbalance of resource distribution between plant growth

and defense. Plant defense is generally beneficial, but not always. A model discussed

by Ellen et al. (1987) and other studies (Simms and Rausher, 1987) showed that energy

invested in the defense against herbivores was rather non-beneficial and increased

plant resource consumption that could have been used for other biological functions.

Whereas, benefits of herbivory-induced defense (Heil, 2004), pathogen-activated

defense (van Hulten et al., 2006) and cost/benefit of defense responses (Mauricio and

Rausher, 1997) were also reported in many plant-pathogen and plant-herbivore

interactions. The benefit or cost of defense is dependent on the environmental

conditions and resource availability (Purrington and Bergelson, 1997; Heil et al., 2000).

Cost and benefits of induced and constitutively expressed defenses are explained by

the optimal defense theory (ODT) (Moreira et al., 2012). The theory of ODT explains the

241

differential allocation of induced and constitutive defense based on the importance of

the organs or tissues in the plant’s survival. According to the ODT, valuable

reproductive organs exhibit constitutive defense, whereas, leaves and roots, which are

the plant’s expandable organs, show the induced defense. It is hypothesized that

induced resistance costs fewer resources and more benefits as compared to the

constitutive defense. However, the calculations of defense-induced costs and benefits in

the plant are not easy and simple to understand. It is an intricate networking between

threat sensing signaling components, defense activation, and nutrient status of the

plant.

Plant growth hormones (PGRs) and nutrients’ status are the critical components

to optimize the balance of energy supply between growth and defense. PGRs, whether

defense-associated salicylic acid (SA), jasmonic acid (JA), ethylene (ET) or growth

related auxins (AU), cytokinins (CT), gibberellic acid (GA), brassinosteroids (BR) and

abscisic acid (ABA), directly or indirectly regulate the plant defense (Huot et al., 2014).

PGRs have an antagonistic or synergistic effect on each other that regulates growth and

defense responses (Nemhauser et al., 2004; Robert-Seilaniantz et al., 2011; Naseem et

al., 2015). Nutrient homeostasis in the plants is also regulated by plant hormones

(Rubio et al., 2009; Krouk et al., 2011). Effect of cultivars and nutrient interactions in the

plant defense was discussed in the Hyaloperonospora parasitica infection in A. thaliana

(Heidel and Dong, 2006). The study reported that systemic acquired resistance (SAR) is

beneficial to the wild phenotype plants exhibiting normal SAR, under a normal or low-

nutrient condition in H. parasitica infected plants. Whereas, the high-nutrient condition

did not show any increased benefit to wild phenotype plants the under H. parasitica

242

infection. Also, the study showed that in the overexpressed NPR1 genotype, constitutive

expression of SAR is beneficial under a strong damage caused by pathogen attack. JA

and ET induced resistance-associated defenses are studied widely in plants attacked

by herbivores and the necrotrophs (Simms and Rausher, 1987; Agrawal, 1999; Elle et

al., 1999). Although JA induced chemical defense is reported to be cost-effective in the

defense against herbivores, it is found that constitutive expression of JA-inducible

responses delay plant phenology (Cipollini, 2010).

Pathogen infection can have a deleterious effect on the plant health due to its

direct or indirect damage. Citrus plants infected with HLB disease, caused by putative

bacteria Candidatus Liberibacter asiaticus (CaLas) infection, suffer severely

compromised growth, immature fruit drop, and leaf senescence. CaLas-infected plant

phloem tissues are found to have callose deposition that barriers the spread of CaLas.

However, the overaccumulation of callose in the phloem also creates blockages for

nutrient and photosynthate flow. There are no reports on CaLas specific immunity or

effector triggered immunity (ETI) exhibited by any infected plants. Therefore, in the

absence of resistance, plants induce different non-host specific defenses to fight against

the CaLas infection. CaLas-infected susceptible citrus scions and rootstocks showed

upregulation of genes involved in different plant defenses (Martinelli et al., 2012;

Martinelli et al., 2013; Zhong et al., 2016). In such a scenario, improved HLB-tolerant

scions and rootstocks are important to increase plant sustainability. The economically

enduring and biological HLB tolerance can be achieved either by developing HLB

tolerant citrus cultivars, and/or modifying cultural practices that can increase plant

sustainability under HLB pressure. Different levels of HLB tolerance are observed in the

243

hybrids of trifoliate orange (Poncirus trifoliata [L.] Raf), rough lemon (Citrus jambhiri

Lush.), pummelos (Citrus maxima Merr.) and lemons (Citrus limon L. [Burm.] f.)

(Folimonova et al., 2009; Fan et al., 2012; Albrecht and Bowman, 2012b; Aritua et al.,

2013; Martinelli et al., 2016). The citrus breeding program at the University of Florida,

Citrus Research and Education Center, Lake Alfred (UF-CREC) has developed putative

HLB-tolerant hybrid rootstocks in response to the need of improved plant sustainability

under HLB disease pressure.

HLB disease development in citrus trees is not only attributed to the lack of

immunity but also the inadequate supply of nutrients (Cimò et al., 2013). The role of

proper nutrition management is vital to reducing the disease severity (Dordas, 2008),

whereas improper nutrition management may not achieve the desirable results

(Gottwald et al., 2012). An understanding of nutrient interactions with disease

development and cultivars is vital to implement effective nutrition management. CaLas-

infected plants are found to have a similarity to those showing zinc (Zn) deficiency

symptoms. Moreover, boron (B), calcium (Ca), magnesium (Mg) and phosphorus (P)

deficiencies are also prominent in the CaLas-infected plants (Spann and Schumann,

2009). Enhanced nutrient foliar application programs (ENPs) have been implemented to

address nutrient deficiencies in the CaLas-infected plants. However, ENPs did not

show a significant role in reducing HLB disease severity (Gottwald et al., 2012). Foliar

application of nutrients does not address the deficiencies in the roots. Soil application of

nutrients in combination with insecticide and hormones was found to be effective in

reducing HLB disease severity (Shen et al., 2013). Therefore, implementation of

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efficient nutrient management focusing more on root health could be a key to achieving

desirable results in CaLas-infected plants.

The interaction between nutrition and citrus rootstocks plays a vital role in HLB

disease development in Citrus. Therefore, to address the issue of nutrient deficiency

and symptoms severity in the CaLas-infected plants, combinations of a putatively HLB-

tolerant tetraploid (4x) rootstock and an improved nutrient formulation were tested in

CaLas-infected plants under greenhouse conditions. The improved nutrient formulation

is a controlled release fertilizer delivering elevated levels of secondary and

micronutrients impacted by HLB In this study, the role of the improved nutrient

formulation was also tested in reducing CaLas infection in the ‘Valencia’ sweet orange

(Citrus sinensis [L.] Osbeck) scion grafted onto HLB-susceptible Swingle citrumelo

rootstock combination (a standard in the Florida industry). The results obtained from the

greenhouse study will be helpful to understand the potential role of nutrition and citrus

rootstocks to relieve the HLB disease severity and enhance the economic tolerance in

the susceptible Citrus plants.


Plant Material and Nutrition Treatment

The greenhouse-based experiment was designed to analyze the effect of

interaction between citrus rootstocks and nutrient formulations on CaLas-infected

‘Valencia’ (VAL) scion. In the rootstock treatment, two different rootstocks were tested

(Table 6-1). The putatively HLB-tolerant 4x hybrid used in this study is a complex

candidate rootstock hybrid that contains a combination of more than four different citrus

species and/or cultivars. The 4x hybrid is a cross of a cybrid autotetraploid [Amblycarpa

(Citrus amblycarpa) + ‘Volkmer’ lemon (Citrus Volkmariana)] and tetrazyg Orange 19

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(released as UFR-4). The somatic cybrid between Amblycarpa mandarin + Volkmer

lemon is an autotetraploid which was created using the protoplast fusion technique. This

unusual combination retained two copies of the ‘Volkmariana’ nuclear genotype in

combination with Amblycarpa cytoplasm. Orange 19 is called a ‘tetrazyg’, which is a

zygotic allotetraploid produced from crossing two tetraploids that can be somatic hybrids

or tetrazygs. Orange 19 (commercially released as UFR-4) is a hybrid of two somatic

hybrids: [‘Nova’ mandarin + ‘Hirado Butan Pink’ Pummelo] x [‘Cleopatra’ mandarin +

Argentine Trifoliate orange]. The 4x hybrid rootstock used in this study will be named

‘Tetr’ here onward. The commercially important and HLB-susceptible Swingle (Sw)

rootstock used in this study is a hybrid between grapefruit (Citrus paradisi [Macf.]) and

Trifoliate orange.

In the nutrition treatment, two formulations of Harrell’s™ POLYON coated

controlled-release fertilizer (CRF) were used. One of the formulations, the nursery mix

(NM), is designed to fertilize greenhouse plants; whereas, the other nutrient formulation

contained increased concentration of micronutrients and 4% Ca compared to the NM. It

will be referred to as Enhanced-CRF (ECRF) (Table 6-2). The ECRF formulation was

designed to compensate for the secondary and micronutrient deficiencies observed in

roots of CaLas infected trees. Multiple plant biological replicates (4-6) and randomized

experimental design were used to test the effects of the interaction between nutrient

formulations and rootstock treatments on VAL scion in the downstream analyses of this

experiment.

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CaLas-Inoculation and Detection

CaLas-infected ‘VAL’ sticks were grafted onto the rootstocks grown using the

nutrient formulation combinations in 4 x 4-inch pots to test the effects of the interaction

between rootstock and nutrient formulation (Figure 6-1). In addition, healthy VAL sticks

were also grafted onto Sw and provided with NM formulation, which served as the

control in this experiment. Two methods of CaLas infection were performed in the

ECRF-applied scion/rootstock combination. In the first type of inoculation, plants were

infected by grafting a 6- to 7-inch-long CaLas-infected VAL bud-sticks onto the healthy

rootstock (stick-grafting infection). In the second type of inoculation, healthy

scion/rootstock grafts were infected by grafting a CaLas-infected VAL blind-bud onto the

rootstock (blind-bud infection) using the standard T-grafting technique. Plants under NM

formulation were infected by only the stick-grafting infection method. Stick grafting

ensures CaLas inoculum availability for the infection and enough plant material for the

sampling. CaLas-infected VAL sticks were collected from the UF-CREC, Block 2B from

commercial VAL trees grown under a conventional nutrition program. These plants were

tested for the detection of CaLas and citrus tristeza virus (CTV) strains. Healthy VAL

sticks were procured from a certified citrus nursery. CaLas-infected VAL sticks and buds

collected from the field were sterilized thoroughly using 10% bleach and 70% alcohol

treatment to avoid any contamination other than HLB, and then immediately used for

the grafting. Grafting of all plants was conducted in the months of September and

October, 2015.

Quantitative real-time PCR (qRT-PCR) based CaLas detection was conducted to

test the cycle threshold (Ct) values and bacterial titer of the VAL sticks and

post-inoculated grafts. The GenElute™ Plant Genomic DNA Miniprep Kit (Sigma

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Aldrich, Woodlands, TX) was used to extract DNA from leaves, and the PowerMax®

Soil DNA Isolation Kit (MOBIO Laboratories, Inc., Carlsbad, CA) was used to isolate

root DNA. The extracted DNA was quantified in a NanoDrop ND-100 spectrophotometer

(Thermo Scientific, Wilmington, DE). CaLas specific CQULA04F and CQULA04R primer

pair and CQULAP10 TaqMan-probe (FAM fluorophore dye) were used for the

amplification of CaLas 16sRNA (Wang et al., 2006). The TaqMan® Gene Expression

Master Mix Kit (Applied Biosystems, Foster City, CA) was used to perform qPCR assay.

All reactions were carried out in 25 µl reaction volume containing 50 ng DNA and 0.3

µM probe and primer concentrations. Amplification was conducted over 40 cycles of

qPCR in StepOnePlus™ PCR system (Applied Biosystems).

Sampling

Three time-dependent leaf samplings were conducted in this experiment. Roots

were collected at the end of the experiment to avoid changes in the leaf gene

expression analysis that may arise because of root damage. The first sampling of

leaves was performed based on the sprouting and availability of enough plant material

for the gene expression and CaLas detection. At the end of 13 weeks after bud sprout

(WAB), enough sampling material was available on the grafted VAL scion in all

rootstock and nutrient combinations. The rest of the time-dependent samplings were

done at 25 and 37 WAB. Roots were collected at the end of 37 WAB to analyze gene

expression and CaLas detection. All three time-dependent leaf samples were used for

gene expression and CaLas detection. At each time point, the same leaves were

collected for CaLas detection to monitor the effect of nutrient and rootstock treatments

to the Ct value changes. Sampling for CaLas detection was performed by collecting leaf

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discs from the leaf midrib portion using a paper punch; whereas, for gene expression

analysis, fully expanded leaves were collected from each biological replicate.

RNA Extraction and Gene Expression Quantification

Total RNA was extracted from the collected leaves or roots, using Qiagen

RNeasy Mini Plant RNA Extraction kit (Qiagen Inc., Valencia, CA). The manufacturer’s

protocol was followed using 100 milligram starting material and on-column DNase

treatment performed with RNase free DNase Free kit (Qiagen). Quantity and quality of

the extracted RNA were analyzed in a NanoDrop spectrophotometer. Quantitative real-

time reverse transcriptase (qRT-RT) PCR technique was used to amplify the selected

genes. A total of six genes were selected to analyze gene expression pattern in

response to nutrient formulation and rootstock treatments. These genes were nutrient

transporters, defense and HLB-response associated genes (Table 6-3). Gene specific

primers and probes were designed using Integrated DNA Technologies (IDT) Primer

Quest Tool (IDT, Coralville, IA). For normalization, GLYCERADEHYDE-3-PHOSPHATE

DEHYDROGENASE C2 (GAPC) forward and reverse primer pairs and GAPC specific

probe were used as the reference gene control. TaqMan® and Power SYBR® green

RNA-to-Ct™ 1-Step RT PCR (Applied Biosystems) master mix was used to convert

total RNA into cDNA and then amplify it with the primers and probe combination. A total

of 50 ng of DNase free RNA and 500 nM of each primer and probe were used to amplify

and quantify the real-time gene expression. Amplification was performed over 40 cycles

in a StepOnePlus™ real-time PCR machine (Applied Biosystems).

Relative gene expression quantification was calculated using ΔΔCt method

(Livak and Schmittgen, 2001). For each sample, the Ct value of GAPC was subtracted

from the Ct value for the gene of interest to get ΔCt value. ΔCt values obtained from

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leaves of healthy VAL grafted onto Sw_NM interaction were used as the control. The

relative gene expression was calculated as Log2 fold change (Log2 FC) scale. Data

analysis of differential gene expression (DGE) in response to rootstock and nutrient

interactions was conducted using the generalized mixed model in the JMP13 statistical

software (SAS Inc., Cary, CA). The significant differences were estimated using HSD

Tukey test at p ≤ 0.05. The visual presentation of the data was created, using Microsoft

Office Excel 10.

Plant Phenotype Analysis

Plant phenotypic analyses were conducted at 37 WAB time point. These

analyses include the presence of HLB symptoms in leaves, plant height, and the

number of branches and diameters of the plants (5, 10 and 15 centimeters above and

below the graft union). Significant differences in the phenotypic observations were

analyzed, using JMP13 (SAS Inc.) and tested using HSD Tukey test at p ≤ 0.05. The

visual presentation of the data was created with Microsoft Office Excel 10.

Results

CaLas-Detection

The Ct value of the tree that used for budsticks collection was in the range of 25-

27, and selected budsticks tested negative for CTV. In the ECRF treatment, VAL/Sw,

and VAL/Tetr plants that were infected by two different HLB inoculation methods, stick-

graft infection and blind-bud infection, did not show a significant difference in their Ct

values. Therefore, stick-graft infected and blind-bud infected plants in each rootstock

treatment were pooled into their respective rootstock and nutrient formulation

combinations, and named as Sw_ECRF and Tetr_ECRF. Real-time qRT- PCR based

CaLas detection results showed that in all combinations, the 13 WAB time point had the

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lowest Ct values compared to the 25 WAB and 37 WAB time point Ct values (Figure 6-

2A). At 13 WAB, VAL grafted onto the Sw_NM combination had the lowest mean Ct

value as compared to 13 WAB Cts in other combinations. At 13 WAB time point, the

mean of Ct values of CaLas-infected VAL grafted onto Sw_NM, Sw_ECRF, Tetr_NM

and Tetr_ECRF were 24.3, 30.4, 26.7 and 31.1, respectively (Figure 6-2A).

At the 25 WAB time point, the mean Ct value of VAL grafted onto Sw_NM

interaction increased non-significantly compared to its 13 WAB. However, the mean Ct

of Sw_NM combination at 25 WAB was significantly low compared to the Ct values of

the rest of three combinations at the 25 WAB (Figure 6-2A). At the 25 WAB, mean Ct of

VAL grafted onto Sw_ECRF, Tetr_NM and Tetr_ECRF combinations increased

significantly compared to their 13 WAB (Figure 6-2A).

At the 37 WAB time point, the mean Ct value of VAL grafted on Sw_NM,

Sw_ECRF, Tetr_NM and Tetr_ECRF combinations changed compared to their Ct at 25

WAB, but indifferently (Figure 6-2A). The mean Ct value of Sw_NM interaction at 37

WAB time point was 25 which was the lowest as compared to the other three

combinations (Figure 62-A). Overall results of CaLas detection showed that the mean

Ct value of VAL grafted onto Sw_NM remained the lowest at all three-time points

compared to VAL grafted onto the Sw_ECRF, Tetr_NM and Tetr_ECRF combinations.

Roots collected from three biological replicates of Sw_NM combination detected

Ct value in the range of 28-32. In Sw_ECRF, Tetr_NM, and Sw_ECRF combinations,

only one biological replicate in each combination detected CaLas in the roots. (Figure 6-

2B).

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DGE Analysis of The Defense and Transporter Genes in Leaves

Differential expression analysis of the NPR1 gene

Comparative gene expression analysis of NPR1 in leaves of CaLas-infected VAL

grafted onto two rootstocks under two different nutrient formulations showed

significantly altered NPR1 transcripts expression levels at three different time points.

The combination of Sw and NM upregulated NPR1 expression level in the CaLas-

infected VAL at the 13 WAB, but the NPR1 expression level decreased significantly in

25 WAB and 37 WAB in the same combination (Figure 6-3A). CaLas-infected VAL

grafted onto Sw_ECRF combination had downregulated NPR1 expression at 13 WAB

and 25 WAB. However, at the 37 WAB time point, NPR1 expression level upregulated

significantly in the same combination (Figure 6-3A). The pattern of NPR1 regulation in

the CaLas-infected VAL grafted onto Tetr_NM and Tetr_ECRF was similar to that of the

Sw_ECRF-NPR1 expression pattern at all three-time points (Figure 6-3A). The

expression level of NPR1 at 13 WAB and 25 WAB was significantly lowered in

Tetr_ECRF as compared to the respective time points in Tetr_NM. There were no

significant differences in the NPR1 expression levels in VAL grafted onto Tetr_NM and

Tetr_ECRF combinations at 37 WAB. Overall, the pattern of Tetr_NM and Tetr_ECRF-

induced NPR1 expression in the CaLas-infected VAL was significantly different from the

VAL/Sw_NM combination.

Differential expression analysis of the NPR3 gene

Differential expression analysis of NPR3 at three-time points showed fluctuations

in the expression levels in all nutrient and rootstock combinations. In all nutrition and

rootstock interaction combinations, CaLas-infected VAL showed either suppression or

negligible expression of the gene at three different time points. However, at 25 WAB,

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the expression level of NPR3 was highly increased in the Sw_ECRF, and slightly

upregulated in the Tetr_NM combinations (Figure 6-3B).

Differential expression analysis of the PP2B gene

Relative expression quantification of PP2B in CaLas-infected VAL under the

influence of different rootstocks and nutrient formulations did not change significantly.

Sw_NM, Sw_ECRF and Tetr_ECRF combinations had higher PP2B expression levels

at the 13 WAB among all three-time points in the CaLas-infected VAL, which was then

downregulated significantly in the later two time points (Figure 6-3C). In the Tetr_NM

combination, the mean of PP2B expression level was close to 0 log2 fold change (log2

FC), which was then increased significantly at 25 WAB and again decreased

significantly at 37 WAB (Figure 6-3C). Overall, regardless of differences in the nutrient

formulations and rootstock, PP2B followed the similar expression pattern over three

different time points in VAL for all rootstock and nutrition combinations.

Differential expression analysis of the ZRT2 gene

ZRT2 expression pattern in VAL scion was different in response to all

combinations of rootstocks and nutrient formulations. At 13 WAB, all combinations

showed downregulated ZRT2 expression levels (Figure 6-3D). In the Sw_NM

combination, the expression levels of ZRT2 in VAL upregulated significantly in 25 WAB

and 37 WAB as compared to 13 WAB, and in Sw_ECRF combination, ZRT2 expression

levels always remained lower compared to Sw_NM at all three-time points (Figure 6-

3D). In the Sw_ECRF, ZRT2 expression level was significantly downregulated at 13

WAB as compared to the 25 WAB and 37 WAB, but the differences between 25 WAB

and 37 WAB time points were non-significant (Figure 6-3D). CaLas-infected VAL grafted

onto 4x Tetr rootstock followed the similar pattern of ZRT2 expression in both nutrition

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formulations. VAL- ZRT2 gene expression always remained downregulated in all three

times points in the Tetr_NM and Tetr_ECRF combinations (Figure 6-3D).

Differential expression analysis of the NRAMP2 gene

Expression analysis of NRAMP2 encoding metal co-transporter in the leaves of

CaLas-infected VAL grafted onto the Sw and Tetr rootstocks under NM nutrient

formulation followed a different pattern as compared to the ECRF nutrient formulation

(Figure 6-3E). The interaction between Sw and NM downregulated NRAMP2 expression

level in VAL at 13 and 25 WAB and upregulated NRAMP2 expression significantly at the

37 WAB time point (Figure 6-4E). Whereas, the effect of Tetr_NM combination

suppressed NRAMP2 expression in VAL at 13 WAB, and then increased significantly at

25 WAB, and again downregulated non-significantly at 37 WAB (Figure 6-3E). ECRF

nutrient formulation had a similar effect on the expression of NRAMP2 in the CaLas-

infected VAL regardless of rootstocks. The expression level of NRAMP2 was

remarkably high at 13 WAB time point in the CaLas-infected VAL under ECRF

formulation, which later decreased significantly in 25 WAB and 37 WAB time points in

the both rootstocks (Figure 6-3E).

Differential expression analysis of the NIP6 gene

CaLas-infected VAL exhibited differential expression of boron transporter NIP6

pattern in the different rootstocks and nutrition formulation interactions. NIP6 expression

level remained upregulated in CaLas-infected VAL under the influence of Sw_NM

combination. In the same combination, NIP6 expression levels were upregulated at all

time points. Among these, the significantly highest expression level of NIP6 was

observed at 13 WAB. (Figure 6-3F). NIP6 expression in the CaLas-infected VAL

followed the zigzag pattern among all three-time points in Tetr_NM, which showed NIP6

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expression upregulated at 13 and 37 WAB, and downregulated at 25 WAB (Figure 6-

3F). ECRF supplemented Sw, and the Tetr rootstock showed the similar pattern of NIP6

expression in the CaLas-infected VAL in which NIP6 expression downregulated at 13

and 25 WAB, but increased significantly at 37 WAB in VAL scion (Figure 6-3F).

DGE Analysis of Defense and Transporter Genes in Roots

Comparative analysis of the selected genes in roots of all the combinations

showed downregulation in their expression levels except NPR3, FRO2 and PP2B genes

in the NM_Tetr combination (Table 6-4). NPR1 expression level downregulated non-

significantly in roots collected from all the combinations except Sw_NM combination.

Root-NPR3 expression level was significantly downregulated in ECRF applied

formulation as compared to the NM formulation in both the rootstocks. Roots collected

from Tetr_NM combination showed increased expression of FRO2, but this increased

expression was insignificant compared to Tetr_ECRF combination. The expression level

of NRAMP2 and NIP5 remained downregulated in roots of all the combinations with no

significant differences. The expression level of PP2B was significantly low in Sw roots

as compared to Tetr roots in both the nutrient formulations.

Plant Phenotype Analysis

Rootstock and nutrient formulation interactions had a significant impact on the

total plant growth and appearance of the HLB-like symptoms at the end of the

experiment. CaLas-infected VAL grafted onto the Sw rootstock showed significant

differences in plant phenotypes under the two different nutrient formulations. CaLas-

infected VAL/Sw plants fertilized with ECRF were significantly taller as compared to the

NM formulation (Figure 6-5A). The average height of VAL/Sw plants under ECRF

nutrition was 96 cm with the significantly lower percentage of HLB-like symptomatic

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leaves at the end of 37 WAB. Whereas, the average height of the VAL/Sw combination

plants grown under NM formulation was 64 cm, and about 33% of a total number of

leaves were showing HLB-like symptoms at the end of 37 WAB (Figure 6-4).

Phenotypic analysis of CaLas-infected VAL grown onto Tetr candidate rootstock

exhibited notably improved plant phenotype as compared to the VAL/Sw under ECRF

and NM nutrition (Figure 6-5B). VAL/Tetr plants were significantly taller; 108 cm and

122 cm in NM and ECRF nutrient formulations, respectively, and only 16% and 10% of

a total number of leaves were showing HLB-like symptoms, respectively, in NM and

ECRF fertilization formulations (Figure 6-4). VAL/Tetr_ECRF combination also showed

significantly fewer branches among all combinations (Table 6-5).

Discussion

Defense is important for plant survival, but growth and development are essential

for plant sustainability. Hence, plants optimize the distribution of energy resources

towards defense and growth. However, some of the biotic and abiotic circumstances

pose a great challenge to plants in maintaining a balance between defense and growth.

Efficient nutrition management is a key component for increasing plant sustainability

under disease and pest pressure (Dordas, 2008). In HLB, it is reported that CaLas-

infected susceptible citrus cultivars have very low economic viability and sustainability

(Folimonova et al., 2009). In addition, conventional nutrition management is inefficient to

reduce the disease pressure and improve plant performance in the CaLas-infected

plants (Xia et al., 2011; Gottwald et al., 2012). Also, DGE analysis of the nutrient

transporters and plant growth associated genes discussed in Chapter 5, which

proposes a hypothesis that a lack of adequate nutrition may increase HLB disease

severity in VAL/SW. Therefore, learning from past experiences and referring to the

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contemporaray work to understand HLB-citrus interaction, an important aim of this

greenhouse study was to investigate the importance of utilizing a 4x putatively HLB-

tolerant rootstock and efficient nutrition management to enhance the plant performance

of CaLas-infected scion and improve the overall HLB tolerance in the grafted trees.

The relative quantification analysis of the micronutrient transporter encoding

genes and defense associated NPR1 gene in this study showed that modifying

conventional NM fertilizer formulation in combination with a newly developed putative

HLB-tolerant rootstock can show differential molecular changes in the CaLas-infected

VAL scion. Moreover, plant phenotypes can also be improved. The changes in the Ct

values over three different time points showed that VAL grafted onto Sw and provided

with ECRF nutrient formula could decrease CaLas population. These results highlight

the effect of improved micronutrient management in the CaLas-infected plants.

Deficiency symptoms of Zn, B, Fe and Cu nutrients in the CaLas-infected plants have

been previously studied (Schumann and Spann, 2009). The results of this greenhouse

experiment suggest that optimal increase of micronutrients content in the fertilizer

formulation is helpful to improve plant phenotypes of HLB-susceptible citrus varieties

such as the VAL/Sw combination. qRT-PCR based Ct values generated in CaLas-

infected VAL/Tetr combination under NM formulation highlight the rootstock inherent

capacity to lower the CaLas bacterial population at three different time points that were

selected in this study. Although there were no significant differences observed between

the Ct values of VAL/Tetr combination provided either with ECRF and NM, the

phenotype of CaLas-infected VAL/Tetr was superior when provided with ECRF

formulation. The improved phenotype of VAL/Tetr, provided with either of nutrient

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formulations, shows the potential of the Tetr rootstock in defending CaLas infection

under field conditions.

NPR1 is a key protein in activating SAR induced plant defense (Kinkema et al.,

2000; Wu et al., 2012). Also, a study on the cloning and transfer of Arabidopsis NPR1

gene into transgenic citrus reported that overexpression of Arabidopsis NPR1 in

transgenic citrus could increase tolerance to HLB (Dutt et al., 2015). The differential

expression patterns of NPR1 in response to the rootstock and nutrient formulations in

this study underline the possibility of manipulation of defense responses in the CaLas-

infected citrus by adopting improved rootstock(s) and nutrition practices. The NPR1 like-

3 (NPR3) is a negative regulator of NPR1 (Shi et al., 2013; Seyfferth and Tsuda, 2014).

The role of NPR3 is to switch off the NPR1 expression under healthy plant conditions.

However, it can affect the NPR1 expression under stressed conditions also. Hence,

there is a negative correlation between NPR1 and NPR3 expression. In the Sw_NM

combination, increased level of NPR1 in the CaLas-infected VAL at the 13 WAB time

point and significant downregulation at later time points suggest that under conventional

fertilizer formulation, Sw can activate the defense by upregulating NPR1 at an early

stage of CaLas spread; but, NPR1-dependent defense lowers gradually as CaLas

infection progresses in the plant. When susceptible Sw rootstock was fertilized with

ECRF, the NPR1 expression pattern reversed in VAL scion. The opposite pattern of

NPR1 expression in VAL grafted onto Sw_ECRF and Sw_NM combinations

emphasizes an important role of micronutrients in altering NPR1 expression in CaLas-

infected and susceptible plants. VAL/Tetr under NM and ECRF application showed

lower expression of NPR1 at 13 WAB, and at later time points, it increased significantly.

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The lower percentage of HLB-like symptoms, higher Ct values and reduced expression

of NPR3 is consistent with the NPR1 induced possible defense at the 25 WAB and 37

WAB time points in VAL/Tetr_NM and VAL/Tetr_ECRF plants.Both greenhouse and

field CaLas-infected plants are found to be deficient in micronutrients such as B, Zn,

Mg, Mn, Fe and Ca, with deficiencies in roots much greater than in leaves (J.W.

Grosser, personal communication). Hence, genes encoding micronutrient transport

ZRT2, NRAMP2, NIP6 were selected to analyze the effect of the interaction of the

enhanced nutrient formulation and improved rootstock on the cellular alterations of

micronutrients in the CaLas-infected VAL. Citrus is a strategy I plant which responds to

Fe deficiency by acidifying rhizosphere Fe3+ ions to Fe2+ ions to make it available for the

plant uptake (Martínez-Cuenca et al., 2013). There are many Fe2+ regulators found in

plants. Iron deficiency or toxicity regulates the expression of Fe2+ regulators (Walker and

Connolly, 2008). The Fe regulators are transcription factors, transporter genes,

enzymes, etc. Fe specific transporters also compete for Zn and Mn mobility in the plants

(Bashir et al., 2016). Especially Fe transporters belong to the ZIP family also regulate

Zn and Mn uptake based on the availability of Fe, Mn and Zn (Guerinot, 2000). Another

group of metal transporters is known as transition metal transporters such as NRAMP.

The NRAMPs are present on the vacuolar membranes and their expression level

changes according to the demand and supply of metals in the plant cytoplasm (Hall and

Williams, 2003). The activity of ZRT2 gene is studied in Saccharomyces cerevisiae

(Zhao and Eide, 1996) suggesting its role in Zn uptake in Zn limiting conditions. The

results of ZRT2 expression changes in Sw_NM did not match the results obtained in S.

cerevisiae which showed that ZRT2 expression increased in Zn replete condition and

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downregulated under Zn deficiency (Bird et al., 2004). The whole scenario of ZRT2 and

NRAMP2 regulation in the case of Sw_NM interaction suggests that under a traditional

nutrition supply, and increasing CaLas infection, VAL/Sw plants experience the Zn

deficiency which was exhibited by a higher percentage of HLB-like symptoms in

VAL/Sw_NM plants; while, the suppression of ZRT2 and fluctuating NRAMP2

expression level in the Tetr_NM interaction suggests the efforts of Tetr rootstock to

maintain Zn homeostasis in the CaLas-infected VAL.

The interaction between ECRF with Sw and Tetr rootstocks influenced the ZRT2

and NRAMP2 expression levels in the CaLas-infected VAL in a similar fashion (Figure

6-3D and E). In VAL/Sw_ECRF and VAL/Tetr_ECRF plants, the expression level of

ZRT2 was strongly downregulated, and NRAMP2 was significantly upregulated at 13

WAB, and in later two-time points, ZRT2 and NRAMP2 levels remained downregulated.

Strong upregulation of NRAMP2 gene expression at 13 WAB time point indicates that

the micronutrient deficiency may compensate by releasing the deficient nutrients from

the storage organelles. The expression level patterns of ZRT2 and NRAMP2 in the VAL

grafted onto two different rootstocks and grown under ECRF nutrition underscore the

positive effect of enhanced micronutrient content on balancing the Zn status in CaLas-

infected plants.

CaLas-infected plants are also lacking adequate B content (Spann and

Schumann, 2009). The deficiency of B is generally exhibited as corky veins in the plants

(Yang et al., 2013). In Arabidopsis, B transporters were identified. These are Nod-like

intrinsic protein (NIP) and boric acid channels. Expression of NIP6 is shoot-specific

(Tanaka et al., 2008), whereas NIP5 is a root-specific transporter (Takano et al., 2006;

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Tanaka et al., 2011). The study of Arabidopsis-NIP6 gene reported that NIP6 facilitates

rapid permeation of boric acid across the membrane. Also, NIP6 mRNA accumulated

about 1.4 FC under B-deprived condition compared to the high B condition (Tanaka et

al., 2008). The increased levels of NIP6 transcripts throughout all three-time points in

the Sw_NM may indicate B deficiency in the CaLas-infected VAL. The pattern of NIP6

expression was quite similar in CaLas-infected VAL grafted onto Sw_ECRF and

Tetr_ECRF at all three-time points. It showed downregulation of NIP6 at 13 WAB and

25 WAB and increased expression of NIP6 at 37 WAB (Figure 6-3F). The pattern of

NIP6 expression in the ECRF provided rootstocks suggests a potential role of improved

micronutrient nutrition practices in lowering the B deficiency. The fluctuations in the

NIP6 expression levels in Tetr_NM combination support the rootstock specific response

to maintain B homeostasis in the CaLas-scion.

Gene expression analysis of roots did not show upregulation of the selected

genes. However, there were significant differences in the expression of NPR1, NPR3,

FRO2 and PP2B in both the rootstocks and nutrient formulation combinations. The

NPR1 expression levels in roots of the Sw_NM combination were significantly

suppressed as compared to the other combinations. A significant decrease of NPR1 in

roots the Sw_NM supports the downregulated expression level of NPR1 in leaves of the

same combination at 37 WAB. Significantly suppressed NPR3 expression in the ECRF

formulation at the end of 37 WAB suggests that ECRF formulation may help to

downregulate NPR3 expression. Suppression of NPR3 expression also supports the

less suppression of NPR1 expression in roots of Sw_ECRF and Tetr_ECRF as

compared to Sw_NM. Iron starvation indicator FRO2 expression changes were studied

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in citrus (Martínez-Cuenca et al., 2013). According to the study, the higher levels of Zn2+

and Mn2+ disturb the balance between Fe, Zn and Mn in the root growth culture media of

citrus. Also, FRO2 overexpression indicates an Fe2+ starved condition (Walker and

Connolly, 2008). FRO2, encoding ferric chelate reductase (FC-R) enzyme, has showed

increased activity in the Fe deficient condition (Robinson et al., 1999). According to

Martínez-Cuenca et al. (2013), the overexpression of FRO2 was a result of Zn2+ and

Mn2+ competatition with Fe2+ that could affected Fe2+ compartmentalization, thereby

induced to Fe2+ starvation. Similarly, FRO2 upregulation in Tetr_NM and in Tetr_ECRF

may be a result of competition between Zn, Mn and Fe uptake that led to possible Fe2+

starved condition in the plants. In our study, there is not adequate evidence to explain

the expression pattern of NRAMP2. However, from the results it can be speculated that

Fe2+ starvation would have increased the expression level of NRAMP2 in roots, but

there was not a significant increase in the NRAMP2 expression level. Therefore, the

expression pattern of NRAMP2 in roots observed in this study is not conclusive with the

data available. NIP5 is a plasma membrane protein and required for B uptake under the

B depleted condition (Takano et al., 2006). The downregulated NIP5 expression level in

roots of all combinations suggest the plants were not experiencing B deficiency. Plants

involve many other nutrient transporters that may influence the expression pattern of the

selected genes in this study, directly or indirectly. However, the primarily results of the

selected genes in this study underscore the importance of efficient micronutrient

management to increase plant sustainability when plants are CaLas-infected. Further

field evaluations and molecular analyses of the combinations are required to confirm the

greenhouse results.

262

Overall, the greenhouse study underscores the potential role of root-applied

improved micronutrient formulations to lessen the HLB disease symptoms that are

caused by micronutrient deficiencies and other disease damage. Also, DGE analysis of

the selected genes suggests that HLB-induced nutrient deficiency or defense

development responses can be identified at the molecular level in the early stage of

HLB infection. Identifying the plant's stress at an early stage of infection may help to

implement preventive disease strategies needed to inhibit severe disease damage. The

positive response of the Valencia/SW_ECRF trees in this study may help explain the

positive turnaround of CaLas-impacted commercial sweet orange/SW trees in various

groves that have been converted to new, evolving nutritional programs. The positive

phenotypic and gene expression analysis results obtained in VAL grafted onto the

putative HLB-tolerant Tetr rootstock opens new avenues for growers and researchers to

test new scion/rootstock combinations utilizing improved HLB-tolerant citrus rootstocks

along with enhanced nutrition programs in the field.

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Table 6-1. Rootstock treatments

Rootstock Rootstock parents Scion

Swingle; 2n (Sw)

Grapefruit X Trifoliate orange Valencia; (VAL)

Putative HLB-tolerant tetraploid;4x (Tetr)

Amblycarpa + Volkmeriana X Orange 19 (UFR-4)

Valencia; (VAL)

Table 6-2. Controlled release fertilizer formulations

Nutrients Harrell’s Nursery Mix

(NM) % Harrell’s enhanced controlled release fertilizer (ECRF) %

N-P-K 16-5-10 15-6-12

Calcium absent 4.5

Boron absent 0.07

Zinc 0.05 0.71

Manganese 0.09 0.92

Iron 0.23 1.09

Magnesium 1.43 0.98

Potassium 10 12

Copper 0.05 0.04 Nutrient’s content is expressed in the percentage concentration.

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Table 6-3. Primer sequences Gene Identification Abbr∞. Primer sequence*

LOC102617188/ C. sinensis_Non-expressor of PR1

NPR1 F- 5’-CCAGAGTTGGTGGCTCTTTAT-3’

R- 5’CTGAGTCTAGTGCTCGATGTATTC-3’

Ciclev10017873m.g/LOC102621158/NPR1 like-3

NPR3 F- 5’-AGGTTCTCAGCCTCCGGATTA-3’

R- 5’-CCATCGGATTCCTCATTTC-3’

Ciclev10003574m.g/Phloem protein 2B PP2B

F- 5’-AAGCAGATGGTGAAGTCAAGAG-3’

R- 5’-CTCTCCCAATTCAACCTCCATC-3’

P- 5’-/56-FAM/ACTGAAGGT/ZEN/GGTGGTGATGAAAGGT/3IABkFQ/-3’

Ciclev10030012m/Zinc-iron regulated 2 ZRT2

F- 5’-AAGTGGGAACCGATGGTAATC-3’

R- 5’-AAACAGAGGGCGAGAATGAG-3’

P- 5’-/56-FAM/CAGGACAAAG/ZEN/TTCCATTGGAGACACCA/3IABkFQ/-3’

Ciclev10025318m.g/Natural resistance-associated macrophage 2

NRAMP2

F- 5’-TGGAGTTGTGGGCTGTATTATC-3’

R- 5’-CTTGGACACGGCCTTTCTTA-3’

P- 5’-/56-FAM/CTTGATTGC/ZEN/ACAAGAGCGGAGTGC/3IABkFQ/-3’

Ciclev10026151m/Nod-like intrinsic protein;6

NIP6

F- 5’-GCTGTCATGGTCGTCATCATCTTAT-3’

R- 5’-TCCATGGAAAGTGCTTTAGGG-3’

P- 5’-/56-FAM/CTGCTGTCA/ZEN/CCATTGCCTTTGCTG/3IABkFQ/-3’

Ciclev10001994m/ Nod-like intrinsic protein;5

NIP5

F- 5’-CTCTCAACAGGACAATCTCTG-3’

R- 5’-TATGTAAGCCGGAACCTGAAC-3’

P- 5’-/56-FAM/TCCGTCCCT/ZEN/TACCATAGCATTTGCG/3IABkFQ/-3’

Ciclev10019053m/Ferric oxide reducatse-2 FRO2

F- 5’- GGGCAGTTACAAACAACATCTC -3’

R- 5’-CCACATGGCAAGACCAGATA-3’

P- 5’-/56-FAM/AATATCAAA/ZEN/CGTGGCCGGAGAGCT/3IABkFQ/-3’

Glyceradehyde-3-phosphate dehydrogenase C2

GAPC

F- 5’- GACATCAACGGTAGGAACTCG -3’

R- 5’-CAATGAAGGACTGGAGAGGTG-3’

P- 5’-/56-FAM/CTTCAACAT/ZEN/CATTCCCAGCAGCACC/3IABkFQ/-3’

*F; forward primer sequence, R; reverse primer sequence; P; probe; /56- FAM/, 5’6 FAM™ FAM dye; /3IABkFQ/, ZEN- 3’Iowa Black® FQ quencher. ∞ abbreviations used in the text and figures.

265

Table 6-4. Gene expression analyses in the roots

Combinations Gene expression analysis changes (Log2 FC)

NPR1 NPR3 FRO2 NRAMP2 NIP5 PP2B

Sw_NM -2.14a -4.22a -5.02b -2.06 a -0.40 a -1.99c

Sw_ECRF -0.61b -14.12c -4.00 b -1.13 a -1.10 a -3.36c

Tetr_NM -1.80b 1.25 b 2.10 a -1.74 a -1.61 a 0.70a

Tetr_ECRF -1.59b -13.78c -0.88 ab -0.40 a -0.99 a -1.25b Significant differences were calculated at P < 0.05 using HD Tukey test. Different letters indicate the significant differences in the Log2 FC value for the same gene among the different combination at 37 WAB.

266

Table 6-5. Phenotypic measurements including tree diameters below and above the graft union, and no.of branches/tree

Combinations Below graft union Above graft union No. of

branches Dia_5cm Dia_10 cm Dia_15 cm Dia_5 cm Dia_10 cm Dia_15 cm

Sw_NM 12.55 12.14 12.13 8.81 8.52 7.89 4.80

Sw_ECRF 9.55 10.24 9.68 8.37 8.42 7.76 4.25

Tetr_NM 8.94 9.08 9.35 6.82 8.16 7.17 3.75

Tetr_ECRF 8.20 8.00 8.41 6.72 6.51 6.37 2.66* *Significant difference between combinations at p < 0.05 using HD Tukey’s test

267

A B Figure 6-1. CaLas-infected Valencia sweet orange (VAL) stick grafts. A) VAL grafted onto SW rootstock. B) VAL grafted onto Tetr rootstock.

268

Figure 6-2. CaLas detection from leaves and roots of different combinations. A) Leaf samples B) Root samples at 37 WAB.

In leaves, different letters indicate significant differences in the Ct values among the 3 time points for each combination. In roots, only the Sw_NM combination had all 3 biological replicates CaLas-infected, whereas only one plant was CaLas-infected in each of the remaining combinations at 37 WAB

B

A

269

Figure 6-3. Gene expression analysis of the selected genes in leaves. A) NPR1, B) NPR3, C) PP2B, D) ZRT2, E) NRAMP2, F) NIP6. All values are expressed in Log2 fold change. Different letters indicate significant differences in the Log2 FC value for the same gene among the three different time points

B

C

A

270

Figure 6-3. Continued

D

E

F

271

Figure 6-4. Phenotypic differences in rootstock-nutrient formulation combinations. Different letters indicate the significant differences in the height and % HLB symptoms within the four different scion/rootstock combinations.

Figure 6-5. Plant phenotype at 37 WAB. A) VAL grafted onto Sw rootstock. B) VAL grafted onto Tetr rootstock. NM: nursery mix; ECRF: enhanced controlled release fertilizer

A B

272

CHAPTER 7 SUMMARY AND CONCLUSIONS

A good understanding of plant-pathogen interactions is important in plant

breeding and plant pathology studies. Plant genetics, environmental conditions, and

pathogenicity play key roles in plant-pathogen interactions. In Florida, prevalent

huanglongbing (HLB) disease has been causing overwhelming economic losses due to

reduced plant sustainability. Koch’s postulates are not proven in case of HLB-causing

putative Candidatus Liberibacter asiaticus (CaLas). Therefore, inadequate knowledge of

CaLas limits researchers to identify the bacteria pathogenicity mechanism, and

therefore find an efficient solution. Therefore, the overall goal of this study is to analyze

the role of improved-rootstock/scion interactions to fight against HLB and to improve

plant sustainability in HLB-affected plants. This dissertation includes a field study, and a

greenhouse study that also includes a nutrition component.

The citrus plant is a two-component system in which rootstock and scion are

crucial to commercial citrus production. Citrus rootstocks and scions have a significant

effect on important horticultural traits and fruit production. Therefore, the aim of the field

study was to understand the differential effect of citrus rootstocks in regulating

‘Valencia’ (VAL) sweet orange scion performance at different stages of HLB disease

development. RNA-sequencing based comparative differential gene expression (DGE)

analysis of two citrus scion/rootstock combinations was conducted. These combinations

were ‘Valencia’/Swingle (VAL/SW) and ‘Valencia’/improved candidate rootstock

(VAL/CAN). The commercially used SW rootstock is HLB susceptible under

conventional nutrition programs. Whereas, the CAN is a putatively HLB-tolerant

273

rootstock developed by the citrus breeding program at the University of Florida, Citrus

Research and Education Centre (UF-CREC).

Fruit juice quality results obtained in the field study showed that irrespective of

the stages of HLB development, the VAL fruit juice Brix and BRIX-acid ratio remained

higher in VAL/CAN -asymptomatic and -symptomatic treatments as compared to the

symptomatic VAL/SW combination. A lower BAR value of symptomatic VAL/SW fruit

juice was possibly the result of a significant higher acidity percentage. The two years

data of fruit juice quality from symptomatic and asymptomatic VAL/CAN suggest that

the CAN rootstocks can produce standard VAL fruit juice quality, and therefore, this

combination has potential to be commercially accepted in the citrus industry.

Comparative DGE analysis of the VAL/CAN and VAL/SW combinations showed

that asymptomatic VAL/SW had many strongly upregulated defense-associated genes

and the defense was stronger in the symptomatic stage; whereas, CAN rootstock

showed more tolerance to HLB by upregulating a smaller number of defense-related

genes. Upregulation of a greater number of defense and immunity-associated genes in

the VAL/SW combination as compared to VAL/CAN suggest the overreaction of

commercial rootstock such as SW to HLB infection. The higher sensitivity of VAL/SW

combination to HLB suggests that these plants may be investing more energy for

defense and create an energy distribution imbalance between defense and routine

metabolism/growth. Studies on the plant growth-defense tradeoff suggested that more

energy investment in the defense comes at the cost of compromised plant growth and

development (Denancé et al., 2013; Huot et al., 2014). The similar theory may support

the gradually deteriorating health of HLB-affected commercial scion/rootstock

274

combinations. CaLas-infected VAL/CAN phenotypes showed that the plants were

healthy and less symptomatic as compared CaLas-infected VAL/SW combination. DGE

analysis of the field grown scion/rootstock combinations in this study showed distinct

differences in the expression level of growth and development associated genes.

VAL/CAN combination showed upregulation of growth factors GRF7 in asymptomatic

leaves, cell wall modifying pectin methyl esterase in asymptomatic leaves, phloem

regenerating transcription factor KANADI in symptomatic leaves and roots, and root

promoting phytosulfokines in the symptomatic roots as compared to respective HLB

stages and plant tissue of VAL/SW suggesting that CaLas-infected VAL/CAN plants

may be improving tolerance to HLB through promoting phloem regeneration, cell wall

modifications and root growth.

Differential expression changes in the hormonal metabolism related genes

between VAL/CAN and VAL/SW highlight the possible reasons of HLB-induced damage

in the VAL/SW combination. Commercial rootstock such as SW has found to have

severe root loss under HLB infection. Therefore, a significant overrepresentation of

water deprivation and salt stress biological functional categories in the asymptomatic

and symptomatic VAL/SW is apparent. Also, upregulation of abscisic acid (ABA)

metabolism genes supports the significant presence of abiotic stress biological category

identified by MapMan, Blast2go and Pathway studio functional analysis in VAL/SW.

Auxin (AU) and ethylene (ET) -metabolism and -response genes were strongly

upregulated in the VAL/SW combination; whereas, brassinosteroid (BR) and AU

response genes were overexpressed in VAL/CAN combination suggesting that AU-ET

crosstalk may favor the CaLas spread and HLB development in the Citrus, and AU-BR

275

interaction is potentially beneficial in balancing defense and growth in VAL/CAN.

Jasmonic acid (JA)-activated MYC gene was strongly upregulated in the symptomatic

VAL/CAN roots indicating the CAN rootstock specific hormonal genes reprogramming in

the advanced stage of HLB development in plants. The activation of MYC branch also

supports the suppression of ET and salicylic acid (SA) signaling pathway in the

symptomatic VAL/CAN.

The research findings of the transcriptome comparison of field grown VAL/CAN

and VAL/SW suggest that CAN and SW rootstock are differentially acting on the VAL

scion by reprogramming hormonal metabolism, defense, and growth-associated genes.

The significant differential expression changes were also observed in the nutrient

transporter and cell wall modification genes. Altogether, the transcriptomic analysis of

CaLas-infected VAL/CAN, and VAL/SW supports that rootstock can differentially

change CaLas-infected scion transcriptome. The significant upregulation of genes

involved in AU-BR interactions, enhanced phloem regeneration ability, cell wall

modifications, transporters regulations, and phytosulfokines signaling suggests possible

role these mechanisms to improve CaLas-infected VAL/CAN sustainability and are

useful to develop testable models for future HLB-citrus interactions studies.

The greenhouse study included a nutrition component, as nutrition is another

crucial aspect of HLB disease management. Nutrition and rootstock interactions are

crucial to deciding the fate of CaLas-infected plants. Therefore, to address the issue of

nutrient deficiency and symptoms severity in CaLas-infected plants, interactions of a

UF-CREC developed putatively HLB-tolerant 4x (Tetr) rootstock, and an enhanced

controlled release fertilizer (ECRF) formulation were studied in CaLas-infected plants

276

under greenhouse conditions. The two scion/rootstock combinations; VAL/Tetr and

VAL/SW, and two nutrient formulations; nursery mix (NM) and enhanced controlled

release fertilizer (ECRF) were tested to analyze the effect of rootstock and nutrient

formulations in mitigating HLB disease severity in the VAL scion. The differential

expression of nutrient transporter genes, improved plant phenotypes and a lower

bacterial titer in the VAL/Tetr_ECRF, VAL/Ter_NM, and VAL/SW_ECRF support the

hypothesis that rootstock and nutrient interactions can differentially modify the response

of VAL scion to CaLas infection. The comparison between VAL/SW_NM and

VAL/SW_ECRF plants showed that ECRF supplied VAL/SW plants had improved plant

phenotype and reduced CaLas titer in the leaves, at the end of the experiment

indicating that root applied ECRF formulation can improve plant phenotype and reduce

CaLas spread in the infected VAL/SW commercial combination under greenhouse

conditions. An improved understanding of rootstock/nutrition interactions should result

in the development of improved production systems (efficient, affordable delivery of

optimal nutrition) that should maximize the performance of improved rootstocks in an

HLB-endemic Florida.

In the situation of resource restrictions, plants must balance the energy

resources spending towards growth and defense. In plants, while the defense is

imperative for survival, it comes at the expense of energy that could use for routine

plant growth/metabolism. As we observed that in the VAL/SW field plants, SA, JA, and

ET hormonal dependent defense and plant immunity genes were strongly upregulated

which suggest that VAL/SW is defending itself against the disease by deploying

different defense mechanisms (Figure7-1). However, this defense may not be long

277

lasting, and resources required for plant growth and development is compromised.

Whereas, in the VAL/CAN combination, overall in both the HLB stages SA, JA, and ET-

activated defense gene responses were not strongly upregulated, but, BR and AU-

responsive genes were significantly upregulated. Therefore, CAN rootstock may have

been operating the defense against HLB at a lower level, but growth/metabolism is not

compromised as to severely impact overall plant health (Figure7-2). Results obtained in

the greenhouse study were supporting the conclusions of the transcriptomic study of

field grown VAL/CAN and VAL/SW combinations. The greenhouse study results

showed that improved root applied nutrition practices may help commercial rootstocks

such as SW to enhance plant phenotype of CaLas-infected VAL scion. The conclusions

of field and greenhouse studies showed that UF-CREC developed CAN and Tetr

rootstocks can be part of the equation to combat the devastating HLB disease in

Florida. Moreover, the combination of evolving, improved nutrition practices and HLB

tolerant rootstocks should play a significant role in obtaining adequate HLB tolerance in

new citrus plantings as necessary to ensure a sustainable and profitable future citrus

industry.

278

Figure 7-1. Graphic presenting summary of CaLas-infected VAL/SW combination. Boxes present possible mechanism (Testable models) those are responsible for each growth and defense regulations in CaLas-infected combination.

Figure 7-2. Graphic presenting summary of CaLas-infected VAL/CAN combination. Boxes present possible mechanism (Testable models) those are responsible for each growth and defense regulations in CaLas-infected combination.

279

APPENDIX A SUPPLEMENTAL DATA FOR CHAPTER 2

Table A-1. Identification of samples for RNA-seq run Experimental Sample ID

Plant tissue

Rootstock Scion Treatment

13B Leaves SW VAL Symptomatic

13D Roots SW VAL Symptomatic

14A Leaves SW VAL Symptomatic


15B Leaves SW VAL Symptomatic


19B Leaves SW VAL Asymptomatic

19D Roots SW VAL Asymptomatic

20B Leaves SW VAL Asymptomatic


21A Leaves SW VAL Asymptomatic


5A Leaves CAN VAL Symptomatic

5C Roots CAN VAL Symptomatic

6A Leaves CAN VAL Symptomatic

6D Roots CAN VAL Symptomatic

7B Leaves CAN VAL Symptomatic

7C Roots CAN VAL Asymptomatic

22B Leaves CAN VAL Asymptomatic





280

Table A-2. Individual RNA-seq library pooling calculations for the sequencing Sampl

e Dilutio

n Avearg

e Concentratio

n Molarity

Pool volume

Pool Pool ng in half

ID factor size (bp)

(pg/ul) (pM) ratio volume volume pool

21A 5 389 1190 5010 0.26546 3.9820 1.99101 2.3693

5C 9 376 1470 6420 0.20716 3.10747 1.55373 2.2839

6D 13 375 1920 8350 0.15928 2.3892 1.19461 2.2936

7C 7 375 1600 6950 0.19136 2.87057 1.43525 2.2964

22C 4 372 1220 5380 0.24721 3.7081 1.85408 2.2619

23C 6 377 1090 4730 0.28118 4.21775 2.10887 2.2986

24D 11 376 2110 9150 0.14535 2.18032 1.09016 2.3002

22B 2 361 723 3310 0.40181 6.02719 3.01359 2.1788

23B 2 397 1050 4320 0.3078 4.61805 2.30902 2.4244

24B 3 397 986 4080 0.32598 4.88970 2.44485 2.410

13B 3 400 1010 4120 0.3228 4.8422 2.42111 2.4453

14A 2 381 808 3480 0.38218 5.73275 2.8663 2.31603

15B 1 386 1220 5210 0.25527 3.82917 1.91458 2.33579

19B 3 382 885 3840 0.34635 5.195 2.5976 2.2989

20B 1 342 1430 6920 0.19219 2.88294 1.44147 2.06130

13D 1 399 2070 8440 0.15758 2.36374 1.18187 2.44647

14D 5 371 684 3030 0.43894 6.58415 3.29207 2.25178

15D 4 401 906 3670 0.3623 5.43596 2.71798 2.46249

19C 1 390 310 1330 1 15 7.5 2.325

20D 2 382 686 2910 0.45704 6.85567 3.42783 2.35149

21D 4 368 878 3920 0.33928 5.08928 2.54464 2.2341

5A 1 461 615 2280 0.58333 8.75 4.375 2.6906

6A 12 375 834 3720 0.35752 5.36290 2.68145 2.23633

7B 20 405 2440 9880 0.13461 2.01923 1.00961

5

The calculations are provided by ICBR sequencing core

281

Figure A-1. Tuxedo pipeline components. (Adapted from Trapnell et al., 2012)

282

Table A-3. Shell Script used to identify DGE between the comparisons mentioned in table 2-1 #!/bin/bash ### Define the project folder PF='/scratch/lfs/aditisatpute/leaves' ### Create the directory structure mkdir $PF/99_qsubs mkdir $PF/01_RNA_seqs/ mkdir $PF/02_quality_trimmed_seqs/ mkdir $PF/03_tophat_align_out/ mkdir $PF/04_cufflinks_assembly_out/ mkdir $PF/06_cuffquant_out/ mkdir $PF/07_cuffdiff_out/ mkdir $PF/00_base_files/ #### Move the file for one folder 01_RNA_seqs ### Inside the folders 01_RNA_seqs for file in $(ls leaves/*); do echo $file ; done for file in $(ls leaves/*/*); do cp $file . ; done for file in $(ls leaves/*/*); do mv $file . ; done ####Constructe the files to use bowtie2 gunzip Cclementina_182_v1.0.transcript.fa.gz module load bowtie2 bowtie2-build -f Cclementina_182_v1.0.transcript.fa Cclementina_182_v1 ### In this case, I already have the bowtie files so I use this commands to cp for file in $(ls /scratch/lfs/isantanna/aditi/00_base_files/*.); do echo $file ; done for file in $(ls /scratch/lfs/isantanna/aditi/00_base_files/*.); do cp $file . ; done cp Cclementina_182_v1.0.gff3.gz ### Merge fastq.gz files #Cat the files #!/bin/bash for file in *L001*R1* do IFS='-' eval 'array=(${file})' echo ${array[0]} base=$()

283

cat *${array[0]}*R1* > ${array[0]}_all_R1.fastq.gz done #!/bin/bash for file in *L001*R2* do IFS='-' eval 'array=(${file})' echo ${array[0]} base=$() cat *${array[0]}*R2* > ${array[0]}_all_R2.fastq.gz done ## create list of files on 01_RNA_seqs/ PF='/scratch/lfs/aditisatpute/leaves/01_RNA_seqs/' ls -1 *_all_R1.fastq.gz | sort >list.txt ####Size of the files wc -l list.txt Trimmomatics #!/bin/bash #PBS -l nodes=1:ppn=8 #PBS -l pmem=300m #PBS -l walltime=01:45:00 #PBS -r n #PBS -N trimmo #PBS -t 1-12 #PBS -o trimmo.log #PBS -j oe #PBS -m abe #PBS -M [email protected] ### qsub running to use trimmomatics PF='/scratch/lfs/aditisatpute/leaves/' IN_FILE1=$(head -n $PBS_ARRAYID /scratch/lfs/aditisatpute/leaves/01_RNA_seqs/list.txt | tail -n 1) IN_FILE2=$(echo $IN_FILE1 | sed -e "s,all_R1,all_R2,g" ) echo $IN_FILE2

284

echo $IN_FILE1 ###IN_FILE2=$(echo $IN_FILE1 | sed s,_R1_,R2_,) OUT1=$IN_FILE1\_PE OUT2=$IN_FILE2\_PE OUT3=$IN_FILE1\_SR OUT4=$IN_FILE2\_SR module load trimmomatic/0.32 #with ILLUMINACLIP# trimmomatic PE -threads $PBS_NUM_PPN -phred33 $PF/01_RNA_seqs/$IN_FILE1 $PF/01_RNA_seqs/${IN_FILE2} $PF/02_quality_trimmed_seqs/$OUT1 $PF/02_quality_trimmed_seqs/$OUT3\ $PF/02_quality_trimmed_seqs/$OUT2 $PF/02_quality_trimmed_seqs/$OUT4\ LEADING:20 TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:36 ## create list of files on 02_quality_trimmed_seqs/ PF='/scratch/lfs/aditisatpute/leaves/' ls -1 *_all_R1.fastq.gz_PE | sort > $PF/02_quality_trimmed_seqs/trimlist.txt ####Tophat #!/bin/bash #PBS -l nodes=1:ppn=8 #PBS -l pmem=2g #PBS -l walltime=20:00:00 #PBS -r n #PBS -N tophat #PBS -t 1-12 #PBS -o tophat.log #PBS -j oe #PBS -m abe #PBS -M [email protected] PF='/scratch/lfs/isantanna/leaves/aditi/' IN_FILE1=$(head -n $PBS_ARRAYID $PF/02_quality_trimmed_seqs/trimmlist.txt | tail -n 1) IN_FILE2=$(echo $IN_FILE1 | sed -e "s,all_R1,all_R2,g" ) BIOREP=$(echo $IN_FILE1| cut -d "_" -f 1,2) #echo $IN_FILE2 cd $PF ### Aligning short reads to the genome with Tophat2 (tophat/2.1.0)

285

module load tophat tophat -p $PBS_NUM_PPN -G $PF/00_base_files/Cclementina_182_v1.0.gene.gff3 \ -o $PF/03_tophat_align_out/$BIOREP\_thout \ $PF/00_base_files/Cclementina_182_v1 $PF/02_quality_trimmed_seqs/$IN_FILE1 $PF/02_quality_trimmed_seqs/$IN_FILE2 ####Cufflinks #!/bin/bash #PBS -l nodes=1:ppn=4 #PBS -l pmem=4g #PBS -l walltime=40:00:00 #PBS -r n #PBS -N cufflinks #PBS -t 1-12 #PBS -o cufflinks.log #PBS -j oe #PBS -m abe #PBS -M [email protected] export MC_CORES=$PBS_NUM_PPN PF='/scratch/lfs/isantanna/aditi/leaves/' cd $PF IN_FILE1=$(head -n $PBS_ARRAYID $PF/02_quality_trimmed_seqs/trimmlist.txt | tail -n 1) IN_FILE2=$(echo $IN_FILE1 | sed -e "s,all_R1,all_R2,g" ) BIOREP=$(echo $IN_FILE1 | cut -d "_" -f 1,2) #Assemble expressed genes and transcripts ### Create a file assemblies.txt that list the assembly file for each sample ####Inside the folders ######ls -1 $PWD/*_clout/transcripts.gtf | cat > assemblies.txt ### Run cuffmerge to create a single merged transcriptome annotation ##cuffmerge -g genes.gtf -s genome.fa -p 8 assemblis.txt cuffmerge -G $PF/04_cufflinks_assembly_out/$BIOREP\_clout/transcript.gtf -s $PF/00_base_files/Cclementina_182_v1.fa -p $PBS_NUM_PPN -o $PF/06_cuffquant_out/$BIOREP\_merged.gtf ### Run cufflinks to identify differentially expressed genes and transcripts ### NOT USING CUFFQUANT

286

module load cufflinks cuffdiff -o $PF/07_cuffdiff_out/ -b $PF/00_base_files/Cclementina_182_v1.fa -u -p $PBS_NUM_PPN -L 13B,5A $PF/05_merged_asm/merged13.gtf $PF/03_tophat_align_out/13B_thout/accepted_hits.bam,$PF/03_tophat_align_out/14A_thout/accepted_hits.bam,$PF/03_tophat_align_out/15B_thout/accepted_hits.bam \ $PF/03_tophat_align_out/5A_thout/accepted_hits.bam,$PF/03_tophat_align_out/6A_thout/accepted_hits.bam,$PF/03_tophat_align_out/7B_thout/accepted_hits.bam \

287

APPENDIX B SUPPLEMENTAL DATA FOR CHAPTER 5

Table B-1. Differentially expressed and significantly upregulated growth associated genes in leaves of the asymptomatic VAL/CAN combination as compared to asymptomatic VAL/SW leaves C. Clementina_ID Arabi ID Gene name Bin Name† Log2 FC Arabidopsis_definition

Ciclev10014887m.g AT1G61820 BGLU46 misc.gluco-, galacto- and mannosidases 0.996597 beta glucosidase 46

Ciclev10020312m.g AT1G17050 SPS2 secondary metabolism. isoprenoids.mevalonate Pathway.geranyl diphosphate synthase

0.97524 solanesyl diphosphate synthase 2

Ciclev10031034m.g AT5G14120 MUA22.12 development.unspecified 0.971787 Major facilitator superfamily protein

Ciclev10030755m.g AT1G70300 KUP6 transport.potassium 0.963527 K+ uptake permease 6

Ciclev10011587m.g AT1G66330 T27F4.8 development. unspecified 0.956597 senescence-associated family protein

Ciclev10008343m.g AT3G13050 MGH6.18 transport. misc 0.955683 Major facilitator superfamily protein

Ciclev10021374m.g AT4G16780 HB-2 RNA.regulation of transcription. HB,Homeobox transcription factor family

0.952059 homeobox protein 2

Ciclev10012332m.g AT5G46800 BOU transport.metabolite transporters at the mitochondrial membrane


Ciclev10025741m.g AT2G17500 MJB20.6 transport.misc 0.926079 Auxin efflux carrier family protein

Ciclev10019080m.g AT1G17840 WBC11 transport.ABC transporters and multidrugresistance systems

0.911355 white-brown complex homolog protein 11

Ciclev10032548m.g AT1G69530 EXPA1 cell wall.modification 0.904924 expansin A1

Ciclev10005245m.g AT2G41290 SSL2 secondary metabolism.N misc.alkaloid-like 0.900795 strictosidine synthase-like 2

Ciclev10005737m.g AT4G15920 DL4000C development.unspecified 0.900184 Nodulin MtN3 family protein

Ciclev10004728m.g AT3G47420 PS3 transporter.membrane system unknown 0.895495 phosphate starvation-induced gene 3

Ciclev10008184m.g AT4G27570 T29A15.60 secondary metabolism.flavonoids.anthocyanins 0.885495 UDP-Glycosyltransferase superfamily protein

Ciclev10007571m.g AT1G55850 CSLE1 cell wall.cellulose synthesis.cellulose synthase 0.862554 cellulose synthase like E1

Ciclev10019511m.g AT1G74780 F25A4.25 development.unspecified 0.847991 Nodulin-like / Major Facilitator Superfamily protein

288

Table B-1. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_definition

Ciclev10032279m.g AT1G69780 ATHB13 RNA.regulation of transcription.HB,Homeobox transcription factor family

0.840293 Homeobox-leucine zipper protein family

Ciclev10016732m.g AT1G70670 F5A18.15 development.unspecified 0.822939 Caleosin-related family protein

Ciclev10014491m.g AT2G32950 COP1 development.unspecified 0.813941 Transducin/WD40 repeat-like superfamily protein

Ciclev10000781m.g AT3G05030 NHX2 transport.unspecified cations 0.796861 sodium hydrogen exchanger 2

Ciclev10031076m.g AT3G26570 PHT2;1 transport.phosphate 0.781768 phosphate transporter 2;1


289

Table B-2. Differentially expressed and significantly upregulated growth associated genes in the leaves of the asymptomatic VAL/SW combination as compared to the asymptomatic VAL/CAN leaves

C. Clementina_ID Arabi ID Name Bin Name† Log2 FC* Arabidopsis_definition

Ciclev10020318m.g AT1G78560 T30F21.11 transport. unspecified cations -0.978515 Sodium Bile acid symporter family

Ciclev10020840m.g AT1G17020 SRG1 secondary metabolism. Flavonoids, flavonols -0.988751 senescence-related gene 1

Ciclev10025677m.g AT4G38250 F22I13.20 transport.amino acids -0.976413 Transmembrane amino acid transporter family protein

Ciclev10028214m.g AT2G17840 ERD7 development.unspecified -0.967868 Senescence/dehydration-associated protein-related

Ciclev10011546m.g AT1G66430 F28G11.11 major CHO metabolism.degradation.sucrose.fructokinase

-0.966871 pfkB-like carbohydrate kinase family protein

Ciclev10000256m.g AT5G03650 SBE2.2 major CHO metabolism. synthesis.starch.starch branching

-0.954209 starch branching enzyme 2.2

Ciclev10028100m.g AT2G28120 F24D13.9 development. unspecified -0.951213 Major facilitator superfamily protein

Ciclev10028425m.g AT2G04305 T23O15.7 transport.unspecified cations -0.940556 Magnesium transporter CorA-like family protein



Ciclev10013854m.g AT3G18110 EMB1270 development.unspecified -0.921116 Pentatricopeptide repeat (PPR) superfamily protein

Ciclev10029088m.g AT3G45970 EXLA1 cell wall.modification -0.890012 expansin-like A1

Ciclev10005827m.g AT2G33430 DAL1 development.unspecified -0.855005 differentiation and greening-like 1

Ciclev10028241m.g AT5G24318 AT5G24318 misc.beta 1,3 glucan hydrolases.glucan endo-1,3-beta-glucosidase

-0.813193 O-Glycosyl hydrolases family 17 protein

Ciclev10012267m.g AT2G37770 T8P21.32 minor CHO metabolism.others -0.812652 NAD(P)-linked oxidoreductase superfamily protein

* The negative sign in the column of Log2 FC indicates comparative downregulation of the gene expression level in VAL/CAN and upregulation in VAL/SW combination. †Classification of the measured parameter into a set a functional category in the MapMan analysis tool

290

Table B-3. Differentially expressed and significantly upregulated growth associated genes in leaves of the symptomatic VAL/CAN combination as compared to the symptomatic VAL/SW leaves C. Clementina_ID Arabi ID Name Bin Name† Log2 FC Arabidopsis_definition

Ciclev10000788m.g AT2G30300 T9D9.11 development. 0.996198 Major facilitator superfamily protein

Ciclev10016312m.g AT2G04780 FLA7 cell wall.cell wall proteins.AGPs 0.994991 FASCICLIN-like arabinoogalactan 7

Ciclev10014333m.g AT5G40390 SIP1-like minor CHO metabolism.raffinose family.raffinose synthases.known

0.99486 Raffinose synthase family protein

Ciclev10018639m.g AT5G44030 CESA4 cell wall.cellulose synthesis.cellulose synthase

0.990095 cellulose synthase A4

Ciclev10016841m.g AT1G53160 SPL4 development. squamosa promoter binding like (SPL)

0.988754 squamosa promoter binding protein-like 4

Ciclev10023723m.g AT4G02320 T14P8.1 cell wall.pectin*esterases.misc 0.988321 Plant invertase/pectin methylesterase inhibitor superfamily

Ciclev10000103m.g AT2G21770 CESA9 cell wall.cellulose synthesis.cellulose synthase

0.95496 cellulose synthase A9


Ciclev10009640m.g AT4G30410 AT4G30410 RNA.regulation of transcription. 0.951305 sequence-specific DNA binding transcription factors

Ciclev10017247m.g AT3G21055 PSBTN PS.liGhtreaction.photosystem II.PSII polypeptide subunits

0.944909 photosystem II subunit T

Ciclev10000393m.g AT5G63810 BGAL10 misc.Gluco-, Galacto- and mannosidases.beta-Galactosidase


Ciclev10014769m.g AT5G12860 DiT1 transport.metabolite transporters AT the mitochondrial membrane

0.925347 dicarboxylate transporter 1

Ciclev10028412m.g AT2G38170 CAX1 transport.calcium 0.921842 cation exchanger 1

Ciclev10031076m.g AT3G26570 PHT2;1 transport.phosphate 0.921254 phosphate transporter 2;1

Ciclev10029316m.g AT1G10200 WLIM1 RNA.regulation of transcription. putative transcription reGulATor

0.917802 GATA type zinc finger transcription factor family protein



Ciclev10019848m.g AT3G18490 AT3G18490 RNA.regulation of transcription. 0.910504 Eukaryotic aspartyl protease family protein



Ciclev10011855m.g AT3G54050 HCEF1 PS.calvin cyle.FBPase 0.892762 high cyclic electron flow 1

Ciclev10021082m.g AT2G45630 F17K2.16 PS.photorespiration. hydroxypyruvate reductase

0.88716 D-isomer specific 2-hydroxyacid dehydrogenase family protein

291

Table B-3. Continued C. Clementina_ID Arabi ID Name Bin Name Log2 FC Arabidopsis_definition

Ciclev10001422m.g AT4G24220 VEP1 development. 0.886658 NAD(P)-binding Rossmann-fold superfamily protein



Ciclev10007993m.g AT4G33220 PME44 cell wall.pectin*esterases.misc 0.881526 pectin methylesterase 44

Ciclev10015315m.g AT2G01940 SGR5 RNA.regulation of transcription.C 2H2 zinc finger family

0.877109 C2H2-like zinc finger protein

Ciclev10009128m.g AT5G56840 MIK19.31 RNA.regulation of transcription. MYB-related transcription factor family

0.874946 myb-like transcription factor family

Ciclev10008452m.g AT4G29240 F17A13.60 cell wall.cell wall proteins.LRR 0.874696 Leucine-rich repeat (LRR) family

Ciclev10001438m.g AT1G75500 WAT1 development. 0.873279 Walls Are Thin 1

Ciclev10004372m.g AT3G57520 SIP2 minor CHO metabolism.raffinose family.raffinose synthases.putative

0.868302 seed imbibition 2

Ciclev10031341m.g KAO2 ent-kaurenoic acid hydroxylase 2 0.87059 gibberellins (GAs) biosynthesis

Ciclev10020440m.g AT1G25440 F2J7.10 RNA.regulation of transcription.C2C2(Zn) CO-like, Constans-like zinc finger family

0.864337 B-box type zinc finger protein with CCT domain

Ciclev10017084m.g AT5G45040 K21C13.23 PS.ligtreaction.other electron carrier (ox/red) 0.863893 Cytochrome c

Ciclev10015683m.g AT5G14700 T9L3.2 secondary metabolism.phenylpropanoids .lignin biosynthesis.CCR1


Ciclev10014341m.g AT3G27170 CLC-B transport. anions 0.861428 chloride channel B

Ciclev10004357m.g AT1G54350 F20D21.17 transport.ABC transporters and multidrugresistance systems

0.851036 ABC transporter family protein

Ciclev10015097m.g AT5G06390 FLA17 cell wall.cell wall proteins.AGPs 0.850866 FASCICLIN-like arabinogalactan protein 17 precursor

Ciclev10021785m.g AT5G15090 VDAC3 transport.porins 0.850468 voltage dependent anion channel 3

Ciclev10033819m.g AT1G14150 PQL2 PS.liGhtreaction.photosystem II.PSII polypeptide subunits

0.848177 PsbQ-like 2

Ciclev10001649m.g AT1G32170 XTH30 cell wall.modification 0.847725 xyloglucan endotransglucosylase /hydrolase 30

Ciclev10024886m.g AT3G13750 BGAL1 misc.Gluco-, Galacto- and mannosidases.beta-Galactosidase

0.843285 beta galactosidase 1

Ciclev10031262m.g AT5G44640 BGLU13 misc.Gluco-, Galacto- and mannosidases 0.840258 beta glucosidase 13

Ciclev10016846m.g AT4G20970 AT4G20970 RNA.regulation of transcription.bHLH,Basic Helix-Loop-Helix family


Ciclev10030847m.g AT5G13550 SULTR4;1 transport.sulphate 0.837309 sulfate transporter 4.1

292


Ciclev10008774m.g AT5G55630 ATKCO1 transport.potassium 0.834926 Outward rectifying potassium channel protein

Ciclev10016045m.g AT3G58040 SINAT2 development. 0.834744 seven in absentia of Arabidopsis 2

Ciclev10031135m.g AT3G07340 AT3G07340 RNA.regulation of transcription. bHLH,Basic Helix-Loop-Helix family


Ciclev10026870m.g AT3G49780 PSK4 development. 0.830314 phytosulfokine 4 precursor

Ciclev10015394m.g AT2G20680 F5H14.35 mGluco-,Galacto- and mannosidases 0.825341 Glycosyl hydrolase superfamily protein

Ciclev10032564m.g AT3G55330 PPL1 PS.liGhtreaction.photosystem II.PSII polypeptide subunits

0.822998 PsbP-like protein 1

Ciclev10016511m.g AT5G54680 ILR3 RNA.regulation of transcription. bHLH,Basic Helix-Loop-Helix family


Ciclev10029911m.g AT1G13790 F16A14.2 RNA.regulation of transcription. putative transcription reGulATor

0.819111 XH/XS domain-containing protein

Ciclev10009335m.g AT4G26220 T25K17.30 secondary metabolism.phenylpropanoids.liGnin biosynthesis.CCoAOMT

0.816262 S-adenosyl-L-methionine-dependent methyltransferases


Ciclev10029689m.g AT4G37720 PSK6 development. 0.806825 phytosulfokine 6 precursor

Ciclev10031699m.g AT1G67340 F1N21.16 RNA.regulation of transcription. 0.80481 HCP-like superfamily protein with MYND-type zinc finger

Ciclev10016289m.g AT1G68810 T6L1.1 RNA.regulation of transcription.bHLH, Basic Helix-Loop-Helix family


Ciclev10011554m.g AT5G17300 RVE1 RNA.regulation of transcription. MYB-related transcription factor family




Ciclev10026007m.g AT1G15950 CCR1 secondary metabolism. phenylpropanoids.

0.770359 cinnamoyl coa reductase 1

Ciclev10011143m.g AT5G06530 F15M7.6 transport. ABC transporters and multidrugresistance systems


Ciclev10018855m.g AT4G00730 ANL2 RNA.regulation of transcription HB,Homeobox transcription factor family

0.754306 Homeobox-leucine zipper family protein

Ciclev10011731m.g AT3G02570 MEE31 cell wall.precursor synthesis. phosphomannose isomerase

0.745654 Mannose-6-phosphate isomerase, type I

Ciclev10016129m.g AT4G03210 XTH9 cell wall. modification 0.74911 xyloglucan endotransglucosylase/hydrolase 9

293


Ciclev10028807m.g AT2G28910 CXIP4 transport. calcium 0.740086 CAX interacting protein 4

Ciclev10012588m.g AT5G03170 FLA11 cell wall. cell wall proteins. AGPs 0.738492 FASCICLIN-like arabinogalactan-protein 11

Ciclev10030794m.g AT1G70610 TAP1 transport.ABC transporters and multidrugresistance systems

0.733327 transporter associated with antigen processing protein 1

Ciclev10025791m.g AT4G37060 PLP5 development. storage proteins 0.732467 PATATIN-like protein 5

Ciclev10002627m.g AT1G78020 F28K19.24 development. 0.732379 Protein of unknown function (DUF581)

Ciclev10004369m.g AT5G49360 BXL1 cell wall. degradation.mannan-xylose-arabinose

0.728261 beta-xylosidase 1

Ciclev10022321m.g AT1G53670 MSRB1 RNA.regulation of transcription. putative transcription reGulATor

0.71928 methionine sulfoxide reductase B 1

Ciclev10004221m.g AT1G04920 SPS3F CHO metabolism.synthesis.sucrose.SPS 0.715703 sucrose phosphate synthase 3F

Ciclev10002429m.g AT5G58260 NDH-N PS.lightreaction. NADH DH 0.711627 oxidoreductases

Ciclev10019267m.g AT5G61150 VIP4 RNA.regulation of transcription. putative transcription regulator

0.703989 leo1-like family protein

Ciclev10031120m.g AT4G18020 APRR2 RNA.regulation of transcription. Psudo ARR transcription factor family

0.697944 CheY-like two-component responsive regulator family protein

Ciclev10014198m.g AT5G37020 ARF8 RNA.regulation of transcription.ARF, Auxin Response Factor family

0.689275 auxin response factor 8

Ciclev10008943m.g AT5G56860 GNC RNA.regulation of transcription. C2C2(Zn) GATA transcription factor family


Ciclev10006161m.g AT3G06740 GATA15 RNA.regulation of transcription. C2C2(Zn) GATA transcription factor family

0.675366 GATA transcription factor 15

Ciclev10031052m.g AT1G67710 ARR11 RNA.regulation of transcription.ARR 0.675231 response regulator 11

Ciclev10008943m.g AT5G56860 GNC RNA.regulation of transcription.C2C2(Zn) GATA transcription factor family


Ciclev10006161m.g AT3G06740 GATA15 RNA.regulation.C2C2(Zn) GATA transcription factor family

0.675366 GATA transcription factor 15

Ciclev10031052m.g AT1G67710 ARR11 RNA.regulation of transcription.ARR 0.675231 response regulator 11

Ciclev10017102m.g AT3G02870 VTC4 minor CHO metabolism. myo-inositol. inositol phosphATase

0.668874 Inositol monophosphatase family protein

Ciclev10007657m.g AT4G31390 F3L17.6 transport.ABC transporters and multidrugresistance systems


†Classification of the measured parameter into a set a functional category in the MapMan analysis

294

Table B-4. Differentially expressed and significantly upregulated growth associated genes in the leaves of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN leaves C. Clementina_ID Arabi ID Gene name Bin Name† Log2 FC* Arabidopsis_define

Ciclev10025462m.g AT3G50740 UGT72E1 secondary metabolism. phenylpropanoids. lignin biosynthesis




Ciclev10013854m.g AT3G18110 EMB1270 development. -0.973641 Pentatricopeptide repeat (PPR) superfamily protein

Ciclev10011715m.g AT5G67150 K21H1.11 secondary metabolism. phenylpropanoids


Ciclev10014094m.g AT4G12750 AT4G12750 RNA.regulation of transcription. putative transcription regulator

-0.945108 Homeodomain-like transcriptional regulator

Ciclev10007293m.g AT1G80490 TPR1 development. -0.93964 TOPLESS-related 1

Ciclev10003745m.g AT2G01170 BAT1 transport.amino acids -0.933943 bidirectional amino acid transporter 1

Ciclev10011531m.g AT5G03720 HSFA3 RNA.regulation of transcription.HSF,Heat T-shock transcription factor family

-0.931311 heat shock transcription factor A3

Ciclev10025727m.g AT4G36970 AP22.30 RNA.regulation of transcription. -0.910655 Remorin family protein

Ciclev10031658m.g AT1G76520 F14G6.12 transport.misc -0.903648 Auxin efflux carrier family protein

Ciclev10031274m.g AT3G26590 AT3G26590 transport.misc -0.881479 MATE efflux family protein

Ciclev10028824m.g AT5G60800 MAE1.5 transport.misc -0.877349 Heavy metal transport/detoxification superfamily protein

Ciclev10012147m.g AT2G36000 F11F19.9 RNA.regulation of transcription -0.873168 Mitochondrial transcription termination factor family protein

Ciclev10004036m.g AT1G77920 F28K19.13 RNA.regulation of transcription. bZIP transcription factor family

-0.868729 bZIP transcription factor family protein

Ciclev10005073m.g AT4G27940 MTM1 transport.metabolite transporters AT the mitochondrial membrane

-0.864684 manganese tracking factor for mitochondrial SOD2

Ciclev10015899m.g AT3G09150 HY2 PS.lightreaction.other electron carrier (ox/red).ferredoxin oxirdoeductase

-0.863078 phytochromobilin:ferredoxin oxidoreductase, chloroplast / phytochromobilin synthase (HY2)

Ciclev10001380m.g AT3G18710 PUB29 RNA.regulation of transcription. PHOR1 -0.859019 plant U-box 29

Ciclev10025961m.g AT1G78080 RAP2.4 RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive element

-0.831904 related to AP2 4

295

Table B-4. Continued C. Clementina_ID Arabi ID Gene name Bin Name Log2 FC Arabidopsis_define


-0.817589 homeobox protein 2

Ciclev10023309m.g AT2G32700 LUH RNA.regulation of transcription.LUG

-0.795593 LEUNIG_homolog

Ciclev10007519m.g AT5G20250 DIN10 minor CHO metabolism.raffinose family.raffinose synthases.putative


Ciclev10032069m.g AT1G14360 UTR3 transport.NDP-sugar at the ER -0.777345 UDP-galactose transporter 3

Ciclev10009180m.g AT2G23810 TET8 development. -0.771434 tetraspanin8


Ciclev10031124m.g AT5G18070 DRT101 cell wall.precursor synthesis -0.755555 phosphoglucosamine mutase-related

Ciclev10002475m.g AT5G27280 F21A20.2 RNA.regulation of transcription.unclassified

-0.746242 Zim17-type zinc finger protein


Ciclev10004676m.g AT2G41190 T3K9.4 transport.amino acids -0.727794 Transmembrane amino acid transporter family protein

Ciclev10015406m.g AT2G26560 PLA2A development.storage proteins -0.727405 phospholipase A 2A



Ciclev10019745m.g AT1G69850 NRT1:2 transport.nitrate -0.715118 nitrate transporter 1:2

Ciclev10007808m.g AT5G53850 K19P17.1 minor CHO metabolism.others -0.698661 haloacid dehalogenase-like hydrolase family protein

Ciclev10031846m.g AT1G68840 RAV2 RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive element binding protein family


Ciclev10015899m.g AT3G09150 HY2 PS.lightreaction.other electron carrier (ox/red).ferredoxin oxirdoeductase

-0.863078 phytochromobilin:ferredoxin oxidoreductase, chloroplast / phytochromobilin synthase (HY2)

Ciclev10001380m.g AT3G18710 PUB29 RNA.regulation of transcription. PHOR1

-0.859019 plant U-box 29

296

Table B-4. Continued



Ciclev10004728m.g AT3G47420 PS3 transporter.membrane system unknown -0.849536 phosphate starvation-induced gene 3

Ciclev10025961m.g AT1G78080 RAP2.4 RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive element

-0.831904 related to AP2 4


-0.817589 homeobox protein 2

Ciclev10023309m.g AT2G32700 LUH RNA.regulation of transcription.LUG -0.795593 LEUNIG_homolog

Ciclev10015269m.g AT1G32330 HSFA1D RNA.regulation of transcription.HSF, Heat-shock transcription factor family

-0.794278 heat shock transcription factor A1D

Ciclev10008965m.g AT5G57330 MJB24.14 minor CHO metabolism.others -0.793661 Galactose mutarotase-like superfamily protein

Ciclev10007519m.g AT5G20250 DIN10 minor CHO metabolism.,raffinose family.raffinose synthases.putative


Ciclev10032069m.g AT1G14360 UTR3 transport.NDP-sugar at the ER -0.777345 UDP-galactose transporter 3

Ciclev10009180m.g AT2G23810 TET8 development. -0.771434 tetraspanin8


Ciclev10031124m.g AT5G18070 DRT101 cell wall.precursor synthesis -0.755555 phosphoglucosamine mutase-related

Ciclev10002475m.g AT5G27280 F21A20.2 RNA.regulation of transcription. -0.746242 Zim17-type zinc finger protein


Ciclev10004676m.g AT2G41190 T3K9.4 transport.amino acids -0.727794 Transmembrane amino acid transporter family protein

Ciclev10015406m.g AT2G26560 PLA2A development.storage proteins -0.727405 phospholipase A 2A



Ciclev10019745m.g AT1G69850 NRT1:2 transport. nitrate -0.715118 nitrate transporter 1:2

Ciclev10007808m.g AT5G53850 K19P17.1 minor CHO metabolism. others -0.698661 haloacid dehalogenase-like hydrolase family protein

Ciclev10031846m.g AT1G68840 RAV2 RNA.regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive element



297

Table B-5. Differentially expressed and significantly upregulated growth associated genes in the roots of symptomatic VAL/CAN combination as compared to symptomatic VAL/SW roots

C. Clementina_ID Arabi ID Name Bin Name† Log2 FC


Ciclev10011096m.g AT3G53720 ATCHX20,CHX20 transport.metal 1.49929 cation/H+ exchanger 20

Ciclev10033625m.g AT3G21420 secondary metabolism. flavonoids.flavonols


Ciclev10014116m.g AT4G20400 JMJ14,PKDM7B RNA.regulation of transcription. JUMONJI family

1.48361 JUMONJI 14

Ciclev10007831m.g AT1G52190 transport.peptides and oligopeptides

1.48348 Major facilitator superfamily protein

Ciclev10012494m.g AT2G29290 secondary metabolism. N misc.alkaloid-like


Ciclev10004839m.g AT5G18840 transporter. sugars 1.46949 Major facilitator superfamily protein

Ciclev10015452m.g AT5G48230 ACAT2,EMB1276 secondary metabolism. isoprenoids. mevalonate Pathway.acetyl-CoA C-acyltransferase

1.46867 acetoacetyl-CoA thiolase 2

Ciclev10008832m.g AT4G17190 FPS2 secondary metabolism. isoprenoids. mevalonate Pathway.farnesyl pyrophosphate synthetase

1.46627 farnesyl diphosphate synthase 2


Ciclev10008031m.g AT3G18830 ATPLT5,ATPMT5, transporter.sugars 1.45494 polyol/monosaccharide transporter 5

Ciclev10026364m.g AT3G21150 BBX32 RNA.regulation of transcription.C2C2(Zn) CO-like, Constans-like zinc finger family

1.45465 B-box 32

Ciclev10023463m.g AT1G63440 HMA5 transport.metal 1.4462 heavy metal atpase 5

Ciclev10028404m.g AT2G28110 FRA8,IRX7 cell wall.hemicellulose synthesis. Glucuronoxylan

1.42099 Exostosin family protein

Ciclev10006898m.g AT5G43360 ATPT4,PHT1;3, transport. phosphate 1.39969 phosphate transporter 1;3



Ciclev10011564m.g AT5G03570 ATIREG2,IREG2 transport.metal 1.38193 iron regulated 2

Ciclev10010546m.g AT2G18950 ATHPT, VTE2 secondary metabolism. isoprenoids. tocopherol biosynthesis.homogentisate phytyltransferase


298


Ciclev10011564m.g AT5G03570 FPN2, IREG2 transport.metal 1.38193 iron regulated 2

Ciclev10031658m.g AT1G76520 transport.misc 1.37629 Auxin efflux carrier family protein

Ciclev10001464m.g AT1G33520 MOS2 RNA.RNA binding 1.37537 D111/G-patch domain-containing protein

Ciclev10029065m.g AT1G27730 STZ,ZAT10 RNA.regulation of transcription.C2H2 zinc finger family

1.36936 salt tolerance zinc finger

Ciclev10000940m.g AT4G08250 development. 1.36524 GRAS family transcription factor

Ciclev10033633m.g AT3G08040 FRD3 metal handling.acquisition 1.36175 MATE efflux family protein

Ciclev10009072m.g AT4G31270 RNA.regulation of transcription.Trihelix, Triple-Helix transcription factor family

1.33777 sequence-specific DNA binding transcription factors

Ciclev10025271m.g AT5G09760 cell wall.pectin*esterases.PME 1.329 Plant invertase/pectin methylesterase inhibitor superfamily

Ciclev10012473m.g AT2G37330 ALS3 development. 1.32721 aluminum sensitive 3

Ciclev10028100m.g AT2G28120 development. 1.32373 Major facilitator superfamily protein

Ciclev10033673m.g AT3G54320 ASML1,ATWRI1 development. 1.32365 Integrase-type DNA-binding superfamily protein

Ciclev10022800m.g AT2G47930 AGP26,ATAGP26 cell wall.cell wall proteins.AGPs 1.32346 arabinogalactan protein 26

Ciclev10019486m.g AT1G73220 1-Oct,AtOCT1 transporter.sugars 1.32097 organic cation/carnitine transporter1

Ciclev10004215m.g AT5G43630 TZP RNA.regulation of transcription.C3H zinc finger family

1.3182 zinc knuckle (CCHC-type) family protein

Ciclev10031126m.g AT1G12940 ATNRT2.5,NRT2.5 transport.nitrate 1.31477 nitrate transporter2.5

Ciclev10004869m.g AT3G57790 cell wall.degradation.pectate lyases and polygalacturonases

1.3051 Pectin lyase-like superfamily protein

Ciclev10025259m.g AT3G13790 ATBFRUCT1, ATCWINV1

major CHO metabolism. degradation. sucrose.invertases.cell wall


299


Ciclev10025259m.g AT3G13790 ATBFRUCT1, ATCWINV1

major CHO metabolism. degradation. sucrose. invertases. cell wall


Ciclev10028932m.g AT5G22650 HD2,HD2B, RNA.regulation of transcription.HDA 1.29785 histone deacetylase 2B

Ciclev10029999m.g AT1G07530 ATGRAS2, SCL14

RNA.regulation of transcription. GRAS transcription factor family

1.29047 SCARECROW-like 14

Ciclev10031948m.g AT1G17020 ATSRG1,SRG1 secondary metabolism. flavonoids.flavonols 1.28935 senescence-related gene 1

Ciclev10002239m.g AT3G50060 MYB77 RNA.regulation of transcription.MYB domain transcription factor family


Ciclev10007519m.g AT5G20250 DIN10 minor CHO metabolism.raffinose family.raffinose synthases.putative

1.25127 Raffinose synthase family protein

Ciclev10000612m.g AT1G34190 anac017,NAC017 development. 1.24959 NAC domain containing protein 17

Ciclev10021699m.g AT2G31180 ATMYB14, MYB14,

RNA.regulation of transcription.MYB domain transcription factor family


Ciclev10033561m.g AT5G48450 sks3 cell wall.pectin*esterases.misc 1.2424 SKU5 similar 3

Ciclev10024068m.g AT1G29800 RNA.regulation of transcription. 1.24057 RING/FYVE/PHD-type zinc finger family protein

Ciclev10013434m.g AT1G16360 transport. misc 1.23413 LEM3 (ligand-effect modulator 3) family protein / CDC50 family protein

Ciclev10022334m.g AT3G48100 ARR5,ATRR2 RNA.regulation of transcription.ARR 1.2182 response regulator 5

Ciclev10004304m.g AT5G04040 SDP1 development. storage proteins 1.21413 Patatin-like phospholipase family protein

Ciclev10006992m.g AT3G28860 ATMDR1, MDR1, GP19

transport.ABC transporters and multidrugresistance systems

1.21118 ATP binding cassette subfamily B19

Ciclev10027681m.g AT5G60040 NRPC1 RNA.transcription 1.20566 nuclear RNA polymerase C1

Ciclev10010379m.g AT2G38290 AMT2,AMT2;1, transport.ammonium 1.20101 ammonium transporter 2

Ciclev10011715m.g AT5G67150 secondary metabolism. phenylpropanoids 1.19836 HXXXD-type acyl-transferase family protein

Ciclev10019941m.g AT4G23500 cell wall.degradation .pectate lyases and polygalacturonases


Ciclev10032029m.g AT2G40340 AtERF48, DREB2C

RNA. regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive

1.17466 Integrase-type DNA-binding superfamily protein

300




Ciclev10032029m.g AT2G40340 AtERF48, DREB2C


1.17466 Integrase-type DNA-binding superfamily protein

Ciclev10029865m.g AT1G59740 transport.peptides and oligopeptides 1.1652 Major facilitator superfamily protein

Ciclev10004432m.g AT4G15560 CLA,CLA1 ,DXPS2,DXS

secondary metabolism.isoprenoids.non-mevalonate Pathway.DXS

1.16461 Deoxyxylulose-5-phosphate synthase

Ciclev10015358m.g AT5G36160 secondary metabolism.isoprenoids.tocopherol biosynthesis

1.1561 Tyrosine transaminase family protein

Ciclev10018737m.g AT3G47950 AHA4,HA4 transport.p- and v-ATPases 1.14843 H(+)-ATPase 4

Ciclev10021887m.g AT2G03090 EXP15,EXPA15 cell wall.modificATion 1.14666 expansin A15

Ciclev10028522m.g AT1G49780 PUB26 RNA.regulation of transcription.PHOR1 1.14472 plant U-box 26



Ciclev10025088m.g AT4G37870 PCK1,PEPCK Gluconeogenese/ cycle.PEPCK 1.13996 phosphoenolpyruvate carboxykinase 1

Ciclev10001679m.g AT5G42630 ATS,KAN4 RNA.regulation of transcription.G2-like transcription factor family, GARP


Ciclev10010902m.g AT2G36380 ATPDR6,PDR6 transport.ABC transporters and multidrugresistance systems


Ciclev10006777m.g AT5G41070 DRB5 RNA.RNA binding 1.12834 dsRNA-binding protein 5

Ciclev10027998m.g AT5G59730 ATEXO70H7, EXO70H7

RNA.regulation of transcription.unclassified 1.12647 exocyst subunit exo70 family protein H7

Ciclev10025927m.g AT1G75500 WAT1 development. 1.12587 Walls Are Thin 1

Ciclev10030052m.g AT2G28160 ATBHLH029, ATBHLH29,

RNA.regulation of transcription.bHLH,Basic Helix-Loop-Helix family

1.12177 FER-like regulator of iron uptake

Ciclev10032093m.g AT1G59960 secondary metabolism. flavonoids. chalcones


Ciclev10000627m.g AT1G76890 AT-GT2,GT2 RNA.regulation of transcription.Trihelix, Triple-Helix transcription factor family

1.11007 Duplicated homeodomain-like superfamily protein

301


Ciclev10000627m.g AT1G76890 AT-GT2,GT2 RNA.regulation of transcription. Trihelix, Triple-Helix transcription factor family


Ciclev10010513m.g AT1G52540 development. 1.10316 Protein kinase superfamily protein

Ciclev10025480m.g AT5G65380 development. 1.10304 MATE efflux family protein

Ciclev10013734m.g AT4G36810 GGPS1

secondary metabolism.isoprenoids.non-mevalonate Pathway.GeranylGeranyl pyrophosphate synthase

1.08799 geranylgeranyl pyrophosphate synthase 1

Ciclev10033853m.g AT5G06710 HAT14 development. 1.08504 homeobox from Arabidopsis thaliana


Ciclev10026034m.g AT3G50820 PSBO-2,PSBO2 PS.lightreaction.photosystem II. PSII polypeptide subunits

1.0783 photosystem II subunit O-2

Ciclev10033331m.g AT5G43360 ATPT4,PHT1;3 transport. phosphate 1.0774 phosphate transporter 1;3

Ciclev10012378m.g AT3G56650 PS.lightreaction.photosystem II. PSII polypeptide subunits

1.07595 Mog1/PsbP/DUF1795-like photosystem II reaction center PsbP family protein

Ciclev10023764m.g AT5G19790 RAP2.11 RNA. regulation of transcription.AP2/EREBP, APETALA2/Ethylene-responsive

1.06993 related to AP2 11

Ciclev10006637m.g AT1G02520 PGP11 transport. ABC transporters and multidrugresistance systems


Ciclev10015315m.g AT2G01940 ATIDD15,SGR5 RNA. regulation of transcription. C2H2 zinc finger family


Ciclev10028824m.g AT5G60800 transport.misc 1.06226 Heavy metal transport/detoxification superfamily protein

Ciclev10033462m.g AT5G16680 RNA.regulation of transcription. 1.05329 RING/FYVE/PHD zinc finger superfamily protein

Ciclev10014849m.g AT5G14940 transport. peptides and oligopeptides 1.04512 Major facilitator superfamily protein

Ciclev10018859m.g AT3G18390 EMB1865 development. 1.02989 CRS1 / YhbY (CRM) domain-containing protein

302


Ciclev10020723m.g AT3G08900 RGP,RGP3 cell wall. cell wall proteins.RGP 1.02968 reversibly glycosylated polypeptide 3

Ciclev10002627m.g AT1G78020 development. 1.01648 Protein of unknown function (DUF581)

Ciclev10033294m.g AT1G66950 ATPDR11, PDR11

transport. ABC transporters and multidrugresistance systems


Ciclev10030419m.g AT4G38460 GGR secondary metabolism. isoprenoids. non-mevalonate Pathway. Geranyl Geranyl pyrophosphate synthase

1.01462 geranylgeranyl reductase

Ciclev10001976m.g AT1G01720 ANAC002, ATAF1

development. 1.00984 NAC (No Apical Meristem) domain transcriptional regulator superfamily protein

Ciclev10009285m.g AT5G57150 RNA. regulation of transcription.bHLH, Basic Helix-Loop-Helix family


Ciclev10031347m.g AT1G67440 emb1688 development. 1.00121 Minichromosome maintenance (MCM2/3/5) family protein

Ciclev10019735m.g AT3G54220 SCR,SGR1 development. 0.986574 GRAS family transcription factor

Ciclev10030860m.g AT4G30080 ARF16 RNA.regulation of transcription.ARF, Auxin Response Factor family


Ciclev10028125m.g AT4G34950 development. 0.980289 Major facilitator superfamily protein

Ciclev10023420m.g AT3G50700 AtIDD2,IDD2 RNA.regulation of transcription.C2H2 zinc finger family

0.978277 indeterminate(ID)-domain 2

Ciclev10026005m.g AT3G14180 RNA.regulation of transcription.Trihelix, Triple-Helix transcription factor family

0.973121 sequence-specific DNA binding transcription factors

Ciclev10027365m.g AT2G18950 ATHPT,HPT1, TPT1,VTE2

secondary metabolism. isoprenoids. tocopherol biosynthesis.homogentisate phytyltransferase


Ciclev10023758m.g AT2G44670 development. 0.965932 Protein of unknown function (DUF581)

Ciclev10031793m.g AT1G25550 RN of transcription. G2-like transcription factor family, GARP

0.958853 myb-like transcription factor family protein

303



RNA.regulation of transcription. bZIP transcription factor family

0.956459 Basic-leucine zipper (bZIP) transcription factor family protein

Ciclev10002059m.g AT5G44080 RNA. regulation of transcription. bZIP transcription factor family

0.956459 Basic-leucine zipper (bZIP) transcription factor family protein

Ciclev10019257m.g AT5G62000 ARF1-BP, ARF2

RNA.regulation of transcription. ARF, Auxin Response Factor family


Ciclev10003094m.g AT3G57230 AGL16 RNA.regulation of transcription. MADS box transcription factor family

0.94146 AGAMOUS-like 16

Ciclev10012263m.g AT3G09600 RNA. regulation of transcription. MYB-related transcription factor family


Ciclev10025318m.g AT1G15960 NRAMP6 transport. metal 0.936005 NRAMP metal ion transporter 6

Ciclev10012248m.g AT5G01340 transport. metabolite transporters at the mitochondrial membrane


Ciclev10006582m.g AT4G25610 RNA.regulation of transcription. C2H2 zinc finger family


Ciclev10020717m.g AT3G04070 anac047,NAC047 development. 0.92048 NAC domain containing protein 47

Ciclev10013219m.g AT3G23325 RNA.processing.splicing 0.917594 Splicing factor 3B subunit 5/RDS3 complex subunit 10

Ciclev10010625m.g AT4G10310 ATHKT1,HKT1 transport. cations 0.907006 high-affinity K+ transporter 1

Ciclev10017841m.g AT3G26570 ORF02,PHT2;1 transport.phosphate 0.893156 phosphate transporter 2;1

Ciclev10007994m.g AT5G39090 secondary metabolism.flavonoids. anthocyanins.


Ciclev10000593m.g AT1G76880 RNA.regulation of transcription. Trihelix, Triple-Helix transcription factor family


Ciclev10030681m.g AT1G29400 AML5,ML5 development. 0.878172 MEI2-like protein 5

Ciclev10010256m.g AT3G28345 transport.ABC transporters and multidrugresistance systems

0.867407 ABC transporter family protein

Ciclev10011572m.g AT4G14605 RNA.regulation of transcription. unclassified

0.850842 Mitochondrial transcription termination factor family protein

Ciclev10030845m.g AT1G27900 RNA.processing.RNA helicase 0.850187 RNA helicase family protein

304


Ciclev10029094m.g AT2G39700 ATHEXP ALPHA 1.6,EXPA4

cell wall.modification 0.848533 expansin A4


Ciclev10011800m.g AT1G05055 ATGTF2H2, GTF2H2

RNA.regulation of transcription.unclassified

0.830538 general transcription factor II H2

Ciclev10018153m.g AT3G54220 SCR,SGR1 development. 0.825398 GRAS family transcription factor

Ciclev10016371m.g AT3G13570 SCL30A RNA.processing.splicing 0.82508 SC35-like splicing factor 30A

Ciclev10011230m.g AT1G06820 CCR2,CRTISO secondary metabolism.isoprenoids.carotenoids

0.823593 carotenoid isomerase

Ciclev10014913m.g AT1G11260 ATSTP1,STP1 transporter.sugars 0.820703 sugar transporter 1

Ciclev10006456m.g AT4G23540 RNA.regulation of transcription. unclassified

0.817905 ARM repeat superfamily protein

Ciclev10001105m.g AT2G17900 SDG37 RNA.regulation of transcription.SET-domain transcriptional regulator family

0.814816 SET domain group 37

Ciclev10019376m.g AT5G53370 ATPMEPCRF, PMEPCRF

cell wall.pectin*esterases.misc 0.808714 pectin methylesterase PCR fragment F

Ciclev10021611m.g AT5G19790 RAP2.11


0.803556 related to AP2 11

Ciclev10014995m.g AT3G15390 SDE5 RNA.transcription 0.795239 silencing defective 5

Ciclev10019143m.g AT5G62620 cell wall.hemicellulose synthesis 0.779805 Galactosyltransferase family protein

Ciclev10014929m.g AT2G32290 BAM6, BMY5 major CHO metabolism.degradation. starch.starch cleavage

0.777807 beta-amylase 6

Ciclev10000444m.g AT5G45380 ATDUR3, DUR3 transport. cations 0.753465 solute:sodium symporters;urea transmembrane transporters


development. 0.74454 Major facilitator superfamily protein

Ciclev10000865m.g AT4G02630 PS.lightreaction.state transition 0.739566 Protein kinase superfamily protein




305

Table B-6. Differentially expressed and significantly upregulated growth associated genes in roots of the symptomatic VAL/SW combination as compared to symptomatic VAL/CAN roots C. Clementina_ID Arabi ID Gene Name Bin Name† Log2 FC* Arabidospsis_define

Ciclev10030930m.g AT1G70370 PG2 cell wall. degradation .pectate lyase and polygalacturonases

-1.49393 polygalacturonase 2

Ciclev10000954m.g AT2G01170 BAT1 transport. amino acids -1.49078 bidirectional amino acid transporter 1

Ciclev10007412m.g AT2G26650 AKT1 transport. potassium -1.48144 K+ transporter 1


transport. anions -1.47314 HCO3- transporter family


Ciclev10032279m.g AT1G69780 ATHB13 RNA.regulation of transcription. HB,Homeobox transcription factor family

-1.45959 Homeobox-leucine zipper protein family


development. -1.45427 GDSL-like Lipase/Acylhydrolase superfamily protein

Ciclev10020081m.g AT1G24100 UGT74B1 Secondary metabolism. Glucosinolates.synthesis.shared. UDP-Glycosyltransferase

-1.45161 UDP-glucosyl transferase 74B1

Ciclev10002142m.g AT4G17030 ATEXLB1 cell wall.modification -1.44633 expansin-like B1

Ciclev10001438m.g AT1G75500 WAT1 development. -1.41316 Walls Are Thin 1


transporter.sugars -1.37628 Major facilitator superfamily protein


development. -1.37345 Protein of unknown function (DUF581)

Ciclev10028195m.g AT4G39210 APL3 major CHO metabolism.synthesis.starch.AGPase

-1.37003 Glucose-1-phosphate adenylyltransferase family protein

Ciclev10011996m.g AT3G02230 RGP1 cell wall.cell wall proteins.RGP -1.36758 reversibly glycosylated polypeptide 1


metal handling.binding, chelATion and storage



secondary metabolism. phenylpropanoids



RNA.regulation of transcription. putative transcription regulator

-1.35339 Methyltransferase MT-A70 family protein


major CHO metabolism.synthesis. starch.AGPase

-1.34951 ADP-glucose pyrophosphorylase family protein

Ciclev10014314m.g AT3G59550 ATRAD21.2 development. -1.34898 Rad21/Rec8-like family protein

Ciclev10014787m.g AT1G11580 PMEPCRA cell wall.pectin*esterases.PME -1.34822 methylesterase PCR A

306

Table B-6. Continued C. Clementina_ID Arabi ID Gene Name Bin Name Log2 FC Arabidospsis_define

Ciclev10014314m.g AT3G59550 ATRAD21.2, development. -1.34898 Rad21/Rec8-like family protein

Ciclev10014787m.g AT1G11580 PMEPCRA cell wall. pectin*esterases.PME -1.34822 methylesterase PCR A

Ciclev10008774m.g AT5G55630 ATTPK1

transport. potassium -1.33862 Outward rectifying potassium channel protein

Ciclev10002725m.g AT3G03270 development. -1.33814 Adenine nucleotide alpha hydrolases-like superfamily protein

Ciclev10012013m.g AT5G07050 development. -1.33779 nodulin MtN21 /EamA-like transporter family protein

Ciclev10011731m.g AT3G02570 MEE31,PMI1 cell wall. precursor synthesis. phosphomannose isomerase

-1.32996 Mannose-6-phosphate isomerase, type I

Ciclev10027299m.g AT4G13420 HAK5 transport. potassium -1.32931 high affinity K+ transporter 5

Ciclev10020350m.g AT1G74910 major CHO metabolism. synthesis. starch.AGPase

-1.32925 ADP-glucose pyrophosphorylase family protein

Ciclev10002438m.g AT1G46480 WOX4 RNA.regulation of transcription. HB,Homeobox transcription factor family

-1.3027 WUSCHEL related homeobox 4

Ciclev10001508m.g AT4G08150 KNAT1 RNA.regulation of transcription. HB,Homeobox transcription factor family

-1.30198 KNOTTED-like from Arabidopsis thaliana

Ciclev10026558m.g AT5G48930 HCT secondary metabolism. phenylpropanoids.lignin biosynthesis. HCT

-1.30046 hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase

Ciclev10020725m.g AT3G61850 DAG1 RNA.regulation of transcription. C2C2(Zn) DOF zinc finger family


Ciclev10004372m.g AT3G57520 ,SIP2 minor CHO metabolism. raffinose family.raffinose synthases.

-1.28919 seed imbibition 2

Ciclev10022253m.g AT2G46680 HB-7 RNA.regulation of transcription. HB, Homeobox transcription factor family

-1.28529 homeobox 7

Ciclev10005068m.g AT1G04420 minor CHO metabolism. others -1.27869 NAD(P)-linked oxidoreductase superfamily protein

Ciclev10020175m.g AT2G47790 RNA.regulation of transcription. putative transcription regulator

-1.27783 Transducin/WD40 repeat-like superfamily protein

Ciclev10007286m.g AT1G19220 ARF19,IAA22 RNA of transcription. ARF, Auxin Response Factor family

-1.27446 auxin response factor 19

Ciclev10025286m.g AT1G80530 development. -1.26395 Major facilitator superfamily protein

Ciclev10019134m.g AT1G12240 BETAFRUCT4 major CHO metabolism. degradation. sucrose. invertases.vacuolar

-1.26225 Glycosyl hydrolases family 32 protein

307



secondary metabolism. flavonoids. anthocyanins.



cell wall. pectin*esterases.PME -1.26008 Plant invertase/pectin methylesterase inhibitor superfamily

Ciclev10018941m.g AT5G40390 SIP1 minor CHO metabolism.r affinose family. raffinose synthases. known



transport. amino acids -1.25226 Transmembrane amino acid transporter family protein

Ciclev10007849m.g AT5G57390 AIL5,CHO1,EMK development. -1.24598 AINTEGUMENTA-like 5


secondary metabolism. flavonoids.chalcones

-1.23187 Chalcone-flavanone isomerase family protein

Ciclev10013465m.g AT2G27880 AGO5 development. -1.21299 Argonaute family protein

Ciclev10000784m.g AT2G03220 ATFT1,ATFUT1 cell wall. hemicellulose synthesis -1.20446 fucosyltransferase 1


Ciclev10033306m.g AT4G24040 ATTRE1,TRE1 minor CHO metabolism .trehalose.trehalase -1.18611 trehalase 1

Ciclev10016171m.g AT4G18910 ATNLM2,NIP1;2 transport.Major Intrinsic Proteins.NIP -1.18577 NOD26-like intrinsic protein 1;2


transport.misc -1.18355 SEC14 cytosolic factor family protein

Ciclev10014642m.g AT4G04450 AtWRKY42, WRKY42

RNA.regulation of transcription. WRKY domain transcription factor family

-1.18222 WRKY family transcription factor

Ciclev10008218m.g AT5G55730 FLA1 cell wall. cell wall proteins.AGPs -1.16654 FASCICLIN-like arabinogalactan 1

Ciclev10025122m.g AT3G13810 AtIDD11,IDD11 RNA.regulation of transcription. C2H2 zinc finger family

-1.16641 indeterminate(ID)-domain 11

Ciclev10007893m.g AT4G32650 ATKC1, KAT3 transport. potassium -1.16602 potassium channel in Arabidopsis thaliana 3

Ciclev10020718m.g AT3G22830 AT-HSFA6B, RNA.regulation of transcription.HSF, Heat-shock transcription factor family

-1.1634 heat shock transcription factor A6B


-1.16013 indole-3-acetic acid inducible 18

Ciclev10007435m.g AT4G32880 ATHB-8,ATHB8, RNA.regulation of transcription. HB,Homeobox transcription factor family

-1.15524 homeobox gene 8

Ciclev10012382m.g AT5G37820 NIP4;2,NLM5 transport. Major Intrinsic Proteins. -1.15521 NOD26-like intrinsic protein 4;2


RNA.regulation of transcription. bHLH,Basic Helix-Loop-Helix family

-1.15479 basic helix-loop-helix (bHLH) DNA-binding superfamily protein

308


Ciclev10027790m.g AT5G60690 IFL,IFL1,REV development. -1.15354 Homeobox-leucine zipper family protein


RNA.regulation of transcription. G2-like transcription factor family, GARP

-1.14421 Homeodomain-like superfamily protein


transport.misc -1.12944 Major facilitator superfamily protein


RNA.regulation of transcription.C2C2(Zn) CO-like, Constans-like zinc finger family

-1.12365 B-box type zinc finger protein with CCT domain

Ciclev10007585m.g AT1G55850 ATCSLE1, CSLE1

cell wall. cellulose synthesis. cellulose synthase

-1.11563 cellulose synthase like E1


minor CHO metabolism.others -1.11395 Galactose mutarotase-like superfamily protein


transporter.sugars -1.11218 Major facilitator superfamily protein


minor CHO metabolism.myo-inositol.poly-phosphatases

-1.09781 DNAse I-like superfamily protein

Ciclev10026012m.g AT1G15950 CCR1,IRX4 secondary metabolism. phenylpropanoids.lignin biosynthesis.CCR1

-1.09732 cinnamoyl coa reductase 1


transport.misc -1.08865 Sec14p-like phosphatidylinositol transfer family protein

Ciclev10011104m.g AT3G56640 SEC15A development. -1.0826 exocyst complex component sec15A

Ciclev10023436m.g AT1G03310 ATISA2, BE2,DBE1,

major CHO metabolism. synthesis.starch.debranching

-1.07458 debranching enzyme 1

Ciclev10018822m.g AT4G01970 AtSTS,STS minor CHO metabolism.raffinose family.stachyose synthases

-1.06324 stachyose synthase

Ciclev10032203m.g AT2G43000 ,NAC042 development. -1.05897 NAC domain containing protein 42

Ciclev10026349m.g AT2G22570 ,NIC1 secondary metabolism.phenylpropanoids

-1.05823 nicotinamidase 1

Ciclev10021916m.g AT5G18290 SIP1;2,SIP1B transport.Major Intrinsic Proteins.SIP

-1.05643 Aquaporin-like superfamily protein

Ciclev10031324m.g AT2G39890 PROT1 transport.amino acids -1.04624 proline transporter 1


secondary metabolism. N misc.alkaloid-like



minor CHO metabolism. others

-1.04126 pfkB-like carbohydrate kinase family protein


minor CHO metabolism.others -1.04126 pfkB-like carbohydrate kinase family protein

309


Ciclev10000924m.g AT4G31500 CYP83B1, SUR2

secondary metabolism. synthesis.shared. CYP83B1 phenylacetaldoxime monooxyGenase



RNA.regulation of transcription. -1.01413 A20/AN1-like zinc finger family protein

Ciclev10031413m.g AT5G01240 LAX1 transport.amino acids -1.01 like AUXIN RESISTANT 1

Ciclev10024806m.g AT2G07680 ATMRP11, MRP11

transport.ABC transporters -1.00811 multidrug resistance-associated protein 11




Ciclev10007571m.g AT1G55850 ATCSLE1,CSLE1 cell wall. cellulose synthesis. -0.994439 cellulose synthase like E1

Ciclev10000528m.g AT1G22150 SULTR1;3 transport. sulphate -0.991938 sulfate transporter 1;3

Ciclev10021174m.g AT2G47260 ATWRKY23, WRKY23

RNA.regulation of transcription. WRKY domain transcription factor family



minor CHO metabolism. others -0.985285 NAD(P)-linked oxidoreductase superfamily protein

Ciclev10000183m.g AT1G19850 ARF5,IAA24,MP RNA.regulation of transcription.ARF, -0.979247 Transcriptional factor B3 family protein / auxin-responsive factor AUX/IAA-related

Ciclev10022841m.g AT3G62410 CP12,CP12-2 PS.calvin cyle -0.977682 CP12 domain-containing protein 2

Ciclev10007592m.g AT2G25930 ELF3,PYK20 RNA.regulation of transcription. ELF3 -0.973833 hydroxyproline-rich glycoprotein family protein


cell wall.degradation.pectate lyases and polygalacturonases



secondary metabolism. flavonoids.flavonols



minor CHO metabolism. others -0.954348 Galactose mutarotase-like superfamily protein


transport.misc -0.952714 Major facilitator superfamily protein

Ciclev10031361m.g AT5G06839 bZIP65,TGA10 RNA.regulation of transcription. bZIP transcription factor family

-0.950505 bZIP transcription factor family protein

Ciclev10021502m.g AT2G45960 ATHH2,PIP1;2 transport.Major Intrinsic Proteins.PIP -0.950058 plasma membrane intrinsic protein 1B

Ciclev10033106m.g AT1G79610 ATNHX6,NHX6 transport. cations -0.941524 Na+/H+ antiporter 6


-0.933771 indoleacetic acid-induced protein 8

310

Table B-6. Continued

C. Clementina_ID Arabi ID Gene Name

Bin Name Log2 FC Arabidospsis_define

Ciclev10020814m.g AT5G54160 OMT1 secondary metabolism. phenylpropanoids. lignin biosynthesis. COMT



transport. cations -0.928885 Sodium Bile acid symporter family

Ciclev10016129m.g AT4G03210 XTH9 cell wall.modification -0.923353 xyloglucan endotransglucosylase/hydrolase 9

Ciclev10007733m.g AT3G21240 4CL2 secondary metabolism. phenylpropanoids. lignin biosynthesis.4CL

-0.917904 4-coumarate:CoA ligase 2

Ciclev10010507m.g AT5G54250 CNGC4, transport.cyclic nucleotide or calcium regulated channels

-0.911337 cyclic nucleotide-gated cation channel 4

Ciclev10016661m.g AT4G20280 TAF11 RNA.transcription -0.905921 TBP-associated factor 11


RNA.regulation of transcription. -0.905681 RNA-binding (RRM/RBD/RNP motifs)

Ciclev10033898m.g AT1G64550 GCN3 transport.ABC transporters and multidrugresistance systems

-0.901822 general control non-repressible 3

Ciclev10026140m.g AT1G79900 BAC2 transport.metabolite transporters at the mitochondrial membrane

-0.895872 Mitochondrial substrate carrier




Ciclev10031035m.g AT5G04360 ATLDA, major CHO metabolism. synthesis.starch.debranching

-0.872316 limit dextrinase

Ciclev10027839m.g AT5G60450 ARF4 RNA.regulation of transcription. ARF, Auxin Response Factor family


Ciclev10001348m.g AT1G76130 AMY2, major CHO metabolism. degradation. starch.starch cleavage

-0.867968 alpha-amylase-like 2

Ciclev10026197m.g AT4G37790 HAT22 development. -0.86767 Homeobox-leucine zipper protein family


RNA.regulation of transcription. -0.863974 CCCH-type zinc fingerfamily protein with RNA-binding domain


development. -0.861646 GRAS family transcription factor

Ciclev10008754m.g AT2G24220 PUP5 transport.nucleotides -0.860078 purine permease 5




Ciclev10004634m.g AT5G51710 KEA5 transport.potassium -0.852118 K+ efflux antiporter 5


RNA.regulation of transcription. putative transcription

-0.848635 zinc ion binding;DNA binding

311



RNA.regulation of transcription.Trihelix, Triple-Helix transcription factor family

-0.84733 sequence-specific DNA binding transcription factors


-0.827998 PHE ammonia lyase 1


minor CHO metabolism.others -0.827008 pfkB-like carbohydrate kinase family protein

Ciclev10007311m.g AT5G20280 SPS1F major CHO metabolism.synthesis.sucrose.SPS

-0.821748 sucrose phosphate synthase 1F


PS.calvin cyle.rubisco small subunit -0.821124 Ribulose bisphosphate carboxylase (small chain) family protein

Ciclev10026028m.g AT5G08640 FLS1 secondary metabolism.flavonoids.flavonols

-0.818553 flavonol synthase 1

Ciclev10008100m.g AT2G24270 ALDH11A3 Glycolysis.cytosolic branch -0.814697 aldehyde dehydrogenase


minor CHO metabolism.sugar alcohols -0.812479 GroES-like zinc-binding dehydrogenase family protein

Ciclev10025462m.g AT3G50740 UGT72E1 secondary metabolism.phenylpropanoids. lignin biosynthesis


Ciclev10027815m.g AT3G46970 PHS2 major CHO metabolism.degradation. starch.starch phosphorylase

-0.806649 alpha-glucan phosphorylase 2

Ciclev10030786m.g AT5G20420 CHR42 RNA.regulation of transcription. Chromatin Remodeling Factors

-0.804047 chromatin remodeling 42



Ciclev10028912m.g AT5G60200 TMO6 RNA.regulation of transcription. C2C2(Zn) DOF zinc finger family

-0.784425 TARGET OF MONOPTEROS 6


RNA.regulation of transcription. Bromodomain proteins

-0.773077 DNA-binding bromodomain-containing protein

Ciclev10030358m.g AT5G60770 NRT2.4 transport.nitrate -0.771382 nitrate transporter 2.4

Ciclev10007305m.g AT5G57110 -ACA8 transport.calcium -0.766576 autoinhibited Ca2+ -ATPase, isoform 8

Ciclev10014198m.g AT5G37020 ARF8,ATARF8 RNA.regulation of transcription. ARF, Auxin Response Factor family


312



RNA.regulation of transcription.DNA methyltransferases

-0.760717 S-adenosyl-L-methionine-dependent methyltransferases superfamily protein


transport.metal -0.759678 high-affinity nickel-transport family protein


transport.metal -0.759678 high-affinity nickel-transport family protein


RNA.regulation of transcription.unclassified

-0.733479 Zinc finger C-x8-C-x5-C-x3-H type family protein

Ciclev10002389m.g AT5G63880 VPS20.1 RNA.regulation of transcription. SNF7 -0.726184 SNF7 family protein


313

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BIOGRAPHICAL SKETCH

Aditi Satpute was born in Pune, a culturally rich city in India. In 2008, she

obtained her baccalaureate degree in Agriculture from College of Agriculture, Pune,

India. In 2012, she graduated from Texas A and M University, Kingsville, USA with

master’s in plant science. Aditi admitted to the Ph.D. program in the horticultural

sciences department at the University of Florida in 2012 Fall semester. She received

her Ph.D (Horticulture Sciences) from the University of Florida in the summer 2017

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