disclaimers-space.snu.ac.kr/bitstream/10371/166623/1/000000160525.pdf · 2 days ago · we...

저 시-비 리- 경 지 2.0 한민

는 아래 조건 르는 경 에 한하여 게

l 저 물 복제, 포, 전송, 전시, 공연 송할 수 습니다.

다 과 같 조건 라야 합니다:

l 하는, 저 물 나 포 경 , 저 물에 적 된 허락조건 명확하게 나타내어야 합니다.

l 저 터 허가를 면 러한 조건들 적 되지 않습니다.

저 에 른 리는 내 에 하여 향 지 않습니다.

것 허락규약(Legal Code) 해하 쉽게 약한 것 니다.

Disclaimer

저 시. 하는 원저 를 시하여야 합니다.

비 리. 하는 저 물 리 목적 할 수 없습니다.

경 지. 하는 저 물 개 , 형 또는 가공할 수 없습니다.

http://creativecommons.org/licenses/by-nc-nd/2.0/kr/legalcode

http://creativecommons.org/licenses/by-nc-nd/2.0/kr/

Master’s Thesis of Science in Agriculture

Functional Analysis of Cellulose Synthase-like Genes

Responsive to Tomato Yellow Leaf Curl Virus

Infection in Tomato

토마토황화잎말림 바이러스 감염에 반응하는

토마토 셀룰로오스 합성 유전자의 기능 연구

February 2020

Siwon Choe

Department of International Agricultural Technology

Graduate School of International Agricultural Technology

Seoul National University



Infection in Tomato

A thesis

submitted in partial fulfillment of the requirements to the faculty

of Graduate School of International Agricultural Technology

for the Degree of Master of Science in Agriculture

By

Siwon Choe

Supervised by

Prof. Jang-Kyun Seo

Major of International Agricultural Technology




December 2019

Approved as a qualified thesis

For the Degree of Master of Science in Agriculture

by the committee members

Chairman Jin-Ho Kang, Ph.D.

Member Jang-Kyun Seo, Ph.D.

Member Choonkyun Jung, Ph.D.

i

Abstract



Infection in Tomato

Siwon Choe

Major of International Agricultural Technology




Tomato (Solanum lycopersicum) is an economically important vegetable

crop worldwide and it has long served as a model system for plant

development, genetics, pathology, and physiology. Virus diseases seriously

affect tomato growth and productivity. In particular, tomato yellow leaf curl

virus (TYLCV) is one of the most destructive viruses in tomato because it

causes severe symptoms, such as stunting, leaf size reduction and curling,

and yellowing. In the previous study, our comparative transcriptome analysis

showed that various genes associated with the cellulose biosynthesis pathway

are responsive to TYLCV infection. Cellulose synthesis genes play critical

ii

roles in cell wall biosynthesis, cell elongation, and plant growth. Thus, we

hypothesized that the symptoms related to plant growth might be associated

with the alteration of expression of cellulose synthesis genes upon viral

infection. In this study, we sought to characterize seven tomato cellulose

synthase-like genes that are significantly regulated by TYLCV infection

using a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS)

systems as a loss-of-function approach. Using the TRV-based VIGS system,

we successfully silenced the target cellulose synthase-like genes and

observed the resulting phenotypes. Silencing of a few cellulose synthase-like

genes resulted in retarded growth of the plants and delayed development of

vascular tissues, suggesting that alteration of these cellulose synthase-like

genes upon TYLCV infection might be associated with stunting symptoms

caused by TYLCV in tomato. Our study can provide new insights on how

host plant genes are specifically associated with disease symptom

development and a molecular basis to facilitate future plant immunity

engineering.

·····························································································

Keywords : Tomato, Tomato yellow leaf curl virus, Cellulose synthase, Virus induced gene silencing, Tobacco rattle virus

Student Number : 2018-24842

iii

Contents

Abstract················································································ i

Contents ··············································································iii

L i s t o f F i g u r e s

·········································································v

List of Tables ·········································································vi

I n t r o d u c t i o n

···········································································1

Materials and Methods······························································4

1 . C o n s t r u c t i o n o f T R V - V I G S c l o n e s

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 4

2 . P l a n t m a t e r i a l s a n d g r o w t h c o n d i t i o n s

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 4

3 . A g r o b a c t e r i u m - m e d i a t e d i n f i l t r a t i o n

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 5

4 . R N A i s o l a t i o n , R T - P C R , a n d q R T - P C R

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 5

5. Microscopy ··········································································6

6. Phylogenetic analysis ······························································6

Result ···················································································7

1. Optimization of a TRV based virus-induced gene silencing system in

t o m a t o .

····························································································7

iv

2. Establishment of a dual gene regulation system using a TRV vectors.

··························································································10

3. TYLCV infection causes dramatic changes in expression of some cellulose

s y n t h a s e - l i k e g e n e s i n t o m a t o

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 1 3

4. Silencing of Solyc07g043390 resulted in a stunting phenotype. ·······17

5. Overexpression of Solyc07g043390 diminished stunting symptoms caused by TYLCV ············································································22

6. Silencing phenotypes of other 6 cellulose synthase-like genes ·············24

Discussion ············································································35

References ···········································································41

A b s t r a c t i n K o r e a n

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 48

v

List of Figures

Figure 1. An efficient TRV-VIGS condition was optimized by testing for

silencing of PDS gene in tomato.

····················································9

Figure 2. GFP expression and PDS silencing at once using the TRV dual

v e c t o r

·························································································11

Figure 3. Alteration of expression levels of 33 cellulose synthase and

cellulose synthase-like genes in tomato when infected with TYLCV or ToCV.

··········14

Figure 4. Confirmation of systemic infection of the TRV recombinants.

······19

Figure 5. Morphological phenotypes of the Solyc07g043390-silenced plant.

20

Figure 6. Alteration of anatomical structures in the Solyc07g043390 silenced

p l a n t

····················································································21

Figure 7. Overexpression of Solyc07g043390 in

tomato·························23

Figure 8. VIGS phenotype of Solyc12g056580

···································25


···································27


·································28

vi


·································30


·································32


·································33

vii

List of Table

Table 1. List of primers used in this study ·······································34

１

Introduction

Tomato (Solanum lycopersicum) is an economically important vegetable

crop worldwide and it has been used as a model system for plant

development, genetics, pathology and physiology. Tomato growth and yield

are often inhibited by various biotic and abiotic stresses (Kissoudis et al.,

2016). A viral disease caused by tomato yellow leaf curl virus (TYLCV) is

one of the most serious biotic factors limiting tomato production in many

countries (Moriones & Navas-Castillo, 2000; Navas-Castillo et al., 2000).

TYLCV, a member of the genus Begomovirus in the family Geminiviridae, is

a DNA virus with a single-stranded circular DNA genome of approximately

2.7–2.8 kb. TYLCV is known to be mediated through whitefly Bemisia

tabaci (Ghanim et al., 1998; Kil et al., 2016), and recent studies have shown

that seed transmission is also possible in tomatoes (Kil et al., 2016). TYLCV

can cause huge yield loss in tomato production, ranging from 50% to 100%

(Makkouk et al., 1979).

Viral infection is a complex procedure involving the interaction between

viruses and their host plants. Understanding the plant host response to viral

infection is important for developing disease control strategies. Alterations in

host plant gene expression due to virus infection are associated with viral

symptoms. For instance, genes involved in the ROS signaling pathway are

associated with necrosis symptoms (Levine et al., 1994; Mochizuki et al.,

2014). Chlorosis symptoms are known to be caused by down-regulation of

photosynthesis-related genes (Lu et al., 2012). While TYLCV induces stunted

growth, severe reduction of leaf size, and yellowing and curling of young

leaves in tomato (Navot et al., 1991), a previous transcriptome analysis

showed that TYLCV infection alteration of several genes involved in the

cellulose biosynthesis pathway (Seo et al., 2018).

２

Cellulose is a polymer composed of beta-1,4-linked glucan chains which is

a major components of plant cell walls. It is synthesized by the cellulose

synthase complex (CSC), located in the plasma membrane of the cells, in

species ranging from bacteria to higher plants (Herth, 1985). Amino acid

sequence revealed that cellulose synthase (CESA) is a member of glycosyl

transferase family II (GT-2) (Campbell et al., 1997; Saxena et al., 1995). The

CESA protein have QXXRW motif, it was originally identified in bacterial

cellulose synthases (Saxena et al., 1995). There are 10 CESA in Arabidopsis

thaliana, 12 CESA in Oryza sativa, 18 CESA in populus trichocarpa, 8

CESA in barley and 9 CESA in corn. In addition to CESA, 30 genes with

similarity to cellulose synthase were found in arabidopsis (Richmond, 2000).

These cellulose synthase-like (CSL) genes are classified according to exon,

intron structures, and there are 8 families in arabidopsis (Hazen et al., 2002;

Richmond, 2000). CESA and CSL together form the cellulose synthase

superfamily. CSL protein also have a QXXRW motif. The major difference

between CSL and CESA is the presence of zinc-binding domain. Most CSL

do not have zinc–binding domain, which can be expected to making a single

polymer chains without forming a complex (Richmond & Somerville, 2000).

Biochemical evidence indicates that single polymers are mostly synthesized

in the Golgi apparatus and exported to the extracellular space (Karr, 1976;

Carpita and McCann, 2000) and the localization of the CSL to the Golgi

has been identified (Richmond & Somerville, 2000). Recent study has found

that there are 38 CESA/CSL proteins in tomato (Song et al., 2019). However,

functional study of cellulose synthase genes in tomato have not been studied

yet.

Virus-induced gene silencing (VIGS) is an easy and fast gene silencing

technique using RNA-mediated defense mechanism. VIGS is a type of post-

transcriptional gene silencing (PTGS) that causes plant transformation within

2-3 weeks. This technique can reduce labor dramatically by eliminating plant

regeneration and transformation steps needed for gene function analysis

using reverse genetics. In addition, the effect of gene silencing is very high

３

and the technique is simple, so that a large amount of genes can be analyzed.

VIGS has several interesting features. VIGS can knock down multiple genes

in the same family simultaneously by targeting conserved sequences among

the genes, this can overcome the problems caused by gene redundancy. VIGS

does not require a full cDNA sequence because if the nucleotide sequences

match more than 21nt, silencing can occur. VIGS has sometimes a short

duration of approximately 3 weeks. The efficiency of silencing can be

reduced and the original phenotype can be recovered. Previous researches

showed that VIGS has been successfully used for functional characterization

of cellulose synthase genes in Nicotiana benthamiana (Burton et al., 2000;

Zhu et al., 2010), Linum usitatissimum (Chantreau et al., 2015).

In this study, we hypothesized that dramatic alteration of cellulose

synthesis genes upon TYLCV infection might be associated with the

symptoms of stunting and leaf size reduction. Therefore, we sought to

examine loss-of-functions of seven tomato CSL genes, which were identified

to be significantly down- or up-regulated by TYLCV infection in tomato,

using the TRV-based VIGS system. In addition, we generated a transgenic

tomato overexpressing a CSL gene that was identified to be highly associated

with the stunting symptom, to examine whether ectopic overexpression of a

CSL gene that is down-regulated by TYLCV infection can compensate the

stunting symptom and support normal growth in tomato.

４

Materials and methods

1. Construction of TRV-VIGS clones

Target sequence for VIGS were determined using the Sol Genomics

Network VIGS tool (https://vigs.solgenomics.net/) based on Solanum

lycopersicum ITAG v.3.2. Seven 400 bp fragments corresponding to

Solyc07g043390, Solyc12g056580, Solyc02g089640, Solyc03g097050,

Solyc07g051820, Solyc08g076320, Solyc11g066820 were PCR-amplified

using Q5 High-Fidelity DNA polymerase (NEB, USA) and appropriate

primer pairs (Table 1). The amplified fragments were cloned into the TRV-

VIGS vector pTRV2 using the KpnI, XbaI (ThermoFisher, USA) sites. A

partial sequence of GUS gene was inserted into pTRV2 and the resulting

construct was used as a negative control for VIGS experiments. The VIGS

constructs were transferred to Agrobacterium strain EHA105 by freeze-thaw

method. The plasmid DNAs for Agro-transformation were prepared using

Plasmid Miniprep Kit (QIAGEN, USA). Clones were selected on Kanamycin

(100 µg/µl) and Rifampicin (50 µg/µl) Luria Bertani agar plate (1.5%) at 28℃

for two days.

2. Plant materials and growth conditions

Solanum lycopersicum (cv. Ailsa Craig) and Nicotiana benthamiana were

grown in a growth chamber under 16 h/24℃ day and 8 h/24℃ night

conditions for 3 weeks. After agroinfiltration with pTRV constructs, plants

were grown in a growth chamber under 16 h/20 day and 8℃ h/20 night ℃

conditions for VIGS. Transformed tomato seeds was germinated in

Murashige-Skoog (MS) solid media and incubated in a growth chamber

under 16 h/24℃ day and 8 h/24℃ night conditions for 7 days.

５

3. Agrobacterium-mediated infiltration

pTRV1 and pTRV2 constructs were transferred to Agrobacterium

tumefaciens strain EHA105 and a TYLCV infectious clone was transferred to

strain GV3101 by freeze-thaw method. These were inoculated to 5 ml LB

broth with Kanamycin (100 µg/µl) and Rifampicin (50 µg/µl) and incubated

in shaking incubator at 220 rpm at 28 for overnight. After transferring to ℃

fresh LB media with antibiotics and 20 uM Acetosyringone, the subculture

media was incubated in shaking incubator at 220 rpm at 28 for 9 h with. ℃

The culture was resuspended to O.D.600 = 0.5 with infiltration buffer (10 mM

MES, 10 mM MgCl2 and 200 µM Acetosyringone, pH 5.6). Resuspended

cultures were incubated in 28℃ shaking incubator at 220 rpm for 4 h.

Cultures were mixed together in equal proportions and infiltrated onto the

abaxial surface of 3 weeks old S.lycopersicum and N.benthamiana leaves

using 1ml needleless syringe.

4. RNA isolation, RT-PCR, and qRT-PCR

Total RNA was isolated from the leaves (4-5 internodes counted from the

bottom in plants) using PureLink RNA mini kit (Invitrogen, USA). First-

strand cDNA was synthesized from 1 µg of total RNA using M-MulV

Reverse Transcriptase (NEB, USA). Total RNA was denatured at 65℃ for

5min with 10 µM reverse primer. The reverse transcription reaction was

incubated at 42℃ for 1 h. To detect virus accumulation in the inoculated

tomato plants and verify insert maintenance in the genomes of viral progenies,

viral cDNA was amplified by PCR using Ex Taq DNA Polymerase (Takara,

Japan) with TRV specific primers (Table 1). The PCR amplification included

a 3min denaturation at 95℃, followed by 35 cycles of 95℃ for 30 sec, 58℃

for 30 sec and 72℃ for 30 sec. Gene expression was determined by AriaMX

Real-time PCR system (Agilent, USA). qRT-PCR was performed in a

reaction volume of 20 µl(2 µl diluted cDNA, 10 µl of Real-time mix, 1 µl

６

20X EvaGreen and primer pairs at 1 µM) using 2X Real-Time PCR Smart

mix (Solgent, Korea). All qRT-PCR reactions were performed on three

technical repetitions. The data were normalized using β–actin as a reference

gene.

5. Microscopy

The stem fragments were fixed in methanol and then embedded in 6.5%

agarose (Zelko et al., 2012). Sections of 80 µm thickness were made by a

microtome, stained with toluidine-blue-O for 1 min and examined by Axio

Observer Z1 (ZEISS, Germany). For Scanning electron microscope

observation, the samples from VIGS and control terminal leaflet of 6th

branches were cut into small pieces of 1cm3. The images were captured using

2 kV to minimize surface charging of the pavement cells. The microscope

TM3030plus (Hitach, Japan) was used this observation.

6. Phylogenetic analysis

Phylogenetic analysis was carried out with amino acid sequences of 17

arabidopsis, and 16 tomato CESA and, CSL genes : AT2G25540, AT4G32410,

AT5G05170, AT4G39350, AT2G21770, AT5G64740, AT5G09870,






Solyc08g082670, Solyc02g089640, Solyc04g077470, Solyc11g066820. CESA

and CSL amino acid sequences were aligned by clustalW and phylogenetic

tree was made by MEGA7 maximum likelihood method. The bootstrap

analysis was performed with 100 replications.

８

Result

1. Optimization of a TRV based virus-induced gene silencing system in

tomato.

TRV has a positive strand RNA and consist in bipartite genome and

particles are rod-shaped. RNA1 is 185 to 196 nm long and RNA2 is

approximately 50 to 115nm long. Proteins encoded by RNA1 are sufficient

for replication and movement within the host plant and proteins encoded by

RNA2 allow virion formation and nematode mediated transfer between

plants (MacFarlane, 1999). The TRV RNA1 construct using the pBINTRA6

vector as the backbone contains full length infectious cDNA clones, wherein

the RNA polymerase ORF is interrupted by intron 3 of the Arabidopsis Col-0

nitrate reductase NIA1 gene for stability in Escherichia coli (Wilkinson &

Crawford, 1993). The RNA2 construct replaced the 29.4k and 32.8k genes

with multiple cloning sites that were not essential for replication, leaving

only the 5’ and 3’ non-translated regions and virus coat proteins (Ratcliff et

al., 2001).

To perform a loss of function study of cellulose synthase genes with TRV-

VIGS (Ratcliff et al., 2001) in tomato, we have optimized the temperature

with the highest silencing effect from TRV-VIGS in tomato (cv. Ailsa Craig,

AC) under various growth conditions. The silencing of Phytoene desaturase

(PDS) in tomato results in a photo-bleaching phenotype due to suppression of

carotenoid biosynthesis (Liu et al., 2002). We use PDS gene to visually

identify the effect of VIGS. The fragment of PDS cDNA was amplified by

RT-PCR with specific primer (Liu et al., 2002) from tomato cv. Alisa Craig.

The tomato PDS gene was inserted into the pTRV2 VIGS vector in reverse

orientation at the BamHI enzyme site in pTRV2 multiple cloning sites (MCS).

Tomato grown below 20℃ after inoculation of TRV showed homogeneous

spread of the virus and high silencing efficiency of the virus (Burch-Smith et

９

al., 2004), (Ekengren et al., 2003). Tomato showed a bleaching phenotype

caused by PDS silencing in large areas (Figure 1A, 1B). Inoculated with TRV

using 3-week-old tomato, it showed that the fourth to fifth branches had the

highest VIGS effect at 18 dpi to 25 dpi (Figure 1C).

１０

Figure 1. An

efficient TRV-VIGS condition was optimized by testing for silencing of PDS

gene in tomato. PDS VIGS tomato showed photo-bleaching phenotype at 21 dpi.

Inoculated with 3 weeks old tomato. Growth condition : Day (16 h) 20℃, Night (8 h)

20℃, Humidity 60%

１１

2. Establishment of a dual gene regulation system a using a TRV vectors.

As a viral vector, TRV has many advantages. First, Since TRV has very

mild symptoms, it is less likely to be confused with VIGS phenotype. Second,

Unlike other viral vector, it spreads large area uniformly (Ratcliff et al., 2001).

In addition, it can infect growing points that no other viral vector can. TRV

can infect more than 400 species(Harrison & Robinson, 1978), including

tomato (Solanum lycopersicum), sunflower (Helianthus annuus), tulip

(Tulipa spp.), barley (Hordeum vulgare), corn (Zea mays), potato (Solanum

tuberosum), beet (Beta vulgaris), spinach (Spinacia oleracea), pepper (Capsicum

annuum), cucumber (Cucumis sativus), bean (Phaseolus vulgaris), brassica

(Brassicaceae). So it can be useful as a viral vector when studying the

genetic function of plants for which no transformation method has been

developed.

TRV has been developed as a VIGS vector and is widely used in many

plants such as Arabidopsis thaliana (Burch-Smith et al., 2006), Nicotiana

benthamiana (Senthil-Kumar & Mysore, 2014), Solanum lycopersicum (Liu

et al., 2002) but not developed as a viral overexpression vector. We have

developed a dual vector using TRV that allows gene silencing and

overexpression at once using 2A, a self-cleavage peptide of foot-and-mouth

disease virus (Liu et al., 2017). 2A was inserted into 3’ of CP gene in TRV-

VIGS vector to act as overexpression vector (Figure 2A). The PDS and GFP

were used to visually identify gene silencing and overexpression caused by

VIGS. When only the PDS gene was silenced, the photo-bleaching phenotype

was observed in the upper leaves and GFP expression was not observed.

Inoculation of TRV dual vector with PDS gene and GFP gene into Nicotiana

benthamiana and tomato showed both GFP expression and PDS silencing

(Figure 2B, 2C). GFP appeared to be expressed in the same region where

photo-bleaching occurred by PDS silencing. The pTRVd vector, which did

not insert any gene fragments into the MCS region, showed necrosis when

inoculated and did not grow well.

１２

Figure 2. GFP expression and PDS silencing at once using the TRV dual vector

(A) Schematic diagram of the pTRV dual, pTRVd-GFP-pds vector. The pTRV dual

vector is a viral vector modified by adding 2A, self-cleavage peptide at the 3’ of the

coat protein of pTRV2-VIGS vector. The pTRVd-GFP-pds was generated by

inserting the PDS gene at KpnI, XbaI enzyme sites at the MCS and inserting the

GFP gene at the 3’ of the 2A. LB; Left Border, RB; Right Border, 35S; Cauliflower

mosaic virus 35S promoter, MCS; Multiple Cloning Sites, Rz; Self-cleaving

ribozyme, NOS; Nopaline synthase terminator.

(B) The TRV dual vector express GFP and silence PDS simultaneously. The white

parts of the leaves were where PDS silencing was induced and the green parts show

GFP expression. The picture was taken at 18 dpi. Growth condition: Day (16 h)

23℃, Night (8 h) 23℃, Humidity 50%, TRV dual vector action on Nicotiana

benthamiana (C) TRV dual vector action on tomato. The GFP was observed by

in vivo image system (fluorescence) FOBI (NeoScience, Korea)

１３

１４

3. TYLCV infection causes dramatic changes in expression of some

cellulose synthase-like genes in tomato

TYLCV induce stunted growth, leaf size reduction and the yellowing and

curling leaves in tomato (Seo et al., 2018). To identify genes associated with

growth retardation, we used RNA-sequencing to analyze gene expression

changes in tomato infected with TYLCV. Several genes involved in the

cellulose biosynthesis pathway, thought to be closely associated with the

stunting phenotype, were responded by TYLCV (Figure 3A). In the

Solgenomics database, there were 33 genes annotated with the tomato

cellulose synthase family. Among the 33 genes, 16 tomato cellulose synthesis

genes which showed significant expression level in RNA-seq data and

Arabidopsis cellulose synthase family genes were analyzed by phylogenetic

tree. Seven genes with large expression changes were selected for functional

study using TRV-VIGS : Solyc02g089640, Solyc03g097050, Solyc07g043390,

Solyc07g051820, Solyc08g076320, Solyc11g066820, Solyc12g056580. Of

these, Solyc12g056580, Solyc08g076320, Solyc03g097050, Solyc02g089640

were up-regulated and Solyc07g051820, Solyc07g043390, Solyc11g0668320

were down-regulated when TYLCV infection. As a result of phylogenetic

tree analysis with Arabidopsis cellulose synthase gene family, it was showed

Solyc12g056580 belongs to CESA, Solyc11g066820 belongs to CSLA,

Solyc07g051820 belongs to CSLB, Solyc02g089640 belongs to CSLC,

Solyc03g097050 and Solyc08g076320 belong to CSLD group.

Solyc07g043390 is located between CSLB group and CSLG group (Figure

3B).

１５

Figure 3. (A) Alteration of expression levels of 33 cellulose synthase and

cellulose synthase-like genes in tomato when infected with TYLCV or ToCV.

(B) Phylogenetic analysis of tomato cellulose synthase-like genes along with

those of A.thaliana cellulose synthase family. The amino acid sequences were

aligned by clustalW and the phylogenetic tree created using neighbor-joining

method using MEGA7 program using full-length amino acid sequences of

Solanum lycopersicum Solyc08g061100, Solyc01g087210, Solyc04g071650,




Solyc11g066820 and Arabidopsis thaliana AtCESA10 (NP_001318288.1),

AtCESA1 (NP_194967.1), AtCESA3 (NP_196136.1), AtCESA2

(NP_195645.1), AtCESA9 (NP_179768.1), AtCESA6 (NP_201279.1),

AtCESA5 (NP_196549.1), AtCSLD3 (NP_186955.1), AtCSLD2

(NP_001318575.1), AtCSLB5 (NP_193264.3), AtCSLB6 (NP_193267.1),

AtCSLG2 (NP_567692.2), AtCSLG1 (NP_194132.3), AtCSLG3

(NP_194130.3), AtCSLC4 (NP_566835.1), AtCSLC12 (NP_192536.1),

AtCSLA2 (NP_197666.1). The bootstrap analysis was performed with 100

replications.

１６

11634.5

7605.5

6584

5597.5

4405

3424.5

2826.5

2754.5

2404.5

2234

1644

855.5

809.5

790

624.5

487.5

434.5

368.5

371.5

306

314

185

145

143.5

89

71.5

68.5

56.5

53

45.5

31.5

21.5

16.5

11624.5

6558

3

5120.5

9123

8172.5

2254

1911.5

426.5

1495

1169.5

833

2283

496.5

2073.5

6394

69

33

379.5

74.5

142.5

12.5

11.5

30.5

7

94

4.5

209.5

29

28.5

95.5

4.5

14

9479.5

5425

4768

2616

5638

3016.5

3603.5

3009.5

1071

3272

1842.5

793.5

2100.5

799.5

1056

1920

150

84.5

327.5

207.5

261

20

16

50.5

8

132

6.5

141.5

49

17

51.5

51

12

Solyc08g061100

Solyc01g087210

Solyc07g043390

Solyc04g071650

Solyc02g089640

Solyc12g056580

Solyc08g082640

Solyc08g082670

Solyc11g066820

Solyc07g051820

Solyc08g082660

Solyc03g005450

Solyc08g076320

Solyc08g082650

Solyc04g077470

Solyc03g097050

Solyc08g006310

Solyc10g083670

Solyc09g057640

Solyc06g074630

Solyc12g015770

Solyc09g009010

Solyc09g072820

Solyc07g005840

Solyc02g072240

Solyc11g005560

Solyc09g008990

Solyc08g005280

Solyc12g014430

Solyc07g065660

Solyc12g088240

Solyc09g075550

Solyc11g007600

Mock

TYLCV

ToCV

Expression level (FPKM)A

１７

B

１８

4. Silencing of Solyc07g043390 resulted in a stunting phenotype.

Solyc07g043390 gene is the largest downregulated cellulose biosynthesis

pathway gene that changes when TYLCV is infected (Figure 3A). To

investigate function of Solyc07g043390 in tomato, We used TRV based virus-

induced gene silencing system. In this study, pTRV2-gus with a similar

insertion size of pTRV2-3390 was used as a viral control. RT-PCR was used

to confirm that the infection of TRV and insert accumulation that indirectly

checking the VIGS progress (Burch-Smith et al., 2006). TRV infection and

insert accumulation were confirmed at 18 dpi in pTRV2-gus and pTRV2-

3390 (Figure 4A, 4B). VIGS efficiency was monitored by mRNA

accumulation level of Solyc07g043390 using quantitative real-time PCR. The

Solyc07g043390 was found to be approximately 90% silenced as compared

to control (Figure 4C). At 21 dpi, the silenced tomato showed stunt

phenotype and leaf size reduction compared to controls (Figure 5).

We examined anatomical analysis by stem sectioning. Before microscopic

observation, staining was performed with toluidine-blue-O for 1 minute to

make it easier to distinguish stem tissue. Stem sections were performed at 80

µm. The first phenotype is thin epidermis. Normal epidermis is thick at the

outermost part of stem, but silenced tomato showed thin epidermis compared

to control. The second phenotype is a thin collenchyma cell wall.

Collenchyma cells are cells found under the epidermis, providing support and

structure of plants. Normal collenchyma cell wall should have a thick cell

wall, but silenced tomato showed thin collenchyma cell wall compared to

control. The third phenotype is retardation of secondary xylem development

in the sixth internode. When staining the stem section with TBO, the part that

is dyed blue is the secondary xylem part. A thick blue secondary xylem was

observed in the control tomato, but a blue part of xylem was not observed in

the tomato stem that silenced Solyc07g043390 (Figure 6A). Therefore,

silencing of Solyc07g043390 affected cell development.

We investigated the cell surface and shape of pavement cells using

１９

scanning electron microscopy. The Solyc07g043390 silenced cells showed

smaller pavement cells and cell wall wrinkles phenotypes compared to

controls (Figure 6B). Previous studies have shown that the cesa9 mutant

phenotype in Arabidopsis has distorted seed coat epidermal cell shapes (Stork

et al., 2010), silencing of the petunia CESA3 gene with VIGS showed that

the epidermal cell size of the leaves decreased (Yang et al., 2017). Therefore,

the silencing of Solyc07g043390 affected cell wall synthesis.

２０

Figure 4. (A) RT-PCR confirmation of systemic infection of the TRV recombinants. (B) RT-PCR confirmation of maintenance of the target inserts in the genomes of TRV progenies (C) qRT-PCR analysis of VIGS efficiency. The expression level of Solyc07g043390 decreased about 90% when silenced using a TRV-VIGS system.

２１

Figure 5. Morphological phenotypes of the Solyc07g043390-silencedplant.

(A) Morphology of the whole plants of Healthy, pTRV2-gus, pTRV2-Solyc07g043390 plants. (B) The picture taken from above. This picture was taken at 21 dpi.

２２

Figure6. Alteration of anatomical

structures in the Solyc07g043390 silenced plant. (A) Sections of 80 µm thickness were made using a microtome, stained with toluidine-blue-O for 1 min, stem structure were examined using a ZEISS Axio Observer Z1. Scale bars 100 µm (B) Scanning electron microscopy of the adaxial side of the

terminal leaflet of the fourth leaf. Scale bars 30 µm.

２３

5. Overexpression of Solyc07g043390 diminished stunting symptoms caused by TYLCV

To investigate the phenotype of Solyc07g043390 overexpression to

infection with TYLCV, we generate transgenic tomato that overexpress

Solyc07g043390 gene using 35S promoter for whole body overexpression.

The full sequence of Solyc07g043390 was inserted into the XbaI, KpnI

enzyme site of pBI-121 vector MCS to generate overexpressing

transformants (Figure 7A). 3 week old plants were infected with TYLCV.

From 2 weeks after infection, TYLCV symptoms were observed in the upper

leaves. As a result of analyzing the Solyc07g043390 expression pattern, NT

plants infected with TYLCV were about 70% down-regulated in

Solyc07g043390 expression compared to healthy plants. Healthy OX plants

not infected with TYLCV had approximately twice as high Solyc07g043390

expression as compared to healthy NT plants. OX plants infected with

TYLCV express Solyc07g043390 at the level of healthy NT plants (Figure

7G). Constitutive overexpression of Solyc07g043390 resulted in growth

retarding phenotype, dark green leaves, curly-leaf phenotype compared to

control (Figure 7D).

Infection of TYLCV with NT plants resulted in stunt symptoms, but when

TYLCV was infected with OX plants, these symptoms were suppressed. By

measuring the height of the plants, NT plants showed a 47% decrease in

TYLCV infection and OX plants showed a 25% decrease (Figure 7H).

２４

Figure 7. Overexpression of Solyc07g043390 in tomato

(A) Schematic diagram of 35S::Solyc07g043390 vector. Pnos : Nopaline

synthase promoter NPTII : Kanamycin selection marker. Morphological

phenotypes of Solyc07g043390 overexpression (B) NT healthy (C) NT

TYLCV (D) OX healthy (E) OX TYLCV (F) Expression patterns of

Solyc07g043390 in healthy NT, healthy OX, TYLCV infected NT, TYLCV

infected OX (G) Plant height measurement

２５

6. Silencing phenotypes of other 6 cellulose synthase-like genes

We silence another 6 cellulose synthase genes that responded greatly by

TYLCV. TRV infection was confirmed using primers of CP gene of TRV.

Insert maintenance was confirmed by RT-PCR using a primer containing

MCS, bands for each insert size. To investigate functions of Solyc12g056580

with a greatly up-regulation with TYLCV infection through TRV-VIGS,

Solyc12g056580 fragments that 643 bp was inserted into MCS of pTRV2

vector. TRV infection (Figure 8A) and insert maintenance (Figure 8B) was

confirmed. Analysis of Solyc12g056580 mRNA expression by qRT-PCR was

showed that significant down-regulation of this gene in VIGS plant as

compared to control and TRV did not affect Solyc12g056580 expression

(Figure 8C). Solyc12g056580 also showed stunt phenotype but slightly

different from Solyc07g043390. The stem was bent, rugged stem surface and

curling branches compared to controls (Figure 8D). The phylogenetic tree

analysis revealed that the Solyc12g056580 gene belongs to the CESA

subfamily of the cellulose synthase superfamily. In Arabidopsis, there are 10

CESA genes were identified. Of these CESAs, CESA1, CESA3 and one of

CESA6-like genes (CESA2, CESA5, CESA6, CESA9) form cellulose

synthase complexes, which are involved in the formation of primary cell wall

components. CESA4, CESA7 and CESA8 play an important role in the

formation of secondary cell walls (Takata & Taniguchi, 2015; Taylor et al.,

2003). Seven of the ten CESA mutation have been studied. The rsw1-1, an

allele of AtCESA1, mutant was originally isolated on the basis of the

temperature sensitive root expansion type (Arioli et al., 1998). At the

nonpermissive temperature, rsw1-1 mutant produce less cellulose and more

β-1,4-glucan compared to wild type. The mutation was suggested to interfere

with the formation of the rosette synthase complex at the nonpermissive

temperature (Arioli et al., 1998). Cellulose reduction has been reported in

other additional alleles of Arabidopsis CESA1 (Beeckman et al., 2002;

Gillmor et al., 2002; Williamson et al., 2001).

２６

Figure 8. (A) RT-PCR confirmation of systemic infection by the TRV constructs. (B) RT-PCR confirmation of maintenance of the target inserts in the genomes of TRV progenies. (C) qRT-PCR analysis of VIGS efficiency. The expression level of Solyc12g056580 decreased about 80% when silenced using a TRV-VIGS system. (D) Morphology of the whole plants of Healthy,

２７

pTRV2-gus, pTRV2-6580 plants. (E) Stem part of the plants. This picture was taken at 20 dpi.

To investigate functions of Solyc02g089640 with a greatly up-regulation

with TYLCV infection through TRV-VIGS, Solyc02g089640 fragments that

663 bp was inserted into MCS of pTRV2 vector. TRV infection (Figure 9A)

and insert maintenance (Figure 9B) was confirmed. Analysis of

Solyc02g089640 mRNA expression by qRT-PCR was showed that significant

down-regulation of this gene in VIGS plant as compared to control and TRV

did not affect Solyc02g089640 expression (Figure 9C). Solyc02g089640 did

not affect plant growth and leaf size but it showed twisted branch phenotype

(Figure 9D) and the twisting direction was not constant. The phylogenetic

tree analysis revealed that the Solyc02g089640 gene belongs to the CSLC

subfamily of the cellulose synthase superfamily. In Pichia pastoris cell,

overexpression of the AtCSLC4 protein in Arabidopsis confirmed the

production of soluble 1,4-β-glucans suggesting that AtCLSC4 has glucan

synthesis activity (Cocuron et al., 2007a). As there is no information on the

phenotype of branching as a phenotype of other genes, the association with

glucan synthesis is considered to be further study.

To investigate functions of Solyc11g066820 with a greatly down-regulation




Solyc11g066820 mRNA expression by qRT-PCR was showed that significant

down-regulation of this gene in VIGS plants as compared to control and TRV

did not affect Solyc11g066820 expression (Figure 10C). Solyc11g066820 did

not affect plant growth and leaf size. It showed partial chlorosis phenotype on

the leaves. The phylogenetic tree analysis revealed that the Solyc11g066820

gene belongs to the CSLA subfamily of the cellulose synthase superfamily. In

Arabidopsis, AtCSLA1, 2, 7, 9 are involved in mannan synthesis (Liepman et

al., 2005). CSLA7 is important for pollen tube growth, embryogenesis

(Goubet et al., 2003). In plants, mannan is structurally and functionally

２８

diverse and serves as a structural element and energy source (Moreira &

Filho, 2008). Various plants store energy in the form of mannan in endoderm

tissue

Figure 9. (A) RT-PCR confirmation of systemic infection by the TRV constructs. (B) RT-PCR confirmation of maintenance of the target inserts in

２９

the genomes of TRV progenies. (C) qRT-PCR analysis of VIGS efficiency. The expression level of Solyc02g089640 decreased about 80% when silenced using a TRV-VIGS system. (D) Morphology of the whole plants of Healthy, pTRV2-gus, pTRV2- 9640 plants. (E) Leaf morphology of plants. Thispicture was taken at 21 dpi.

３０

Figure 10. (A) RT-PCR confirmation of systemic infection by the TRV constructs. (B) RT-PCR confirmation of maintenance of the target inserts in the genomes of TRV progenies. (C) qRT-PCR analysis of VIGS efficiency. The expression level of Solyc11g066820 decreased about 90% when silenced using a TRV-VIGS system. (D) Morphology of the whole plants of Healthy, pTRV2-gus, pTRV2- 6820 plants. (E) Leaf morphology of plants. This picture was taken at 18 dpi.

３１

(Buckeridge, 2010). In addition to carbohydrate storage and rescue,

mannan performs a variety of other functions. In fern roots, mannan performs

a variety of other functions. In fern roots, mannan is deposited as a

component of cell wall layout as a defense mechanism to limit microbial

invasion (Leroux et al., 2011). And also mannan polysaccharides have been

shown to cause folic acid and necrosis at very low concentrations, suggesting

that they have been identified by plant cells as a signal of pathogen attack or

environmental perturbation (de Pinto et al., 2003). Thus, the partial chlorosis

phenotype of the leaf, which appears to be the result of silencing the

Solyc11g066820 gene, is thought to be associated with this function of

mannan.





Solyc08g076320 mRNA expression by qRT-PCR showed a slight decrease in

the expression levels of Solyc08g076320 when TRV was infected, and

showed a significant down regulation in VIGS plants (Figure 11C). But no

obvious phenotypic changes in Solyc08g076320. The phylogenetic tree

analysis revealed that the Solyc08g076320 gene belongs to the CSLD

subfamily of the cellulose synthase superfamily. The phylogenetic tree

analysis revealed that the Solyc03g097050 gene belongs to the CSLD

subfamily of the cellulose synthase superfamily. CSLD is the most

homologous to CESA among all CSL families at the gene and protein levels

(Richmond & Somerville, 2001). In arabidopsis, expression of members of

the CSLD family is diverse, AtCSLD2 is expressed in old, expanded leaves,

while AtCSLD5 is expressed in flowers and young leaves. AtCSLD2 and

AtCSLD3 are highly expressed in roots. (Hamann et al., 2004). CSLD

expression was observed in the growing pollen tube of tobacco (Doblin et al.,

2001). CSLD3 mutant showed that did not develop root hair in arabidopsis

(Favery et al., 2001). Thus, the enzyme is proposed to play a role in plant tip-

３２

growth. In Arabidopsis, CSLD2 and CSLD3 involved female gametophyte

development (Yoo et al., 2012).

Figure 11. (A) RT-PCR confirmation of systemic infection by the TRV constructs. (B) RT-PCR confirmation of maintenance of the target inserts in the genomes of TRV progenies. (C) qRT-PCR analysis of VIGS efficiency.

３３

The expression level of Solyc08g076320 decreased about 90% when silenced using a TRV-VIGS system. (D) Morphology of the whole plants of Healthy, pTRV2-gus, pTRV2- 6320 plants. The picture was taken at 21 dpi.

To investigate functions of Solyc07g051820 with a down-regulation with

TYLCV infection through TRV-VIGS, Solyc07g051820 fragments that 401

bp was inserted into MCS of pTRV2 vector. TRV infection (Figure 12A) and

insert maintenance (Figure 12B) was confirmed. Analysis of Solyc07g051820

mRNA expression by qRT-PCR showed a slight decrease in the expression

levels of Solyc07g051820 when TRV was infected and showed a significant

down regulation in VIGS plants (Figure 12C). But no obvious phenotypic

changes in Solyc051820. The phylogenetic tree analysis revealed that the

Solyc07g051820 gene belongs to the CSLB subfamily of the cellulose

synthase superfamily. In arabidposis, AtCSLB1, AtCSLB2, and AtCSLB6

were shown to be negatively regulated by ethylene, which may play a role in

cell expansion (Hamann et al., 2004).





Solyc03g097050 mRNA expression by qRT-PCR was showed that up-

regulation of this gene in pTRV-gus and pTRV-7050 plants as compared to

control, healthy (Figure 13C). There are no obvious phenotypic changes in

Solyc03g097050. Repeated experiments with Solyc03g097050-VIGS with

inserts using other regions of the Solyc03g097050 gene did not silence the

Solyc03g097050 gene. The exact cause is unknown at this time, but other

approaches are needed to study the Solyc03g097050 gene.

３４

Figure 12. (A) RT-PCR confirmation of systemic infection by the TRV constructs. (B) RT-PCR confirmation of maintenance of the target inserts in

３５

the genomes of TRV progenies. (C) qRT-PCR analysis of VIGS efficiency. The expression level of Solyc07g051820 decreased about 80% when silenced using a TRV-VIGS system. (D) Morphology of the whole plants of Healthy, pTRV2-gus, pTRV2- 1820 plants. The picture was taken at 20 dpi.

Figure 13. (A) RT-PCR confirmation of systemic infection by the TRV

constructs. (B) RT-PCR confirmation of maintenance of the target inserts in

the genomes of TRV progenies. (C) qRT-PCR analysis of VIGS efficiency.

The expression level of Solyc03g097050 increased about 180% when using a

TRV-VIGS system. (D) Morphology of the whole plants of Healthy, pTRV2-

gus, pTRV2- 7050 plants. The picture was taken at 21 dpi.

３６

Table 1.

List of primers used in this study

３７

３８

Discussion

Infection of plants by viruses requires the virus to modify host cells to

facilitate infections. Such modifications include induction of host factors

necessary for replication, propagation and movement, suppression of host

immune responses that may associated with changes in host gene expression

(Whitham et al., 2003). These host plant gene expression regulation causes

various disease symptoms. TYLCV symptoms, such as stunt, reduced leaf

size, yellowing and leaf curl symptoms that occur when TYLCV is infected,

are also associated with host genes.

We used TRV based VIGS to loss of function study of target genes that

responded significantly to TYLCV infection and developed a dual vector that

enables simultaneous overexpression and silence by adding FMDV 2A to the

TRV VIGS vector (Figure 2A). Many attempts have been made to only

overexpress gene using TRV dual vector, but failed. The biggest problem was

the necrosis symptoms of the TRV vector. Many previous studies have

observed severe necrosis symptoms when inoculated with TRV empty

vectors, which interfered with normal growth. This could be alleviate

necrosis symptoms by inserting partial sequences of GFP or GUS that the

plants do not have. We also observed that the TRV empty vector caused

necrosis symptoms, and when GFP was expressed only, GFP did not spread

to the upper leaves of plants due to strong necrosis symptoms (data not

shown). In order to alleviate necrosis symptoms, a partial sequence of GUS

was inserted into the dual vector. However, TRV was insufficient for

systemic infection. We tried to suppress necrosis symptoms by deleting 16K

protein of TRV RNA1, which is known as silencing suppressor of TRV, and

replacing it with silencing suppressor of other viruses such as cucumber

mosaic virus 2b, B2 of Flock house virus, or P19 of tomato bushy stunt virus.

TRV clone from 16K protein was also found to cause necrosis symptoms.

３９

Therefore, further studies are needed to proceed with overexpression of gene

using TRV dual vector.

Stunt and leaf size reduction, one of the major symptoms of TYLCV are

associated with cellulose biosynthesis. The cellulose is the main component

of the cell wall and cell wall is very important for plant growth and

development, but the complete synthesis mechanism is unknown. The

complexity of cellulose structure implies that many genes are involved in

cellulose biosynthesis (Mohnen, 2008) and their expression is specifically

regulated in different cell types, tissues or organs and species in response to

developmental and environmental cues (Farrokhi et al., 2006). Cellulose is an

aggregate of unbranched polymer chains made of β-1,4-linked glucose

residues that make up the majority of primary and secondary cell walls

(Campbell et al., 1997; Lei et al., 2012; Olek et al., 2014; Richmond, 2000).

Cellulose microfibrils are made on the surface of the cell membrane to

strengthen the cell wall. They regulate cell morphogenesis and work with

many other components, including lignin, hemicellulose and pectin in cell

wall, in strong structural supports and cell shape (Cutler & Somerville, 1997).

In the absence of such support structures, cells can lose shape, expand and

spread due to cell growth (Hogetsu & Shibaoka, 1978). In plants, cellulose is

synthesized by rosette complex, cellulose synthase complex. The synthesized

cellulose is then combined by other proteins around it to form microfibrils.

Subunits of the rosette complex were found to be encoded by the CESA

(Cellulose synthase) gene. The rosette complex is thought to comprise six

complexes of five or six enzymes. The enzymes containing conserved N-

terminal Zn-binding domain indicating a mechanism for association of the

catalytic subunits (Kurek et al., 2002). These enzymes have several

transmembrane domains, which is consistent with previous microscopic and

biochemical data indicating that cellulose synthase is an integral membrane

protein and that cellulose biosynthesis occurs in the plasma membrane

(Delmer, 1999; Mueller & Brown, 1980; Ross et al., 1991). Symptoms of

TYLCV not only stunting but also yellowing of leaves. Yellowing of leaves is

４０

a chlorosis because of chlorophyll deficiency. Chlorophyll is made from δ‐

amino levulinic acid (ALA)‐ . This intermediate ALA was formed via

primary biosynthetic pathways in higher plants that require 5-carbon

substrate such as ketoglutarate or glutamate (Miller et al., 1984). The

yellowing of leaves by TYLCV is thought to be caused by several genes

involved in chlorosis development and photosynthesis of plants (Lu et al.,

2012).

The Solyc07g043390 showed significant down-regulation in TYLCV-

infected plants (Figure 3A). Solyc07g043390 belonging to a group between

cellulose synthase-like B (CSLB) group and cellulose synthase-like G (CSLG)

group (Figure 3B). In addition to the Arabidopsis, there was no gene close to

Solyc07g043390 in rice or populous, which has been studied for cellulose

synthase. The CSLB and CSLG group were reported to be specific to dicots

(Dhugga, 2012). Unfortunately, the CSLB and CSLG group features are still

unknown. Using the TRV-VIGS system, the Solyc07g043390 gene was

silenced nearly 90% compared to control, showing stunting phenotype

(Figure 4, 5). The leaf size was reduced and the number of nodes on the stem

was reduced, and the length was shortened. Overexpression of

Solyc07g043390 diminished stunt phenotype by TYLCV (Figure 8B).

Anatomical analysis reveals that Solyc07g043390 silenced tomato have a thin

epidermis at 4th internode. Thin epidermal phenotype is expected to be

affected by cell wall synthesis due to Solyc07g043390 gene silencing. The 6th

internode of silenced tomato showed that thin collenchyma cell wall

compared to control (Figure 6A). Collenchyma cells are found under

epidermal cells, outer layer of cells in young stems, in leaf veins. The main

function of a collenchyma cells is support the structure of growing plants.

Collenchyma cells have thick deposition of extra cellulose in cell wall. This

gives the longitudinal strength of the plant (Leroux, 2012). Solyc07g043390

silence affected cellulose biosynthesis, presumably thinning cellulose

deposits in collenchyma cells. And also found retardation of secondary xylem

development (Figure 6A). The Solyc07g043390 may associated with vascular

４１

tissue development. SEM observation of the Solyc07g043390 silenced leaf

showed that the epidermal cells were observed when the amount of

hydroxyproline, which plays a major role in cell wall structure, was reduced

(Fragkostefanakis et al., 2014). This suggest that Solyc07g043390 affects cell

wall structure and Solyc07g043390 could be associated with TYLCV stunt

symptoms. The Solyc12g056580 belonging to the CESA group (Figure 3B)

showed stunt, stem bent phenotype compared to control when silenced by

TRV-VIGS. The function of tomato CESA has not been studied yet, The

Arabidopsis CESA plays a major role in cellulose biosynthesis. Arabidopsis

CESA mutants have shown that interfere with assembly to rosette synthase

complex and aggregation of the β-1,4-glucan into microfibrils (Arioli et al.,

1998). In previous study, silencing cellulose synthase with potato virus X

resulted in a decrease in cellulose content and showed dwarf phenotype

(Burton et al., 2000). This suggests that Solyc12g056580 will play a major

role in tomato cellulose synthesis as CESA of tomato. The Solyc02g089640

belonging to the CSLC group (Figure 3B) showed branch twisting phenotype

compared to control when silenced by TRV-VIGS. The CSLC proteins have

been implicated in the synthesis of the 1,4-β-glucan backbone of xyloglucans

(Cocuron et al., 2007b) and other polysaccharides (Dwivany et al., 2009).

The Solyc11g066820 belonging to the CSLA group (Figure 3B) showed

partial leaf chlorosis phenotype compared to control when silenced by TRV-

VIGS. The many CSLA genes have been shown to encode mannan synthase

enzyme that polymerize the 1,4-β-linked backbone of mannan and

glucomannan (Dhugga et al., 2004; Gille et al., 2011; Popper et al., 2011;

Suzuki et al., 2006; Yin et al., 2011). Mannan is known to cause immune

responses in pathogen reactions (Zang et al., 2019) and previous studies have

shown that mannan causes chlorosis in nicotiana tabacum and rice (Shetty et

al., 1966). This may be related to the chlorosis symptoms of Solyc11g066820,

a cellulose synthase like gene. The Solyc08g076320 belonging to the CSLD

group (Figure 3B) showed no obvious phenotypic changes. The CSLD

proteins have been thought to be involved in the synthesis of β-glucan

４２

polymers (Doblin et al., 2001). Recent studies suggest that CSLD is involved

in mannan synthesis (Goubet et al., 2009; Liepman & Cavalier, 2012). As a

result of recent studies, the Solyc08g076320 is thought to be involved in fruit

ripening due to the increased expression level in tomato breaker stage (Song

et al., 2019). The Solyc07g051820 belonging to the CSLB group (Figure 3B)

showed no obvious phenotypic changes. The CSLB proteins functions are

still unknown. Solyc07g051820 has shown a large amount of expression in

green parts of tomato such as leaf, stem and young fruits (). The

Solyc03g097050 belonging to the CSLD group (Figure 3B) showed up-

regulation when TRV infected. Since the qRT-PCR amplification of region of

the Solyc03g097050 gene is different from that of the VIGS fragment, (Table

1), we can rule out possibility of increased expression values due to

replication of VIGS vector. The expression level through RNA-seq shows

that the expression level of Solyc03g0497050 gene is greatly increased not

only by TYLCV but also by ToCV (Figure 3B). This suggests that the

Solyc03g097050 is up-regulated by virus in response to infection. Therefore,

there is a limitation to study the function of Soly03g097050 gene using TRV-

VIGS. Four genes showed significant phenotype as a result of silencing with

TRV-VIGS. These genes are genes with high levels of expression regardless

of whether they are up or down regulated when TYLCV is infected (Figure

3A). To provide functional information, we successfully applied a VIGS

approach and demonstrated that the stunt symptom on tomato was obtained

by targeting Solyc07g043390 previously described as dramatically down-

regulation upon TYLCV infection. This may suggest that Solyc07g043390

can play an important role in the establishment of the cell walls. These data

will also contribute to our overall knowledge about the function of cellulose

synthase genes. VIGS could be used as a tool to investigate the functional

study for cellulose synthase gene in tomato. Our study can provide new

insights on how host plant genes are specifically associated with disease

symptom development and a molecular basis to facilitate future plant

immunity engineering.

４３

４４

４５

References

Arioli T., Peng L., Betzner A. S., Burn J., Wittke W., Herth W., Camilleri C., Hofte H., Plazinski J., Birch R., Cork A., Glover J., Redmond J., Williamson R. E. (1998) Molecular analysis of cellulose biosynthesis in Arabidopsis. Science. 279, 717-720.

Beeckman T., Przemeck G. K., Stamatiou G., Lau R., Terryn N., De Rycke R., Inze D., Berleth T. (2002) Genetic complexity of cellulose synthase a gene function in Arabidopsis embryogenesis. Plant Physiol. 130, 1883-1893.

Buckeridge M. S. (2010) Seed Cell Wall Storage Polysaccharides: Models to Understand Cell Wall Biosynthesis and Degradation. Plant Physiology. 154, 1017-1023.

Burch-Smith T. M., Anderson J. C., Martin G. B., Dinesh-Kumar S. P.(2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J. 39, 734-746.

Burch-Smith T. M., Schiff M., Liu Y., Dinesh-Kumar S. P. (2006) Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol. 142, 21-27.

Burton R. A., Gibeaut D. M., Bacic A., Findlay K., Roberts K., Hamilton A., Baulcombe D. C., Fincher G. B. (2000) Virus-induced silencing of a plant cellulose synthase gene. Plant Cell. 12, 691-706.

Campbell J. A., Davies G. J., Bulone V., Henrissat B. (1997) A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities. Biochem J. 326 ( Pt 3), 929-939.

Chantreau M., Chabbert B., Billiard S., Hawkins S., Neutelings G. (2015) Functional analyses of cellulose synthase genes in flax (Linum usitatissimum) by virus-induced gene silencing. Plant Biotechnol J. 13, 1312-1324.

Cocuron J. C., Lerouxel O., Drakakaki G., Alonso A. P., Liepman A. H., Keegstra K., Raikhel N., Wilkerson C. G. (2007a) A gene from the cellulose synthase-like C family encodes a beta-1,4 glucan synthase. P Natl Acad Sci USA. 104, 8550-8555.

４６

Cocuron J. C., Lerouxel O., Drakakaki G., Alonso A. P., Liepman A. H., Keegstra K., Raikhel N., Wilkerson C. G. (2007b) A gene from the cellulose synthase-like C family encodes a beta-1,4 glucan synthase. Proc Natl Acad Sci U S A. 104, 8550-8555.

Cutler S., Somerville C. (1997) Cellulose synthesis: Cloning in silico. Current Biology. 7, R108-R111.

de Pinto M. C., Lavermicocca P., Evidente A., Corsaro M. M., Lazzaroni S., De Gara L. (2003) Exopolysaccharides produced by plant pathogenic bacteria affect ascorbate metabolism in Nicotiana tabacum. Plant Cell Physiol. 44, 803-810.

Delmer D. P. (1999) CELLULOSE BIOSYNTHESIS: Exciting Times for A Difficult Field of Study. Annu Rev Plant Physiol Plant Mol Biol. 50, 245-276.

Dhugga K. S. (2012) Biosynthesis of non-cellulosic polysaccharides of plant cell walls. Phytochemistry. 74, 8-19.

Dhugga K. S., Barreiro R., Whitten B., Stecca K., Hazebroek J., Randhawa G. S., Dolan M., Kinney A. J., Tomes D., Nichols S., Anderson P. (2004) Guar seed beta-mannan synthase is a member of the cellulose synthase super gene family. Science. 303, 363-366.

Doblin M. S., De Melis L., Newbigin E., Bacic A., Read S. M. (2001) Pollen tubes of Nicotiana alata express two genes from different beta-glucan synthase families. Plant Physiol. 125, 2040-2052.

Dwivany F. M., Yulia D., Burton R. A., Shirley N. J., Wilson S. M., Fincher G. B., Bacic A., Newbigin E., Doblin M. S. (2009) The CELLULOSE-SYNTHASE LIKE C (CSLC) family of barley includes members that are integral membrane proteins targeted to the plasma membrane. Mol Plant. 2, 1025-1039.

Ekengren S. K., Liu Y., Schiff M., Dinesh-Kumar S. P., Martin G. B.(2003) Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato. Plant J. 36, 905-917.

Farrokhi N., Burton R. A., Brownfield L., Hrmova M., Wilson S. M., Bacic A., Fincher G. B. (2006) Plant cell wall biosynthesis: genetic, biochemical and functional genomics approaches to the identification of key genes. Plant Biotechnol J. 4, 145-167.

Favery B., Ryan E., Foreman J., Linstead P., Boudonck K., Steer M., Shaw P., Dolan L. (2001) KOJAK encodes a cellulose synthase-like protein

４７

required for root hair cell morphogenesis in Arabidopsis. Gene Dev. 15, 79-89.

Fragkostefanakis S., Sedeek K. E., Raad M., Zaki M. S., Kalaitzis P.(2014) Virus induced gene silencing of three putative prolyl 4-hydroxylases enhances plant growth in tomato (Solanum lycopersicum). Plant Mol Biol.85, 459-471.

Ghanim M., Morin S., Zeidan M., Czosnek H. (1998) Evidence for transovarial transmission of tomato yellow leaf curl virus by its vector, the whitefly Bemisia tabaci. Virology. 240, 295-303.

Gille S., Cheng K., Skinner M. E., Liepman A. H., Wilkerson C. G., Pauly M. (2011) Deep sequencing of voodoo lily (Amorphophallus konjac): an approach to identify relevant genes involved in the synthesis of the hemicellulose glucomannan. Planta. 234, 515-526.

Gillmor C. S., Poindexter P., Lorieau J., Palcic M. M., Somerville C.(2002) Alpha-glucosidase I is required for cellulose biosynthesis and morphogenesis in Arabidopsis. J Cell Biol. 156, 1003-1013.

Goubet F., Barton C. J., Mortimer J. C., Yu X., Zhang Z., Miles G. P., Richens J., Liepman A. H., Seffen K., Dupree P. (2009) Cell wall glucomannan in Arabidopsis is synthesised by CSLA glycosyltransferases, and influences the progression of embryogenesis. Plant J. 60, 527-538.

Goubet F., Misrahi A., Park S. K., Zhang Z. N., Twell D., Dupree P. (2003) AtCSLA7, a cellulose synthase-like putative glycosyltransferase, is important for pollen tube growth and embryogenesis in Arabidopsis. Plant Physiology.131, 547-557.

Hamann T., Osborne E., Youngs H. L., Misson J., Nussaume L., Somerville C. (2004) Global expression analysis of CESA and CSL genes in Arabidopsis. Cellulose. 11, 279-286.

Harrison B. D., Robinson D. J. (1978) The tobraviruses. Adv Virus Res.23, 25-77.

Hazen S. P., Scott-Craig J. S., Walton J. D. (2002) Cellulose synthase-like genes of rice. Plant Physiol. 128, 336-340.

Herth W. (1985) Plasma-membrane rosettes involved in localized wall thickening during xylem vessel formation of Lepidium sativum L. Planta.164, 12-21.

Hogetsu T., Shibaoka H. (1978) Effects of Colchicine on Cell-Shape and on

４８

Microfibril Arrangement in Cell-Wall of Closterium-Acerosum. Planta.140, 15-18.

Kil E. J., Kim S., Lee Y. J., Byun H. S., Park J., Seo H., Kim C. S., Shim J. K., Lee J. H., Kim J. K., Lee K. Y., Choi H. S., Lee S. (2016) Tomato yellow leaf curl virus (TYLCV-IL): a seed-transmissible geminivirus in tomatoes. Sci Rep. 6, 19013.

Kissoudis C., Sunarti S., van de Wiel C., Visser R. G., van der Linden C. G., Bai Y. (2016) Responses to combined abiotic and biotic stress in tomato are governed by stress intensity and resistance mechanism. J Exp Bot. 67, 5119-5132.

Kurek I., Kawagoe Y., Jacob-Wilk D., Doblin M., Delmer D. (2002) Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via oxidation of the zinc-binding domains. Proc Natl Acad Sci U S A. 99, 11109-11114.

Lei L., Li S., Gu Y. (2012) Cellulose synthase complexes: composition and regulation. Front Plant Sci. 3, 75.

Leroux O. (2012) Collenchyma: a versatile mechanical tissue with dynamic cell walls. Ann Bot-London. 110, 1083-1098.

Leroux O., Leroux F., Bagniewska-Zadworna A., Knox J. P., Claeys M., Bals S., Viane R. L. L. (2011) Ultrastructure and composition of cell wall appositions in the roots of Asplenium (Polypodiales). Micron. 42, 863-870.

Levine A., Tenhaken R., Dixon R., Lamb C. (1994) H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell. 79, 583-593.

Liepman A. H., Cavalier D. M. (2012) The CELLULOSE SYNTHASE-LIKE A and CELLULOSE SYNTHASE-LIKE C families: recent advances and future perspectives. Front Plant Sci. 3, 109.

Liepman A. H., Wilkerson C. G., Keegstra K. (2005) Expression of cellulose synthase-like (Csl) genes in insect cells reveals that CslA family members encode mannan synthases. P Natl Acad Sci USA. 102, 2221-2226.

Liu Y., Schiff M., Dinesh-Kumar S. P. (2002) Virus-induced gene silencing in tomato. Plant J. 31, 777-786.

Liu Z., Chen O., Wall J. B. J., Zheng M., Zhou Y., Wang L., Ruth Vaseghi H., Qian L., Liu J. (2017) Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep. 7, 2193.

４９

Lu J., Du Z. X., Kong J., Chen L. N., Qiu Y. H., Li G. F., Meng X. H., Zhu S. F. (2012) Transcriptome analysis of Nicotiana tabacum infected by Cucumber mosaic virus during systemic symptom development. PLoS One.7, e43447.

MacFarlane S. A. (1999) Molecular biology of the tobraviruses. J Gen Virol.80 ( Pt 11), 2799-2807.

Makkouk K. M., Shehab S., Majdalani S. E. (1979) Tomato Yellow Leaf Curl - Incidence, Yield Losses and Transmission in Lebanon. Phytopathol Z.96, 263-267.

Miller G. W., Pushnik J. C., Welkie G. W. (1984) Iron Chlorosis, a World Wide Problem, the Relation of Chlorophyll Biosynthesis to Iron. J Plant Nutr.7, 1-22.

Mochizuki T., Ogata Y., Hirata Y., Ohki S. T. (2014) Quantitative transcriptional changes associated with chlorosis severity in mosaic leaves of tobacco plants infected with Cucumber mosaic virus. Mol Plant Pathol. 15, 242-254.

Mohnen D. (2008) Pectin structure and biosynthesis. Curr Opin Plant Biol.11, 266-277.

Moreira L. R. S., Filho E. X. F. (2008) An overview of mannan structure and mannan-degrading enzyme systems. Appl Microbiol Biot. 79, 165-178.

Moriones E., Navas-Castillo J. (2000) Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide. Virus Res. 71, 123-134.

Mueller S. C., Brown R. M., Jr. (1980) Evidence for an intramembrane component associated with a cellulose microfibril-synthesizing complex in higher plants. J Cell Biol. 84, 315-326.

Navas-Castillo J., Camero R., Bueno M., Moriones E. (2000) Severe Yellowing Outbreaks in Tomato in Spain Associated with Infections of Tomato chlorosis virus. Plant Dis. 84, 835-837.

Navot N., Pichersky E., Zeidan M., Zamir D., Czosnek H. (1991) Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component. Virology. 185, 151-161.

Olek A. T., Rayon C., Makowski L., Kim H. R., Ciesielski P., Badger J., Paul L. N., Ghosh S., Kihara D., Crowley M., Himmel M. E., Bolin J. T.,

５０

Carpita N. C. (2014) The structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers. Plant Cell. 26, 2996-3009.

Popper Z. A., Michel G., Herve C., Domozych D. S., Willats W. G., Tuohy M. G., Kloareg B., Stengel D. B. (2011) Evolution and diversity of plant cell walls: from algae to flowering plants. Annu Rev Plant Biol. 62, 567-590.

Ratcliff F., Martin-Hernandez A. M., Baulcombe D. C. (2001) Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J. 25, 237-245.

Richmond T. (2000) Higher plant cellulose synthases. Genome Biol. 1, REVIEWS3001.

Richmond T. A., Somerville C. R. (2000) The cellulose synthase superfamily. Plant Physiol. 124, 495-498.

Richmond T. A., Somerville C. R. (2001) Integrative approaches to determining Csl function. Plant Mol Biol. 47, 131-143.

Ross P., Mayer R., Benziman M. (1991) Cellulose biosynthesis and function in bacteria. Microbiol Rev. 55, 35-58.

Sabba R. P., Vaughn K. C. (1999) Herbicides that inhibit cellulose biosynthesis. Weed Sci. 47, 757-763.

Saxena I. M., Brown R. M., Jr., Fevre M., Geremia R. A., Henrissat B.(1995) Multidomain architecture of beta-glycosyl transferases: implications for mechanism of action. J Bacteriol. 177, 1419-1424.

Senthil-Kumar M., Mysore K. S. (2014) Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana. Nat Protoc. 9, 1549-1562.

Seo J. K., Kim M. K., Kwak H. R., Choi H. S., Nam M., Choe J., Choi B., Han S. J., Kang J. H., Jung C. (2018) Molecular dissection of distinct symptoms induced by tomato chlorosis virus and tomato yellow leaf curl virus based on comparative transcriptome analysis. Virology. 516, 1-20.

Song X., Xu L., Yu J., Tian P., Hu X., Wang Q., Pan Y. (2019) Genome-wide characterization of the cellulose synthase gene superfamily in Solanum lycopersicum. Gene. 688, 71-83.

Stork J., Harris D., Griffiths J., Williams B., Beisson F., Li-Beisson Y., Mendu V., Haughn G., Debolt S. (2010) CELLULOSE SYNTHASE9 serves a nonredundant role in secondary cell wall synthesis in Arabidopsis epidermal testa cells. Plant Physiol. 153, 580-589.

５１

Suzuki S., Li L., Sun Y. H., Chiang V. L. (2006) The cellulose synthase gene superfamily and biochemical functions of xylem-specific cellulose synthase-like genes in Populus trichocarpa. Plant Physiol. 142, 1233-1245.

Takata N., Taniguchi T. (2015) Expression divergence of cellulose synthase (CesA) genes after a recent whole genome duplication event in Populus. Planta. 241, 29-42.

Taylor N. G., Howells R. M., Huttly A. K., Vickers K., Turner S. R. (2003) Interactions among three distinct CesA proteins essential for cellulose synthesis. P Natl Acad Sci USA. 100, 1450-1455.

Whitham S. A., Quan S., Chang H. S., Cooper B., Estes B., Zhu T., Wang X., Hou Y. M. (2003) Diverse RNA viruses elicit the expression of common sets of genes in susceptible Arabidopsis thaliana plants. Plant J. 33, 271-283.

Wilkinson J. Q., Crawford N. M. (1993) Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2. Mol Gen Genet. 239, 289-297.

Williamson R. E., Burn J. E., Birch R., Baskin T. I., Arioli T., Betzner A. S., Cork A. (2001) Morphology of rsw1, a cellulose-deficient mutant of Arabidopsis thaliana. Protoplasma. 215, 116-127.

Yang W., Cai Y., Hu L., Wei Q., Chen G., Bai M., Wu H., Liu J., Yu Y.(2017) PhCESA3 silencing inhibits elongation and stimulates radial expansion in petunia. Sci Rep. 7, 41471.

Yin L., Verhertbruggen Y., Oikawa A., Manisseri C., Knierim B., Prak L., Jensen J. K., Knox J. P., Auer M., Willats W. G., Scheller H. V. (2011) The cooperative activities of CSLD2, CSLD3, and CSLD5 are required for normal Arabidopsis development. Mol Plant. 4, 1024-1037.

Yoo C. M., Quan L., Blancaflor E. B. (2012) Divergence and redundancy in CSLD2 and CSLD3 function during Arabidopsis thaliana root hair and female gametophyte development. Frontiers in Plant Science. 3.

Zelko I., Lux A., Sterckeman T., Martinka M., Kollarova K., Liskova D.(2012) An easy method for cutting and fluorescent staining of thin roots. Ann Bot. 110, 475-478.

Zhu X., Pattathil S., Mazumder K., Brehm A., Hahn M. G., Dinesh-Kumar S. P., Joshi C. P. (2010) Virus-induced gene silencing offers a

５２

functional genomics platform for studying plant cell wall formation. Mol Plant. 3, 818-833.

Abstract in Korean

토마토황화잎말림 바이러스 감염에 반응하는

토마토 셀룰로오스 합성 유전자의 기능 연구

최시원

서울대학교 국제농업기술대학원 국제농업기술학과

지도교수 서장균

토마토는 경제적으로 매우 중요한 작물이며 식물 발달, 유전학, 병

리학, 생리학 분야에서 오랜 기간 모델 식물로 사용되어왔다. 바이

러스질병은 토마토 성장과 생산에 많은 영향을 미친다. 특히 토마

토황화잎말림 바이러스는 심각한 발육 저하, 잎 크기 감소, 잎 말림,

황화 병징을 동반하기에 토마토의 가장 해가 되는 바이러스 중 하

나이다. 이전 연구에서 우리는 전사체 비교 분석을 통하여 셀룰로

오스 생합성 경로에 연관된 여러가지 유전자들이 토마토황화잎말림

바이러스에 의해 반응하는 것을 보여주었다. 셀룰로오스 합성 유전

자는 세포벽 생합성, 세포 신장 그리고 식물 성장에 중요한 역할을

한다. 이 연구에서 우리는 기능 소실 접근법으로써 담배얼룩바이러

５３

스를 이용한 바이러스 기반 유전자 침묵 기술로 토마토황화잎말림

바이러스에 의해 두드러지게 조절되는 7개의 토마토 셀룰로오스 합

성 유사 유전자들의 특성을 분석한다. 담배얼룩바이러스를 이용한

바이러스 기반 유전자 침묵 기술을 사용하여 우리는 목표로 하는

셀룰로오스 합성 유사 유전자들을 성공적으로 침묵시켰고 표현형을

관찰하였다. 몇몇 셀룰로오스 유사 유전자들의 침묵은 성장을 지연

시키고 관 조직의 발달을 지연시켰으며 이는 토마토황화잎말림 바

이러스 감염 시 변화하는 셀룰로오스 합성 유전자의 변화가 토마토

황화잎말림 바이러스에 의해 발생하는 토마토 발육 저하 증상과 연

관되어 있음을 시사하고 있다. 우리 연구는 숙주 식물 유전자가 어

떻게 질병 발생과 어떻게 연관되어 있는지, 미래 식물 면역 공학을

용이하게 하는 분자적 기반에 대한 새로운 이해를 제공한다.

·······························

주요어: 토마토, 토마토 황화잎말림바이러스, 셀룰로오스,

바이러스 기반 유전자 침묵, 담배 얼룩바이러스

학번: 2018-24842

５４

감사의 글

많은 분들의 도움으로 이 논문을 완성할 수 있었기에 감사의 인

사를 전합니다. 성심성의껏 지도해주신 지도교수 서장균 교수님 감

사드립니다 그리고 저의 연구에 아낌없는 조언을 해주신 김주곤 교

수님, 강진호 교수님, 정춘균 교수님께도 감사드립니다.

2년 동안 같은 연구실에서 지내며 자신의 일처럼 많은 도움과 관

심을 준 보람누나와 경재, 명휘 모두 감사합니다. 가족처럼 지내며

항상 함께했던 작물유전체육종 연구실, 작물분자생물학 연구실 구

성원 모두에게 감사합니다.

그리고 연구실 생활에 많은 도움을 준 수정누나, 논문 제작과 발

표에 많은 도움을 준 누리, 많은 시간을 할애하여 저의 실험을 도

와준 서원이형에게도 감사합니다.

마지막으로 항상 아낌없는 지원을 해주며 응원해주시는 부모님과

누나에게 감사합니다. 모든 분들 덕분에 석사학위 과정을 마칠 수

있었습니다.

석사학위 기간 동안 훌륭한 교수님들과 종자연구소의 뛰어난 학

생들과 함께 생활할 수 있어서 영광이었고 행복했습니다. 언제나

겸손한 자세로 계속해서 배움을 이어나가도록 하겠습니다. 올바른

방향으로 지식을 사용하고 인류발전에 기여하는 사람이 되겠습니다.

disclaimers-space.snu.ac.kr/bitstream/10371/166623/1/000000160525.pdf · 2 days ago · we...

Documents