rna interference based resistance against chili leaf curl
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RNA interference based resistance against chili leaf curl disease complex
A dissertation submitted to Quaid-i-Azam University, Islamabad in
partial fulfilment of requirements for the degree of
DOCTOR OF PHILOSPHY
IN
BIOTECHNOLOGY
By
Muhammad Shafiq
National Institute for Biotechnology and Genetic Engineering
(NIBGE), Faisalabad
and
Quaid-i-Azam University Islamabad, Pakistan
2013
NIBGE |School of Biotechnology NIBGE Faisalabad [QAU Islamabad]
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The Controller of Examinations,
Quaid-i-Azam University
Islamabad
This thesis submitted by Mr. Muhammad Shafiq is accepted in its present form by
Quaid-i-Azam University Islamabad as satisfying the thesis requirements for the
award of the degree of Doctor of Philosophy in Biotechnology.
Supervisor: -------------------------------------------- (Dr. Yusuf Zafar T.I) Minister (Technical)
Permanent Mission of Pakistan to the IAEA
Hofzeile 13, A-1190
Vienna – Austria
Co-Supervisor: ---------------------------------- (Dr. Shaheen Aftab) National Institute for Biotechnology and Genetic
Engineering, Faisalabad.
External Examiner 1 ------------------------------------
Dr. Asif Ali Khan Professor, Department Plant Breeding and Genetics, University of Agriculture Faisalabad
External Examiner 2 ------------------------------------
Dr. Sultan Habibullah Khan Assistant Professor, Centre for Agricultural Biochemistry and Biotechnology University of Agriculture Faisalabad
Director: ------------------------------------
(Dr. Shahid Mansoor S.I) National Institute for Biotechnology and Genetic
Engineering, Faisalabad.
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ABSTRACT
Chilli (Capsicum annuum), a member of the family Solanaceae, is an important spice
crop cultivated in tropical and subtropical countries. Chilli leaf curl disease (ChLCD)
is a limiting factor for chilli yield across Pakistan and India. Symptoms of ChLCD
include severe upward leaf curl with cup-shape, yellowing and stunted plant growth.
This disease is caused by begomoviruses (single-stranded DNA viruses (family
Geminiviridae) that are transmitted by whiteflies). All three different types of
begomoviruses are already reported from chillies. In this study chilli samples showing
typical disease symptoms were collected from Faisalabad in the Province of Punjab
(Pakistan) during the year 2006. All samples were positive for begomoviruses and
Pepper leaf curl Lahore virus (PepLCLV) along with Tomato leaf curl New Delhi
virus DNA B and Chilli leaf curl betasatellite (ChLCB) were identified. The DNA of
Pepper leaf curl Lahore virus consisted of 2747 nucleotides and had the highest
sequence identity (99%) with PepLCLV-[PK: Lah: 04] AM404179). Agrobacterium-
mediated inoculation of the partial repeat construct of PepLCLV clone obtained in
this study to Nicotiana benthamiana induced very mild symptoms and very low flow
of viral DNA were detected in infected plant leaves. Co-inoculation of ChLCB with
PepLCLV to N. benthamiana did not affect the symptoms severity or the virus titre.
However neither the PepLCLV alone or with ChLCB was able to induce any
symptoms on N. tabacum L. and C. annuum. Inoculation of PepLCLV with DNA B
of ToLCNDV induced very severe symptoms in N. benthamiana, N. tabacum and
symptoms typical of ChLCD in C. annuum. Southern hybridization analysis showed
very high DNA accumulation for PepLCLV and DNA B of ToLCNDV in all three
plant species. Sequence analysis showed that predicted rep-binding iterons in
PepLCLV (GGGGAC) was different with two nucleotides from that of ToLCNDV
DNA B (GGTGTC). This indicated tolerance of two nucleotide differences in iterated
elements for replication. Based on this study, it is proposed that PepLCLV has been
recently mobilized into chillies upon its interaction with DNA B of ToLCNDV. This
is the first experimental demonstration of infectivity for a bipartite begomovirus
causing ChLCD in chillies from Pakistan and suggests that component capture may
contribute to the emerging complexity of begomovirus diseases in the region.
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The purpose of this study was to develop a broad-spectrum resistance against ChLCD
complex based on the concept of pathogen-derived resistance. A hairpin RNAi
construct (peAC1-AC2dsRNA/pFGC) based on overlapping region of highly
conserved region of Rep and TrAP of PepLCLV was produced in a binary vector
pFGC5941. In order to study silencing efficiency of peAC1-AC2dsRNA/pFGC, the
construct was transiently challenged with PepLCLV along with DNA B ToLCNDV.
Results showed that the RNAi construct was successful in blocking the viral infection
as all tested plants were symptomless. Transgenic tobacco plants expressing this
construct challenged with PepLCLV and DNA B of ToLCNDV by agroinoculation
and with viruliferous whiteflies showed variable resistance ranging from 6.6% to
93.3%. Lines showing resistance more than 75% were ranked resistant/tolerant while
lines showing resistance less than 50% were ranked susceptible. One line TA14
showing 93.3% was ranked as highly resistant/tolerant while the line TA 3.2 showing
6.6 % resistance/tolerance was ranked as highly suscepteible. These lines also
exhibited significant resistance against ToLCNDV. The relatively conserved nature of
Rep and TrAP and their ability to help in development of resistance against
heterologous virus suggested that the technology may be useful to develop broad-
spectrum resistance. Plants need broad spectrum resistance because they were often
infected with multiple begomoviruses in the field.
Some viral proteins interfere with different cell signalling pathways and induce
symptoms in plants. For example expression of P6 protein of CaMV in Arabidopsis
induced dwarfness in transgenic plants. It is reported that Arabidopsis plants with
TIR3 gene mutated (tir3) are also dwarf. P6 transgenic (A7, B6) and tir3 Arabidopsis
plants which were resistant to auxin and ethylene also showed resitance to 2,3,5-
Triiodobenzoic Acid (TIBA) treatment. It indicates that P6 interacts with a pathway
overlapped with TIR pathway. Symptoms in Arabidopsis expressing the P6 protein of
CaMV probably comes by disturbance of auxin response factor 10 (ARF10), ARF16,
and ARF17 also. Also P6-expressing transgenic Arabidopsis plants showed reduced
accumulation of miR160 which is known to regulate ARF10, ARF16 and ARF17.
A protocol was also developed for chilli plant regeneration using hypocotyl and
cotyledon explants. The study was conducted to observe the effect of genotypes,
culture conditions and growth regulators on plant regeneration of chili pepper (C.
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annuum) genotypes grown in Pakistan including Seedex Pepper (SP), Loungi,
Tatapuri and Sanam. Of the evaluated genotypes, SP was found to be the most
responsive for both hypocotyl and cotyledon explants. Hypocotyl and cotyledon
explants were tested for transformation by A. tumefactions LBA4404 having the 35S
GFP/pFGC construct and A. tumefaciens EHA105 with peAC1-AC2dsRNA/pFGC
construct. Co-cultivation at different temperatures (22 and 25ºC), photoperiods (16h
light 8h dark, and complete darkness) as well as co-cultivation time periods, were
evaluated. GFP assays showed that putative transgenic calli had not been transformed
and calli died after 40-60 days. The experiment was repeated ten times.
The data presented in this thesis should help in devising novel control strategies
against Begomoviruses. A combination of novel sources of resistance with natural
sources of resistance may help to exploit the technology in the field conditions.
However, because most pepper varieties are recalcitrant to genetic transformation,
control of diseases caused by the ChLCD complex using this strategy awaits future
progress.
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Acknowledgment
All the praise and thanks to almighty Allah, who is the compassionate, the merciful,
the creator of the universe and source of all knowledge and wisdom. I offer my
humblest thanks to „The Holy Prophet Hazrat Mohammed” (Peace Be Upon Him)
who ordained every Muslim to yearn for knowledge from the cradle to grave.
It is my pleasure to thank Dr. Shahid Mansoor S.I Director NIBGE who kindly gave
me permission and access to facilities available at NIBGE to carry out this research
work. I wish to express my deep sense of gratitude for my supervisor, Dr. Yusuf
Zafar T.I Minister (Technical); Permanent Mission of Pakistan to the IAEA
Vienna - Austria who has made a great contribution for the successful completion of
this work.
I am extremely thankful to my co-supervisor Dr. Shaheen Asad (PSO). It was indeed
an honor to work under his guidance. Her personal interest, extremely amicable
encouraging behavior and ample support helped me in the successful completion of
this work.
It is my pleasure to thank Dr. Rob. W Briddon for his help, suggestions and all kind
support.
Special thanks to Mr. Muhammad Arshad (SS) for his encouraging attitude and
moral support. I am also thankful to my Dr. Zahid Mukhtar for his valuable help
open heart and cooperation.
I am at short for worlds to express my gratitude to my friends Muhammad Ikram
Anwar, Dr. Farooq, Muhammad AAmir Mehmood, Khadim Hussain,
Muhammad Arshad, Muhammad Asif Habib, Muhammad Mubin, and Mutahar
Mansoor Qaisrani for his all time available help and sincere cooperation.
I am also grateful to my other lab fellows Ghulam Raza, Kazim Ali Muhammad
Ismail Lashari, Jamil Amjad Hashmi and Arif Khan for their cooperation and nice
company.
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No acknowledgment would ever adequetly express my obligation to my parents,
brother Ghulam Abid, my wife and my son Muhammad Faizan Rasul (late) and
my daughter Maryam Naseem who‟s love and prays will always remain with me.
Muhammad Shafiq
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Dedicated
To
Prophet Muhammad
(Peace be upon him)
My mother
And
Son
(Muhammad Faizan Rasul)
ABBREVIATIONS
µL microlitre
AAP acquisition access period
AD Anno Domini
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asRNA anti-sense RNA
AVRDC Asian Vegetable Research and Development Centre
AZPs artificial zinc finger proteins
BC Before Christ
BND benzoylated naphthoylated DEAE BSA bovine serum albumin
CaCl2 calcium chloride cccDNA covalently closed circular DNA
CIAP calf intestine alkaline phosphatase
CLCuD cotton leaf curl disease
CP coat protein
CR common region
CTAB cetyl trimethyl ammonium bromide
DEAE diethylaminoethyl cellulose
DNA deoxyribonucleic acid
DNAi DNA interference
dNTP deoxyribonucleotide triphosphate
dsDNA double-stranded DNA
dsRNA double-stranded RNA
DTT dithiothreitol
EDTA ethylene diamine tetraacetic acid
EU European Union
FeSO4.7H2O ferrous sulphate hepta hydrate
GFP green fluorescence protein
GUS beta-glucuronidase
hpRNA hairpin RNA
HR hypersensitive response
ICTV International Committee on Taxonomy of Viruses
IPTG isopropyl-beta-D-1-thiogalactopyranoside
IR intergenic region
IRD iteron related domain K2HPO4 dipotassium phosphate
KCl potassium chloride kDa kilo Dalton
kV kilo Volt LB Luria broth
LIR large intergenic region LYMV legume yellow mosaic virus
MCS multiple cloning site
mg milligram
MgSO4 magnesium sulphate
MgSO4.7H2O magnesium sulphate heptahydrate
miRNA microRNA
mM milli molar
MP Movement Protien
mRNA messenger RNA
NaCl sodium chloride
NaH2PO4 sodium phosphate
NH4Cl ammonium chloride
NLS nuclear localization signals
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NSP nuclear shuttle protein
nt. Nucleotide
NW New World
OD optical density
ORF open reading frame
OW Old World
PCNA proliferating cell nuclear antigen
PCR polymerase chain reaction
PDR pathogen derived resistance
pH paviour of hydrogen
Pre-miRNA precursor miRNA
PVP polyvinyl pyrrolidone
RCA rolling circle amplification
RCR rolling circle replication
RDR recombination-dependent replication
RdRP RNA dependent RNA polymerase
REn replication enhancer protein
Rep replication associated protein
RISC RNA-induced silencing complex
RNA ribonucleic acid
RNAi RNA interference
rpm revolutions per minute
SBS School of Biological Sciences
SCR satellite-conserved region
SDS sodium dodecyl sulphate
SIR small intergenic region
siRNA small interfering RNA
SSC standard sodium citrate
ssDNA single-stranded DNA
TAE tris-acetate EDTA
Taq Thermus aquaticus
ta-siRNAs trans-acting siRNAs
TGS transcriptional gene silencing
TrAP transcriptional activator protein
T-Rep truncated Rep
UV ultra violet
VIGS virus induced gene silencing
X-Gal 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside
YMD yellow mosaic disease
VIRUSES AND SATELLITES
Abutilon mosaic virus (AbMV)
African cassava mosaic virus (ACMV)
Ageratum yellow vein virus (AYVV)
Bean dwarf mosaic virus (BDMV)
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Bean golden yellow mosaic virus (BGYMV) Bean leaf curl China betasatellite (BLCCNB)
Bean yellow dwarf virus (BeYDV) Beet curly top virus (BCTV)
Beet severe curly top virus (BSCTV)
Cabbage leaf curl virus (CabLCuV)
Cestrum yellow leaf curling virus (CmYLCV)
Chilli leaf curl betasatellite (ChLCB)
Chilli leaf curl virus (ChiLCV)
Citrus tristezia virus (CTV)
Corchorus golden mosaic virus (CoGMV)
Corchorus yellow vein virus (CoYVV)
Cotton leaf curl Kokhran virus (CLCuKV)
Cotton leaf curl Multan betasatellite (CLCuMB) Cotton leaf curl Multan virus (CLCuMV)
Cowpea golden mosaic virus (CPGMV)
East African cassava mosaic Cameroon virus (EACMCV)
East African cassava mosaic Zanzibar virus (EACMZV)
Erectites yellow mosaic betasatellite (ErYMB)
Euphorbia leaf curl virus (EuLCV)
Horesgram yellow mosaic virus (HgYMV)
Indian cassava mosaic virus (ICMV)
Kenaf leaf curl betasatellite (KLCuB)
Kudzu mosaic virus (KuMV)
Maize streak virus (MSV)
Mesta yellow mosaic betasatellite (MeYMB) Mungbean yellow mosaic India virus (MYMIV)
Mungbean yellow mosaic virus (MYMV)
Okra leaf curl betasatellite (OLCuB)
Papaya leaf curl betasatellite (PaLCuB) Papaya leaf curl China virus (PaLCuCNV)
Papaya leaf curl Guangdong virus (PaLCuGuV)
Papaya leaf curl virus (PaLCuV)
Pedilanthus leaf curl virus (PedLCV)
Pepper leaf curl Bangladesh virus (PepLCBDV)
Pepper leaf curl Lahore virus (PepLCLV)
Potato virus X (PVX)
Squash leaf curl virus (SqLCV) Sri Lankan cassava mosaic virus (SLCMV)
Tobacco curly shoot betasatellite (TbCSB) Tobacco leaf curl betasatellite (TbLCB)
Tobacco mosaic virus (TMV)
Tobacco yellow dwarf virus (TbYDV)
Tomato golden mosaic virus (TGMV)
Tomato leaf curl Bangalore betasatellite (ToLCBB)
Tomato leaf curl Bangalore virus (ToLCBV)
Tomato leaf curl Bangladesh betasatellite (ToLCBDB)
Tomato leaf curl Bangladesh virus (ToLCBDV)
Tomato leaf curl China betasatellite (ToLCCNB)
Tomato leaf curl China virus (ToLCCNV)
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Tomato leaf curl Gujrat virus (ToLCGV)
Tomato leaf curl Karnatka virus (ToLCKV) Tomato leaf curl New Delhi virus (ToLCNDV)
Tomato leaf curl virus (ToLCV)
Tomato mottle virus (ToMoV)
Tomato pseudo-curly top virus (TPCTV)
Tomato yellow leaf curl China betasatellite (TYLCCNB)
Tomato yellow leaf curl China virus (TYLCCNV)
Tomato yellow leaf curl virus (TYLCV)
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Table of Contents
Chapter 1 Introduction and review of literature
1.1 Plant viruses
1.2 Geminiviruses
1.3 Begomoviruses
1.4 Satellites
1.5 DNA replication of geminiviruses
1.6 Evolution of geminiviruses
1.7 Strategies for engineering broad spectrum resistance to geminiviruses
1.7.1 Resistance by the expression of proteins
1.7.2 Defective interfering DNA
1.7.3 Antisense RNA-mediated resistance
1.7.4 RNA interference
1.8 Chilli pepper
1.8.1 Plant transformation in chili pepper
Chapter 2 Materials and methods
2.1 Collection of viral infected plant samples
2.2 Isolation of total genomic DNA
2.3 DNA quantification
2.4 PCR amplification
2.5 Agarose gel electrophoresis of PCR products
2.6 Cloning of genes in a TA cloning vector pTZ57R
2.6.1 Purification and ligation of PCR product in TA vector
2.6.2 Preparation of compeent E. coli cells
2.6.3 Transformation of ligation products into E. coli strain DH5α
2.6.4 Screening of the trans-conjugant clones
2.6.5 Plasmid isolation from recombinant E. coli
2.6.6 Verification of clones
2.6.7 Agarose gel electrophoresis
2.7 DNA sequencing
2.8 Sequence analysis
2.9 Transient Assays
2.10 Plant transformation through Agrobacterium -mediated in Nicotiana tabacum
(CV. Samsun).
2.11 Isolation of total genomic DNA from Nicotiana tabacum plants
2.12 Southern blot hybridization to check the integration of the transgene
2.13 Basta sensitivity test
2.14 Isolation of total RNA
2.15 siRNA analysis of the transgenic plants
2.16 Virus resistance evaluation
2.17 Viral replication in transgenic plants
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Chapter 3 Component captured by begomovirus; Pepper leaf curl Lahore
virus requires DNA B of Tomato leaf curl New Delhi to cause leaf curl
symptoms
3.1 Introduction
3.2 Material and methods 3.2.1 Collection of virus infected plant samples
3.2.2 Isolation, cloning and sequencing
3.2.3 Agrobacterium-mediated inoculation of plants
3.3 Results 3.3.1 Detection of begomovirus components in chilli samples showing leaf curl
symptoms 3.3.2 Analysis of the sequence of PGL1
3.3.3 Infectivity and symptoms of PepLCLV
3.3.4 PepLCLV trans-replicates ToLCNDV DNA B and induces leaf curl
symptoms
3.4 Discussion
Chapter 4 Resistance against chilli leaf curl disease complex (ChLCD) using
RNA interference
4.1 Introduction
4.2 Material and Methods
4.2.1 Cloning of RNAi based gene constructs
4.2.2 Transient assays
4.2.2 Plant transformation
4.2.3 Challenge with virus
4.3 Results
4.3.1 RNAi constructs silence ChLCV-M Rep in transient assays
4.3.2 RNAi constructs for PepLCLV in transient assays
4.3.3 Plant Transformation
4.3.4 Transgenic tobacco resistant to ChLCD complex
4.3.5 Transgenic tobacco resistant to heterologus virus
4.4 Discussion
Chapter 5 The role of Cauliflower mosaic virus (CaMV) defence and silencing
suppressor protein 6 (p6) in modulating auxin signaling
5.1 Introduction
5.2 Material and Methods.
5.2.1 TIBA plate Experiment
5.2.2 miRNA detection from A7, B6 and Ler gl1
5.3 Results:
5.4 Discussion
Chapter 6 Response of different (Capsicum annuum L) genotypes for callus
induction, plant regeneration and plant transformation
475bp
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6.1 Introduction
6.2 Materials and Methods
6.2.1 Plant material, Seed Germination and Explant preparation
6.2.2 Culture Medium and condition
6.2.3 Agrobacterium-mediated genetic transformation in chilli pepper (Capsicum
annuum L)
6.3 Results
6.3.1 Callus Induction
6.3.2 Plant regeneration from calli
6.3.3 Effect of different factors on Agrobacterium-mediated plant transformation in
chilli pepper (Capsicum annuum L)
6.4 Disscussion
Chapter 7 General discussion
Chapter 8 References
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TABLES
Table 1.1 Approximate protein size and function of different ORF of geminivirus
Table 1.2 Plant RNAi transformation vectors and their attributes
Table 2.1 Names, sequences and brief description of primers used in this study.
Table 3.1 Features of the begomovirus isolated from Capsicum annuum
Table 3.2 Pairwise percent of nucleotide identities between the genomic
components and protein sequence identities of encoded genes from the
virus isolate PGL1 with the components and genes of all isolates
available in the databases of selected other begomovirus species.
Table 4.1 Primers designed for the amplification of AC1-AC2 in the sense and
antisense orientation. The restriction sites included in primers are
underlined.
Table 4.2 Regeneration and transformation efficiencies of transformed tobacco
leaf discs
Table 4.3 ChLCD resistance/tolerance pattern for peAC1-AC2dsRNA/pFGC plants at T1
stage.
Table 6.1 Chilli callus induction medium (ChC)
Table 6.2 Chilli shoot regeneration medium (ChSR)
Table 6.3 Growth regulators and their stock preparation
Table 6.4 Analysis of Variance Table for chilli callus induction
Table 6.5 Effect of genotype on chilli callus induction.
Table 6.8 Interaction of genotype and explants (hypocotyl and cotyledon)
response on chilli callus induction.
Table 6.8 Effect of combination of genotype, explants and callus induction
medium response on chilli callus induction.
Table 6.9 Analysis of variance table for plant regeneration
Table 6.10 Effect of genotype on chilli plant regeneration.
Table 6.11 Effect of combination of genotype, explant and shoot regeneration
medium on plant regeneration from chilli calli.
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FIGURES
Figure 1.1 Maize streak virus particle (Geminivirus Cryo-electron microscopy
image)
Figure 1.2 Genetic organization of begoviruses
Figure 1.3 General mechanism of RNAi
Figure 3.1 Phylogenetic dendrograms based upon an alignment of selected
complete sequences (or DNA A components) of begomoviruses.
Figure 3.2 Alignment of the N-terminal amino acid sequences of the Rep protein of
PepLCLV (PGL1) clone with the sequences of other begomoviruses infecting
chilli on the Indian subcontinent.
Figure 3.3 Alignment of the intergenic region sequences of PepLCLV (PGL1),
ToLCNDV DNA A and DNA B.
Figure 3.4 Symptoms induced by PepLCLV clone PGL1 in N. benthamiana, N. tabacum
and C. annuum.
Figure 3.5 Virus replication in systemic leaves of inoculated N. benthamiana plants
probed with PGL1.
Figure 4.1 Sliding window plot showing the distribution of genetic variation
estimated by nucleotide diversity (Pi) for geminivirus infecting
Capsicum annuum in Pakistan.
Figure 4.2 A diagram demonstrating the binary vector (pFGC5941) engineered
with the silencing trigger construct.
Figure 4.3 Transient assays through agro co-infiltration in young Nicotiana
benthamaiana plants.
Figure 4.4 Transient assays of construct peAC1-AC2dsRNA/pFGC in Nicotiana
benthamiana with PepLCLV and ToLCNDV DNA B.
Figure 4.5 Inhibition of virus replication in systemic leaves of agroinfiltrated N.
benthamiana plants probed
Figure 4.6 Plant transformations in N. tabacum
Figure 4.7 Confirmation of the transgenic plants
Figure 4.7 Virus resistance assay.
Figure 4.8 Dot Blot analysis of replication of PepLCLV in RNAi transgenic plant
following exposure to viruliferous whitefly
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Figure 4.9 Virus resistance assay.
Figure 4.10 Southern blot analysis of transgenic plants (peAC1 -
AC2dsRNA/pFGC) inoculated with PepLCLV (PGL1) and ToLCNDV
DNA B.
Figure 4.11 Virus resistance assay
Figure 5.1 Effect of 3,5-triiodobenzoic acid (TIBA, auxin transport inhibior) on
Arabidiopsis lines
Figure 5.2 Expression of ath-MIR160 (A) and ath-MIR167 (B) miRNA probe on
P6 transgenic and wild type plant
Figure 6.1 Effect of genotype on chilli callus induction
Figure 6.2 Interaction of genotype and explants (hypocotyl and cotyledon)
response on chilli callus induction
Figure 6.3 Combination of genotype, explants and callus induction medium
response on chilli callus induction.
Figure 6.4 Effect of genotype on chilli plant regeneration
Figure 6.5 Effect of combination of genotype, explant and shoot regeneration
medium on plant regeneration from chilli calli.
Figure 6.6 Different steps in chili tissue culture
Figure 6.7 Plant transformations in chilies and transgene analysis.
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1
1.1 Plant viruses
Viruses are small and tiny infectious and obligate intracellular pathogen and emerged
as the most difficult, formidable and complex plant pathogens [1-7]. Tobacco
mosaic virus (TMV) was identified as the first virus which was discovered [8]. ICTV
in their 9th
meeting has approved ~2284 species of viruses that infect plants. Some
plant viruses have single stranded ssDNA or double-stranded dsDNA genome,
whereas others have dsRNA genomes. Approximately 90 % of plant viruses contains
ssRNA genome. A group of viruses belongs to family Caulimoviridae are Double
stranded DNA (dsDNA) viruses whereas Nano viruses and Gemini viruses are ssDNA
viruses [9].
Plant virus genomes characterization at molecular level has been recently focused
with a particular interest in determining virus movement replication and infection in
plants. Understanding the virus gene functions has been used to explore the potential
for commercial use by biotechnology companies. Viral-derived sequences have been
particularly used to provide an understanding of novel forms of resistance [9].
1.2 Gemini viruses
Gemini viruses are ssDNA plant viruses characterized by small geminate particles
(18×20 cm) which are transmitted by insects, particularly leafhoppers and whiteflies
[10]. Gemini viruses can wipe out entire crops of many different plants [4, 6, 7, 11,
12], making them a serious threat to farming industry worldwide [12, 13]. Climate
change, food trade and agricultural practices; are major factors for the dissemination
of insect vectors and spread of geminiviruses into more temperate regions from
tropical region. [2, 14, 15]. Geminiviridae constitutes the second largest family [2,
16].
Gemini viruses replicates in the infected cells nuclei by a rolling circle mechanism
(RCR) and do not encodes for their own polymerases [17, 18,19]. However,
Gemoniviruses depends on plant factors to support their replication [20, 21]. Maize
streak virus (MSV), localize to differentiated, quiescent cells [22, 23]. By contrast,
Abutilon mosaic virus (AMV) is restricted to vascular tissues [24, 25]. Gemini viruses
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2 are important tools for studying virus–plant interactions, cell cycle and DNA
replication in plants [2, 26, 27]. There are seven genera in the family Geminivirdae
Gemini viruses are classified into seven major genera based on their genome structure
(mono or bi partite), insect vector, and plant host range [7, 28-31].
Nomenclature and taxonomy of geminivirus is complex because the number of
different genomic sequences in data bases are increasing [8, 32, Fauquet, 2003
#1587]. The ICTV introduced a new species demarcation criteria for which 89 %
sequence homology proposed for new species using the DNA star software clustal V
package [9] and 85–94 % nucleotide (nt) identity correspond to strain of the same
virus species and 92–100 % nucleotide identity is proposed for the new variant.
Figure 1.1
Maize streak virus particle (Geminivirus Cryo-electron microscopy image). Image reproduced from [33].
Genus Mastreviruses are leafhopper transmitted geminiviruses and has monopartite
genome and infect both dicots and monocots plants [34]. These are mostly found in
the Old World. Maiz streak virus (MSV) are the most widely characterized member
[34, 35]. Mastrevirus genome contains a small (SIR) and large (LIR) intergenic
region, which is located at opposite sides of the circular genomes. The LIR contains
ori for the synthesis of virion-strand DNA [35].
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3 Genus Curtovirus have leafhopper-transmitted dicot infecting geminivirus with
monopartite 3.0 kb ssDNA circular genomes. [36]. The known type member of this
genus is Beet curly top virus (BCTV). Genome arrangements of curtovirus is quite
similar to that of monopartite begomoviruses. Coat protein (ORF V1) of BCTV is
necessary for systemic movement [37]. [38] recognized different regions of the beet
mild curly top virus (BMCTV) capsid protein involved in formation of virion,
systemic infection, transmission through leaf hopper and its most important role in
systemic infection [39].
The genus topocuvirus contain the sole treehopper transmitted geminivirus tomato
pseudo-curly top virus (TPCTV). This genus has a monopartite ssDNA circular
genome and its genetic organization is similar to that of curtoviruses and represents
the well characterized genus of geminiviruses [40, 41].
Genus Becurtovirus contains only one genome segment that codes for five proteins
(two reverse with spliced replication and involved in initiation of protein formation
while three forward), on virion sense strand origion of replication (TAAGATTCC)
[42]. These viruses are transmitted through leaf hoppers in to Dicotyledonous plants
[43].
Similarly Eragrovirus also contains one fragments of genome that codes for four
proteins and origin of replication TAAGATTCC on virion strand. These viruses
infects mostly monocotyledonous plants and their mode of transmission through
vector is not known.
Turncurtovirus also contain one genome fragment with six proteins (four reverse and
two forward), their vector is not still known. These viruses infect dicots and distantly
related to curtoviruses.
1.3 Begomoviruses
Begomoviruses are the whitefly transmitted dicot infecting geminivirus (WTG),
which comprises more than 200 species [3, 6, 44]. There are three types of
Begomoviruses i.e bipartite, monopartite as well as monopartite requiring beta
satellite. Bipartite begomoviruses contains Coat protein, Rep and Open reading frame
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4 (ORF) for replication for replication and virion formation. DNA B components of
begomovirus genome contains two proteins that are involved in inter- and intra-
cellular movement of virion within the plants [26]. In genome of begomoviruses there
is conserved motif that control gene expression and replication, in the form of a stem
loop putative structure which contain a conserved nonanucleotide TAATATTAC
[26].
Bipartite begomovirus has been mostly identified in new world but now recently
bipartite begomoviruse is isolated and characterized from the old-world. ToLCNDV
is the best example of bipartite found in the old-world.and contain both DNA A and
DNA B componenet[4, 45]. However, some begomoviruses has a monopartite
genome which is associated with a single-stranded DNA betasatellite and also termed
as DNA β [46-49]. DNA A has 6-7 gene and DNA B has 2 gene and function of each
is summarized in table 1.1.
1.4 Satellites
Satellites molecules are ssDNA viruses that depend on a helper DNA A virus
component for replication and encapsidation. These satellite molecules lack sequence
homology with helper virus “genome” [50]. This type of molecules are found
commonly with RNA viruses. Satellites lessen the severity of symptoms in infected
plants by interfering with the replication of their helper viruses and [51]. Few viruses
are known that are not associated with helper viruses and produced different
symptoms [52, 53].
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Table 1.1
Approximate protein size and function of different ORF of geminiviruses
ORF Appr.
Sizes
Functions
AC1 40.2 kDa, Required for replication, site-specific nuclease, ligase,
auto suppressor, and DNA sequence-specific binding
protein. Cell cycle regulatory protein [54, 55].
AC2 19.6 kDa Transactivator for AV1 and BV1 gene expression and
also suppressor of RNA silencing [56-61].
AC3 15.6 kDa Increases the replication efficiency of geminiviruses.
[56]
AC4 12.0 kDa Disease symptom and movement? [57]
AV1 29.7 kDa Capsid protein, vector specificity and may regulate the
ss/ds DNA ratio [62].
AV2 12.8 kDa Functions in virus movement in groups II [63-65].
BC1 29.6 kDa Virus movement (N.A. cell to cell trafficking), host
ranges and symptom development [66] [67, 68].
BV1 33.1 kDa Virus movement (Transport N.A. out of nuclei) and host
ranges [66-68].
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Figure 1.2
Begomoviruses genetic organization
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Tomato leaf curl virus satellite (ToLCV-sat); a single begomovirus approximately 682
nt was shown to be associated with a ToLCV. ToLCV-sat had no apparent effects on
viral replication and symptoms caused by ToLCV [69, 70]. ToLCV-sat is not exactly
related to ToLCV in terms of genome sequence length and needs ToLCV for
replication and insect transmission in plants.
Several satellite molecules i.e, Tomato leaf curl virus satellite were identified from
other monopartite begomoviruses and these molecules are known as beta satellites
that‟s are 1350 nt. In length. These molecules required a helper viruses for their
replication and movement in plants as well as transmission into insect vectors.
However, beta satellite effects the replication of their helper begomoviruses hence,
altered the symptoms in plants [47, 71].
Sequence analysis of beta satellite DNA molecules discovered that they are
approximately the half the size of helper virus DNA A and has only homology with
conserved hairpin structure and a TAATATTAC loop sequence, and has have little or
no resemblance to either DNA A or DNA B molecules of begomoviruses. Beta
satellite entails begomovirus DNA A helper component for replication, encapsidation,
movement in plants and insect transmission [6, 47, 51, 71]. Beta satellite has three
structural features: a 115 bp highly conserved region, βC1 gene and an adenine-rich
region, however βC1 gene, [69] which is located on complementary sense strand, is
conserved both size and position in all beta satellite species [47, 72]. βC1 gene has
the capability to encode a 13- to 14-kDa protein containing 118 amino acids [73]; [69,
74]. The accurate job of beta satellite and its C1 protein in pathogenesis is
unidentified although it has been projected that beta satellite may show a straight or
an unplanned role in replication, assisting movement, or counteracting host defense
response [75]. C1 protein binds DNA in size and a sequence specific manner. It also
exploits the host plant immune system as a suppressor of RNA silencing [75].
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Alpha satellite, another types of satellite like molecules [76-78] which encode only a
single product with resemblance to the Rep protein of Nano viruses [79]; another
family of plant infecting single-stranded DNA viruses [80]. Alpha satellite has the
ability to self-replicate in the plant cell, but require the helper component of ssDNA
begomovirus for movement in plants and encapsidation for whitefly transmission.
Alpha satellite seems to have no part in the disease development, being unnecessary
both for infection and symptom stimulation in host plants [81, 82].
Begomovirus associated satellite molecules are mostly present in the old world and
are important components of plant diseases. This was a matter of urgency due to the
large number of such molecules which have been identified, characterized and
submitted to data bank [78, 83]. The ICTV Geminiviridae Study Group has recently
proposed a system of nomenclature and taxonomy for the beta satellites and alpha
satellites associated with geminiviruses. Pairwise comparisons of all available full-
length DNA beta satellite and alpha satellite nt. sequences shows that the minimum
numbers of pairs occur at a sequence homology of 78 % for beta satellites and 83 %
for alpha satellite are recommended as the species demarcation threshold (DMT) for a
dissimilar species [49].
1.5 DNA replication of geminiviruses
Rolling circle replication (RCR) is the mechanism adopted by the geminiviruses to
replicate in the nucleus of infected cells [84]; [2, 85]. Geminivirus has Rep binding
sites in the intergenic region (IR) [26].
1.6 Gemini virus Evolution
It is assumed that these viruses are evolved as episomal DNA replicons from most
ancient groups of prokaryotes that are altered from the primitive ancestor of
eukaryotes of modern plants [90]. This hypothesis could be supported with the ability
of wide range of geminivirus to replicate in Agrobacterium tumefaciens [93], and
there are several evidences of the conserved features of rep protein in prokaryotic and
eukaryotic DNA replicons, polycistronic mRNAs [91, 92]. Although in the process of
co-evolution of viruses along with their hosts, these DNA molecules attains new
properties by recombination process and formed recombinant DNA with host genome
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9 [90]. According to phylogenetic studies [94, 95], the ancestors of geminiviruses had
single DNA A components which were involved in infection of monocots and
transmitted though leaf hopper. This trend were shifted and transmission of these
viruses‟ starts with white flies as well as infection of dicots happened due to ancestral
old world begomoviruses [96]. The attainment of second component of genome DNA
B is occurred later on the evolutionary scale, however this might be happened before
the separation of contents as new world and old word both contains the
begomoviruses [51, 90]. Monopartite begomovirus that have associated helper viruses
as beta satellites have opened a new possibility to infect new host and cause different
symptoms [76, 77, 97-100]. Recombination events between the begomovirus and
mastrevirus might have created the new one genus curtovirus [101]. Another genus
Topocovirus have emerged after the recombination of ancient curtovirus and this
virus is not related to another two genera of geminivirus [102]. Recombination is
main reason behind the diversity of geminivirus. This process of recombination
dependent replication strategy [12, 103] is most probably happened naturally by the
mixing of two different begomoviruses infection in the same cell of the plant [104].
Hence as a result of recombination large number of begomovirus species have been
evolved.
1.7 Engineering broad spectrum resistance to geminiviruses
Conventional plant breeding play an important for controlling plant pathogen [105].
Increased knowledge of plant DNA science at molecular level and the study of host
plant interaction at molecular level have stemmed in the development of a variety of
strategies to control virus disease epidemic in plants and enhancing plant virus
resistance using transgenic approach over the recent past. There are two groups of
gene sources: pathogen derived genes and non-pathogen derived genes so for virus
resistance one concept is the use of non-pathogen source which is non-pathogen
derived resistance (non-PDR) [106] and other is pathogen derived resistance (PDR)
[107]. Most strategies based on the concept of PDR [107].
1.7.1 Resistance by the expression of proteins
Transgenic virus resistant plant expressing the coat protein of TMV was produced in
1986. Some viral or non-viral proteins can be used to engineered resistance for viruses
in plants where as in Gemini viruses rep protein is very important for the replication
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10 of viruses. Transgenic plant producing N-terminally (T-Rep) truncated Rep showed
enhanced resistance against geminivirus infection (T-Rep; [108, 109], and T-Rep
transgenic plants showed a degree of resistance [108, 110]. Same strategies using T-
Rep was used to produced tomato and this plant shows resistance to homologous as
well as heterologous Gemini viruses [111]. Similar TGMV MP, ACMV TrAP, and
ToMoV MP [112] expressing transgenic also showed resistance to ToMoV and
CaLCuV infection [113]. The effect of virus on plants could be neutralized due to the
production of antibodies, because Plant virus particles have immunogenic properties.
However antibody producing immune system is absent in plants. [114] produced
transformed tobacco plant with a functional single-chain Fv antibody against
Artichoke mottled crinkle virus (AMCV). The antibody displayed high affinity for
AMCV coat protein of both intact virions and disrupted subunits.
1.7.2 Defective interfering DNA
Gemini virus sub genomic DNA molecule is actually defecting inferring molecule
which interferes with the replication of helper molecules. Accumulation of defective
interfering (DI) RNAs were also found in Tombus viruses and carmoviruses [115].
Transgenic N. benthamiana plant expressing DI RNA derived from RNA 2 of
Tobacco rattle virus (TRV) showed enhanced resistance against against tobacco rattle
virus disease complex [116].
1.7.3 Antisense RNA-mediated resistance
Basically transgene is negative strand that is complementary to the viral mRNA [117].
After the transcription of the complementary strand, RNA/RNA duplex formed and as
a result translation of viral protein formation stopped. Only limited resistance has
been observed by the use of this method against RNA plant viruses so far. However,
this method is preferable in case of DNA viruses that transcribe their mRNA in the
nucleus. Lindbo and Dougherty was first recognized the RNA-mediated gene
silencing in plants, who described that untranslatable viral RNA sequences could
trigger specific, post transcriptional RNA degradation of the transgene mRNA and are
linked to viral protection [118].
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11 1.7.4 RNA interference
According to molecular biology central dogma is formation of the protein from RNA,
that predicts RNA as source of information but it is not a regulatory molecule
(universal component of gene expression). Stop of further viral replication and their
transposable elements silencing in nematodes, insects, fungi and plants by si RNAs
that are 21 nucleotide bases of dsRNA can be achieved through a technique RNA
interference (RNAi) Small interfering RNA are processed from dsRNA viral
replication intermediates [119-123].Viral RNAs translation could be suppressed
though the silencing complex that is formed with RISC and target the complementary
viral strand and chopped it in to small pieces [124]. Different pathways and
mechanisms are involved in the formation of siRNA. Two RNA three type enzymes
Drosha and DICER –LIKE (DSL) in plants, catalyze the precursor of siRNA into 21-
24nt. Duplexes [122]. siRNAs associated with hetero chromatin (24nt) that are
produced by the activities of RDR2 (RNA polymerase IV), DCL3 and also required
AGO4 to direct the methylation of DNA through cytosine and histone H3 at Lys-9
[125] [126]. The formation of post transcriptional active siRNAs from exogenous
sources (viral and transgenic), that may involve RDR1/RDR6 and DCL2 in case of
some other viruses [119, 126]. In case of endogenous small trans acting small
interfering (ta-si) RNAs from polymerase III gene that guides the cleavage of target
mRNAs and (ta-si) RNAs also need RDR6 for precursor formation as suppressor of
gene silencing 3 (SGS3) [125-129]. Also this ta-siRNA formation needs DCL1 (its
role ay be indirect)[126].
Although almost all the classes of known endogenous small RNAs required HEN1 (an
RNA methyl transferase enzyme that modifies th 3‟ end) in Arabidopsis plant [130].
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Figure 1.2
General mechanism of RNAi
TAS1, TAS2, and TAS3 are known families of ta-siRNA encoding genes in A.
thaliana [131]. The TAS1 family contain three genes that encode ta-siRNAs (siR255
and siR480) that target four messenger RNAs encoding proteins of unidentified
function [132]. TAS2-derived ta-siRNAs (siR1511) targets a set of messenger RNAs
encoding pentatricopeptide repeat proteins [132]. Whereas, TAS3 locus identifies two
ta-siRNAs that target a set of messenger RNAs for several Auxin response factors
(ARFs), including ARF3 (ETTIN) and ARF4 [132].
Generally Micro RNAs are small non-coding RNAs that are expressed as primary
microRNAs in which dicer and Drosha act in a ~70 nt the stem-loop precursor and
mature 21-25nt. micro RNA respectively also it is a part of RISC complex [134].
Micro RNA targets the RISC complex to messenger RNAs with complementary
sequences and triggers the mRNA cleavage or completely inhibit translation process.
The mode of microRNA regulation can be altered in association with other proteins
and that can activate the gene expression of protein [135-137]. However the exact
mechanism of target identification is not yet clear. Although the pairing of 7-8 nt. at
5‟ end of the microRNA for multiple sites in the 3‟ end (untranslated region) is
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13 sufficient to trigger inhibition of the translation process [138]. MicroRNAs
(miRNAs), also target several mRNAs concerned with auxin responses. MiR160
targets (ARF10, ARF16 and ARF17), out of twenty three Arabidopsis ARF genes
[139, 140]. Plants expressing a miRNA-resistant version of ARF17 have
increased ARF17 mRNA levels and altered accumulation of auxin-inducible GH3-like
mRNAs, YDK1/GH3.2, GH3.3, GH3.5, and DFL1/GH3.6, which encode auxin-
conjugating proteins [139, 140]. As a result of these defects expression changes occur
in developmental defects such as embryo, abnormalities of emerging leaves, shape of
leaf, premature inflorescence development etc. The defects suggests the importance
of miR160-directed ARF17 regulation and indicate ARF17 as a regulator (GH3-like)
of auxin response gene [139]. Several defects related to the phenotypes have been
previously observed in plants expressing viral suppressors of RNA silencing. Those
plants which have mutations in their genes are important for miRNA function and
providing a molecular rationale for phenotypes of previously studied of miRNA
function [141]. RNA silencing is a general antiviral defense mechanism in plants
[121, 134]. Pathogen derived resistance (PDR) in plants is type of resistance against
particular virus in which transgene of virus is engineered and stably transformed to
plant [118]. Pathogen derived resistance developed in plants as a result of RNA
silencing and all the RNAs with homologues sequence of the transgene degraded the
mRNA of the virus and prevent plant from infection [142; 143].
Similarly in another work it has been demonstrated that the plant viruses could also
induced RNA silencing by itself in plants termed as VIGS (virus induced gene
silencing) can be targeted to either transgenes or endogenous genes [144]. VIGS
technique has been used to screen gene function by preparing the endogenous
sequence libraries cloned into a viral vector [145].
Transient expression of reported gene from Discosoma encoded with GFP was
reduced to 47% after 24h of insertion of cognate siRNAs in BY2 protoplasts. Rep
protein of the ACMV were targeted through the co delivery of siRNA that was
designed to destroy the mRNA and stopped the accumulation by 91%. However it
also inhibits the accumulation of ACMV genomic DNA to 66% during transfection at
36 and 48 h. In case of siRNA-induced reporter gene silencing was specific for
ACMV rep and did not showed any effects on the replication of EACMCV. [145]
have made a generic vector, pHANNIBAL to take single PCR product from gene of
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14 insert and could easily converted into a highly effective (hpRNA) silencing construct
(Table 1.2).
Table 1.2
Plant RNAi transformation vectors and their attributes
Vector
pHannibal [146]
pFGC594
1 chromdb.or
g
pMCG161 chromdb.org
pHellsgate [146]
Target
Organism
dicots dicots monocots dicots
Cloning
Method
restriction
digest/ligation
restriction
digest /
ligation
restriction
digest /
ligation
Gateway
recombination
Bacterial
Selection
spectinomycin kanamycin chloramphe
nicol
spectinomycin
Plant
Selection
chloramphenicol Basta Basta chloramphenicol
dsRNA
promoter
CaMV 35S CaMV 35S CaMV 35S CaMV 35S
inverted
repeat
spacer
Pdk intron ChsA intron Waxy intron Pdk intron
1.8 Chili pepper
Chili (Capsicum annuum L.) is among the most economically important fruits and vegetables
worldwide [147-149]. The name is perhaps derived from the Latin "capsa," or box for the
pod-like fruit [150]. Pepper originated in Mexico (Southern Peru and Bolivia; [151]
and are now grown worldwide under various climatic conditions. Chili pepper
belongs to the family Solanaceae, and is closely related to the tomato, nightshade, and
potato. These are also known as Capsicum, Sweet Pepper, Red Pepper, Chili pepper
and Paprika depending upon the species and also the manner in which it is prepared
and used [148]. C. annuum is the predominant species cultivated, encompassing both
hot and sweet Pepper. Seeds straw is colored. Many different forms are known within
the capsicum species [152]. Chromosome number is 2n=24, with two pairs of
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15 acrocentric chromosomes [153]. They are very important in almost every Asian
country. There exists a great scope for its export from Pakistan. Chilies are used as a
condiment, either green or dry, in all preparations of vegetables. The bulk of chilies in
European countries are used up in the food industry, where it is used as a colorant and
for flavoring. Chilies form an indispensable condiment in every household. The
pungency of chili is due to the presence of the active principal” Capsaicin” which is
contained in the skin and the septa of fruit [154]. It is used in homeopathy. Chilies can
be used for curing gout, paralysis, black vomit and tropical fever.
Chilies are grown all over Pakistan and India. [155]. The time of sowing of chilies in
Pakistan is largely determined by the time of transplanting in the field. In plains
having frost, nurseries are raised up in Oct-Nov and transferred in mid-April/May. In
frost-free areas nurseries are raised in July-August and transferred in Sep-Oct [156]. It
is cultivated in all four provinces of Pakistan on a total area of 56.400 hectares but
mainly grown in Punjab and Sindh. According to Agriculture statistics of Pakistan, In
2003-04 its production in Punjab was 10.8000 tonnes on a total area 6.4000 hectares
[157]. Among commercial varieties grown in Pakistan, the important ones are
Tatapuri, Gola Peshawari, Neelam, Talhar, Longi, Skyline and Sanam.
Chilies are infected by a large number of bacterial, fungal and viral diseases. In
Pakistan, diseases of viral nature are the major cause for serious yield losses of chilies
[51]. It is reported that there are 45 different viruses that infect chilies/peppers
worldwide. However the most frequently mentioned viruses that infect chilies are
begomoviruses [158, 159]. Chili leaf curl disease is a significant factor for chilies
production in the Indian subcontinent. ChLCD is transmitted by whitefly [28] and
become the cause of spread of begomoviruses [158].
The synergistic action of geminivirus disease complex comprising a monopartite and
a bipartite begomoviruses along with beta satellites attributed to incidence and
symptom severity of chili leaf curl disease [160]. Previously, Chili leaf curl virus-
Multan (ChLCuV-M) full-length DNA A was found from infected samples of chilies.
Chili leaf curl betasatelite (1390 bp) have found in a huge collection of chili samples
exhibiting leaf curl symptoms from Pakistan. These betasatelite components are
similar to that of nib-16 clone [51, 161, 162].
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1.8.1 Plant regeneration and transformation in Chili
Regeneration is the biological process by which organs were formed by
differentiating a cell or a group of cells. Most often direct regeneration occurs through
shoot proliferation from previous meristems as an alternative of de novo formation of
a meristem. Usually in callus mediated regeneration organ forming capacity is
inadequate for primary callus, that specifies the potential existence of meristems
rooted in the explant [163]; [164]; [165]. First attempt to regenerate chili pepper in
vitro was made by [166]. They examined the effect of different cytokinins and
observed 6-benzyl amino purine (BAP) was more effective in producing shoots from
cotyledonary leaf. Pepper is highly recalcitrant plant and is very difficult plant to
transform [163]. Chili pepper showed a variable genetic sources. There are some
reports of genetic transformation of chilies has been reported but with low efficiency
and no reproducible results has been produced. [167-169].
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Chapter 2
Material and Methods
2.1. Plant sample collection
Virus infected plant showing symptoms were observed in fields and photographed.
Symptomatic and non-symptomatic plants leaves were collected, labelled and
transported on ice. Samples were brought to Plant Genetic engineering lab NIBGE
Faisalabad and stored at -80ºC till the extraction of DNA.
2.2 Isolation of total genomic DNA
Total DNA was extracted from N. tabacum (leaves), N. benthamiana (leaves) and C.
annuum (leaves and calli), plants using CTAB [170].
2.3 DNA quantification
DNA was quantified by determined its concentration by spectrophotometer
(SmartSpec BIORAD, USA). The absorbance readings were taken at 260 nm
wavelength and the conversion factor was O.D260 1=50 µg ml-1
. Each sample was
diluted to a certain level before loading the sample in the cuvette and dilution factor
was set in the machine. The reading of machine was blanked by loading the water,
which was used for DNA dilution to subtract the background reading.
2.4 PCR amplification
2X dream Taq master mixture (Fermentas) was used to amplify desired fragment
from 10 pg-1µg template DNA with specific primers as described in (Table 2.1).
Following conditions were set on machine for amplification; 94˚C for 5 min, 94˚C
for 1 min followed by 35 cycles of, anneal ing temperature 48˚C to 52˚C for 1
min and 72˚C for 3 min with final extension of 10 min at 72 ˚C.
2.5 Agarose gel electrophoresis
1 % agarose gel containing ethidium bromide stain was used to check amplified DNA.
10 µl of PCR product was mixed with 6X loading dye and loaded to gel along with
1kb Ladder. Gel was run at 80 volts for 40 min and visualized in UV trans-
illuminator. Gel image was taken through Gel documentation system.
Table 2.1: Names, sequences and brief description of primers used in this study.
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Primer name Sequence Used for
Pe AC1-AC2 SF
CCCTCGAGTAAATACCCTTAAGAAATGA To produce sense construct of AC1-AC2
Pe AC1-AC2 SR CCCCATGGTTTAAGGAATTCATGGGGGC -do-
Pe AC1-AC2 ASF GGGGATCCTCATTTCTTAAGGGTATTTA To produce antisense construct of AC1-AC2
Pe AC1-AC2 ASR GGTCTAGAAAATTCCTTAAGTACCCCCG -do-
Begomo F ACGCGT GCCGTGCTGCTGCCCCCATTGTCC For amplification of DNA-A of begomovirus
Begomo R ACGCGT ATGGGCTGYCGAAGTTSAGAC -do-
β01 GGTACCACTACGCTACGCAGCAGCC For amplification of betasatelite of begomovirus
β02 GGTACCTACCCTCCCAGGGGTACAC -do-
BC1F CACCATGGCAATAGGAAATGATGGTATGGG For amplification of DNA-B (MP) of begomovirus
BC1R AAGGATCCTCTTAATTTTTTGAATAAATTTGGC -do-
BAR Partial L GAAGTCCAGCTGCCAGAAAC For amplification and
identification of BAR gene
BAR Partial R CTCTACACCCACCTGCTGAAG -do-
35S Partial L CTACGCAGCAGGTCTCATCA For amplification and
identification of 35S
sequence
35S Partial R GAAGCAAGCCTTGAATCGTC -do-
2.6 CLONING
2.6.1 Cloning of amplified PCR product
Amplified PCR product was cloned using an InsTA clone PCR Cloning Kit
(Fermentas) according to the instructions provided by the manufacturer.
2.7 Preparation of competent cells
2.7.1 Preparation of heat shock competent E. coli cells
A single colony from the freshly grown pure cultures of E.coli was inoculated in to
20mL of LB broth medium and incubated at 37oC for overnight. Then 2 mL of
overnight culture was inoculated into 250mL LB broth and allow to grow at 37oC in a
shaking incubator until an OD600 of 0.5-1. The culture was put on ice for 50 minutes
then shifted to sterile falcon tube and centrifuge at 8,000 rpm at 4oC for 5 min.
The ce l l pe l l e t s was d i sso lved in 20 mL of 0.1 M MgCl2 and centrifuged.
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Pellet was again washed with 20 mL of 0.1 M CaCl2 and incubated on ice for 30
minutes and centrifuged. At the end cells were re suspended in 4 mL of 0.1 M CaCl2
and m i x e d w i t h filter-sterilized cold glycerol (3:1). The cells were stored
in aliquots of 200 μL at -80oC.
2.7.2 Preparation of electro competent A. tumefaciens cells
A single colony from a freshly grown plate of A. tumefaciens (strain LBA4404)
was picked using a sterile toothpick and inoculated into 20 mL LB liquid medium
with 25 µg/mL rifampicin in a sterile 50 mL flask and placed in a shaker (160 rpm)
at 28oC for 2 days. 5 mL of the culture was inoculated into 250 mL of LB broth
medium with 25 µg/mL rifampicin and placed in a shaker at 28oC until the OD600 of
cells was 0.5-1. The cells were transferred to falcon tubes and kept on ice for 10
min and centrifuged at 8,000 rpm for 10 min at 4oC. The pellet was
dissolved in 50 mL of cold (SDW) and centrifuged. Same step was repeated.
Cells were suspended again in 10 mL SDW with 10% glycerol and centrifuged . Then
cells were suspended in 4mL of 10% cold sterile glycerol, aliquoted in
Eppendorf tubes and stored at –80oC.
2.8 Transformation of competent cells
2.8.1 Transformation of E. coli competent cells by heat-shock
Transformation of competent E. coli cells was carried out by the methods
described by [171].
2.8.2 Transformation of A. tumefaciens competent cells
2 µL of ligation mixture was added with electro-competent (A. tumefaciens) cells at
40C and shifted to electroporation cuvette. Electric shock was given to cells at
1.44 kV and 1mL of LB broth medium was mixed with cells and placed in a shaker at
28 ˚C for 2 h. The cells were spread on plates containing LBA medium with suitable
antibiotics placed in a 28˚C incubator for 48 h.
2.9 Plasmid isolation from recombinant E. coli
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Recombinant plasmids from over-night grown cultures of E. coli at 37oC were
isolated according to the following protocol. A single colony was picked up and
cultured in LB broth medium (25ml) having 50 µg/ml kanamycin and allowed to
grow at 37oC overnight with vigorous shaking. The E. coli culture was then
centrifuged at 14000 rpm in 1.5 ml eppendorf tubes for 5 minutes. The pellet was
allowed to dry for two minutes following the decantation of the supernatant. To this
pellet 100 µl of (solution I) was added and the pellet was suspended in it by means of
vortex. Mixed well by inverting gently after addition of 150 µl of solution II.
Centrifugation was done at 14000 rpm for 5 minutes after addition of 200 µl of
solution III after thorough mixing. The supernatant was shifted to fresh Eppendorf
tube and two volumes of 100% ethanol was added to the eppendorf tube. The
eppendorf was left for 2 minutes at -20oC and centrifuged at 14000 rpm for 5 minutes.
The resultant pellet was washed with 70% ethanol and dried after centrifugation. The
pellet was suspended in 20 µl of SDW and stored at -20oC.
Plasmids were extracted using a Plasmid Miniprep Kit (Fermentas). Overnight
cultures of E. coli were transferred to 1.5 mL Eppendorf and centrifuged for 2 min
and the upper aqueous phase was removed with pipette (repeat this step to remove all
culture medium). The pellet was re suspended in 250 µL Resuspension Solution.
Cells were lysed with 250 µL Lysis Solution, neutralized with 350 µL
Neutralization Solution and the tube was centrifuged at 12000 rpm for 5 min. A
mini-column was inserted into a collection tube and the supernatant was transferred
to the column and centrifuged for 1 min and the flow- through was discarded. The
matrix ( p l a s m i d s t i c k ) was washed twice with 500 µL of Wash Solution and
centrifuged for 1 min to remove residues of Ethanol. Then the column was
transferred to new tube and the DNA was eluted in 50 µL distilled water.
2.10 Restriction Digestion of plasmid
Restriction of plasmid DNA was accomplished with enzymes and their
matching buffers according to manufacturer instructions (Fermentas). A total
volume of 10 μL (3units restriction enzyme, buffer, 500 ng DNA and sterile
distilled water) was used to confirm the insert size and 20 μL (10 units‟ restriction
enzyme, buffer, 2 µg DNA and sterile distilled water) for digestions of clone.
Reaction mixtures were incubate for 1-3 h at optimum temperatures. DNA
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21
fragments size were determined on Agarose gel stained with Ethidium bromide.
2.11 Glycerol stocks Preparation
Bacterial cultures or cloned plasmids were preserved through glycerol stocks.
B r i e f l y , In Eppendorf tubes (700 µL broth culture and 300 µL sterile glycerol)
was added and mixed well, then stored at -80 ˚C freezer for future use. For the
recovery of cultures, a sterile loop was inserted on glycerol stock and streaked on
LBA plate with antibiotics and kept at 37oC.
2.12 Purification of DNA
2.12.1 PCR product purification
The amplified PCR product was p u r i f i e d b y using a Wizard SV Gel and PCR
purification kit (Promega) as described by the manufacturer.
2.12.2 Phenol-chloroform extraction of DNA
Phenol: chloroform (1:1) extraction was used to get rid of the proteins i m pu r i t i e s
from DNA. An equal volume of phenol: chloroform was added into DNA and vortex
until it turns milky and centrifuged for 10 minutes and supernatant was shifted to new
tube. 3 M sodium acetate (1/10 volume) and 2.5 volumes chilled A.E was
added and mixed with DNA solution and kept at -20˚C for o n e h o u r . Then this
mixture was centrifuged a n d D N A p e l l e t w a s washed with 70% ethanol, air
dried and dissolved in SDW.
2.13 Sequencing and sequence analysis
Cloned samples were sent to Macrogen (South Korea) for sequencing with
universal primers (M13F/ M13R [-20]). Sequence specific primers were designed to
enhance the sequence length. The sequence file were assembled and analyzed with
the help of the Lasergene package of sequence analysis software (DNAStar Inc.,
Madison, WI, USA). Sequence similarity searches (BLAST) and phylogenetic
were performed i n N C B I d a t a b a s e a n d u s i n g c l u s t a l x to compare the
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sequence to others reported ones. [172-173]. All sequences were deposited in the
databank using EBI website
2.14 Agro inoculation
All Clones which are prepared in the binary vector pFGC4901 were electroporated
into Agrobacterium strain EHA105 whereas clones in pGreen0029 or PVX
vector pGR107 were electroporated to Agrobacterium strain LBA4404. For
agro inoculation to chilies glycerol stocks of Agrobacterium strain with required
clones were streaked on solid LB medium plates containing 12.5 µg/mL rifampicin
and 50 µg/mL kanamycin and incubated at 28 ˚C for 48 h. A single colony of
bacteria was picked with a sterile wire loop and inoculated into 50 mL LB
containing the required antibiotics and placed in a shaker (160 RPM) at 28 ˚C until
the O.D600 of the culture was 1. The cells were harvested by centrifugation at 8000
rpm for 8 min and re suspended in LB (pH 7.0) containing acetosyringone
(final concentration 100 µM). Chili seeds were surface sterilized by dipping in
70% ethanol for 1 min followed by soaking them in 0.1% HgCl2 and 1% SDS for 6
minutes. Then seeds were thoroughly rinsed three times with double distilled water
and were sown in MSO [MS salts and vitamins (Phyto Technology USA, Prod NO:
M404), Sucrose 3.0%, 0.8% agar (Sigma) and pH 5.7-5.8].
G30 syringe needle was used to puncture the hypocotyl of germinating seedlings (3-4
times) and inoculated with Agrobacterium by soaking, kept in dark at 25 ˚C for
overnight. Inoculated seedlings were washed twice with distilled water prior to
grown in pots containing silt and sand in equal proportion with a small amount
of compost. Plants were kept at 25˚C for 10 days and later on moved to a
growth chamber at 30-35˚C with 16 h light period. N. benthamiana and N. tabacum,
plants were used for agro infiltration at t 4 - 5 leaf stage. Inoculum o f
Agrobacterium was injected into broad expended leaves with 5 mL syringe.
Plants were grown at 25˚C with 16 h light in a growth room.
2.15 Plant Transformation
2.15.1 Plant transformation through Agrobacterium -mediated in N. tabacum
(CV. Samsun).
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Seeds were surface sterilized by soaking them in 5% bleach, followed by a dip in 70%
ethanol for 1 min. Seeds were thoroughly rinsed three times with double distilled
water to remove ethanol. Seeds were sown in MSO medium (described in section
2.14). Two weeks later, plantlets were transferred to fresh MSO medium. Fully
expanded leaves were used for cutting leaf discs after 3-4 weeks.
Single colony of A. tumefaciens from EHA105 peAC1-AC2dsRNA/pFGC clone
(section 4.2) was picked by sterile tooth pick and inoculated in 25 ml of LB broth
medium (100 g/ml Rifampicin and 50 g/ml Kanamycin) and kept in shaker
incubator at 250 rpm for 2 days under dark conditions. Top three leaves were Cut in
the form of disc under aseptic conditions and placed on MS1 medium [MSO, 0.1mg/l
NAA and 1 mg/l BAP], having 15-20 discs per plate. Leaf discs were place in plate
upside down and incubated at 25 ºC with 16/8 light and dark cycles, also explants
were left for pre incubation for 24h.
Agrobacterium culture was centrifuged at 3000 rpm for 10 min and re suspended in
MSO medium with 3% sucrose to an OD of 0.4-0.5 at 590 nm. 30 ml of bacterial
suspension was placed in sterile falcon tube and leaf discs were placed in bacterial
suspension. Tube was inverted gently for about 2-5 min. leaf discs were dry on sterile
filter paper and placed on co culture medium for 48-72 h at 25 ºC with light
conditions.
Leaf discs were removed from co-culture medium and placed on MS1 selection
medium [MS1 medium, glufosinate ammonium (10mg/l) and cefotaxime (50mg/l)]
and incubated as described above. Leaves were subcultures after every 2-3 weeks and
placed on MS1 selection medium. Regeneration started after 20-25 days in tobacco.
Shoots were cut from callus when at least 1 internode was formed. Shoots were
transferred into rooting media with reduced MS2 rooting medium [MS0 medium,
glufosinate ammonium (10mg/l) and cefotaxime (50mg/l)]. Seedling plants were
washed with tap water to remove the media. Selected transgenic plants were shifted to
sterile soil pots and placed at 25 ºC under 16h photoperiod (hardening) and covered
with polyethylene bags to retain humidity for 7-10 days. After that envelops were
removed to reduce humidity slowly and plants were allowed to acclimatized with
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humidity and temperature. For Self-fertilizations paper bags were used to enclose
flower buds.
2.16 Hybridization
2.16.1 Immobilize DNA onto a permanent substrate (membrane)
DNA samples were checked in 1% agarose gel at 70 volts for 30 min. The gel was
treated with different solutions i.e., depurination solution for 15 min,
denaturation solution for 30 min and neutralization solution for 30 min. Gel was
washed with sterile distilled water after each step and shake gently in shaker.
DNA from gel was shifted to nylon membrane in 5X SSC by capillary action,
and UV crosslinker (Crosslinker-UVP) at 120 mJ/cm2 energy was used to
crosslinked DNA on the nylon membrane .
2.16.2 Probe synthesis
2.16.2.1 Biotin labeled Probe
Biotin Deca Label DNA Labeling kit (Fermentas) was used to prepared DNA probe.
2.16.2.1 DIG labeled Probe
DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Applied Science,
Indianapolis, IN, Cat. No. 11 745 832 910) was used to prepared the DIG labeled
probe followed by protocol [174].
2.16.3 Hybridization
2.16.3.1 Hybridization using Biotin labeled probe
Biotin labeled Membrane was washed with 0.1X SSC, 0.5% SDS solution for 45
min at 65˚C. Then membrane was treated with 0.2 mL/cm2 pre-hybridization
solution, 0.5% SDS, 50% deionized formamide and 50 µg/mL denatured salmon
sperm DNA) for 2-4 h in a hybridizer at 42 ˚C. Prehybridization of the
membrane was done by the manufacturer instruction (Biotin Chromogenic
Detection Kit by Fermentas).
2.16.3.2 Hybridization using DIG labeled probe
The hybridization of the biotin DIG labeled probe on the nylon memebrane was was
done according to manufacturer‟s instructions (DIG Northern Starter Kit, Roche). The
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membrane was equibrated by equibration buffer and exposed to x-ray film (Fuji film)
after soaking in CDP-Star solution (Applied Biosystems, USA).
2.17 Basta sensitivity test
To evaluate for basta (glufosinate ammonium) sensitivity, MS0 medium plates were
prepared with 50mg/l glufosinate basta. These seeds were allowed to grow at 26 ±
2oC with 16/8 hours day/night in growth chamber. The basta resistant plantlets were
then transferred to pots/soil for glass house study. Self-fertilizations were facilitated
using paper bags to enclose flower buds
2.18 Isolation of total RNA
Concert Plant Trizol Reagent (Invitrogen USA) was used to extract the total RNA
from transgenic plant following the manufacturer‟s instructions. Infected leaves (400
mg) were cut from each transgenic plant and ground with liquid nitrogen in very fine
powder in pestle and mortar and transferred to 1.5 ml Eppendorf tube. Each sample
was added with 0.5 ml cold Concert Plant RNA Reagent and mixed by tapping and
incubated at RT for 5 min. this mixture was centrifuged at RT for 2 min at 13000 rpm
to take clear supernatant. Supernatant was taken into separate tube and 5 M NaCl (100
µl) and Chloroform (300 µl) was mixed in each tube and centrifuged at 4oC for 10
min at 13000 rpm. Supernatant was shifted to new tube and same volume of
isopropanol was mixed with it and kept for 10 min at room temperature. The tubes
were again centrifuged at 4oC for 10 min at 13000 rpm. Pellet was washed with 70 %
ethanol and centrifuged for 2 min. Dried pellet was dissolved in 30 µl RNase free
water. The quality and concentration of RNA was observed on agarose gel (1 %) and
stored at -80oC.
2.19 siRNA analysis of the transgenic plants
Total nucleic acids of transgenic and untransformed plants were extracted with the
help of Concert Plant Trizol Reagent (Invitrogen USA) (section 2.14). Twenty
micrograms of total RNAs were separated by denaturing Urea polyacrylamide gel
electrophoresis (PAGE) (Denaturing PAGE gel: 15% polyacrylamide, 7 M Urea, 0.5
× TBE) at 200 V for 2.5 h and transfer to Hybond-N+
membrane (Amersham
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Pharmacia) by capillary blotting [171]. The RNA was fixed on the membrane by
crosslinking in UV crosslinker (CL-1000 Ultraviolet Crosslinker-UVP) at 120 mJcm-2
energy.
Sense AC1-AC2 PCR amplified (575bp) fragment (section 4.2.2) was cloned into
pTZ57R (Fermentas, Arlington, Canada) using the same protocol as described in
section 2.6 and resulting clone was Sense AC1-AC2/PTZ . The RNA probe was
prepared by Invitro transcription using a Sense AC1-AC2/PTZ and Roche Dig RNA
Labeling Kit (SP6/T7) Cat. No. 11 175 025 910 according to the manufacturer‟s
instructions followed by alkaline hydrolysis buffer treatment (60 mM Na2CO3; 40
mM NaHCO3; pH 10.2) and Hydrolysis-neutralization buffer (3 M sodium acetate;
1% (v/v) acetic acid; pH 6.0). Alkaline hydrolysis regulates the size of RNA probes.
The membrane were soaked in pre-hybridization solution for 1 hour at 40oC. The
probe was denatured by heating and chilling on ice and added to the pre-hybridization
solution. Hybridization of the membrane was carried out at 40 0C for 12 hour
followed by washing twice with washing buffer for 15 minutes at 50 0C. All the steps
including membrane blocking, reacting hybridized probes with Anti-Digoxigenin-AP
Fab fragments, and membrane washing was performed following the instructions of
manufacturer (DIG Northern Starter Kit, Roche). The membrane was equibrated by
equibration buffer and exposed to x-ray film (Fuji film) after soaking in CDP-Star
solution (Applied Biosystems, USA).
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Component captured by begomovirus; Pepper leaf curl Lahore virus
requires DNA B of Tomato leaf curl New Delhi to cause leaf curl
symptoms
3.1 Introduction
Viruses that belongs to geminiviridae family have single stranded circular (ss) DNA
genomes and are divided into 4 genera on the basis of their genome organization,
insect vectors and host range. Among all four genera begomoviruses are more
destructive group of viruses which are transmitted through white fly B. tabaci and
infect dicots [175, 176]. Begomoviruses are distributed throughout world where
environmental conditions support the population of white fly and has become a most
important limitation in the crops production [14, 177, 178].
An important factor that limit the chili production on the Indian subcontinent is Chili
leaf curl disease (ChLCD) which is caused by begomoviruses [179]; [180]; [181].
These viruses often cause severe symptoms such as upward and downward leaf
curling, mottlings, yellowing, vein thickening and stunted growth. Formerly, chili leaf
curl beta satellite has been reported from the chili samples collected from all over the
Pakistan [161]. Similarly in other reports one species of beta satellite (ChLCB) was
found to be associated with this disease complex due to the same geographical
segregation [179].
One of the major cause of spread of begomoviruses in pepper could be infected
tomato crops that are growing in close relevance with pepper and become source of
infection. It was experimentally test that white flies carry this virus from infected to
healthy seedlings of tomato and chili [158]; [182], because inoculation of healthy
plants developed same typical symptoms of leaf curl disease of tomato caused by
(ToLCNDV) [182]; [160].
Diversity of the begomoviruses from large sample of chili leaf samples in Pakistan
have been studied [180]. Pepper leaf curl Lahore virus (PepLCLV) has been also
reported from Pakistan although its infectivity is not confirmed yet through
experiments [51]. However, the isolation and characterization of PepLCLV from chili
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plant has been also studied with the interaction beta satellites and the DNA B
component of ToLCNDV as
DNA B is essential to infect plants and induction of disease symptoms but its
interaction with ChLCB is not clearly understood.
3.2 Material and methods
3.2.1 Collection of virus infected plant samples
A chili plant showing distinctive symptoms of begomovirus infection was detected in
2004 and symptomatic or healthy leaves of chili were collected from the field located
near Faisalabad, Pakistan. Samples were transported to laboratory and stored at -80ºC
for further analysis. This isolate was previously shown to harbor ChLCB–
(AM279673) [161].
3.2.2 Isolation, cloning and sequencing
Genomic DNA was extracted from healthy and symptomatic chili samples using
CTAB method described by [170]. Required fragments full-length begomovirus, beta
satellite and alpha satellite molecules were amplified through PCR using universal
primers as described in (Table 2.1) [183]; [184]; [185].
Two set of primers, BC1-F/R [158], were used to detect DNA B component of
ToLCNDV. PCR product were purified and cloned into pTZ57R/T vector
(Fermentas). PGL1 (clone) was sequenced from (Macrogen, Korea) from both sides.
DNA sequences were analyzed with Lasergene package software and multiple
sequence alignments were performed using Clustal X [172]. Phylogenetic trees were
constructed by neighbor-joining method and printed using Tree view [173].
3.2.3 Agrobacterium-mediated inoculation of plants
Partial direct and tandem repeat constructs for Agrobacterium-mediated inoculation
were produced in the binary vector pGreen standard protocol were followed [186].
PGL1 (1498 nt) fragment from the intergenic region was removed with XbaI and
EcoRI restriction endonucleases and sub cloned into the XbaI–EcoRI sites of pGreen
to produce the clone pGPeA. icPGL1 clone was produced by restriction of PGL1(full
length) with XbaI and inserted into pGPeA at its unique site XbaI and it was
confirmed by digestion with EcoRI. This partial direct repeat of PGL1 was
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transformed into A. tumefaciens strain GV3103 electro competent cells by
electroporation.
The infectivity analysis of PGL1 was performed alone/combination with the DNA B
component of TLCNDV [31], (ChLCB-AJ316032) [179] and (CLCuMB-
AJ298903)[187] in N. benthamiana, N. tabacum cv Samsun, and C. annuum cv
Loungi by Agrobacterium-mediated inoculation (section 2.14). Ten plants were
inoculated per crop and kept at 25°C with 70% RH and 16 h/day light in greenhouse.
Daily observations of plants were made to detect appearance of symptoms.
Figure 3.1
Phylogenetic dandrogram constructed by alignment of selected complete
sequences of begomoviruses (or DNA A components)
DNA A sequences used for comparison are as follows; Cucurbit leaf crumple virus(CuLCrV),
Tomato golden mosaic virus (TGMV), Cowpea golden mosaic virus(CPGMV), Cabbage leaf
curl Jamaica virus (CabLCuJV), Okra yellow crinkle virus (OYCrV), Indian cassava mosaic
virus (ICMV), Pepper leaf curl Lahore virus (PepLCLV), Pepper leaf curl Bangladesh virus
(PepLCBDV), Cotton leaf curl Kokhran virus-Manisal (CLCuKV-Man), Chili leaf curl virus-
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Khanewal (ChLCVKh), Bean golden yellow mosaic virus (BGYMV), Papaya leaf curl China
virus-Ageratum (PaLCuCNVAge), Chili leaf curl virus-India (ChLCV-IN),Chili leaf curl
virus-Multan(ChLCV-MU), Cotton leaf curl Multan virus-Rajasthan (CLCuMVRaj),Cotton
leaf curl Multan virus-Bhatinda (CLCuMV-Bha), Cotton leaf curl Multan virus-Faisalabad
(CLCuKV-Fai), Mungbean yellow mosaic India virus (MYMIV), Tobacco curly shoot virus
(TbCSV), Radish leaf curl virus (RaLCV), Indian cassava mosaic virus-India (ICMV-IN).
The tree was arbitrarily rooted on the sequence of Tomato leaf curl New Delhi virus
associated DNA B which is distinct sequence with same size. The database accession number
in each case is given. Isolate and strain descriptors are as given in [188].
3.3 Results
3.3.1 Detection of begomovirus components in chili samples
Leaf curl symptom in chili plants are linked with monopartite begomoviruses along
with betasatelite and ToLCNDV [160]. The presence of a virus in the symptomatic
sample were analyzed through PCR using universal primers (BegomoF/R) that could
amplify begomovirus of DNA A [185]. A PCR product of 2.8 kb was amplified from
the symptomatic chili plant, while no amplification event was found with healthy chili
plants that confirmed the relation of begomovirus with induction of disease symptom.
The DNA B of ToLCNDV was detected using primer pair BC1F/BC1R [158].
Specific primers of movement protein (MP) gene of ToLCNDV were used to amplify
a fragment of 850 nucleotides which is the indication of virus in plants. Similarly the
presence of a beta satellite in samples was detected using the universal primer pair
(Beta01/Beta02) [184] with amplified product length 1350 nucleotide. Earlier
betasaltelite was characterized as an isolate of ChLCB [161].
3.3.2 Analysis of the sequence of PGL1
PGL1 clone (2747 nt) sequence is available in the databases with accession number
AM691745. Comparison of this Sequence with other reported sequences revealed that
the genome had the highest sequence identity (99%) with PepLCLV-[PK: Lah: 04]
(AM404179) followed by 89% with PepLCuBDV-PK [PK: Kha: 04] (DQ116881).
This showed that PGL1 is an isolate of PepLCLV -[PK:Fai:04] [188]. Phylogenetic
dandrogram were constructed to group PGL1 with other isolates of PepLCLV for
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which a full-length sequence is available in the databases (PepLCLV-[PK:Lah:04])
and to be closely related to PepLCBDV (Figure 3.1).
The clone shows the typical genome organization of begomoviruses with 2 ORFs in
the virion-sense strand (V1 and V2) and 4 ORFs (C1, C2, C3 and C4) in the
complementary-sense [176]. Genetic characters of this Begomovirus are described in
table 3.1. Minute different were found in gene that encode for replication-associated
protein (Rep).Uptil now the Rep protein of the PepLCLV isolate is larger than that of
other isolates of this viruse characterized [51], as well as from other begomoviruses.
Mainly the Rep protein has fourteen amino acid leader sequence at the N-terminal end
that is absent in other closely related begomoviruses that infects pepper (Figure 3.2).
The intergenic region (IR) of PepLCLV contains nearly 241 nucleotides and is similar
to those are found in isolates of ToLCNDV (Figure 3.3, Table 3.2). In all
begomoviruses a nine nucleotide long (TAATATTAC) conserved region is found in
stem loop and which is characterized as the origin of virion-strand DNA replication
[189]. Inside the IR region partial direct repeats of an iterons (GGGGAC) were
identified that lies near TATA box of the Rep promoter and generally these sequences
are species specific Rep binding motifs [190]; [96].
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Table 3.1 Features of the begomovirus isolated from C. annuum
*Gene
s are
indicat
ed as
coat
protein
(CP),
replicat
ion-associated protein (Rep), transcriptional activator protein (TrAP), and replication enhancer (REn).
The products encoded by ORFs V2 and C4 have yet to be named.
ORF* Start codon
(nucleotide
coordinates)
Stop codon
(nucleotide
coordinates)
Predicted size of
ORFs
(nt)
Predicted size of
protein
(no. of amino acids)
V2 510 145 365 122
CP 1075 305 770 257
Rep 2651 1524 1127 376
TrAP 1621 1217 404 134
REn 1476 1072 404 134
C4 2452 2195 257 86
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Table 2
Pairwise Nucleotide identities between the components of genome and protein
encoded genes sequence identities from isolate PGL1 in comparison with the
components and genes of all isolates available in the databases.
* Numbers of sequences from the databases used in the comparisons.
# Gene names are as indicated in Table 1.
Begomovirus Complet
e
sequence
(percent
age
nucleoti
de
sequence
identity)
Intergen
ic region
(percent
age
nucleoti
de
sequence
identity)
Gene#
(percentage amino acid sequence identity)
AV2 CP REn TrAP Rep AC4
ChLCV [11]* 84–87.2 77.3-
88.3
87.9-
92.4
94.4-
98.4
75.8-
91.7
82.6-
97.7
75.2-
91
39.3-
92.9
CLCuMV [10]* 74–74.6 59.4-
63.8
63-
74.1
82.3-
94.4
63.6-
76.9
57.6-
68.2
69.8-
72.4
40.5-
46.4
EACMV [10]* 69.6-
69.9
62.4-64 62.1-
62.9
75.4-
76.2
68.9-
70.5
63.6-
65.2
53.6-
65.8
26-
27.3
PapLCV [5]* 72.3-87 61.9-
85.9
69.1-
90.5
92.5-
97.6
73.5-
90.9
70.5-
95.5
61.3-
91
40.5-
94
PepLCV [4]* 74-86.7 51.7-
80.4
75-
91.4
78.1-
98
66.7-
90.9
71.2-
91.7
69.6-
75.8
34.5-
35.7
PepLCBDV [3]* 88.6-
89.4
78.7-
85.8
89.7-
92.2
94.8-
96
90.2-
95.4
93.9-
97.7
90.7-
93.5
92.9-
92.9
PepLCLV [2]* 98.9-99 96.2-
96.7
97.4-
98.3
98-
98.4
99.2-
99.2
100-
100
97.5-
98.3
97.6-
100
ToLCNDV-
Chili [4]*
71.4-
71.8
64.8-66 68.2-
70
92.1-
93.3
63.6-
67.4
54.5-
56.8
67.2-
68.6
39.7-
41.4
ToLCGV[7]* 77.8-
78.5
73.5-
75.1
85-
86.7
78.6-
78.6
79.5-
81.8
87.9-
90.9
74.4-
75.6
34.5-
36.9
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34
3.3.3 Infectivity and symptoms of PepLCLV
The infectivity of clone PGL1 (PepLCLV) was studied in N. tabacum Samsun,
N. benthamiana and C. annuum by Agrobacterium-mediated inoculation (Table 3.3).
Low infectivity (2/10) in N. benthamiana was observed after the inoculation of
PepLCLV and mild leaf curl were observed in infected plants (Figure 3.4). Presence
of virus was detected through PCR with specific primers but it was absent in case of
Sothern blot hybridization as shown in (figure 3.5), representing very a minor virus
DNA accumulation rate in inoculated plants. ChLCB along the helper virus Cotton
leaf curl Multan virus was found infectious to N. benthamiana and produced severe
leaf curling among infected plants (Table 3.3), demonstrating that the ChLCB clone is
infectious and responsible for symptoms development through helper virus.
Conversely, when inoculated in the presence of ChLCB and PepLCLV, plant
produced mild symptoms in N. benthamiana (Figure 3.4) and virus level were below
the detection limit when assessed through Southern blot hybridization (Figure 3.5).
Inoculation of PepLCLV to N. tabacum, either alone or with ChLCB did not cause
infection. Further, the interaction of PepLCLV with beta satellites, infectious clone
was inoculated beta satellite of CLCuMB [179]. However, no infection was found
after the inoculation with beta satellites so CLCuMB and ChLCB were found to be
infectious in N. benthamiana along with helper virus CLCuMV as described in (Table
3.3).
3.3.4 PepLCLV trans-replicates ToLCNDV DNA B and induces leaf curl
symptoms
Agro inoculation of plants were performed with partial repeats of PepLCLV along
with the DNA B of ToLCNDV [31] which induced symptoms leaf curl in N. tabacum
Samsun, N. benthamiana, and C. annuum. The symptoms in N. benthamiana were
consist of severe stunting and upward leaf curling. Southern hybridization was used to
detect the typical begomovirus replication intermediates using PepLCLV as probe
(Figure 3.5). However the level of virus accumulation in plants inoculated alone with
PepLCLV were not detectable by Southern hybridization (Figure 3.6, lane 2).
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35
Inoculation of PepLCLV with ToLCNDV DNA B and ChLCB resulted in disease
symptoms but the virus levels were lower (Figure 3.6, lanes 3 and 4) as compared to
plants inoculated with PepLCLV and ToLCNDV DNA B (Figure 3.6, lanes 5-7). In
case of N. tabacum and chili plants downward leaf curling and yellowing were found
as most prominent symptom.
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36
Table 3.3 Infectivity and symptoms induced by Pepper leaf curl Lahore virus
(PepLCLV)
Plant
species
Inoculums Infectivity (plants
infected/inoculated)
Symptoms
Experiment
I II III IV Total
N. benthami
ana
PepLCLV 2/10 1/6 0/7 1/5 4/28 Very mild leaf curling
PepLCLV + ChLCB 1/10 0/6 1/7 0/5 2/28 Very mild leaf curling
PepLCLV + ToLCND +
DNA B
9/10 6/6 6/7 4/5 25/28 severe downward leaf
curling
PepLCLV + ToLCND
DNAB + ChLCB
8/10 5/6 7/7 4/5 24/28 severe downward leaf
curling
PepLCLV + CLCuMB 0/10 - - - 0/10 No symptoms
PepLCLV + TbLCB . - - 0/5 0/5 No symptoms
CLCuMV + ChLCB 4/5 5/6 - - 9/11 Severe leaf curling
CLCuMV + CLCuMB 5/5 - - - 5/5 Severe leaf curling
N. tabacum PepLCLV + ChLCB 0/10 0/4 - - 0/14 No symptoms
PepLCLV+ToLCND DNA
B
8/10 3/6 - - 11/16 Leaf curling
C. annuum PepLCLV + ChLCB 0/10 0/5 - - 0/15 No symptoms
PepLCLV + ToLCND
DNA B
`5/10 3/6 - - 8/16 Leaf curling
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Figure 3.2
Alignment of the N-terminal amino acid sequences of the Rep protein of PepLCLV with
other reported sequence of begomoviruses that infects chili on the Indian subcontinent.
Alignment were optimized by the introduction of Gaps (-) into the sequences. So Conserved
sequences are marked (*). The begomovirus (DNA A component) sequences of (ChLCV),
(CLCuKV), (PaLCuV), (PepLCBDV), (PepLCLV) and (ToLCNDV) were used for the
alignment. The database accession number of each is given in [188].
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Figure 3.3
Alignment of the IR regions of PepLCLV, ToLCNDV DNA A and DNA B. Conserved
sequences in the alignment are marked (*). The positions of the stem (light orange color) and
conserved nona nucleotide (TAATATTAC) sequences (lime color) of the predicted stem-loop
structure, the TATA box of the Rep promoter (violet color) and predicted iterons (dark green
color) for PepLCLV, while blue for ToLCNDV (DNA A) component and red for ToLCNDV
(DNA B) are designated.
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Figure 3.4
Symptoms induced by PepLCLV clone PGL1 in N. benthamiana, N. tabacum and C.
annuum. a) N. benthamiana plant infected with PepLCLV at 20 dpi b) N. benthamiana
plant infected with PepLCLV and ChLCB at 20 dpi. c) A N. benthamiana plant infected with
PepLCLV and ToLCNDV DNA B at 14 dpi. d) A N. benthamiana plant infected with
PepLCLV, ChLCB and ToLCNDV DNA B at 14 dpi. e) A N. tabacum plant infected with
PepLCLV and ToLCNDV DNA B at 30 dpi. f) A C. annuum plant infected with PepLCLV
and ToLCNDV DNA B at 40 dpi.
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Figure 3.5
Virus replication in inoculated leaves of N. benthamiana plants probed with PGL1.
(Lane 1) Plants were agro inoculated with PepLCLV, (lane 2) PepLCLV and ChLCB, (lanes
3, 4) PepLCLV, ChLCB and ToLCNDV DNA B, (lanes 5-7) PepLCLV and ToLCNDV DNA
B. lane 8 represent DNA that was isolated from chili plants infected with PepLCLV taken
from fields. 10 μg of genomic DNA was loaded on gel for each sample.
3.4 Discussion
Geminiviruses are on the top of rapidly emerging plant viruses that are cause of
destructive diseases in crops, including several factors such as increase population of
vectors as well as presence of an alternate host. Geminiviruses are rapidly evolving
group of plant virus due to changed climatic conditions such as abrupt change of
environment and frequency of vector population. The success rate of association of
beta satellites with district group of begomoviruses could be determined by the ability
of beta satellites to replicate [191]. The driving force ahead for rapid emergence and
resistance breakdown through begomovirus-beta satellite complexes are due to the
interaction of begomoviruses with their diverse beta satellites, the movement of
components of begomovirus to alternate hosts and recombination among them [14].
Several reported have been published on the mixed infection of geminiviruses and
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about their complex interaction with host plants i.e. Cassava plant were infected by
two different strains of begomovirus species and the synergistic interaction cause the
severe disease [57]. It is very interesting fact about ToLCNDV (bipartite
begomovirus) has been also detected in other hosts because this virus has ability to
interact with other components of begomovirus and help to increase its host range.
(160) reported the interaction of ToLCNDV with ChLCB in open field environment
consequently severe symptoms appear, in another such type of report Tomato leaf curl
Gujarat virus (ToLCGV) interact with ToLCNDV (DNA B component) subsequently
sever symptoms appears [192]; [193]. Recently it has been found that ToLCGV lacks
DNA B and over winter an alternate host in weeds (manuscript in preparation), which
suggests that this virus was move to tomato crop from weeds after interaction with
DNA B component of ToLCNDV and formed a complex that results in severity of
viral symptoms and ultimately loses in crops . Geminiviruses replicate through the
process of rolling circle replication that is initiated by Rep protein (replication
enhancer protein) (26). Rep bind to specific sequence in the intergenic region in loop
that contains nonanucleotide sequence (TAATATTAC) where Rep initiate replication
of geminivirus through nicking. In case of bipartite virus which contains two
components DNA A and DNA B in which Rep protein is responsible for the
replication and maintain reliability of the split genome.
Although the sequence and mutational analysis of viral isolates recommends that
these viruses can tolerate the changes in the intergenic region which showed no
effects on the Rep recognition. But in this study predicted iteron sequence of
PepLCLV (GGGGAC) as compare to the iteron sequence of ToLCNDV (GGTGTC)
as shown in figure 3.5. It means that first three bases are crucial for the recognition of
Rep protein in iteron sequence of begomovirus. One of the remarkable finding of this
research work is that PepLCLV isolate has limited ability to trans-replicate beta
satellites.
These finding are comparable with the results of [51], revealed the association
ChLCB with PepLCLV from Pakistan. The inability of the virus to trans replicate beta
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satellites is might be due to N terminal leader sequence of the putative Rep protein of
virus as shown in figure 3.2. However, there is need of an evidence to support this
hypothesis so to confirm through mutagenesis. So other possible reason could be that
natural variant of PepLCLV may lack leader peptide sequence in Rep that is might be
responsible for the interaction of this virus with beta satellites. Moreover, these effort
to characterize begomoviruses are not complete to understand hidden mechanisms
involved and perhaps another begomovirus strain could interact with beta satellite in a
naturally infected chili plant in field conditions. In chili plant monopartite, bipartite
and monopartite associated with beta satellite has been previously identified [158,
160, 161]. The full length clone PGL1of begomovirus associated ChLCD, has 2747
nt. In sequence which showed (99%) with (PepLCLV) refer to it an isolate of
PepLCLV on the basis of 89% SDT (species demarcation threshold) for
begomoviruses [188].
Inoculum of this clone was introduced into N. benthamaiana that produced only mild
symptoms. However, inoculation of beta satellite and clone of ChLCD infected plant
produced similar mild symptoms whereas virus titer were not detected through
Southern hybridization. Although, when plants were inoculated with DNA B
component of ToLCNDV typical symptoms of ChLCD were found in N.
benthamiana, N. tabacum and C. annuum. These results provides the evidence that
this indentified virus might be bipartite. Plants inoculated PepLCLV, ToLCNDV
DNA B and ChLCB showed surprising results with low viral DNA load as compared
to the plant where ChLCB was absent as shown in figure 3.5. This experiment first
time provides the demonstrations of infectivity analysis for a bipartite begomovirus
which is responsible for ChLCD [51]. The exact mechanism of interaction between
the rep and beta satellite replication is not clearly understood as beta satellite lack the
iteron sequence responsible for the replication and encoded by the helper virus
component [191], so it required further confirmation through experiments.
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Due to the variations in to the expected iteron sequence of ToLCNDV and PepLCLV
Both has ability to trans replicate DNA B of ToLCNDV and produced chili leaf curl
disease in experimental host and chili crop. Despite the interaction of PepLCLV with
beta satellites, both has ability to reduce the virus load and symptoms severity, due to
the interference this happened.
Future studies will focus on interference to combat the viruses and may offer a tool
for attaining resistance to viruses causing chili leaf curl disease. The complex nature
of these ChLCD complex in Indian subcontinents would be a great task for the
development of resistant varieties from old and new methods. High yield loses due to
the chili leaf curl disease complex are imposing serious threat to chili cultivation and
forcing farmers to grow alternate crops instead of chili to avoid these viruses.
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Resistance against chili leaf curl disease complex (ChLCD)
using RNA interference
4.1 Introduction
Chili leaf curl disease (ChLCD); whitefly transmitted begomoviruses disease complex
is major root cause for reduction of chili production in the Pakistan [158, 182, 194].
The higher incidence of chili leaf curl disease with severe symptoms may be due to
the synergistic effect of Gemini virus disease complex that includes monopartite,
bipartite begomoviruses and beta satellite [194]. Due to the higher loses, chili
cultivation is badly effected and is forcing farmers to grow other crops.
RNA silencing mediated by short-interfering RNA (siRNA) is used by plants as a
defense against viruses [195, 196]. The RNA silencing phenomenon was first
discovered in plants [197]. In the case of Gemini viruses, viral DNA is targeted at the
transcriptional level, and viral mRNA is targeted at post transcriptional level [196].
Several reports are now available on application of RNAi for developing resistance
against Gemini viruses with different levels of success [117, 198-201]. Here RNA
interference had been used for developing resistance against this disease. The most
conserved regions among the begomoviruses infecting chili crop prevailing in this
region were dissected out and targeted through RNAi.
4.2 Material and Methods
4.2.1 Cloning of RNAi based gene constructs
The extent of variation and highly conserved region of begomoviruses causing
ChLCD in subcontinent was evaluated using DNASP version 4.10.3. The level of
variation of seven begomoviruses infecting C. annuum isolates was estimated by the
average number of nucleotide differences per site (Pi) at all sites along the genome.
The hairpin gene constructs based on overlapping and the conserved region of C1, C2
and C3 region (575 bp) of Pepper leaf curl Lahore virus DNA A (accession
AM691745) was constructed as described by [202]. The construct was designated as
AC1-AC2dsRNA/pFGC based on overlapping regions of AC1 and AC2. The primers
(Table 2.1) were designed to amplify the AC1-AC2 region (575 bp) of DNA A in
sense and antisense orientations on the basis of sequences of PepLCLV (AM691745).
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The primer sequences used in this study are given in Table (2.1). In forward and
reverse primers for sense cloning, XhoI and NcoI restrictions sites were incorporated,
respectively. Whereas BamHI and XbaI were introduced forward and reverse primers
for cloning in antisense, respectively.
4.2.2 Transient assays
To check the efficiency of peAC1-AC2dsRNA/pFGC transient assays were done
along with Ch Rep/PVX, (already available in the lab) and infectious clone of
PepLCLV with DNA B components of ToLCNDV (Section 3.2.3) through agro
infiltration in N. benthamaiana, N. tabacum and C. annuum. Respective
Agrobacterium suspension carrying clones were pelleted and resuspended separately
in a solution containing 10 mM MgCl2 and 150 g/ml acetosyringone to an optical
density of 1 at 600 nm. Co-infiltration experiments were performed in the following
combinations.
1. Ch Rep/PVX + AC1-AC2dsRNA/pFGC (mixed in a ratio 1:1)
2. Ch Rep/PVX
3. PepLCLV A + ToLCNDV B + peAC1-AC2dsRNA/pFGC (mixed in a ratio
1:1:1)
4. PepLCLV A + ToLCNDV B (mixed in a ratio 1:1)
4.2.2 Plant transformation
The construct peAC1-AC2dsRNA/pFGC was transformed into A.tumefaciens strain
EHA105 by electroporation (section 2.15). N. tabacum was transformed by the
Agrobacterium-mediated leaf disk method (section 2.15). The putative peAC1-
AC2dsRNA/pFGC transgenic plants were selected on ammonium glufosinate (10
mg/l) respectively. PCR analysis (section 2.4) was carried out to confirm the presence
of transgene as well as selection marker gene using respective pair of primers (Table
2.1). The transgene integration in plants was determined by Southern blot
hybridization. A DIG labeled probe of respective gene was prepared (Section 2.16).
The transgene specific siRNA was also detected (section 2.19).
4.2.3 Challenge with virus
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15 T1 plants from 9 transgenic lines of N. tabacum (peAC1-AC2dsRNA/pFGC) plants
were exposed to viruliferous whiteflies in glasshouse. The 10 plants in each line of T1
generation of these transgenic lines with peAC1-AC2dsRNA/pFGC construct were
also agro infiltrated with infectious clones of PepLCLV DNA A and ToLCNDV B
[194]. Then the resistant lines (peAC1-AC2dsRNA/pFGC construct) were also
injected with infectious clones of ToLCNDV DNA A and ToLCNDV B. Non-
transgenic tobacco plants of the same age were used as control [194] (Chapter 3). The
replication of begomoviruses was detected through Southern or Dot Blot
hybridization using full length viruses as biotin or DIG labeled probe (section 2.16).
Relative level of viral DNA were quantified through semi quantitative PCR as
southern blot hybridization do not give quantitative estimation (section 2.16).
Figure 4.1
Plot showing the distribution of genetic variation estimated by nucleotide diversity
(Pi) for Gemini virus infecting c. annuum in Pakistan. The relative positions of the
ORFs of viral DNA genome are illustrated above the plot in linear DNA format.
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Figure 4.2
A diagram demonstrating the binary vector (pFGC5941) engineered with the
silencing trigger construct.
4.3 Results
In the present research, a study was conducted on silencing of ChLCD complex
through RNAi. Gene constructs; peAC1-AC2dsRNA/pFGC based on region
overlapping Rep and TrAP of DNA A (Figure 4.1, 4.2) was cloned. RNAi gene
construct was transformed in N. tabacum through Agrobacterium-mediated plant
transformation (Figure 4.6). The silencing efficiency of construct was checked
through transient assays in N. benthamaiana, N. tabacum and C. annuum.
4.3.1 RNAi constructs silence ChLCV-M Rep in transient assays
There are many draw backs of transgenic plant use to test the viral constructs
including time span, labor work required for transformation and regeneration. Thus
transient assay system has been developed by [203]; [204] co-inoculation of virus
with and hairpin construct targeting the virus in a leaves, suspension culture or
protoplast. Replication protein of ChLCV-M (chili leaf curl Multan virus Rep/PVX)
produces downward leaf curling in N. benthamaiana plants when expressed through
PVX. Silencing efficiency of peAC1-AC2dsRNA/pFGC, were checked in N.
benthamiana plants using transient assays. Agrobacterium culture harboring peAC1-
AC2dsRNA/pFGC and chili Rep/PVX were grown at 28°C for 48 hours and activated
with MgCl2 and acetosyringone. The activated cultures were mixed with different
O.Ds of the cultures and co-infiltrated in N. benthamiana plants. The plants infiltrated
with only Chili Rep/PVX were used as positive control (Figure 4.3). After 10 days of
post inoculation, the plants used as positive control developed severe leaf curl
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symptoms in systemic leaves while those co-inoculated with peAC1-
AC2dsRNA/pFGC did not produce typical symptoms but only developed PVX
symptoms. These results clearly show that the gene constructs targeting Rep-TrAP of
PepLCLV efficiently silence Rep when expressed through PVX, respectively.
Figure 4.3
Transient assays through agro co-infiltration in N. benthamaiana and C. annuum
plants. A) peAC1-AC2dsRNA/pFGC co-infiltrated with Chili Rep/PVX, only PVX
symptoms were visible on the N. benthamaiana and no symptoms were produced by
Rep of PepLCLV. B) Control N. benthamaiana agro infiltrated with Chili Rep/PVX
construct. Plants exhibited leaf curl symptoms after 7 days. C) peAC1-
AC2dsRNA/pFGC co-infiltrated with Chili Rep/PVX, only PVX symptoms were
visible on the C. annuum plants and no symptoms were produced by Rep of
PepLCLV. D) Control C. annuum plant agro infiltrated with Chili Rep/PVX
construct. Plants exhibited leaf curl symptoms after 15 days.
4.3.2 RNAi constructs for PepLCLV in transient assays
475bp
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When plants were injected with infectious clone of PepLCLV (section 3.2.3) beside
DNA B component of ToLCNDV [94], leaf curl in N. benthamiana, N. tabacum and
C. annuum [194] were observed. The symptom vary in plants as in case of N.
benthamiana severe upward leaf curling and stunting were observed as compared to
chilies and tobacco where, downward leaf curling and yellowing were most prominent
[194].
Inoculated plants accumulated higher levels of viral DNA in systemic as compared to
when those co-inoculated with peAC1-AC2dsRNA/pFGC and also did not produce
typical symptoms (Figure 4.4). This displays that constructs targeting Rep and Trap
gene of the virus efficiently silenced PepLCLV. Southern hybridization was used to
confirm the viral replication in infected leaves. The virus was not detected in plants
infiltrated with RNAi constructs showing inhibition or reduction in viral replication
(Fig 4.4), (Fig 4.5). These results suggested that RNAi construct (peAC1-
AC2dsRNA/pFGC) targeting Rep-Trap sequences were able to block systemic
infection of the virus. These results suggested that RNAi construct (peAC1-
AC2dsRNA/pFGC) targeting Rep-Trap sequences were able to block systemic
infection of the virus.
Figure 4.4
Transient assays of construct peAC1-AC2dsRNA/pFGC in N. benthamiana with
PepLCLV and ToLCNDV DNA B. A) N. benthamiana agro inoculated with
PepLCLV and ToLCNDV DNA B along with construct peAC1-AC2dsRNA/pFGC.
B) N. benthamiana injected with PepLCLV and ToLCNDV DNA B as a control.
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Figure 4.5
Replication of virus infected leaves of N. benthamiana plants probed with PepLCLV
Inhibition of virus replication agro infiltrated leaves of N. benthamiana plants probed
with PGL1. N. benthamiana agro inoculated with PepLCLV and ToLCNDV DNA B
along with construct peAC1-AC2dsRNA/pFGC in systemic leaves (Lane 1-3). N.
benthamiana agro inoculated with PepLCLV and ToLCNDV DNA B along with
construct peAC1-AC2dsRNA/pFGC in inoculated leaves (Lane 4-5) N. benthamiana
agro inoculated with PepLCLV and ToLCNDV DNA B as a control in systemic
leaves (Lane 6-7).
4.3.3 Plant Transformation
Invitro regeneration and genetic transformation of Pepper is extremely difficult [163].
Major problem that arises during gene transfer in chili are i) difficulty of regeneration
of plants from tissue culture; ii) procurement of transformed pepper tissues; iii)
regeneration of plants following transformation [164]. Due to the problem of stable
transformation, RNAi studies are difficult in C. annuum (section 2.10.2). The
infectivity of PepLCLV to N.tabacum (Chapter 3) concludes using this model host for
investigating the use of RNAi to obtain resistance to ChLCD. The gene constructs
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AC1-AC2dsRNA/pFGC was transformed in N. tabacum through Agrobacterium-
mediated transformation by leaf disc method (Section 2.10). From the transformation
experiment, 17 T1 independent transformations were obtained, but nine of them were
apparently normal/healthy plants were selected randomly for further assessment using
PCR, southern and Northern blot techniques. These analyses showed that RNAi
construct was integrated into the plant genome (Figure 4.7 A) and transgene produce
Rep-Trap specific siRNA (Figure 4.7 B).
Figure 4.6
Plant transformation in N. tabacum transgene analysis.
A) Callus induction B) shoot regeneration C) root formation on selection media D)
Putative transgenic plants shifted in the soil.
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Table 4.1
Regeneration and transformation efficiencies of transformed tobacco leaf discs
Sr.
No
No.of explants
placed on
shoot
induction
medium (A)
No. of explants
producing shoot
(B)
Regeneratio
n efficiency
(%)
explants No
producing
roots
(C)
Transform
ation
efficiency
(%)
1 15 10 67 8 53
2 15 11 73 9 60
Tot
al
30 21 70 17 57
A) B)
Figure 4.7
Confirmation of the transgenic plants
A) Southern blot analysis of transgenic plants for integration events (Lane 1-5)
transgenic tobacco with peAC1-AC2dsRNA/pFGC construct probed with AC1-AC2
B) RNA gel blot analysis of AC1-AC2 specific small interfering RNAs (siRNAs) in
AC1-AC2dsRNA/pFGC transgenic plant lines . (Lane 1-5) transgenic tobacco with
peAC1-AC2dsRNA/pFGC construct (Lane 6) Negative control plant (Lane 7)
positive control (primer AC1-Ac2)
4.3.4 Transgenic tobacco resistant to ChLCD complex
Gene constructs; PeAC1-AC2dsRNA/pFGC was found to be the best for silencing of
PepLCLV in transient assay. N. tabacum transgenic lines were developed with this
construct (Figure 4.6). 15 T1 plants from 9 lines of peAC1-AC2dsRNA/pFGC
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construct were positive for 35S promoter and specific gene , with similar number of
control plants which were assessed for virus resistance. Young T1 plants were
exposed to around 30 white flies (acquired virus) in each plant under controlled glass
house kept for 120 days. These white files were infected with ChLCD and reared on
chili plants for experimental use.
For the whole season in glass house the high number of white flies (30/plant) was
retained. Plants were observed regularly on weekly basis for presence of disease
symptoms and the percentage of disease incidence was calculated according to the
data. Our data specified that non-transgenic plants developed infection in early growth
stage of plant. Due to a high amount of virus acquired white-flies, disease occurrence
was relatively high as shown in figures. Plant that can be host of virus, could be
resistant if it inhibit the virus multiplication and subsequently stopped disease
symptoms development [205]. Our results showed that some transgenic N.
benthamiana lines were not produce symptoms after inoculation through large
number of white flies. However, our results suggested that all peAC1-
AC2dsRNA/pFGC lines showed variable resistance pattern against chili leaf curl
disease ranging from 6.6 - 93.3 %. Those lines which showed resistance above 75%
were consider as resistant or tolerant whereas lines with 50% resistance were ranked
as susceptible. One line peAC1-AC2dsRNA/pFGC TA14 line was categorized as
extremely tolerant display 93.3% resistance against chili leaf curl disease whereas,
the line peAC1-AC2dsRNA/pFGC TA 3.2 exhibits 6.6 % resistance was classified as
highly susceptible line. However, non-transgenic control plants presented no disease
tolerance as shown in table 4.2. DNA from all the transgenic lines of peAC1-
AC2dsRNA/pFGC TA14 and investigated for virus replication and multiplication
using dot blot hybridization as shown in figure 4.8. PCR reaction with specific
primers were performed to observe the presence or absence of particular gene in T1
generation of transgenic lines. According to results transgenic peAC1-
AC2dsRNA/pFGC plant showing significantly high resistance against chili lead curl
disease (table 4.2 and figure 4.10).
In another experiments N. tabacum 20 plants each of transgenic lines (TA 14.1 and
TA 2.2) and control plant were agro infiltrated with PepLCLV and DNA B
component of ToLCNDV and kept under controlled conditions. 25 days after agro
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infiltration, virus replication was checked by Southern blot hybridization (Fig 4.10).
In control plants, there was a very strong signal of virus but virus was not detected in
transgenic plants. Thus, the constitutive formation of dsRNA in transgenic plants
efficiently blocked virus replication. Virus resistance evaluation experiments with
PepLCLV and DNA B component of ToLCNDV were repeated with T2 plants as
well.
Figure 4.7
Virus resistance assay. A) Tobacco control showing ChLCD symptoms infected with
ChLCD viruliferous white flies B) Transgenic peAC1-AC2dsRNA/pFGC tobacco
resistant to ChLCD infected viruliferous white flies
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Table 4.1 ChLCD resistance/tolerance pattern for peAC1-AC2dsRNA/pFGC plants at T1
stage.
Transgenic
event.
Transgenic
Plants*
Plants with
symptoms
Plants without
symptoms
%age of
resistanc
e
TA 13.1 15 4 11 73.3 TA 14.1 15 1 14 93.3 TA 8.3 15 9 6 40.0 TA 3.2 15 14 1 6.66
TA 15.1 15 3 12 80.0 TA 1.1 15 14 1 6.66
TA 2.2 15 2 13 86.6
TA 10.1 15 14 1 6.66
TA 11.2 15 5 10 66.6
Control 15 15 0 0
* Plant PCR positive with 35 S promoter and AC1-AC2 primers were exposed to
ChLCD infected white-flies
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Figure 4.8
Dot Blot analysis of replication of PepLCLV in RNAi transgenic plant following
exposure to viruliferous whitefly
(A1-A2) Transgenic tobacco plant of peAC1-AC2dsRNA/pFGC without exposure to white
fly (A3-A4, B1-B4, C1-C4) Transgenic tobacco plant (peAC1-AC2dsRNA/pFGC) after
exposure to white -fly (20dpi) (D1-D4) Control untransformed tobacco plant after exposure to
white-fly (20dpi)
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Figure 4.9
Virus resistance assay. A) Tobacco control showing symptoms of PepLCLV and
ToLCNDV DNA B. B) Transgenic peAC1-AC2dsRNA/pFGC tobacco resistant to
PepLCLV and ToLCNDV DNA B.
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Figure 4.10
Southern blot analysis using biotin labeled PepLCLV probe of transgenic plants
(peAC1 -AC2dsRNA/pFGC) inoculated with ChLCD (PepLCLV and DNA B of
ToLCNDV). Transgenic plants without inoculation (Lane 1), Plasmid Control (Lane
2), Transgenic plants inoculated with (PepLCLV and DNA B of ToLCNDV) (Lane
3-4) Non transgenic inoculated (PepLCLV and DNA B of ToLCNDV) (Lane 5-7).
4.3.5 Transgenic tobacco resistant to heterologous virus
ToLCNDV produce stunting with leaf curling in N. Tabacum within twenty one days
of inoculation [64]. 15 transgenic plant line TA 14.1 T2 plants was infiltrated with
ToLCNDV. The 10 control plants infiltrated with only ToLCNDV were used as
positive control. Symptoms appeared on all control plants (10/10) after 25-30 days.
Different phenotypes in the transgenic plants were observed, including (7/15) showed
mild symptoms after 35-40 days and 8/15 delay in symptoms with no modification of
symptoms in two lines. So it was found that the hairpin construct also block
replication of ToLCNDV (Fig 4.11). So this construct shows resistance against
homologus as well as Heterologous plant viruses.
1 2 3 4 5 6 7
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Figure 4.11
Virus resistance assay A) Transgenic tobacco plant with construct peAC1-
AC2dsRNA/pFGC agro inoculated with both components of ToLCNDV B) Tobacco
agro inoculated with bi partite ToLCNDV as a control.
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4.4 Discussion
Chili pepper is an important vegetable crop cultivated through Pakistan. Chili
genotypes commonly grown in Punjab are susceptible to ChLCD complex [194].
Whenever a plant is confronted with virus, some complex changes in the host
undergoes to activate defense mechanism and also these changes need for the
replication as well as virus movement inside plant. RNA silencing has key role in the
defense against plant viruses to the regulation of gene expression and chromosome
organization [15, 206, 207]. At present, RNA silencing seems to be the most
favorable choice for developing resistance against Gemini viruses [200, 206-208].
Some sources especially genetically engineered resistance in plant has been reported
as a potential source of control against viruses. Most of the work on the use of RNA
silencing have been reported against against TGMV, TYLCSV and TYLCV, where
expression of sense and antisense RNA in transgenic plants have been employed
successfully to control these viruses [209-213] produced transgenic plants with
expression of siRNAs against TYLCSV and ToCMoV, respectively. Hence thèse
plants often exibited delay in symptoms development, mainly with low inoculum in
comparaison to the situation with RNA viruses [214], entirely resistant lines were not
observed. In conclusion of this whole process viral mRNA of all RNA viruses are
main target of RNA silencing so the success of this strategy depends on the relevance
of the target genes. RNAi technique has been investigated as a source of delivering
resistance to plants against begomoviruses [199, 200].The first successful field tests
of RNAi-based resistant plant lines has been conducted for the first time [215]. In this
study we selected the region (overlapping region of Rep and TrAP) that are the
potential target for siRNA in the field infected plants. This region is also conserved
among the begomoviruses [201, 216].
Furthermore, it was found that Rep protein is responsible for the replication and
symptom determinant, when expressed through PVX in N. benthamaiana [217]. Rep
protein perform multiple function [218-220]. For the rolling circle replication this
protein identifies the common region (CR) that are
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61 facilitated by the presence of four highly sequence-specific Rep binding sites [220,
221]. AC2 which is also know as TrAP protein and is also known to control the
function of host genes expression [222]. Thus, these sequences provide a better
target for providing better protection against the disease. Based on all these work, it
was concluded that both of these regions can be an important target for developing
RNAi based resistance against this virus. So, in this research work, chili Rep-TrAP
expression was silenced by making RNAi constructs targeting Rep-TrAP (AC1-AC2)
in transient assays. Hairpin RNAi construct AC1-AC2 dsRNA/pFGC targeting Rep-
TrAP (575 bp fragment containing 400 bp region of Rep at C-terminal and 175 bp of
TrAP at N-terminal). Then this construct AC1-AC2 dsRNA/pFGC was analyzed by
co-infiltration with Ch.Rep/PVX. RNAi construct blocks rep expression and the
plants had developed only PVX symptoms, Rep of ChLCV-M is different from other
Gemini viruses when expressed through PVX because in this particular case Rep
produces downward leaf curling in N. benthamaiana plants.
Another strategy termed as Post transcriptional gene silencing was established to
control the viruses when plant cells at once transfected with ACMV [CM], so
therefore siRNA was designed synthetically to target the AC1 gene of virus. As a
result reduction in the accumulation levels of AC1 mRNA by more than 90% and
viral DNA by 70% were observed in comparison to control plants [223]. Resistance
against CLCuV in tobacco were observed in response to transgenic expression of
AC1-antisense [117] and for BGMV in case of bean [224]. Therefore it has been
concluded that RNAi-based resistance against geminiviruses appears to be highly
encouraging for developing resistance when AC1 gene was targeted.
In another experiments transient assays were used to check the effect of RNAi gene
constructs on virus levels in inoculated and systemic leaves. N. benthamiana and C.
annuum were used for transient assay. The southern analysis of these inoculated
plants have shown the replication of the virus in the inoculated leaves but the titer of
the virus in the systemic leaves has been found to be very reduced. The detection of
the virus in inoculated leaves confirmed reliable infiltration. The decrease in the virus
titer in systemic leaves shows that silencing signal generated in the inoculated leaves.
Furthermore, RNAi constructs were also transformed stably in N. tabacum to see if
transgenic plants were protected from the disease. Transgene plants looks
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62 morphologically normal as compared to non-transgene plants and showed no adverse
effects on plant growth. The work carried out here showed that targeting of these
conserved regions through RNAi significantly reduced the level of viral DNA in
transgenic plant when these transgenic plant was inoculated with ChLCD infected
viruliferous whitefly. The virus replication was reduced to undetectable level in some
cases when checked by Southern blot hybridization but could be detected by PCR.
Thus, silencing of these sequences could effectively control chili leaf curl disease
component through RNAi. In further it would be interesting to see silencing of whole
virus complex is because of siRNA generated from AC1 or AC2 in transgenic plant.
If plant suppress the multiplication and accumulation of virus and subsequently
disease symptoms appearance that it could be known as resistant [205]. There are
several levels of resistance in plants such as high to moderate in which no virus
accumulates in the plant rendering it. Whereas in case of low resistance virus
accumulate in plant at low level as compared to susceptible and mild symptoms could
be found. However in tolerance plant expresses mild symptoms but looks healthy and
have normal level of virus in it [205].
Here some lines showed resistance above 75% (resistant) where as other lines show
less than 50% resistance were consider as susceptible. Only TA14 with 93.3% level of
resistance were ranked as highly resistant against ChLCD (Table 4.2). Although virus
transmitted through whiteflies led to the milder infection as compared to the artificial
infiltration of virus into plants for experimental purpose [225].
Plants (20 of each) of transgenic lines TA 14.1 and TA 2.2 (ranked as highly
resistant/tolerant in whitefly infected with CLCD exposure) and twenty healthy plants
of N. tabacum were inoculated with (PepLCLV + DNA B component of ToLCNDV)
and kept under controlled conditions. As a results after three weeks of inoculation
typical symptoms of PepLCLV, upward leaf curling and stunting were found in all
twenty non-transgenic plants. As a comparison no such type of symptoms were
observed on TA 14.1 and TA 2.2 transgenic plants as shown in figure 4.13. Twenty
five days after agro infiltration of non-transgenic leaves Southern blot hybridization
were performed to check the presence of viral DNA. So the results was clearly
showed the presence of viral DNA in non-transgenic plants as compared to transgenic
ones as shown in figure 4.14a.
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63
To further investigate the presence of virus in transgenic plants PCR technique was
used in which primers designed to target DNA A. This study helps to explore the
presence of virus in (13 out of 20 plants) in TA 2.2 while in (10 out of 20) in TA14.1
transgenic plants, but only at low dilutions; whereas in non-transgenic infected plants
viral DNA was detected at all dilutions (Figure 4.14b). RNA silencing is sequence
homology based and is important in case of engineering resistance. ToLCNDV is a
bipartite begomoviruses [31], and it infects chilies, tomato and watermelon crops in
the Indian subcontinent [158, 226]. ToLCNDV produced typical of leaf curling and
stunting in N. tabacum within three weeks of inoculation [64]. In order to understand
that how much sequence homology is required for the efficient gene silencing and
also to see that whether the transgenic peAC1-AC2dsRNA/pFGC plant can block
heterologus viruses. 15 TA 14.1 transgenic plants were also infiltrated with
ToLCNDV. The 10 control plants infiltrated with only ToLCNDV were used as
positive control. Symptoms appeared on control plants after 25-30 days. The
transgenic plants inoculated with ToLCNDV also showed mild symptoms after 35-40
days. So it was found that the hairpin construct can efficiently block ToLCNDV. (Fig
4.9). Rep-TrAP 575 bp fragment (containing 400 bp region of Rep at C-terminal and
175 bp of TrAP at N-terminal was used in hairpin construct) of PepLCLV showed
89% resemblance to ToLCNDV (Accession No DQ629102) Rep-TrAP region. These
findings revealed that transgenic plants with peAC1-AC2dsRNA/pFGC constructs
were capable to prevent the replication and movement of virus; as 3 out of 9
transgenic lines were found immune for the virus.
The basic objective of this study was to search the possibility of attaining resistance to
ChLCD complex by silencing the Rep and Trap gene of PepLCLV. As these genes
are conserved among begomoviruses that belongs to Old World [57, 216, 227].
However this strategy could be used to develop in different crops against particular
begomoviruses specie which may cause sever diseases.
In this research plant transformation system in a local variety of chilies through
Agrobacterium mediated plant transformation as RNAi gene constructs are working,
these can inhibited the replication of ChLCD complex in transgenic chili plants. This
work can lead to the development of virus resistant chili crop in Pakistan.
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64 The role of Cauliflower mosaic virus (CaMV) defense and
silencing suppressor protein 6 (p6) in modulating auxin
signaling
5.1 Introduction
Introduction
Cauliflower mosaic virus (CaMV) is the type member of the genus
caulimoviruses, which is one amongst the six genera of the family Caulimoviridae.,
including Para retroviruses that infect plants and replicate by reverse transcription of a
circular ds DNA genomes (1). Para retroviruses replicate just like retroviruses through
reverse transcription while the viral particles contain DNA instead of RNA (2).
Genome of CaMV comprises of six genes and it is approximately of 8kb in size (3).
Gene VI of CaMV, encodes a multifunctional P6 protein with 62 kD polypeptide,
which is main genetic cause of symptom appearance and viral pathogenicity (4) and
can also influence the compatibility and host range (5). As P6 stores in the form of
inclusion bodies in the cytoplasm of infected cells (6), and has numerous functional
domains, comprising of an RNA binding domain, a translational transactivator, and a
zinc finger domain, these all domains are essential for viral infectivity (7 Four main
role of P6 protein of CaMV has been identified so far which includes interaction with
two other proteins of CaMV that involved in aphid transmission, P2 and P3 (8). P6
form cytoplasmic inclusion bodies of different sizes; among them smaller one move
dynamically through endoplasmic reticulum along actin filaments (9). This movement
is most important for virus trade in intracellularly in plant cells and P6 also interacts
with P1 (movement protein) of CaMV (10) and CHUP1, which intermediates
association between chloroplasts and the cytoskeleton (11). In case of Arabidopsis
and N. benthamiana P6 affects signaling pathways mediated by Salicylic acid,
Jasmonic acid, Ethylene and auxin synthesis (12).
Isolates of CaMV Bari-1 or Cabb B-JI display minor and severe symptoms in
Arabidopsis but isolate Baji-31 which is recombinant P6 protein of CaMv in
Arabidopsis transgenic lines showed very severe symptoms (14). 14) studied 41
transgenic lines of Arabidopsis out of which 17 lines (A7 & B6) showed minor to
severe vein chlorosis and stunting. In another study 15) were mutagenized the A7 line
seeds by gamma radiation and seedlings were screened for inhibited indications of
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65 restricted growth and chlorosis. Mutants having wild type phenotypes were labeled as
(b4-2 and b2-3) but had functional transgene. When these two mutant lines (b4-2 and
b2-3 were backcrossed to Arabidopsis col-o (wild type) to obtain plants with -
irradiated mutation without P6 transgene.
Auxin is an important and multifunctional hormone that can effect in plant growth
(16). Even though auxin-dependent growth is observable among plant tissues,
produced primarily in apical regions of the shoot and move in a polar manner to
different locations in plant (17). Auxin is reallocated to root tips from root apex
through cortical and epidermal tissues (18). Plant hormone auxin is transported into
plants through a dynamic process of polar auxin transport in cell to cell manner and
polarity is its main feature (19, 20). Although polar auxin transport has coordinative
role for plant development (21). BIG/TIR3 gene from Arabidopsis encode a huge
calossin-like protein which is responsible for polar auxin transport (22). Arabidopsis
mutant tir3 (transport inhibitor response 3), have a pleotropic phenotype, containing
fewer and shorter siliques, reduced inflorescence height, reduced petiole and root
length, and decreased apical dominance (24). TIBA which is known as 2, 3,5-
triiodobenzoic acid is an inhibitor of polar auxin transport (PAT) system (23). TIBA
blocked the process of embryo formation from embryogenic cells but cell division
remain unaffected (25). Also TIBA inhibition could induced abnormal development
of embryos subsequently result as plantlets without shoots and roots (26).
Systemic virus infection did some morphogenetic modifications in plants as well as
weakens the state of auxin hormone. Hence it‟s suggested by some reports that auxin
activity reduced in infected plants and also become cause of stunting growth in plants
(25).The molecular mechanisms involved in the alteration of auxins metabolism and
its transport is still not known (27). The previous studies described that signaling
pathway of auxin is involved in symptoms appearance during viral infection (28). It is
clearly known that auxin response factors are synchronized through gene silencing
mechanism (29) and P6 protein is the pathogenicity determined and suppressor of
RNA silencing in case of (30). So the basic aim of current study was to examine the
effects of cauliflower mosaic virus P6 protein on auxin signaling pathways and also
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66 its involvement in assembly of ARF (siRNA/miRNA) species and its subsequent
effects on plant growth.
Material and Methods
TIBA plate Experiment
The seeds of the 21), b4-2, b2-3 [f], CSE 1, CSE 2 (Geri 2004) and Col-O, Ler
Gl1 (wild type) were taken. ½ strength MS medium was prepared and autoclaved.
For each genotypes, different plates were prepared which comprised of MS media and
(0, 5, 10, 35, 50 and 70 um TIBA). Seeds of each genotype were kept on each
individual conc. of TIBA separately and incubate at 4ºC for 4 days and then shifted to
Growth room. After that data for the seed germination was noted after 15 days from
each plate.
Detection of miRNA from (A7, B6 and Ler gl1)
Arabidopsis seeds A7, B6 and wild type Ler gl1 were grown under a 9h photoperiod
and at 21 2C temperature was maintained (32). Tri Reagent (Sigma) was used to
extract total RNA from A7, B6 and wild type plants and separated on 15%
polyacrylamide gels electrophoresis 33) and ath-MIR167 (Wu, 2006) were prepared
by annealing primers sets (ath-MIR160) and (ath-MIR167). These were used at 250
nM as templates for in vitro transcription with α-
32P UTP by T7 RNA polymerase at
22°C.
Results:
Five different concentrations of TIBA (i.e., 0, 5, 10, 35, 50 and 70 um) and
Arabidopsis lines A7 and B6, TIR3 mutant seeds were grown on MS media, similarly
transgenic P6 (b4-2 and b2-3) with gamma radiation mutation, (CSE 1A and CSE 2A)
mutants without P6 gene and (Col-O and LerG1) control plants. Data was collected
after two weeks and it was found that (0, 5 and 10 um conc.) of TIBA had no
influence on the development of wild type Arabidopsis as well as control plants.
However, conc. of TIBA (50 and100 um) were lethal for all types of plants used.
However 35 um conc. suppressed the growth of wild plants while A7, B6 and tir3
mutants were unaffected. Though wild type Arabidopsis (b4-2, b2-3, CSE1A and
CSE2A) lines were killed by 35um conc. of TIBA as shown in figure 1.
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67
Aforementioned results revealed that there might be a strong relationship among P6
protein of CaMv to modulate auxin signal and also termed as ARF, which are
synchronized by gene silencing (34). To observe either P6 controls the manufacture of
auxin response factors with regulation of siRNA/miRNA species in Arabidopsis
microRNAs ath-MIR160 a, b,c and ath-MIR167 a, were tested in wild type (lerge1)
and A7 and B6 lines by Northern blot analysis (Figure 2). No significant effect was
found on ath-MIR167 and the level of ath-MIR160 was reduced in A7 and B6 lines.
Discussion
Various proteins of virus could disturb signaling pathways of cell. Certain studies on
the direct relations between auxin signaling and gibberellin levels for Tobacco mosaic
virus (TMV) and Rice dwarf virus (RDV) have been conducted (35). The rep protein
of TMV altered the localization and stability of interacting auxin/indole acetic acid
(Aux/IAA) proteins in Arabidopsis, it is also responsible for the alteration of auxin-
mediated gene regulation as well as promotes disease development (33. Similar kind
of replicase-Aux/IAA interaction in tomato plant was identified that could affect
disease development (36).
Arabidopsis plants of P6 transgenic (A7, B6) and TIR mutant (tir) showed resistance
to toxic effect of TIBA after treatment with TIBA. P6 gene expression provokes
symptoms like phenotype without virus infection in both host (N. tabacum) and non-
host (Arabidopsis) plants of CaMV (37). There are reports on the P6-transgenic (38)
and tir3 plants that are entirely unresponsive to ethylene and auxin (29) transgenes
although, gamma radiation mutation that suppress the gene product are quite sensitive
to TIBA.These test transgene plants were also found to be susceptible for ethylene
(suppress gene product formation), perhaps its role is to mediate characteristics of P6
gene of CaMV with host during infection (30). P6-transgenic and CaMV-infected
plants showed symptoms of chlorosis and stunting are mainly dependent on
interaction of P6 and different components involved in ethylene signaling, also it may
function to extend these interactions as ethylene suppress gene products formation
(31). The suppressor gene product may act as a complementary helpful regulator of
ethylene signaling, maybe towards downstream region (35 whereas, P6 gene also
stimulates chlorosis and suppression of plant defense mechanism.
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68
Perhaps this interaction will be abolish with the deletion of this suppressor gene and
leads to reduced susceptibility in plants against viral infection (36). Different proteins
of virus can also affect miRNA pathway and could effect on improvement after
expression in plants (37). Plants that are expressing viral proteins (P1/HC-Pro, p19,
p15, and p21) showed small amount of miRNA-directed ARF proteins can activate or
suppress transcription that depends on the nature of middle domain of protein (38). In
present study P6-expressing transgenic plants exhibited less buildup of MiR160 that
could targets ARF10, ARF16, and ARF17 (39). Whereas auxin response factors are
plant-specific family of DNA (40).
Aux/IAA proteins are nuclear proteins that survive for short span and can
heterodimerize with activating (41). Domains of C-terminal Aux/IAA proteins
mediate heterodimerization and preserved with the CTD of Auxin response factors
proteins (41). Increase level of auxin enhance the proteolysis of Aux/IAA proteins
and allow these proteins to homodimerize and also induced early gene expression of
ARF gene.
MiRNA-resistant ARF17 plants which showed increase level of mRNA expression
and improved buildup of auxin-inducible GH3-like mRNAs comprising
(YDK1/GH3.2, GH3.3, GH3.5, and DFL1/GH3.6), that code for auxin-conjugating
proteins (42). Basically theses alteration in expression correlates with growth related
imperfections, containing embryo and emerging leaf symmetry irregularities, leaf
shape defects and root growth defects etc. (45). These defects determine the
importance of miR160-directed ARF17 regulation and implicate ARF17 as a regulator
of GH3-like early auxin response genes (44).
Mutations in DCL1, AGO1, HYL1, and HEN1 genes of Arabidopsis plant, damage
the miRNA pathway and lead to developmental defects that overlap with those
exhibited by ARF17 plants. Particularly hypo morphic ago1 rosette leaves are serrated
and ago1, hyl1, and hen1 null mutant‟s show upward curled rosette leaves and a
dwarfed stature.
Indeed, miR160 accumulation is reduced and ARF17 mRNA accumulation is
increased in dcl1, ago1, hyl1, and hen1 mutants (46), there is a probability of reduced
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69 miR160-directed ARF17 regulation responsible for growth related anomalies of
mutants.
Figure 1: Effect of 3, 5-triiodobenzoic acid (TIBA) on Arabidopsis lines, P6
transgenic with gamma radiation mutation, mutants which lacks P6 gene and control
plants.
Figure 2: Expression of ath-MIR160 (A) and ath-MIR167 (B) miRNA probe on P6
transgenic and wild type plant (Lane 1) Lrg1 (Lane 2) A7 (Lane 3) B6
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70 Response of different (Capsicum annuum L) genotypes for
callus induction, plant regeneration and plant
transformation
6.1 Introduction
Chili pepper belongs to the genus Capsicum, which is from the family Solanaceae,
subfamily Solanoideae and tribe Solaneae [163, 274, 275]. The genus Capsicum
comprises of 5 cultivated and 26 wild species [274]. C. annuum is the most broadly
cultivated species and economically important crop of Pakistan. Chili occupies 19%
of the total area among vegetable cultivation and is grown on an area of 38.4 thousand
hectares with 90.4 thousand tones yield [276]. In Pakistan, Sindh province is the main
producer of chilies followed by Punjab and Baluchistan. Different types of sweet
pepper, pungent chili peppers are cultivated and in common used in the world [274]
as, or a source of dried powders of various colors, yet it suffers great losses due to
infection by various viruses. It has been reported that there are 45 different viruses
that infect chilies/peppers world-wide [276]. Genetic engineering holds great promise
for the effective control of plant viruses [277, 278]. Therefore, it was deemed
necessary to genetically engineer the pepper plant, since it has many useful traits.
Genetic transformation is now a routine procedure to insert genes into diverse plant
species, containing vegetable and fiber crops [279, 280].
Current progress in plant genetics and biotechnology is extremely dependent upon the
use of in-vitro tissue culture, hence the establishment of effective plant regeneration
system [281]. Among the various systems applied somatic embryogenesis (SE)
through callus induction is of special value [281]. It also offers opportunities for In
vitro production of plants through clonal propagation and genetic modification
through genetic transformation. As a result, increasing number of protocols describing
efficient in-vitro regeneration are being published [163, 164, 282, 283].
In chili pepper, several protocols are available for inducing in-vitro plant regeneration
[163, 164, 168, 284, 285]. However, some of these reports suggest a strong influence
of genotype, [286, 287] culture medium composition, explants source, and
environment on the regeneration process. Among them the genotype [287] and
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71 nutrient composition [288] are regarded to be the major sources of variation in in-
vitro culture [164] and had been studied.
Pepper belongs to the family Solanaceae, whose members are certainly responsive to
tissue culture and transformation practices, however pepper is considered to be an
tremendously problematic and resistant species for in-vitro regeneration and genetic
transformation [163].
The gene transfer in chili is difficult and there are several obstacles i) whole plant
regeneration through tissue culture ii) procurement of transformed tissues iii) plant
regeneration after transformations [163]. In last ten years scientists have made great
progress in chili transformation worldwide [163, 164, 168, 284, 285 ]. Transgenic
pepper expressing the coat protein gene of CMV [289] and plants that expressed
CMV satellite RNA [290] were obtained with low regeneration and transformation
efficiencies. However, the published protocols could not be repeated in other
laboratories. RNAi has been used to engineer resistance against ChLCD (section 4.3).
Transgenic peAC1-AC2dsRNA/pFGC tobacco plant showed considerable
resistance/tolerance against ChLCD (Table 4.2 and Figure 4.10). Due to the problem
of stable transformation, RNAi studies are difficult in C. annuum.
This study was therefore conducted to screen Pakistani chili commercial genotypes
for callusing response and to develop an efficient in-vitro clonal propagation protocol.
This protocol demonstrates the genotype independent response for morphogenic
callus formation and genotype dependent response for plant regeneration. Different
factors influencing genetic transformation in local chili pepper genotype were also
investigated.
6.2 Materials and Methods
6.2.1 Plant material, Seed Germination and Explant preparation
The main objectives of this study was to examine the effects of various plant growth
regulators, genotypes and explants on chili tissue culture as no reports of such type of
effects on chili regeneration was available from Pakistan so far. Commercial
genotypes of C. annuum (Loungi and Sanam) seeds were taken from Ayub
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72 Agricultural Research Institute, Faisalabad Pakistan and C. annuum genotypes
[Seedex pepper (SP) and Tatapuri (TP)] seeds from Sindh Horticulture Research
Institute, Mirpurkhas were obtained and used in this study. Chili seeds were surface
sterilized (section 2.14) and were sown in MSO medium (section 2.15). The
hypocotyls and cotyledons were excised from seedlings after 12 days of germination
and used as explants for callus induction. The hypocotyl explants were cut into 3.5-
4.5 mm long pieces and cotyledons were transversely cut into two parts. The explants
were cultured immediately in order to prevent the drying of cut edges of the explants.
6.2.2 Culture Medium and condition
The hypocotyl and cotyledon explants were placed on three different compositions of
chili callus induction media [(ChC1, ChC2 and ChC3) (Table 6.1)] at a temperature of
25 ± 2°C under 16/8 h photoperiod. For plant regeneration, calli obtained from each
combination were divided into three parts and cultured on three different compositions of
(ChSR1, ChSR2 and ChSR3) shoot regeneration media (Table 6.2). All the media were
solidified with 0.8% (w/v) tissue culture grade agar. The pH was adjusted to 5.8 prior
to autoclaving. Sterilized medium was poured into Petri plates and the plates were
sealed with parafilm. The elongated multiple shoots (3–4 cm long) were excised
individually and cultured on MSO chili root proliferation medium (section 2.10.1) for
fifty days and allow to developed roots. After the rooting agar was removed from
plantlets through washing and plantlets were shifted to pots containing soil:
Vermiculite (1:1) mixture. Plats were kept in shade for 15 days and watered regularly
and after that transferred to green house. Fifty hypocotyl explants and fifty cotyledon
explants (10 explants in 5 replicates) of each genotype were used in these
experiments. The mean ± SE values have been calculated from all the data of
experiments using Statistix 8.1 and were presented in the results.
6.2.3 Agrobacterium-mediated genetic transformation in chili pepper (C. annuum
L)
Hypocotyl and cotyledon explants of C. annuum SP (section 6.2.1) were obtained
(Section 6.2.2). A single clone of A. tumefaciens LBA 4404 35S GFP/pFGC
(Provided by Molecular virology and Gene silencing Lab, NIBGE) and A.
tumefaciens EHA105 peAC1-AC2dsRNA/pFGC (section 4.2.6) was inoculated in 20
ml LB liquid medium supplemented with 50 mg/l kanamycin, 100 mg/l streptomycin
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73 (section 2.10.1) and cultured on a rotary shaker at 28°C, 180 rpm, for 24 h. The
bacterial cells were then centrifuged and the pellet suspended in MS0 liquid medium
[MS0 (section 2.10.1) without agar] to O.D.600=0.38–0.42. Hypocotyl and cotyledon
explants were inoculated with the cultures of A. tumefaciens LBA4404 having 35S
GFP/pFGC construct and A. tumefaciens EHA105 peAC1-AC2dsRNA/pFGC for 8–
10 min, followed by co-culture on ChC2 medium (Section 6.2, Table 6.1) at different
temperatures (22 and 25ºC), photoperiod (16h light 8h dark, and complete darkness)
and co-cultivation time periods. Explants were separated from co-culture medium and
kept on ChC2 selection medium [ChC2 medium, glufosinate ammonium (Basta, 4
mg/l) and cefotaxime (50 mg/l)] and incubated (Section 2.14). Explants were
subcultures after every 2-3 weeks and placed on ChC2 selection medium. GFP
fluorescence was observed in putative transgenic calli using 100-W long wave UV
lamp (Blak-Ray Model B 100YP; UV Products). Total genomic DNA of callus of
chilies was extracted by CTAB method (section 2.2). The transgene in callus was
confirmed through PCR with specific primers of the transgene. Calli became
brownish and dead after 40 days. 200 hypocotyl and 150 cotyledon explants of both
genotypes in 10 different batches (with different experimental condition) were
evaluated in these experiments.
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Table 6.1
Chili callus induction medium (ChC)
Chili Callus
induction media
Basal media
Φ
Sucrose
(g/L)
BA
(mg/L)
IAA
(mg/L)
NAA
(mg/L)
Agar
(mg/L)
ChC1 MS salts +
vitamins
30 2.0 - 1.0 9.0
ChC2 MS salts +
vitamins
30 3.0 0.5 - 8.5
ChC3 MS salts +
vitamins
30 4.0 0.5 - 9.0
Φ MS salts and vitamins (Phyto Technology USA, Prod NO: M404)
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Table 6.2
Chili shoot regeneration medium (ChSR)
Chili Shoot
Regeneration media
Basal
medium Φ
Sucrose
(g/l)
BA
(mg/l)
NAA
(mg/l)
GA3
(mg/l)
Agar
(mg/l)
ChSR1 MS salts +
vitamins
30 5.0 0.05 - 9.0
ChSR2 MS salts +
vitamins
30 4.0 0.05 2 8.5
ChSR3 MS salts +
vitamins
30 3.0 0.05 - 9.0
Φ MS salts and vitamins (Phyto Technology USA, Prod No: M404)
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76
Table 6.3
Growth regulators and their stock preparation
Growth
Regulator
Stock
Conc.
Preparation* Storage
IAA 1,000
ppm
Dissolved 100 mg IAA in 10 ml
water then made the volume with
water to 100 ml
Below 0°C
NAA 1,000
ppm
Dissolved 100 mg NAA in 5 ml of IN
NaOH then made the volume with
water to 100 ml
4°C
BA 1,000
ppm
Dissolved 100mg BA in 5 ml of 1N
NaOH, then made the volume with
water to 100 ml
4°C
GA3 1,000
ppm
Dissolved 100mg GA3 in 5 ml of IN
NaOH then made the volume with
water to 100 ml
4°C
*Used double distilled deionized water
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77 6.3 Results
6.3.1 Callus Induction
The data for the callus induction was recorded after 30-32 days. Callus was induced
from both hypocotyl and cotyledon explants from all chili genotype. Factorial analysis
of variance (ANOVA) test was carried out to detect differences among the factors
tested. The data were analyzed and statistical analysis revealed that there is no
significant difference on type of explants and medium used in this study on frequency
of callus induction (Table 6.6). For all four genotypes, callus induction from
cotyledons was quicker than hypocotyls. Calli appeared from the cut edges of both
hypocotyls and cotyledon explants after about 3-4 weeks culture with variable
proliferation rates. Most of the calli obtained were compact with opaque or yellowish
color and some were soft, watery, morphogenic calli with translucent or light yellow
in color. Callus was formed in both explants tissues (hypocotyl and cotyledon)
segments of 2 spinach cultivars, but the percentage of callus formation were not
variable in different explants.
Loungi showed the lowest callus induction potential (52.6%), while SP showed
maximum callus induction growth. Among the media composition, callus induction
medium having IAA 1 mg/l + BA 3 mg/l was found to be most effective for callus
induction but the differences were statistically non-significant (Table 6.6). Callus
obtained from this medium were quite good in texture and friable in nature than other
medium, SP hypocotyl explants showed maximum callus induction on medium
containing IAA 1 mg/l + BA 3 mg/l while Sanam cotyledons showed maximum callus
induction on medium having IAA 1 mg/l and BA 2 mg/l. Rhizogenic calli were
obtained from hypocotyl explants of Loungi that were non-regenerable in ChC1
medium.
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78
Table 6.4
ANOVA showing the effect of explants, genotypes and Media interaction (%) for
chili callus induction
Source DF SS MS F P
Explant 1 7.008 7.0083 2.30 ns
Genotype 3 44.758 14.9194 4.90 s
Media 2 5.600 2.8000 0.92 ns
Explant *Genotype 3 98.692 32.8972 10.80 s
Explant *Media 2 11.667 5.8333 1.92 ns
Genotype*Media 6 25.867 4.3111 1.42 ns
Explant *Genotype*Media 6 91.133 15.1889 4.99 s
Error 96 292.400 3.0458
Total 119 577.125
Grand mean 5.8750 CV 29.71
Significant value (P >0.05), significant difference (s), non-significant difference (ns)
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79
Table 6.5
Effect of genotype on chili callus induction.
Genotype Mean
(%)
Homogeneous
Groups
SP 68.3 A
TP 59.6 Ab
Sanam 54.3 B
Loungi 52.6 B
Figure 6.1
Effect of genotype on chili callus induction
Callus Induction After 32 days
0
20
40
60
80
1
Genotype
Mean
Call
us I
nd
ucti
on
(%)
SP TP Sanam Loungi
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80 Table 6.6
Interaction of genotype and explants (hypocotyl and cotyledon) response on chilli
callus induction.
Figure 6.2
Interaction of genotype and explants (hypocotyl and cotyledon) response on chilli
callus induction.
Callus Induction After 32 days
0
20
40
60
80
100
1
Genotypes
Mean
Call
us
Ind
ucti
on
(%
)
Hypocotyl SP Hypocotyl TP Cotyledon Loungi Cotyledon SP
Cotyledon Sanam Hypocotyl Sanam Cotyledon TP Hypocotyl Loungi
Explants Genotype Mean
(%)
Homogeneous
Groups
Hypocotyl SP 76.6 A
Hypocotyl TP 73.3 Ab
Cotyledon Loungi 62.0 Bc
Cotyledon SP 60.0 C
Cotyledon Sanam 57.3 CD
Hypocotyl Sanam 51.3 CDE
Cotyledon TP 46.0 DE
Hypocotyl Loungi 43.3 E
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81 Table 6.7 Effect of combination of genotype, explants and callus induction medium
response on chili callus induction.
Explant Genotype Media Mean
(%)
Homogeneous
Groups
Hypocotyl SP ChC2 80.0 a
Hypocotyl SP ChC3 80.0 a
Hypocotyl TP ChC1 80.0 a
Hypocotyl TP ChC3 80.0 a
Cotyledon Sanam ChC2 76.0 ab
Cotyledon Loungi ChC3 70.0 abc
Hypocotyl SP ChC1 70.0 abc
Cotyledon Loungi ChC2 68.0 abcd
Hypocotyl Sanam ChC3 68.0 abcd
Cotyledon SP ChC1 66.0 abcde
Cotyledon SP ChC3 62.0 abcde
Hypocotyl TP ChC2 60.0 abcde
Cotyledon Sanam ChC1 60.0 abcde
Hypocotyl Sanam ChC1 56.0 bcdef
Cotyledon TP ChC1 56.0 bcdef
Cotyledon SP ChC2 52.0 cdefg
Hypocotyl Loungi ChC1 50.0 cdefgh
Cotyledon TP ChC3 48.0 defgh
Cotyledon Loungi ChC1 48.0 defgh
Hypocotyl Loungi ChC2 46.0 efgh
Cotyledon Sanam ChC3 36.0 fgh
Hypocotyl Loungi ChC3 34.0 gh
Cotyledon TP ChC2 34.0 gh
Hypocotyl Sanam ChC2 30.0 h
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82
Figure 6.3 Combination of genotype, explants and callus induction medium response
on chili callus induction.
Chili Callus Induction After 32 days
0
20
40
60
80
100
Mean(%)
Explant, Genotype and Media
Mean
Callu
s In
du
cti
on
(%
) Hypocotyl SP ChC2
Hypocotyl SP ChC3
Hypocotyl TP ChC1
Hypocotyl TP ChC3
Cotyledon Sanam ChC2
Cotyledon Loungi ChC3
Hypocotyl SP ChC1
Cotyledon Loungi ChC2
Hypocotyl Sanam ChC3
Cotyledon SP ChC1
Cotyledon SP ChC3
Hypocotyl TP ChC2
Cotyledon Sanam ChC1
Hypocotyl Sanam ChC1
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83 6.3.2 Chili Plant regeneration
Three different chili shoot regeneration media (ChSR1, ChSR2 and ChSR3) based on
MS salts supplemented with 1% (w/v) sucrose, 0.05 mg/l NAA, 3-5 mg/l BA alone or
in combination of GA3 2 mg/l (Table 5.4) were used to define suitable medium for
chili plant regeneration from calli. Significant difference at (P >0.05) in plant
regeneration system was observed in the genotypes. All the 4 genotypes showed
different regeneration response on three different combinations but none of them gave
any regeneration response on MS without any hormones. SP showed the highest
regeneration efficiency and TP did not response to plant regeneration. Interestingly
the genotype SP which showed a high regeneration potential also performed better in
callus induction. Shoot regeneration was started 30-40 days after culturing on shoot
regeneration medium (Figure 6.1). Regeneration frequency varied between 0 and 16
% in hypocotyl explants. Highest regeneration frequency (16 %) was obtained from
genotype SP. In cotyledon explants, maximum regeneration frequency (8.0 %) was
obtained in genotype SP.
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84
Table 6.8
Analysis of variance table for plant regeneration
Source DF SS MS F P
Genotype 3 16.7583 5.58611 29.79 s
Media 2 0.6167 0.30833 1.64 ns
Explant 1 0.0750 0.07500 0.40 ns
Genotype*media 6 0.9167 0.15278 0.81 ns
Genotype*Explant 3 1.2917 0.43056 2.30 ns
Media*Explant 2 0.1500 0.07500 0.40 ns
Genotype*Media*Explant 6 0.9833 0.16389 0.87 ns
Error 96 18.0000 0.18750
Total 119 38.7917
Grand mean 0.2917 CV 148.46
Significant value (P >0.05), significant difference (s), non-significant difference (ns)
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85 Table 6.9
Effect of genotype on chili plant regeneration.
Genotype Mean
(%)
Homogeneous Groups
SP 9.33 A
Loungi 1.33 B
Sanam 1.00 B
TP 0.00 B
Figure 6.4
Effect of genotype on chili plant regeneration.
Chilli Plant Regeneration
0
2
4
6
8
10
1
Genotypes
Mean
Pla
nt
Reg
en
era
tio
n %
SP Loungi Sanam TP
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86
Table 6.10
Effect of combination of genotype, explant and shoot regeneration medium on plant
regeneration from chili calli.
Genotype Explant Media Mean(%) Homogeneous
Groups
SP
Hypocotyl
ChSR1 10.0 b
ChSR2 8.0 bc
ChSR3 16.0 a
Cotyledon
ChSR1 8.0 bc
ChSR2 6.0 bcd
ChSR3 8.0 bc
Loungi
Hypocotyl ChSR1 2.0 de
ChSR2 0.0 e
ChSR3 0.0 e
Cotyledon ChSR1 2.0 de
ChSR2 0.0 e
ChSR3 4.0 cde
Sanam Hypocotyl ChSR1 0.0 e
ChSR2 0.0 e
ChSR3 2.0 de
Cotyledon ChSR1 2.0 de
ChSR2 2.0 de
ChSR3 0.0 e
TP Hypocotyl ChSR1 0.0 e
ChSR2 0.0 e
ChSR3 0.0 e
Cotyledon ChSR1 0.0 e
ChSR2 0.0 e
ChSR3 0.0 e
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87
Figure 6.5
Effect of combination of genotype, explant and shoot regeneration medium on plant
regeneration from chili calli.
Chilli Plant Regeneration
0
5
10
15
20
1
Genotype, Explant, Media
Mean
Pla
nt
Reg
en
era
tio
n
(%)
SP Hypocotyl ChSR1
SP Hypocotyl ChSR2
SP Hypocotyl ChSR3
SP Cotyledon ChSR1
SP Cotyledon ChSR2
SP Cotyledon ChSR3
Loungi Hypocotyl ChSR1
Loungi Hypocotyl ChSR2
Loungi Hypocotyl ChSR3
Loungi Cotyledon ChSR1
Loungi Cotyledon ChSR2
Loungi Cotyledon ChSR3
Sanam Hypocotyl ChSR1
Sanam Hypocotyl ChSR2
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88
Figure 6.6
Different step in chili tissue culture
A) Callus induction from cotyledon explants B) Callus induction from hypocotyl
explants C) Rhizogenic callus D) Shoot regeneration E) Multiple shoot induction F)
regenerated plant
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89
Figure 6.7
Plant transformation in chilies and transgene analysis.
A-C) Chili callus after inoculation with A. tumefaciens LBA4404 35S GFP/pFGC and
A. tumefaciens EHA105 peAC1-AC2dsRNA/pFGC. D) PCR confirmation of the
transgene using specific primers of the transgene in calli.
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90 6.3.4 Effect of different factors on Agrobacterium-mediated plant transformation
in chili pepper (C. annuum L)
One of our research goals was to develop a transformation system that allows
homogeneous transgene expression of local chili genotype with GFP/pFGC. The
ultimate goal was to transform local cultivar with peAC1-AC2dsRNA/pFGC35S
GFP/pFGC construct. The hypocotyl and cotyledon explants (SP genotypes) from 10-
15 days old seedlings were placed on ChC2 (Table 6.1) medium supplemented with
different concentration (0, 2, 3, 4, 5 and 6 mg/l) of glufosinate ammonium (Basta).
After 25 days the callus differentiation rates of the explants were investigated. The
explants tested induced callus on basta free medium. But only the cotyledon explants
could tolerate a basta concentration of 4 mg/l. When the level of basta was 5 mg/l or
higher, calli could not be induced for any explants. Hence, 5 mg/l Basta considered to
be the minimal lethal dose.
In the preliminary experiments of this study various factors that influence the
efficiency of T-DNA delivery in Chili plant was assessed. Factors includes two
explant types A. tumefaciens cells inoculation at 22 and 25ºC, as well as photoperiod
(16h light/ 8h dark and complete darkness) and time spans of co-cultivations.
Explants were inoculated and co-cultured with A. tumefaciens LBA4404 having 35S
GFP/pFGC construct and A. tumefaciens EHA105 peAC1-AC2dsRNA/pFGC for 8–
10 minutes. Transient GFP expression was observed with very low frequencies in
hypocotyl explants after 2 or 3 days of co-culture under complete darkness (data not
shown). Transient GFP gene expression was lost completely from the tissue 20 days
after transformation. Calli became brownish and dead after 40-45 days (Figure 6.7C).
PCR analysis showed that the transgene was present in the putative transgenic callus
(Figure 6.7).
5.4 Discussion
The concept of In vitro culture which exploits the ability of plant cells to regenerate
was proposed by [291] and demonstrated for the first time by [292]. Previous studies
have revealed the several aspects of inherent problems that are associated with in vitro
studies of chili i.e, non-availability of morphogenic calli, severe recalcitrance, less
defined shoot buds, genotypic requirement which can expose to tissue culture and
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91 plant improvement through genetic transformation system [163]. Chili has less ability
to regenerate while other Solanaceae crops such as tomato, tobacco, potato are
frequently used as model systems due to their ability to regenerate plants.
Regardless of the fact many findings had been conducted for the relative success of
regeneration system in different crops [164, 293, 294 ] on shoot morphogenesis in
chili but genetic engineering is still restricted by the low morphogenetic potential of
these species [168 , 295, 296]. Proper explants selection at specific stage of plant ,
alteration of different constituents in nutrient media and additives could help to lessen
recalcitrance [163].
Effort has been made to identify suitable explant in chili for morphogenic callus
induction and subsequent plant growth. Different types of explants comprises of
cotyledons, hypocotyls, leaves, shoot tips, and roots etc. have been employed for plant
regeneration in chili [164, 294, 297-299]. In this study hypocotyl and cotyledon
explants were used from 10-15 days old seedlings. No significant difference was
observed for type of explants for callus formation and plant regeneration. But in this
study by defining a suitable medium composition, morphogenic callus induction was
achieved from hypocotyl explants of SP chili genotype (Table 6.8, Figure 6.4 and
Table 6.11). The results of this study were supported by the finding of [167] and [283]
which also showed that callus induction and shoot initiation was higher in hypocotyls
and embryos than cotyledons.
Auxins and cytokinins are mandatory to induce cell division and growth in tissue
cultures system [163]. In this study four different chili genotypes were tested for
morphogenic callus induction on modified MS and B5 media containing different
concentration of BA (2-4 mg/L) in combination with IAA (0.5 mg/L) or BA 2.0mg/L
in combination with NAA (1.0 mg/L) (Table 6.4). Callus induction from hypocotyl
explant ranged from 70-80 % for SP; 30-60 % for Sanam; 60-80 % for TP and 34-70
% for Loungi (Table 6.8) on three medium. Callus induction from cotyledon explant
ranged from 52-66 % for SP; 36-76 % for Sanam; 34-56 % for TP and 48-70 % for
Loungi (Table 6.8) on three different media. It can be concluded from these results
that increasing BA concentration in the callus induction media generally have no
effect on chili callus induction from hypocotyl as show in Table 6.8. The ChC2
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92 (medium containing BA 3 mg/L and IAA 0.5 mg/L) is the best medium for callus
induction from both hypocotyl and cotyledon explants. these findings match with the
results of [300] who also favored the role of BA over Kin for induction of shoot
formation in chili. [297] found that IAA and BA produced best results for shoot bud
formation, when applied in combination. Of the four genotypes evaluated, only SP
developed watery, green fluffy and morphogenetic calli. [274, 301] also reported
similar observation with other genotypes of C. annuum viz., Americano and Dulce
Italiano.
Plant regeneration was achieved in 3 chili genotype but with very low plant
regeneration frequency. Three different mixtures of regeneration media were tested
(Table 6.2). A combination of high cytokinin to auxin ratio in the regeneration media was
found to be effective for chili plant regeneration [302]. Plant regeneration frequency varied
between 0-16 percent (Table 6.11). Maximum shoot regeneration was observed in SP (16.0) genotype
calli (Calli obtained from hypocotyl explants), While TP (Calli obtained from both hypocotyl and
cotyledon explants) did not showed any response to plant regeneration potential. ChSR3 (medium
containing BA 3mg/L and IAA 0.05 mg/L) the best medium for plant regeneration from
hypocotyl calli but there is no effect of different concentrations of BA (3-5mg/L) on
plant regeneration from cotyledon explants (Table 6.11). It was thus noted that the
hypocotyl explant gave maximum regeneration potential on low concentration of BA
(Table 6.2) but hypocotyl callus transformed to brownish-black and non-regenerable
upon increasing the concentration of BA up to 5 mg/l. Regenerated plantlets were
rooted in nutrients and hormone free MS medium. 3-4 weeks later, rooted plants were
shifted to soil for acclimatization.
Plant regeneration of pepper plants via callus is not common due to the problems during callus
induction and its development into plant [287, 299]. In case of other genotypes the shoot buds either do
not elongate or may produce distorted leaves [303, 304]. Same difficulties were experienced in our
present study and very low shoot elongation was obtained. Attempts to elongate these shoot buds, such
as culture in high BA and low IAA [299] and addition of GA3 or AgNO3 were unsuccessful.
In this study the more callus formation but low rate of regeneration was found higher
that might be attributed to comparatively higher doses of auxin which is used to
induced callus in medium. However, the exact level of hormones in callus initiation
medium need compromise between callus induction and regeneration frequency.
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93 Transfer of material from callus induction medium to plant regeneration medium
promoted the regeneration capability of the genotype otherwise prolonged culturing
on the callus induction medium made callus compact and non-regenerable. So it is
advisable to transfer the material soon after induction of callus to shoot regeneration
medium.
Dependence of genotype is critical factor that could impacts the organogenesis in chili
tissue cultures. As different cultivars of chili have strong genotype specificity for
regeneration and considered as critical factor for previously employed regeneration
protocols for specific cultivars.
Superficial regeneration of shoots from genetically manipulated cells is among the
two strategies used commonly. However, selection of responsive genotype and
explant source is first strategy while optimization of cultural and environmental
conditions for the enhance genetic potential is second one. Before deciding the
genotype for tissue culture, there should be comparison with other genotypes to
establish an efficient regeneration system. The logic behind this could be different
parts of genotypes could be more adaptive in contrast to former ones while not in the
case of others. In our study, the results have shown that SP responded best on
hypocotyl explants than cotyledon while TP did not in any combination of hormone
used, so TP is highly recalcitrant genotype. Thus, C. annuum genotype SP was found
to be the most suitable among the four genotypes studied for subsequent genetic
transformation studies. Its calli will be used as recipients of exogenous DNA in
genetic manipulation.
Classical plant breeding techniques have been widely used to rise chili yields with
improved varieties selection [305, 306] which are tolerant to abiotic stress [305-308]
Unluckily, some important factors i.e., resistance to herbicides and pathogens, and
absent from the genetic pools of chili genotypes [309-311]. The use of plant
transformation techniques to introduce resistance genes into plant genomes may have
a
significant effect on quality and yield of chili. Establishment of a successful
transformation system for chili plant for the regeneration of particular specie is critical
step for its transformation.
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94 A protocol for chili plant regeneration was developed (section 6.3 and 6.4). Among
the four genotypes, calli of genotype SP displayed morphogenetic potential and
capacity to regenerate complete plantlets. Thus, C. annuum genotype SP was found to
be the most suitable among the four genotypes studied for subsequent genetic
transformation studies (section 6.4). SP explants (both hypocotyl and cotyledon) were
used as recipients of exogenous DNA in genetic manipulation. The first most
important step to be considered is optimization of Agrobacterium mediated plant
interaction which includes the reliability of the bacterial strain that can developed a
localized necrosis process in wounded tissues [312]. The effects of explant and
different conditions for co-cultivation with A. tumefaciens, on the transformation
efficiency of SP genotypes were examined. Explants were inoculated and co-cultured
with two different Agrobacterium strain (A. tumefaciens LBA4404 having 35S
GFP/pFGC construct and A. tumefaciens EHA105 peAC1-AC2dsRNA/pFGC) for 8–
10 minutes. A. tumefaciens cell inoculate at different temperatures (22 and 25ºC),
photoperiod (16h light 8h dark and complete darkness) and co-cultivation time
periods (01, 02 and 03 minutes). Acetosyringone – used as indicator of A. tumefaciens
vir genes improve the transformation efficiency. Absolute acetosyringone -
dependency has been observed in C. annuum [295], where acetosyringone was one of
the essential components for transformation. However, after inoculating explants with
A. tumefaciens, transient expression of the green florescent protein (GFP) reporter
gene was very low. GFP activity was only exhibited by explants that were inoculated
with A. tumefaciens culture having acetosyringone. Reporter gene expression
generally was lost completely from the tissue 20 days after transformation and calli
become dead after 40-45 days.
The problems of poor survival rate of calli during Agrobacterium-mediated
transformation were due to hypersensitive response. In-vitro recalcitrance of plants
have been related to reactive oxygen species (ROS) production [313]. Higher levels
of free radical activity were found in resistant genotypes of potato and grape as well
as in non-embryogenic calli of rice crop [314, 315]. The use of an antioxidant silver
oxide in sugarcane transformation caused an 80 % cell death which was reduced in
comparison to controls, and the quality of the callus was not disturbed in any phase
of tissue culture [316]. Silver nitrate (2mg/l) in ChC2 selection medium, attempted to
reduce ROS response and it also did not affect the transformation efficiency but in the
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95 presence of silver nitrate calli remained green and watery up to 50-60 days. The
transgene in callus was confirmed through PCR with specific primers of the transgene
(Figure 6.7). The main objective of this work was to control ChLCD through genetic
engineering techniques. However, pepper genotypes are recalcitrant to genetic
transformation, control of diseases caused by ChLCD complex using this strategy
awaits future progress. This procedure could be applied to other cultivars of pepper to
induced genetic resistance aiming to produced resistance for pathogen and metabolic
engineering
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96 Chapter 7
General Discussion
Chili pepper is an important spice and vegetable economic crop in Pakistan. It is one
of the World‟s staple vegetables and classified among the Solanaceae. However,
losses are high due to susceptibility of the crop to plant viruses [158, 160, 194, 317].
Whitefly transmitted ssDNA viruses (family Geminiviridae, genus Begomovirus) are
the causal agents of severe diseases of crops in Indian subcontinent [191, 195]. The
modern agriculture management ways and human activity are the major cause of
begomoviruses emergence in different countries of world.
To fulfil the hunger need of people, newly introduced crops and changing of cropping
patterns, use of susceptible genotype, insecticides and introduction of exotic viruses
with their vectors have been concerned in the etiology of geminiviruses spread in
crops [4, 177, 195]. Although inherent characters of begomoviruses such as their
evolutionary aptitudes by recombination events, can give rise to novel species or
variants which can spread new disease epidemics [318]. In cassava crop there was a
mixed infection of two different species of bipartite begomoviruses [57].
Both components of bipartite begomoviruses demonstrates that the two molecules
(DNA A and B) have diverse molecular evolutionary histories based on phylogenetic
analysis [193]. ToLCNDV, has been regularly identified in numerous plant species
across India, Bangladesh and Pakistan and suggest that this bipartite begomoviruses
help other begomoviruses to expand host range. ToLCGV exists without DNA B in
certain weeds [319] and therefore suggested that ToLCGV was spread to tomato from
a weed host on its interaction with ToLCNDV (DNA B component). However due to
the mixing of viral components on weed hosts results in new disease complexes were
found with increased level of virulence to agricultural crops.
Chili leaf curl disease complex is the most damaging factor that effects the pepper
production in Pakistan [320], as this disease has been established during the 1960s in
Indian subcontinent [321]. Although this virus has become prevalent in the chili
cultivated areas of Asia including India and Pakistan from past decades [322], and till
today [161, 323].Increase incidence of this disease in chili found in Indian sub
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97 continents demanded the use of some suitable strategy to minimize losses and to
managed it through resistant cultivars.
This manuscript is part of this effort aiming to understand and manage the chili
begomoviruses complex in Pakistan. Characterization of viruses is the most important
step to initiate the suitable disease control strategy through the development of
resistant cultivars. Pepper leaf curl Lahore virus (PepLCLV) is a newly reported
begomoviruses along with betasatelite from the chili field near Lahore [51]. Identity
and genetic diversity of begomoviruses was performed through the establishment of
ChLCD associated with chili field in Faisalabad, Pakistan. During current study a
novel new variant of Pepper leaf curl Lahore virus was found which gives maximum
(99%) homology with PepLCLV (AM404179). As this distinct variant of virus
(PGL1) of PepLCLV was unable to replicate betasatelite and gives leaf curl symptom
only with the interaction of DNA B component of ToLCNDV as shown in figure 3.4.
Putative rep protein analysis revealed that protein sequence has a leader sequence at
its N-terminal (Figure 3.3) which may be remarkably significant to determine the
failure of the virus to replicate beta satellites.
Begomovirus has typical genome organization with four open reading frames (Rep,
TrAP, REn and C4) in the complementary sense while two (CP and pre CP) ORF in
the virion sense. Sequence analysis of PGL1 revealed that the iteron on PepLCLV
(GGGGAC) differ from the iteron found on ToLCNDV (GGTGTC) with two bases
only as shown in figure 3.4. Therefore, it is assumed that the first 2 -3 bases might
have some role in recognition of rep binding site. Most interesting finding of this
study is that pepper leaf curl Lahore virus (PepLCLV) is not capapble to replicate beta
satellites, however this hypothesis need confirmations through mutagenesis.
Information of the host range of particular virus is so important to understand the
epidemiology and could be useful for identification of virus and effective control
stretegy [324]. The experimental host range data imply that can infect N. tabaccum,
N. benthamiana and C. annuum. Mild symptoms on the tobacco plant was found after
agroinfiltration of cloned virus. When PepLCLV clone with beta satellites was
inoculated into tobacco plants similar kind of symptoms were observed. However
when agroinfiteration of the DNA B component of ToLCNDV was performed in N.
benthamiana, and N. tabacum plants it induced typical symptoms of ChLCD.
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98 This finding suggests that virus characterized here may be bipartite. This is first
attempt to experimentally test the infectivity for a bipartite begomoviruses producing
ChLCD. This indicates that isolate probable represents a new species of Begomovirus.
However for the first time this experiment was performed to confirm Koch‟s
postulates with the help of cloned of viral DNA that is associated with chili leaf curl
disease along with DNA B. The management of begomoviruses disease complex is
quite difficult [325] , expensive [326]. Use of chemicals and cultural practices is not
an efficient method to control the viruses. The loss of natural enemies due to
extensive use of insecticide could developed resistance against insecticides and
contribute to ineffective control and environmental problems. However the most
effective strategy for begomoviruses could be the use of integrated management with
resistant cultivars along with used of cultural practices and pesticides aim to reduce
the viral titer. So the long term control strategy could be the breeding of
begomoviruses resistant plants. The breeding for developing virus resistance in
commercial varieties of chili is problematic and so far managed to some extent by
cultural practices [327]. On the other hand genetic engineering/biotechnology
provides the understanding of the broad-spectrum and stable resistance which may be
pathogen derived resistance (PDR) or non-pathogen based. RNA (dsRNA) mediated
interference, through a complex process protects plants from invasive viruses.
Recently, RNA interference has been reported as a natural defense system against
virus infection [328]. RNA interference has emerged as a robust and breakthrough
technology for engineering virus resistance in plants. RNA interference is a
mechanism that involved mRNA degradation of specific sequence, which is
conserved across the kingdom [329]. [212] produced transgenic plants based on RNA
interference against TYLCSV. However, delay of symptoms were found in these
plant mainly at low level of inoculum.
Begomoviruses encode limited no. of genes and mostly depend on host factors for
successful infection. Replication associated protein encoded by members of family
Geminiviridae is the only essential protein required by virus for successful infection
[40]. It is a pleiotropic gene with several function including sequence specific
binding, cleavage, oligomerization, helicase activity, transcriptional regulation,
replication activity, ATP dependent topoisomerase activity and several interactions
with host proteins [330]. AC2 is suppressor of RNA silencing [2]. Data base search
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99 results indicate that the region selected is not only conserved (89%-100%) in different
ChLCD causing begomoviruses i.e PepLCLV, ChLCV, CLCV, and ToLCNDV, but
also in heterologous viruses including malvestrum, tomato, papaya, okra, cassava,
hollyhock infecting begomoviruses (85-95%). This region also produced maximum
siRNA in naturally begomoviruses infected plant. Pathogen-derived resistance” has
been focused on the overlapping region of AC1 and AC2 using hairpin construct.
Thus, these sequences provide a better target for providing better protection against
the disease. So, in this research work, we have silenced chili Rep-AC2 expression by
making RNAi constructs targeting Rep-TrAP (AC1-AC2) in transient assays. We
amplified 575 bp fragment containing 400 bp region of Rep at C-terminal and 175 bp
of TrAP at N-terminal. The amplified fragment was cloned in a dsRNA binary vector
(pFGC5941) in sense and antisense orientation flanked by intron cloning gene
constructs in such a way when introduced within plant cell produces inverted repeat
dsRNA which can more efficiently block the gene of homologous sequence [331].
Then this construct AC1-AC2 dsRNA/pFGC was analyzed by co-infiltration with
Ch.Rep/PVX. RNAi construct blocks rep expression and the plants had developed
only PVX symptoms, we have seen the Rep of ChLCV-M is different from other
geminiviruses when expressed through PVX because in this particular case Rep
produces leaf curling in N. benthamaiana plants.
RNAi gene constructs and PepLCLV were also transiently co-expressed in N.
benthamiana and C. annuum and virus levels was checked in inoculated and systemic
leaves. The Southern analysis of these inoculated plants have shown the replication of
the virus in the inoculated leaves but the titer of the virus in the systemic leaves has
been found to be reduced (Figure 4.5). The detection of the virus in inoculated leaves
confirmed reliable infiltration. The decrease in the virus titer in systemic leaves shows
that silencing signal generated in the inoculated leaves. The reason for this
improvement may be the target sequence difference, different assay approach,
conditions and the fact that large amount of siRNA could be generated in vivo through
RNAi construct. The construct AC1-AC2 dsRNA/pFGC was transformed in tobacco
and evaluation of transgenic plants showed that targeting of these conserved regions
through RNAi significantly reduced the level of viral DNA in transgenic plant when
these transgenic plants was inoculated with ChLCD infected viruliferous whitefly.
The virus replication was reduced to undetectable level in some cases when checked
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100 by Southern blot hybridization but could be detected by PCR. Thus, silencing of these
sequences could effectively control ChLCD component through RNAi. The two
transgenic line TA14 line and TA15.1 was ranked as highly resistant/tolerant showing
93.3% and 80.0% resistance/tolerance against ChLCD (Table 4.2). Both lines showed
resistance against homolougus (PepLCLV) and heterologous (ToLCNDV) virus.
AC1-AC2 575 bp fragment (containing 400 bp region of Rep at C-terminal and 175
bp of TrAP at N-terminal used peAC1-AC2dsRNA/pFGC hairpin construct) of
PepLCLV showed 71.8% resemblance to same ToLCNDV AC1-AC2 region. These
results revealed that peAC1-AC2dsRNA/pFGC transgenic plants were capable of
prevention of replication and movement whereas, 3 out of 9 transgenic lines were
found resistant. The basic aim of current investigation was to explore the ChLCD
complex resistance by silencing the Rep-Trap gene of PepLCLV. Hence, it is
suggested that AC1-AC2 silencing is a useful strategy for the development of broad-
spectrum resistance to cope with various other disease of begomoviruses.
Results presented in this project show that the two components essentially required
for the disease can be silenced successfully through RNAi. Results reported in this
thesis have added several novel concepts in generating durable resistance against
begomoviruses that cause important diseases on several crops in Pakistan and other
parts of the world. The spread of silencing is an important area that requires further
investigation and can be addressed by determining the origin of siRNA from resistant
plants challenged with the virus. siRNA originating from non-target viral sequences
would suggest spread of gene silencing.
Viruses are the natural artist and have been determinant in helping us to unravel
mechanisms used not only by the viruses themselves, but also by cell systems [144,
332, 333]. P6 protein expression in cauliflower mosaic virus in Arabidopsis induced
the dwarfness in transgenic lines of plants [236]; [252]. However, Arabidopsis plants
with mutated (tir3) are also exhibit dwarfness [260]. P6 transgenic (A7, B6) and tir3
Arabidopsis plants were found resistant to Auxin, ethylene and TIBA treatment. This
study revealed that P6 interacts with a pathway overlay with TIR pathway. Symptoms
appearance in Arabidopsis with P6 protein of CaMV is due to the disruption of Auxin
response factors (ARF10, ARF16, and ARF17). Hence Arabidopsis plants exhibited
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101 less accumulation of miR160 which is responsible for the regulation of ARF10,
ARF16 and ARF17 [139].
Classical plant breeding techniques have been widely used to rise chili yields with
improved varieties selection [305, 306] which are tolerant to abiotic stress. Unluckily,
some important factors i.e., resistance to herbicides and pathogens, and absent from
the genetic pools of chili genotypes [309-311]. Use of genetic engineering may be
helpful to introduced resistance genes into plants could be a better remedy for these
biotic stress and will impart beneficial impacts on chili yield. Introduction of genes to
Pepper plant is difficult and has resisted the efforts from many years [163, 295]. In
this à Protocol was developed for chili plant regeneration (section 6.3 and 6.4). In-
vitro response of four genotypes; Tata puri, SP, Sanam and Lungi was studied to find
out a suitable genotype for genetic transformation experiments section 6.3 and 6.4).
Among the four chili genotypes. C. annuum genotype SP showed reproducible plant
regeneration ability. The effects of explant and different conditions for co-cultivation
with A. tumefaciens, on the transformation efficiency of SP genotypes were also
examined. The transgene in callus was confirmed through PCR with specific primers
of the transgene (Figure 6.7) but the plant was unable to regenerate after
transformation. The main aim of this thesis is to control ChLCD through genetic
engineering techniques. However, pepper genotypes are recalcitrant to genetic
transformation, control of diseases caused by ChLCD complex using this strategy
awaits future progress.
This study represents the first report that RNAi could be used for inhibition of pepper
leaf curl virus thus also provides evidence that RNAi has the potential to be developed
into a novel antiviral approach provided that efficient plant transformation system in a
local variety of chilies through Agrobacterium mediated plant transformation for
RNAi constructs into local cultivars (chili) through Agrobacterium mediated plant
transformation system. RNAi gene constructs are working; these can inhibit the
replication of ChLCD complex in transgenic chili plants. Research work from this
thesis could provide important information for the development of virus resistant chili
crop in Pakistan.
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