Establishment of Cotton leaf curl virus

infectivity system

Umair Bashir

2015

Department of Biotechnology
Pakistan Institute of Engineering and Applied Sciences
Nilore, Islamabad, Pakistan

This page intentionally left blank

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Registration Number:
Thesis Title:

Umair Bashir
Department:
NIBGE
10-6-1-019-2013
Date of Registration: 16-09-2013
Establishment of cotton leaf curl virus infectivity system

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b. Do Recommend that the candidate be certified to the faculty for the degree of
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3. Head of Department (Name): Dr. Shahid Mansoor (SI)
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Thesis Submission Approval

This is to certify that the work contained in this thesis entitled Establishment of
cotton leaf curl virus infectivity system, was carried out by Umair Bashir, and in
my opinion, it is fully adequate, in scope and quality, for the degree of M.Phil.
Furthermore, it is hereby approved for submission for review and thesis defense.

Supervisor:_________________
Name: Dr. M. Saeed
Date: October 2, 2015
Place: NIBGE, Faisalabad.

Head, Department of Biotechnology: _________________

Name: Shahid Mansoor
(SI)
Date: October 2, 2015
Place: NIBGE, Faisalabad

Establishment of Cotton leaf curl virus

infectivity system

Umair Bashir

Submitted in partial fulfillment of the requirements

for the degree of M.Phil
2015

Department of Biotechnology
Pakistan Institute of Engineering and Applied Sciences
Nilore, Islamabad, Pakistan

Dedications

I dedicate this thesis to my beloved

Parents and to Dr. Ishtiaq Hassan

Declaration of Originality

I hereby declare that the work contained in this thesis and the intellectual content of
this thesis are the product of my own work. This thesis has not been previously
published in any form nor does it contain any verbatim of the published resources
which could be treated as infringement of the international copyright law. I also
declare that I do understand the terms copyright and plagiarism, and that in case of
any copyright violation or plagiarism found in this work, I will be held fully
responsible of the consequences of any such violation.

Umair
Bashir
October 2, 2015
NIBGE, Faisalabad.

Copyrights Statement

The entire contents of this thesis entitled Establisment of cotton leaf curl virus
infectivity system by Umair Bashir are an intellectual property of Pakistan Institute
of Engineering & Applied Sciences (PIEAS). No portion of the thesis should be
reproduced without obtaining explicit permission from PIEAS.

Acknowledgements

In the name of Almighty ALLAH, the most merciful, kind and beneficent. All
praises to Allah (S.W.T.) for all the bounties He bestowed on me to enable me to
successfully finish this thesis. In moments of distress, He guided me, showed me what
to do, removed all obstacles from and lighted my path, inspired me and eased the
tedious task of writing.
I am tremendously grateful to my Supervisor Dr.M. Saeed (Pricipal Scientist) for his
kind supervison, cooperative attitude, sharp insights, guidance of this research and
great efforts in explaining things in a simple and clear way that inspired and enriched
my growth as a student, researcher and as a scientist.
I would like to express my deepest appreciation to Dr. Shahid Mansoor (S.I.),
Director NIBGE, for providing excellent research facilities and friendly environment.
I provide my esteem gratitude to Dr. Imran Amin (Pricipal Scientist), Dr. Fazli Rabbi
Awan (Pricipal Scientist), Dr. Munir A. Anwar (Principal Scientist), Dr. Amir (Senior
Scientist), Dr. Muhammad Afzal (Pricipal Scientist) for guiding me and helping me
whenever I needed their help.
I heartily acknowledge all my fellows at Molecular virology Laboratory specially Mr.
Dr. Ishtiaq Hassan, Mr. Rahim Ullah, Mr. Muhammad Zubair, , Mr. Hasssan Jameel,
Mr. Hamza and Mr. Bilal Haider for their help, support, encouragement, and unique
collegium to endure the difficulties in my MPhil research work.
I want to dedicate this thesis and my MPhil research to Dr. Ishtiaq Hassan. Without
his supervision I would not have been able to complete these tasks. He has been really
kind and helpful through out my research. I wish him very best of luck for his career
as a professor. I know he will do a great job there as he got every thing that needs to
be a good teacher.
My Special appreciation goes to my respected seniors, Mr. Rahim Ullah, Mr.
Muhammad Zubair, Dr. Akhtar Rasool, Dr. Ikram Anwar, Mr. Tawaf Ali Shah, Mr.
Muhammad Jameel Mohmand, Mr. Zafar Ali Khan, Mr. Fawad Khan, Mr. Tahir Khan,
Mr. Tahir Naqash, Mr. Abdul Tawab, Mr. Niaz Khan, Mr. Kamal Khan, Mr, Jamrood
Khan, Mr. Waqas Ashraf, Mr. Imran Sohail, Mr. Mohsin Ali for their tremendous
assistance and priceless ideas and suggestions when I needed them the most.
I am blessed to have a great group of class fellows. Each and every one having his/her
own special attributes. I am grateful to all my class fellows specially Noor Akbar, ZiaUr-Rehman, Saif-ur-Rehman, Muhammad Sher, Waqar Afridi, Omar Khan, Bilal
Haider, Ismail Chugtai (Senior Scientist), Hassaan Khan, Ali Tahir for all the
emotional support, entertainment, and care which really motivated me during hard
times.
9

Many thanks to my friends at hostel specially Fazal Sattar, Saeed Ahmad, Hazrat
Ismail, Arif Ullah, Imtiaz Khan, Saleem Ur Rahman, Haq Nawaz Khan, Fareed Ullah,
Ayaz Khan, Wajid, Malik Ihsan, Momin Khan, Omar, Ali for for providing a
Last but not the least, my deepest gratitude goes to my beloved parents for their
endless love, prayers and encouragement at every stage of life to achieve my goals
and convert my dreams into reality and without whom I would never have been able
to achieve so much.
Umair Bashir

10

Table of Contents

Dedications....................................................................................................................ii
Declaration of Originality.............................................................................................iii
Copyrights Statement....................................................................................................iv
Acknowledgements........................................................................................................v
Table of Contents.........................................................................................................vii
Table of Figures..............................................................................................................x
List of Tables.................................................................................................................xi
List of Abbreviations and Symbols..............................................................................xii
Abstract.......................................................................................................................xiii
Chapter 1.

Introduction..............................................................................................1

1.1

Cotton..............................................................................................................1

1.2

Diseases of Cotton...........................................................................................1

1.3

Virus................................................................................................................2

1.4

Geminiviruses..................................................................................................3

1.5

Classification of geminiviruses.......................................................................4

1.5.1

Mastrevirus...............................................................................................4

1.5.2

Curtovirus.................................................................................................5

1.5.3

Topocuvirus..............................................................................................5

1.5.4

Becurtovirus.............................................................................................5

1.5.5

Turncurtovirus..........................................................................................6

1.5.6

Eragrovirus...............................................................................................6

1.5.7

Begomovirus............................................................................................7

1.6

Satellites..........................................................................................................9

1.7

Satellites associated with geminiviruses.........................................................9

11

1.7.1

Betasatellites............................................................................................9

1.7.2

Alphasatellites........................................................................................10

1.8

Proteins encoded by Geminiviruses and their functions................................11

1.8.1

Replication associated protein (Rep)......................................................11

1.8.2

Transcriptional activator protein/C2 protein (TrAP)..............................11

1.8.3

Replication enhancer protein (REn).......................................................12

1.8.4

C4 protein...............................................................................................12

1.8.5

Coat protein (CP)...................................................................................12

1.8.6

V2 protein...............................................................................................13

1.8.7

Nuclear shuttle protein (NSP)................................................................13

1.8.8

Movement protein (MP).........................................................................13

1.9

Movement of geminiviruses within host.......................................................13

1.10

Agroinfection.............................................................................................14

1.10.1

Agroinoculation and agroinfiltration......................................................15

1.10.2

Biolistic inoculation...............................................................................17

1.11

Objective of the study................................................................................18

Chapter 2.

Materials and Methods...........................................................................19

2.1

Solutions........................................................................................................19

2.2

Preparation of electro competent Agrobacterium tumefaciens cells.............19

2.3

Transformation of electro competent Agrobacterium tumefaciens cells.......19

2.4

Preparation of inoculum................................................................................20

2.5

Plant materials...............................................................................................20

2.6

Agroinoculation.............................................................................................20

2.7

Preparation of glycerol stocks.......................................................................21

2.8

DNA extraction..............................................................................................21

2.9

Quantification of DNA..................................................................................21

2.10

Polymerase chain reaction (PCR).................................................................22

12

2.11

Gel electrophoresis....................................................................................22

2.12

Restriction Analysis...................................................................................22

Chapter 3.
3.1

Results....................................................................................................24

Infectivity of Cotton leaf curl Kokhran virus with Cotton leaf curl Multan

betasatellite in G. hirsutum Variety Coker...............................................................24

3.2

Infectivity of Cotton leaf curl Kokhran virus with Cotton leaf curl Multan

betasatellite and Tomato leaf curl New Delhi Virus in G. hirsutum Variety Coker..27
3.3

Confirmation of Cotton leaf curl Kokhran Virus through restriction

endonuclease digestion.............................................................................................31
3.4

Infectivity of Cotton leaf curl Kokhran virus with Cotton leaf curl Multan

betasatellite in G. hirsutum Variety NIBGE 3701 and G. hirsutum Variety NIAB

Karishma..................................................................................................................33
3.5

Graft inoculation of G. hirsutum Variety NIBGE 3701................................36

Chapter 4.

Discussion..............................................................................................38

Chapter 5.

References..............................................................................................41

13

Table of Figures

Figure 1.1: Structure of Geminivirus. Geometrical representation of icosahedral

structure [37]..................................................................................................................3
Figure 1.2 The genome organization of Tomato leaf curl New Delhi virus, a bipartite
begomovirus...................................................................................................................4
Figure 1.3 Genome organization of Mastrevirus, curtovirus, topocovirus, eragrovirus,
turncurtovirus and becurtovirus.....................................................................................6
Figure 1.4 The genome organisation of a typical monopartite begomovirus alongwith
alphasatellite and betasatellite........................................................................................8
Figure. 1.5 Agroinfection: Transfer of viral DNA into T-DNA either for inoculation or
development of transgenic plants [200].......................................................................15
Figure 3.1 Symptoms induced by CLCuKoV-CLCuMB in G. hirsutum variety coker
......................................................................................................................................26
Figure 3.2 The PCR amplification of CLCuKoV in the symptomatic Coker plants....27
Figure 3.3 Symptoms induced by the CLCuKoV, ToLCNDV and CLCuMB in Coker.
......................................................................................................................................30
Figure 3.4 The comparison of restriction maps of CLCuKoV and CLCuKoV-Bu using
pDRAW32....................................................................................................................31
Figure 3.5 Digestion of PCR product of CLCuKoV and CLCuKoV-Bu.....................32
Figure 3.6 The agroinoculation of cotton varieties NIBGE 3701 and NIAB Karishma
with CLCuKoV and CLCuMB....................................................................................35
Figure 3.7 Grafting of NIBGE 3701 and Coker...........................................................37

14

List of Tables

Table 3.1 Infectivity of CLCuKoV with CLCuMB in G. hirsutum variety Coker......25

Table 3.2 Infectivity of CLCuKoV, ToLCNDV with CLCuMB in G. hirsutum variety
Coker............................................................................................................................29
Table 3.3 Infectivity of CLCuKoV with CLCuMB in G. hirsutum variety NIBGE
3701 and G. hirsutum Variety NIAB Karishma...........................................................34
Table 3.4 Graft inoculation of G. hirsutum variety Coker and G. hirsutum variety
NIBGE 3701.................................................................................................................36

15

List of Abbreviations and Symbols

CLCuD Cotton leaf curl disease

CTAB

cetyl trimethyl ammonium bromide

DNA

deoxyribonucleic acid

dNTP

deoxyribonucleotide triphosphate

dsDNA

double-stranded DNA

GFP

green fluorescence protein

HR

hypersensitive response

ICTV

international committee on taxonomy of viruses

IR

intergenic region

kDa

kilo Dalton

miRNA microRNA
MP

movement protein

NLS

nuclear localization signals

NSP

nuclear shuttle protein

NW

New World

OD

optical density

RCA

rolling circle amplification

SCR

satellite conserved region

Abstract

Cotton leaf curl disease has severely affected the cotton production in South Asia. The
genus Begomovirus comprises of viruses that infect economically important crops
16

throughout the world. The genome of begomoviruses consist of monopartite or

bipartite DNA components and are transmitted by insect vector whitefly Bemisia
tabaci. The cotton leaf curl disease appeared in 1980s in Multan and spread
epidemically in Pakistan and was caused by monopartite/satellite complex of
begomoviruses. Through the efforts of plant breeders resistant cotton varieties were
introduced and the disease was controlled by late 1990s. Soon in 2001, a resistance
breaking virus appeared in Burewala District of Punjab which was named as Cotton
leaf curl Burewala virus (CLCuBuV).This new virus is a recombinant of Cotton leaf
curl Multan virus (CLCuMV) and Cotton leaf curl Kokhran virus (CLCuKoV).
Recently CLCuBuV has been renamed as Cotton leaf curl Kokhran virus strain
Burewala (CLCuKoV-Bu). Tomato leaf curl New Delhi virus (ToLCNDV) has also
been found to infect cotton plants. Agroinoculation deliver the viral DNA (either
dimer or partial dimer) cloned in binary vector using Agrobacterium tumefaciens into
host cells. In the study decribed over here, different varities of cotton were
agroinoculated with CLCuKoV, CLCuMB and ToLCNDV.
The infectious clones of CLCuKoV, ToLCNDV (DNA A and B) and CLCuMuB were
already available in the lab and were used for Agrobacterium-mediated inoculation. A
single colony was picked from a fresh streaked plate and grown for 48 hours at 28 oC.
For each culture the inoculum were prepared having bacterial population of optical
density (OD) = 1.0 and OD = 4.0. Equal amount of inoculum were mixed for each
combination and then inoculated to various cotton varieties. In first batch of
experiment a single plant got infected, but in the second and third batches, three plants
in each batch showed typical cotton leaf curl disease symptoms. Although, the
inoculation efficiency was very low as we were able to infect only seven cotton plants
but was reproducible. This is a significant achievement as for the first time in Pakistan
cotton plants were successfully infected through agro-inoculation. In all the infected
plants CLCuKoV was confirmed through restriction fragment length polymorphism.
As neither of the plants inoculated with low inoculum (having OD = 1) were showing
symptoms, therefore it is suggested that OD = 4.0 is the optimum inoculum
concentration for infecting cotton plants through agro-inoculation.

17

Chapter 1. Int
rod
ucti
on

Chapter 2.

Cott
on

Cotton is one of the most important economic crop used for its fiber and oil seed.
Each year around 25 million metric tons of cotton is produced (International Cotton
Advisory Committee, ICAC) [1]. Cotton belongs to genus Gossypium and family
Malvaceae. Approximately 50 species includes in genus Gossypium out of which four
(G. barbadense, G. hirsutum, G. arboreum and G. herbaceum) are used for cotton
fiber production [1]. The genus is divided into eight diploid genomes (AG and K)
and one tetraploid genome [2]. G. hirsutum and G. barbadense are allotetraploids
(AADD) that results from the natural hybridization process [3]. The full length
sequencing of the A genome of G. arboreum and the D genomes of G. raimondii and
G. hirsutum have been completed [4-6]

Chapter 3.

Dise
ases
of
Cott
on

Among the different diseases of cotton, cotton leaf curl disease (CLCuD) is the most
serious one. Farquharson reported the first evidence of cotton leaf curl disease in 1912
in Nigeria in Gossypium peruvianum and Gossypium vitifolium with characteristic
symptoms of leaf curling and vein thickening [7]. The issue came to light during the
epidemic break of this disease in Sudan during 1927-28 [8]. It was thought at that
time whitefly was linked to the leaf curl symptom development [9-11].
18

In 1967 cotton leaf curl disease was observed in Pakistan near Multan district [12]. In
1988 sever infection was observed in Multan district near Moza Khokran on variety
S-12. The native cotton species Desi Cotton (G. arboreum) is resistant to leaf curl
disease but has low quality lint, so farmers have to turn towards American
Cotton (G. hirsutum) which is susceptible to CLCuD. Disease incidence has been
much lower in Sindh than in Punjab [13]. By using conventional breeding resistant
varieties like NIBGE 3701 and NIAB Karishma were developed that reduces the
losses during late 1990s [14]. In 2001 the disease symptoms were observed in
resistant varieties at Burewala, district Vehari [15, 16]. Through molecular analyses
and grafting techniques the resistance breaking strain of cotton leaf curl virus was
confirmed [16]. It was characterized as a specie Cotton leaf curl Burewala virus
which has been recently classified as strain of CLCuKoV (CLCuKoV-Bu), and was
found to be associated with Cotton leaf curl Multan betasatellite (CLCuMB)[17, 18].
Since 2001 the diversity of viruses causing cotton leaf curl disease is more in Sindh
[19, 20]. Recent data of Next Generation Sequencing (NGS) showed that bipartite
virus like Tomato leaf curl New Delhi virus has been found in cotton with other
viruses (unpublished data).

Chapter 4.

Viru
s

Virus is parasite that is functionally active only in host. They totally depend on their
host and use their biochemical machinery. The important processes of replication,
transcription and translation of viruses are carried by host machinery. Viruses lives in
the cells having no metabolic activity of their own. Virus particle have nucleic acid
(either ribonucleic acid or deoxyribonucleic acid) that is enclosed in protective
coating [21]. From plants to archaea, viruses infect every form of life [22]. Viruses are
generally classified on the basis of genome, range of hosts, vectors and viral proteins
[23]. Baltimore (1971) proposed classification system of viruses based on genomic
nucleic acid that results in seven groups of viruses. These are as follows; singlestranded (ss) DNA, double-stranded (ds) DNA, double-stranded RNA, positive sense
single-stranded RNA, negative sense single-stranded RNA and reverse transcribing
viruses (either ssRNA or dsDNA genomes). The viruses are classified into six orders,
87 families, 19 subfamilies, 349 genera and approx. 2284 species [24]. Viruses are
19

found in every ecosystem and environment that allow the horizontal gene transfer
[25]. Viruses causes various diseases in plants, humans and animals. Viruses spread in
animals and humans through blood sucking by insects, fecal- oral route and sexual
contact. In plants majority of viruses need vector to move from one plant to another.
Adolf Mayer proved in 1886 that plant pathogen can be transferred from symptomatic
plant to healthy plant [26]. Tobacco mosaic virus (TMV) was the first to be
discovered by Martinus Beijerinck [27]. Plant viruses having DNA genomes are
classified into two group circular dsDNA and circular single- stranded DNA ssDNA.
Caulimoviruses have circular double-stranded dsDNA genome while nanoviruses and
geminiviruses have circular ssDNA genome [28].

Chapter 5.

Ge
mini
viru
ses

The most economically important plant viruses belongs to the family Geminiviridae
infecting either monocotyledons or dicotyledonous plants [29, 30]. These are
transmitted by arthropod vectors [29, 31-33]. In a poem written by Japanese Empress
Koken in 752 A.D, that possibly for the first time described viral symptoms. The poet
admires the beautiful yellowish coloration of grass (taken to mean Eupatorium
plants) which has since been attributed to a geminivirus [34]
Geminiviruses are circular ssDNA viruses with one or two components. Their size is
approximately 2.6 to 3.0 kb. Their DNA is encapsulated into a twinned icosahedral
protein coat. These viruses replicate through ds intermediates in the host plant using
their replication machinery and also through rolling circle replication [35]. These
viruses is about 22 nm in diameter and 38 nm long as shown in figure 1.1 [36].

Figure 1.1: Structure of Geminivirus. Geometrical representation of icosahedral

structure [37]

20

Because of small genome size and DNA genomes these can easily manipulated by
molecular approaches [30]. These viruses are found in (sub-) tropical and temperate
areas of the world [38].

Figure 1.2 The genome organization of Tomato leaf curl New Delhi virus, a
bipartite begomovirus. Position and orienation is represented by arrows. The
genes in clockwise direction are virion-sense while the ones in anti-clockwise are
complementary-sense.
21

Chapter 6.

Clas
sific
atio
n of
gem
inivi
ruse
s

The family Geminiviridae is classified into seven genera based on genome

organization, insect vector and host range. These are as follows; Begomovirus,
Becurtovirus, Curtovirus, Turncurtovirus, Topocuvirus, Mastrevirus and Eragrovirus
[39]. Three genera of viruses (Mastrevirus, Begomovirus, Curtovirus) have been of
greater interest to the scientists because of their economic importance.

Chapter 7.

Mas
trev
irus

These are transmitted by leafhopper and have monopartite genomes [40, 41]. The
Mastrevirus name is derived from Maize streak virus (MSV); that infects
monocotyledon plants across Africa resulting in devastating losses [37]. These viruses
like Tobacco yellow dwarf virus (TYDV), Bean yellow dwarf virus (BYDV) [42] and
Chickpea chlorotic dwarf virus can also infect dicotyledon plants (dicots) [43-45].
The genome of Mastrevirus range in size from 2.6-2.8 kb and encodes four conserved
proteins; two in complementary-sense orientation (the replication associated protein
(Rep A) and Rep) and two in virion-sense orientation (the movement protein (MP)
and the coat protein) as shown in figure 1.3. Two intergenic regions, the small IR
(SIR) and large IR (LIR), separates complementary- and virion-sense genes. SIR and
LIR contain regulatory elements [46-48]. The LIR form stem-loop structure that

22

contains nonanucleotide motif TAATATTAC within the loop which is the origin of
replication [49].

Chapter 8.

Cur
tovi
rus

The transmission vector of these viruses are leafhopper. These are monopartite
viruses. Curtovirus infect only dicots. Beat curly top virus (BCTV) of this genus can
infect more than 300 species [50]. There are three known species of curtovirus BCTV, Spinach severe curly top virus (SSCTV) and Horseradish curly top virus
[51].The genome range in size from 2.9-3.1 kb and has 7 genes on it as shown in
figure 1.3 [52, 53].

Chapter 9.

Top
ocu
viru
s

Tomato pseudo-curly top virus (TPCTV) is a single species of this genus and is
transmitted through treehopper. It has only been found in new world (NW) and these
infects dicots [54-56]. TPCTV has monopartite genome and encodes six genes as
shown in figure 1.3.

Chapter 10.

Bec
urt
ovir
us

The first identified becurtovirus was Beat curly top Iran virus (BCTIV) and induced
almost same symptoms as induced by curtovirus [57-59]. The transmission vector is
leafhopper Circulifer hematoceps [58, 60]. The second becurtovirus has been
identified in the Unites States Spinach curly top Arizona virus (SCTA) [61]. Unlike
curtovirus five genes are encoded by becurtovirus genome (CP, V2, V3, Rep and C2)
as shown in figure 1.3.

23

Chapter 11.

Tur
ncu
rtov
irus

Turnip curly top virus (TCTV) is the only species of newly established genus [62]. It
is transmitted by leafhopper Circulifer hematoceps [63]. The genome encodes six
genes as shown in figure 1.3.

Chapter 12.

Era
gro
viru
s

It is also newly established genus. Eragrostis curvula streak virus (ECSV) is only
species of this genus which was isolated from wild grass in South Africa [64]. The
insect vector has not been identified yet. The genome encodes four genes (CP, V2,
Rep and C2) as shown in figure 1.3.

24

Figure 1.3 Genome organization of Mastrevirus, curtovirus, topocovirus,

eragrovirus, turncurtovirus and becurtovirus. Arrow represents the postion and
orientation. The genes like CP, V2, V3 and MP are in virion-sense orientation while
Rep,TrAP, C4 and Ren are in complementary-sense orientaion. The intergenic region
are represented as long intergenic region (LIR) and small intergenic region (SIR).the
hairpin structure is in intergeinc region having nonanucleotide.

Chapter 13.

Beg
om
ovir
us

This genus of family geminiviridae is economically the most important and infects a
wide range of dicots in tropical and sub-tropical part of world [65, 66]. The insect
vector of Begomovirus is whitefly Bemisia tabaci (Gennadius). The virus occur in
both Old World (OW) and New World (NW) but the viruses of these two regions are
distinct from each other [29]. The main difference is that NW begomoviruses lack
gene V2. Two species of OW begomoviruses, Corchorus yellow vein virus (CoYVV)
and Corchorus golden mosaic virus (CoGMV), also lack V2 gene [67, 68]. In NW
majority of begomoviruses have genome consisting of two components while in OW
most of them have monopartite and few have bipartite genomes. Recently, a
monopartite NW begomovirus has been identified [69, 70]. This genus has
approximately 300 species [29]. There are two genomic components in bipartite
begomoviruses named as DNA A and DNA B as shown in figure 1.2 [71, 72]. These
components are circular ssDNA. Their size ranges from ~2.6-2.8 kb. Transcription is
bidirectional. The monopartite begomovirus have the genome that is homolog of the
DNA A component of bipartite begomovirus as shown in figure 1.4. The DNA A
genome of monopartite encodes four genes; Rep, transcriptional activator protein,
replication enhancer protein and the C4. Two genes are encoded in the virion-sense
orientation (CP and V2 protein) [29]. The DNA B encodes one gene in
complementary-sense, MP, and one gene, nuclear shuttle protein in virion-sense
orientation. A short sequence of around 200 nt is common in DNA A and DNA B
components of begomoviruses known as common region (CR) [73-76]. It has

25

important role in replication and transcription of both components [35]. The CR

sequence is unique among different begomoviruses [77].

26

Figure 1.4 The genome organisation of a typical monopartite begomovirus

alongwith alphasatellite and betasatellite. The virion-sense genes CP and V2
while complementary-sense genses C4,Rep,REn and TrAP are shown by arrows.
SCR represents satellite conserved region and A-rich is adenine rich region.

Chapter 14.

Sate
llites

Monopartite viruses are often found associated with additional DNA molecules.
Satellites are nucleic acids or virus that depends on helper virus for some or all of the
process like replication, encapsidation and movement in plants [78]. Satellite like
nucleic acids are also known that does not fall under the criteria of satellites. These
27

may or may not depend on the helper virus for their replication. Kassanis first used
the term satellite in 1962 [79]. The first identified satellite RNA was associated with
Tobacco ringspot virus, a Nepovirus [80]. Some satellite RNA have no effects on
symptoms severity while some enhance the symptoms induced by helper virus [81,
82].The circular DNA molecule (~0.64 kb) satellite was first found to be associated
with Tomato leaf curl virus (ToLCV) in northern Australia and was called sat-DNA
[83].

Chapter 15.

Sate
llites
asso
ciate
d
with
gem
inivi
ruse
s

There are two type of satellite molecules known as alphasatellite and betasatellite.
These are mostly found associate with monopartite begomovirus in OW. In NW only
alphasatellite have been reported to be associated with bipartite virus [84, 85].
Recently alphasatellite and betasatellite have been found in association with
mastrevirus in wheat [86].

Chapter 16.

Bet
asa
telli
tes

Betasatellites were previously known as DNA-. These are half the size of
begomovirus genome. Betasatellite are small ssDNA molecules mostly associated
with OW monopartite begomoviruses that are often associated with the symptoms
induction [87-89]. More than 400 complete sequences of betasatellites have been
28

submitted to the sequence databases after its discovery in 2000 and mostly these are
identified in the OW [101-103]. Betasatellites have also been associated with bipartite
begomoviruses [90, 91]. Betasatellite have a highly conserved region (known as the
satellite conserved region [SCR]) containing ~150 nt that are conserved between all
betasatellite and a region which is rich in adenine residues. This region also have
conserved ORF that encodes C1 protein (118 amino acids) in the complementarysense orientation [90, 92-95]. Betasatellites are completely dependent on the helper
virus for the replication and insect transmission [92].
The C1 is involved in important functions like symptoms development, suppressor
of gene, may be involve in virus movement in the host plant [93, 94, 96-99]. DNA A
component of monopartite begomoviruses contains C4 which is a symptom
determinant. C4 deletion mutants of DNA A along with betasatellite induced
symptoms indicating C1 can induce disease symptoms [100]. For the helper virus
infections betasatellite may or may not be required. In some cases betasatellites may
enhance the virus DNA level and the symptoms. According to a study CLCuMB along
with Tomato leaf curl New Delhi virus DNA A could induce symptomatic infections in
absence of the DNA B [101]. In another study DNA levels of betasatellites were
reduced in the presence of DNA B relative to the plants that were infected with DNA
A and betasatellite only [102].

Chapter 17.

Alp
has
atel
lite
s

Alphasatellites do not fall under the definition of satellites. They have their own Rep
so are capable to replicate in the host cells [103]. These are however like betasatellite
dependent on the helper virus for movement, encapsidation and transmission by insect
vector [103, 104]. These are satellite-like molecules formerly called as DNA-1. As
contrast to betasatellites, alphasatellites are highly conserved. Alphasatellites are
circular ssDNA molecules having around 1380 nucleotides. There is highly conserved
structure that has a single gene (Rep) in the virion-sense orientation similar to the Rep
of nanoviruses [105, 106]. These are associated with monopartite begomovirusesbetasatellites complexes in only OW [105]. Alphasatellites have been identified in
29

NW bipartite virus complexes. Examples are Melon chlorotic mosaic virus

(MeCMV), Euphorbia mosaic virus (EuMV) and Cleome leaf crumple virus (ClLCr)
[84, 85]. Alphasatellites have also been found with mastrevirus infecting wheat [86].
Recent studies have shown that alphasatellites are more diverse than previously
thought [107, 108]. It is found that alphasatellites may lower the virus and/or
betasatellites titer in infected plant thus prolongs the survival of infected plant and
provides more time for the transmission of the complex by its insect vector [109]. The
Rep of Gossypium mustelinium symptomless alphasatellite (GMusSLA) and
Gossypium darwinii symptomless alphasatellite (GDarSLA) has post-transcriptional
gene silencing (PTGS)

activity [110]. So these are essential for their helper

begomoviruses to overcome RNAi-based host defenses. Cotton leaf curl Kokhran

virus strain Burewala (CLCuKoV-Bu) and its associated recombinant betasatellite
were found after the resistance breakdown but no alphasatellite were identified [111]

30

Chapter 18.

Prot
eins
enco
ded
by
gem
inivi
ruse
s
and
their
func
tion
s

Chapter 19.

Rep
lica
tion
ass
ocia
ted
pro
tein
(Re
p)

The replication associated protein also named as AC1, C1 or AL1 is approx. 41 kDa
protein that is encoded in complementary-sense orientation of DNA A component by
all geminiviruses [29]. Geminiviruses depend on the host for the replication process
because they do not have any gene product that carry on polymerase activity. The
protein is similar to the rolling-circle replication initiator protein found in
bacteriophages [112]. Rep is only viral protein that is required for replication of virus
31

and its multiple functions [113, 114]. The functional domains of Rep protein have
been identified; the domain for activities like DNA binding, nicking, religation of
separated strands and oligomerization is on the N-terminal while C-terminal has the
domains with ATA-binding and ATPase activity [115-117]. There are two methods of
replication in geminiviruses, one is through rolling circle replication (RCR) [118] and
the other is through recombinant dependent replication [119]. The IR has sequences
specific (iterons) to the Rep binding sites important for replication initiation and
regulating complementary-sense gene expression [35]. In RCR the Rep nicks at origin
of replication (TAATATTAC) resulting in 3-OH end which act as primer for viral
DNA synthesis [120]. Recent studies showed that Rep also suppress transcriptional
gene silencing [121].

Chapter 20.

Tra
nsc
ript
ion
al
acti
vat
or
pro
tein
/C2
pro
tein
(TrA
P)

The TrAP is approximately 15 kDa (~ 134 amino acids). It is encoded by three genera;
curto-, topocu- and begomo-viruses [122]. It is responsible for the up-regulation of
virion-sense genes [123, 124]. The promoter of this gene is within the Rep gene [125].
The TrAP of Cotton leaf curl Multan virus cannot transactivate (up-regulate) its
virion-sense promoter but can do it in other begomovirus [111]. Cotton leaf curl
Burewala virus has fragment of TrAP gene that consist of 35 amino acids [111, 126129]. It also involves in nuclear localization [130] and suppressor of PTGS [131-133].
It is also pathogenicity determinant [134-136].

32

Chapter 21.

Rep
lica
tion
enh
anc
er
pro
tein
(RE
n)

REn protein (also known as C3, AC3 or AL3) has ~132 amino acids and mostly found
in geminiviruses that infect dicots. It enhances the viral replication but is not essential
[137-139]. Mastrevirus and some Curtovirus lack this protein [59, 140]. Same level of
REn is found in the cells of infected plants along with Rep showing its involvement in
viral replication [141]. REn might be involved in the replication initiation by
enhancing Rep binding to the iterons [142]. The interaction of REn with itself and
with proliferating cell nuclear antigen (PCNA) of host is important for the replication
of geminiviruses whereas its interaction with retinoblastoma-related (RBR) protein
might be involved in the infection [143]. In mastreviruses Rep A perform the function
of REn [116, 118].

Chapter 22.

C4
pro
tein

C4 gene unlike Rep is not much conserved and is present within Rep gene having
different reading frame in some genera of geminiviruses [35]. The geminiviruses
infecting dicots have this protein, with only exception of mastreviruses. The function
of this protein is still unclear in bipartite virus as the mutation analyses were
inconclusive [144-148]. In monopartite begomoviruses and curtoviruses the
symptoms severity decreases and the viral DNA levels lower in the plants infected
with mutated C4 relative to the wild types [149-153]. The C4 mutant of Tomato
yellow leaf curl virus failed to have systemic infection showing C4 is essential for
viral movement [150].

33

Chapter 23.

Coa
t
pro
tein
(CP
)

Coat protein (V1 or AV1) is located on virion-sense. It is the only structural protein
encoded by geminiviruses and is involved in the function like encapsidation,
movement within the host and increasing the levels of viral ssDNA [154] also allows
transmission of virus through insect vector [36, 155]. Hatta and Francki (1979)
showed that there are 22 capsomeres to form two incomplete T=1 icosahedra and each
particle has one ssDNA molecule [156]. In monopartite begomovirus CP is involved
in symptoms induction and systemic spread [157-159] while in bipartite begomovirus
it is dispensable for systemic infection [160, 161]. Studies has shown that CP is
important for viral movement [162]. CP is present near the nucleus and cell periphery
[163]. Movement across nucleus is more likely dependent on ssDNA binding to CP
[164]. CP also has the role in the viral movement between nucleus and cytoplasm
[162, 163, 165, 166]. By interacting with V2 it facilitate intercellular movement
[167]. CP also determines the type of insect vector for the viral transmission [168].
This protein is also an important component for infectivity [158].

Chapter 24.

V2
pro
tein

The V2 gene is present in OW begomoviruses on the virion-sense strand and is

involved in systemic movement [169]. The protein has ~112 amino acids. V2 is
important for symptoms induction and viral movement [167, 170, 171]. The ORF of
V2 gene overlaps CP gene. Any changes in this gene effects the expression of CP,
experimental studies showed mutation in AV2 induces very mild symptoms and low
level of viral DNA [159, 170-173].

34

Chapter 25.

Nuc
lear
shu
ttle
pro
tein
(NS
P)

In bipartite begomovirus this protein is present on the virion-sense (V-sense) strand of

DNA B. The DNA B also encode MP that allows viral movement between cells
through plasmodesmata while NSP is responsible for the movement within the cells.
NSP synthesis is regulated by TrAP at transcriptional level [123]. Virus infectivity lost
after the mutation in NSP [174, 175]. NSP and MP recognize the DNA on selective
manner [176, 177]. According to study NSP amino acids domains present at Cterminal interact with MP [178].

Chapter 26.

Mov
em
ent
pro
tein
(MP
)

The gene is present on complementary strand of DNA B in bipartite begomovirus.

Movement protein is essential for long distance movement of dsDNA from cell to cell
[179]. It is involved in the viral movement via plasmodesmata, when the gene was
mutated the cell to cell movement ceased [180]. The NW bipartite begomovirus need
it for infectivity [181, 182]. The size exclusion limit of plasmodesmata increases due
to MP [179]. From plasma membrane MP enters to plasmodesmata [183-185]. MPNSP-DNA association occurs at the C-terminal of MP [185-187].

35

Mo

Chapter 27.

vem
ent
of
gemi
nivir
uses
withi
n
host
Plants cells have an additional layer, the cell wall. Cells being separated have a
connecting channel known as plasmodesmata (PD) that control many process in
plants by regulating transport of molecules in and out of the cell and also provide
defense against pathogens [188]. PD regulate the movement of macromolecules [189].
Through phloem virus move and infect the plant [190, 191]. Both DNA A and B are
required in bipartite begomovirus to infect the plant [192, 193], still there are cases
where DNA A of bipartite begomovirus lacking DNA B component induces infection
[194] [195]. MP and NSP proteins of DNA B are involved in the movement of virus.
Two models are given on the way bipartite begomovirus move in plants. The relayrace model suggest that NSP is involved in the viral movement between nucleus and
cytoplasm while MP is involved in the transport of viral DNA from cell-to-cell
through plasmodesmata [177, 179]. The second is couple-skating model, according
to this NSP form a complex NSP-DNA and transport viral DNA outside the
nucleus. Then MP joins the complex, transport it to the microsomes for cell-to-cell
movement [176]. The V2 protein of monopartite begomovirus transport viral DNA
within cell while C4 functions at cell periphery, both these act together in the same
manner as MP of bipartite begomovirus [163]. It is thought that CP is functional
analog of NSP and MP [164, 187]. Monopartite begomoviruses are limited to phloem
while bipartite begomoviruses can penetrate other tissues (like in epidermal cells
hence may cause mechanical infections) along with phloem [163]. The DNA B allows
wider tissue penetration of bipartite begomoviruses [196].
36

Chapter 28.

Agr
oinf
ectio
n

Plant viruses are mostly transmitted by insects or nematodes vectors from infected
plants to healthy one. The natural way of viral transmission is helpful in
understanding the virus ecology or epidemiology. These vectors are difficult to
maintain and utilize in the experiments in controlled manner. It is also difficult to
determine how much viral particles transmitted by insect vectors while mechanical
inoculation is quantitative. Though mechanical inoculation also have some advantages
like virus being transmitted poorly that may result in the virus inability to enter the
cell, replicate or move in the cells where viral particles are targeted during mechanical
abrasion of leaf [197]. Through Agrobacterium tumefaciens Ti plasmid vectors it is
possible to transfer transgene/foreign DNA into plant cells [198]. This experimental
technique is termed as agroinfection [199]. It can be defined as introduction of viral
genetic material into plants using Agrobacterium vector.

Figure. 1.5 Agroinfection: Transfer of viral DNA into T-DNA either for
inoculation or development of transgenic plants [200]
37

Agrobacterium has natural way of viral delivery into plants thus making it more
preferable to mechanical inoculation. The viral genome is easy to maintain in the
bacteria rather in insect vector.

Chapter 29.

Agr
oin
ocul
atio
n
and
agr
oin
filtr
atio
n

With the help of binary vectors gene function analysis in plants become easier.
Agroinoculation is now mostly applied for the delivery of viral genome in plants
through binary vectors. It was first used to study plant-virus interaction by cloning
viral genome in binary vectors and transferring into plants through agroinoculation
[199]. It is also used for transient expression of proteins in target tissue using different
expression vectors. Because of high copy number of viral transcript transient
expression of that gene is quite high. Gene expression cab be studied in
agroinoculation by passing the need of transgenic plants for that gene [201].
Agroinoculation was modified by using visual reporter proteins to a technique called
agroinfiltration for transient gene expression. Vacuum-aided agroinfiltration along
with GUS was used to determine the efficiency of this process in tobacco [202]. In
agroinfiltration Agrobacterium transferred into plant leaves through needless syringe.
This method has been modified for many plant species [203]. It is also used for
transient silencing in target tissues [204]. Silencing through this method can be
induced in the non-infiltrated cells adjacent to infiltrated cells [205]. Agroinoculation
is effective way of transferring viral genomes that unable understand viral replication,
their assembly and viral movement. Using this method Cauliflower mosaic virus and
Maize streak virus were the first viruses to be studied for the plant-virus interaction
[199, 204]. Tobacco golden mosaic virus, Tomato yellow leaf curl virus and Potato
38

yellow mosaic geminivirus were the first to show infectivity in the plant they were
inoculated. Through mutation analyses different viral genes involved in symptom
development and viral accumulation has been determined [206]. Agroinoculation has
been helpful in screening out the resistant germplasms [207]. Heterologous protein
can be produced by this method [208-210]. Agroinoculation has been used to identify
functions and behaviours of many unidentified genes [211, 212].
The rate of expression of a transgene is high within 2-3 days following
agroinoculation, then the expression level decreases because of RNA silencing that
block the expression of that gene [213, 214]. Agroinoculation has also been useful in
inducing infections with RNA viruses [215]. This method is useful in carrying out
infection in bipartite virus [216]. Agroinoculation of infectious clones produce copies
that are identical genetically [217] [218, 219]. In recent studies an alternate method
adopted in which the shoots of tomato soaked into Agrobacterium suspension
containing Tomato yellow leaf curl virus allowed efficient replication of the virus
[220]. Agroinoculation of stems is effective according to various studies performed
[217-219]. Through agroinoculation geminiviruses can enter the leaf disks and whole
plant [207, 217]. Through agroinoculation it has been determined that leafhopper is
vector of wheat dwarf geminivirus [221]. Potato yellow mosaic virus was infected in
Nicotiana benthamiana, tomato and potato via agroinoculation [222]. Similarly
through agroinoculation Mungbean yellow mosaic virus and Strawberry mild yellow
edge potexvirus shown to be the cause of disease in blackgram and strawberry,
respectively [223, 224]. It is also used as virus induced gene silencing (VIGS) in
plants [225].

Chapter 30.

Bio
listi
c
inoc
ulat
ion

In this process the foreign DNA is introduced in living cells through high speed
microprojectiles in a vacuum chamber having compressed helium gas [226]. This
method was developed for species which are recalcitrant to Agrobacterium mediated
plant transformation [226]. This method is quite effective but requires equipment that
39

are highly expensive. In the different tissues of different plants species successful
transformation has been possible through this method. It has overcome the barriers of
transformations in different plant species. This method has also been useful in
transforming the microbes. The biolistic has been used to transform animal cell lines
and intact animals. The main components of biolistic are particle accelerator,
microprojectile, biological parameter and experimental design. Tungsten and gold
particles are mostly used but gold particles are preferred over tungsten because these
are much rounder and of uniform size. The size of particle is in accordance to the size
of the target cell. Biological parameters do effect the biolistic transformation. The
gene construct being transformed must express itself in that tissue. There must be
maximum penetration of particles in the cell in such a way that the cell keep
performing normal after the bombardment. Vectors used in the biolistic
transformation must be replicating or integrative. For efficient transformation through
biolistic the target cells should be young dividing cells [227].
By using particle bombardment method cloned begomovirus DNA has been
introduced in plants [228, 229]. Both monomeric and dimeric form of cloned
begomovirus DNA gives good results inoculated through biolistic [230]. The viral
clone separated from the vector gives higher inoculation rate [230]. The viral titer of
begomoviruses is usually low in the infected plant. High levels of viral DNA of
begomovirus can be obtained through a method named as rolling circle replication
(RCA) that uses bacteriophage phi29 DNA polymerase [231]. The phi29 DNA
polymerase acts on circular plasmid and as the DNA of begomovirus is circular so it
act as template for that enzyme [232]. Through RCA and restriction analyses viral
DNA is confirmed [233]. The method of RCA is very efficient as it can amplify the
viral DNA without any information of sequence or any primer as in case of PCR thus
can be used for cloning and identification purposes [233, 234]. After the RCA,
through partial digestion a dimer is obtain which then can be cloned in binary vectors
like pCambia thus developing agroinfectious begomovirus clone [235, 236]. To
increase the inoculation efficiency the components of cloned viral genome can be
excised from the plasmids then ligate to amplify through RCA. This product can then
be used for biolistic inoculation for development of successful and more efficient way
of symptom induction [237]. When Squash leaf curl virus (SLCV) was inoculated
through particle bombardment method it was revealed that inoculation carried out
40

under vacuum and RCA amplified viral nucleic acid gave highest rate of inoculation
[238]. The transmission rate of Tomato yellow leaf curl virus through biolistic
inoculation is low [239, 240]. The partial dimer of monopartite Tomato leaf curl
Karnataka virus was inoculated through biolistic for the first time in 2002
(Chatchawankanphanich and Maxwell, 2002). The first attempt of biolistically
inoculating Tomato leaf curl virus using dimeric construct of TYLCV isolate from
Cuba and Tomato yellow leaf curl Sardinia virus (TYLCSV) [239], though it was not
successful. While successful inoculation of dimeric form of TYLCV-[Alm] (Almeria
isolate) DNA in 2005 [241]. According to a study the inoculation efficiency using
particle bombardment varies with plants, as in tomato plants it was about 40% while
in datura plants the efficiency was 85% [240]. It was demonstrated that using biolistic
inoculation normal disease symptoms appeared in infected plants but were delayed as
compared to whiteflies transmission [240]. Through biolistics inoculation of viral
DNA may speed up the development of the tolerance/resistance of cassava cultivars in
breeding programs [242].

Chapter 31.

Obj
ectiv
e of
the
stud
y

For the analyses of viral infectivity in cotton different methods has been used.
Grafting, whitefly mediated viral transfer, agro biolistic and agroinoculation are the
most used method in the infectivity study. In grafting and whitefly mediated
infections there should be a symptomatic plant in order to transfer the infection. In the
field we do not have CLCuKoV as this virus has evolved into CLCuBuV. The
objective of this were to establish the infectivity system for cotton plants in order to
understand the CLCuD in a better way.

41

Chapter 32. Ma
teri
als
and
Me
tho
ds

Chapter 33.

Solu
tion
s

Distilled water was used for the preparation of chemical solutions. These were
autoclaved where it was necessary. These chemical solutions contained reagents that
were analytical and molecular biology grade.

42

Chapter 34.

Prep
arat
ion
of
elect
ro
com
pete
nt
Agr
obac
teriu
m
tum
efac
iens
cells

With the help of a sterile wire loop a single colony from a freshly grown plate of
Agrobacterium tumefaciens (strain GV3101) was picked and grown into 20ml LB
liquid medium with 25g/ml rifampicin in a 50ml autoclaved flask. The culture was
incubated in a shaker (160 rotations per minute) at 28 oC for 48 hours. After 48 hours
5ml of the culture was transferred into a 1 liter flask containing 250ml of LB medium
with 25g/ml rifampicin and placed in a shaker at 28 oC until the OD600 of cells was
0.5-1. This culture was transferred aseptically to ice cold 50ml falcon tubes, incubated
on ice for 10 minutes and centrifuged at 3000g for 10 minutes at 4 oC. The pellet was
resuspended in 50ml of ice-cold sterile distilled water (SDW) and centrifuged. Cells
were again resuspended in cold SDW and centrifuged again. Then the cells were
resuspended in 10ml cold SDW containing filter sterilized cold 10% [v/v] glycerol
43

and were centrifuged. This wash was also repeated. Finally the cells were resuspended in 3-4ml of filter sterilized cold 10% [v/v] glycerol. Then cells were taken
in 1.5ml microcentrifuge tubes and stored at -80 oC for future use.

Chapter 35.

Tra
nsfo
rma
tion
of
elect
ro
com
pete
nt
Agr
obac
teriu
m
tum
efac
iens
cells

Eectro-competent A. tumefaciens cells were taken on ice and added 500ng of the
plasmid DNA and transferred to a chilled electroporation cuvette. The electroporator
(ECM 600, BTX, and U.S.A) was set at 1.44kV. Cuvette was properly cleaned so that
no water drop remained on its surface. Then the cuvette was inserted into the device
and the start button was pressed for an electric shock for 6 milli seconds. 1 ml of LB
liquid medium was added to the cells after the electric shock and the tube was placed
44

in a shaker at 28 oC for 1-2 hours. After incubation cells were short spinned to make
pellet. Almost 900 l supernatant was discarded and the pellet was resuspended in
remaining supernatant. The cells were finally spread on Petri plates containing AB
minimal medium (3g of K 2HPO4, 1g of NaH2PO4, 1g of NH4Cl, 0.15g of
MgSO4.7H2O 0.3 g, 0.005g of KCl, 0.0025g CaCl2, FeSO4.7H2O, and 20% [w/v]
glucose [pH 7.2] in a 1 liter volume) with agar (14g) and appropriate antibiotics,
wrapped with parafilm and placed in a 28 oC incubator for 48 hours.

Chapter 36.

Prep
arat
ion
of
inoc
ulu
m

The infectious clones of Cotton leaf curl Kokhran virus (CLCuKoV; accession no)
and Cotton leaf curl Multan betasatellite [17, 243] has been described previously. The
clones were transformed to Agrobacterium tumefaciens strain GV3101. Single colony
was grown separately in 50ml L-broth medium containing 50 g g/ml kanamycin,
tetracycline 10 g/ml and 50 g/ml of rifampicin. The cultures were incubated in a
shaker at 160 rpm for 48 hours at 28 C. After incubation the cultures were transferred
separately into 50 ml sterile falcon tubes and centrifuge at 4000 rpm for 15 min, at 28
C. The supernatant fluid was discarded and the cells pellet was suspended and
dissolved completely slowly through tapping in 10 mM MgCl2, 10 mM MES pH 5.7,
and 150 M acetosyringone. The inoculum having optical density (OD) 1.0 and 4.0
were prepared for both, CLCuKoV and CLCuMB. Both the inoculum having same
OD were mixed in equal volume (1:1) and were kept overnight in dark at room
temperature.

45

Chapter 37.

Pla
nt
mat
erial
s

Three varieties of G. hirsutum; NIBGE 3701, NIAB Karishma and Coker 312 along
with Nicotiana benthamiana were used for the infectivity analyses.

Chapter 38.

Agr
oino
cula
tion

CLCuKoV and CLCuMB were co-infiltrated to the cotyledonary leaves of cotton

plants. The leaves of cotton plants were superficially wounded with needle prior to the
infiltration through needless syringe. The process was repeated till the leaves were
fully soaked with the inoculum. The stem agroinoculation, i.e. injecting of inoculum
into axillary bud using 1-ml insulin syringe was also performed along with
infiltration. N. benthamiana plants were infiltrated with the 1-ml needless syringe at
2-3 leaf stage. The agroinoculated plants were transferred to glasshouse having
optimum growth conditions (~25C, 16 hour light/8 hour dark cycle and 65 % relative
humidity) for 48 hrs. After 48 hrs the cotton plants were shifted to a glasshouse where
the temperature was kept 32 C.

46

Chapter 39.

Prep
arat
ion
of
glyc
erol
stoc
ks

In purpose to preserve the bacterial cultures, glycerol stocks were prepared of clones
that showed infectivity in the plants. 700 L of bacterial culture was mix with 300 L
filtered sterile glycerol in a 1.5 mL microcentrifuge tube. These stocks were stored at
-80 0C for longer preservation. To retrieve from this stock, a very small amount of
culture was streaked with the help of sterile wire loop on freshly made growth media
plate which had appropriate antibiotic selection.

Chapter 40.

DN
A
extr
acti
on

DNA was extracted from infected plants by using the cetyl trimethyl ammonium
bromide (CTAB) method (Doyle and Doyle, 1990). Leaf tissues (100-200mg) were
grounded to powder form in a autoclaved pestle and mortar with liquid nitrogen
then mixed with 700l pre-warmed CTAB buffer (100mM Tris-HCl [pH 8.0], 20mM
EDTA, 1.4M NaCl, 2% [w/v] CTAB and 0.2% [v/v] -mercaptethanol). After that
transferred into a 1.5mL micro-centrifuge tube and were incubated at 65 0C for 40
minutes on a dry bath. To facilitate the mixing tubes were inverted after every 10
minutes. Then 650L of chloroform isoamyl alcohol (24:1 [v/v]) was added, mixed
well and centrifuged for 10 minutes in a microfuge. The upper aqueous layer was
transferred to a new tube, mixed with 0.6 volume of isopropanol and placed in -20 0C
47

for an hour. Then centrifuge for 10 minutes. The supernatant was discarded and the
pellet was washed with chilled 70% ethanol. After that the tube was centrifuged for 5
minutes, the supernatant was discarded, the pellet was dried then dissolved in sterile
distilled water (SDW) and stored at -20 0C.

Chapter 41.

Qua
ntifi
cati
on
of
DN
A

The concentration of dsDNA in each sample was determined by visual observation on

the ethidium bromide 1 % (w/v) agarose gel under UV light as well as using
Nanodrop Spectrophotometer 2000c. Each sample was quantified followed by the
blank sample containing SDW in which the DNA was dissolved. The values were
exported in ng/L and the dilutions were prepared for further procedure.

Chapter 42.

Pol
yme
rase
chai
n
reac
tion
(PC
R)

Polymerase chain reaction (PCR) was performed to amplify a specific DNA sequence
using short oligonucleotides as primers and DreamTaq DNA polymerase master mix.
48

For PCR amplification of DNA, a reaction mixture of 25 L containing 40 ng of

template DNA, 12.5 L of DreamTaq polymerase master mix, both primers 0.5 M
each and then made up to a volume of 25 L with SDW in 0.2 mL PCR tubes. The
reaction mixture was incubated in thermal cycler. The PCR profile was run on the
machine with an initial denaturation temperature at 95 0C for 5 min followed by 35
cycles of denaturation temperature at 94 0C for 1 min, annealing temperature at 50 0C
for 30 sec and extension temperature at 72 0C (optimum temp for Taq DNA
polymerase) for 3 min. Then the final extension temp was given at 72 0C for 10
minutes. For diagnostic PCR the volume of reaction mixture was 25L per tube. The
volume of 3 L of amplified product was loaded on 1% agarose gel containing
ethidium bromide.

Chapter 43.

Gel
elect
roph
oresi
s

The isolated or restricted DNA fragments were resolved by electrophoresis on 1 %

[w/v] agarose gels containing 0.5X TAE (20 mM Tris-acetate and 0.5 mM EDTA [pH
8.0]) buffer. TAE gels were electrophoresed at approximately 100 volts. DNA was
mixed with 5X loading dye and run on gel containing ethidium bromide (0.5 g/mL).
The ethidium stained DNA was viewed using a gel documentation system and
photographed. Fragment sizes were estimated by comparison with a coelectrophoresed 1 kbp DNA ladder.

49

Chapter 44.

Res
trict
ion
Ana
lysis

For the confirmation of PCR products restriction analysis has been done through
single digestion with restriction enzymes. For this, the product was digested with
appropriate enzymes using their corresponding buffers in accordance with the
suppliers guidelines. 20 L volumes of mixture (total reaction mixture contains 1 g
DNA, 10 units restriction enzyme, buffer and SDW) for digestions. The reaction
mixtures were given the optimum temperatures for 1-3 hours as the incubation time
and temperature could be different for different enzymes so it was adjusted according
to the guidelines of manufacturer. The restriction pattern of the restricted clones was
observed on 1 % (w/v) agarose gels stained with ethidium bromide along with the
standard 1,000 bp DNA ladder

50

Chapter 45. Res

ults

51

Chapter 46.

Infe
ctivi
ty of
Cott
on
leaf
curl
Kok
hra
n
viru
s
with
Cott
on
leaf
curl
Mul
tan
beta
satel
lite
in
G.
hirs

52

utu
m
Vari
ety
Cok
er
G. hirsutum Variety Coker plants (n=26) following co-agroinoculation of CLCuKoV
and CLCuMB (each having inoculum concentration OD=1) did not show any
symptoms of infection, neither the CLCuKoV was detected through PCR
amplification (Table 3.1). The non-agroinoculated (healthy control) plants also did not
show any symptoms. However, symptoms were observed in the plants coagroinoculated with the same virus and betasatellite having inoculum concentration
OD=4. These plants were infiltrated through a needleless syringe in the leaf as well as
inoculated through a needle in stem. Out of twenty six agro-inoculated (OD=4)
plants, six plants symptoms started appearing after 18-23 days of inoculation.
Symptoms started appearing in the newly developing young leaves in all the infected
plants. In early stages of symptom development, vein darkening was observed which
got more severe at later stages and resulted in severe vein thickening. With the
passage of time symptoms severity increased and the plants were showing downward
leaf curling, upward leaf curling, abnormal shoots development, stunted growth and
leaf-like enations on the main and lateral veins on the lower side of the leaves. After
thirty five days of inoculation the plants were fully symptomatic and were showing
typical cotton leaf curl disease symptoms (Figure 3.1).
All the N. benthamiana plants co-inoculated with CLCuKoV and CLCuMB exhibited
symptoms like leaf curling, stem twisting, vein thickening, vein darkening, abnormal
shoots development and stunted growth (Figure 3.1).
In all the symptomatic coker plants the presence of CLCuKoV was confirmed through
PCR (Figure 3.2) while in none of the non-symptomatic plants nor in the healthy
control plants the virus was detected.

53

Methods of
inoculation

AIF
AIF + AIN

Optical
density of
inoculum
(OD)

1.0

AIF
AIF + AIN

4.0

Germplas
m

No. of plants
infected/ No.
of plants
inoculated
0/13

G.
hirsutum
Variety
Coker

0/13

Symptoms
appearanc
e in
Symptoms*
infected
plants
(dpi)
NS
NS

0/26

NS

6/26

18-23

LC, VD, EN, VT,

ASG,SG

N.
AIF
1.0
benthamia 12/12
14
LC,ST,SG, ASG
na
G.
hirsutum

0/4
NS
Variety
Coker
Table 3.1 Infectivity of CLCuKoV with CLCuMB in G. hirsutum variety Coker

Methods of inoculation denoted by Agroinfiltration (AIF) and Agroinoculation (AIN)

The experiment was repeated at least 3 times
* Symptoms denoted as leaf curling (LC), vein thickening (VT), vein darkening (VD), enations (EN), stem twisting (ST),
stunted growth (SG), no symptoms (NS) and abnormal shoot growth (ASG)

In each experiment healthy plants were used as control

54

Figure 3.1 Symptoms induced by CLCuKoV-CLCuMB in G. hirsutum variety

coker. The coker plants shown were either inoculated with CLCuKoV-CLCuMB (AF), non-inoculated (G and F). N. benthamiana plant (I) was inoculated with
CLCuKoV and CLCuMB.

55

Figure 3.2 The PCR amplification of CLCuKoV in the symptomatic Coker

plants. Amplification of size 2.8 kb was observed in all samples. The lane 10
represent negative control. 1 kb ladder used as marker in the lane represented by L

56

Chapter 47.

Infe
ctivi
ty of
Cott
on
leaf
curl
Kok
hra
n
viru
s
with
Cott
on
leaf
curl
Mul
tan
beta
satel
lite
and
Tom
ato

57

leaf
curl
New
Del
hi
Viru
s in
G.
hirs
utu
m
Vari
ety
Cok
er
A total of 48 plants of G. hirsutum Variety Coker were either agroinoculated or
agroinfiltrated with CLCuKoV, CLCuMuB and Tomato leaf curl New Delhi Virus
(ToLCNDV) in different combinations. N. benthamiana plants were also inoculated
with CLCuKoV and CLCuMB (3 plants) and ToLCNDV DNA A and ToLCNDV
DNA B (3 plants) as shown in table 3.2. The same numbers of plants were kept as
healthy control.
In almost all the agroinoculated N. benthamiana plants the symptoms started
appearing after 14 days of inoculation, confirming that the inoculum of CLCuKoV,
CLCuMB and ToLCNDV are working properly. All the coker plants either coagroinfiltrated or co-agroinoculated behaved just like the healthy control except one
plant which was agro-inoculated with CLCuKoV, CLCuMB and ToLCNDV DNA A.
In this plant the symptoms appearance started at twenty days post-inoculation. After
35 days of inoculation the plant was showing typical cotton leaf curl disease
58

symptoms i.e. vein darkening, vein thickening, abnormal new shoots development,
stunted growth and enations on the lower side of leaves (Figure 3.3).
In none of the non-symptomatic G. hirsutum Variety Coker plants the CLCuKoV was
detected. Whereas, CLCuKoV was detected through PCR in the single symptomatic
plant (Figure 3.2).

Inoculum

CLCuKoVCLCuMB-

Methods of
inoculation

Germplasm

No. of plants

Symptoms

density of

infected/ No.

appearance in

inoculum

of plants

infected

(OD)

inoculated

plants (dpi)

AIF

0/8

ToLCNDV
DNA A-

Optical

4.0

Symptoms
*

NS
-

AIF + AIN

0/8

NS

AIF

0/8

NS

ToLCNDV
DNA B
CLCuKoVCLCuMB-

4.0

ToLCNDV
DNA A

20

AIF + AIN

1/8

LC, VD,
EN, VT,
ASG,SG

CLCuKoV-

AIF

CLCuMBToLCNDV
DNA B
CLCuKoVToLCNDV
DNA B
ToLCNDV
DNA AToLCNDV

G. hirsutum
Variety Coker

0/8
4.0

NS
-

AIF + AIN

0/8

AIF

NS

0/8
4.0

AIF + AIN

0/8

AIF

NS
-

0/8
4.0

NS
NS

AIF + AIN

0/8

NS

DNA B
ToLCNDV

AIF

0/8

DNA ACLCuMB
CLCuKoVCLCuMB
ToLCNDV
DNA AToLCNDV

4.0
AIF + AIN
AIF

AIF

NS
-

0/8
N.
benthamiana
N.
benthamiana

NS

1.0

3/3

14

1.0

3/3

14

0/6

59

LC,ST,SG,
ASG
ULC. SG,
ASG

DNA B

G. hirsutum
Variety Coker

NS

Table 3.2 Infectivity of CLCuKoV, ToLCNDV with CLCuMB in G. hirsutum

variety Coker

Methods of inoculation denoted by Agroinfiltration (AIF) and Agroinoculation (AIN)

The experiment was repeated at least 3 times
* Symptoms denoted as leaf curling (LC), upper leaf curling (ULC), vein thickening (VT), vein darkening (VD),
(EN), stem twisting (ST), stunted growth (SG), no symptoms (NS) and abnormal shoot growth (ASG)

In each experiment healthy plants were used as control

enations

Figure 3.3 Symptoms induced by the CLCuKoV, ToLCNDV and CLCuMB in

Coker. The coker plants were either inoculated with CLCuKoV, ToLCNDV and
CLCuMB (A-D) or non inoculated (E and F). The N. benthamiana plants shown
60

were inoculated with CLCuKoV with CLCuMB (G and H) or ToLCNDV-DNA and

ToLCNDV-B (I).

61

Chapter 48.

Con
firm
atio
n of
Cott
on
leaf
curl
Kok
hra
n
Viru
s
thro
ugh
restr
ictio
n
end
onu
clea
se
dige
stio
n

62

The sequences of Cotton leaf curl Kokhran Virus and Cotton leaf curl Kokhran VirusBu were analyzed through pDRAW32 software, comparison of the restriction maps
showed that BamHI cuts once CLCuKoV and twice CLCuKoV-Bu as

shown in

Figure 3.4. Plasmid DNA of CLCuKoV-Bu was used as a positive control in every
PCR reaction. The amplified PCR products from the infected coker plants genomic
DNA and the PCR product from positive control sample were digested with BamHI.
The restriction endonuclease digestion confirmed that the infected plants contain the
Cotton leaf curl Kokhran Virus (Figure 3.5)..

Figure 3.4 The comparison of restriction maps of CLCuKoV and CLCuKoV-Bu

using pDRAW32. It shows the position of restriction endonuclease digestion of
BamH1.

63

Figure 3.5 Digestion of PCR product of CLCuKoV and CLCuKoV-Bu. The

endonuclease (BamHI) digestion of the PCR product obtained from the genomic DNA
of the infected coker plants (lane 1, 2, 4 and 5) and plasmid DNA of CLCuKoV-Bu
(lane 3) has been shown.

64

Chapter 49.

Infe
ctivi
ty of
Cott
on
leaf
curl
Kok
hra
n
viru
s
with
Cott
on
leaf
curl
Mul
tan
beta
satel
lite
in
G.
hirs

65

utu
m
Vari
ety
NIB
GE
3701
and
G.
hirs
utu
m
Vari
ety
NIA
B
Kari
shm
a
A total of 52 plants of G. hirsutum Variety NIBGE 3701 were either agroinoculated or
agroinfiltrated with CLCuKoV and CLCuMB having OD=1 or OD=4 (Table 3.3). Just
to insure that the inoculum of CLCuKoV and CLCuMB is working properly, 9 plants
of N. benthamiana plants were also inoculated.
In almost all the N. benthamiana plants the symptoms initiated 14 days post
inoculation. After 22 days of inoculation all the N. benthamiana plants were showing
down ward leaf curling, vein thickening, vein darkening, abnormal shoots
development and stunted growth. In none of the agroinoculated G. hirsutum Variety
66

NIBGE 3701 and G. hirsutum Variety NIAB Karishma plants symptoms appeared
(Figure 3.6) nor the CLCuKoV was detected through PCR in these plants.

Table 3.3 Infectivity of CLCuKoV with CLCuMB in G. hirsutum variety NIBGE

3701 and G. hirsutum Variety NIAB Karishma

67

Methods of
inoculation
AIF
AIF + AIN
AIF
AIF + AIN

Optical
No. of plants
density of
infected/ No.
Germplasm
inoculum
of plants
(OD)
inoculated
0/13
1.0
G. hirsutum
0/13
Variety
NIBGE 3701 0/13
4.0
0/13

AIF
1.0
AIF + AIN
AIF
4.0

G. hirsutum
Variety NIAB
Karishma

AIF + AIN
AIF

1.0

N.
benthamiana
G. hirsutum
Variety
NIBGE 3701
G. hirsutum
Variety NIAB
Karishma

Symptoms
appearance
in infected
plants (dpi)
-

Symptoms
*
NS

NS

NS
NS

NS

0/13

0/13

0/13

0/13

9/9

14

ULC,ST,S
G, ASG

0/4

NS

0/4

NS

NS
NS
NS

Methods of inoculation denoted by Agroinfiltration (AIF) and Agroinoculation (AIN)

The experiment was repeated at least 3 times
* Symptoms denoted as upper leaf curling (ULC), vein thickening (VT), stem twisting (ST), stunted growth (SG), no symptoms
(NS) and abnormal shoot growth (ASG)

In each experiment healthy plants were used as control

68

Figure 3.6 The agroinoculation of cotton varieties NIBGE 3701 and NIAB
Karishma with CLCuKoV and CLCuMB. The NIBGE 3701 (A and B), NIAB
Karishma (C and D) and N. benthamiana (E & F) plants were inoculated with
CLCuKoV and CLCuMB developed the infection with the same inoculum.

69

Chapter 50.

Gra
ft
inoc
ulati
on
of
G.
hirs
utu
m
Vari
ety
NIB
GE
3701

To administer high dose of virus and betasatellite graft-inoculation was performed.

For this purpose scions of coker plants infected with CLCuKoV and CLCuMB have
been grafted to root stocks of healthy cotton plants (Variety NIBGE 3701; Figure 3.7).
As a control scions from the same infected plants have also been grafted to root stocks
of G. hirsutum Variety Coker. In addition, scions from field infected (plants infected
with CLCuKoV-Bu and CLCuMB) have been grafted to root stocks of G. hirsutum
Variety NIBGE 3701 and G. hirsutum Variety Coker. These plants are under
investigation in glass house and further molecular analysis (Southern blot
hybridization, for comparison of the virus and betasatellite titer and also whitefly
transmission assays) will be performed.

70

Table 3.4 Graft inoculation of G. hirsutum variety Coker and G. hirsutum variety
NIBGE 3701
Scion
Scion infected with
Rootstock
Source
CLCuKoV-BuField
CLCUMB
G. hirsutum
Variety Coker
CLCuKoVGlasshouse
CLCUMB
Field
Glasshouse

CLCuKoV-BuCLCUMB
CLCuKoVCLCUMB

71

G. hirsutum
Variety
NIBGE 3701

Figure 3.7 Grafting of NIBGE 3701 and Coker. The grafting of NIBGE 3701 with
field scion infected with CLCuKoV-Bu (A-B) and scion having CLCuKoV with
CLCuMB (C). The grafting of Coker was done with scion infected with
CLCuKoV+CLCuMB (D and E) and infected field scion (E)

72

Chapter 51. Dis

cus
sio
n

Pakistans economy, depending largely upon the production of cotton for its industrial
use and foreign exchange earnings, is severely threatened by CLCuD since the early
90s. In the mid-90s losses to the crop were slightly reduced due to the introduction of
resistant varieties through conventional breeding. The resistance was breached in
2001 with the appearance of resistance breaking Burewala strain. The losses to
cotton crop since then were almost exclusively due to Burewala strain of CLCuKoV
(CLCuKoV-Bu) and its associated betasatellite, CLCuMuB. The most useful
procedure to study how cotton crop is infected by the virus would indeed be an
efficient infectivity system of cloned viruses to investigate the molecular basis of
resistance. It is very unfortunate, though several efforts were made to achieve
infectivity in cotton, that no such system is available as yet. Agro-inoculation of
CLCuKoV, along with its cognate betasatellite cloned as dimers in binary vector, was
used with different optical densities (OD) as an attempt to achieve infectivity in
different varieties of cotton.
To prove the Koch`s postulates and carry on infectivity analyses in cotton several
procedures have been applied. Whitefly-, biolistic and Agrobacterium tumefaciens(agroinoculation) and graft-inoculation mediated transmissions are amongst the most
frequently used procedures. In case of graft- and whitefly mediated transmission there
must be a live infected plant with no vigorous manipulation of viral DNA [244].
Agroinoculation deliver the viral DNA (either as full dimer or partial dimer) cloned in
binary vector into host cells [245, 246]. It is important that the infectious clone must
have tandem repeats (dimer or partial dimer) that provides two origin of replication
necessary to carry on viral replication. This approach allows us to perform
experiments in planta aimed to study interactions between different viruses, protein
expression and virusvector interaction or virus resistance [247]. Most geminiviruses
73

are dependent on specific insect vectors for their transmission and agroinoculation has
greatly simplified the study of this group of viruses. In cases where cloned viral
genomes are being studied, agroinoculation has been used to conduct infectivity
analysis and to determine the host range of geminiviruses [248]. Agroinoculation
develops localized infections in phloem cells in and at the site of inoculation
providing systemic infection [249]. Agroinoculation has also proven to be successful
for several phloem-limited viruses such as Beet western yellows virus, Potato leaf roll
virus and Beet mild yellowing virus [215, 250-256]
Agroinoculation was used successfully to induce infection with respective viral clones
in Physalis floridana at six-leaf stage, Nicotiana benthamiana at six to eight-leaf
stages and in Capsicum annuum at four-leaf stage [215]. The tandem repeats of
Tomato yellow leaf curl virus were successfully agro-inoculated into Lycopersicon
esculentum [207, 217]. Cotton is highly recalcitrant to agro-inoculation; however,
during this study efforts were made to induce infectivity in different cultivars of
cotton through agro-inoculation of CLCuKoV/CLCuMuB complex (as dimers in
binary vector) with varying ODs. The successful infections were reported at OD 4 in
Coker (a cultivar of cotton susceptible to CLCoKoV) using A. tumefaciens strain
GV3101. Symptoms in N. benthamiana are mostly induced at a lower OD equal to 1
[247] which is much lower in comparison to cotton. However, the cultivar of cotton
NIBGE 3701 never developed any symptoms of the disease following several repeats
of agro-inoculations with the same clone using the same OD. The possible reason
might be its resistance to CLCuKoV since this cultivar was developed prior to the
appearance of resistance breaking Burewala strain [257]. For further confirmation
graft-inoculations in NIBGE 3701 was initiated using infectious scions from both
experimentally infected (CLCuKoV) and field infected (CLCuKoV-Bu) plants. The
grafted plants are under observation and will be studied for accumulation of virus by
PCR and/or Southern blot hybridization and also the whitefly transmission assays of
virus should be performed for both the cultivars.
The temperature also plays a key role in the development of symptoms in cotton. All
the agro-inoculated Coker plants which developed symptoms were provided an
optimum temperature in the range of 35-37 0C. Previously, Briddon (2001) has
showed that the optimum temperature for the development of symptoms is 35 0C at
the very least.
74

The NGS data showed the incidence of ToLCNDV in cotton. ToLCNDV is a bipartite
begomovirus which is widely prevalent in Indian subcontinent. [258]. The coinoculation of tomato plants with ToLCNDV DNA-A and CLCuMB resulted in
successful systemic infection [259]. In the present study CLCuKoV and CLCuMB
were inoculated along with ToLCNDV (DNA A and DNA B) components in different
combinations (see Table 3.2) but only a single plant got infected. This might be
because of the reason that agroinoculation is not that much optimized to successfully
develop the infection with ToLCNDV. For this purpose more efforts are required for
optimizing the condition and inoculation. The efficiency of this method is still less
than 50%. Several factors may be responsible that make this method less efficient. It
may be due to the inability of agrobacterium tumefaciens strain to interact in
productive way with the viral component [248]. A. tumefaciens strain has been shown
to effect the viral infection in case of Lettuce infectious yellows virus (LIYV) in N.
benthamiana via agroinoculation [260]. For the systematic infection the monopartite
begomoviruses require coat protein [163, 261]. As we know that betasatellites are
dependent on the helper virus for replication, systematic infection and insect
transmission [262]. The co-inoculation of CLCuKoV and CLCuMB might have
delivered both components in same cells but CLCuMB might not have efficiently
transreplicated by the helper virus in those inoculated cells. Thus could not develop
the systematic infection. Further study is required to understand the factors that can
make agroinoculation a more efficient way of viral delivery and subsequent viral
infection.
CLCuKoV-Bu has devastated most of the cotton crops throughout the cotton belt in
Punjab and Sindh provinces of Pakistan and also in the North-Western parts of India.
In the present study the protocol of agro-inoculations of cotton plants has been
optimized. Therefore, it will be interesting to make an infectious clone of CLCuKoVBu and study its infectivity in cotton plants. Moreover, the study described here was
conducted with the aim to understand the begomovirus and its betasatellite interaction
in their natural host that may allow us to design better control methods

75

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nce
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