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5.

1 EMERGING BEGOMOVIRAL DISEASES IN HIMACHAL PRADESH


Begomoviruses (family Geminiviridae) are causal agents of the most devastating diseases
of vegetable, grain and fiber crops particularly in tropical and sub-tropical regions of the
world (Varma and Malathi, 2003). Begomoviruses are whitefly-transmitted, infect
dicotyledonous plants and comprise the largest genus with more than 800 complete DNA-A
sequences available in EMBL database, representing 200 different species based on the
89% cut-off identity for species demarcation (Fauquet and Stanley, 2005; Fauquet et al.,
2008). These viruses are now intruding into more temperate parts of the world. Human
activity and modern day agriculture are one of the key factors in the emergence of
begomoviruses in various parts of the world. Changing crop systems (introducing new
crops) to accommodate the growing need of consumers due to increasing population,
cultivation of susceptible genotypes in extensive areas, increasing use of insecticides,
movement of plant material across borders and introduction of exotic viruses and their
vectoring insects into new environments have been implicated in the outbreaks of
begomoviruses in different crops (Varma and Malathi, 2003; Morales and Anderson, 2001).
Green-house cultivation, introduction and increase in the population of whiteflies and
global warming are possible reasons for the spread of begomoviruses into more temperate
areas. Evolutionary capabilities of begomoviruses through recombination and pseudorecombination are the intrinsic variability features that can give rise to new species or
variants which can start new disease epidemics in previously unaffected regions (Harrison
and Robinson, 1999). The present work is one of the pioneer studies on begomoviral
diseases in Himachal Pradesh, in which several begomovirus-related diseases have been
described. First begomoviral disease in Himachal Pradesh was reported in 2001 on the
weeds A. conyzoides and A. houstonianum which are found widely along the road sides,
field bunds, water channels, tea gardens, orchards, pastures and grasslands (Sharma et al.,
2001). Later in 2006, first molecular evidence of tomato leaf curl disease was reported from
different areas of Solan district of Himachal Pradesh at different altitudes ranging from
600-1650 amsl (Sambyal et al., 2006). In the present study, initially we focused on the
emerging begomoviral diseases on solanaceous crops cultivated in sub-temperate regions of

Discussion
Kangra district but later on we worked out the scenario of these diseases on other important
crops and weeds. Genomes of the infecting begomoviruses were completely characterized
(Table 5.1). Both monopartite and bipartite begomoviruses were found in this region and
association of betasatellite and alphasatellite molecules was also evident.

5.1.1 Tomato leaf curl disease


In 2006, leaf curl disease was observed on tomato plants growing in green houses in
Palampur area of Himachal Pradesh. Association of whiteflies with the symptomatic plants
pointed towards begomovirus infection. The symptoms were distinct than the typical leaf
curling and comprised of downward leaf curl, yellowing, thickening and crumpling of the
leaves. As expected, a distinct begomovirus species was diagnosed to cause this disease.
RCA technique and cloning of RCA products into suitable vector were standardized and
complete bipartite genome of the infecting begomovirus was molecularly characterized
(accession numbers AM884015 and AM992534). Complete DNA-A component showed a
maximum of 86% nucleotide identity with all other known begomoviruses, below the cutoff limit of 89% for distinct begomovirus species demarcation (Fauquet and Stanley 2005,
Fauquet et al., 2008). A name tomato leaf curl Palampur virus was proposed for this new
species. No satellite molecule was detected from the symptomatic plants. Tomato-infecting
begomoviruses are widespread in India (Reddy et al., 2005) and till date, eleven distinct
begomoviruses (both mono and bipartite) have been reported to infect tomato (Table 5.2).
ToLCPMV has an additional ORF AC5 in DNA-A, similar to some recently
described begomoviruses. It was observed that the coat protein gene is the most conserved
and the IR is the most variable part of begomovirus genome. The virus showed closest
relationship with ToLCNDV and some cucurbit-infecting begomoviruses. ToLCPMV was
later reported to cause up to 100% loss of cucurbit crops growing in greenhouses in Iran
(Heydarnejad et al., 2009). ToLCPMV has also been reported to cause a severe disease in
bitter gourd in Pakistan (Ali et al., 2010). ToLCPMV along with Pepper leaf curl
betasatellite has recently been reported to naturally infect pumpkin in India (Namrata et al.,
2011). ToLCPMV has also been reported to cause severe yellow leaf curl disease on

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Discussion

Table 5.1 List of completely sequenced begomoviruses in this study


Virus

Acc. no.

Tomato leaf curl Palampur virus: A distinct bipartite


begomovirus species infecting tomato

DNA-A: AM884015
DNA-B: AM992534

Chilli leaf curl Palampur virus: A distinct begomovirus


species infecting chilli and associated with betasatellite

DNA-A: FM877858
Beta: FM877803

An isolate of Tomato leaf curl New Delhi virus associated


with potato leaf curl disease

DNA-A: AM850115
DNA-B: FN356024

A tentatively distinct strain of Ageratum enation virus


associated with DNA1 and causing Zinnia leaf curl disease

DNA-A: FN543099
DNA 1: FN543100

AEV associated with DNA1 causing vein clearing disease


in Crassocephalum crepidioides

DNA-A: FN794201
DNA 1: FN794202

AEV associated with DNA1 causing vein clearing disease


in Ageratum conyzoides

DNA-A: FN794198
DNA 1: FN794199

An isolate of Mungbean yellow mosaic India virus


infecting common beans

DNA-A: FN794200
DNA-B: FR714861

Table 5.2 List of begomoviruses infecting tomato in India


S. No.
1
2
3
4
5
6
7
8
9
10
11

Virus name
Tomato leaf curl New Delhi virus
Tomato leaf curl Karnataka virus
Tomato leaf curl Bangalore virus
Tomato leaf curl Gujarat virus
Tomato leaf curl Kerala virus
Tomato leaf curl Pune virus
Tomato leaf curl Joydebpur virus
Tomato leaf curl Palampur virus
Tomato leaf curl Patna virus
Papaya leaf curl virus
Pepper leaf curl Bangladesh virus

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Genome
Bipartite
Monopartite
Monopartite
Bipartite
Monopartite
Monopartite
Monopartite
Bipartite
Monopartite
Monopartite
Monopartite

Discussion
muskmelon in Pakistan (Malik et al., 2011). All these reports suggest that ToLCPMV has
some ancestral link with cucurbit-infecting begomoviruses as this virus has been reported
from a number of cucurbit hosts. This speculation was further strengthened by
recombination analysis, which suggested that this new begomovirus could have descended
from a sequence that might have arisen as a result of recombination between ToLCNDV
and SLCCNV-like ancestors. Recombination and pseudo-recombination are major factors
contributing to begomovirus evolution and diversity, and might help in emergence of new
extremely virulent virus variants (Rojas et al., 2005a, b; Harrison and Robinson, 1999;
Moffat, 1999; Padidam et al., 1999). It appeared that ToLCPMV nucleotides between
genome coordinates 220 and 839 have originated from SLCCNV-like ancestor. This region
includes partial pre-coat protein and coat protein genes. But in phylogenetic analysis of the
recombinant region, ToLCPMV branched separately indicating that this region is highly
variable than all other known viruses. DNA-B of ToLCPMV showed very distant
relationship with DNA-B of any other begomovirus and it showed maximum of 73%
nucleotide identity with ToLCNDV and ToLCGV. Any significant recombination event
was not detected in DNA-B component. Analysis of CR of DNA-A and DNA-B
component of all ToLCPMV isolates revealed that it is not completely conserved. A 17 nt
deletion was observed in CR of DNA-B of all ToLCPMV sequences just upstream to the
origin of replication.
ToLCPMV could not be transmitted mechanically. We tried to clone dimers of the
viral genome by partial restriction digestion method described by Wu et al., (2008) and
Ferreira et al., (2008) but were not able to clone the dimers successfully into a binary
vector. To prove Kochs postulates, agro-infectious clones of the virus consisting of 1.4 and
1.8 mer direct tandem repeats of DNA-A and DNA-B components respectively were
constructed in pCAMBIA-1300 in a two-step cloning procedure. Avoiding the use of EtBr
in agarose gel electrophoresis of restriction digested viral DNA showed higher efficiency of
cloning. The infectious clones could produce typical downward curling, yellowing and
wrinkling of the tomato leaves. Downward leaf curl was also observed on N. benthamiana
inoculated with these clones. The viral clones did not produce any symptoms on chilli and
C. sativus plants. Both DNA-A and DNA-B were found to be essential for systemic
infection and induction of leaf yellowing and downward curling symptoms, which
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Discussion
confirmed the bipartite nature of the virus. Elemental analysis using CHNS analyzer
revealed that ToLCPMV infection reduces the quantity of essential elements (carbon,
hydrogen, nitrogen and sulfur) in stem, leaves and roots of the infected tomato plants in
comparison to the non-infected plants. Quantity of CHNS was more in leaves, followed by
shoots and roots. Overall 15-30% decrease in CHNS quantity was estimated in ToLCPMV
infected tomato leaves, roots and shoots.
Malik et al., (2011) also constructed infectious clones of muskmelon isolate of
ToLCPMV. However, the gene encoding the NSP was truncated in comparison to
previously characterized ToLCPMV isolates. Infiltration of agro-infectious clones (with
defective DNA-B) on N. benthamiana did not result in any symptoms. However, DNA-A
component of ToLCPMV when inoculated with DNA-B component of ToLCNDV, lead to
systemic infection with leaf curl symptoms. This data again suggested that ToLCPMV is a
bipartite begomovirus and functional DNA-B is essential for its infectivity. This also shows
that ToLCPMV DNA-A can trans-replicate DNA-B of ToLCNDV and points towards a
possible pseudo-recombination between these two viruses (Malik et al., 2011). The DNA-B
of this ToLCPMV isolate was able to move systemically when inoculated with any of the
two DNA-A components. Agro-infiltration of DNA-A and DNA-B components of
ToLCPMV did not produce symptomatic infection on muskmelon, whereas inoculation of
ToLCPMV DNA-A with the DNA-B of ToLCNDV resulted in a hypersensitive response
along the veins (Malik et al., 2011). Bipartite begomoviruses usually have similar Rep
binding sequences (iterons) in the CR of DNA-A and DNA-B because of which, the DNAA encoded Rep can recognize and initiate replication of the two components (HeyraudNitschke et al., 1995; Laufs et al., 1995b; Orozco and Hanley-Bowdoin, 1996). The iteron
sequences are generally species-specific, thus the Rep of one species usually does not
recognize the iteron sequences of another species (Choi and Stenger 1995; Jupin et al.,
1995). ToLCNDV and ToLCPMV have similar iteron sequences and probably because of
that ToLCPMV DNA-A was able to trans-replicate ToLCNDV DNA-B and produce
symptomatic infection (Malik et al., 2011). Authors also suggested that DNA-B of
ToLCPMV might be non-functional and maintained by DNA-A as a satellite. But our
studies on infectivity of ToLCPMV clearly indicate that DNA-B is an essential component
of ToLCPMV genome and it cannot be considered as a satellite molecule. They also
146

Discussion
provided evidence that ToLCPMV is a part of ToLCNDV and ToLCGV disease complex
(Chakraborty et al., 2003b). Till date, fifteen complete genome and some partial sequences
of ToLCPMV infecting tomato, melon, cucumber, pumpkin, muskmelon and bitter gourd
have been submitted to EMBL database from different parts of the world by various
research groups. This shows that the virus has a wide natural host range and geographical
distribution and it can emerge as one of the most serious begomovirus problems in future.

5.1.2 Chilli leaf curl disease


Chilli is an important and widely cultivated vegetable and spice crop in India. From the
economic point of view, chilli is no longer considered as a minor vegetable crop. Chilli has
been found to be susceptible to various types of pathogens and viruses are considered as
one of the most important factors (Villalon, 1975; Kang et al., 1973). During the past
several years, chilli leaf curl disease (ChLCD) has become a serious problem in most of the
chilli-growing areas of India (Chattopadhyay et al., 2008). Begomoviruses have been a
major threat in chilli for a long time, and disease incidence and severity has greatly
increased in past few years. Chilli leaf curl etiology was established during the 1960s in
India (Mishra et al., 1963; Dhanraj et al., 1968). In India, chilli had been reported to be
infected by begomoviruses namely ChiLCV, ToLCNDV, ToLCGV, ToLCBV and Tomato
leaf curl Joydebpur virus (Chakraborty et al., 2003; Reddy et al., 2005; Khan et al., 2006;
Senanayake et al., 2007; Shih et al., 2007). Previously, there had not been any report of
begomovirus infecting chilli in Himachal Pradesh. Symptoms of upward leaf curling,
stunting and shortening of internodes were observed in chilli fields in Palampur region. A
distinct begomovirus species associated with Chilli leaf curl betasatellite was found to
cause the disease (Kumar et al., 2011a). Chilli leaf curl virus was also detected from
different chilli samples showing leaf curl symptoms but mixed infections were not found. A
total of eighteen samples were collected, out of which four were found positive for a
distinct begomovirus species (ChiLCPaV), whereas six samples were positive for ChiLCV.
In the present study we identified a previously unknown monopartite begomovirus
along with Chilli leaf curl betasatellite, causing leaf curl disease of chilli in Palampur
region of Himachal Pradesh. Studying the emergence of these diseases and analyzing the
causes for the spread of the begomoviruses into more temperate areas is very important for
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Discussion
future disease management. The begomoviral genome (FM877858) and the betasatellite
(FM877803) consisted of 2775 and 1376 nucleotides (nt) respectively. The virus appeared
to be monopartite as no other component was detected with PCR and restriction of the RCA
products. The genome sequence had less than 87.9% identity with all other reported
begomovirus sequences, below the threshold level of 89% for species demarcation. This
suggested that the isolate is a member of a distinct begomoviral species, for which the name
chilli leaf curl Palampur virus was proposed. The additional ORF (ORF5) was not found
in ChiLCPaV genome. The present betasatellite sequence is composed of 1376 nt, encoding
a single 120 aa long C1 protein in complementary orientation and its complete nucleotide
sequence showed 91-94 % identity with various ChLCB sequences, reported mainly from
India and Pakistan. From the previous studies, it is evident that the single protein encoded
by the betasatellites in multifunctional. For most of the viruses (including chilli leaf curl
Palampur virus), the associated betasatellite encodes a dominant pathogenicity determinant
(Gopal et al., 2007; Kon et al., 2007; Cui et al., 2005). Infectious clones consisting of
partial tandem repeats of the viral genome (1.9 mer) and the betasatellite (1.7 mer) were
constructed in binary vector pCAMBIA-1300 and agro-inoculated to chilli and N.
benthamiana. Kochs postulates were fulfilled by agroinoculation of the infectious
constructs of the virus and associated betasatellite. The virus could alone replicate and
systemically infect chilli and N. benthamiana plants producing stunting symptoms in chilli
plants rather than the typical leaf curl. Betasatellite was essential for the induction of leaf
curl symptoms and the single betasatellite-encoded protein might be the major
pathogenicity determinant.
We also described that the present begomovirus sequence might have descended from
a sequence that probably arose through a recombination between ToLCKV and CYVMVlike ancestors. The potential recombination breakpoints were predicted at nt positions 360
and 1214, respectively. The recombinant region (nt 360 to 1214) includes partial V1, V2
and C3 genes and is very closely identical to the corresponding region of ToLCKV.
However some part of ChiLCPaV genome (partial C1 and C2 genes) is very different from
all other known begomoviruses which suggests that there might a third recombination
partner that has not been characterized till date. The study provides another evidence of
interspecific recombination involved in the evolution of begomoviruses. No significant
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Discussion
recombination event was detected in the betasatellite sequence.
For an agricultural country like India, begomoviral diseases have become one of the
major limiting factors for the cultivation of numerous economically important crops.
Further studies on detection and characterization of hitherto unknown viruses are required
to develop a clearer picture of diversity and geographical distribution of these viruses. It
would be important to specifically look for ChiLCPaV infections in surrounding weeds and
other plantations exhibiting geminivirus like symptoms. The number of begomoviruses
infecting chilli is increasing very rapidly. Till 2000, ten begomoviruses were known to
infect chilli throughout the world but this number has increased to thirty since then. Out of
these, twelve viruses are tomato-infecting begomoviruses, which indicate that number of
begomovirus-related diseases of chilli might increase significantly in coming years (Raj et
al., 2010). Vector management is very important aspect for minimizing the losses caused
by begomoviruses. The crop should be carefully noticed for whiteflies from planting to
harvesting period and spraying of efficient insecticides should be done as soon as possible.
Elimination of weed plants in the vicinity of the growing crop is also an important step for
the control of chilli leaf curl disease (Raj et al., 2010). Understanding host-pathogen
interactions and mechanism of RNA silencing suppression of this virus is also an important
future area of research.

5.1.3 Emerging begomoviral diseases of other important crops


ToLCNDV infection has emerged as a serious virus disease in India. Currently it is
widespread throughout India and other neighboring countries like Pakistan, Bangladesh and
Thailand. Till date, about eleven distinct strains of ToLCNDV have been reported to infect
a wide range of agricultural crops like potato, tomato, chilli, bitter gourd, watermelon,
pigeon pea, cowpea, Eclipta spp. and Croton spp. (Raj et al., 2010). Potato is worlds one
of the most important vegetable crops and apical leaf curl disease caused by ToLCNDV
(potato strain) has become a major limiting factor to its cultivation. Delayed setting in
winter in subtropical countries due to global warming and changed cropping pattern might
have helped ToLCNDV to establish on potato (Garg, 2005). In potato growing areas of
Kangra district of Himachal Pradesh (2006-2007), apical leaf curl disease was observed to
be quite common. In slot-blot hybridization using radioactive probe against ToLCNDV CP
149

Discussion
gene, ten out of twenty collected samples were found positive. Based on these results and
visual observations of number of symptomatic plants, a disease incidence of approximately
50% was estimated. The infecting begomovirus was molecularly characterized and
ToLCNDV was found to be associated with the diseased plants. Complete genome of the
isolate (accession numbers AM850115 and FN356024) had closest identity with other
isolates of potato strain of ToLCNDV. Any significant recombination event was not
detected in either DNA-A or DNA-B of this isolate. In phylogenetic analysis of all
available ToLCNDV complete DNA-A sequences, the isolates reported from common
hosts clustered together regardless of their geographical origin. This was the first molecular
study of a begomovirus infecting potato in Himachal Pradesh (Kumar et al., 2010a).
Common bean is another important crop grown in this region. Yellow mosaic
disease of grain legumes is a major constraint to their productivity across the Indian
subcontinent (Varma et al., 1992; Varma and Malathi, 2003). This disease affects the
majority of legume crops viz. mungbean (Vigna radiata), blackgram (Vigna mungo),
pigeonpea (Cajanus cajan), soybean (Glycine max), mothbean (Vigna aconitifolia) and
common bean (P. vulgaris) and yield losses per annum are estimated to be $300 million for
blackgram, mungbean and soybean together (Varma and Malathi, 2003). Yellow mosaic
disease in southern Asia is mainly caused by Mungbean yellow mosaic virus (MYMV),
MYMIV, Dolichos yellow mosaic virus (DoYMV) and Horsegram yellow mosaic virus
(HgYMV) (Qazi et al., 2007b). MYMV is more predominant in southern and western
regions of India, where as MYMIV is widespread in northern, central and eastern parts
(Usharani et al., 2004b; Karthikeyan et al., 2004; Girish and Usha, 2005). A yellow mosaic
disease on common beans was observed in experimental fields of CSK Himachal Pradesh
Agricultural University, HP and the causal pathogen was identified as an isolate of
MYMIV. On visual observation, 60-65% infection was estimated with plants showing
yellow mosaic, downward leaf curling and vein swelling symptoms. Complete bipartite
genome of the isolate was sequenced (accession numbers FN794200 and FR714861) and
complete sequences of DNA-A and, DNA-B showed a maximum of 98-99% identity with
respective sequences of other MYMIV isolates reported from India and Pakistan.
Surprisingly, DNA-A and DNA-B component of this isolate did not share any significant
common region. No satellite component was detected with MYMIV. This study represents
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Discussion
first molecular evidence on MYMIV infecting P. vulgaris in India. This is also a first
record of a begomovirus infecting beans in Himachal Pradesh.
Z. elegans is a popularly cultivated flowering plant in India. A leaf curl disease of
zinnia was described in 1933 (Mathur, 1933) but there was no molecular evidence of the
virus associated with this disease in India. Previously, AlYVV and a betasatellite complex
have been reported to infect zinnias in Vietnam (Ha et al., 2008). Betasatellites have also
been reported to be associated with leaf curl disease of zinnia in Pakistan (Briddon et al.,
2003). Zinnia plants showing upward leaf curl symptoms in this area were found to be
associated with AEV and a nanovirus-like alphasatellite component. Complete genome of
this monopartite begomovirus (FN543099) and the alphasatellite component (FN543100)
was molecularly characterized. Viral genomic sequence had 93% identity with all other
AEV sequences available in GenBank, suggesting that it might be a tentative distinct strain
of AEV. Besides AEV, the sequence showed close identity with isolates of Euphorbia leaf
curl virus (EU194914) and Radish leaf curl virus (EF175733), reported from India. Earlier
it was believed that alphasatellite is always present along with a betasatellite, but in this
study any betasatellite was not detected from the infected samples. Therefore this
association of a begomovirus with an alphasatellite component without any betasatellite
was novel. This report was first molecular evidence of any begomovirus infection in Zinnia
spp. in India and AEV infection throughout the world (Kumar et al., 2010b). The
commonly occurring weeds in this region viz. C. crepidioides and A. conyzoides showing
vein yellowing symptoms were also screened for begomoviruses. It is believed that during
non-cropping seasons the weeds may act as their reservoirs of begomoviruses. AEV and an
alphasatellite were found to be associated with these weeds. Complete AEV and
alphasatellite sequences from C. crepidioides (accession numbers FN794201 and
FN794202) and ageratum (accessions numbers FN794198 and FN794199) are 99%
identical with each other and are closely related to the zinnia isolates. This study provides
additional evidence of weeds acting as reservoirs of begomoviruses. This finding was first
on any begomovirus infection in C. crepidioides in India; the first on AEV infecting C.
crepidioides worldwide and A. conyzoides in India (Kumar et al., 2011b). Any other
begomovirus was not detected from these weeds.

151

Discussion
5.2 ADVANTAGES OF ROLLING CIRCLE AMPLIFICATION IN GEMINIVIRUS
GENOMICS
In the present study, we have amplified whole genomes of the begomoviruses by the
emerging technique i.e. rolling circle amplification. This technique has many advantages
over traditional polymerase chain reaction amplification method. No prior sequence
information of the target is required, which increases the possibility of discovering novel
viruses. As RCA takes place at isothermal conditions, costly thermocyclers are not required
for the amplification. Minimal reagents are required in RCA and it avoids the generation of
false-positive results (Rector et al., 2004; Wang et al., 2005b). The only natural source of
false positive in plants is small mitochondrial plasmids (Homs et al., 2008). RCA also
avoids artificial recombination of DNAs, which may arise during PCR. Proof-reading
activity of -29 DNA polymerase makes RCA a very reliable method. Direct sequencing
of the RCA products can be performed with high precision, which helps in quick detection
of the virus. All infecting circular DNA components can simultaneously be amplified in a
single RCA reaction, including satellites and defective interfering DNAs as well as other
circular DNA viruses present in mixed infections. This feature of RCA makes it much more
informative than PCR (Haible et al., 2006). Another advantage of RCA is that it can work
dried leaf samples and mixed-infected plants (Schubert et al., 2007). Last but not the least;
RCA can be used to construct infectious clones of geminiviruses in a simple one-step
cloning approach (Wu et al., 2008; Ferreira et al., 2008).

5.3 BEGOMOVIRUSES AND RNA SILENCING


RNA silencing is a regulatory as well as a host-defense mechanism, participating in a
number of essential eukaryotic cellular processes and defense against viruses and
transgenes. At first glance, the relevance of RNA silencing for geminiviruses seems limited
because they have a circular DNA genomes, which replicate in the nucleus and lack dsRNA
replicative forms/intermediates. Despite these features, geminiviruses can trigger the host
RNA silencing mechanism and their mRNAs are targeted in infected plants (Lucioli et al.,
2003; Chellappan et al., 2004). It has been shown that during the infection process, siRNAs
are generated corresponding to all regions of the viral genome, including the IR (containing
promoter sequences) and the ori, which are not even transcribed (Chellappan et al., 2004).
152

Discussion
Viruses have developed several mechanisms to evade RNA silencing like evolution of
satellite genomes resistant to siRNAs, defective interfering RNAs, high mutation rates
causing loss of target sequences, formation of secondary structures which are not accessible
to RISC, partitioning of replicating cycles in vesicles, nucleus and chloroplasts, and
encapsidation (Voinnet, 2005). Most of the viruses encode RNA silencing suppressors as
an adaptive response against RNA silencing. Available evidence suggests that in different
viruses, the suppressors have evolved independently as they do not share any significant
feature or sequence identity with one another and they are structurally as well as
functionally different. The first hint about viral suppression of RNA silencing came from
the studies on increased disease severity due to synergistic effect of co-infection of a
potexvirus and potyvirus. In this experiment, potyviral HcPro was discovered as synergism
determinant, which was subsequently identified as a silencing suppressor (Anandalakshmi
et al., 1998; Kasschau et al., 1998). Cucumber mosaic virus 2b protein was soon identified
as another suppressor of RNA silencing. HcPro and 2b were already known to be
pathogenicity determinants, and interestingly most of the suppressors identified from
diverse viruses till date, have been characterized as pathogenicity factors (Brigneti et al.,
1998; Voinnet et al., 1999).
Till date, begomoviruses are known to encode five distinct silencing suppressors
(AC2/C2, C1, AC4/C4, AV2/V2 and Alpha-Rep proteins). In sub-cellular localization
studies of suppressor proteins of different begomoviruses, AC2 protein has always been
reported to localize in the nucleus (Wang et al., 2003; Trinks et al., 2005; Dong et al.,
2003; Kon et al., 2007) whereas AC4/C4 protein binds preferentially to the plasma
membrane as well as to cytosolic membranes including the perinucleus (Fondong et al.,
2007). V2 protein of TYLCV-Is localizes in the cytoplasm and suppresses RNA silencing
in its early stages (Zrachya et al. 2007) whereas C1 is known to suppress systemic RNA
silencing and localizes in cytoplasm as well as in nucleus (Gopal et al., 2007; Kon et al.,
2007; Cui et al., 2005). This implies that these proteins play different roles in the
interaction with the host, and might be targeting different steps in RNA silencing pathway
or might be interacting with different host proteins. In future, the miRNA and siRNA
pathways should thoroughly be explored for antiviral defense mechanisms against
emerging geminiviruses. Identification of host proteins interacting with a viral suppressor
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Discussion
should be a very useful aspect to utilize viral suppressors for a better understating of the
silencing pathway and ultimately virus management.

5.3.1 Suppressors of RNA silencing encoded by ToLCPMV


Due to the growing importance of ToLCPMV, study of the mechanism by which this virus
overcomes the natural RNA silencing was defined as one of the objectives. To investigate
the ability of various proteins of ToLCPMV to suppress RNA silencing, two approaches
were used. ToLCPMV AV2 and AC4 proteins were identified as suppressors of RNA
silencing in these two different assays. Local transient assay was performed to identify a
suppressor involved in early events of establishing RNA silencing (Zrachya et al., 2007). In
this approach, co-infiltration of two A. tumefaciens strains was performed in N. tabacum cv.
Xanthi leaves, one strain carried a binary vector expressing GFP reporter and other carried
a vector harboring viral ORF, as described by Voinnet et al., (2000). Expression of GFP
was monitored in infiltrated leaves 2 dpi onwards to 10 dpi. GFP expressed strongly till 2
dpi (not shown) but it was almost completely silenced at 7 dpi, as anticipated. However, in
the presence of AC4 protein of ToLCPMV, inhibition of RNA silencing was observed as
GFP expression could be detected even at 10 dpi. Results were confirmed by RT-PCR
analysis of GFP transcript. At 10 dpi, GFP transcript could only be amplified in case of
ToLCPMV-AC4 and the positive control. Specificity of the amplified products was
confirmed by direct sequencing of the gel-purified DNA. Constitutively expressed ACTIN
gene was taken as an internal control. The results suggest that AC4 protein of ToLCPMV is
a suppressor of early events of RNA silencing. Sub-cellular localization studies of
AC4:GFP protein revealed that ToLCPMV AC4 is a membrane protein. This finding is
consistent with the localization results of EACMCV AC4 protein (Fondong et al., 2007).
Interestingly in previous studies, begomovirus AC4 protein has been reported to suppress
systemic silencing but in the present study it was detected in an assay that focuses on local
silencing suppression. Begomovirus AC4 protein is known to selectively bind singlestranded small RNAs (including siRNAs miRNAs) (Chellappan et al., 2005). It is believed
that the siRNA as well as the miRNA pathway might restrict virus replication and it has
been demonstrated for a mammalian retrovirus (Lecellier et al., 2005). This hypothesis is
supported by the fact that most of the viral silencing suppressors, when over-expressed in
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Discussion
transgenic plants, interfere with miRNA production and/or its action, which leads to
abnormalities in plant development, often resembling viral symptoms (Voinnet, 2005).
EACMV AC4 protein is also reported to be the determinant of viral pathogenicity
(Fondong et al., 2007). These results suggest that AC4 might be suppressing RNA
silencing by binding to and inactivating siRNAs and interfering with the spread of RNA
silencing signal.
The second approach was reversal of transgene silencing (Voinnet et al., 2000), in
which transgenic N. tabacum cv. Xanthi plants expressing GFP gene as well as siRNAs
against GFP (GFP silent line; Karjee et al., 2008) were inoculated with individual viral
ORFs for their transient expression. This assay identifies a suppressor that acts at later
events of RNA silencing (established RNA silencing). Surprisingly in this assay, AV2
protein of ToLCPMV showed a strong reversal of GFP expression whereas AC4 showed
very mild activity. These results are consistent with the findings on suppression mechanism
of ToLCNDV (Basu et al., 2011). AV2:GFP fusion protein localized exclusively in the
nucleus and occasionally a very weak membrane localization was observed. Weak
membrane localization of AV2 might be due to its possible interaction with AC4 protein
(the other suppressor). AC2 protein of various geminiviruses is known to localize in the
nucleus and induce a host suppressor gene (WEL1; Sharma and Ikegami, 2008). This might
be the case with nuclear localizing AV2 protein of ToLCPMV. These results suggest that
ToLCPMV encodes AC4 and AV2 for suppression of RNA silencing. AC4 acts in the early
stages of RNA silencing whereas AV2 might be acting in the later stages (established RNA
silencing). AC4 is a membrane protein and might be interfering with cell-to-cell spread of
RNA silencing signal but in contrast AV2 is a nuclear and might be involved in suppression
of established RNA silencing. Testing these hypotheses is an important future prospect.

5.4 PROSPECTIVE OF BEGOMOVIRUS MANAGEMENT


In the battle between host defense mediated by RNA silencing and the begomoviruses
equipped with silencing suppressors, the viruses seem to have an edge, as huge losses of
economically very important crops have been recorded in recent past (Yadava et al., 2010).
Keeping this in view, development of management strategies to efficiently contain the virus
becomes a necessity. Use of insecticides to get rid of the whitefly vector, is neither an
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Discussion
effective nor an eco-friendly approach. In modern-day biotechnology, focus is required on
engineering begomovirus resistance through transgenic approach (Dasgupta et al., 2003).
Expression of various full length or truncated or defective proteins of the virus has been
effective in achieving pathogen-derived resistance (Faria et al., 2006). Antisense RNA and
RNAi technology have also been used with some success (Day et al., 1991; Aragao et al.,
1998; Bonfim et al., 2007). Expression of AC1-double-stranded RNA (dsRNA) showed
reliable resistance to Cotton leaf curl virus and Tomato yellow leaf curl virus in transgenic
tobacco and tomato respectively (Asad et al., 2003; Yang et al., 2004). A promising latest
approach using artificial microRNAs to achieve virus resistance has proved very effective
for some viruses (Qu et al., 2007). Apart from these pathogen-derived resistance strategies,
many other approaches like use of DNA binding proteins, ribozymes, GroEL and peptide
aptamers have been attempted to engineer resistance (Chilakamarthi et al., 2007; Akad et
al., 2007; Takenaka et al., 2007; Lopez-Ochoa et al., 2006). Artificial zinc finger proteinmediated resistance has been achieved in case of TYLCV, in which binding activity of viral
Rep was blocked (Takenaka et al., 2007). A hammerhead ribozyme engineered against
Rep showed significant cleavage activity on synthetic Rep transcript (Chilakamarthi et al.,
2007). GroEL gene of Bemisia tabaci when expressed in tomatoes under the control of a
phloem-specific promoter protected the plants from TYLCV infection (Akad et al., 2007).
Peptide aptamer mediated-resistance involves the expression of recombinant proteins that
bind to and inactivate target proteins. Rep-binding peptide aptamer proteins have been
identified for geminiviruses and their potential for providing broad-spectrum resistance has
been demonstrated in plant protoplasts (Lopez-Ochoa et al., 2006). Transgenic resistance
against begomoviruses has shown limited success despite the use of a number of strategies.
Ability of begomoviruses to evolve rapidly by recombination and mutations is the major
limitation to all these strategies. The available evidence suggests that durable resistance
against begomoviruses can only be achieved by combined use of more than one strategies
involving different mechanisms of action; for example artificial microRNA technology
coupled with non-pathogen derived resistance such as GroEL is likely to be much more
efficient. In future, identification and silencing of host genes involved RNA silencing
suppression can be an attractive aspect. Identification of viral suppressors of RNA silencing
becomes important for discovering host genes involved in suppression.
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Discussion
There are many factors for emergence of begomovirus problems in newer areas
including changing cropping systems, increased green-house agricultural practices,
movement of infected planting material, introduction of susceptible plant varieties and
changing weather events. Introductions of new crops or varieties in a country must first be
assessed for their susceptibility on a small scale for a few growing seasons. The exchange
of vegetative plant material from one area to the other should be highly cautious step.
Quarantine practices and testing of imported plating materials should be extremely
thorough and accurate. In future crop management, focus should be made on reducing
vector populations and factors leading to evolution of more virulent begomovirus variants
need to be well understood. Research focus is also needed on interactions between various
satellite molecules and begomoviruses, since there has been a clear affect of such
interactions on disease severity. Availability of natural host resistance should be explored
more and cropping practices leading to reduce the evolution of resistance-breaking virus
variants should be adopted.

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