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Abstract
Begomoviruses
(family Geminiviridae) are single-stranded DNA viruses transmitted
by the whitefly Bemisia tabaci. Many economically important diseases
of crops are caused by begomviruses, particularly in developing
countries.These include African cassava mosaic virus (ACMV), which
causes significant losses to the subsistence crop cassava in
sub-Saharan Africa, and the betasatellite-associated begomoviruses
causing cotton leaf curl disease (CLCuD) that causes significant
losses to the mainstay of the economy of Pakistan, cotton.The study
presented here was designed to provide us with a better
understanding of begomovirus pathogenicity, virus host interactions
and the roles of individual virus-encoded genes in these
interactions, as well as the effects of environmental factors on
pathogenicity. Ultimately the information gained may be useful in
allowing the design of better control strategies using either
natural or engineered resistance.
To investigate, on a wider scale than has previously been conducted,
which gene products encoded by begomoviruses are involved in symptom
induction/pathogenicity, all genes encoded by ACMV, Cotton leaf curl
Multan virus (CLCuMV)/Cotton leaf curl Multan betasatellite (CLCuB),
Cabbage leaf curl virus (CbLCuV) and Tomato yellow leaf curl virus (TYLCV),
representing four different classes of begomoviruses, were expressed
in Nicotiana benthamiana using a Potato virus X (PVX) vector. All
efforts to transform the PVX construct with the TYLCV replication
associated protein gene (rep) into Agrobacterium were unsuccessful,
indicating that this Rep may be toxic to Agrobacterium and
precluding its use in all further studies. With the exception of the
REn proteins, the TrAP proteins of ACMV and CLCuMV, the CPs of
CLCuMV and CbLCuV, and the DNA B encoded MP and NSP of CbLCuV and
MSP of ACMV, all other begomovirus proteins, when expressed from PVX,
induced a phenotype above and beyond the mild symptoms induced by
PVX in N. benthamiana.All three Rep proteins induced a severe
symptom phenotype, however, for CLCuMV and CbLCuV, after the initial
severe symptoms plants gradually recovered. Although all (A)V2
proteins induced severe symptoms, that of CLCuMV additionally
induced a necrotic response in both inoculated leaves and leaves
developing subsequent to inoculation that was reminiscent of a
hypersensitive response. PVX-mediated expression of CLCuMB βC1
induced the most unusual symptoms in N. benthamiana.These symptoms
resembled those induced by CLCuD in cotton and consisted of leaf
curling, vein yellowing, stunting and the formation of enations on
the undersides of leaves.These results indicate that, even for quite
closely related viruses within a single genus, each virus interacts
with plants (or at least N.benthamiana) in a distinct fashion and
that the ultimate outcome (the visible symptoms) are likely a
complex interaction of multiple virus-encoded genes with distinct
host factors, or affecting the same host factors to varying degrees.
Overall it is clear that the findings with one virus do not set a
precedent for the other viruses.
MicroRNAs (miRNAs) are small endogenous RNAs that regulate gene
expression in plants and animals. miRNAs are involved in a variety
of activities, including plant development, signal transduction,
protein degradation, response to environmental stress, and pathogen
invasion. Several studied have shown that miRNA-directed processes
might be a general feature of virus pathogenicity.In order to study
this phenomenon, in relation to begomoviruses, infections of N.
benthamiana by four distinct virus species were assessed for their
effects on ten miRNAs known to be important in plant
development.Additionally, the effects of the expression of all genes
encoded by the four begomoviruses from a PVX vector on miRNA levels
were studied. Northern blot analysis using specific oligonucleotide
probes for miRNAs showed that, in general, begomovirus infection
increases the accumulation of miRNAs. However, there was no general
consistency between the viruses, each affecting different miRNAs and
to varying extents.The analysis showed that, essentially, all
begomovirus-encoded gene products have the ability to influence host
miRNA levels, the first time this has been demonstrated.It was also
found that genes encoding suppressor of gene silencing affect the
miRNA level in a significant way when compared with non
suppressors.Again there was a lack of consistency, each virus
appearing to use a different protein, or proteins, to influence
miRNA levels, although there were some minor trends apparent such
as, for example, the
TrAP protein of all viruses analyzed being the strongest
up-regulator of the widest range of miRNAs. Furthermore, the results
suggest that miRNA binding by begomovirus suppressors such as TrAP
and (A)C4, and by implication also those of other phytopathogenic
viruses, are collateral damage; thus that they are unintentional and
result merely from the overlap of the siRNA and miRNA pathways.
RNA interference (RNAi) is a natural defense response of plants
against invading viruses. As a counter-defense viruses encode
suppressors of gene silencing that allow them to effectively invade
plant hosts. Using a novel quantitative real time PCR (qPCR) assay
and conventional northern blot analysis, the ability of all genes
encoded by the begomovirus CLCuMV and its associated betasatellite,
CLCuMB (which together cause CLCuD), were assessed for their ability
to suppress RNAi. The analysis showed that the V2, TrAP, C4 and βC1
proteins exhibit suppressor activity. Although each of these
proteins has, for other viruses, been previously shown to have
suppressor activity, this is the first time all proteins encoded by
a geminivirus (or begomovirus-betasatellite complex) have been
examined and also the first for which four separate suppressors have
been identified. Since all the proteins were examined in a single
experiment, this also allowed comparison of the relative suppressor
activity of each protein, which showed that the strongest activity
was for V2.
Environmental conditions have a marked effect on the infectivity and
spread of viruses in plants.To investigate the effects of
temperature and light intensity on virus spread and gene silencing
in plants, the CLCuMV-encoded V2 protein and the green fluorescent
protein (GFP) were used as markers of PVX infection. Both light and
temperature were found to have profound effects on PVX infection,
with lower light and lower temperatures enhancing virus symptoms and
gene silencing.The enhanced symptoms and silencing were associated
with higher levels of viral RNA and GFPspecific siRNA.Thus siRNA
accumulation and viral transcript accumulation has a positive
correlation.The presence of high levels of siRNA under low
temperature and low light also showed that the RNA silencing pathway
is more active under these conditions.These findings suggest that
RNA silencing has no role in the differential behavior of PVX in
response to varying environmental conditions.
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