Molecular Characterization of Melon Leaf Curl Disease and Development of Control Strategies
2009
Begomoviruses as
well as potyviruses infect cucurbits in different parts of the world
but are shown to be independent pathogens able to cause severe
disease on their own. A devastating disease suspected to be of viral
origin was found in traditional muskmelon growing-areas of Punjab in
the districts of Sahiwal, Pakpatten and Vehari during 2003.
Characteristic symptoms of the disease in Punjab province were
mosaic and chlorotic spots, leaf distortion and deformation, vein
thickening, enations on the upper side of the leaves, leaf yellowing
and stunting of affected plants. Initial screening of diseased
samples revealed the presence of a bipartite begomovirus and the
potyvirus Zucchini yellow mosaic virus (ZYMV). For comparison with
Punjab, surveys were also conducted in Mardan district of North
Western Frontier Province (NWFP) where plants showed leaf curling,
mosaic and enations on the upper side of the leaf. Results showed
that the disease found on muskmelon in Mardan district was caused by
multiple infections of two viruses, ZYMV and Cucumber mosaic virus
(CMV, family: Cucumoviridae). Dual infection of a potyvirus and a
bipartite begomovirus associated with a severe yellow leaf curl
disease of muskmelon in Punjab was a novel phenomenon and was
investigated in detail. Analysis of the complete nucleotide sequence
of the DNA A and DNA B of the begomovirus showed it to be a new
species that is closely related to Tomato leaf curl new Delhi virus
(ToLCNDV), another bipartite begomovirus widely distributed across
the Indian subcontinent. The intergenic region of DNA A showed a
high level of nucleotide identity with DNA A of ToLCNDV and was
infectious when inoculated with DNA B of ToLCNDV to Nicotiana
benthamiana. The DNA B of this new species named as Muskmelon yellow
leaf curl virus (MYLCV) shows unusual features such as an intergenic
region that did not match with ToLCNDV or any other begomovirus but
retained some of features required for trans-replication, including
the hairpin structure with nonanucleotide sequence conserved in all
geminiviruses, and the rep-binding domains identical to those found
on ToLCNDV. The movement protein (MP) shows high level (87%) of
identity to ToLCNDV but the nuclear shuttle protein gene was
truncated. The DNA B of MYLCV was unable to move systemically when
inoculated with DNA A of either viruses but was maintained when
co-inoculated with DNA A and DNA B of ToLCNDV. Inoculation of
ToLCNDV DNA A and DNA B on muskmelons resulted in localized cell
death, shown previously in tobacco and tomato to be induced by the
NSP. The expression of NSP under the control of the 35S promoter
also caused cell death on muskmelons. To understand the possible
role of ZYMV in the movement of MYLCV, the helper component protein
(HC-Pro) of ZYMV was cloned in a PVX expression vector because
HC-Pro of potyviruses is a multifunctional protein mainly involved
in viral synergistic activities and movement. Inoculation of DNA A
with PVX-HC-Pro resulted in severe leaf curling in N. benthamiana
suggesting a synergistic interaction between potyvirus and MYLCV
where the movement of DNA A of MYLCV was supported by HC-Pro of ZYMV.
The data suggests that the defective DNA B of MYLCV is complemented
by the potyvirus in cucurbits and that this dual infection results
in the severe disease phenotype. The synergistic interaction between
an RNA virus and a DNA virus suggests a novel mechanism to avoid
host defense and complementation of movement by an unrelated viral
protein.
The strategy to engineer a broad based resistance against the
disease complex was based on breaking the synergism between these
viruses by targeting ZYMV using RNA interference (RNAi) technology.
The novel RNAi approach targeted HC-Pro and coat protein (CP) genes
of ZYMV with a single construct. Both tranient assays in wild type
and transgenic N. benthamiana showed successful resistance and
blocking of synergism between viruses. Application of the technology
in melons may provide a durable solution to this important disease
complex that is devastating melon crops in Pakistan.
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