Molecular Characterization of Melon Leaf Curl Disease and Development of Control Strategies

Abstract

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 XIIIto 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.