Cucurbit yellow stunting disorder virus and watermelon chlorotic stunt virus induced gene silencing in tobacco plants

Abstract

Cucurbit yellow stunting disorder virus (CYSDV) and Watermelon chlorotic stunt virus (WmCSV) are the most widespread and damaging viruses to cucurbits in the Middle East. CYSDV and WmCSV are cucurbit-infecting bipartite whitefly-transmitted begomoviruses. Post-transcriptional gene silencing (PTGS) is a universal mechanism by which plants are able to systemically switch off the expression of targeted genes via the reduction of steady-state levels of specific RNAs. PTGS was used in this study to control the two viruses. In this study, the efficiency of the dsRNA for the ability to trigger resistance against the CYSDV and WmCSV was investigated. Three regions of three genes of CYSDV genome were selected; the coat protein gene (CP), heat shock gene (Hsp70) and ORF3, while the two regions of two genes of WmCSV genome were selected; CP gene and rep gene. Bioassay, dot-blot hybridization and polymerase chain reaction (PCR) methods were capable to evaluate the resistance against viruses. Clear symptoms on tobacco plants took two to three weeks to appear and all non-infiltrating tobacco plants (positive control) showed viral symptoms after inoculation. Most of the agro-infiltrating sense/antisense constructs did not yield symptoms of the viruses. Dot-blot hybridization, showed that negative hybridization was obtained with infiltrating tobacco plants with prepared constructs compared to those non-infiltrating tobacco plants used as the control. Only one out of five gave positive signals with the construct pasCYSDV-Hsp70. Using PCR, positive reactions of the expected size of 500 bp fragment with WmCSV and 800 bp with CYSDV were obtained with the infiltrating tobacco plants with sense constructs, which pointed out the existence of viral genome in challenging tobacco plants. Infiltrating tobacco plants with sense/antisense constructs gave negative PCR pointed out the lack of the viral genome.