Screening of a multi-virus resistant RNAi construct in cowpea through transient vacuum infiltration method.

Abstract

Plant viruses are the most devastating pathogens causing substantial economic losses in many crops. Current viral disease management relies on prophylactics, roguing and insect vector control, since in most crops resistant gene pools for resistance breeding are unavailable. RNA interference, a sequence dependent gene silencing mechanism holds great potential in imparting virus resistance. In this study, the efficacy of a RNAi gene construct developed against four viruses commonly infesting tomato and chilli viz., capsicum chlorosis virus, groundnut bud necrosis virus, cucumber mosaic virus and chilli veinal mottle virus was evaluated. A 3546bp dsRNA-forming construct comprising sense-intron-antisense fragments in binary vector pBI121 (hpRNAi-MVR) was mobilized into Agrobacterium tumefaciens. Cowpea (Vigna unguiculata) was used as an indicator plant for GBNV agroinfiltration to evaluate the efficacy of hpRNAi-MVR construct in conferring GBNV resistance. The type of agroinfiltration, bacterial concentration and incubation-temperatures were optimized. Vacuum infiltration of three pulses of 20-30s at 66.66kPa were effective than syringe infiltration. Of the five Agrobacterial concentrations, OD600 0.5 was more efficient. Incubation temperature of 31 ± 1°C was favorable for development of disease symptoms than 20 ± 1°C and 26 ± 1°C. ELISA revealed a 35% decline in viral load in hpRNAi-MVR infiltrated plants compared to vector control plants. Quantitative real time PCR results have shown a viral gene silencing to the extent of 930-990 folds in hpRNAi-MVR infiltrated plants compared to vector control. This approach is simple, rapid and efficient to screen the efficacy of RNAi constructs developed for the RNAi mediated plant virus management.