A novel protocol to identify the siRNA hotspots for creating RNAi-based Begomovirus resistance

Begomoviruses cause leaf curl disease, a global threat to crop yield, productivity and food security. Researchers face a significant challenge in developing resistance strategies against these begomoviruses. Here, a novel protocol with a simplified, generic approach has been proposed to target dicot infecting Begomoviruses' DNA-B. In this protocol, small interfering RNA (siRNA) producing regions are identified followed by a search for suitable recombination-free regions among the above hotspot regions. We calculated the efficacy ratio (E-ratio) from the available siRNA parameters, to predict siRNA-producing hotspot regions. Among the resulting 50 highly conserved and recombination-free putative hotspot regions, the majority were derived from the nuclear shuttle protein (NSP)-encoding region. These hotspot regions are promising candidates for deriving recombination-free, specific, effective siRNAs with the least amount of off-target effect. The prospective application of this strategy allows its' integration with any RNAi-based strategy; to gain efficacy when predicted regions of NSP (DNA-B) and prospective DNA-A candidate regions are targeted together against leaf curl causing bipartite begomoviruses. Therefore, we present a protocol for designing siRNAs that meet the criteria of the parameters like conservation, off-target effect, and recombination-free events for long-term generic resistance against leaf curl-causing bipartite begomoviruses.