RNA interference (RNAi) mediated Tomato leaf curl virus gene silencing | Development of RNAi based transgenic resistance in tomato by silencing ToLCV replicase gene

RNA interference (RNAi) is an evolutionarily conserved, homology dependent gene silencing mechanism; an area of upfront basic research, further gaining importance in applications like development of virus resistant transgenics. To explore the possibility of transgenic resistance to the Tomato leaf curl virus,a constraint in tomato production, sub-region of viral replicase gene (truncated rep) was chosen as target for RNA based silencing. In silico analysis of rep gene with siRNA design algorithms and appraisal of rep mRNA, disclosed potent siRNAs and the accessibility of mRNA for gene silencing. Four different RNAi constructs viz., antisense T-rep and three hairpin RNA constructs, were developed and tomato transformed to obtain transformants. Transformation and regeneration of ToLCV infected leaves resulted in regeneration of recovered plants. Interestingly, tomato transformants showed phenotypic anomalies, attributed to the off-target silencing by siRNAs. Thus the book caters to the plant molecular biologists and virologists working on the arena of gene silencing with information on basic criteria for target region selection, construct design strategies and silencing studies.