Amplicon-based RNAi construct targeting beta-C1 gene gives enhanced resistance against cotton leaf curl disease

Cotton leaf curl disease (CLCuD) is one of the major limiting factors affecting cotton production in Pakistan for the last three decades. The disease is caused by begomoviruses of the family Geminiviridae. RNA interference (RNAi) is a promising tool that has been proved effective against several pathogens. Using RNAi, different genomic regions of geminiviruses have been targeted to attain sustainable resistance. However, the silencing of the transgene upon virus infection is a limiting factor. Here, we have developed for the first time an amplicon-based RNAi construct to target beta C1 gene of betasatellite associated with cotton leaf curl begomoviruses. In addition to producing short interfering (si) RNAs, Rep-based activation or looping out of the construct induced upon virus infection produces multiple copies of transgene that results in accumulation of defective molecules of betasatellite. Subsequent transcription gives rise to increased number of siRNAs that gives enhanced resistance. Transgenic Nicotiana benthamiana plants having RC beta (RNAi construct for betasatellite) were challenged against Cotton leaf curl Khokran virus (CLCuKV) and Cotton leaf curl Multan betasatellite (CLCuMB). Reduced titer of the virus and betasatellite were detected through Southern blot hybridization. Significance of the study has been discussed.