Resistance to cotton leaf curl disease in transgenic tobacco expressing β<i>C1</i> gene derived intron-hairpin RNA

56-62RNA silencing is an adaptive, inducible antiviral defence mechanism in the host against invading viruses. The adaptive antiviral function is characterized by the formation of virus-derived small interfering RNAs (siRNAs) during viral infection. As a counter defense strategy, a number of plant viruses evolve viral suppressors to target antiviral silencing. Cotton leaf curl disease (CLCuD) is a disastrous complex disease caused by presently known five distinct monopartite begomovirus species in association with disease specific betasatellite (DNA-β), which is essential for induction of disease symptoms. Betasatellites are circular, ssDNA molecules that depend on helper viruses for their replication, encapsidation, insect transmission and movement in plants. They possess no appreciable sequence identity to their respective helper viruses except for a conserved hairpin structure necessary for their replication. In this study, siRNA-mediated strategy was applied to generate transgenic tobacco (Nicotiana tabacum) against CLCuD infection. A hairpin (hp) RNAi construct capable of expressing dsRNA homologous to the βC1 gene of Cotton leaf curl Multan betasatellite (CLCuMuB) was designed and developed. A total of eighteen (T0) and seven (T1) independent lines of transformed N. tabacum plants were developed following Agrobacterium tumefaciens-mediated transformation with the βC1 gene-derived intron-(i)hpRNAi construct. Presence of the potential stretch of βC1 was confirmed by PCR coupled with Southern hybridization. The copy numbers of transgene varied between one and three. The transgenic N. tabacum plants of both T0 and T1 lines showed high level of resistance following inoculation with viruliferous whiteflies (Bemisia tabaci). No symptoms were developed on the five (T0) and two (T1) transgenic lines, and remained symptoms free even 90 d post inoculation. The present study has demonstrated that βC1 gene based RNAi-mediated resistance strategy possesses potential to silence CLCuMuB implicated in the induction of CLCuD symptoms