Title : Engineered disease resistance in cotton using gene silencing to knock down Cotton leaf curl Kokhran virus-Burewala and Cotton leaf curl Multan betasatellite
Abstract:
Cotton leaf curl virus disease (CLCuD) is caused by a suite of whitefly-transmitted begomoviruses that infect cotton annually in Pakistan, resulting in extensive losses. RNA-interference (RNAi) is proven technology for knocking down gene expression in viral pathogens by targeting vital genes. In this study, a siRNA construct was designed, transformed into cotton plants, and plants were tested for efficacy to silence the expression of the Cotton leaf curl Khokran virus-Burewala (CLCuKoV-Bu) AC1 gene, essential for replication initiation, the Cotton leaf curl Multan betasatellite (CLCuMB) coding region, βC1, and non-coding satellite conserved region, having a role in suppression of plant host immunity. Results of the fluorescence in situ hybridization and karyotyping assays for six T1 plants indicated the transgene was present as a single copy on chromosome number six. Transgenic cotton plants (n=13) and non-transformed, positive control (n=13) were subjected to challenge-inoculation by whitefly transmission of CLCuKoV-Bu and CLCuMB using ten adult viruliferous whiteflies per plant. The absence of characteristically severe leaf curl symptoms on transgenic plants, accompanied by greatly reduced begomoviral-betasatellite accumulation (confirmed by real-time PCR analysis) in transgenic cotton plants, provided evidence of knock down indicative of abatement of CLCuKoV-Bu/CLCuMB biological activity.
