RNAI MEDIATED RESISTANCE AGAINST GEMINIVIRUSES

The main objective of this study was to develop resistance against cotton leaf curl virus (CLCuV) using a novel strategy based on RNAi. Cotton leaf curl disease (CLCuD) is associated with several distinct geminivirus of the genus Begomovirus, which are able to replicate and spread in susceptible host plants including cotton when inoculated with DNA satellite called DNA beta. DNA beta is a novel single stranded DNA satellite of approximately 1350 nucleotides in length that has been isolated from infected cotton plants is found to be essential for induction of the characteristic symptoms in cotton. DNA β is biologically functional, being required for amplification of DNA A to high levels and for the efficient systemic spread of infection throughout susceptible cotton plant. Two strategies (transient assay and stable transformation) were used to block beta molecule through RNAi technology. The β Cl-C2 genes encoded by CLCuV/DNA β (U89) were targeted in transient assay and β Cl CLCuV/DNA β (U77-4) for stable transformation to developed resistance against CLCuD. Since β Cl protein is a virulence determinant that suppresses RNA silencing and hypersensitive response (HR), the targeting of this gene is likely to protect plants against virus infection. Results confirmed that DNA beta encodes a virulent factor that suppresses host defense responses. The Cl-C2 region was cloned in RNAi vector (pFGC5941 dsRNA) and vector containing the same in the sense or antisense and both orientations in the same vector were also developed. For stable transformation beta Cl along with promoter region was cloned in plant expression vector pBin61 for producing dsRNA through double promoter (one side of Cl gene is 35S promoter and other side is β promoter). These constructs were transformed in A. tumefaciens strain LBA 4404. The RNAi vector containing Cl-C2 region of DNA β was then transiently expressed in N. bethamiana in combinations of infectious clones {DNA A (PK3) + DNA β and DNA A (H12) + DNA β }. The Cl region of DNA β was then transformed in N. tabacum through Agrobacterium mediated leaf disc transformation. The presence of transgene was confirmed through PCR and southern hybridization in transgenic plants. Evaluation of transgenic plants through whitefly inoculation and infectious clones showed the complete blockage of β replication and developed resistance against CLCuV. Results showed that RNA mediated silencing of a virulence gene derived from a DNA satellite can interfere with virus infection in a sequence-specific manner. Our results support the view that RNAi can be successfully used to control plant DNA viruse