Conserved sequence of replicase gene mediated resistance in Nicotiana tabacum L. cv Abirami through RNA silencing

Tobacco streak virus (TSV) is economically the most important virus infecting several crop plants in India. Symptomatic samples collected from sunflower and okra fields were subjected to RT-PCR with TSV replicase gene specific primers. The replicase (Rep) genes of these isolates were sequenced in order to develop transgenic resistance to Tobacco streak virus (TSV) in tobacco (Nicotiana tabacum L.) cv Abirami by expressing hairpin RNA transcript (hpRNA). The sequence analysis of replicase gene of these isolates showed a sequence identity of around 99% at nucleotide level with Tamil Nadu okra isolate. The hairpin construct was generated using pHANNIBAL vector with the conserved nucleotide sequence corresponding to position 3065 to 3405 of the TSV replicase gene. The Rep hairpin construct was cloned into pART27 and mobilized into Agrobacterium tumefaciens LBA4404 and introduced into tobacco by Agrobacterium mediated transformation. The T₀ plants obtained were screened by PCR and Southern blot analysis using the genomic DNA from transformed tobacco plants. The transformants produced ~299 bp and 340 bp amplicons corresponding to nptII gene and Rep gene respectively. The Southern blot analysis showed single and multiple-copy integration of the transgenes. The transgenic T₀ tobacco plants showed resistance against TSV upon mechanical inoculation of TSV without producing any visible symptoms; resistance was also confirmed by DAC-ELISA.