Begomovirus solanumdelhiense (tomato leaf curl New Delhi virus, ToLCNDV), is member of the genus Begomovirus, family Geminiviridae, is a prolific bipartite whitefly transmitted begomovirus in the Indian sub-continent has a wide host range, including solanaceous, cucurbitaceous and other plants. Recently, dsRNA-mediated non-transgenic approaches have been promising in managing plant viruses. Such an approach could be effective if the pathogenicity determinants of a virus are targeted. In the case of ToLCNDV, viral pathogenicity has been demonstrated with coat protein (AV1), pre-coat protein (AV2), transcription activator protein (AC2) and nuclear shuttle protein (NSP). In the present study, we investigated the involvement of the three RNA silencing suppressor proteins (AV2, AC2, AC4) encoded by ToLCNDV in pathogenicity determinants through transient overexpression and hairpin RNAi-based knockdown assays in Nicotiana benthamiana plants. Further, we showed that the transcripts of AV2, AC2, and AC4 genes can systemically move and express their proteins. Hairpin RNAi constructs targeting each pathogenicity determinant could effectively reduce symptom development and virus titer upon inoculation of ToLCNDV in N. benthamiana plants. Exogenous application of dsRNA individually (dsAV2/dsAC2/dsAC4) or together (cocktail dsRNA: dsCk) against the pathogenicity determinants showed a significant reduction of viral load and reduced severity of disease in plants treated with dsCk followed by dsAC4. The present report reconfirms that the RNA silencing suppressor proteins encoded by DNA-A genomic component of ToLCNDV, can also act as pathogenicity determinants. Further, we demonstrated for the first time that exogenous application of dsRNA targeting those pathogenicity determinants reduces ToLCNDV load and limits symptom development in tomato plants.

Groundnut bud necrosis virus (GBNV) is the most significant member of the genus Orthotospovirus occurring in the Indian subcontinent. There is hardly any effective measure to prevent GBNV in crop plants. In order to develop GBNV infection prevention procedure, we examined the effect of the direct foliar application of double-stranded RNA (dsRNA) derived from the full-length NSs gene (1,320 nucleotides) of GBNV. The bacterially expressed dsRNA to the non-structural (dsNSs) gene of GBNV was purified and delivered to plants as an aqueous suspension containing 0.01% Celite for evaluating its efficacy in preventing GBNV infection in systemic host, Nicotiana benthamiana as well as in local lesion and systemic host, cowpea cv. Pusa Komal (Vigna unguiculata). The dsNSs application and challenge-inoculation were conducted in three different combinations, where plants were challenge-inoculated with GBNV a day after, immediately, and a day before the application of dsNSs. N. benthamiana plants, which were not treated with dsRNA showed severe systemic wilting and death by 9–16 days post-inoculation (dpi). The non-treated cowpea plants exhibited many chlorotic and necrotic lesions on the cotyledonary leaves followed by systemic necrosis and death of the plants by 14–16 dpi. The dsNSs treated plants in all the combinations showed significant reduction of disease severity index in both N. benthamiana and cowpea. The treatment combination where the GBNV inoculation was conducted immediately after the dsNSs treatment was found to be the most effective treatment in preventing symptom expression. The viral RNA analysis by real time PCR also showed 20 and 12.5 fold reduction of GBNV in cowpea and N. benthamiana, respectively. Our results suggest that the foliar application of dsRNA derived from the full-length NSs gene of GBNV through Celite is successful in delivering long dsRNA leading to effective prevention of GBNV infection.