Doubling down on groundnut bud necrosis virus: combined dsRNAs targeting non-structural (NSs) and nucleocapsid (NP) genes confer superior resistance.

Abstract

Main conclusion: This study demonstrates that dsRNA targeting NSs and NP genes of GBNV effectively reduces viral titre and symptoms, strengthening RNAi defenses and offering a sustainable, eco-friendly approach for crop protection. Groundnut bud necrosis virus (GBNV), a highly destructive Orthotospovirus endemic to the Indian subcontinent, presents a major challenge due to the lack of resistant cultivars and rising insecticide resistance in vector populations. In this study, we evaluated the effectiveness of exogenously applied double-stranded RNAs (dsRNAs) targeting the non-structural (NSs) and nucleocapsid protein (NP) genes of GBNV in inducing RNA interference (RNAi)-mediated resistance. Two dsRNAs were expressed in Escherichia coli HT115 and exogenously applied to Nicotiana benthamiana and Vigna unguiculata (cowpea cv. Pusa Komal) as aqueous suspensions (10 µg/seedling), either individually or in combination (5 µg each). Following GBNV inoculation, untreated controls displayed severe local and systemic symptoms, while dsRNA-treated plants exhibited delayed symptom onset and reduced disease severity. Combined dsRNA treatment resulted in an 87% reduction in disease severity in cowpea and 54% in N. benthamiana. Quantitative real-time PCR revealed significant reductions in viral transcript levels: ~ 35-fold in cowpea and ~ 38-fold in N. benthamiana with combined dsRNAs, compared to 15- and 20-fold (dsNP alone) and 5- and 13-fold (dsNSs alone), respectively. The GBNV Protection Index (GBNVPI) was highest for the combined dsRNA (0.98 in cowpea, 0.90 in N. benthamiana), followed by dsNP (0.92 and 0.78) and dsNSs (0.75 and 0.70). Northern-blot analysis confirmed the generation of virus-derived siRNAs, indicating activation of the RNAi pathway. Overall, cowpea exhibited stronger protection than N. benthamiana. These results demonstrated that co-application of dsRNAs targeting dual genes of GBNV significantly enhanced RNAi-mediated resistance, representing a promising strategy for managing this virus in susceptible crops.