Topical application of conserved dsRNA confers sustainable broad-spectrum resistance against leaf curl disease causing begomoviruses in crops

Abstract

Leaf curl disease caused by begomoviruses is a significant global threat to economically important crops. RNA interference (RNAi), activated by double-stranded RNA (dsRNA) has been proved as an effective strategy to control plant viruses. This study proposes a novel RNAi-mediated approach to combat a broad spectrum of begomoviruses. The conserved regions identified from various DNA-A components of begomoviruses were targeted to design the dsRNA molecules in silico. Further, these putative dsRNAs were synthesized in vitro from the hybrid regions of the AV1/AV2 and AC1/AC4 genes of the papaya leaf curl virus (PaLCuV). A single topical application of these dsRNAs conferred broad spectrum resistance against both PaLCuV (a monopartite begomovirus) and tomato leaf curl Palampur virus (ToLCPalV, a bipartite begomovirus). Semi-quantitative PCR revealed a negligible presence of the virus in dsRNA-treated plants compared to untreated controls. Real-time PCR analysis at 15 days post-inoculation (dpi) showed a significant reduction in virus load and an alleviated expression of AV1, AV2, AC1, and AC4 gene transcripts by approximately 9.6, ∼6.1, ∼4.5, and ∼9.3 fold, respectively, against ToLCPalV in Nicotiana benthamiana. Similarly, in papaya plants dsRNA application reduced virus gene expression by ∼6.0, ∼7.7, ∼6.1, and ∼7.7 fold for AV1, AV2, AC1 and AC4, respectively, compared to untreated controls. These results suggested that the antiviral immunity induced by dsRNA was sustained in plants over time. The study highlights the potential of dsRNA molecules synthesized from conserved regions of begomovirus genome, to confer resistance against leaf curl disease. This is the first report to demonstrate that topical application of dsRNA, can induce broad-spectrum resistance in plants against leaf curl disease-causing begomoviruses, offering a promising tool for managing virus diseases in crops.