Hairpin-RNA Spray Confers Resistance to Mungbean Yellow Mosaic India Virus in Mungbean

The prevalence of Begomovirus diseases poses a significant threat to legume crops, necessitating the exploration of innovative control measures. This investigation explores the utilization of dsRNA molecules to initiate RNA interference (RNAi) targeting begomovirus, particularly focusing on Mungbean yellow mosaic India virus (MYMIV) and its potential threat to mungbean crops. Given the lack of genetic resistance in commercially available mungbean varieties, the study endeavors to employ RNAi as a strategic method for the effective control of MYMIV. The approach involves the preparation of vectors for the transient expression of three dsRNA targeting multiple overlapping ORFs of MYMIV DNA A through agroinoculation, and the selection of a highly efficient construct for dsRNA expression in bacteria, enabling topical application to mungbean plants in growth chamber experiments. Agroinoculation assays demonstrate effective resistance against MYMIV, as confirmed by reduced symptom severity, limited virus accumulation, and the presence of viral mRNAs. The stability of the prepared dsRNA against nucleases is confirmed, showcasing its ability to enter plant cells, move to non treated trifoliate leaves, and form siRNA when sprayed onto mungbean leaves, as validated by qRT-PCR and northern blotting. Varied combinations of the timing of dsRNA spray and virus infection reveal differential resistance against the virus. Notably, spraying two days before or on the same day as virus exposure emerges as the most suitable time to achieve optimal resistance against virus infection. In light of these findings, the topical application of dsRNAs stands out as a promising and effective strategy for MYMIV control in mungbean crops.