Artificial miRNA-based silencing of begomovirus gene confers resistance to cotton leaf curl disease in cotton

Background Cotton leaf curl disease (CLCuD), caused by a complex of begomoviruses and their associated betasatellites, severely reduces cotton productivity. Conventional breeding approaches have shown limited success due to the rapid evolution of viral populations and the involvement of the long duration breeding cycles. To address this challenge, the present study explores an artificial microRNA (amiRNA)-based strategy to confer durable resistance against CLCuD in Gossypium hirsutum cv. HS6. Methods A computationally predicted endogenous miR2949a was engineered to generate the artificial miRNA (amiR2949a), which was designed and computationally validated in silico to simultaneously target key genes ( C1, C3, C4, V1 , and V2 ) of Cotton leaf curl Multan virus (CLCuMuV, a begomovirus), as well as the β C1 gene of the betasatellites collectively associated with CLCuD. The engineered pre-amiR2949a construct was transformed into cotton via Agrobacterium tumefaciens -mediated transformation. Putative transgenic plants were subsequently inoculated with viruliferous whiteflies, and resistance was assessed through molecular approaches and phenotypic evaluation. Results The transgenic lines exhibited strong resistance to CLCuD showing 71-81% disease suppression, and a complete absence of disease symptoms and virus accumulation up to 60 days post-inoculation (dpi). Quantitative expression analysis of CLCuMuV, although limited to the C1 gene, revealed a significant reduction (69%-90%) in its transcript levels. This reduction was accompanied by an inverse correlation between amiR2949a accumulation and C1 expression, indicating efficient amiRNA-mediated silencing of viral gene. Conclusion These findings demonstrate that targeted suppression of the viral C1 gene effectively restricts CLCuMuV accumulation and symptom development in cotton. Furthermore, the study establishes amiRNA-mediated RNA silencing as a robust and scalable strategy for engineering resistance against the cotton-infecting begomovirus complex, with potential applicability to other economically important crops.