amiR414e-mediated silencing of overlapping C1/C4 genes confers resistance to Cotton leaf curl Multan virus in Gossypium hirsutum cv. HS6

Background Cotton leaf curl disease (CLCuD), caused by monopartite begomovirus in association with satellite molecules, remains a serious threat to cotton production in the Indian subcontinent. Conventional control approaches have shown limited success, whereas RNA interference (RNAi) has proven an effective antiviral approach. Methods A synthetic artificial (a) miR414e was engineered to target the overlapping regions of the C1 and C4 genes of Cotton leaf curl Multan virus (CLCuMuV). The pre-amiR414e construct was cloned into the pBI121 vector and transformed into Gossypium hirsutum cv. HS6. Molecular analyses were conducted using PCR and quantitative real-time PCR (qRT-PCR). Results Transgenic cotton plants expressing amiR414e were successfully developed, and the transgene showed expected Mendelian inheritance in the T ₁ and T ₂ generations. Majority of the plants remained asymptomatic following CLCuMuV inoculation, while a few developed mild symptoms. At 60 days post-inoculation (dpi), PCR assays failed to detect CLCuMuV in all the asymptomatic plants, demonstrating a high degree of resistance in the T ₁ and the T ₂ lines. Additionally, transgenic (T ₂ ) lines overexpressing amiR414e effectively downregulated C1 gene transcripts, showing a negative correlation between amiR414e and the C1 gene accumulation. Conclusion In this study, expression of amiR414e in G. hirsutum cv. HS6 conferred stable resistance against CLCuMuV, with majority of the plants remaining asymptomatic. These findings demonstrate the potential of amiRNA-mediated gene silencing as a viable strategy for developing CLCuMuV resistant cotton.