amiR414e-mediated silencing confers resistance to Cotton leaf curl Multan virus in cotton.

backgroundCotton leaf curl disease (CLCuD), caused by monopartite begomoviruses in association with satellite molecules, remains a serious threat to cotton production in the Indian subcontinent. Conventional control strategies have shown limited success, whereas RNA interference (RNAi) has emerged as an effective antiviral approach.

methodsA 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 in the pBI121 vector and transformed into Gossypium hirsutum cv. HS6. Molecular analyses were conducted using PCR and quantitative real-time PCR (qRT-PCR).

resultsTransgenic cotton plants expressing amiR414e were successfully developed, and the transgene exhibited expected Mendelian inheritance patterns in the T1 and T2 generations. At 60 days post inoculation (dpi), PCR assay failed to detect CLCuMuV in all the asymptomatic plants, demonstrating a high degree of resistance in both T1 (80–90%) and T2 (60–72%) lines. Additionally, transgenic (T2) lines overexpressing amiR414e showed significant downregulation of C1 gene transcripts (51–78%), revealing a negative correlation between amiR414e and the C1 gene accumulation.

conclusionIn this study, expression of amiR414e in G. hirsutum cv. HS6 conferred heritable resistance against CLCuMuV, with most of the transgenic plants remaining asymptomatic. These findings demonstrate the potential of amiRNA-mediated gene silencing as a viable and precise strategy for developing CLCuMuV-resistant cotton cultivars.