Enhanced exogenous RNAi by loop-ended double-stranded RNA in plants

Exogenous RNA interference (exoRNAi) has emerged as a promising tool for gene silencing in plants, yet a robust and efficient exoRNAi technology remains elusive. Here we evaluated a Loop-ended dsRNA (ledRNA) design for enhancing exoRNAi efficacy. ledRNA has a dumbbell-like structure, consisting of a long dsRNA stem flanked by single-stranded loops, with a nick site at the dsRNA stem. When topically applied to leaves, cotyledons, meristems or roots, the ledRNA accumulated at higher levels than traditional hairpin RNA (hpRNA) both in the treated tissues (leaf, cotyledon, roots) and untreated tissues (adjacent leaf areas and remote cotyledons and roots). This suggests that ledRNA possesses greater stability and more efficient uptake and movement in plants than the traditional RNAi molecules. Topically delivered ledRNA induced stronger silencing of the endogenous FAD2 gene in N. benthamiana leaves, leading to increased accumulation of monounsaturated fatty acids. Beyond plants, ledRNA also exhibited RNAi activity in diverse kingdoms of life. When introduced into cultures of the fungus Fusarium oxysporum, ledRNA effectively and specifically downregulated expression of the FoCYP51A, FoCYP51B, FoCYP51C, FoSDHB, FoSDHC and FoSDHD genes in the fungal cells. Additionally, oral ingestion of ledRNA targeting the critical MpRack-1 gene by green peach aphids (Myzus persicae) significantly reduced their fecundity, leading to a 75 % decrease in progeny production. These findings highlight ledRNA as a promising tool for enhancing the efficacy of exoRNAi-based manipulation of gene expression.