Smith Neil A, Corral Max, Shu Canwei, Chen-Wang Jiaozi, Zhang Daai, Zhong Chengcheng, Singh Karam, Wood Craig C, Gao Ling-Ling, Anderson Jonathan P, Greaves Ian K, Wang Ming-Bo
CSIRO Agriculture and Food, Clunies Ross Street,Canberra, ACT 2601, Australia.
CSIRO Agriculture and Food, Floreat, WA, Australia.
J Biotechnol. 2025 Oct;406:198-210. doi: 10.1016/j.jbiotec.2025.07.010. Epub 2025 Jul 14.
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.
外源RNA干扰（exoRNAi）已成为一种在植物中进行基因沉默的有前景的工具，但强大而高效的exoRNAi技术仍难以实现。在这里，我们评估了一种用于增强exoRNAi功效的环状末端双链RNA（ledRNA）设计。ledRNA具有哑铃状结构，由双链RNA茎和两侧的单链环组成，双链RNA茎上有一个切口位点。当局部应用于叶片、子叶、分生组织或根时，ledRNA在处理过的组织（叶片、子叶、根）和未处理的组织（相邻叶区域以及远处的子叶和根）中积累的水平均高于传统的发夹RNA（hpRNA）。这表明ledRNA在植物中比传统RNAi分子具有更高的稳定性、更有效的摄取和移动能力。局部递送的ledRNA在本氏烟草叶片中诱导了内源性FAD2基因更强的沉默，导致单不饱和脂肪酸积累增加。除了植物，ledRNA在不同的生命王国中也表现出RNA干扰活性。当引入尖孢镰刀菌的培养物中时，ledRNA有效且特异性地下调了真菌细胞中FoCYP51A、FoCYP51B、FoCYP51C、FoSDHB、FoSDHC和FoSDHD基因的表达。此外，绿色桃蚜（烟蚜）口服靶向关键MpRack-1基因的ledRNA显著降低了它们的繁殖力，导致后代产量下降75%。这些发现突出了ledRNA作为一种有前景的工具，可用于增强基于exoRNAi的基因表达操纵的功效。
