利用微生物诱导基因沉默技术靶向大丽轮枝菌VdEno基因防控棉花黄萎病

Microbe-induced gene silencing targeting VdEno of Verticillium dahliae for the control of cotton Verticillium wilt

摘要:

小RNA (small RNAs, sRNAs)是RNA干扰(RNA interference, RNAi)的关键因子，不仅能够介导细胞自主基因沉默，也可以作为信号分子跨物种发挥作用。基于种间RNAi的微生物诱导基因沉默(microbe-induced gene silencing, MIGS)技术已经被证实能够有效防控作物真菌病害。本研究以哈茨木霉(Trichoderma harzianum, Th)为底盘菌株，表达致病真菌大丽轮枝菌(Verticillium dahliae)生长发育必需基因VdEno的同源双链RNA (double-stranded RNA, dsRNA)，构建了工程菌株Th-VdEnoi。结果表明，工程菌株Th-VdEnoi能够产生目标sRNA (siVdEno)。进一步实验证实，Th-VdEnoi菌株上清液能够在翻译水平上诱导大丽轮枝菌V592的VdEno基因沉默。盆栽防控实验结果显示，与单独接种V592或者同时接种V592和Th-GFPi工程菌株的棉花相比，同时接种V592和Th-VdEnoi的棉花黄萎病病情指数明显降低，棉花根中病原菌的生物量明显减少。综合以上结果，本研究证实了VdEno基因是防控棉花黄萎病的有效靶标；MIGS技术是防控作物土传真菌病害的有效方法。MIGS技术为减少传统化学农药使用、开发环保型生物农药、促进农业可持续发展提供了技术支撑。

Abstract:

Small RNAs (sRNAs), the key components of RNA interference (RNAi) or RNA silencing, can mediate cell-autonomous gene silencing and function as signaling molecules across species. Microbe-induced gene silencing (MIGS), which is based on interspecies RNAi, is an effective approach for controlling fungal diseases in crops. The enolase gene VdEno is essential for the growth and development of the fungal pathogen Verticillium dahliae, which causes cotton Verticillium wilt. In this study, we engineered Trichoderma harzianum (Th) to express the double-stranded RNA (dsRNA) targeting VdEno. The engineered strain Th-VdEnoi successfully generated VdEno-specific small interfering RNA (siVdEno). We further confirmed that Th-VdEnoi effectively induced VdEno silencing at the translational level. The results of crop protection assays revealed that the cotton plants co-inoculated with V. dahliae (strain V592) and Th-VdEnoi presented significantly reduced disease severity and lower fungal biomass in their roots than the control plants inoculated with V. dahliae alone or with V. dahliae and Th-GFPi (a control strain expressing GFP-targeting dsRNA). Collectively, our findings demonstrate that VdEno is an effective target for controlling cotton Verticillium wilt and confirm that MIGS is a promising strategy for managing soil-borne fungal pathogens in crops. MIGS provides strong technical support for reducing the application of conventional chemical pesticides, developing eco-friendly biopesticides, and facilitating the sustainable development of agriculture.