寄主诱导核盘菌SscipC基因沉默增强油菜的菌核病抗性

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most devastating diseases in rapeseed, but the resistance breeding of rapeseed is hampered due to the lack of resistant source. In our previous study, transcriptome sequencing was conducted in S. sclerotiorum before and after infecting the host Brassica oleracea and non-host species rice (Oryza sativa), we found a S. sclerotiorum gene SscipC which involved in mycelium and infection cushion development showed significantly suppressed expression in rice. RNAi-based gene silencing of SscipC in S. sclerotiorum resulted in reduced virulence on host plant. Special sRNA in rice targeting SscipC was detected by Bioinformatics analysis and sRNA sequencing, but there were no such sRNA in Brassicacea. We hypothesis that sRNA targeting the pathogenicity gene may involve in the immune mechanism of rice against S. sclerotiorum. In this project, we will knockout the special sRNA in rice via CRISPR/Cas9 system, conduct the HIGS vector and express the sRNA targeting SscipC in HIGS transgenic rapeseed. This project endeavor to elucidate the resistance mechanism of sRNA in rice against S. sclerotiorum, and the mechanism will be utilized in improving the Sclerotinia resistance of rapeseed via HIGS. This project will theoretically and practically be of great benefit for the Sclerotinia resistance breeding of rapeseed.

核盘菌引起的菌核病是危害我国油菜生产的一大重要病害，而目前油菜抗病育种受制于抗病资源的缺乏。我们前期对核盘菌侵染寄主甘蓝和非寄主水稻进行转录组测序，发现与菌丝生长和侵染垫形成相关的核盘菌基因SscipC的表达在水稻中被显著抑制，沉默该基因后的核盘菌致病力显著下降。生物信息学预测和sRNA测序发现水稻能产生靶向SscipC基因的sRNA，而芸苔属作物中则没有。我们推测水稻能产生靶向核盘菌致病基因的sRNA可能是其对核盘菌免疫的一个原因。本研究拟利用CRISPR/Cas9技术敲除水稻sRNA的关键序列，并根据SscipC基因序列和水稻sRNA序列构建寄主诱导的基因沉默（HIGS）载体转化油菜，在油菜中特异表达靶向SscipC基因的sRNA。本研究试图阐明sRNA在水稻抗菌核病中的分子机制，并以此为线索利用HIGS改良油菜的菌核病抗性。该项目的执行将对油菜菌核病抗病育种具有重要的理论和实践意义。

核盘菌引起的菌核病是危害我国油菜生产的一个重要病害，而目前油菜抗病育种受制于抗病资源的缺乏。sRNA在植物-病原菌互作中可跨物种进行基因沉默，寄主诱导的基因沉默（HIGS）已被广泛用于寄主的抗病性改良。我们前期对侵染核盘菌的材料进行转录组测序分析，筛选到一个真菌特异基因SscipC在侵染寄主甘蓝中高表达，同时水稻中存在一个特异sRNA靶向SscipC。本项目研究发现沉默该基因后，转化子菌丝生长异常，致病力显著下降。构建靶向SscipC的水稻sRNA载体转化拟南芥后，转基因株系的菌核病抗性显著增强。将SscipC-HIGS转入油菜中，能显著增强转基因油菜的菌核病抗性。转录组分析、共表达分析及酵母互作分析发现核盘菌SscipC基因通过影响核盘菌核糖体蛋白表达来参与核盘菌菌丝生长、侵染垫形成及致病过程。本项目研究拓宽了植物菌核病抗性育种的抗源筛选范围，为菌核病的安全防控提供了一个重要的靶标基因。