基于激发防御反应的霜霉菌质外体效应因子PvNLP7发掘圆叶葡萄抗病相关基因的研究

葡萄霜霉病是危害葡萄最为严重的世界性病害之一。发掘葡萄抗病相关基因进行抗病新种质的创制成为霜霉病防治的新策略，但如何准确鉴定出抗病相关基因是目前的新挑战。本申请拟通过研究激发PTI反应的葡萄霜霉菌质外体效应因子NLPs来解决该问题。根据前期转录组分析数据，我们目前已筛选出能够激发植物防御反应的PvNLP7，并明确了它在细胞膜上激活的抗病信号通路。将进一步分析它作为MAMP分子激发植物PTI反应，验证其结构与功能之间的关系。并以之为诱饵，从霜霉菌免疫品种圆叶葡萄‘Noble’中筛选出识别蛋白，利用双分子荧光互补及免疫共沉淀进行互作验证。最后将识别基因稳定转化至欧洲葡萄中，鉴定转基因植株的抗病性，借助蛋白质组学技术分析识别蛋白在葡萄抗病反应中的调控网络。本研究将初步揭示圆叶葡萄识别霜霉菌PvNLP7效应因子的分子机制，为鉴定葡萄抗霜霉病基因提供新思路，同时为葡萄抗病分子育种提供重要的基因资源。

Grape downy mildew, caused by the oomycete pathogen Plasmopara viticola, is one of the most devastating grapevine diseases worldwide. It has been a novel strategy for downy mildew control to dig out resistance related genes for developing resistant grape cultivars. However, how to accurately identify downy mildew resistance related genes from grapevine is the current pivotal challenge. In this proposal, the study on the apoplastic effectors PvNLPs from P. viticola, which can trigger PTI response in plants will be vital to fundamentally solve this issue. According to our previous transcriptomic analysis, we have isolated eight PvNLP effectors and found that only PvNLP7 can induce plant cell death and defence responses. Furthermore, PvNLP7 was found to be localized to the cell membrane and could trigger complicated signaling pathways involved in plant innate immune responses. In the following study, we will investigate the role of PvNLP7, acting as a MAMP molecule, in triggering PTI response in plants, to reveal the relationship between the structure and function of PvNLP7. Subsequently, screening the recognition proteins from downy mildew early-infected Muscadine rotundifolia cv. ‘Noble’ will be performed using PvNLP7 as the bait. The interaction between candidate recognition proteins and PvNLP7 will be verified by bimolecular fluorescence complementation (BiFC) and Co-Immunoprecipitation (Co-IP) assays. Finally, these recognition genes will be stably transformed into European grapevine cultivar‘Thomson Seedless’ to evaluate their roles in resistance to P. viticola. The regulatory network controlled by recognition genes (resistant genes) will be further explored by means of proteomics analysis. This study will preliminarily reveal the molecular mechanism of the perception of PvNLP7 by M. rotundifolia, which will lay a solid theoretical foundation for identification of new resistant genes from grapevine, and also provide crucial gene resources for molecular breeding for grape disease resistance.