基于葡萄白粉菌关键效应因子鉴定野生华东葡萄抗白粉病基因

Grapevine powdery mildew is the most important fungal disease of worldwide grape growing regions. The wild Chinese grapevine Vitis pseudoreticulata accession Baihe-35-1 was demonstrated to be highly resistant to multiple diseases, including powdery mildew and downy mildew in the past forty years. Recent studies of powdery mildew effectors have provided a good idea for isolating new disease resistance genes in plants. Based on our previous results of the genome of Erysiphe necator isolate, NAFU1,which was isolated by our group in 2011 at the vineyards of Norhwest of China. In this project, we will further analyze the characterization of NAFU1 effector comapred with other powdery mildew pathogens using genomics and bioinformation methos. We will identify the key effectors which could induce hypersensitive response cell death in Vitis pseudoreticulata accession Baihe-35-1 inoculation with E. necator isolate NAFU1. We will try to illustrate the expression profile of the key effectors of NAFU1 during key infection time point after inoculation with NAFU1. To identify the disease resistance proteins interacted with that key effectors in the wild Chinese grapevine Vitis pseudoreticulata accession Baihe-35-1, we will screen a yeast cDNA bank induced by powdery mildew pathogen by a yeast two-hybrid using that key effectors as baits. In addition, we will also investigate the role of the candidate powdery mildew resistance gene(s) in over-expression European grape, and in CRISPR/Cas9 editing wild Chinese grapevine accession Baihe-35-1. The obtained results will not only provide useful information for further understanding the molecular mechanism of wild Chinese grape Vitis pseudoreticulata accession Baihe-35-1 disease resistance to powdery mildew, but also provide more interesting clues to identity disease resistance genes in plant. Also, it can provide more gene resources of resistance to powdery mildew for improvement European grape or other fruit trees in future.

葡萄白粉病是一种危害最严重的世界性葡萄病害。中国野生葡萄是很好的抗病基因资源库。最新的白粉菌效应因子研究结果为挖掘植物抗病基因提供了新思路。本项目是在前期自主获得葡萄白粉菌菌株NAFU1基因组序列的基础上，利用比较基因组学及生物信息学技术，进一步深入分析NAFU1效应因子特征，筛选能在野生华东葡萄上产生过敏性细胞坏死的白粉菌关键效应因子，明确其关键效应因子在白粉菌侵染关键阶段的表达特性，构建白粉菌诱导的酵母cDNA文库，利用筛选出的关键效应因子，鉴定野生华东葡萄白河-35-1抗白粉病基因，在感病葡萄中超量表达，在抗病华东葡萄中进行基因编辑，分析候选抗病基因功能。项目的完成不但有利于加深理解野生华东葡萄抗白粉病分子机理，也可以为挖掘植物抗病基因提供新的线索，还可以为定向改良欧洲葡萄或其他果树抗病性提供优良的基因资源，最终为培育抗病优质葡萄或其他果树新品种（系）提供理论依据和技术支撑。

葡萄白粉病是一种危害最严重的世界性葡萄病害。中国野生葡萄是很好的抗病基因资源库。最新的白粉菌效应因子研究结果为挖掘植物抗病基因提供了新思路。本项目基于本课题组前期保存的葡萄白粉菌生理小种NAFU1，对其基因组进行测序，获得高质量、完整的基因组，其大小为61.12Mb；克隆了NAFU1基因组候选效应因子51个，通过建立的葡萄叶片瞬时转化体系，鉴定到关键效应蛋白2个，分别为CSEP118和CSEP087。沉默CSEP118削弱白粉菌致病性；CSEP118靶向葡萄硫氧还蛋白VviTrxz，促进VviTrxz蛋白积累；而VviTrxz负调控葡萄对白粉菌的抗性；VviTrxz通过调控过氧化氢酶活性调节过氧化氢的积累；VviTrxz与葡萄LRR受体蛋白RLP互作；RLP增强葡萄对白粉菌的抗性；抗病葡萄和感病葡萄中的RLP启动子存在明显差异。基于以上研究结果，提出了抗病葡萄和感病葡萄与白粉菌互作模型。此外，还研究了CSEP087的功能。建立了葡萄基因编辑体系，通过编辑白粉菌负调控基因MLO，提高了葡萄对白粉菌抗性。以上研究结果为生产上利用该基因提供了理论依据。在国内外学术刊物上发表论文6篇，其中SCI源刊论文4篇，中文核心期刊论文2篇；申请国家发明专利2项，获得实用新型专利1项；培养博士1名，培养硕士4名，正在培养博士研究生1名，硕士研究生6名。完成预定研究目标。