一个水稻类病斑突变体的基因克隆及其增强抗病的机理分析

Plant diseases and pests cause significant losses to agriculture and pose a serious threat to global food security. Genetic resistance is the most effective and economical approach to control plant diseases and pests. However， as new pathotypes of pathogens emerge, most resistant cultivars often become susceptible with a few years the cultivar released. Therefore, a thorough understanding of plant defense mechanisms is urgently needed. Lesion mimic mutants form necrotic lesions in the absence of pathogens, which is usually due to consistent activation of defense responses. Lesion mimic mutants typically display broad-spectrum resistance to a wide variety of pathogens. Therefore, these mutants constitute important materials in studying plant disease resistance. Before this proposal, a lesion mimic mutant was identified from the newly established whole-genome sequenced fast-neutron-induced mutant population in the Japonica rice variety Kitaake. This mutant displays enhanced resistance to Xanthomonas oryzae pv. oryzae. The gene, encoding a protein with 7 transmembrane domains, has been efficiently cloned using the cosegregation assays with the genome sequence available in a small segregating population. In this proposal, the CRISPR/Cas9 approach or genetic complementation will be used to further study functions of the gene. Omics approaches, including transcriptomics, proteomics and metabolomics, will be used to elucidate the molecular mechanisms of enhanced resistance of the lesion mimic mutant. This proposed study will promote our understanding of molecular mechanisms of plant disease resistance and facilitate optimization of resistance in breeding.

植物病虫害严重影响农业生产，能造成作物的大规模减产，严重威胁了世界粮食安全。培育和种植抗病品种是防治植物病虫害最经济有效的措施。然而新的病原菌生理小种的形成往往导致专化抗性的失效，因此全面深入解析植物抗病防御机制显得尤为重要。植物类病变突变体是在没有病原物侵染情况下就能自发产生病斑的突变体，这类突变往往表现出较好的广谱抗病性，是研究植物抗病机制的重要材料。本项目前期申请人已从水稻快中子突变体库中鉴定出一个类病斑突变体，此突变体表现出对白叶枯病较强的抗性。类病变基因已被克隆，是一个跨膜蛋白。本申请将进一步分析此类病斑突变体的抗性，利用CRISPR/Cas9或者遗传互补验证基因功能，并综合转录组学、蛋白组学和代谢组学技术对类病斑突变体增强抗病的分子机理进行研究，为全面深入解析水稻防御反应及抗病基因的优化奠定基础。

稻瘟病造成水稻严重减产，是一种毁灭性的真菌病害，因此解析水稻抗病防御机制，创制优良种质资源尤为重要。前期研究中，我们筛选到一个水稻类病斑突变体rbl1，对稻瘟病抗性显著增强，rbl1叶片中ROS积累，抗病基因表达上调，抑制了侵染菌丝在细胞间的扩散。我们利用全基因组测序克隆到目的基因RBL1 (RESISTANCE TO BLAST1)，并通过遗传互补和基因敲除验证了鉴定结果。RBL1编码CDP-DAG合成酶，定位在内质网，在所有植物中高度保守；脂质组分析发现，rbl1中PI的含量显著降低，感病因子PI(4,5)P2 细胞膜定位丢失，过表达PIS1可以部分恢复rbl1表型，揭示RBL1通过调节水稻体内的PI生物合成，调控水稻免疫反应的分子机制。进一步通过CRISPR技术编辑RBL1基因，我们筛选到一个广谱抗病株系RBL12，产量没有受到显著影响，为水稻抗病分子育种提供了新的理论切入点。