假单胞菌S58植物表层免疫激发子的鉴定与功能初探

Plant surface immunity is the first layer barrier to defend against phytopathogens, which is activated by plant cell-surface pattern recognition receptors (PRRs) upon perception of microbe general elicitors to provide a broad-spectrum, durable and robust resistance mechanism. Exploiting surface immunity elicitors and plant defense mechanisms are important strategies for the green management of plant disease and can greatly reduce the application of agrochemicals. Pseudomonas sp. S58 was isolated from tobacco rhizosphere in Yunnan province. It could trigger typical surface immune responses in Nicotiana benthamiana and defend tobacco wild fire disease caused by P. syringae. Preliminary results show that a Tn5 mutant being inserted in a lipopeptide encoding gene fails to trigger such immunity. In this study, we would focus on this target and aim to 1) combine genetic mutations, genome mining and comparative metabolomics to isolate and characterize the potential lipopeptide elicitor; 2) deploy comparative transcriptomics (RNA-seq) and quantitative expression analysis (qPCR) to screen and identify surface immunity-related genes triggered by the lipopeptide elicitor; 3) arrange virus-induced gene silencing (VIGS) to exploit the involvement of the gene candidates in lipopeptide-triggered surface immunity and define specific immunity pathways. This project is potent to receive a breakthrough in discovering novel surface immunity elicitors and immune receptors and understanding their activation and regulation mechanisms. The achievement would also enrich the immunity elicitor library and the resistance gene bank, thereby pave the way for the deployment of microbial agents and disease-resistant breeding for plant disease management.

植物表层免疫是植物抵抗病原微生物侵染的第一道防线，具有广谱、持久、高效的特点。发掘和利用表层免疫激发子及其信号传递关键基因是实施绿色防控战略的重要途径。假单胞菌S58是一株分离自云南烟草种植区的生防细菌，可以引起本氏烟的表层免疫反应从而抑制野火病的发生。前期利用Tn5随机突变获得一个免疫抗性丧失的突变体，位点解析表明转座子破坏了一种脂肽合成相关基因。本项目以此为靶标：1）综合利用遗传突变、Genome mining和比较代谢组分析，分离鉴定脂肽激发子，明确其种类和性质；2）通过比较转录组和定量基因表达谱分析，筛选激发子特异的抗病免疫相关基因；3）利用病毒诱导基因沉默对候选基因逐一进行抑制，解析激发子参与的免疫信号途径，获得抗病相关基因。本项目的实施不仅可以在新型表层免疫激发子及其信号途径研究领域实现突破，同时还将丰富免疫抗性激发子资源和抗病基因材料，为微生物免疫制剂开发和抗病育种奠定基础。

利用有益微生物防控植物病害是绿色高效、环境友好的防控手段。假单胞菌是目前研究中最为常见的一类植物益生菌，其代谢产物丰富、功能多样，极具应用潜力。本研究针对云南烟草根际土中分离到的一株广谱高效的病原拮抗菌株地中海假单胞菌S58（Pseudomonas mediterranea），该菌株及其发酵上清液接种本氏烟能够引起不依赖于III型分泌系统的细胞坏死类的免疫反应。利用组学、化学及遗传学手段探究菌株S58潜在的免疫激发子及其诱导本氏烟免疫反应的信号通路。主要结果如下：.1. 利用Tn5转座子随机突变，从14,976个转化子中筛选得到3株丧失激发细胞坏死能力的突变体，插入位点解析证明Tn5破坏了一个脂肽合成基因和一个AHL信号分子合成基因。通过PRISM在线预测发现菌株S58基因组中可能存在9个次生代谢合成基因簇，经同源重组逐一敲除发现cluster6和cluster7突变体丧失激发细胞坏死的能力，且Tn5插入突变破坏的基因正位于cluster6和cluster7。而cluster6主要负责合成AHL信号分子，则cluster7合成的脂肽是激发本氏烟表层免疫的关键物质。.2. 利用制备液相分离纯化，并通过nanoLC-MS/MS结合在线预测，确定该物质为含有22个氨基酸残基的环状脂肽，其结构为C10H19O2-Dhb-Pro-Ala-Ala-Pro-Val-Val-Dhb-Thr-Val-Ile-Dha-Ala-Ala-Ala-Val-Dhb-Thr-Ala-Dab-Ser-Val，并命名为medpeptin。.3. P. mediterranea S58能够诱导本氏烟表层免疫相关基因的上调表达，激发ROS爆发、胼胝质沉积、引起离子渗漏、抑制P. syringae的增殖，而敲除cluster7后除了ROS累积量未受影响，其它免疫相关指标均显著下降。基于转录组测序筛选并验证得到28个可能参与免疫通路的候选基因，并利用VIGS手段构建相应基因的沉默表达植株，最终获得两个可能直接参与medpeptin激发本氏烟表层免疫通路的关键基因C25（Receptor-like protein kinase）和C28（Leucine-rich repeat-containing protein 7）。.综上，本研究从P. mediterranea S58中分离获得一个新的环脂肽类免疫激发子