根结线虫病原相关分子模式（PAMPs）Mg-eEF1A和MjEF-TU的发现及其触发植物免疫反应的机制

Molecules of Plant pathogens, i.e. pathogen associated molecular patterns (PAMPs), can be recognized by host cell surface localized pattern-recognition receptors (PRRs), which would trigger plant immunity. Currently, several recognition mode from plant bacteria and fungi between PAMPs and PRRs have been characterized. However proteinaceous PAMPs from plant-parasitic nematodes (PPNs) and PRRs haven’t been found yet. Before the project, one elongation factor Mj-eEF1A from Meloidogyne graminicola was found to interact with the PRR CERK1 by using yeast two-hybrid system. Based on this, the other elongation factor MjEF-TU from PPNs was found to match the bacteria PRR EF-TU by using bioinformatics analysis. Moreover, the experiments showed that Mg-eEF1A and MjEF-TU can induce up-regulation of several defense gene expressions in host plants, suggesting that Mg-eEF1A and MjEF-TU are possibly PAMPs of root-knot nematodes. Therefore, the following research contents of this project would be: to analyze the influences of Mg-eEF1A and MjEF-TU on PTI; to study the secretion characteristics of Mg-eEF1A and MjEF-TU; to study their characteristics of induction immunity and action pathway in plants, etc.; then to demonstrate whether Mg-eEF1A and MjEF-TU from Meloidogyne are PAMPs, and their recognition mode with the PRRs. The present project is the first time to provide a new method to get the PPNs PAMPs and it is hopeful to obtain the first proteinaceous PAMPs from PPNs and PRRs, laying foundation for controlling PPNs by using PAMPs/PRRs.

植物模式识别受体（PRRs）可识别病原物的病原相关分子模式（PAMPs），从而激发免疫反应。已报道多种植物病原细菌和真菌的PAMPs/PRRs识别模式，但还未发现植物线虫的蛋白类PAMPs及PRRs。我们前期用植物受体CERK1为诱饵蛋白，通过酵母双杂获得与CERK1互作的拟禾本科根结线虫翻译延长因子Mg-eEF1A，并用生物信息学分析获得与细菌的PAMPs—EF-TU同源的爪哇根结线虫线粒体翻译延长因子MjEF-TU。实验表明它们均可诱导寄主防卫基因上调，暗示它们可能是根结线虫的PAMPs。本项目拟研究Mg-eEF1A和MjEF-TU的分泌特性、对PTI反应的影响、诱导植物抗性及作用通路等，从而明确它们的PAMPs特性及触发植物免疫反应的机制。本项目提供一个鉴定植物线虫PAMPs的新方法，首次发现植物线虫的蛋白类PAMPs及PRRs，为今后利用PAMPs/PRRs防治植物线虫奠定基础。

植物模式识别受体（PRRs）可识别病原物的病原相关分子模式（PAMPs），从而激发免疫反应以抵抗病原物入侵。人们已发现植物病原细菌、真菌和卵菌的PAMPs/PRRs，但植物线虫的蛋白类PAMPs及PRRs还未见报道。在本研究中，我们发现了两个植物线虫的蛋白PAMPs，分别是拟禾本根结线虫的翻译延长因子Mg-eEF1A和爪哇根结线虫的翻译延长因子MjEF-TU。免疫检测分析表明它们可诱导植物活性氧爆发、胼胝质沉积、防卫基因表达提高和MAPK激活等PTI反应，且病原接种发现Mg-eEF1A和MjEF-TU可诱导植物抵抗不同类的病原物。RT-qPCR分析发现Mg-eEF1A和MjEF-TU在线虫寄生阶段表达上调，进一步免疫杂交显示它们均可被线虫释放到体外，其中MjEF-TU被分泌到寄主植物细胞外质体中。酵母双杂、基因编辑和基因突变等分析显示OsCERK1和EFR分别是Mg-eEF1A和MjEF-TU的模式识别受体。此外MjEF-TU的N端具有线粒体前导肽，western blot分析确认MjEF-TU是爪哇根结线虫线粒体蛋白，且EFR可介导线虫线粒体提取物触发植物的免疫反应。我们还通过序列比对、酵母双杂和免疫检测分析探明了Mg-eEF1A和MjEF-TU诱导植物PTI反应的作用位点，分别是Mg-eEF1A的300-464 aa片段，而MjEF-TU N端的18个保守氨基酸（Nelf18）。转录分析和线虫接种表明Mg-eEF1A和MjEF-TU分别影响了OsCERK1和EFR相关的防卫通路，从而影响了线虫的侵染。这些结果表明根结线虫侵入植物根后，其翻译延长因子Mg-eEF1A和MjEF-TU被释放出来，分别被植物的模式识别受体OsCERK1和EFR识别，从而激活了OsCERK1和EFR相关的防卫信号，进而抵抗线虫的侵染。本项目首次发现了植物线虫的蛋白类PAMPs及PRRs，明确了Mg-eEF1A和MjEF-TU被植物OsCERK1和EFR分别识别，从而影响了相关的防卫通路，进而触发植物抗病的机制。本研究还表明EF-TU是一个横跨细菌和线虫的PAMP，线虫的EF-TU定位在线粒体，这是发现的首个在微生物和动物间跨界的PAMP，也首次揭示了线虫可将线粒体蛋白释放到寄主中，为进一步研究病原和寄主的互作提供了新理论和新思路，也为今后利用PAMPs及PRRs防治植物线虫病奠定了基础。