组氨酸激酶在辣椒疫霉致病过程中的功能及作用机制分析

Phytophthora are the destructive pathogens on a variety of crops. In recent years, researches on the molecular mechanisms of pathogenesis has been developed rapidly, while the molecular mechanisms of signal regulation is remain only partly known. Histidine kinases (HKs) sense and transduce via phosphorylation events many intra- and extracellular signals, which play the important roles in the pathogenicity of pathogenic microbes, but their functions and regulation mechanism in oomycetes remain unknown. Using bioinformatics methods, we identified three histidine kinases in P. capsici and found that the novel characteristics in system components. Analysis of the expression level of PcHHK2, the results showed that it was significantly up-regulated during the infection, indicating that the gene may be involved in the regulation of pathogen infection process. And, it could respond to different stress. Based on these preliminary results, the project will attempts to validate the function of histidine kinase in the development and pathogenicity by obtaining the knock out mutants and complement mutants, to indentify the genes regulated by histidine kinase and analysis the regulated genes related metabolic and signal pathways through RNA-Seq. The downstream signal pathway of histidine kinase in P. capsici will be analyzed by interactional proteins identify, which explain the regulation mechanisms of histidine kinase in the pathogenicity of pathogenic oomycete. Results of this project will provide the theoretical basis for expanding the cognition of biological TCSs and exploring the pathogenesis of Oomycetes in-depth.

疫霉菌是多种主要农作物的重大致灾病原菌，近年来，其致病机制研究取得了重大突破，但是对其信号调控机制缺乏深入的了解。组氨酸激酶是一类负责信号感应与传导的蛋白，其在病原细菌和真菌的致病性过程中起着重要作用，但是在卵菌中尚未有相关研究。前期，我们在辣椒疫霉中鉴定了3个组氨酸激酶基因，发现其具有独特的结构特征，进一步通过表达分析发现PcHHK2基因在侵染阶段显著上调表达，并且能够对不同的胁迫产生应答。本研究拟通过获得敲除突变体，明确组氨酸激酶在辣椒疫霉生长发育和致病过程中的功能；通过RNA-Seq，鉴定其调控基因，进一步鉴定下游互作蛋白，解析组氨酸激酶在辣椒疫霉感应寄主环境信号和调控基因表达过程中的作用机制。预期研究结果将扩展对组氨酸激酶的认知，深化对卵菌致病机制的理解。

双组分信号传导系统是微生物感知外界环境信号的一种常见机制。组氨酸激酶（HK，Histidine Kinase）是系统中负责信号收集与传导的关键组成元件，广泛参与细胞生理生化过程，但是在卵菌中的功能尚未有相关研究。本研究通过生物信息学在卵菌中鉴定了组氨酸激酶的同源基因，并对其功能域进行预测分析，发现卵菌的组氨酸激酶在结构组成上与真菌的存在显著差异，其是由杂合型组氨酸激酶与磷酸转移蛋白融合形成，这在真菌中是两个独立且存在上下游关系的基因。因此，推测卵菌中的组氨酸激酶在功能上可能会出现一定的分化。表达分析发现，辣椒疫霉的PcHK2在生长发育和侵染阶段显著上调表达，并且能够响应KCl和山梨醇形成的高渗透胁迫及H2O2引起的氧化胁迫。利用CRISPR/Cas9技术在辣椒疫霉中成功敲除了PcHK2，获得2个稳定转化子，通过对转化子进行表型验证发现，PcHK2的缺失导致辣椒疫霉菌丝出现畸形、生长显著减慢，产孢量减少，对不同的胁迫表现出高敏感性，并且致病性显著降低。此外，研究发现PcHK2是调控辣椒疫霉杀菌剂敏感性的重要基因，并对不同类型杀菌剂敏感性的调控途径不同。以上结果表明PcHK2是辣椒疫霉中一个特异的组氨酸激酶，其对辣椒疫霉的生长发育、致病性、抗逆应答以及耐药性都发挥重要的调控作用。进一步通过转录组分析，初步解析了组氨酸激酶PcHK2可通过磷脂酰肌醇信号途径在疫霉抗逆及生长发育中发挥功能；通过调控RxLR效应子及分解酶类等致病相关基因的表达参与疫霉菌的致病；利用荧光素酶互补试验，筛选到1个与组氨酸激酶PcHK2互作的蛋白，该蛋白含有6个WD40基序，并显著响应氧化胁迫反应，为深入探究组氨酸激酶的分子作用机制奠定了基础。受本项目资助，在《Ecotoxicology and Environmental Safety》、《植物保护学报》及《植物病理学报》期刊发表论文4篇。