蛋白激酶A调控辣椒疫霉寄主侵染过程的分子机制

Phytophthora capsici is a highly destructive invasive pathogen that attacks pepper. Based on BLAST searches with representative proteins from well-characterized model organisms, the major subunits of PKA were identified in Phytophthora capsici. The PcPkaR1 gene encodes the regulatory subunit of PKA and PcPkaC1 gene encodes the catalytic subunit of PKA. According to the results of quantitative real-time PCR, PcPkaR1 and PcPkaC1 were up-regulated during early stages of infection, suggesting that PKA plays a role during infection. Molecular mechanisms of PKA’s contributing to P. capsici pathogenicity are interesting and will be learned in this study. 1) For the functional analysis of PcPkaR1 and PcPkaC1, gene-silenced and -overexpression transformants will be generated using CRISPR/Cas9 system and polyethylene glycol (PEG)-mediated protoplast transformation of P. capsici. 2) To identify the cellular component to which the PcPkaR1 and PcPkaC1 proteins will be targeted, fusion proteins with Green Fluorescent Protein (GFP) will be introduced into wild-type strain and examined via epifluorescence microscopy. 3) We will use affinity purification to identify interacting proteins of PcPkaC1 and confirm the associations by co-immunoprecipitation (co-IP) and glutathione S-transferase (GST) pull-down assays. The detailed functions of interacting proteins will be learned via phenotypic analysis of gene-silencing transformants. The functional assignment of PKA and discovery of its target proteins may help to understand the pathogenicity factors affecting plant defense systems and provide clues for identification of new fungicide targets.

辣椒疫霉引起的疫病是辣椒生产上的一种毁灭性病害，但目前针对疫霉菌致病机理的研究较为薄弱。前期研究发现辣椒疫霉蛋白激酶A调控亚基PcPkaR1和催化亚基PcPkaC1均在侵染阶段上调表达，为了进一步解析其调控侵染过程的分子机理，本项目拟在前期研究基础上，利用CRISPR/Cas9基因组编辑技术及原生质体转化技术分别获得PcPkaR1和PcPkaC1敲除突变体及过量表达菌株，通过表型分析明确PcPkaR1和PcPkaC1的生物学功能；采用与荧光蛋白的融合表达和细胞学观察的方法分析其亚细胞定位；通过亲和纯化、co-IP和GST pull-down等方法筛选并验证PcPkaC1的互作蛋白，解析PcPkaC1的下游关键基因及功能，从而揭示蛋白激酶A的功能和信号网络，阐明其参与调控辣椒疫霉生长发育及致病过程的分子机制。该研究结果有望为设计新型植物疫病控制策略和新农药的开发提供重要理论依据。

cAMP信号通路已被证实在调节植物病原菌的形态变化和致病性方面起着重要作用，但在辣椒疫霉中尚缺乏相关研究。在本项目中，利用CRISPR/Cas9基因组编辑技术及原生质体转化技术分别在辣椒疫霉中获得了103个蛋白激酶A调控亚基PcPkaR1候选转化子及和365个蛋白激酶A催化亚基PcPkaC1候选转化子，经筛选后，PcPkaR获得2个异核体敲除转化子，PcPkaC获得1个异核体敲除转化子，进行单孢分离后均无法获得双倍体纯合敲除突变体。鉴于无法对获得的杂合转化子进行生物学功能分析，故同时针对cAMP信号通路的另一个关键基因磷酸二酯酶PcPdeH进行了敲除及功能分析。为了阐明PcPdeH的功能，我们使用了II型CRISPR/Cas9系统在辣椒疫霉中获得了PcPdeH敲除突变体。PcPdeH敲除转化子表现出营养生长缺陷和芽管萌发异常。侵染实验结果表明，与野生型相比，PcPdeH敲除突变体对辣椒和烟草叶片的致病力显著降低，侵染菌丝的扩展受到显著抑制。此外，PcPdeH敲除突变体对外源氧化压力的敏感度显著上升。同时，与野生型和对照菌株相比，PcPdeH的敲除导致胞内cAMP水平增加了1.5-2倍。基于以上结果，我们推测PcPdeH对调控辣椒疫霉的营养生长、芽管萌发、致病性和胞内cAMP水平起着至关重要的作用。该研究为阐述cAMP信号通路在辣椒疫霉致病过程中的作用机制提供了重要的理论基础。