苹果树腐烂病菌CAP蛋白超家族成员VmPR1c的致病机理研究

Apple Valsa canker, caused by Valsa mali, is one of the most devastating diseases and key problem in China and difficult to manage problems. Therefore, better understanding of their pathogenicity mechanisms is crucial for developing more effective disease management strategies. During the pathogen-host co-evolution, mutual recognition or avoiding recognition will lead to pathogenic or immune response. Pathogen has evolved several strategies to successfully infect their hosts to obtain nutrients such as secreting some virulence factors to suppress the host defense. CAP family members are mostly secreted proteins with a highly conserved CAP domain and have been implicated in a variety of physiological contexts, including immune defense in plants and mammals and toxicity of insect and reptile venoms. CAP proteins have recently emerged as novel virulence factors in pathogenic fungi. However, their mode of action has remained elusive. Previous studies have shown that the transcripts level of VmPR1c, a member of CAP proteins, was above 9-fold higher at 6 hpi than 0 hpi. The virulence of VmPR1c deletion mutant was drastically impaired, suggesting that VmPR1c is required for the virulence of V. mali. Therefore, this project plan to characterize the feature of VmPR1c, identify the host target proteins using yeast two hybrid, Co-IP and BiFC, and understand their function in transgenic apple. These results may reveal the novel mechanism of pathogenicity causing by VmPR1c, which will help us develop more precisely targeted disease management strategies.

由子囊菌Valsa mali引起的苹果树腐烂病造成皮烂枝枯树死，一直是苹果生产上的重点防治对象和难题，因此，尽快解析病原菌的致病机理，将为研发新的高效防治技术提供理论依据和物质基础。在病原菌与寄主长期协同进化过程中，不断的相互识别和躲避识别导致了致病或免疫，病原菌必须利用多种策略和手段克服植物的防御屏障方可成功入侵，其中外泌毒性因子发挥着重要功能。CAP蛋白具有高度保守的结构域，大多数可外泌，其功能和靶标多样。真菌CAP蛋白主要参与致病，但作用机理未知。本项目前期研究发现苹果树腐烂病菌CAP蛋白VmPR1c基因只在侵染初期6h上调表达9倍以上，其敲除突变体致病力几乎丧失，是关键致病因子但作用机理不明。为此，本项目拟首先揭示VmPR1c蛋白特征，并利用Y2H、Co-IP和BiFC筛选鉴定互作靶标，进而分析靶标功能，明确VmPR1c致病机理，为加快该重大病害防控理论研究提供新的思路和策略。

由子囊菌苹果黑腐皮壳（Valsa mali）引起的苹果树腐烂病严重制约我国苹果产业的健康发展。解析病原菌的致病机理，将为研发新的高效防治技术提供理论依据和物质基础。在病原菌与寄主长期协同进化过程中，病原菌利用多种策略和手段克服植物的防御屏障方可成功入侵，其中外泌毒性因子发挥着重要功能。本项目前期从V. mali强致病力菌株03-8的基因组中鉴定到三个编码CAP超家族蛋白的基因（VmPR1a、VmPR1b和VmPR1c），其中VmPR1c基因敲除突变体致病力显著降低。为了揭示VmPR1c在腐烂病菌中致病分子机理，本项目围绕VmPR1的毒力功能和蛋白特征、互作靶标及调控机制开展研究。取得的主要结果如下：1. VmPR1c是腐烂病菌中CAP超家族蛋白中致病关键因子，其关键的毒力功能和蛋白特征结构域为CTE和CAP结构域以及第211位组氨酸。2. VmPR1c与苹果VQ家族蛋白MdVQ29互作，促进MdVQ29蛋白通过26S泛素蛋白酶体途径降解，从而抑制其下游转录因子MdWRKY23的转录激活和MdCOI1介导的JA信号转导通路，最终导致寄主抗病性降低。3. 苹果RING型E3泛素连接酶MdULE3蛋白作为VmPR1c的互作靶标，通过抑制VmPR1c的毒力功能提高苹果的抗病性。4. VmPR1c也可与苹果细胞分裂S期蛋白激酶MdSkp1互作，其通过影响 MdSKIP14与MdSkp1的互作，抑制苹果对V. mali的抗病性，从而促进病原菌的侵染。5. VmPR1c与苹果病程相关蛋白MdPR1a及其同源蛋白MdPR1b、MdPR1c互作，其毒力功能受到这3个蛋白的影响。通过本项目的实施，揭示了VmPR1c参与病菌与寄主互作的分子机理，同时创制了MdVQ29转基因苹果抗病材料，研究结果将为加快该重大病害防控理论研究和广谱抗病育种提供新的思路、策略和材料。