葡萄溃疡病菌效应蛋白LtEpg1调节植物免疫的机理解析

Analysing the role of effectors in phytopathogenic fungi during infection and colonization of host plants is critical for understanding plant-pathogen interactions. However, effectors of grape canker pathogen Lasiodiplodia theobromae has not been reported to date. Interestingly, we have identified an effector named as LtEPG1 in L. theobromae, which was induced during the infection process on grapes. Protein encoded by this gene was predicted to have secretion characteristics and was annotated to be endopolygalacturonase. Our aim of this project is to explore the biological function of LtEpg1 during the process of fungal infection in grapes, to reveal the target receptor in grapevine which interact with LtEpg1, and clarify the response pathways to LtEpg1 in grapevine and the enzyme activity by performing expression pattern analysis, subcellular tissue localization, plant cell transient expression, yeast two-hybrid system analysis, plant transgenics, omics analysis via many biochemical, molecular and phytopathological approaches. Based on above, we can uncover how LtEpg1 helps the fungus to invade the host's immune response to establish a parasitic relationship and cause the disease. This would help us to understand the mechanism of grapevine immunity suppressed by the effector LtEpg1 during the infection process in Lasiodiplodia theobromae. The results can also provide evidence for understanding the interaction between plant opportunistic pathogenic fungi and woody hosts, and ultimately provide a theoretical basis and perspective for controlling similar diseases caused by plant opportunistic pathogenic fungi Lasiodiplodia theobromae.

解析植物病原真菌效应蛋白在病原菌侵染和定殖寄主过程中的作用，对深入理解病原菌与寄主的互作十分关键。葡萄溃疡病菌是（可可毛色二孢菌）重要的植物机会病原真菌，至今还未见到该菌中效应蛋白生物学功能的报道。我们前期分离鉴定到一个在可可毛色二孢菌侵染葡萄过程中诱导表达的效应因子基因LtEPG1，具有外泌特点，注释为糖基水解酶，过表达菌株毒力增强。本项目拟利用表达模式分析、亚细胞组织定位、植物细胞瞬时表达、酵母双杂交、植物转基因和组学分析等系列生物化学、分子生物学和植物病理学手段来探究LtEpg1在病原菌侵染葡萄过程中的生物学功能，寄主的互作蛋白与应答调控途径，效应蛋白的酶活性；以明确LtEpg1如何调节寄主的免疫反应来帮助病原真菌与寄主建立寄生关系而导致病害发生，为揭示葡萄溃疡病菌中效应因子的作用模式和在致病过程中的作用提供实验证据，并为制定植物机会病原真菌引起的同类病害防控技术提供理论与视角。

葡萄座腔菌引起的葡萄溃疡病是世界范围内普遍发生的、且造成严重损失的一种枝干病害。该病害目前在全国20多个省份普遍发生，年损失超10亿元，且有加重趋势。近年来，葡萄溃疡病得到研究人员的高度关注，但有关该病害的发生机制报道较少，特别是有关病原菌效应蛋白如何与寄主互作，协助病原菌突破透寄主的防御并成功进行定殖和扩展而导致病害发生的机理未见公开报道。人们对病原菌致害机制认知上的局限性导致实际生产中尚未构建高效的防控技术。. 糖苷水解酶是植物病原菌侵染寄主时向胞外分泌的一类胞外酶，这类蛋白酶能够破坏寄主的细胞壁结构，获得自身生存需要的营养,识别寄主分泌的抗病因子，调控寄主的免疫反应。葡萄溃疡病菌作为一种长期潜伏在植物内部的机会病原真菌，全基因组包含大量假定的糖苷水解酶，这些糖苷水解酶是否有利于病原菌的营养获取及侵染致病并不清楚。. 本研究围绕葡萄溃疡病菌一个糖苷水解酶LtEPG1开展功能分析。酵母蔗糖酶分泌系统揭示了LtEPG1蛋白的信号肽能够引导蛋白的分泌。基因超表达及RNAi干涉，结合病原菌侵染实验揭示了LtEPG1蛋白能够促进葡萄溃疡病菌的致病。烟草瞬时表达试验发现LtEPG1蛋白能够引起烟草细胞的免疫反应，点突变实验发现LtEPG1蛋白保守的氨基酸位点并不影响其激发活性。蛋白纯化及酶解试验表明LtEPG1蛋白能够以果胶为底物，发挥酶解活性。酵母双杂交及免疫共沉淀试验证明LtEPG1蛋白与寄主中SNF1相关蛋白激酶调节亚基KINβ1互作。表达量检测显示KINβ1基因的表达受到葡萄溃疡病菌的侵染诱导。烟草瞬时表达显示KINβ1蛋白定位在烟草叶片细胞膜和胞内泡状结构。葡萄溃疡病菌侵染LtEPG1和KINβ1转基因植株的转录组测序，揭示了LtEPG1和KINβ1参与调控的寄主免疫应答途径。. 研究结果明晰了LtEPG1蛋白在葡萄溃疡病菌侵染致病过程中的功能及作用机理，揭示了LtEPG1蛋白通过靶标KINβ1调控寄主免疫反应的分子机理。研究结果有助于明晰糖苷水解酶在葡萄溃疡病菌潜伏性生长中发挥的作用，为全面揭示葡萄溃疡病菌的致病机制及其与葡萄的互作机制提供线索，最终为制定植物机会病原真菌引起病害的防控提供一定理论依据。