效应蛋白LtGAPR1在葡萄溃疡病菌致病中的作用机理

Lasiodiplodia theobromae is an important plant opportunistic fungus that causes grapevine canker disease. As it spread rapidly around the world and caused serious economic losses, the disease has attracted much attention in recent years. Previous studies have shown that effectors are essential pathogenic factors of grapevine Botryosphaeria pathogens. However, their roles in the pathogenesis of grapevine Botryosphaeria pathogens have not been reported. A putative effector gene, LtGAPR1, which was induced during infection, was identified in our previous transcriptome data. The encoding product of this gene is a secreting protein, and overexpressing LtGAPR1 significantly reduced the virulence of L. theobromae. This study intends to adopt a series of biochemistry, molecular biology and plant pathology method to explore the biological function of LtGAPR1, in order to uncover the functions of LtGAPR1 in the infection process of grapevine Botryosphaeria pathogens and its virulence targets in grapevine. Our results will reveal LtGAPR1 and the orthologous genes’ roles in the plant opportunistic fungi, which will create a theoretical foundation for further characterizing the pathogenic mechanism of grape Botryosphaeria pathogens, and provide a theoretical basis for the ultimate control of this disease.

葡萄溃疡病是一种重要的葡萄枝干病害，近年来在全球迅速蔓延并造成严重损失。已有研究表明，效应蛋白是葡萄溃疡病菌的重要致病因子，但其在葡萄溃疡病菌致病过程中起的具体作用尚无相关报道。我们的前期研究发现，葡萄溃疡病菌中一个推定效应因子基因LtGAPR1在其侵染葡萄过程中显著上调表达，该基因编码的蛋白具有外泌性质，且过量表达菌株的致病力较野生型降低。本项目拟采用表达模式分析、亚细胞定位、酵母双杂交和转基因等一系列生物化学、分子生物学和植物病理学手段来探究葡萄溃疡病菌中推定效应因子LtGAPR1的生物学功能，并分析LtGAPR1在病原菌侵染寄主过程中的作用及其在寄主中的互作靶标。研究结果将有助于揭示LtGAPR1类效应蛋白在病原真菌侵染木本植物中的作用机制，为进一步解析葡萄溃疡病菌的机会性致病机制及其与木本植物的互作机制奠定理论基础，为最终控制该类病原菌提供依据

由葡萄座腔菌科真菌引起的葡萄溃疡病是近年来世界范围内普遍发生的一种重要的葡萄枝干病害。据统计该病害在我国20个葡萄主产省市均有发生，年均发病面积超100万亩，年损失超10亿元人民币，且有逐年加重趋势。虽然葡萄溃疡病得到了广泛关注，但在实际生产中尚未获得高效环保的防控措施，根本原因在于目前对葡萄溃疡病菌的致病机理和葡萄的抗病机理的认识仍然有限。.过敏原类蛋白在调控人和动物中的过敏性反应及机体免疫功能方面发挥了重要作用。前期工作中，本课题组成员通过葡萄溃疡病菌全基因组功能注释，从该病原菌中鉴定了7个假定的过敏原类蛋白，但这些蛋白在葡萄溃疡病菌侵染致病过程中的作用及机制并不清楚。.本研究围绕葡萄溃疡病菌一个假定的过敏原类蛋白LtGAPR1开展功能分析。酵母蔗糖酶分泌系统揭示了LtGAPR1蛋白是一个外泌蛋白，能够分泌至胞外。基因超表达及干涉，结合病原菌侵染实验揭示了LtGAPR1蛋白能够负调控葡萄溃疡病菌的致病力。烟草中瞬时表达实验发现LtGAPR1蛋白能够引起植物的免疫反应。显微观察发现LtGAPR1蛋白全长及信号肽缺失突变体定位在烟草细胞核和细胞质。质谱分析、免疫共沉淀及双分子荧光实验筛选并验证LtGAPR1蛋白与寄主中的靶标蛋白NbPsQ2互作。超表达和干涉NbPsQ2基因能分别提高和降低烟草对葡萄溃疡病菌的免疫力。这些研究结果揭示了LtGAPR1蛋白通过寄主靶标NbPsQ2调控病原菌侵染致病及植物免疫反应的分子机理。.研究结果明晰了LtGAPR1蛋白在葡萄溃疡病菌侵染致病过程中的功能及作用机理，有助于揭示过敏原类效应蛋白在植物机会病原真菌侵染木本植物中的作用，为解析葡萄溃疡病菌的机会性致病机制及其与木本植物的互作机制奠定理论基础，为控制该类病原菌引起的病害提供理论依据。