纹枯病菌效应蛋白AGLIP1调控水稻免疫的分子机制

Rhizoctonia solani is one of the most severe necrotrophic pathogens which threat to rice production.The effect of effectors which is secreted by necrotrophic pathogens during infection has growing attention recently. However, little is known about the virulence and mechanisms of these proteins. Previous studies have shown that R. solani AG1 IA effector protein AGLIP1 causes cell death when transiently expressed in Nicotiana benthamiana. In addition, AGLIP1 can inhibit BAX, INF1-induced cell death in Nicotiana benthamiana. Further research has revealed AGLIP1 was up-regulated during R. solani infection. Yeast two-hybrid assay showed that AGLIP1 interacts with rice casein hydrolase OsClpP6. AGLIP1 decreased the accumulation of OsClpP6 in Nicotiana benthamiana. In this study, we will observe the secretion and transportation of AGLIP1 during the infection of rice blast by heterologous expression in Magnaporthe oryzae. Also verify the interaction between AGLIP1 and OsClpP6 in rice by Co-IP assay. Exploring the molecular mechanism of AGLIP1 to reduce the stability of OsClpP6. Initial gene knockout assays will be used to investigate biological characteristics in plant immune response of osclpp6. The results will provide new outlooks in understanding the molecular mechanisms of R. solani pathogenesis and rice resistance.

水稻纹枯病菌是威胁水稻生产的重要死体营养型病原菌。效应蛋白对该类病原菌致病力的影响受到关注，但对其发挥功能的机制还知之甚少。前期研究表明，纹枯病菌效应蛋白AGLIP1在烟草瞬时表达时引起细胞死亡；此外，该蛋白还能抑制BAX、INF1诱导的烟草细胞死亡；该基因在纹枯病菌侵染水稻时上调表达；酵母双杂交试验表明AGLIP1与水稻酪蛋白水解酶OsClpP6互作；并且该蛋白在烟草中降低OsClpP6的稳定性。本研究将在稻瘟病菌中异源表达AGLIP1，观察该蛋白在稻瘟菌侵染水稻时的分泌与转运；通过免疫共沉淀等技术验证AGLIP1与OsClpP6在水稻中的互作；探索AGLIP1降低OsClpP6稳定性的分子机理；构建AGLIP1转基因水稻和osclpp6缺失突变体，鉴定AGLIP1对水稻免疫的影响，明确OsClpP6在水稻免疫中的功能。预期结果有助于深入揭示纹枯病菌操纵水稻免疫进而增强其致病力的分子机理。

水稻纹枯病菌被列为“死体营养型病原菌”，效应子对其致病力贡献的分子机制尚未报道。本项目在13个候选的分泌蛋白中，发现AGLIP1强烈诱导烟草叶片和水稻原生质体细胞死亡，并且S174和D230是其行使毒力功能所必须的关键位点；拟南芥中诱导表达AGLIP1抑制病原细菌侵染早期的免疫应答，并促进病原菌繁殖。水稻中异源表达AGLIP1强烈诱导细胞死亡，主要表现为：愈伤褐化，苗期叶片坏死，活性氧积累，细胞死亡标记基因MT2B和HLM1显著下调，成熟期结实率降低近50%。人工接种纹枯病菌后，异源表达AGLIP1的水稻抗病性显著降低。AGLIP1与抗坏血酸过氧化物酶OsAPX8互作。OsAPX8对叶绿体活性氧稳态起关键作用。本研究又证实了OsAPX8调控水稻对纹枯病的抗性，异源表达和功能缺失OsAPX8的水稻，分别表现为抗病和感病。过量表达OsAPX8可降低AGLIP1诱导细胞死亡的能力。AGLIP1通过干扰OsAPX8的叶绿体定位，促使水稻细胞活性氧积累，进而诱导细胞死亡。研究结果可通过基因编辑技术创制互作位点突变体水稻品种，为抗病分子育种等水稻病害绿色防控技术提供支撑。