驱动蛋白MoKar3及其互作蛋白调控稻瘟病菌生长发育及致病的分子机制研究

The blast fungus Magnaporthe oryzae recognizes host surface, forms appressorium and uses invasive nails to invade host cells. Heterotrimeric GTP-binding protein (G protein) is involved in recognizing host surface and transmits them to the downstream cAMP-PKA and MAPK signaling pathway to regulate the formation and development of appressorium. Mitosis within cells regulates the morphological development and differentiation of appressorium. However, the relationship between G protein, MAPK signaling pathway that are involved in signal transmission from outside to inside and mitosis that regulates morphological development and differentiation of appressorium within cells is not clear. Kinesin Kar3 is a key protein regulating mitotic process, and also receives signals transmitted by G protein and MAPK signaling pathway. However, to date the function and mechanism of kinesin Kar3 in plant pathogenic fungi is still unclear. Here, we use genetics means to obtain the deletion mutant of MoKAR3 that plays an important role in mitosis, and examine a series of biological phenotypes including mitosis, growth, development, appressorium formation and pathogenicity. Furthermore, we identify the interactional protein of MoKar3 and elucidate the biological functions of two or three interactional proteins in the mitosis, growth and pathogenesis of Magnaporthe oryzae. These results will not only reveal the molecular mechanism that MoKar3 and its interactional proteins link G protein, MAPK signaling pathway and mitosis to regulate growth, development and pathogenicity in M.oryzae, but also provide a new clue for designing novel fungicides targeting MoKar3 and its interactional proteins.

稻瘟病菌识别寄主表面信号，形成附着胞通过侵入钉侵入寄主细胞。G蛋白信号途径调控对胞外信号的识别，向胞内cAMP-PKA和MAPK信号途径传递，调控附着胞的形成及发育。有丝分裂在细胞内部调控附着胞的形态发育及分化。而由外到内的G蛋白及MAPK信号途径与内部的有丝分裂之间的关联并不清楚。驱动蛋白Kar3是调控有丝分裂的关键蛋白，同时，Kar3能够接收G蛋白及MAPK信号途径传递的信号。但是Kar3在植物病原真菌中的功能以及作用机制仍不清楚。本研究拟阐明MoKar3在稻瘟病菌有丝分裂、生长发育、附着胞形成及致病过程中的功能；进一步鉴定MoKar3的互作蛋白，研究2-3个互作蛋白在有丝分裂、生长发育和致病过程中的作用。研究结果有助于阐明该驱动蛋白及其互作蛋白关联的有丝分裂和信号传导途径在稻瘟病菌生长、发育及致病相关过程中的分子机制，同时为开发以MoKar3及其互作蛋白为靶标的新型药剂提供新线索。

稻瘟病菌识别寄主表面信号，形成附着胞通过侵入钉侵入寄主细胞。G蛋白信号途径调控对胞外信号的识别，向胞内MAPK信号途径传递，调控附着胞的形成及发育。有丝分裂在细胞内部调控附着胞的形态发育及分化。而由外到内的G蛋白及MAPK信号途径与内部的有丝分裂之间的关联并不清楚。本项目通过基因敲除获得了驱动蛋白MoKar3、其互作蛋白微管末端结合蛋白MoBim1编码基因缺失突变体以及MAPK信号途径上游的感受器蛋白MoWsc1、MoWsc2、MoMid2的编码基因单缺失、双缺失、三缺失突变体。亚细胞定位发现，MoKar3定位在细胞核，MoBim1定位在细胞质及细胞核，而MoWsc1、MoWsc2、MoMid2定位于质膜及囊泡中。表型分析发现这些基因共同调控稻瘟病菌的功能性附着胞的形成和致病力。MoBIM1缺失突变体附着胞形成减少、致病力下降。MoWSC1、MoWSC2、MoMID2的双缺失突变体、三缺失突变体孢子萌发、附着胞形成过程中有丝分裂异常，附着胞形成减少，致病力下降。同时发现生防菌枯草芽孢杆菌能够扰乱稻瘟病菌的微管微丝细胞骨架及有丝分裂过程，而MoWsc1调控MAPK途径、有丝分裂及细胞自噬等过程响应生防菌的毒杀作用。本研究结果有助于阐明稻瘟病菌功能性附着胞的形成机制，为开发以信号传导以及有丝分裂相关基因为靶标的新型杀菌剂提供新线索。