F-box蛋白MoGrr1调控稻瘟病菌细胞壁发育的分子机制研究

F-box protein MoGrr1 was identified as key regulator involving in cell wall integrity and pathogenicity in M.oryzae. To further investigate the regulatory mechanism that regulated by the MoGrr1 protein during fungal development, yeast two hybrid and Co-IP methods were used to identify candidate proteins interacting with MoGrr1.Meanwhile, the deletion mutants of candidate protein encoding genes were constructed and phenotypes of each mutants, especially on conidia development, cell wall integrity and pathogenicity, were analyzed and compared with Mogrr1 mutant to identify the similarities and differences. Base on the results above, 1 to 2 candidate mutants, together with wild type and Mogrr1 mutant, were used to investigate the key regulator and potential pathway regulated by MoGrr1 during fungal development and pathogenicity by the usage of RNA-seq, iTRAQ and bioinformatics tools. Our data will facilitate the understanding of signal pathway mediated by MoGrr1 during fungal cell wall development and pathogenesis in M. oryzae.

F-box蛋白MoGrr1已被发现是稻瘟病菌细胞壁发育及致病的重要因子，但其调控机制尚不清楚。本项目将以F-box蛋白MoGrr1为材料，采用酵母双杂交和Co-IP技术共同鉴定与MoGrr1互作的蛋白。分别构建互作蛋白编码基因的敲除突变体，并对其生物学功能进行解析，重点比较候选基因突变体与Mogrr1突变体在调控分生孢子发育、细胞壁完整性及致病性等方面的差异。选择1-2个表型与Mogrr1突变体较为一致的材料，结合高通量测序、定量磷酸化蛋白质组和生物信息学分析等手段综合分析野生型与Mogrr1突变体、野生型与候选突变体及Mogrr1突变体与候选突变体在基因与蛋白质表达谱及信号通路上的异同点，鉴定MoGrr1与互作蛋白共同调控的下游信号通路及关键调控因子，进而初步明确F-box蛋白MoGrr1介导的信号通路是如何调控稻瘟病菌细胞壁发育及致病的分子机理。

分别采用酵母文库筛选、免疫共沉淀（Co-IP）结合液相色谱-质谱联用（LC-MS）方法，筛选到8个潜在（MoPP2Ac、Mouga1、Moppe1、MoPP2Ab、Moppg1、Moskp1、MoPP2B、Morts1）与MoGrr1互作的蛋白。利用酵母双杂交或Western Blot技术，确定Mogrr1与Moppg1、MoPP2Ac、Mouga1、Moskp1存在较强相互作用，而与Moppe1、MoPP2Ab存在弱相互作用。同时，MoPP2Ac与Moppe1、MoPP2Ab、Mouga1、Morts1、MoPP2B之间亦存在互作。进一步试验发现，F-box结构域是决定Mogrr1与互作蛋白相互作用的关键结构域，而LRR结构域不参与Mogrr1与其它蛋白间的相互作用。. 通过同源重组方法，获得了稻瘟菌Moppg1和Morts1基因敲除突变体。其中，Moppg1突变体与Mogrr1突变体表型相似，菌丝生长速率减慢，孢子形态异常且产孢量大幅下降。Moppg1还参与分生孢子萌发和附着胞发育，对胞内糖原转移具有调节作用。突变体对过氧化氢耐受性降低，对细胞壁胁迫因子CFW、CR敏感，且不能侵染寄主，对大麦及水稻丧失致病力。Morts1主要定位在隔膜和胞质中，该基因敲除突变体营养菌丝生长显著减慢，分生孢子梗分化受阻，产孢量显著下降；突变体色素合成能力减弱；对过氧化氢和SDS耐受性增强，对细胞壁抑制剂刚果红敏感，但对CFW耐受性增强；Morts1突变体原生质体释放速度显著下降，对大麦和水稻致病力明显下降。 . 综上述，本项目鉴定到9个与MoGrr1互作的蛋白，并系统研究了其中2个互作蛋白Moppg1与Morts1的生物功能，发现它们是调控稻瘟菌生长发育、细胞壁完整性及致病的重要因子。