稻瘟病菌转运蛋白MoMfs1调控物质转运及致病过程的功能分析

The major facilitator superfamily transporter play crucial role in transport of nutrition and metabolites, drug resistance and neural signaling process in organism. In our early study, we found a MFS transporter-coding gene MoMFS1 in the rice blast fungus was high expressed in infection stage by RNA-Seq, and 12 TMs (transmembrane-spanning helices) are found in the MoMfs1 amino acid sequence. Then we found that MoMFS1 gene was involve in the conidiation and pathogenesis by gene knockout. Moreover, exogenous spore germination fluid of Guy11 can restore the pathogenesis of MoMFS1 deletion mutant. In this study, the MFS transporter-coding gene MoMFS1 will be analyzed in growth and pathogenesis in comparison with the wild type Guy11. For another, the transshipment substance are identified by flight tandem mass spectrometry, liquid phase mass spectrometry and affinity chromatography. To further explore the binding proteins of MoMfs1, Co-IP and yeast two-hybrid system will be adopted using MoMfs1 as a bait protein. The candidate binding proteins will be analyzed by bioinformatics and the interaction relationship with MoMfs1 will be further confirmed by Co-IP. The confirmed candidates (1-2) will be selected to be analyzed by knockout technology. These results will not only reveal the MFS transporter involving in transport, growth, development and virulence of M. oryzae, but also provide insight into the design novel disease management strategies.

质子依赖型MFS转运蛋白在生物体营养物质和代谢产物的转运、病原菌抗药性以及神经信号传导等生物过程中起着重要作用。稻瘟病菌转录组测序结果表明基因MoMFS1（MFS转运蛋白的编码基因）在侵染阶段表达上调，采用基因敲除技术发现该基因影响稻瘟病菌分生孢子产生和致病过程，外源添加野生型孢子萌发液能够恢复敲除突变体致病能力的缺陷。本项目拟以MoMFS1为研究对象，阐明该基因在稻瘟病菌生长发育及致病过程中的调控作用；同时采用飞行串联质谱、液相质谱和亲和层析技术对其转运物质进行鉴定；其次利用Co-IP和Y2H等技术鉴定MoMfs1的互作蛋白，并分析1-2个互作蛋白的生物学功能；从而深入解析MoMFS1调控稻瘟病菌物质转运、生长发育及致病过程的分子机制。研究结果不仅有助于深入揭示MFS转运蛋白的作用机制，而且可为开发新型杀菌剂提供理论依据。

质子依赖型MFS转运蛋白在生物体营养物质和代谢产物的转运、病原菌抗药性以及神经信号传导等生物过程中起着重要作用。本项目通过基因敲除方法对稻瘟病菌中三个关键MFS转运蛋白功能进行分析。研究发现（1）MoMFS3敲除突变体致病性降低，但MoMFS1敲除突变体没有变化；同时，两者均能影响稻瘟病菌对杀菌剂的外排和漆酶，过氧化物酶的转运。对MoMFS3敲除突变体代谢组测定显示不管是菌丝培养液还是附着胞培养液，鉴定到的代谢物多集中于有机酸及其衍生物，脂类和类脂分子，有机杂环化合物等；同时发现附着胞阶段下降的代谢物中有2种真菌毒素。2）构建了MFS转运蛋白编码基因MoMFS2（MGG_01301）敲除载体，并成功获得敲除突变体。进一步分析发现该基因调控稻瘟病菌细胞壁完整性和对杀菌剂的外排，不参与稻瘟病菌的致病性。3）利用中国的7个水稻稻瘟病鉴别寄主对收集的2003-2017年的1584个稻瘟病菌单孢菌株的致病性进行分析，从多样性、优势和常见种群以及抗性等方面评价江苏省稻瘟病菌种群结构特征。4）利用国际水稻所32个水稻单基因系对2019年分离的江苏省224个稻瘟病菌单孢菌株进行致病性分析，Pii、Pik-h、Pi5和Piz均表现出较高的抗性，基因组合Piz/Pii、Piz/ pi5和Piz/Pi5抗性较好，适合在江苏种植区使用。上述研究深入解析MFS转运蛋白调控稻瘟病菌物质转运、生长发育及致病过程的分子机制，为开发新型杀菌剂提供理论依据，且对江苏省稻瘟病菌的种群结构进行了分析，有助于抗病水稻品种的科学布局和抗病分子育种。