基于代谢组学研究玉米大斑病菌漆酶影响病菌发育及致病的生化机制

Laccases is a kind of polyphenol oxidase with a wide range of substrates, and play an important role in fungal development and pathogenicity. In our previous study, Stlac1, Stlac2 and Stlac6 are the major active laccase in the Setosphaeria turcica . The knock-out mutants of StLAC1 and StLAC2 leads to abnormal morphology, no sporulation and loss of pathogenicity. In this study, based on the construction of StLAC6 knock-out mutants, the function difference of the major genes will be studied on the development and pathogenesis. Metabolomics will be used to analyze the different metabolites and metabolic pathways caused by laccase gene knock-out. For screening metabolites directly caused by laccase, molecular docking and fluorescent titration will be used to identify laccase substrates. The substrates and inhibiters of metabolic pathways will be used to verified the relationship between phenotypic changes and metabolite differences, which analyze the biochemical mechanisms of laccase affecting the development and pathogenicity. This study will bring insight of the biological functions of laccase from the perspective of metabolism, expand the understanding of the pathogenic mechanism of plant pathogenic fungi. At the same time, the identification of the endogenous metabolites affecting pathogenesis, will provide the information for design the new fungicides for fungal diseases control.

漆酶是多酚氧化酶，作用底物广泛，在真菌发育及致病中扮演重要角色。前期研究发现Stlac1、Stlac2 和Stlac6为玉米大斑病菌中主要表达的具有漆酶活性的蛋白，其中StLAC1和StLAC2基因缺失后导致菌丝形态异常、不产孢且致病性丧失。本研究在分别创制这3个基因敲除突变体的基础上，分析其在生长发育及致病等方面的功能差异；利用代谢物组学技术，鉴定基因敲除导致的代谢物及代谢途径差异。利用分子对接技术及荧光滴定技术筛选与漆酶稳定结合的差异代谢物，通过体外酶促反应验证同工酶的天然作用底物；进而通过验证漆酶作用底物及代谢途径抑制剂对病菌表型的影响，明确漆酶导致的代谢物差异与突变体表型差异之间的关联。本研究从代谢的角度对漆酶参与影响病菌发育及致病的生化机制进行解析，拓展对植物病原真菌致病机制的认识，同时影响病菌致病的内源性代谢物的鉴定为开发新型杀菌剂和真菌病害防治的新途径提供参考依据。

漆酶作为多酚氧化酶，在真菌发育及致病中扮演重要角色。玉米大斑病菌是玉米叶部病害重要致病真菌之一，前期研究发现玉米大斑病菌基因组编码9个漆酶基因，其中Stlac1、Stlac2 和Stlac6为主要表达的具有漆酶活性的蛋白。通过创制这3个基因的敲除突变体，发现StLAC1和StLAC2敲除后病菌的黑色素合成受阻，进而影响了附着胞萌发，导致致病性降低，StLAC6主要影响过氧化物酶体功能，敲除后导致病菌对杀菌剂的敏感性降低。利用代谢物组学分析了StLAC1、StLAC2和StLAC6敲除突变体细胞内外代谢物及代谢途径的差异，结果显示StLAC1和StLAC2缺失导致细胞内DHN黑色素前体化合物含量发生变化，同时影响了几丁质等细胞壁成分；StLAC1缺失影响了脂类化合物的合成，从而影响附着胞发育、致病性及菌丝形态；StLAC6基因缺失导致真菌毒素含量增加。通过蛋白与差异代谢物分子对接及荧光滴定法证实，25-羟基维生素D2、桔霉素、核黄素等是漆酶的天然底物，而漆酶不直接作用于磷脂等脂类化合物。同时发现Stlac1和Stlac6可以降解玉米木质纤维素中的木质素。黑色素调控转录因子StMR1直接调控StLAC2的表达，参与黑色素合成及致病。本项目结果从代谢的角度对漆酶参与影响病菌发育及致病的生化机制进行了深入的解析，拓展对植物病原真菌致病机制的认识，同时为开发新型杀菌剂和真菌病害防治的新途径提供参考依据。