Data_Sheet_1_The GTPase-Activating Protein FgGyp1 Is Important for Vegetative Growth, Conidiation, and Virulence and Negatively Regulates DON Biosynthesis in Fusarium graminearium.pdf

Ypt1 is a small Rab GTPase in yeast, Gyp1 functions at the Golgi as a negative regulator of Ypt1. Gyp1 homologs are conserved in filamentous fungi. However, the roles of Gyp1 in phytopathogenic fungi are still unclear. Herein, we investigated the functions of FgGyp1 in the wheat pathogen Fusarium graminearum by live-cell imaging, genetic, and pathological analyses. Targeted gene replacement method was used to delete FgGYP1 in F. graminearum. Phenotypic analyses showed that FgGyp1 is critically important not only for the vegetative growth of F. graminearum but also its conidiation. The mutant’s vegetative growth was significantly reduced by 70% compared to the wild type PH-1. The virulence of FgGYP1 deletion mutant was significantly decreased when compared with the wild type PH-1. We further found that FgGyp1 negatively regulates DON production of the fungus. Live-cell imaging clearly demonstrated that FgGyp1 mainly localizes to the Golgi apparatus. Moreover, the TBC domain, C-terminal, and N-terminal regions of FgGyp1 are found to be indispensable for its biological functions and normal localization. The Arg357 residue of FgGyp1 is essential for its functions but dispensable for the normal localization of the protein, while the Arg284 residue is not required for both the functions and normal localization of the protein. Furthermore, we showed that FgGyp1 essentially hydrolyzes the GTP-bound FgRab1 (activated form) to its corresponding GDP-bound (inactive) form in vitro, suggesting that FgGyp1 is a GTPase-activating protein (GAP) for FgRab1. Finally, FgGyp1 was found to be important for FgSnc1-mediated fusion of secretory vesicles from the Golgi with the plasma membrane in F. graminearum. Put together, these data demonstrate that FgGyp1 functions as a GAP for FgRab1 and is important for vegetative growth, conidiation and virulence, and negatively regulates DON biosynthesis in F. graminearum.

Ypt1是酵母中的小型Rab GTP酶（Rab GTPase），Gyp1在高尔基体中作为Ypt1的负调控因子发挥功能。Gyp1的同源蛋白在丝状真菌中具有保守性，但目前Gyp1在植物病原真菌中的作用仍不明确。本研究针对小麦病原真菌禾谷镰孢（Fusarium graminearum）中的FgGyp1功能，通过活细胞成像、遗传学与病理学分析展开了系统研究。我们采用靶向基因替换法敲除了禾谷镰孢中的FgGYP1基因。表型分析结果显示，FgGyp1不仅对禾谷镰孢的营养生长至关重要，同时也参与其分生孢子形成过程。与野生型菌株PH-1相比，突变体的营养生长显著降低70%。FgGYP1基因缺失突变体的致病力相较于野生型PH-1显著下降。我们进一步发现，FgGyp1负调控该真菌的脱氧雪腐镰刀菌烯醇（deoxynivalenol, DON）合成。活细胞成像实验清晰证实，FgGyp1主要定位于高尔基体。此外，FgGyp1的TBC结构域、C端与N端区域对其生物学功能与正常定位均不可或缺。FgGyp1的Arg357残基对其功能至关重要，但不影响该蛋白的正常定位；而Arg284残基既不影响其功能发挥，也不影响其正常定位。体外实验证实，FgGyp1可将结合GTP的活化形式FgRab1水解为结合GDP的非活化形式，这表明FgGyp1是FgRab1的GTP酶激活蛋白（GTPase-activating protein, GAP）。最后，本研究发现FgGyp1在禾谷镰孢中对FgSnc1介导的高尔基体分泌囊泡与质膜的融合过程具有重要调控作用。综上，本研究数据表明，FgGyp1作为FgRab1的GAP发挥功能，且对禾谷镰孢的营养生长、分生孢子形成与致病力至关重要，并负调控该真菌的DON生物合成。