A Type 2C Protein Phosphatase FgPtc3 Is Involved in Cell Wall Integrity, Lipid Metabolism, and Virulence in Fusarium graminearum

Type 2C protein phosphatases (PP2Cs) play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8) exhibited reduced aerial hyphae formation and deoxynivalenol (DON) production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum.2C型蛋白磷酸酶（Type 2C protein phosphatases，PP2Cs）在真核生物的诸多生物学过程调控中发挥重要作用。目前，学界对丝状真菌中PP2Cs的功能尚缺乏深入了解。引发小麦赤霉病的病原菌禾谷镰孢菌（Fusarium graminearum）含有7个假定的PP2C基因：FgPTC1、-3、-5、-5R、-6、-7与-7R。本研究为探究这些PP2Cs的功能，构建了全部7个PP2C基因的敲除突变体。FgPTC3敲除突变体ΔFgPtc3-8的气生菌丝形成与脱氧雪腐镰刀菌烯醇（deoxynivalenol，DON）合成能力均出现显著下降，但分生孢子产量有所提升。该突变体在马铃薯葡萄糖琼脂（PDA）平板上表现出对渗透胁迫与细胞壁损伤剂的抗性增强。致病力测定结果显示，ΔFgPtc3-8无法侵染开花期的小麦穗部。当利用野生型FgPTC3基因对ΔFgPtc3-8进行互补实验时，所有缺陷表型均得到完全恢复。此外，在多种胁迫条件下，FgPTC3可部分挽救酿酒酵母PTC1敲除突变体的生长缺陷。超微结构与组织化学分析表明，ΔFgPtc3-8的分生孢子中含有异常大量的大型脂滴。进一步检测发现，该突变体的甘油基础水平高于野生型亲本菌株。实时荧光定量PCR（quantitative real-time PCR）检测结果显示，突变体中FgOS2、FgSLT2与FgMKK1的基础表达量显著高于野生型菌株。对ΔFgPtc3-8的基因表达系列分析（Serial analysis of gene expression）显示，FgPTC3参与多种代谢通路的调控。与FgPTC3突变体不同，FgPTC1、FgPTC5、FgPTC5R、FgPTC6、FgPTC7或FgPTC7R的敲除突变体在PDA培养基上培养或接种小麦穗部时，均未表现出异常表型。上述结果表明，FgPtc3是关键的PP2C，在禾谷镰孢菌的多种细胞与生物学功能中发挥关键作用，包括细胞壁完整性调控、脂类与次级代谢，以及致病力。