AP-2 complex is widely distributed in eukaryotes in the form of heterotetramer that functions in the uptake of membrane proteins during mammalian/plant clathrin-mediated endocytosis. However, its biological function remains mysterious in pathogenic fungi. In this study, the wheat scab fungus, Fusarium graminearum, was used to characterise the biological function of the AP-2 complex. Our study shows that FgAP-2 complex plays a critical role in the maintenance of hyphal polarity.Lack of any subunit (FgAP2 α , FgAP2 β , FgAP2 σ , and FgAP2 mu ) of the FgAP-2 complex significantly affects the fungal vegetative growth, conidial morphology, and germination. Remarkably, FgAP-2 complex is important for the fungal pathogenicity, especially during colonisation and extension after infecting the host. The FgAP-2 complex is expressed ubiquitously at all developmental stages but having more concentrated protein distribution at the subapical collar and septa in young growing hyphae. Although FgAP-2 complex displays similar dynamic behaviour to the actin patch components and accumulates at endocytic sites, it is dispensable for general endocytosis. We further demonstrated that FgAP-2 complex is required for polar localisation of the lipid flippases FgDnfA and FgDnfB, which led to the proposal that FgAP-2 functions as a cargo-specific adaptor that promotes polar growth and colonising ability of F. graminearum.

The membrane trafficking system is important for compartmentalization of the biosynthesis pathway and secretion of deoxynivalenol (DON) mycotoxin (a virulence factor) in Fusarium graminearum. Flippases are transmembrane lipid transporters and mediate a number of essential physiological steps of membrane trafficking, including vesicle budding, charging, and protein diffusion within the membrane. However, the roles of flippases in secondary metabolism remain unknown in filamentous fungi. Herein, we identified five flippases (FgDnfA, FgDnfB, FgDnfC1, FgDnfC2, and FgDnfD) in F. graminearum and established their specific and redundant functions in the development and pathogenicity of this phytopathogenic fungus. Our results demonstrate that FgDnfA is critical for normal vegetative growth while the other flippases are dispensable. FgDnfA and FgDnfD were found crucial for the fungal pathogenesis, and a remarkable reduction in DON production was observed in ΔFgDNFA and ΔFgDNFD. Deletion of the FgDNFB gene increased DON production to about 30 times that produced by the wild type. Further analysis showed that FgDnfA and FgDnfD have positive roles in the regulation of trichothecene (TRI) genes (TRI1, TRI4, TRI5, TRI6, TRI12, and TRI101) expression and toxisome reorganization, while FgDnfB acts as a negative regulator of DON synthesis. In addition, FgDnfB and FgDnfD have redundant functions in the regulation of phosphatidylcholine transport, and double deletion of FgDNFB and FgDNFD showed serious defects in fungal development, DON synthesis, and virulence. Collectively, our findings reveal the distinct and specific functions of flippase family members in F. graminearum and principally demonstrate that FgDnfA, FgDnfD, and FgDnfB have specific spatiotemporal roles during toxisome biogenesis.

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