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ABSTRACT Reversible phosphorylation by protein kinases and phosphatases plays a central role in regulating cellular processes. However, knowledge of the functions of protein phosphatase 2C (PP2C) S/T phosphatases in Aspergillus flavus has been unreported until now. Here, we have identified seven members of the PP2C family of protein phosphatases in A. flavus. Evolutionary and functional analyses indicated that two redundant PP2C phosphatases, Ptc1 and Ptc2, are highly conserved and regulate conidia development, aflatoxin synthesis, seed infection, and autophagic vesicle formation. The cytoplasmic proteins Ptc1 and Ptc2 exhibit nuclear infiltration after DNA damage-induced autophagy. Their degradation is closely linked to autophagy induction. The Asp residue coordinated with Mg2+ is critical for phosphatase Ptc1 and Ptc2 activity, thermal stability, and biofunction in A. flavus. An immunoprecipitation-mass spectrometry proteomic investigation indicated that 133 proteins co-interact with Ptc1 and Ptc2. Among these proteins, phosphoglycerate kinase 1 (PGK1) interacts with Ptc1 and Ptc2 and shows high levels of phosphorylation in Δptc1, Δptc2, and Δptc1/ptc2 mutants. Furthermore, PGK1 S203 phosphorylation levels are associated with aflatoxin synthesis and autophagic vesicle formation. Overall, these findings contribute to our understanding of the roles and mechanisms of PP2C family phosphatases in A. flavus and highlight their importance in various cellular processes. Furthermore, it reveals a new regulation model in A. flavus, where Ptc1 and Ptc2 activate autophagy and aflatoxin synthesis by regulating PGK1. IMPORTANCE Aspergillus flavus is a model filamentous fungus that can produce aflatoxins when it infects agricultural crops. This study evaluated the protein phosphatase 2C (PP2C) family as a potential drug target with important physiological functions and pathological significance in A. flavus. We found that two redundant PP2C phosphatases, Ptc1 and Ptc2, regulate conidia development, aflatoxin synthesis, autophagic vesicle formation, and seed infection. The target protein phosphoglycerate kinase 1 (PGK1) that interacts with Ptc1 and Ptc2 is essential to regulate metabolism and the autophagy process. Furthermore, Ptc1 and Ptc2 regulate the phosphorylation level of PGK1 S203, which is important for influencing aflatoxin synthesis. Our results provide a potential target for interdicting the toxicity of A. flavus. |
