Hydrogen peroxide as a multifaceted regulator of the Atg4 protease and autophagy in the pathogenic fungus Alternaria alternata
Introduction Autophagy-related protease AaAtg4 was previously identified as a key regulator in the pathogenic fungus Alternaria alternata, orchestrating a complex interplay among autophagy, oxidative stress resistance, iron homeostasis, and ACT toxin biosynthesis. The underlying mechanisms of AaAtg4 in relation to oxidative stress response remain unknown. Methods Genetic and biochemical analyses. Results In this study, we examined the effect of hydrogen peroxide (H₂O₂) on AaAtg4. Functioning as a cysteine protease, AaAtg4 directly interacts with the AaAtg8 ubiquitin-like protein and is indispensable for AaAtg8 processing and autophagosome formation, with its enzymatic activity modulated by oxidative cues. H₂O₂ differentially impacts AaAtg4 activity, phosphorylation, binding with AaAtg8, AaAtg8 lipidation/delipidation, and autophagy. H₂O₂ has biphasic effects on AaAtg4. Moderate H₂O₂ levels enhance AaAtg4 activity and autophagy, whereas excessive H₂O₂ suppresses both, revealing a threshold-dependent redox regulation. Furthermore, AaAtg4 interacts with the stress-responsive mitogen-activated protein kinase AaHog1, which modulates its phosphorylation under conditions less conducive to autophagy and thus, reinforces a dynamic signaling axis. Discussion These findings indicate that H₂O₂ has multifaceted effects on AaAtg4 in a dosage-dependent or threshold-specific manner. These regulatory mechanisms may position AaAtg4 as a central integrator of cellular stress responses and secondary metabolism, thereby advancing our understanding of fungal pathogenicity and environmental adaptation.