Dynamic regulation of Atg4 protease and autophagy by dithiothreitol and iron in Alternaria alternata

Autophagy is a critical cellular process regulated by Atg proteins, yet its modulation by redox-active compounds and iron remains poorly understood. Here, we investigated the effects of dithiothreitol (DTT) and iron on autophagy, AaAtg4 protease activity, and modifications in the plant-pathogenic fungus Alternaria alternata . Using GFP-tagged AaAtg8, fluorescence microscopy, and proteolysis assays, we found that DTT markedly increased autophagic vacuole formation and GFP release, indicating increased autophagic flux. Western blot analyses showed that DTT promoted AaAtg8 lipidation, while co-treatment with hydrogen peroxide (H₂O₂) suppressed this modification. AaAtg4 was constitutively active and could process AaAtg8 regardless of DTT supplementation, whereas moderate DTT concentrations elevated AaAtg4 protein abundance and phosphorylation. Bimolecular fluorescence complementation assays showed that DTT, but not iron, facilitated AaAtg4–AaAtg8 interactions and vacuolar localization, whereas H₂O₂ counteracted these effects. Notably, combined DTT and H₂O₂ sustained autophagy at a low but stable level, suggesting that their interactions affect autophagic regulation. Iron supplementation enhanced autophagy, reduced AaAtg8 abundance, and modulated AaAtg4 phosphorylation without substantially altering protease activity. Collectively, these findings show that DTT promotes AaAtg8 lipidation, AaAtg4 phosphorylation, AaAtg4–AaAtg8 complex formation, and autophagy in a concentration-dependent manner, without increasing AaAtg4 protease activity. In contrast, ferric and, to a lesser extent, ferrous iron affect autophagic flux and modulate AaAtg4 phosphorylation and AaAtg8 abundance. However, their impact is weaker than that of DTT because they do not significantly alter AaAtg4 protease activity, AaAtg8 lipidation, or AaAtg4–AaAtg8 interactions. Altogether, these findings highlight the intricate interplay among redox signaling, nutrient cues, and autophagy regulation. in A. alternata .