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 incompletely understood. Here, we investigated the effects of dithiothreitol (DTT) and iron on autophagy and on AaAtg4 protease activity in the plant-pathogenic fungus Alternaria alternata. Using GFP-tagged AaAtg8, fluorescence microscopy and proteolysis assays revealed that DTT markedly enhanced autophagic vacuole formation and GFP release, indicating increased autophagic flux. Western blot analyses confirmed 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 demonstrated 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 a redox balance in autophagic regulation. Iron supplementation selectively destabilized AaAtg8 and modulated AaAtg4 phosphorylation in a concentration-dependent manner, without altering autophagy or protease activity. Collectively, these findings demonstrate that DTT enhances autophagy primarily by promoting AaAtg8 lipidation, AaAtg4 phosphorylation, and AaAtg4-AaAtg8 complex formation, while exerting minimal influence on AaAtg4 protease activity. In contrast, ion regulates autophagy flux through its effects on AaAtg4 phosphorylation and AaAtg8 stability, without significantly altering AaAtg4 protease activity, AaAtg8 lipidation, or AaAtg4-AaAtg8 interactions. Together, this work underscores the intricate interplay between redox signaling, nutrient cues, and autophagy regulation in A. alternata. IMPORTANCEThis study provides critical new insights into how redox-active compounds and iron modulate autophagy in the plant-pathogenic fungus Alternaria alternata, a pathogen of agricultural relevance. By dissecting the distinct roles of DTT, hydrogen peroxide, and iron in regulating AaAtg8 lipidation, AaAtg4 phosphorylation, and AaAtg4-AaAtg8 interactions, our findings reveal that autophagy is not simply a constitutive process but is finely tuned by redox balance and nutrient cues. This work advances the fundamental understanding of autophagy regulation in filamentous fungi, highlights the interplay between oxidative stress and protease activity, and establishes a framework for exploring how environmental factors shape fungal pathogenicity. Ultimately, these insights may inform novel strategies to mitigate crop fungal diseases by targeting autophagic pathways.