Pexophagy is critical for fungal development, stress response, and virulence in Alternaria alternata

Alternaria alternata can resist high levels of reactive oxygen species (ROS). The protective roles of autophagy or autophagy-mediated degradation of peroxisomes (termed pexophagy) against oxidative stress remain unclear. The present study, using transmission electron microscopy and fluorescence microscopy coupled with a GFP-AaAtg8 proteolysis assay and an mCherry tagging assay with peroxisomal targeting tripeptides, demonstrated that hydrogen peroxide (H₂ O₂ ) and nitrogen depletion induced autophagy and pexophagy. Experimental evidence showed that H₂ O₂ triggered autophagy and the translocation of peroxisomes into the vacuoles. Mutational inactivation of the AaAtg8 gene in A. alternata led to autophagy impairment, resulting in the accumulation of peroxisomes, increased ROS sensitivity, and decreased virulence. Compared to the wild type, ΔAaAtg8 failed to detoxify ROS effectively, leading to ROS accumulation. Deleting AaAtg8 down-regulated the expression of genes encoding an NADPH oxidase and a Yap1 transcription factor, both involved in ROS resistance. Deleting AaAtg8 affected the development of conidia and appressorium-like structures. Deleting AaAtg8 also compromised the integrity of the cell wall. Reintroduction of a functional copy of AaAtg8 in the mutant completely restored all defective phenotypes. Although ΔAaAtg8 produced wild-type toxin levels in axenic culture, the mutant induced a lower level of H₂ O₂ and smaller necrotic lesions on citrus leaves. In addition to H₂ O₂ , nitrogen starvation triggered peroxisome turnover. We concluded that ΔAaAtg8 failed to degrade peroxisomes effectively, leading to the accumulation of peroxisomes and the reduction of the stress response. Autophagy-mediated peroxisome turnover could increase cell adaptability and survival under oxidative stress and starvation conditions.