Autophagy-related protein 4 contributes to siderophore biosynthesis, toxin production, and virulence in Alternaria...

The tangerine pathotype of Alternaria alternata infects multiple citrus cultivars, causing brown spot disease. The role of autophagy in toxin production and siderophore biosynthesis remains to be further confirmed. This study identifies autophagy-related protein 4 (AaAtg4) as a critical regulator of fungal growth, development, stress resistance, iron homeostasis, and virulence, as determined by genetic and biochemical analyses. Targeted deletion of the AaAtg4 gene using split-marker recombination generated two ΔAaAtg4 mutants, which displayed reduced growth on minimal medium, impaired conidiation, delayed germination, and diminished formation of appressorium-like structures compared with the wild-type strain. The mutants were hypersensitive to hydrogen peroxide and iron stress, highlighting the roles of AaAtg4 in oxidative stress tolerance and iron metabolism. ΔAaAtg4 mutant strains failed to produce detectable siderophores and exhibited downregulation of siderophore production-related genes (AaHapX, AaNps6, AaMirB) alongside upregulation of the AaSreA gene encoding a siderophore repressor. Toxin profiling further revealed altered host-selective toxin production, with distinct shifts in retention times in ΔAaAtg4 compared to the wild-type strain. However, AaAtg4, but not autophagy itself, was required for toxin production, as deletion of other autophagy-related genes had no effect. Reintroduction of functional AaAtg4 into a ΔAaAtg4 mutant rescues all defects, confirming that the loss of AaAtg4 function directly causes the observed phenotypes. These findings demonstrate that AaAtg4 is important for spore germination, siderophore biosynthesis, iron acquisition, oxidative stress resistance, and toxin production, thereby establishing its critical role in A. alternata virulence.