Supplementary file 1_The adenylate cyclase-mediated signaling pathway required for regulating siderophore and toxin biosynthesis and pathogenicity in Alternaria alternata.docx

The role of cyclic AMP–protein kinase A (PKA) signaling in siderophore-mediated iron uptake and its connection to virulence remains poorly understood in phytopathogenic fungi. Genetic studies demonstrate that the A. alternata adenylate cyclase (AaAC) regulates diverse cellular processes, including growth, conidiation, iron homeostasis, autophagy, siderophore biosynthesis, and toxin production. Deletion of AaAC results in impaired siderophore secretion, disrupted expression of iron-responsive genes, and a complete loss of ACT toxin biosynthesis, leading to markedly reduced virulence. Transcriptomic analysis under iron-deficient conditions reveals that AaAC deletion induces widespread changes in gene expression, notably the downregulation of genes involved in siderophore biosynthesis and ACT toxin production. These findings indicate that AaAC regulates metabolic pathways essential for fungal survival and pathogenicity. Mutants lacking the GTP-binding protein alpha subunit (Gα), the PKA catalytic subunit, or its regulatory subunit also reduce siderophore production. The findings suggest that environmental cues influencing siderophore biosynthesis are transmitted via a signaling cascade from Gα to AaAC and then to PKA. Additionally, AaAC negatively affects autophagy under nutrient-rich conditions. Gene ontology analysis reveals upregulation of autophagy-related genes, suggesting that AaAC may contribute to cellular energy preservation and physiological stability. These results indicate that AaAC is a key integrator of environmental signals, vital for maintaining iron homeostasis, controlling toxin biosynthesis, and driving virulence in A. alternata.