Role of Antioxidant Molecules in the Oxidative Stress Response Networks in the Tangerine Pathotype of Alternaria alternata

Antioxidant molecules play a crucial role in maintaining redox homeostasis, eliminating oxidative damage, and regulating pathogenesis in phytopathogenic fungi. These antioxidants function through a complex regulatory network involving enzymatic scavengers such as the superoxide dismutases (SODs), catalases, thioredoxins, and glutathiones, as well as non-enzymatic molecules. Specifically, the thioredoxin and glutathione systems (AaTrr1, AaTsa1, AaGpx3, and AaGlr1) mediated by NADPH oxidase represent vital thiol antioxidant systems utilized to regulate the detoxification of reactive oxygen species (ROS) in Alternaria alternata. This present study investigated the regulatory roles of AaBemA, a component of the fungal NADPH oxidase (Nox) complex, in the tangerine pathotype of A. alternata. Loss-of-function genetic analysis demonstrated that AaBemA is essential for the accumulation of cellular hydrogen peroxide (H2O2). Mutant strains with defective AaBemA displayed higher sensitivity to H2O2 and the ROS-generating oxidant tert-butyl-hydroperoxide. These phenotypes closely resembled those previously observed in AaNoxB, AaTrr1, AaTsa1, AaGlr1, and AaYap1 mutants, suggesting a potential interconnection among them. Notably, the defective phenotype of Delta AaBemA could be restored through genetic complementation with wildtype AaBemA gene. Subcellular localization of a functional AaBemA fused with green fluorescent protein (GFP) gene under confocal microscope revealed a widespread distribution of green fluorescence in the cytoplasm and cell membrane, indicating high expression of AaBemA during fungal growth in A. alternata. Intriguingly, inactivation of AaBemA did not affect the morphological phenotype and pathogenicity, indicating that the AaBemA is dispensable for the aforementioned phenotypes. To explore the molecular mechanisms underlying the regulation of ROS stress response, we sequenced the whole transcriptomes of A. alternata wildtype and ROS-sensitive mutants (Delta AaBemA, Delta AaNoxB, Delta AaGlr1, Delta AaTrr1, Delta AaTsa1, Delta AaYap1) which displayed considerable sensitivity to oxidants. Comparative transcriptome analysis revealed significant influence on the gene-expression pattern of numerous genes related to glutathione metabolism, cellular oxidant detoxification, cellular response to abiotic stimulus, and cellular response to osmotic stress. The gene-expression data and pathways related to various essential metabolic processes and ROS tolerance enabled us to propose a NADPH oxidase-mediated regulatory network involving NADPH oxidase (AaNoxB, AaBemA), AaYap1, glutaredoxin (AaGPx3, AaGlr1), and thioredoxin systems (AaTrr1, AaTsa1) in responding to ROS stress in A. alternata.