Abstract

Aspergillus flavus is a major foodborne fungus capable of producing aflatoxins that threaten food quality and safety. Nitrogen metabolism is closely associated with fungal development and secondary metabolism; however, the roles of nitrate-responsive regulators in aflatoxin biosynthesis remain unclear. In this study, the biological functions of AfnirA and AfcrnA , two regulators involved in nitrate utilization, were investigated in A. flavus through gene deletion and overexpression analyses. Transcriptional profiling showed that both genes responded to changes in nitrogen sources and were induced under nitrogen imbalance conditions. Functional analyses demonstrated that AfnirA and AfcrnA differentially affected fungal growth, conidiation, conidial germination, and sclerotial development. Deletion of AfnirA abolished sclerotia formation, whereas disruption of AfcrnA significantly increased sclerotia production. In addition, the Δ AfcrnA mutant exhibited enhanced sensitivity to osmotic, oxidative, and cell wall stresses, accompanied by increased intracellular reactive oxygen species accumulation. Notably, deletion of AfcrnA markedly promoted aflatoxin B1 production, with an approximately 2.3-fold, and upregulated the expression of aflatoxin biosynthetic genes, including aflC , aflD , and aflP . Subcellular localization analysis revealed nuclear localization of AfnirA , whereas AfcrnA displayed vacuolar-associated cytoplasmic localization. These findings indicate that AfnirA and AfcrnA play distinct roles in regulating fungal development, oxidative stress adaptation, and aflatoxin biosynthesis in A. flavus .