FphA Positively Regulates Fungal Development and AFB₁ Biosynthesis and Phosphorylates Ypd1 in Aspergillus flavus

Aspergillus flavus is a ubiquitous fungus capable of producing aflatoxin B₁ (AFB₁), a potent carcinogen that threatens food safety and human health. Here, we identified and characterized histidine kinase FphA as a positive regulator of fungal development and AFB₁ biosynthesis. Deletion of fphA impaired mycelial growth and markedly reduced AFB₁ production, accompanied by downregulation of cluster regulators aflR and aflS and multiple structural genes. Transcriptomic and phosphoproteomic analyses revealed significant transcriptional and phosphorylation changes in MAPK signaling components (e.g., Hog1 and SskA) in the ΔfphA strain. Although yeast two-hybrid assays showed no direct interaction between FphA and MAPK proteins, in vitro kinase assays demonstrated that FphA phosphorylates phosphotransfer protein Ypd1, suggesting regulation via the FphA-Ypd1-MAPK cascade. These findings establish FphA as a key regulator linking environmental sensing with fungal development and toxin biosynthesis, providing potential molecular targets for controlling aflatoxin contamination in agricultural products.