HsfA-driven gene transcription regulates fungal morphogenesis and pathogenicity

Aspergillus flavus, a ubiquitous filamentous fungus, severely compromises global food safety and public health by producing carcinogenic aflatoxins. Heat shock factor 1 (HSF1) orchestrates stress responses in eukaryotes, yet the functional role and regulatory mechanisms of its homolog, HsfA, in A. flavus remain elusive. Here, we demonstrate that A. flavus encodes two hsfA copies, whose knockdown or expression of a dominant-negative variant abrogates spore germination, a prerequisite for fungal development and colonization. Through integrated reverse genetics, ChIP-qPCR, electrophoretic mobility shift assay, and transcriptional profiling, we identify six non-chaperone targets—brlA, fksP, flbC, sntB, velB, and vosA—directly regulated by HsfA via binding to conserved heat shock elements (HSEs) in their promoters. Ectopic expression of each target partially restores germination, developmental progression, and pathogenicity in HsfA-deficient strains, confirming HsfA’s central role in driving these processes via transcriptional activation. Structural divergence between the A. flavus HsfA DNA-binding domain and human HSF1 explains the ineffectiveness of three HSF1 inhibitors against the fungus. Our findings establish HsfA as a pivotal regulator of A. flavus virulence and uncover a novel HSF regulatory pathway, highlighting HsfA as a promising target for mitigating aflatoxin contamination.