Forkhead Box Proteins Modulate Fusarium Development, Pathogenicity, and Fungicide Sensitivity in Transcription-Dependent and -Independent Ways

The forkhead box (FOX) proteins are a conserved family of eukaryotic transcription factors with diverse biological functions. However, the role and evolution of FOX proteins in filamentous fungi have not been fully explored. In this study, we identified and characterized four FOX transcription factors and two forkhead-associated domain (FHA) proteins in the fungal pathogen Fusarium graminearum, the causative agent of Fusarium head blight on cereal crops. Our results reveal that the FOX transcription factors FgHcm1 and FgFoxO4, along with the FHA domain-containing protein FgFha1, are essential for the full virulence of F. graminearum . These proteins regulate fungal penetration of wheat spikelets by modulating the expression of penetration-related genes. In addition, FgHcm1, FgFoxO4, and FgFha1 influence both asexual and sexual reproduction. Deletion of these genes also confers increased sensitivity to various stressors including fungicides. Notably, the gene deletion mutants Δ FgHCM1, Δ FgFOXO4, and Δ FgFHA1 exhibited heightened sensitivity to tebuconazole, and these proteins regulate the inducible expression of the FgCYP51A gene in response to tebuconazole treatment. Furthermore, Δ FgHCM1 mutants showed increased sensitivity to rapamycin with impaired autophagic responses. This study represents the first comprehensive analysis of FOX and FHA proteins in a filamentous fungal pathogen, highlighting their critical roles in virulence, stress tolerance, and fungicide resistance in F. graminearum .