The Basic Helix–Loop–Helix Transcription Factors <scp>UvBhlh1</scp> and <scp>UvBhlh6</scp> Coordinate Virulence in the Rice False Smut Fungus <i>Ustilaginoidea virens</i>

Ustilaginoidea virens is the causal agent of rice false smut, a devastating and globally widespread fungal disease. Transcription factors are crucial regulators of fungal development and pathogenicity. In this study, we demonstrated that the bHLH transcription factor UvBhlh6 in U. virens plays a critical role during plant infection. Using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays, we showed that UvBhlh6 physically interacts with UvBhlh1, UvBhlh4 and UvBhlh7, and also forms homodimers. Among these, UvBhlh1 was also essential for full virulence. Notably, UvBhlh1 and UvBhlh6 form a heterodimer, and UvBhlh1 mediates the nuclear localisation of UvBhlh6, enabling their cooperative regulation of downstream target genes. Phenotypic analysis revealed that ΔUvbhlh1 and ΔUvbhlh6 mutants displayed reduced tolerance to H₂O₂-induced oxidative stress. Moreover, ΔUvbhlh6 showed severely impaired growth under osmotic stress, with inhibition rates exceeding 90% on media containing sorbitol or NaCl. Affinity capture followed by mass spectrometry identified several UvBhlh6-interacting proteins, including the carbohydrate metabolic enzymes UDP galactose 4-epimerase (UvGal10) and isocitrate dehydrogenase (NAD) subunit 1 precursor (UvIdh1), as well as Pot1 (3-ketoacyl-CoA thiolase), a key enzyme in the fatty acid β-oxidation pathway. These findings suggest that bHLH transcription factors play conserved roles in modulating carbohydrate and lipid metabolism in fungi. Taken together, our results demonstrate that UvBhlh1 and UvBhlh6 are key regulators of early infection and virulence in U. virens.