Relief of bHD1 autoinhibition and modular coupling support MAT-A pathway activation in Flammulina filiformis

Flammulina filiformis is a commercially important edible mushroom whose sexual compatibility is determined by genes at two unlinked mating-type loci, A and B. However, the molecular basis of MAT-A activation in this species remains unclear. Here, we established a pair of compatible monokaryotic strains, F11 and F22, derived from the dikaryotic parent ACCC50407, and used them to investigate the genomic and functional basis of MAT-A activation. Genome analysis showed that the MAT-A locus is bipartite, comprising the Aa and Ab subloci. Five HD genes were identified, including one Aa-encoded gene (aHD2-1) and four Ab-encoded genes. In yeast assays, aHD2-1 showed no detectable partner interaction or transcriptional activation activity, whereas specific interactions were detected between the Ab-encoded bHD1 and bHD2 proteins. Deletion analysis further indicated that bHD1-1 contains a functional C-terminal transcriptional activation domain whose activity is constrained in the full-length protein. In vivo, introduction of the compatible allele bHD1-2 into F11 induced pseudoclamp formation and increased clp1 expression. Guided by these results, we constructed a chimeric HD gene, F11ChiHD, by combining the N-terminal region of bHD2-1 with the C-terminal activation region of bHD1-1, which similarly promoted pseudoclamp formation and clp1 upregulation in F11. Together, these findings indicate that MAT-A activation in F. filiformis depends primarily on the Ab sublocus and provide a genetic and functional framework for understanding mating-pathway regulation in this species. KEY POINTS: • MAT-A pathway activation in Flammulina filiformis is driven primarily by the Ab sublocus. • Relief of bHD1 autoinhibition is associated with pseudoclamp formation and clp1 upregulation in monokaryotic F11. • A chimeric HD protein supports modular coupling between HD2-associated DNA targeting and bHD1-associated transcriptional activation.