Homeodomain protein Sxi1α independently controls cell-cell fusion and gene expression during sexual reproduction in Cryptococcus deneoformans.

Sex-specific homeodomain (HD) proteins are key regulators of cell identity and sexual development in fungi, typically functioning as heterodimers to govern transcription. In the human fungal pathogens Cryptococcus neoformans and Cryptococcus deneoformans, the HD proteins Sxi1α and Sxi2a (Sex-inducer 1α and 2a) have been characterized as interacting components that play critical roles in sexual development during α x a sexual reproduction. α cells are the predominant mating type in natural populations of Cryptococcus, and unisexual (same-sex) mating can also occur in certain genetic backgrounds. The roles of Sxi1α and Sxi2a in unisexual reproduction are not fully understood. To elucidate the functions of Sxi1α and Sxi2a, we first applied AlphaFold3 prediction, which identified potential heterodimeric and homodimeric complexes. Formation of a Sxi2a homodimer was then experimentally validated through yeast two-hybrid assays. We subsequently deleted SXI1α and SXI2a in the hyper-filamentous self-fertile C. deneoformans strains XL280α and XL280a. Disruption of these genes did not result in noticeable defects in vegetative growth, virulence-associated traits, colony morphology, sporulation, or competitive fitness during unisexual crosses. Interestingly, both bilateral (mutant x mutant) and unilateral (mutant x wild type) crosses involving the sxi1αΔ mutant showed significantly increased α-α cell fusion efficiency, suggesting a previously unrecognized inhibitory role for Sxi1α in regulating same-sex cell fusion. Consistently, genes encoding mating pheromones and the α-pheromone receptor Ste3 were upregulated in the sxi1αΔ fusion assays. Transcriptomic analysis of sxi1αΔ and sxi2aΔ mutants led to the identification of unique subsets of genes negatively regulated by each transcription factor during unisexual reproduction. Additionally, α x a crosses between sxi1αΔ and sxi2aΔ mutants revealed differential regulation of mating-type (MAT) loci genes dependent only on Sxi1α or Sxi2a. Together, our findings reveal a novel role for Sxi1α in governing cell fusion and demonstrate that Sxi1α and Sxi2a have distinct transcriptional control during unisexual and α x a sexual reproduction, potentially exerting opposing regulation of sex-specific MAT genes.