Homeodomain protein Sxi1α regulates cell-cell fusion during distinct sexual reproduction modes in Cryptococcus deneoformans

Sex-specific homeodomain ( HD ) proteins are key regulators of cell identity and sexual development in fungi, typically functioning as heterodimers to regulate transcription. In the human fungal pathogens Cryptococcus neoformans and Cryptococcus deneoformans , the HD proteins Sxi1α and Sxi2 a (sex-induced 1α and 2 a ) have been characterized as interacting partners that play critical roles in sexual development during a x α sexual reproduction. Given the dominance of α cells in natural populations of Cryptococcus , the roles of Sxi1α and Sxi2 a in unisexual reproduction, which predominantly involves same-sex (α x α) mating, remain unclear. To elucidate the functions of Sxi1α and Sxi2 a in unisexual reproduction, we first used AlphaFold3 to predict their structures, which revealed the potential for both heterodimeric and homodimeric complexes. We subsequently deleted SXI1 α and SXI2 a in the hyperfilamentous self-fertile C. deneoformans strains XL280 a and XL280α. Disruption of these genes did not result in noticeable defects in vegetative growth, virulence-associated traits, colony morphology, sporulation, or competitiveness during either a x α or α x α crosses. Surprisingly, the absence of SXI1 α significantly increased the cell–cell fusion rate during both α x α and a x α mating, suggesting a novel inhibitory role for Sxi1α, independent of the partner Sxi2 a . Together, our findings revealed an unexpected function of Sxi1α in regulating cell fusion, which may contribute to the predominance of MAT α isolates in global Cryptococcus populations and the conservation of SXI1 α in a population that is predominantly α mating type.