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 Sxi2a (sex-induced 1α and 2a) have been characterized as interacting partners that play critical roles in sexual development during a × α sexual reproduction. Given the dominance of α cells in natural populations of Cryptococcus, the roles of Sxi1α and Sxi2a in unisexual reproduction, which predominantly involves same-sex (α × α) mating, remain unclear. To elucidate the functions of Sxi1α and Sxi2a in unisexual reproduction, we first used AlphaFold3 to predict their structures, which revealed the potential for both heterodimeric and homodimeric complexes. We subsequently deleted SXI1α and SXI2 a in the hyperfilamentous self-fertile C. deneoformans strains XL280a 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 × α or α × α crosses. Surprisingly, the absence of SXI1α significantly increased the cell-cell fusion rate during both α × α and a × α mating, suggesting a novel inhibitory role for Sxi1α, independent of the partner Sxi2a. Together, our findings revealed an unexpected function of Sxi1α in regulating cell fusion, which may contribute to the predominance of MATα isolates in global Cryptococcus populations and the conservation of SXI1α in a population that is predominantly α mating type.
Keywords: Cell-cell fusion; Homeodomain protein; Unisexual reproduction.