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 govern transcription. In the human fungal pathogens Cryptococcus neoformans and Cryptococcus deneoformans, the HD proteins Sxi1 and Sxi2a (Sex-inducer 1 and 2a) have been characterized as interacting components that play critical roles in sexual development during x a sexual reproduction. 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 Sxi1 and Sxi2a in unisexual reproduction are not fully understood. To elucidate the functions of Sxi1 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 SXI1 and SXI2a in the hyper-filamentous self-fertile C. deneoformans strains XL280 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 wildtype) crosses involving the sxi1{Delta} mutant significantly increased - cell fusion efficiency, suggesting a previously unrecognized inhibitory role for Sxi1 in regulating same-sex cell fusion. Consistently, genes encoding mating pheromones and the -pheromone receptor Ste3 were upregulated in the sxi1{Delta} fusion assays. Transcriptomic analysis of sxi1{Delta} and sxi2a{Delta} mutants led to the identification of unique subsets of genes negatively regulated by each transcription factor during unisexual reproduction. Additionally, x a crosses between null mutants of sxi1{Delta} and sxi2a{Delta} revealed differential regulation of mating-type (MAT) loci genes dependent only on Sxi1 or Sxi2a. Together, our findings reveal a novel role for Sxi1 in governing cell fusion and demonstrate that Sxi1 and Sxi2a have distinct transcriptional control during unisexual and x a sexual reproduction, potentially exerting opposing regulation of sex-specific MAT genes. Author SummaryAnalogous to processes observed in many eukaryotic organisms, fungal sexual reproduction reshuffles genetic material, producing novel genetic variants with increased fitness and/or adaptive potential. The human fungal pathogen Cryptococcus deneoformans is widespread in the environment and can cause severe infections in immunocompromised individuals. Sexual reproduction in this species occurs between cells of either opposite mating types ( x a mating) or the same mating type ( x and a x a unisexual reproduction). The mating type-specific homeodomain transcription factors Sxi1 and Sxi2a are known to form a complex that broadly controls sexual development following and a cell fusion. Here, we find that Sxi1 inhibits cell-cell fusion during unisexual reproduction, while its partner, Sxi2a, shows only limited inhibition in certain genetic contexts. Further, we show that Sxi1 and Sxi2a regulate transcription of distinct gene sets during both x a and unisexual reproduction, including many within the mating-type (MAT) loci, often in opposing manners. Together, these findings reveal that Sxi1 and Sxi2a function both cooperatively and independently, thereby allowing for a more flexible and finely-tuned mating control system than previously recognized, providing insight into how this organism generates genetic variation and adapts to changing environments.