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

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 in unisexual reproduction and downstream transcriptional regulation, we first applied AlphaFold3 to predict their structures, which revealed the potential for both heterodimeric and homodimeric complexes. We subsequently deleted SXI1α and SXI2 a 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 competitiveness during unisexual crosses. Interestingly, both bilateral (mutant x mutant) and unilateral (mutant x wildtype) crosses involving the sxi1αΔ mutant significantly increased α-α cell fusion efficiency, suggesting a previously unrecognized inhibitory role for Sxi1α in regulating same-sex cell fusion. Transcriptomic analysis of sxi1αΔ and sxi2 aΔ 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 deletion mutants for SXI1α, SXI2 a, or both revealed differential regulation of mating-type (MAT) loci genes dependent only on SXI1α or SXI2 a. Together, our findings revealed an unexpected function of Sxi1α in regulating cell fusion, highlighting the versatile roles of HD transcription factors in fungal sexual reproduction. Additionally, our data suggest that SXI1α and SXI2 a have distinct regulatory roles in both unisexual and α x a sexual reproduction and may function as homodimeric complexes to exert opposing regulatory effects on sex-specific MAT genes.