Homeodomain protein Sxi1α independently controls cell-cell fusion and gene expression during sexual reproduction in Cryptococcus deneoformans
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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 Sxi2 a (Sex-inducer 1α and 2 a ) 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 Sxi2 a in unisexual reproduction are not fully understood. To elucidate the functions of Sxi1α and Sxi2 a 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 SXI1 α and SXI2 a in the hyper-filamentous self-fertile C. deneoformans strains XL280α and XL280 a . 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 sxi1 αΔ 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 sxi1 αΔ and sxi2 a Δ 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 SXI1 α, SXI2 a , or both revealed differential regulation of mating-type ( MAT ) loci genes dependent only on SXI1 α 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 SXI1 α 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.
Author Summary
Like 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. This species reproduces between cells of either opposite mating types (α- a mating) or the same mating type (α-α and a - a unisexual reproduction). The mating type-specific homeodomain transcription factors Sxi1α and Sxi2 a are known to form a complex that broadly controls sexual development upon α and a cell fusion. Here, we found Sxi1α inhibits cell fusion during unisexual reproduction, while its usual partner, Sxi2 a , shows only limited inhibition in certain genetic contexts. Further, we show that Sxi1α and Sxi2 a regulate the transcription of distinct gene sets during both α- a and unisexual reproduction, including many within the mating-type ( MAT ) loci, and often act in opposing manners. Together, these findings reveal that Sxi1α and Sxi2 a function both collectively and independently, thereby generating a more flexible and fine-tuned mating control system than previously recognized and advancing our understanding of how this organism produces genetic variation and adapts to changing environments.
