Dissecting the homeodomain MAT locus and engineering novel tripolar and bipolar mating systems in Cryptococcus...

Sex in fungi is governed by the mating-type (MAT) locus, which exists as a bipolar, pseudobipolar, or tetrapolar system. The significance and impact of MAT on sexual reproduction, however, remain understudied. Furthermore, the evolution of fungal MAT loci shares features with the evolution of plant and animal sex chromosomes. Pathogenic Cryptococcus species harbor a bipolar system with a large contiguous MAT locus, whereas closely related species, such as the non-pathogen C. amylolentus, possess a tetrapolar system with unlinked P/R and HD loci. The P/R locus encodes pheromones and pheromone receptors that mediate partner recognition and cell-cell fusion, while the HD locus encodes homeobox domain-containing proteins (Sxi1 and Sxi2) that play important and evolutionarily conserved roles in sexual reproduction. Here, we explored the roles of HD genes in sexual reproduction and determined the implications of a tetrapolar to bipolar MAT transition. Using a CRISPR-Cas9 system we developed for C. amylolentus, we generated gene deletion mutants and demonstrated that a single compatible pair of heteroallelic Sxi1 and Sxi2 is both necessary and sufficient for mating. By relocating the HD genes to the P/R locus, we found that the artificially generated bipolar configuration led to defective sexual development, which could be partially alleviated through additional rounds of sexual reproduction. Transcriptomic profiling revealed the Sxi1-Sxi2 heterodimeric complex drives expression of genes required for DNA replication and ergosterol biosynthesis during sexual reproduction. These findings provide the first experimental demonstration of a tetrapolar-to-bipolar transition in a tetrapolar species, illuminating MAT locus evolution and homeodomain protein functions.IMPORTANCESexual reproduction is critical for fungal survival and adaptation, yet the mechanisms driving transitions between mating systems remain unclear. With Cryptococcus amylolentus, we provide the first experimental validation of a mating system transition from its original tetrapolar state, through an intermediate tripolar state, to a derived bipolar state in a tetrapolar species. We show that homeodomain (HD) protein heterodimers phenotypically govern dikaryotic filamentation and also transcriptionally modulate DNA replication. These findings establish a mechanistic basis for how MAT locus reorganization drives bipolar evolution from an ancestral tetrapolar state and reinforce that fertility depends on the coordinated control of MAT locus architecture and regulatory functions.