Dissecting the homeodomain <i>MAT</i> locus and engineering novel tripolar and bipolar mating systems in <i>Cryptococcus amylolentus</i>

Sex in fungi is governed by the mating-type (<i>MAT</i>) locus, which exists as a bipolar, pseudobipolar, or tetrapolar system. The significance and impact of <i>MAT</i> on sexual reproduction, however, remain understudied. Furthermore, the evolution of fungal <i>MAT</i> loci shares features with the evolution of plant and animal sex chromosomes. Pathogenic <i>Cryptococcus</i> species harbor a bipolar system with a large contiguous <i>MAT</i> locus, whereas closely related species, such as the non-pathogen <i>C. amylolentus</i>, possess a tetrapolar system with unlinked <i>P/R</i> and <i>HD</i> loci. The <i>P/R</i> locus encodes pheromones and pheromone receptors that mediate partner recognition and cell-cell fusion, while the <i>HD</i> 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 <i>HD</i> genes in sexual reproduction and determined the implications of a tetrapolar to bipolar <i>MAT</i> transition. Using a CRISPR-Cas9 system we developed for <i>C. amylolentus</i>, 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 <i>HD</i> genes to the <i>P/R</i> 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 <i>MAT</i> 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 <i>Cryptococcus amylolentus</i>, 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 <i>MAT</i> locus reorganization drives bipolar evolution from an ancestral tetrapolar state and reinforce that fertility depends on the coordinated control of <i>MAT</i> locus architecture and regulatory functions.