Function of Cryptococcus neoformans KAR7 (SEC66) in karyogamy during unisexual and opposite-sex mating

The human basidiomycetous fungal pathogen Cryptococcus neoformans serves as a model fungus to study sexual development and produces infectious propagules, basidiospores, via the sexual cycle. Karyogamy is the process of nuclear fusion and an essential step to complete mating. Therefore, regulation of nuclear fusion is central to understanding sexual development of C. neoformans. However, our knowledge of karyogamy genes was limited. In this study, using a BLAST search with the Saccharomyces cerevisiae KAR genes, we identified five C. neoformans karyogamy gene orthologs: CnKAR2, CnKAR3, CnKAR4, CnKAR7 (or CnSEC66), and CnKAR8. There are no apparent orthologs of the S. cerevisiae genes ScKAR1, ScKAR5, and ScKar9 in C. neoformans. Karyogamy involves the congression of two nuclei followed by nuclear membrane fusion, which results in diploidization. ScKar7 (or ScSec66) is known to be involved in nuclear membrane fusion. In C. neoformans, kar7 mutants display significant defects in hyphal growth and basidiospore chain formation during both a-α opposite and α-α unisexual reproduction. Fluorescent nuclear imaging revealed that during kar7 × kar7 bilateral mutant matings, the nuclei congress but fail to fuse in the basidia. These results demonstrate that the KAR7 gene plays an integral role in both opposite-sex and unisexual mating, indicating that proper control of nuclear dynamics is important. CnKAR2 was found to be essential for viability, and its function in mating is not known. No apparent phenotypes were observed during mating of kar3, kar4, or kar8 mutants, suggesting that the role of these genes may be dispensable for C. neoformans mating, which demonstrates a different evolutionary trajectory for the KAR genes in C. neoformans compared to those in S. cerevisiae.