Function of Cryptococcus neoformans KAR7 ( SEC66 ) in Karyogamy during Unisexual and Opposite-Sex Mating

The human basidiomycetous fungal pathogenCryptococcus 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 ofC. neoformans . However, our knowledge of karyogamy genes was limited. In this study, using a BLAST search with theSaccharomyces cerevisiae KAR genes, we identified fiveC. neoformans karyogamy gene orthologs:CnKAR2 ,CnKAR3 ,CnKAR4 ,CnKAR7 (orCnSEC66 ), andCnKAR8 . There are no apparent orthologs of theS. cerevisiae genesScKAR1 ,ScKAR5 , andScKar9 inC. 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. InC. neoformans ,kar7 mutants display significant defects in hyphal growth and basidiospore chain formation during botha -α opposite and α-α unisexual reproduction. Fluorescent nuclear imaging revealed that duringkar7 ×kar7 bilateral mutant matings, the nuclei congress but fail to fuse in the basidia. These results demonstrate that theKAR7 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 ofkar3 ,kar4 , orkar8 mutants, suggesting that the role of these genes may be dispensable forC. neoformans mating, which demonstrates a different evolutionary trajectory for theKAR genes inC. neoformans compared to those inS. cerevisiae .