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

Abstract

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.

Fig 1

Illustration of two modes of C. neoformans sexual development and two distinct stages involved in karyogamy. (A) Opposite-sex mating. (I) Cells of two different mating types recognize each other and undergo cell-cell fusion to form dikaryotic hyphae. (II) At the apex of aerial hyphae, a basidium forms and karyogamy occurs, representing “late” diploidization. (III) Meiosis occurs, and four basidiospore chains are produced from the surface of the basidium by repeated rounds of mitosis of the postmeiotic nuclei. (IV) Progeny of two mating types disperse. (A) Unisexual reproduction. (I) Only α mating type cells undergo hyphal growth, where “early” diploidization might contribute to filamentation. (II) At the apex of aerial hyphae, a basidium forms and “late” diploidization can occur. (III) Meiosis occurs, and four basidiospore chains are formed. (IV) Haploid progeny disperse. Thus, during unisexual reproduction, two different hypothetical timings for diploidization exist. The figure depicts the hypothesis of “late” diploidization. (B) Nuclear congression and nuclear fusion. First, two nuclei migrate toward each other (nuclear congression): Kar1, Kar3, Kar4, and Kar9 are involved in this process in S. cerevisiae. Second, nuclear membrane fusion occurs and Kar2, Kar5, Kar7, and Kar8 are involved in this process. Karyogamy components that are unique to S. cerevisiae compared to C. neoformans are shown in gray.

Fig 2

KAR7 is required for opposite-sex mating in C. neoformans. (A) Wild-type (WT) mating produces abundant dikaryotic hyphae decorated with fused clamp connections, basidia, and chains of spores. A unilateral mating between wild-type and kar7 mutant strains also produces mating hyphae that are less abundant than those in wild-type crosses. In the bilateral kar7 × kar7 mutant crosses, few hyphae were observed. (B) For both wild-type × wild-type and wild-type × kar7 crosses, four complete chains of basidiospores were observed; however, in the bilateral kar7 × kar7 mutant crosses, bald basidia lacking any spores or occasionally basidia with only a single immature spore attached were observed. Scale bars = 100 μm for the upper row and 10 μm for the bottom row in panel A and 5 μm for panel B.

Fig 3

Nuclear positioning during mating in the wild type (A) and kar7 mutant (B). DIC, differential interference contrast. a mating type cells producing Nop1-GFP and α mating type cells producing Nop1-mCherry were cocultured on MS mating medium. The two fluorescent signals were observed to merge, indicating that cytosolic fusion occurs during the process of a-α opposite-sex mating. The two nuclei remain separated in the dikaryotic hyphae. In the basidium, the two nuclei migrate toward each other and karyogamy occurs, resulting in diploidization. Meiosis follows, and chains of basidiospores are produced (A). In the crosses between two kar7 mutants, the dikaryotic hyphae produced are similar to those in the wild type; however, in the mutant basidia, the two congressed nuclei do not undergo fusion, resulting in a failure to proceed into meiosis or produce spore progeny (B). Scale bar = 5 μm.

Fig 4

KAR7 is necessary to complete unisexual reproduction in C. neoformans. The edges of colonies of wild-type (WT) strain XL280 exhibit abundant monokaryotic hyphae; however, two independent sets of kar7 mutants (the first and second ones are independent from the third and fourth from left to right) produced considerably less hyphae (A). The wild type produces four intact chains of basidiospores, whereas in the kar7 mutant, unisexual reproduction was arrested (possibly before meiosis), resulting in no spore progeny production. Images in panels B and C show kar7 mutant basidia with a single attached immature spore. Scale bars = 10 μm for panel B and 5 μm for panel C.

Fig 5

Nuclear positions during unisexual reproduction in the wild type and kar7 mutant. Nuclei were visualized with the GFP-Nop1 protein. (A) In the wild-type XL280 strain, sexual hyphae are monokaryotic with unfused clamp connections. Two nuclei congress in the basidia and undergo karyogamy, meiosis occurs, and basidiospores are produced. DIC, differential interference contrast. (B) In contrast, in the kar7 mutants, the two nuclei congress but do not undergo karyogamy, resulting in a failure to produce the diploid nucleus or undergo meiosis, and consequently, no spore chains are produced. Scale bars = 5 μm.

Fig 6

Unisexual reproduction of a diploid kar7/kar7 strain. In the diploid strain, both alleles of the KAR7 gene were disrupted. The wild type and the heterozygous KAR7/kar7::HYG mutant undergo complete same-sex mating, including abundant sexual hypha formation and basidium formation followed by spore chain production. However, kar7::HYG/kar7::URA5 mutants display mating defects, as seen in the haploid kar7 mutants: for example, less abundant sexual hyphae formation and bald or single spore-attached basidium formation as a consequence of a failure to enter into meiosis or produce spore chains. Scale bars = 100 μm for the upper row and 10 μm for the bottom row.

Fig 7

KAR3 and KAR4 are dispensable for mating in C. neoformans. (A) Serotype A kar3 mutants complete opposite-sex mating without any marked defects observed in either unilateral or bilateral mutant crosses. (B) Unisexual reproduction of kar4 mutants produces four chains of basidiospores. V8 (pH 7) and MS media for serotypes D and A, respectively, were used for mating and incubated at room temperature in the dark for 3 weeks before observation. Scale bars = 50 μm for the upper row and 10 μm for the bottom row in panel A and 10 μm for panel B.

Fig 8

KAR8 is not required for opposite- or same-sex mating. (A) Serotype D kar8 mutants exhibit no apparent defects in sexual development during a-α opposite-sex mating. (B) An XL280-derived kar8 mutant also displays no apparent mating defects. V8 medium (pH 7) was used for mating and incubated at room temperature in the dark for 3 weeks before observation. Scale bars = 100 μm for the upper row and 5 μm for the bottom row in both panels A and B.