Dataset Information

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-? opposite and ?-? 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.

SUBMITTER: Lee SC

PROVIDER: S-EPMC3370462 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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

Lee Soo Chan SC Heitman Joseph J

Eukaryotic cell 20120427 6

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 cere ...[more]

PMID: 22544906

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Project description:Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1alpha/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell-cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence.

Project description:The fungus Cryptococcus neoformans can undergo a-α bisexual and unisexual reproduction. Completion of both sexual reproduction modes requires similar cellular differentiation processes and meiosis. Although bisexual reproduction generates equal number of a and α progeny and is far more efficient than unisexual reproduction under mating-inducing laboratory conditions, the α mating type dominates in nature. Population genetic studies suggest that unisexual reproduction by α isolates might have contributed to this sharply skewed distribution of the mating types. However, the predominance of the α mating type and the seemingly inefficient unisexual reproduction observed under laboratory conditions present a conundrum. Here, we discovered a previously unrecognized condition that promotes unisexual reproduction while suppressing bisexual reproduction. Pheromone is the principal stimulus for bisexual development in Cryptococcus. Interestingly, pheromone and other components of the pheromone pathway, including the key transcription factor Mat2, are not necessary but rather inhibitory for Cryptococcus to complete its unisexual cycle under this condition. The inactivation of the pheromone pathway promotes unisexual reproduction despite the essential role of this pathway in non-self-recognition during bisexual reproduction. Nonetheless, the requirement for the known filamentation regulator Znf2 and the expression of hyphal or basidium specific proteins remain the same for pheromone-dependent or independent sexual reproduction. Transcriptome analyses and an insertional mutagenesis screen in mat2Δ identified calcineurin being essential for this process. We further found that Znf2 and calcineurin work cooperatively in controlling unisexual development in this fungus. These findings indicate that Mat2 acts as a repressor of pheromone-independent unisexual development while serving as an activator for a-α bisexual development. The bi-functionality of Mat2 might have allowed it to act as a toggle switch for the mode of sexual development in this ubiquitous eukaryotic microbe.

Project description:Sexual reproduction is critical for successful evolution of eukaryotic organisms in adaptation to changing environments. In the opportunistic human fungal pathogens, the Cryptococcus pathogenic species complex, C. neoformans primarily undergoes bisexual reproduction, while C. deneoformans undergoes both unisexual and bisexual reproduction. During both unisexual and bisexual cycles, a common set of genetic circuits regulates a yeast-to-hyphal morphological transition, that produces either monokaryotic or dikaryotic hyphae. As such, both the unisexual and bisexual cycles can generate genotypic and phenotypic diversity de novo. Despite the similarities between these two cycles, genetic and morphological differences exist, such as the absence of an opposite mating-type partner and monokaryotic instead of dikaryotic hyphae during C. deneoformans unisexual cycle. To better understand the similarities and differences between these modes of sexual reproduction, we focused on two cellular processes involved in sexual reproduction: cell-cell fusion and karyogamy. We identified orthologs of the plasma membrane fusion protein Prm1 and the nuclear membrane fusion protein Kar5 in both Cryptococcus species, and demonstrated their conserved roles in cell fusion and karyogamy during C. deneoformans α-α unisexual reproduction and C. deneoformans and C. neoformans a-α bisexual reproduction. Notably, karyogamy occurs inside the basidum during bisexual reproduction in C. neoformans, but often occurs earlier following cell fusion during bisexual reproduction in C. deneoformans. Characterization of these two genes also showed that cell fusion is dispensable for solo unisexual reproduction in C. deneoformans. The blastospores produced along hyphae during C. deneoformans unisexual reproduction are diploid, suggesting that diploidization occurs early during hyphal development, possibly through either an endoreplication pathway or cell fusion-independent karyogamy events. Taken together, our findings suggest distinct mating mechanisms for unisexual and bisexual reproduction in Cryptococcus, exemplifying distinct evolutionary trajectories within this pathogenic species complex.

Project description:In fungi, unisexual reproduction, where sexual development is initiated without the presence of two compatible mating type alleles, has been observed in several species that can also undergo traditional bisexual reproduction, including the important human fungal pathogens Cryptococcus neoformans and Candida albicans. While unisexual reproduction has been well characterized qualitatively, detailed quantifications are still lacking for aspects of this process, such as the frequency of recombination during unisexual reproduction, and how this compares with bisexual reproduction. Here, we analyzed meiotic recombination during α-α unisexual and a-α bisexual reproduction of C. neoformans. We found that meiotic recombination operates in a similar fashion during both modes of sexual reproduction. Specifically, we observed that in α-α unisexual reproduction, the numbers of crossovers along the chromosomes during meiosis, recombination frequencies at specific chromosomal regions, as well as meiotic recombination hot and cold spots, are all similar to those observed during a-α bisexual reproduction. The similarity in meiosis is also reflected by the fact that phenotypic segregation among progeny collected from the two modes of sexual reproduction is also similar, with transgressive segregation being observed in both. Additionally, we found diploid meiotic progeny were also produced at similar frequencies in the two modes of sexual reproduction, and transient chromosomal loss and duplication likely occurs frequently and results in aneuploidy and loss of heterozygosity that can span entire chromosomes. Furthermore, in both α-α unisexual and a-α bisexual reproduction, we observed biased allele inheritance in regions on chromosome 4, suggesting the presence of fragile chromosomal regions that might be vulnerable to mitotic recombination. Interestingly, we also observed a crossover event that occurred within the MAT locus during α-α unisexual reproduction. Our results provide definitive evidence that α-α unisexual reproduction is a meiotic process similar to a-α bisexual reproduction.

Project description:Meiotic recombination of sex chromosomes is thought to be repressed in organisms with heterogametic sex determination (e.g. mammalian X/Y chromosomes), due to extensive divergence and chromosomal rearrangements between the two chromosomes. However, proper segregation of sex chromosomes during meiosis requires crossing-over occurring within the pseudoautosomal regions (PAR). Recent studies reveal that recombination, in the form of gene conversion, is widely distributed within and may have played important roles in the evolution of some chromosomal regions within which recombination was thought to be repressed, such as the centromere cores of maize. Cryptococcus neoformans, a major human pathogenic fungus, has an unusually large mating-type locus (MAT, >100 kb), and the MAT alleles from the two opposite mating-types show extensive nucleotide sequence divergence and chromosomal rearrangements, mirroring characteristics of sex chromosomes. Meiotic recombination was assumed to be repressed within the C. neoformans MAT locus. A previous study identified recombination hot spots flanking the C. neoformans MAT, and these hot spots are associated with high GC content. Here, we investigated a GC-rich intergenic region located within the MAT locus of C. neoformans to establish if this region also exhibits unique recombination behavior during meiosis. Population genetics analysis of natural C. neoformans isolates revealed signals of homogenization spanning this GC-rich intergenic region within different C. neoformans lineages, consistent with a model in which gene conversion of this region during meiosis prevents it from diversifying within each lineage. By analyzing meiotic progeny from laboratory crosses, we found that meiotic recombination (gene conversion) occurs around the GC-rich intergenic region at a frequency equal to or greater than the meiotic recombination frequency observed in other genomic regions. We discuss the implications of these findings with regards to the possible functional and evolutionary importance of gene conversion within the C. neoformans MAT locus and, more generally, in fungi.

Dataset Information

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

Publications

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

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