A constitutively active GPCR governs morphogenic transitions in Cryptococcus neoformans

Sex in fungi is driven by peptide pheromones sensed through seven‐transmembrane pheromone receptors. In Cryptococcus neoformans, sexual reproduction occurs through an outcrossing/heterothallic a ‐ sexual cycle or an inbreeding/homothallic – unisexual mating process. Pheromone receptors encoded by the mating‐type locus ( MAT ) mediate reciprocal pheromone sensing during opposite‐sex mating and contribute to but are not essential for unisexual mating. A pheromone receptor‐like gene, CPR2 , was discovered that is not encoded by MAT and whose expression is induced during a ‐ mating. cpr2 mutants are fertile but have a fusion defect and produce abnormal hyphal structures, whereas CPR2 overexpression elicits unisexual reproduction. When heterologously expressed in Saccharomyces cerevisiae , Cpr2 activates pheromone responses in the absence of any ligand. This constitutive activity results from an unconventional residue, Leu²²², in place of a conserved proline in transmembrane domain six; a Cpr2^L222P mutant is no longer constitutively active. Cpr2 engages the same G‐protein activated signalling cascade as the Ste3 a /α pheromone receptors, and thereby competes for pathway activation. This study established a new paradigm in which a naturally occurring constitutively active G protein‐coupled receptor governs morphogenesis in fungi.     

Sex-Induced Silencing Operates During Opposite-Sex and Unisexual Reproduction in Cryptococcus neoformans

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from yeast to hyphae during a-α opposite-sex mating and α-α unisexual reproduction (same-sex mating). Infectious spores are generated during both processes. We previously identified a sex-induced silencing (SIS) pathway in the C. neoformans serotype A var. grubii lineage, in which tandem transgene arrays trigger RNAi-dependent gene silencing at a high frequency during a-α opposite-sex mating, but at an ∼250-fold lower frequency during asexual mitotic vegetative growth. Here we report that SIS also operates during α-α unisexual reproduction. A self-fertile strain containing either SXI2a-URA5 or NEO-URA5 transgene arrays exhibited an elevated silencing frequency during solo and unisexual mating compared with mitotic vegetative growth. We also found that SIS operates at a similar efficiency on transgene arrays of the same copy number during either α-α unisexual reproduction or a-α opposite-sex mating. URA5-derived small RNAs were detected in the silenced progeny of α-α unisexual reproduction and RNAi core components were required, providing evidence that SIS induced by same-sex mating is also mediated by RNAi via sequence-specific small RNAs. In addition, our data show that the SIS RNAi pathway also operates to defend the genome via squelching transposon activity during same-sex mating as it does during opposite-sex mating. Taken together, our results confirm that SIS is conserved between the divergent C. neoformans serotype A and serotype D cryptic sibling species.     

Unisexual Reproduction Enhances Fungal Competitiveness by Promoting Habitat Exploration via Hyphal Growth and Sporulation

Unisexual reproduction is a novel homothallic sexual cycle recently discovered in both ascomycetous and basidiomycetous pathogenic fungi. It is a form of selfing that induces the yeast-to-hyphal dimorphic transition in isolates of the α mating type of the human fungal pathogen Cryptococcus neoformans. Unisexual reproduction may benefit the pathogen by facilitating sexual reproduction in the absence of the opposite a mating type and by generating infectious propagules called basidiospores. Here, we report an independent potential selective advantage of unisexual reproduction beyond genetic exchange and recombination. We competed a wild-type strain capable of undergoing unisexual reproduction with mutants defective in this developmental pathway and found that unisexual reproduction provides a considerable dispersal advantage through hyphal growth and sporulation. Our results show that unisexual reproduction may serve to facilitate access to both nutrients and potential mating partners and may provide a means to maintain the capacity for dimorphic transitions in the environment.     

Dynamic genome plasticity during unisexual reproduction in the human fungal pathogen Cryptococcus deneoformans

Genome copy number variation occurs during each mitotic and meiotic cycle and it is crucial for organisms to maintain their natural ploidy. Defects in ploidy transitions can lead to chromosome instability, which is a hallmark of cancer. Ploidy in the haploid human fungal pathogen Cryptococcus neoformans is exquisitely orchestrated and ranges from haploid to polyploid during sexual development and under various environmental and host conditions. However, the mechanisms controlling these ploidy transitions are largely unknown. During C. deneoformans (formerly C. neoformans var. neoformans, serotype D) unisexual reproduction, ploidy increases prior to the onset of meiosis, can be independent from cell-cell fusion and nuclear fusion, and likely occurs through an endoreplication pathway. To elucidate the molecular mechanisms underlying this ploidy transition, we identified twenty cell cycle-regulating genes encoding cyclins, cyclin-dependent kinases (CDK), and CDK regulators. We characterized four cyclin genes and two CDK regulator genes that were differentially expressed during unisexual reproduction and contributed to diploidization. To detect ploidy transition events, we generated a ploidy reporter, called NURAT, which can detect copy number increases via double selection for nourseothricin-resistant, uracil-prototrophic cells. Utilizing this ploidy reporter, we showed that ploidy transition from haploid to diploid can be detected during the early phases of unisexual reproduction. Interestingly, selection for the NURAT reporter revealed several instances of segmental aneuploidy of multiple chromosomes, which conferred azole resistance in some isolates. These findings provide further evidence of ploidy plasticity in fungi with significant biological and public health implications.     

Unisexual Reproduction Reverses Muller’s Ratchet

Cryptococcus neoformans is a pathogenic basidiomycetous fungus that engages in outcrossing, inbreeding, and selfing forms of unisexual reproduction as well as canonical sexual reproduction between opposite mating types. Long thought to be clonal, >99% of sampled environmental and clinical isolates of C. neoformans are MATα, limiting the frequency of opposite mating-type sexual reproduction. Sexual reproduction allows eukaryotic organisms to exchange genetic information and shuffle their genomes to avoid the irreversible accumulation of deleterious changes that occur in asexual populations, known as Muller’s ratchet. We tested whether unisexual reproduction, which dispenses with the requirement for an opposite mating-type partner, is able to purge the genome of deleterious mutations. We report that the unisexual cycle can restore mutant strains of C. neoformans to wild-type genotype and phenotype, including prototrophy and growth rate. Furthermore, the unisexual cycle allows attenuated strains to purge deleterious mutations and produce progeny that are returned to wild-type virulence. Our results show that unisexual populations of C. neoformans are able to avoid Muller’s ratchet and loss of fitness through a unisexual reproduction cycle involving α-α cell fusion, nuclear fusion, and meiosis. Similar types of unisexual reproduction may operate in other pathogenic and saprobic eukaryotic taxa.     

Regain of sex determination system and sexual reproduction ability in a synthetic octoploid male fish

Polyploids in vertebrates are generally associated with unisexual reproduction, but the direct consequences of polyploidy on sex determination system and reproduction mode remain unknown. Here, we synthesized a group of artificial octoploids between unisexual gynogenetic hexaploid Carassius gibelio and sexual tetraploid Carassius auratus. The synthetic octoploids were revealed to have more than 200 chromosomes, in which 50 chromosomes including the X/Y sex determination system were identified to transfer from sexual tetraploid C. auratus into the unisexual gynogenetic hexaploid C. gibelio. Significantly, a few synthetic octoploid males were found to be fertile, and one octoploid male was confirmed to regain sexual reproduction ability,which exhibits characteristics that are the same to sexual reproduction tetraploid males, such as 1:1 sex ratio occurrence, meiosis completion and euploid sperm formation in spermatogenesis, as well as normal embryo development and gene expression pattern during embryogenesis. Therefore, the current finding provides a unique case to explore the effect of sex determination system incorporation on reproduction mode transition from unisexual gynogenesis to sexual reproduction along with genome synthesis of recurrent polyploidy in vertebrates.     

Early developmental,meiosis-specific proteins — Spo11, Msh4-1, and Msh5 — Affect subsequent genome reorganization in Paramecium tetraurelia

Developmental DNA elimination in Paramecium tetraurelia occurs through a trans-nuclear comparison of the genomes of two distinct types of nuclei: the germline micronucleus (MIC) and the somatic macronucleus (MAC). During sexual reproduction, which starts with meiosis of the germline nuclei, MIC-limited sequences including Internal Eliminated Sequences (IESs) and transposons are eliminated from the developing MAC in a process guided by noncoding RNAs (scnRNAs and iesRNAs). However, our current understanding of this mechanism is still very limited. Therefore, studying both genetic and epigenetic aspects of these processes is a crucial step to understand this phenomenon in more detail. Here, we describe the involvement of homologs of classical meiotic proteins, Spo11, Msh4-1, and Msh5 in this phenomenon. Based on our analyses, we propose that proper functioning of Spo11, Msh4-1, and Msh5 during Paramecium sexual reproduction are necessary for genome reorganization and viable progeny. Also, we show that double-strand breaks (DSBs) in DNA induced during meiosis by Spo11 are crucial for proper IESs excision. In summary, our investigations show that early sexual reproduction processes may significantly influence later somatic genome integrity.     

Variation in the sexual behaviour of Achnanthes longipes (Bacillariophyta). III. Progeny of crosses between monoecious and unisexual clones

The allogamous raphid diatom Achnanthes longipes C. A. Agardh possesses a complex breeding system involving interactions between three types of clone: monoecious, unisexual and bisexual. Previous studies showed that these three types can be crossed with each other, with a tendency for sexual characteristics to be inherited: inbred monoecious lineages gave rise to monoecious or, very rarely, to bisexual clones, while inbred unisexual lineages yielded unisexual and bisexual clones. The current paper reports on the progeny of crosses between monoecious and unisexual clones and their inbred offspring. All three types of clone appeared in the F₁ and F₂, although unisexual clones of opposite sex to the parental clone were not found. Inbreeding depression was observed and also a tendency for ‘normal’ auxosporulation (producing two auxospores per pair of gametangia) to be replaced by ‘reduced’ or ‘intermediate’ auxosporulation (producing one auxospore per pair). In addition, patterns of incompatibility were observed that were not seen during earlier studies of clones isolated directly from nature. These included the inability of some F₁ clones to mate with each other, in spite of compatibility with all other clones examined (unisexual, bisexual and monoecious).     

Unisexual Reproduction of Cryptococcus gattii

Cryptococcus gattii is a basidiomycetous human fungal pathogen that typically causes infection in tropical and subtropical regions and is responsible for an ongoing outbreak in immunocompetent individuals on Vancouver Island and in the Pacific Northwest of the US. Pathogenesis of this species may be linked to its sexual cycle that generates infectious propagules called basidiospores. A marked predominance of only one mating type (α) in clinical and environmental isolates suggests that a-α opposite-sex reproduction may be infrequent or geographically restricted, raising the possibility of an alternative unisexual cycle involving cells of only α mating type, as discovered previously in the related pathogenic species Cryptococcus neoformans. Here we report observation of hallmark features of unisexual reproduction in a clinical isolate of C. gattii (isolate 97/433) and describe genetic and environmental factors conducive to this sexual cycle. Our results are consistent with population genetic evidence of recombination in the largely unisexual populations of C. gattii and provide a useful genetic model for understanding how novel modes of sexual reproduction may contribute to evolution and virulence in this species.     

Cryptococcus neoformans Hyperfilamentous Strain Is Hypervirulent in a Murine Model of Cryptococcal Meningoencephalitis

Cryptococcus neoformans is a human fungal pathogen that causes lethal infections of the lung and central nervous system in immunocompromised individuals. C. neoformans has a defined bipolar sexual life cycle with a and α mating types. During the sexual cycle, which can occur between cells of opposite mating types (bisexual reproduction) or cells of one mating type (unisexual reproduction), a dimorphic transition from yeast to hyphal growth occurs. Hyphal development and meiosis generate abundant spores that, following inhalation, penetrate deep into the lung to enter the alveoli, germinate, and establish a pulmonary infection growing as budding yeast cells. Unisexual reproduction has been directly observed only in the Cryptococcus var. neoformans (serotype D) lineage under laboratory conditions. However, hyphal development has been previously associated with reduced virulence and the serotype D lineage exhibits limited pathogenicity in the murine model. In this study we show that the serotype D hyperfilamentous strain XL280α is hypervirulent in an animal model. It can grow inside the lung of the host, establish a pulmonary infection, and then disseminate to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is usually observed in the highly virulent sibling species C. gattii, responsible for the Pacific Northwest outbreak. These studies provide a technological advance that will facilitate analysis of virulence genes and attributes in C. neoformans var. neoformans, and reveal the virulence potential of serotype D as broader and more dynamic than previously appreciated.     

White Cells Facilitate Opposite- and Same-Sex Mating of Opaque Cells in Candida albicans

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTL a) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.