Unisexual Reproduction Drives Meiotic Recombination and Phenotypic and Karyotypic Plasticity in Cryptococcus neoformans

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

The wtf meiotic driver gene family has unexpectedly persisted for over 100 million years

Meiotic drivers are selfish elements that bias their own transmission into more than half of the viable progeny produced by a driver+/driver− heterozygote. Meiotic drivers are thought to exist for relatively short evolutionary timespans because a driver gene or gene family is often found in a single species or in a group of very closely related species. Additionally, drivers are generally considered doomed to extinction when they spread to fixation or when suppressors arise. In this study, we examine the evolutionary history of the wtf meiotic drivers first discovered in the fission yeast Schizosaccharomyces pombe. We identify homologous genes in three other fission yeast species, S. octosporus, S. osmophilus, and S. cryophilus, which are estimated to have diverged over 100 million years ago from the S. pombe lineage. Synteny evidence supports that wtf genes were present in the common ancestor of these four species. Moreover, the ancestral genes were likely drivers as wtf genes in S. octosporus cause meiotic drive. Our findings indicate that meiotic drive systems can be maintained for long evolutionary timespans

Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence

Broad antifungal resistance mediated by RNAi-dependent epimutation in the basal human fungal pathogen Mucor circinelloides.

Mucormycosis-an emergent, deadly fungal infection-is difficult to treat, in part because the causative species demonstrate broad clinical antifungal resistance. However, the mechanisms underlying drug resistance in these infections remain poorly understood. Our previous work demonstrated that one major agent of mucormycosis, Mucor circinelloides, can develop resistance to the antifungal agents FK506 and rapamycin through a novel, transient RNA interference-dependent mechanism known as epimutation. Epimutations silence the drug target gene and are selected by drug exposure; the target gene is re-expressed and sensitivity is restored following passage without drug. This silencing process involves generation of small RNA (sRNA) against the target gene via core RNAi pathway proteins. To further elucidate the role of epimutation in the broad antifungal resistance of Mucor, epimutants were isolated that confer resistance to another antifungal agent, 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in PyrF or PyrG, enzymes which convert 5-FOA into the active toxic form. Using sRNA hybridization as well as sRNA library analysis, we demonstrate that these epimutants harbor sRNA against either pyrF or pyrG, and further show that this sRNA is lost after reversion to drug sensitivity. We conclude that epimutation is a mechanism capable of targeting multiple genes, enabling Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis. Furthermore, it improves our understanding of fungal pathogenesis and adaptation to stresses, including the evolution of drug resistance

Generators of phenotypic diversity illustrated as a game of chance.

<p>Evolution is a multifactorial process that depends on numerous complex factors, including routes to diversity that can be deleterious, neutral, or advantageous depending on the environment. Here we depict these evolutionary trajectories using the analogy of a roulette wheel in which one possible path yields a winner that develops a new trait that allows the microorganism to survive in novel conditions, such as a host treated with antimicrobial agents. However, as in roulette, the odds are against the gambler and the other routes and outcomes are deleterious.</p

Harvesting life's energy: increase in the aerotolerence of the electrogenic anaerobe geobacter sulfurreducens due to over-expression of superoxide dismutase and catalase

Gemstone Team iGEMGeobacter-based microbial fuel cells are becoming increasingly viable as a source of alternative energy. Current research and commercial application have been slowed by the inability of Geobacter species to tolerate the presence of oxygen. Oxidative stress protection enzymes normally play a key role in protecting cells from oxygen damage. This project hypothesizes that the over-expression of two important oxidative stress protection enzymes, catalase and superoxide dismutase, can increase aerotolerance. These genes were amplified from the genome of Geobacter sulfurreducens and cloned into plasmid pRG5 behind the ptaclac promoter. This plasmid was transformed into both E. coli and G. sulfurreducens to examine oxygen tolerance, gene expression, and enzyme activity. Preliminary data show increases in enzyme activity in E. coli and an increase in aerotolerance in G. sulfurreducens for both genes. This seems to be the first example of an increase in an obligate anaerobe's aerotolerance due to the intentional introduction of oxidative stress genes

Summary of basidia dissected from opposite sex mating between strains 431α and XL280a.

1<p>: The strain names correspond to those in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004849#pgen.1004849.s009" target="_blank">S4 Table</a>.</p>2<p>: Mixture of basidiospores from two basidia.</p>3<p>: No spore germinated.</p><p>Summary of basidia dissected from opposite sex mating between strains 431α and XL280a.</p