Unisexual and Heterosexual Meiotic Reproduction Generate Aneuploidy and Phenotypic Diversity De Novo in the Yeast Cryptococcus neoformans
Min Ni1.¤a, Marianna Feretzaki1., Wenjun Li1¤b, Anna Floyd-Averette1, Piotr Mieczkowski2, Fred S. Dietrich1, Joseph Heitman1* 1 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America, 2 Department of Genetics, School of Medicine, University of North Carolina, Chapel Hill, North Carolina, United States of America
Abstract Aneuploidy is known to be deleterious and underlies several common human diseases, including cancer and genetic disorders such as trisomy 21 in Down’s syndrome. In contrast, aneuploidy can also be advantageous and in fungi confers antifungal drug resistance and enables rapid adaptive evolution. We report here that sexual reproduction generates phenotypic and genotypic diversity in the human pathogenic yeast Cryptococcus neoformans, which is globally distributed and commonly infects individuals with compromised immunity, such as HIV/AIDS patients, causing life-threatening meningoencephalitis. C. neoformans has a defined a-a opposite sexual cycle; however, .99% of isolates are of the a mating type. Interestingly, a cells can undergo a-a unisexual reproduction, even involving genotypically identical cells. A central question is: Why would cells mate with themselves given that sex is costly and typically serves to admix preexisting genetic diversity from genetically divergent parents? In this study, we demonstrate that a-a unisexual reproduction frequently generates phenotypic diversity, and the majority of these variant progeny are aneuploid. Aneuploidy is responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Phenotypic/ genotypic changes were not observed following asexual mitotic reproduction. Aneuploidy was also detected in progeny from a-a opposite-sex congenic mating; thus, both homothallic and heterothallic sexual reproduction can generate phenotypic diversity de novo. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments.
Citation: Ni M, Feretzaki M, Li W, Floyd-Averette A, Mieczkowski P, et al. (2013) Unisexual and Heterosexual Meiotic Reproduction Generate Aneuploidy and Phenotypic Diversity De Novo in the Yeast Cryptococcus neoformans. PLoS Biol 11(9): e1001653. doi:10.1371/journal.pbio.1001653 Academic Editor: Aaron P. Mitchell, Carnegie Mellon University, United States of America Received April 23, 2013; Accepted August 1, 2013; Published September 10, 2013 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: MN was supported by the Molecular Mycology and Pathogenesis Training Program T32-AI52080 from the NIH/NIAID. This work was supported by NIH/ NIAID grants R37 AI39115-15 and R01 AI50113-10. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: CFU, colony forming unit; CGH, comparative genome hybridization; CHEF, contour-clamped homogenous electric field; Chr, Chromosome; DSBs, double strand breaks; FACS, fluorescence-activating cell sorting; FLC, fluconazole; NGS, next generation sequencing; NS, niger seed; PFGE, pulsed field gel electrophoresis; RFLP, restriction fragment length polymorphism; SNP, single-nucleotide polymorphism; TS, temperature sensitive. * E-mail: [email protected] ¤a Current address: Regeneron Pharmaceuticals, Inc., Tarrytown, New York, United States of America. ¤b Current address: Natcher Building, National Center for Biotechnology Information, National Institutes of Health, Bethesda, Maryland, United States of America. . These authors contributed equally to this work.
Introduction However, aneuploidy can be advantageous in fungi by conferring antifungal drug resistance and enabling rapid adaptive Aneuploidy, a condition in which cells have an abnormal evolution. Aneuploidy evokes transcriptomic and proteomic number of chromosomes, can cause deleterious effects in changes in the model yeast Saccharomyces cerevisiae [8,10]. Mutations organisms throughout the eukaryotic tree of life. Aneuploidy in a deubiquitinating enzyme of S. cerevisiae, which arose during the underlies several common human genetic diseases, including evolution of an aneuploid isolate, leads to improved proliferation trisomy 21 in Down syndrome and trisomy 13 in Patau syndrome of many aneuploid strains, likely by promoting degradation of [1,2], and is detected in more than 90% of solid tumors [3,4]. The aberrant proteins produced in perturbed stoichiometric ratios presence of even a single extra chromosome in primary mouse [11]. Rancanti et al. found that aneuploidy facilitated adaptive embryonic fibroblasts (MEFs) results in proliferative defects and evolution in yeast cells lacking the conserved motor protein Myo1 metabolic aberrations [5]. In Caenorhabditis elegans and Drosophila involved in cytokinesis [12]. Moreover, it has been found that melanogaster, aneuploidy is often lethal [6,7], and in budding and chromosomal duplication may confer a selective advantage to S. fission yeasts, aneuploidy can inhibit cellular proliferation [8,9]. cerevisiae under stress conditions by promoting genomic instability
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Author Summary chains of infectious spores decorating each basidium. These spores are readily aerosolized and cause infections in humans and Aneuploidy refers to increases or decreases in the copy animals when inhaled [32]. number of individual chromosomes (rather than of the While C. neoformans has a defined a-a opposite sexual cycle, entire haploid or diploid genome). In humans, aneuploidy .99% of natural isolates are of the a mating type [33]. C. is well known to be deleterious, causing genetic disorders neoformans var. neoformans (serotype D) a cells can undergo a-a such as Down syndrome (trisomy 21), and frequently unisexual reproduction to generate spores under laboratory occurring during mitosis in the genesis of cancer. By conditions [34]. Recent population genetic studies provide contrast, aneuploidy in fungi can be advantageous, evidence that multiple pathogenic lineages of C. neoformans var. conferring antifungal drug resistance and enabling rapid grubii (serotype A) and the sibling species responsible for the adaptive evolution. Cryptococcus neoformans is a globally Vancouver outbreak Cryptococcus gattii (serotype B and C) undergo distributed human pathogen that often infects patients a a with compromised immunity. It accounts for significant - sexual reproduction in nature, but this remains to be morbidity and mortality associated with HIV/AIDS and is documented under laboratory culture conditions [30,35–41]. linked to more than one million infections and .600,000 Under nutrient-limiting conditions a haploid cells form a diploid deaths per year world-wide. Although C. neoformans has a or a haploid monokaryotic hyphae. The haploid hyphae grow to defined heterosexual cycle involving a and a cells, more form basidia where a late diploidization event occurs. In the than 99% of clinical and environmental isolates are a. diploid hyphae diploidization of the nuclear content may be Interestingly, C. neoformans a cells undergo a-a unisexual induced early and results in a diploid monokaryon that initiates reproduction to generate diploid intermediates and hyphal growth and the formation of apical basidia. Early infectious haploid spores. Sex is costly, though, and the diploidization may occur through endoreplication or cell–cell question therefore arises as to why C. neoformans would fusion. Cell fusion, followed by nuclear fusion, may be induced in undergo selfing unisexual, meiotic reproduction as op- me´nage a`trois matings where a cells serve as pheromone donors posed to more efficient asexual, mitotic reproduction. We to stimulate a-a fusion between genetically different or clonal cells show here that unisexual, meiotic reproduction in C. [34]. Endoreplication may occur in cells that undergo DNA neoformans results in aneuploidy, creating advantageous replication without cell division or they may undergo nuclear genetic diversity de novo. division and then fusion. In all cases, the resulting diploid nucleus undergoes meiosis and multiple rounds of mitosis generate the and mutation, and although it is a transient solution, the short- meiotic progeny, the basidiospores. In previous studies, we found lived aneuploid intermediate may also serve as a capacitator of that the key meiotic regulators, the endonuclease Spo11 and the evolution [13,14]. In the human pathogenic fungi Candida albicans meiotic recombinase Dmc1, are required for spore production and and Cryptococcus neoformans, aneuploidy can confer resistance to germination during unisexual reproduction [34,42]. Deletion of commonly used antifungal drugs such as fluconazole [15–18]. In SPO11 or DMC1 severely impairs sporulation and the fewer C. albicans, haploids can even arise from concerted chromosome meiotic progeny generated are largely inviable, providing further loss from diploid progenitors [19]. In C. albicans, an isochromo- evidence that unisexual reproduction is a meiotic process [34,42]. some 5 can arise in response to fluconazole treatment and confers Remarkably, unisexual reproduction has been recently discov- drug resistance because the left arm of Chr 5 encodes Erg11 ered to occur in another common systemic human fungal (lanosterol 14a demethylase), the target of fluconazole, and Tac1, pathogen, C. albicans [43]. C. albicans is known to undergo a a transcription factor that activates expression of drug export parasexual cycle involving heterothallic fusion of a and a mating pumps [15,17]. Similarly in C. neoformans, Chr 1 disomy confers type cells followed by stochastic, concerted loss of chromosomes azole resistance because this chromosome harbors ERG11 and [44]. Similarly, fusion of a-a and a-a cell unions via homothallic AFR1 (which encodes the major azole efflux pump) and parasexual reproduction occurs when a pheromone-degrading aneuploidy can also influence the virulence of this pathogen protease (Bar1) is inactivated or in me´nage a` trois matings, in [16,20,21]. Interestingly, a similar drug-resistance phenotype has which a third mating partner serves only as the pheromone donor been observed in the protozoan parasite Leishmania, in which [43]. resistance to front line antimonial-based drugs similarly emerges Sex is costly, and thus the question arises as to why C. neoformans via aneuploidy [22,23]. or C. albicans would undergo inbreeding/selfing unisexual repro- C. neoformans is a globally distributed human fungal pathogen duction, which would limit the amount of genetic diversity that causes life-threatening meningoencephalitis [24]. Cryptococcus inherited from the parents, as opposed to a-a sexual reproduction, predominantly infects individuals with compromised immunity, which promotes outcrossing and genetic exchange. We hypothe- such as HIV/AIDS patients. The United States Centers for size that unisexual reproduction is a hypermutagenic process that Disease Control (CDC) has reported that C. neoformans causes more generates genetic diversity de novo and that the resulting progeny than one million cases of cryptococcosis annually with more than can thereby more rapidly adapt to changing environments than 620,000 attributable mortalities, resulting in approximately one- cells produced asexually by mitosis. third of all AIDS-associated deaths [25]. This fungal pathogen has To test this hypothesis, we isolated progeny generated via selfing now surpassed tuberculosis as a common cause of death in Africa. a-a unisexual reproduction and subjected them to phenotypic and C. neoformans is a basidiomycetous fungus that usually grows in genotypic analyses. Remarkably, we found that a-a unisexual the environment as a haploid, budding yeast. This species has a reproduction generates frequent phenotypic diversity, including bipolar mating system with two mating types: a and a [26–28]. In temperature sensitivity, fluconazole resistance or sensitivity, and response to a variety of environmental conditions, a and a cells increased melanin production among other novel traits. Further secrete lipid-modified pheromones that induce cell–cell fusion, and comparative genomic hybridization (CGH) analysis revealed that the resulting dikaryon undergoes a dimorphic transition to hyphal the majority of phenotypically diverse progeny are aneuploid. growth [28–31]. Ultimately, the hyphal tips form basidia fruiting Phenotypic diversity is caused by this aneuploidy, as loss of the bodies wherein nuclear fusion and meiosis occur, and multiple aneuploid chromosomes restored both euploidy and the wild-type rounds of mitosis and budding result in the production of four long parental phenotype. Aneuploidy and phenotypic variation was
PLOS Biology | www.plosbiology.org 2 September 2013 | Volume 11 | Issue 9 | e1001653 Unisexual Reproduction Generates Genetic Diversity also found to occur at a similar rate following a-a bisexual nine in tRNA genes, and 3,849 in intronic regions (31 of which reproduction but not as a result of mitotic asexual vegetative differed in the 59 splice-site and 23 in the 39 splice-site) (Table S2). growth. Our findings show that sex can generate phenotypic and Among the exonic SNPs and indels, 107 SNPs resulted in the genotypic diversity de novo in the pathogenic yeast C. neoformans with introduction or deletion of stop codons, 68 indels resulted in a implications for other eukaryotic microbes and pathogens, frame shift, 14 indels resulted in the loss of amino acids without a including other fungi and parasites that are common pathogens frame shift, and 3,943 SNPs resulted in nonsynonymous amino of humans. acid substitutions in 960 ORFs (Table S2). Further analysis of the modified ORFs will enable the elucidation of the phenotypic Results differences between XL280 and JEC21, for example with respect to the hypersexual phenotype [45]. Genomic Comparison of Parental Strain JEC21 and Its CGH was performed using a 70-mer oligonucleotide micro- Hyperunisexual Selfing Progeny XL280 array covering all predicted ORFs of the JEC21 genome [49] to To investigate whether phenotypic and genotypic changes are detect chromosome copy number alterations. Based on CGH, the generated by selfing unisexual reproduction, we analyzed the genomes of XL280 and JEC21 (Figure 2A) are quite similar and meiotic progeny produced via this process. To this end, we solo span 14 conserved, linear chromosomes. This enabled use of cultured the promiscuous, hypersexual haploid strain XL280a JEC21-based microarray slides for CGH analysis of XL280 under conditions that support robust a-a unisexual reproduction unisexual progeny. Because this microarray covers only ORFs, no (V8 media or FA (filament agar) for ,2 weeks) and isolated spores differences could be detected in centromeric or telomeric regions. by microdissection. The hyperfilamentous haploid strain XL280a There is an ,28 kb region absent in XL280; in JEC21 this [34,45] generates abundant hyphae, homozygous diploid inter- sequence lies near the right end of Chr 5 and is conserved in many mediates, and haploid meiotic spores when grown on mating- C. neoformans strains [53]. In addition, XL280 is missing one copy inducing media all by itself. of the left end of either Chr 8 or 12, which is a 63-kb duplicated Xl280 is a haploid F1 progeny descended from a cross between region located on both Chr 8 and 12 of JEC21 [54]. NGS data two exceptionally well-validated haploid sibling parental strains, further confirmed these findings (Figure 2B). whose genomes have been both sequenced, B3501a and JEC20a The chromosome content of strain XL280 was compared to (which is isogenic to the sequenced strain JEC21a, with the JEC21 by clamped homogenous electric field (CHEF) gel exception of the mating-type locus) (Figure 1A) [34,45–47]. Thus, electrophoresis to detect any chromosomal translocations. Chr 5 while XL280a shares markers with both parents, it only has one and Chr 8/9 of XL280 are smaller than those of JEC21, while Chr copy of each chromosome (it is haploid) and is not heterozygous 13 of XL280 is larger (Figure 2C). To ascertain where the anywhere in its genome. When it undergoes unisexual reproduc- sequence similarity of these chromosomes lies, we excised each tion with itself, it forms a transient homozygous diploid (generated chromosomal band and performed band array analysis. The through either a-a cell fusion between mother and daughter cell or configurations of Chr 9 and 12 differed between XL280 and via endoreplication) that is identical throughout the diploid, JEC21 (Figure S2). Chr 9 of XL280 hybridized to Chr 8 and Chr euploid genome, in which each gene is present in two identical 12 of JEC21; Chr 12 of XL280 hybridized to Chr 8 of JEC21. copies. These findings were confirmed by Southern hybridization Hyphae produced by unisexual reproduction grow to form (Figure 2D). Previous studies reported that this chromosomal terminal basidia wherein the diploid nucleus undergoes meiosis to translocation and duplication occurred during generation of the generate abundant recombinant progeny. That this is a meiotic JEC21a/JEC20a congenic strain pair [54]. During the cross of process has been established in previous studies [34,42] that strains B3501a6B3502a, Chr 9 and Chr 12 formed a dicentric showed both key meiotic genes SPO11 and DMC1 are required for chromosome via telomere–telomere fusion and then broke to form spore production and viability. The parental strains B3501a and new versions of Chr 12 and Chr 8 in strains JEC21/JEC20 [54]. JEC20a share ,50% genetic identity [46]; thus, their F1 progeny Our results indicate that Chr 9 and Chr 12 of XL280 are more (XL280) would be expected to share ,75% genetic identity with similar to those of the B3501a parent, whereas other chromosomes both B3501a and JEC21a, whose genomes have been sequenced are more similar to the JEC20a parent (Figure 2E). This [46]. Therefore, we used the available JEC21 genomic sequence comprehensive analysis of the XL280 genome reveals how its and genome microarray slides as a foundation to study the genome genome is derived from two well-validated parental reference of XL280 and its a-a unisexual reproduction [48,49]. strains and allowed detailed molecular analyses of unisexual We performed next-generation sequencing (NGS) using high- progeny. throughput Illumina sequencing to compare the genomes of XL280a and JEC21a [50]. In total, 47,891,302 sequences (paired- Frequent Phenotypic and Genotypic Changes Following end reads) of 75 bp in length were generated, providing 160-fold C. neoformans Unisexual Reproduction coverage of the XL280 genomic sequence, which was then Solo culture of strain XL280 on V8 sexual reproduction- analyzed using the known JEC21 genome as the reference for the inducing media resulted in robust production of F1 meiotic spore sequence assembly (Figure S1 and Table S1). All but 4,535,426 progeny via selfing a-a unisexual reproduction. This process sequence reads (,10%) were used in the genome assembly, and of involves ploidy changes (1nR2nR1n) via self cell–cell fusion or these remaining reads, the majority were of low quality (90%). We endoreplication, meiosis, and sporulation to produce haploid assembled the Illumina reads using a combination of de novo meiotic progeny. In total, 90 a-a unisexual reproduction meiotic assembly (Velvet, [51]) and reference genome assembly (BWA, progeny were isolated by spore micromanipulation and germina- [52]) (see Figure S1 and Materials and Methods for detailed tion and were examined in a battery of phenotypic analyses. We procedure). assessed major virulence factors of C. neoformans (growth at 37uC XL280 shares 100% identity with JEC21 over 81% of the and melanin production on niger seed (NS) or L-DOPA media), overall genome and is 99.88% identical at the sequence level sensitivity to the antifungal drugs fluconazole (FLC) or FK506, overall (Figure 1C). Among the SNPs and small indels identified, and unisexual reproduction (self-filamentous growth) (Figure S3). 9,021 were located in exonic regions, 10,254 in intergenic regions, In these analyses, six of 90 F1 progeny (,7%) showed a distinct
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Figure 1. Phenotypic and genomic comparison between XL280 and JEC21. (A) The origin of XL280. The parents are NIH12 (MATa), a clinical isolate from the United States, and NIH433 (MATa), an environmental isolate from Denmark. * indicates that genome sequences are available. (B) XL280 is hyperfilamentous. a-a unisexual reproduction generates hyphae, basidia, and meiotic spores along the periphery of yeast colonies incubated on filament agar (FA) at room temperature for 11 d. (C) Whole genome comparison between XL280 and JEC21. Distribution of sequence identity (y-axis) across the genomes averaged over 10 kb intervals. Blue bars represent the 14 chromosomes of JEC21 [46], and red regions represent the candidate centromeres [69]. doi:10.1371/journal.pbio.1001653.g001 phenotype compared to the haploid parental strain XL280 and S4) are clearly evident in even a relatively small sample of progeny the remaining 84 progeny showed no phenotypic differences from produced by this process, which indicates that a-a unisexual the wild-type (Figure 3A and Figure S3). The six isolated variant reproduction generates phenotypic plasticity de novo since there is F1 progeny strains all exhibited temperature-sensitive (TS) growth no preexisting genetic diversity to admix. (Figure 3A). Two progeny (MN77 and MN89) were sensitive to the We next tested if standard mitotic growth might also lead to calcineurin inhibitor FK506 and produced more melanin, phenotypic changes like meiotic unisexual reproduction. A similar indicating they may have similar genetic changes. MN35 was set of 96 isolates derived from XL280 by mitotic asexual growth FLC-sensitive, while the other four progeny (MN27, MN55, exhibited no phenotypic variation from wild-type in the same MN77, and MN89) were relatively FLC-resistant (Figure 3A). On battery of phenotypic tests (Figure S5) in which phenotypic V8 mating-inducing media (V8 agar), MN27 underwent more variation was readily detected in meiotic unisexual reproduction. robust unisexual development, while MN35, MN55, MN77, and Thus, we conclude that phenotypic variation occurs following MN89 generated fewer hyphae (Figure 3A). Although the two meiotic unisex but not standard mitotic asexual growth. genomes of cells undergoing a-a unisexual reproduction are Phenotypic variation is often attributable to genetic changes. To identical, phenotypic changes to these and other conditions (Figure establish the molecular basis of phenotypic variation of the
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Figure 2. Genome comparison of XL280 and JEC21. (A) CGH of XL280 versus JEC21. Coloring indicates gene dosage as follows: gray, no significant change; red, more abundant; green, less abundant. (B) Coverage analysis of XL280 Chr 5 and Chr 8 with JEC21 as the reference. The red circle indicates that XL280 lacks ,28 kb near the right end of Chr 5 compared to JEC21. The blue circle indicates that XL280 has one copy of the 63 kb region that is duplicated in JEC21 and located on the left ends of Chr 8 and Chr 12. (C) Chromosome configuration of XL280 versus JEC21. Black arrows indicate the size differences of Chr 5, 8/9, and 13 between the two strains. (D) Two probes located before and after the break in Chr 8 of JEC21 hybridized to XL280 Chr 9 and 12, respectively. (E) Chr 9 and 12 of XL280 versus JEC21. doi:10.1371/journal.pbio.1001653.g002 unisexual progeny, we examined changes in ploidy, whole- previous findings that diploid strains are (1) intermediates or chromosome aneuploidy, chromosomal translocations, and products of unisexual reproduction and (2) hyperfilamentous single nucleotide polymorphisms (SNPs) by FACS, CGH, (Figure 3B) [34]. None of the other 90 F1 a-a unisexually CHEF, and Illumina NGS, respectively. FACS analysis revealed produced progeny were diploid (Figure 3B and unpublished that the hypersexual progeny MN27 was diploid, in accord with data).
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Figure 3. Unisexual reproduction generates phenotypic diversity. (A) Progeny produced by a-a unisexual reproduction of strain XL280 in solo cultures were grown in 10-fold serial dilution assays and under the following conditions: YPD at 30uC for 2 d; YPD at 37uC for 2 d; YPD plus 1 mg/ mL FK506 at 30uC for 2 d; YPD plus 8 mg/mL fluconazole (FLC) at 30uC for 6 d; NS (melanin-inducing media) at 30uC for 3 d; and V8 pH = 7 (mating-
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inducing media) at room temperature for 11 d. (B) Diploid progeny were generated infrequently following unisexual reproduction. Flow cytometry profiles of cells stained with the fluorescent dye propidium iodide. JEC21 (1n, haploid control); XL143 (2n, diploid control); XL280 (1n); MN27 (2n, the only diploid strain identified among the 90 XL280 a-a unisexual reproduction progeny (1.1%). All other XL280 a-a unisexual progeny were haploid (or aneuploid) (e.g., MN55). Nuclear DNA content is indicated by 1n (haploid) and 2n (diploid). The x-axis indicates fluorescence intensity reflecting DNA content, and the y-axis indicates cell counts. (C) Three SNPs identified by NGS from strains MN7 and MN55 and were confirmed by Sanger sequencing. (D) The WT allele of HSC20 was cloned in two independent plasmids, and these were used to transform strain MN7 and independent transformants were analyzed. This complementation test shows that the TS phenotype of MN7 is attributable to the recessive hsc20-1 mutation. doi:10.1371/journal.pbio.1001653.g003
Three progeny (MN7, MN55, and MN89) were analyzed by These findings indicate that a-a unisexual reproduction induces Illumina NGS to examine the presence of SNPs. In total, only phenotypic and genotypic plasticity. However, a common genetic three SNPs were identified in the ,20 Mb genomes of the three change responsible for the phenotypic changes remained to be isolates based on 48,644,802 sequences of 75 bp generated for identified. MN7, 46,089,962 for MN55, and 44,650,780 for MN89 (Figure 3C). One SNP located at position 878,429 on Chr 7 of a-a Unisexual Reproduction Frequently Generates MN55 resulted in an L318P coding substitution in the hypothet- Aneuploidy ical protein CNJ03170. A second SNP was located at position CGH analysis of the phenotypically variant progeny revealed 491,632 on Chr 8 in both MN7 and MN55, resulting in a cytosine that four out of six isolates (66.7%) were aneuploid. Isolates 9 to thymine change in the 5 UTR of the CNH02880 gene. The MN35, MN55, MN77, and MN89 contained an extra copy of third SNP was located at position 704,393 on Chr 9 of MN7 and chromosome 13, 9, 10, and 10, respectively, indicating that a high resulted in an H66A amino-acid substitution in the co-chaperone rate (.4%) of aneuploidy is generated as a consequence of a-a Hsc20 (CNI02610), a heat shock protein that functions in iron– unisexual reproduction (Figure 5A). Whole genome sequencing of sulfur cluster assembly [55]. To examine whether this SNP is isolates MN55 and MN89 confirmed the presence of an extra copy responsible for the TS phenotype of isolate MN7, we performed a of Chr 9 or Chr 10, respectively, in a majority of cells in the complementation test by introducing the WT allele of HSC20 into population, as ,2-fold higher levels of sequence reads were the MN7 TS strain. We first documented that the hsc20-1 mutant obtained for these chromosomes compared to other genomic allele is recessive based on analysis of an hsc20-1/HSC20 diploid regions (Figure 5B). Interestingly, Sionov et al. previously fusion product of MN7 and XL280a. While the MN7 strain is TS, associated Chr 10 disomy with FLC resistance [16]. the MN76XL280a diploid fusion product was temperature As a complementary approach to CGH, we developed a facile resistant, similar to the XL280a parent. The WT allele of HSC20 was cloned in a plasmid under control of its native multiplex PCR-based method to detect aneuploidy. In this promoter and terminator and introduced into strain MN7 by approach, 14 primer pairs (Table S3) were combined into a single biolistic transformation. In multiple independent transformants, PCR reaction such that each primer pair represents a separate we found that a single copy of the WT HSC20 allele chromosome and amplifies a specific region of different sizes to complemented the TS phenotype of MN7 and restored WT generate a ladder of 100 to 1400 bp (Figure 5C). PCR products of growth similar to the XL280a parent at higher temperature greater intensity reflect the presence of extra chromosomes (red (Figure 3D). Based on these findings we conclude that the TS arrows in Figure 5C). Bioanalyzer analysis further confirmed a phenotype of progeny MN7 is attributable to the H66A amino- ,2-fold increase in PCR product yield from the additional acid substitution in the co-chaperone Hsc20. aneuploid chromosomes present in 1n+1 isolates (Figure S6). Based on CHEF analysis of the chromosomal karyotype, Using multiplex PCR we screened all of the XL280 meiotic (90) chromosome size differences were detected in the a-a unisexual and mitotic (96) progeny and we did not detect any additional F1 progeny MN27, MN55, and MN89 (Figure 4A). For MN27 aneuploid strains from the meiotic progeny and none from the and MN55, an extra chromosomal band was present and migrated mitotic progeny (Figure S7 and Figure S8). more rapidly than Chr 8/9. For MN89, an extra chromosomal In S. cerevisiae, strains aneuploid for different chromosomes all band was observed to migrate more rapidly than Chr 6/7. To share several common phenotypes, including temperature sensi- further analyze these anomalous chromosomes, we excised the tivity [8]. Therefore, we determined whether aneuploid strains of novel chromosomal bands from the CHEF gel and extracted and Cryptococcus exhibit a similar phenotype. Consistent with the fluorescently labeled the DNA to generate probes. Band CGH observed consequences of aneuploidy in S. cerevisiae, all aneuploid analysis revealed that the excised chromosomal bands covered all C. neoformans strains in our study exhibited a TS phenotype ORF regions (the JEC21 expression array covers ORF and not (Figure 3A). centromeric or telomeric regions) of Chr 9, Chr 9, or Chr 7 in Aneuploidy can compromise growth unless it provides a fitness isolates MN27, MN55, or MN89, respectively (Figure 4B) (XL280 benefit under stress or other distinct conditions. We found that Chr 9 hybridizes to Chr 8 and 12 on the JEC21 array). Because all aneuploid strains exhibit a variety of phenotypes potentially ORFs were recognized despite a shortened chromosomal length, associated with virulence, including antifungal drug resistance. To we hypothesized that centromeric or telomeric regions (not test if the progeny have a competitive advantage relative to the represented on the array) may have suffered significant deletions parental strain, we co-incubated an aneuploid progeny (MN55, in the respective strains. To test this hypothesis, we chose MN89 as MN77, or MN89) with the wild-type parent XL280a (marked with an example and designed specific probes that hybridize to the the NAT drug resistance marker) in the presence of fluconazole. centromeric and telomeric regions of Chr 7 (Figure 4C). As expected, the fluconazole-resistant aneuploid progeny exhibited Restriction enzyme digestion of whole chromosomes and CHEF an increased competitive fitness and outgrew the parental strain in electrophoresis followed by Southern hybridization revealed that competitive growth assays by a ratio of ,4:1 (Figure 6). Chr 7 of MN89 has the same telomere lengths as those of XL280, Moreover, to investigate the fitness of these aneuploid unisexual but a shortened centromeric region (Figure 4C). Further whole progeny, we tested their ability to infect and cause disease in a genome sequencing determined that the size of the deleted murine inhalation model. Mice were infected with the aneuploid centromeric region is 10,257 bp (Figure 4D). strains via intranasal instillation and monitored for signs of
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Figure 4. Chromosome deletions arise during unisexual reproduction. (A) PFGE molecular karyotyping of parental strain XL280 and 7 a-a unisexual produced progeny. Chromosome numbers are indicated on the left. Arrows emphasize the chromosomes that differ in size in progeny strains MN27, MN55, and MN89 compared to parental strain XL280. Sc represents the 0.225 to 2.2 Mb S. cerevisiae CHEF DNA Size Markers (BioRad 170-3605). (B) Band array data for chromosomes indicated with black arrows in panel A. DNA of the labeled chromosome band was extracted from the PFGE gel and subjected to CGH. Coloring indicates gene dosage as follows: gray, no significant change; red, more abundant; green, less abundant. (C) A shorter centromere is observed in progeny strain MN89 based on Southern blot analysis using restriction enzymes SwaI and HpaI. (D) The shorter centromere of MN89 was confirmed by genome sequencing. An ,10,257-bp region (blue line) is missing in progeny MN89 compared with parent XL280. doi:10.1371/journal.pbio.1001653.g004 pulmonary cryptococcosis or meningoencephalitis. In contrast to chromosome 13 exhibit a melanin defect and impaired virulence the view that aneuploidy is deleterious, the aneuploid unisexually in mice [21]. However, we found that disomy 13 in C. neoformans generated isolates MN35 (n+113) and MN55 (n+19) were as var. neoformans (serotype D) did not affect melanin production and virulent as the euploid wild-type parent, despite their modest TS the MN35 strain was equally virulent as the wild-type. Thus, the growth at 37uC on YPD rich media in vitro (Figure 7). Previous specific consequences of aneuploidy may differ between even studies have shown that in the clinically important sibling species closely related lineages. Two other aneuploid strains (MN77 and C. neoformans var. grubii (serotype A) strains that are disomic for MN89) were both attenuated compared to wild-type and,
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Figure 5. Aneuploidy is generated at a high rate by unisexual reproduction. (A) CGH array data for four progeny with novel phenotypes. Progeny strains MN35, MN55, MN77, and MN89 contain an extra copy of Chr 13, Chr 9, Chr 10, and Chr 10, respectively. The colors indicate gene dosage as follows: gray, no significant change; red, more abundant; green, less abundant. (B) NGS is consistent with the CGH data in that 26coverage was observed for each aneuploid chromosome and no others. MN55 (yellow) has an extra copy of Chr 9, which corresponds to the left arm of Chr 8 and the right arm of Chr 12 of strain JEC21 (as shown above). MN89 (blue) has an extra copy of Chr 10. (C) Multiplex PCR was conducted to detect aneuploidy. Multiplex PCR reactions used the primer sets listed in Table S3. Amplicons (i.e., chromosomes or chromosome regions) that differed in abundance between samples were identified in this assay and quantified by scanning (arrowheads). Chromosome numbers are indicated on the left. doi:10.1371/journal.pbio.1001653.g005 interestingly, both are aneuploid for chromosome 10 (Figure 7). stressful gauntlet of the host, several aneuploid progeny appeared Cells isolated from lungs of infected animals exhibited relatively as fit as the euploid parent whereas others were attenuated. stable aneuploid phenotypes and were found to retain their aneuploid chromosome in 70–80% of isolates based on multiplex Aneuploidy Is Responsible for Phenotypic Changes PCR [20 isolates were analyzed from each strain, 15 were To examine whether the observed phenotypic changes are a aneuploid for MN35 (75%), 16 for MN55 (80%), 14 for MN77 consequence of aneuploidy, we compared the phenotype of (70%), and 15 for MN89 (75%) (Figure S9)]. Thus, under the aneuploid strains and euploid derivatives obtained following loss
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Figure 6. Aneuploid strains are fluconazole resistance. (A) XL280 meiotic progeny were grown in YPD, diluted 10-fold in water, spotted, and incubated under the following conditions: YPD at 30uC for 2 d, YPD at 37uC for 2 d, and YPD plus 8 mg/mL fluconazole (FLC) at 30uC for 6 d. (B) Three aneuploid strains exhibited increased competitive fitness in competition assays. An XL280 strain marked with a NAT resistance marker integrated in the SPO11 genetic locus was mixed together at equal abundance with XL280, MN55 (n+19), MN77 (n+110), or MN89 (n+110). The mixtures were
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incubated on YPD and YPD plus 8 mg/mL fluconazole (FLC) at 30uC for 3 d. The cells were scraped from the plates, serially diluted, and plated on YPD and YPD plus NAT. The ratios of survival of each strain are shown for one representative experiment, and * represents significance at p,0.005 from three independent experiments. We note that the NAT marker was neutral in this analysis and served to mark and detect the WT competitor cells. (C) The three aneuploid strains exhibited higher growth rates in the presence of fluconazole. Growth curves of the aneuploid strains were similar to the wild-type in rich media. However, in the presence of fluconazole the growth rate of the aneuploid strains was significantly higher than the wild-type. doi:10.1371/journal.pbio.1001653.g006 of the extra chromosome. Aneuploid strains are relatively unstable To test a-a sexual reproduction, we first investigated 88 F1 and frequently lose the 1n+1 extra chromosomal copy to return to progeny from the cross between strains XL280a and JEC20a.To the 1n haploid euploid state. Here, we analyzed isolate MN77, reduce the chance that a-a unisexual progeny from XL280 were which contains an extra copy of Chr 10 and produces increased mixed with the a-a sexual reproduction progeny from the cross, amounts of melanin, a readily scorable phenotype. We grew three times more yeast cells from the a parent JEC20 were mixed MN77 at 37uC to promote loss of the extra chromosome and with the XL280a cells in the cross. As these strains are ,81% isolated 22 randomly selected colonies for phenotypic and congenic, but not isogenic, they exhibit different phenotypes under genotypic testing (Figure 8). Of these 22 isolates, 14 lost the extra various conditions, and their progeny (88 were examined) showed copy of Chr 10 based on multiplex PCR analysis (Figure 8B), and a range of phenotypes (Figure S11). Among 11 strains that had the in all cases concomitantly lost the enhanced melanin production most overt phenotypic changes, four were aneuploid and no other phenotype (Figure 8A), therefore suggesting that Chr 10 disomy is aneuploids were detected in the larger progeny set (Figure 9A). responsible for the higher level of melanin produced. The Further PCR and RFLP tests confirmed that these F1 progeny remaining eight isolates that retained the Chr 10 aneuploidy all strains were products of meiosis (Figure S12). continued to produce higher levels of melanin (Figure 8A). To test In addition, as a previous study showed that the SXI2a whether Chr 10 disomy is also correlated with other phenotypes, homeodomain factor gene is sufficient to drive sexual development we performed growth dilution assays with fluconazole (at 30uC), of haploid a cells [56], we introduced the SXI2a gene into strain FK506 (at 30uC), and for growth at 37uC. Similar to the melanin XL280a to mimic a-a sexual reproduction, generating strain phenotype, all isolates that had lost the extra copy of Chr 10 MN140.23 (Figure S13). Among 90 isolates generated from the exhibited WT phenotypes similar to the XL280 parent, and all selfing of this a+SXI2a self-fertile strain, seven strains exhibited isolates that retained the extra copy of Chr 10 exhibited the phenotypic changes compared to the parental strain (Figure S14). variant phenotype (Figure 8C). Similar experiments were per- Further CGH analysis showed that two of these strains were formed for isolates MN35 (n+113) and MN55 (n+19), and we found aneuploid (Figure 9B). These results provide evidence that that loss of the extra chromosome was linked with loss of the aneuploidy is also generated by a-a sexual reproduction. variant phenotypes (fluconazole sensitivity and fluconazole resis- To further examine this hypothesis, we analyzed a-a progeny tance, respectively) (Figure S10). These results indicate that from the cross between the isogenic C. neoformans var. grubii strains aneuploidy causes the phenotypic changes observed in disomic KN99a and KN99a. Among 88 progeny, 14 strains exhibited strains. phenotypic changes (Figure S15), and eight were aneuploid (Figure 9C), indicating that a-a sexual reproduction between isogenic strains in the serotype A lineage can also generate Aneuploidy Is Generated During Sexual Reproduction in phenotypic and genotypic diversity frequently involving aneuploid C. grubii and C. neoformans progeny. In summary, aneuploidy is generated during both a-a To test whether the generation of aneuploidy is specific to a-a unisexual and a-a congenic sexual reproduction in both C. unisexual reproduction, we isolated progeny from asexual mitotic neoformans var. grubii (serotype A) and C. neoformans var. neoformans reproduction and a-a opposite-sexual reproduction. We isolated (serotype D). 96 single colonies from yeast extract-peptone-dextrose (YPD) media following asexual mitotic reproduction and from spores Discussion produced on V8 agar following a-a sexual reproduction. No phenotypic or genotypic changes were detected among asexual While sexual reproduction serves to admix genetic diversity mitotic progeny (0/96) (Figure S5 and Figure S8). from two distinct parents to produce progeny with a diverse genetic repertoire, sex also comes with a series of costs. Such costs include: (1) metabolic energy that must be devoted to mating and meiosis; (2) energy and time expended locating a mating partner; (3) that only 50% of parental genes are transmitted to any given progeny or that two individuals are required to produce one progeny (resulting in the so-called 2-fold cost of sex); and (4) the fact that two genomes that have run the gauntlet of adaptive selection are shuffled during the process, breaking apart well- adapted genomic configurations [57]. A central question then is: Why would an organism engage in unisexual reproduction? In some examples, unisex involves two genetically distinct a mating partners, and this leads to genetic exchange and the production of Figure 7. Aneuploid strains are pathogenic in the murine recombinant progeny, similar to a-a sexual reproduction. Also, a- inhalation model. Ten female DBA mice per group were infected with a 6 mating lowers the barrier to finding a rare mate in a 10 cells of aneuploid strains MN35, MN55, MN77, and MN89. The mice predominantly a population, enabling outcrossing even if no a were anesthetized by intraperitoneal injection of phenobarbital, and a they were infected through intranasal instillation. The animals were partners are available. But in other cases, an isolate undergoes monitored daily for clinical sighs of cryptococcal infection and sacrificed unisexual reproduction all by itself (via cell–cell fusion or at predetermined clinical points that predict imminent mortality. endoreplication), and in these cases there is no pre-existing genetic doi:10.1371/journal.pbio.1001653.g007 diversity to admix. If there is no heterozygosity of the diploid
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Figure 8. Aneuploidy causes the observed phenotypic changes. Strain MN77, which contains an extra copy of Chr 10 and has increased melanin production, was grown on YPD at 37uC for 3 d to promote aneuploidy loss. The genotypes and phenotypes of 22 resulting mitotic progeny were analyzed. (A) Melanin production of 22 mitotic progeny on NS media at 30uC for 2 d. (B) Multiplex PCR of 22 progeny detected an extra Chr 10 in all of the strains with the increased melanin phenotype and loss of the extra copy of Chr 10 in all progeny with a wild-type phenotype. (C) Phenotypic analysis of 22 mitotic progeny at high-temperature growth (37uC for 2 d) and with drug treatment (FLC and FK506). XL280 (X) and MN77 (77) are the WT and aneuploid controls, respectively. doi:10.1371/journal.pbio.1001653.g008 undergoing meiosis, why undergo sex if the genome is homozy- genotypes by allowing mating between genetically identical cells gous everywhere? (i.e., mother and daughter cells) and adding a limited amount of As shown here, our studies provide direct experimental support genetic diversity (including aneuploidy, chromosomal size poly- for the hypothesis that unisexual reproduction can generate morphisms/deletions, and SNPs) to a well-adapted genotype. In phenotypic and genotypic diversity de novo. Why might this be of essence, unisexual reproduction provides a mechanism by which a selective and adaptive benefit? In the context of considering the well-adapted genotype can be changed much more subtly than costs of sexual reproduction, unisexual reproduction is one strategy standard sexual outcrossing, and in the case of well-adapted by which several of the costs normally associated with sex can be genotypes has the capacity to provide a more parsimonious route lessened or mitigated entirely. First, the cost of finding a mating to progeny that are enhanced in competitive fitness in response to partner is considerably decreased in the case of mother–daughter subtle changes in environmental conditions. cell mating in which the two cells are physically juxtaposed or Organisms that reproduce sexually have an advantage over eliminated entirely, such as in endoreplication, in which a single organisms that reproduce asexually, as preexisting genetic diversity cell transitions from haploid to diploid as a prelude to meiosis. will generate novel combinations in the population. Meiosis, Second, during selfing unisexual reproduction, ,100% of the through homologous recombination, will generate distinct genetic parental genes are directly transmitted to the F1 progeny, thus compositions that may have a selective advantage over the parents reducing the 2-fold cost of sex. Further, if one considers in a new hostile environment. Unisexual reproduction is a sexual aneuploidy, .100% of the parental genes are transmitted to cycle that can occur between genetically identical cells and, as we progeny. Third, unisexual reproduction eliminates the cost of sex observed here, allows the introduction of limited de novo genetic associated with breaking apart well-adapted genomic configura- diversity through meiosis. As in most species, meiosis in Cryptococcus tions. Instead, unisexual reproduction preserves well-adapted is a highly regulated process. Comparative genomics reveals that
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Figure 9. Aneuploidy is generated during a-a sexual reproduction. Genotypic analysis of representative progeny from (A) XL280a crossed with JEC20a; (B) XL280a ‘‘a-a’’ self-sexual reproduction generated via expression of SXI2a; and (C) C. neoformans var. grubii strain KN99a crossed with congenic strain KN99a [70]. doi:10.1371/journal.pbio.1001653.g009
Cryptococcus species contain the meiotic genes involved in homol- found to be critical for sporulation and spore germination during ogous recombination and synaptonemal complex formation unisexual reproduction [34,42]. [46,58]. The genes that support the presence of a meiotic pathway Sex-induced phenotypic and genotypic variation in a clonal (termed the meiotic toolkit genes: DMC1, MND1, MSH4, MSH5, population is not restricted to the Cryptococcus genus. Aspergillus SPO11, HOP1, HOP2, and REC8) are highly conserved in nidulans is a filamentous ascomycete that, along with its well- Cryptococcus, and two of these (SPO11 and DMC1) have been established sexual cycle, also has a parasexual cycle wherein
PLOS Biology | www.plosbiology.org 13 September 2013 | Volume 11 | Issue 9 | e1001653 Unisexual Reproduction Generates Genetic Diversity haploid mycelia fuse and then their nuclei fuse to form diploid Given the finding that aneuploidy can underlie and drive nuclei [59]. In the parasexual cycle, the diploid mycelium adaptive evolution in S. cerevisiae, C. albicans, C. neoformans, and undergoes a transient aneuploid state by repeated loss of whole Leishmania, it seems likely that beneficial impacts of aneuploidy chromosomes to ultimately regenerate haploid progeny. A may be even more ubiquitous and remain to be discovered in recent study has shown that the A. nidulans parasexual cycle other saprobic and pathogenic eukaryotic microbes. can drive adaptive evolution. Hoekstra and colleagues generated homozygous diploid strains that were isogenic with their haploid Materials and Methods progenitor [60]. In a laboratory setting, faster growing variants frequently arose from the homozygous diploid strains but not Ethics Statement from their haploid parents. Remarkably, all of the faster All of the animal studies were conducted in the Division of growing variants derived from a diploid parent were found to Laboratory Animal Resources (DLAR) facilities at Duke Univer- be haploids that arose through the parasexual cycle. As few as sity Medical Center (DUMC). All of the animal work was 3,000 generations were sufficient for the emergence of more performed according to the guidelines of NIH and Duke rapid growth. Through genetic analysis, this study supported a University Institutional Animal Care and Use Committee model wherein the diploid serves as a capacitator for evolution. (IACUC). The animal experiments were reviewed and approved by the DUMC IACUC under protocol number A266-08-10. This enables recessive mutations to arise sequentially in the sheltered state of the diploid where they are complemented, and these mutations do not survive in the haploid because they are Strains and Media individually deleterious and swept from the population before a Strains and plasmids used in this study are listed in Table S4. second mutation can arise [60]. The parasexual state assorts and Yeast cells were grown on YPD media. Mating of C. neoformans was releases these mutations into the haploid state, where they conducted on 5% V8 juice agar medium (pH = 7). exhibit reverse epistasis and only when in combination confer a benefit (i.e., faster growth), whereas individually each recessive Sequencing of C. neoformans mutation was deleterious. If parasexual cycles can function as DNA sequencing was performed on a Genome Analyzer IIx capacitators to generate, store, and then release genotypic and using Illumina Paired Ends technology. The genomic DNA phenotypic diversity de novo, we propose that sexual cycles, samples were fragmented with a Bioruptor sonicator (Diagenode). including unisexual reproduction, might also serve such a role in Fragmented DNA was used for size selection by extraction which meiosis rather than parasexual chromosome loss is (Qiagen) of 300 to 400 bp DNA fragments from 2% agarose gels involved. after electrophoresis. Size-selected DNA was used for standard A similar unisexual selfing mechanism has been observed in the Illumina library preparation protocols. Prepared libraries were sequenced using Paired Ends 2676 cycles. This approach human fungal pathogen C. albicans. Alby et al. found that a/a cells provided the most suitable sequencing data for SNP and small of C. albicans lacking the Bar1 protease that destroys the a-factor indel detection and deep sequencing coverage. mating pheromone undergo a/a-a/a unisexual mating, yielding tetraploids that can undergo concerted chromosome loss to complete a parasexual cycle [43]. In addition, the parasexual Genomic Sequence Assembly Protocol cycle is induced between two a/a mating partners when mixed in The Illumina-Solexa data were assembled using a combination me´nage a trois matings with a limited number of a/a cells that of de novo assembly (Velvet) and reference genome assembly (BWA) Cryptococcus serve as pheromone donors to trigger unisexual a-a reproduction to assemble the genomic sequence. We used BWA and SAMtools to determine the depth of sequence coverage and [43]. However, the parasexual progeny of C. albicans are identify polymorphisms, particularly SNPs and indels. In addition characterized by high rates of aneuploidy [44]. Although this to the use of BWA for reference genome assembly, we checked the chromosomal assortment process is imprecise, it may be retained completeness of the assemblies by extracting all sequence reads due to a selective pressure that favors aneuploid strains under that BWA was unable to align to the reference genome and certain environmental conditions, such as in patients receiving assembled these reads using Velvet. The resulting contigs were fluconazole, a situation in which an isochromosome 5 derivative compared back to the genome sequence and also used to search that confers drug resistance often arises [15]. Aneuploidy has been GenBank using Blast. This method allows identification of regions linked to phenotypic changes, including resistance to antifungal with multiple polymorphisms that cannot be identified by BWA drugs in both C. albicans and C. neoformans, which could provide a due to the inability of the short reads to be aligned to the reference selective advantage during infection [15–17]. sequence. In addition, these contigs typically originate from highly The positive impact of aneuploidy on evolution and genetic repetitive sequence, such as the rDNA and telomeres. With this diversification extends beyond the fungal kingdom to other Cryptococcus dataset, more than 90% of the unaligned reads were unicellular eukaryotes. The parasitic protozoan Leishmania is the low-quality sequence reads. Overall, the assembly was an iterative etiological agent behind one of the most common neglected process. After polymorphisms were identified in an initial round of diseases, leishmaniasis, a global cause of morbidity and mortality. sequence assembly with BWA and Velvet, a new consensus No vaccine is available, and treatment relies heavily on sequence was generated, and the assembly was repeated. In the pentavalent antimonial compounds with diminishing efficacy due second and subsequent rounds of assembly, polymorphisms were to emerging drug resistance [61]. Recent studies have shown that identified in regions where there were multiple polymorphisms. aneuploidy is widespread in natural and clinical populations, with After several rounds, no additional polymorphisms were identified, variation in the ploidy state (monosomic, disomic, or trisomic) for and the assembly was considered complete (Figure S1). different chromosomes even within the same isolate [62,63]. Mosaic aneuploidy in Leishmania generates dynamic genome Ploidy Determination by FACS plasticity, is well tolerated, and can confer drug resistance The ploidy of progeny was determined by flow cytometry as [22,62], analogous to fungal azole resistance conferred by described previously [64]. Briefly, cells were collected from aneuploidy. overnight YPD liquid medium, washed once in 16 PBS buffer,
PLOS Biology | www.plosbiology.org 14 September 2013 | Volume 11 | Issue 9 | e1001653 Unisexual Reproduction Generates Genetic Diversity and fixed in 1 mL of 70% ethanol overnight at 4uC. Fixed cells (Washington University, St. Louis, MO). After hybridization, were washed once with 1 mL of NS buffer (10 mM Tris-HCl arrays were scanned with a GenePix 4000B scanner (Axon [pH = 7.6]; 250 mM sucrose; 1 mM EDTA [pH = 8]; 1 mM Instruments). Data analysis was performed with Genespring GX MgCl2; 0.1 mM CaCl2; and 0.1 mM ZnCl2) and then stained with v7.3 (Agilent Technologies) and CGH-miner. Band array was propidium iodide (0.3 mg/mL) in 200 mL of NS buffer containing performed as described previously [66]. Briefly, chromosomes RNaseA (1 mg/mL) at room temperature for 4 h. Then, 50 mLof were separated by PFGE and the bands of interest were excised the stained cell preparation was diluted into 2 mL of 50 mM Tris- and treated to extract the DNA. Band DNA was labeled with Cy5 HCl (pH = 7.5) and sonicated for 1 min. Flow cytometry was and hybridized to microarrays with Cy3-labeled whole genome performed on 10,000 cells and analyzed on the FL1 channel of a DNA. Becton-Dickinson FACScan. Multiplex PCR Complementation of hsc20-1 TS Mutation in MN7 Genomic DNA was extracted using the CTAB protocol as The strain MN7 was transformed with the dominant selectable described previously [67]. We utilized the Qiagen multiplex PCR drug marker NEO, amplified from plasmid pJAF1 using the kit for multiplex PCR. PCR reaction mixtures (25 mL) contained universal primers M13F and M13R. The strain MN7 NEO was 12.5 mL of Qiagen multiplex PCR master mix, 2 mM equimolar mixed with strain XL280a NAT and incubated on mating media primer mixture (Table S3), and 100 to 300 ng of genomic DNA. for 48 h. The mating culture was scraped off the plate, washed in Thermal cycling conditions included an initial heat activation of water, serially diluted, and plated on YPD+NAT+NEO medium. 15 min at 95uC followed by 30 cycles of denaturation for 30 s at The ploidy of the fusion products was determined by FACS. 94uC, annealing for 90 s at 58.7uC, and extension for 10 min at Multiple diploid isolates were phenotypically analyzed at 30uC 72uC. For bioanalyzer analysis, 1 mL of PCR reaction was and 37uC to determine the recessive nature of the TS mutation in examined with the Experion DNA 12K Analysis Kit (Bio-Rad). strain MN7. Multiplex PCR reactions were analyzed by gel electrophoresis The wild-type HSC20 gene with its native promoter (269 bp) using 1.8% agarose gels in 16TBE buffer (Tris/Borate/EDTA and terminator (246 bp) was amplified from XL280a genomic buffer). 2 mL of PCR reaction were loaded in the gels that were DNA using the primer pair JOHE38840/JOHE38841 and cloned run overnight at 30 volts or for 5 h at 100 volts. The intensity of in the pJAF12 plasmid using KpnI restriction enzyme (NEB) [65]. the desired bands, which represent the aneuploid chromosomes, Two independently derived plasmids, pMF86 and pMF89, were was quantified relative to the control band using the Gel Doc XR+ obtained and confirmed by sequencing. The plasmids were system of BIO-RAD and Image Lab version 4 software. introduced into strain MN7 via biolistic transformation, and multiple isolates were obtained and analyzed phenotypically. Restriction Fragment Length Polymorphism (RFLP) RFLP analysis was performed as described previously [68]. Pulsed-Field Gel Electrophoresis (PFGE) of C. neoformans Based on the SNPs in XL280 and JEC20, we designed two RFLP A single colony was inoculated into 5 mL of liquid YPD markers, RFLP3 and RFLP7 (primers are shown in Table S5), and medium and grown overnight at room temperature (RT) while digested with NdeI and XbaI, respectively. Other markers shaking. Then, 1 mL of the culture was added to 50 mL of yeast included the STE20a and STE20a mating type locus genes to nitrogen base minimal medium (YNB) with 1 M NaCl (to suppress assign the mating type as a or a. capsule formation), which was then grown overnight at RT on a shaking platform incubator. Yeast cells were then washed three times in 0.5 M NaCl/50 mM EDTA (pH = 8.0). Then, 50 mg of Virulence Studies cells were mixed with 450 mL of low-melting point agarose (Bio- Virulence assays were conducted using a murine inhalation Rad 0.5% in 0.1 M EDTA [pH = 8.0]) and 20 mL of cell wall model of cryptococcosis. Cohorts of 4- to 8-wk-old female DBA mice were anesthetized through intraperitoneal injection of lysing buffer (25 mg/mL Zymolase in 10 mM KPO4 [pH = 7.5]) 6 and poured into molds. The plugs were solidified for 15 min at Nembutal (37.5 mg/kg) and infected intranasally with 5610 cells RT, transferred into 700 mL of lysing solution (0.5 M EDTA, diluted in sterile PBS. The cells of the inoculum were diluted and 10 mM Tris-HCl [pH = 8]), and lysed overnight at 37uC. The plated onto YPD medium to determine CFU and viability. Mice next morning, 400 mL of proteinase K solution (5% sarcosyl, were monitored twice daily, and moribund individuals were 5 mg/ml proteinase K, 0.5 M EDTA [pH = 8.0]) were added, and euthanized with CO2. The survival rates were plotted against time the plugs were further incubated at 50uC for 5 h. The plugs were using Kaplan-Meier survival curves, generated with Prism 4.0 then washed three times for 1 h each with wash solution (50 mM (GraphPad software, La Jolla, CA, USA). The p values were EDTA, 10 mM Tris-HCl [pH = 8.0]). Then, 1% gels made with evaluated by a Log-rank (Mantel-Cox) test. A p value of ,0.05 was pulse field certified agarose in 0.56 TBE buffer were run with a considered significant. The lungs and brains of the euthanized BioRad CHEF Mapper XA system. Running conditions were animals were removed, weighed, and homogenized in 2 ml sterile according to the CHEF Mapper calculation, with appropriate PBS. The samples were serial diluted and plated on YPD media to modifications. count CFUs. Twenty random isolates were colony purified and subjected to multiplex PCR to detect aneuploidy. CGH and Band Array Genomic DNA was ultrasonicated to generate ,500-bp Competitive Fitness and Growth Curve Assays fragments and purified with a DNA Clean and Concentrator kit XL280, MN55, MN77, MN89 (all NAT sensitive), and XL280 (Zymo Research, CA). Then, 2.5 mg of DNA was used for Cy3 marked with the NAT resistance marker integrated at the SPO11 dUTP or Cy5 dUTP labeling reactions using the Random genetic locus (XL280 NAT) were used to measure the competitive Primer/Reaction Buffer mix (BioPrime Array CGH Genomic fitness of the aneuploid strains in the presence of fluconazole. Labeling System, Invitrogen). Labeled DNA was then hybridized Strains were grown overnight in liquid cultures in YPD, washed to microarray slides of 70-mer oligonucleotides for the C. neoformans with sterile water, and diluted to a density of 16107 cells/ml. The JEC21 [49] or the C. neoformans JEC21 and H99 genomes following strains were mixed in equal ratios: XL280 with XL280
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NAT, MN55 with XL280 NAT, MN77 with XL280 NAT, and Figure S6 Bioanalyzer profiles of multiplex PCR reac- MN89 with XL280 NAT. The mixtures were spotted on YPD and tions. Genomic DNA from XL280 (A, control strain) and YPD plus 8 mg/mL fluconazole (FLC) and incubated for 3 d at aneuploid progeny MN77 (n+110) (B) served as template. PCR 30uC. Then, the cells were recovered from the plates, washed products were analyzed via the BioRad Experion Bioanalyzer with sterile water, and plated on YPD to count CFUs. Following System, which identified amplicons that differed in abundance 2 d incubation at 30uC, 300 isolates were replica plated onto between the strains (blue arrows). NAT selective media and survival rates were calculated. The (TIF) experiments were conducted in triplicate. For the growth curve assays, the strains were grown overnight in liquid cultures, Figure S7 Multiplex PCR on XL280 meiotic progeny. washed with sterile water, and diluted into 46104 cells in 200 ml Genomic DNA from 90 unisexual reproduction meiotic progeny of YPD and YPD plus 2 mg/mL fluconazole (fluconazole was isolated and subjected to multiplex PCR to detect aneuploidy. concentration was determined experimentally). The cells were All four aneuploid strains are marked with red. All other 86 progeny were found to be euploids in two replicates of this incubated at room temperature in 96-well plates, and OD600 was measured every 2 h for 48 h using a Tecan-Sunrise analysis. microplate reader. All of the experiments were performed in (TIF) triplicate. Figure S8 Multiplex PCR on XL280 mitotic progeny. Ninety-six isolates of XL280 derived from mitotic asexual Supporting Information growth on YPD were selected. DNA was isolated and Figure S1 NGS data analysis pipeline. Approximately 48 subjected to multiplex PCR to search for aneuploidy. No million 75 bp reads were aligned to the reference genome (JEC21) aneuploids were detected among the 96 progeny, which were representing ,1606 coverage. By performing BWA and SAM all euploid. tools analysis, we detected SNPs and small indels. Unaligned reads (TIF) were assembled de novo by Velvet analysis and blasted against the Figure S9 Aneuploid strains were stable inside the reference genome. The new genome was assembled by replacing murine host. Cells were isolated from homogenized lungs of SNPs. The reads were again aligned to newly assembled genome infected animals with MN35 (n+113), MN55 (n+19), MN77 assemblies to further detect any SNPs that escaped the previous (n+110), and MN89 (n+110), and DNA was isolated from 20 round of analysis. The whole cycle was repeated 30 times to colony-purified isolates and subjected to multiplex PCR to detect assemble the XL280 genome. aneuploidy. The abundance of the aneuploid/euploid chromo- (TIF) some was quantified by estimating the relative intensity of the Figure S2 Band array data for XL280 chromosomes. desired chromosomal bands compared to the respective control. DNA of each band (Chr 5, Chr 6/7, Chr 8/9, Chr 10, Chr 11, Aneuploid strains are marked with red, whereas strains that Chr 12, Chr 13, and Chr 14) was extracted from the PFGE gel (A) reverted to euploid are marked black. and analyzed by CGH (B). Coloring indicates gene dosage as (TIF) follows: gray, no significant change; red, more abundant; green, Figure S10 Aneuploidy also causes the observed less abundant. Sc represents the 0.225–2.2 Mb S. cerevisiae CHEF 13 phenotypic changes in MN35 and MN55. MN35 (n+1 ) DNA Size Markers (BioRad 170-3605). and MN55 (n+19) were grown on YPD for 3 d to promote the (TIF) loss of the extra chromosome. Twenty isolates were picked and Figure S3 Unisexual reproduction progeny of XL280 they were subjected to phenotypic and genotypic analysis. (A) exhibit novel phenotypes. Strains were spotted in 10-fold MN35 (n+113) and 20 isolates were grown, 10-fold serially serial dilutions and grown under the following conditions: (A) YPD diluted, and spotted on YPD at 30uC for 2 d, YPD at 37uCfor at 30uC for 2 d, (B) YPD at 37uC for 2 d, and (C) YPD plus 8 mg/ 2d,andYPDplus8mg/mL fluconazole (FLC) at 30uCfor4d. mL fluconazole (FLC) at 30uC for 4 d. Red labels indicate the Multiplex PCR detect an extra Chr 13 in all of the strains with progeny with variant phenotypes compared to the parental strain. the fluconazole sensitivity observed in MN35. (B) The MN55 (TIF) (n+19) and 20 isolates were spotted in 10-fold serial dilutions and Figure S4 Proliferative capacity of aneuploid strains in incubated under the same conditions. Multiplex PCR confirmed the presence of drugs or chemicals. Aneuploid strains were that the isolates with the fluconazole resistant phenotype carry 10-fold serially diluted, spotted, and grown under the following an extra chromosome 9. conditions: (A) YPD at 30uC for 2 d, (B) YPD plus 10 mg/mL (TIF) hygromycin at 30uC for 2 d, (C) YPD plus 0.1 mg/mL Figure S11 Opposite sexual reproduction progeny of cycloheximide at 30uC for 3 d, (D) YPD plus 0.1 mg/mL XL280 crossed with JEC20 exhibit phenotypic variation. cycloheximide at 30uC for 4 d, (E) YPD plus 200 mM rapamycin Strains were spotted in 10-fold serial dilutions and grown under at 30uC for 3 d; (F) YPD plus 0.1 mM benomyl at 30uC for 2 d; the following conditions: (A) YPD at 30uC for 2 d, (B) YPD at and (G) YPD plus 0.5 mM H2O2 at 30uC for 4 d. 37uC for 2 d, (C) YPD plus 8 mg/mL fluconazole (FLC) at 30uC (TIF) for 4 d, and (D) YPD plus 1 mg/mL FK506 at 30uC for 2 d. a and Figure S5 XL280 mitotic progeny grown on YPD did not a represent the parental strains XL280 and JEC20, respectively. exhibit any phenotypic changes. Strains were 10-fold serially Red labels indicate the progeny whose phenotypes differed diluted, spotted, and grown under the following conditions: (A) compared to the parental strains. YPD at 30uC for 2 d, (B) YPD at 37uC for 2 d, and (C) YPD plus (TIF) 8 mg/mL fluconazole (FLC) at 30uC for 4 d. In no case was any Figure S12 Aneuploid strains from the cross between progeny produced by mitosis found to differ phenotypically from XL280 and JEC20 are recombinants. Four markers were the XL280 parental strain. analyzed: STE20a, STE20a, RFLP3, and RFLP7. MM10, MM16, (TIF)
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MM51, and MM67 are four aneuploid progeny from the cross (TIF) between strains XL280 and JEC20. Table S1 Summary of reads from Illumina sequencing. (TIF) (DOC) Figure S13 SXI2a is overexpressed in MN140.23. North- ern blot analysis using the SXI2a gene as a probe detected the Table S2 XL280 versus JEC21 exonic and intronic overexpressed SXI2a gene in strain MN140.23, which contains a changes. (XLS) PGPD1::SXI2a transgene in the XL280 background. EtBr staining of rRNA served as a loading control. Table S3 Primers used in the multiplex PCR assay. (TIF) (DOC) Figure S14 Unisexual reproduction progeny of Table S4 Strains and plasmids used in this study. MN140.23 (XL280, PGPD1::SXI2a) exhibit phenotypic (DOC) variation. Strains were spotted in 10-fold serial dilutions and grown under the following conditions: (A) YPD at 30uC for 2 d, (B) Table S5 Primers used in this study. YPD at 37uC for 2 d, (C) YPD plus 8 mg/mL fluconazole (FLC) at (DOC) 30uC for 4 d, and (D) YPD plus 1 mg/mL FK506 at 30uC for 2 d. C1, C2, and C3 represent strains XL280, XL566 (XL280 ura5), Acknowledgments and MN140.23. Red labels indicate the progeny with phenotypes We thank Juan Lucas Argueso, Pat Greenwell, and Tom Petes for advice. that differed compared to the parental strain. We thank Cecelia Shertz Wall, Tom Petes, Blanche Capel, and Danny (TIF) Lew for critical reading of the manuscript. Figure S15 Opposite sexual reproduction progeny of isogenic serotype A strains KN99a and KN99a exhibit Author Contributions phenotypic variation. Strains were 10-fold serially diluted, The author(s) have made the following declarations about their spotted, and grown under the following conditions: (A) YPD at contributions: Conceived and designed the experiments: MN MF JH. 30uC for 2 d, (B) YPD at 37uC for 2 d, (C) YPD plus 8 mg/mL Performed the experiments: MN MF WL AF. Analyzed the data: MN MF fluconazole (FLC) at 30uC for 4 d, and (D) YPD plus 1 mg/mL WL FSD. Contributed reagents/materials/analysis tools: PM FSD. Wrote FK506 at 30uC for 2 d. The progeny that phenotypically differed the paper: MN MF JH. from the wild-type is marked red.
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Primer whereas others are not yeast cells undergo meiosis and (heterothallic). The mechanisms sporulate to produce two a and that give rise to only two versus two α haploid progeny. many mating-types, and those A notable feature of budding Fungal mating- that enable mating-type switching yeast is the capacity to switch type loci are now understood in mating-type, which makes it a considerable molecular detail. homothallic organism. The S. The mating-type locus of fungi cerevisiae MAT locus (Figure 1) James A. Fraser and Joseph has evolved into two different resides on chromosome III, and Heitman organizational paradigms: the the unique region of the active bipolar and tetrapolar systems. In MAT locus spans only 642 bp in a Fungi form one of the most organisms with a bipolar mating- cells and 747 bp in α cells, a small ubiquitous and successful type system, cells are of two fraction of the 316,613 bp kingdoms of life on Earth. There opposite mating-types — chromosome on which they are more than 100,000 known commonly a and α or plus and reside. Chromosome III also species of fungi, divided into four minus — and cell identity is contains two other copies of the phyla — ascomycetes, dictated by a single MAT locus mating-type cassette at the basidiomycetes, zygomycetes that has two alternative alleles. telomeric HML and HMR loci, and and chytrids — and these have For sexual reproduction to occur, these MAT alleles are populated a vast array of mating partner cells must have transcriptionally repressed by a biological niches, including those different MAT alleles. Bipolar process known as silencing. that are pathogens of animals systems promote inbreeding, and Mating-type switching is restricted (including humans and insects), can lead to the rapid homozygosis to cells that have budded at least plants and even other of recessive mutations in once (mother cells), and is microorganisms. The organisms that are predominantly effected by an endonuclease, HO, morphological diversity among diploid, such as S. cerevisiae. which cleaves MAT and triggers species is staggering, from the Fungi with a tetrapolar system, recombination between the active budding yeasts and filamentous by contrast, arrange their mating- and silent cassettes. This process fungi to the mushrooms and wood type information in two distinct is thought to promote mating of rotting organisms. Fungi also unlinked regions of the genome, siblings following meiosis to serve as powerful tools for our commonly known as the a and b rapidly re-establish the diploid understanding of cell biology, and loci. Both of these regions must state and possibly also several have emerged as model differ for mating to occur. In many homozygose recessive mutations. systems, including most notably tetrapolar fungi, the mating-type Much of what we know about the budding yeast loci are multiallelic, giving rise to the fungal bipolar system is based Saccharomyces cerevisiae. We thousands of mating-types in the on a detailed understanding of the shall review here our current most extreme examples. S. cerevisiae MAT locus, which understanding of the role of a Tetrapolar systems promote encodes either one or two key cell specialized region of the fungal outbreeding, as any given meiotic fate determinants: a1 or α2, genome known as the mating- segregant can only interbreed with homodomain proteins which can type locus, which plays a central 25% of its siblings from a cross: homodimerize (α2–α2) or role in the sexual cycle. a1 b1 can mate with a2 b2, but not heterodimerize (a1–α2) to repress The mating-type, or MAT, locus with a1 b1, a1 b2 or a2 b1. The a-specific or haploid-specific is a unique region of the fungal evolution of these paradigms in genes, respectively (Figure 2). A genome that governs the part reflects the evolution of third product is α1, another DNA- establishment of cell-type identity different phyla of fungi, in that binding protein that activates α- and orchestrates the sexual cycle. ascomycetous fungi have bipolar cell specific genes in α cells. The It is this region that differs in DNA mating-type systems, whereas central feature is that cell identity sequence between cells of basidiomycetous fungi may be is controlled by only one or two opposite mating-type. The MAT either bipolar or tetrapolar. principal determinants, locus encodes global transcription homeodomain or α-domain factors, which establish cell-type Saccharomyces cerevisiae — the proteins which bind DNA to identity by controlling the yeast MAT paradigm control expression of other genes. expression of developmental The first paradigm for This theme has been revisited in cascades, and this commonly understanding fungal cell identity many other fungi in which cell involves homeodomain or other came from studies of the mating- identity determinants are classes of transcriptional type locus of S. cerevisiae. This homeodomain or HMG box regulatory elements. Some fungi organism has two haploid mating- transcriptional regulators exist in only two mating-types types, a and α, which signal via (sometimes both). (bipolar), whereas others occur in secreted peptide pheromones that hundreds or thousands of mating- trigger cell and then nuclear fusion Candida albicans — the MAT types (tetrapolar). Some fungi are to produce stable a/α diploid locus of a pathogenic yeast also endowed with the capacity to cells. In response to adverse The recent discovery that the switch mating-type (homothallic), nutritional conditions, a/α diploid genome of Candida albicans Current Biology Vol 13 No 20 R793
Saccharomyces cerevisiae Candida albicans Ustilago maydis Homothallic, bipolar ascomycete Heterothallic, bipolar ascomycete Heterothallic, tetrapolar basidiomycete bW1 bE1 α2 α1 a1 a1 α2 OBP1 α PIK1 α α1 PAP1 α α mfa2 pra2 b1 HMLα MATa HMRa MTLα a2 bW2 bE2 α2 α1 α2 α1 a1 PAP1 a OBP1 a PIK1 a a1 mfa1 pra1 b2 HMLα MATα HMRa MTLa a1
Cryptococcus neoformans Heterothallic, bipolar basidiomycete
α α α α α α α α α α α α α α α α α α α α α α SXI1 CAP1 SPO14 RPL22 IKS1 STE3 ETF1 MF 4 MFα3 ZNF1 PRT1 MF 2 MF 1 MYO2 STE20 STE11 RPO41 LPD1 CID1 RUM1 RPL39 STE12 MATα
STE12 a STE3 a MFa2 RPO41 a STE11 a SPO14 a CAP1 a RPL39 a PRT1 a ZNF1 a LPD1 a CID1 a MFa1 IKS1 a RPL22 a ETF1 a STE20 a α RUM1 a MYO2 a MATa Current Biology
Figure 1. The structure of fungal MAT loci. Fungi have either bipolar or tetrapolar mating-type systems which direct the interaction of partners during the sexual cycle. S. cerevisiae is a homothallic ascomycete with a bipolar mating system. Cells have the ability to undergo mating-type switching in response to cleav- age by the HO endonuclease, allowing the active MAT cassette to be replaced with the silent cassette of opposite mating-type. The closely related pathogenic yeast C. albicans also has a bipolar system, but lacks silent cassettes or the HO endonuclease, and does not undergo mating-type switching. The phytopathogen U. maydis is a tetrapolar, heterothallic fungus with two mating-type loci: one, b, encodes homeodomain transcription factors, while the other, a, encodes pheromones and pheromone receptors; these are multi-allelic loci, giving rise to many different mating-types. In contrast, the pathogenic basidiomycete C. neoformans has a bipolar system with only two mating types, though it is also heterothallic. The C. neoformans mating-type locus encodes a homeodomain transcription factor, pheromones and pheromone receptors, and other elements of the pheromone activated MAP kinase cascade in addition to many genes whose role in mating, if any, is unknown. encodes a mating-type locus high efficiency, but this requires a sequence of C. albicans of any (MTL) overturned the long-held developmental switch in which the silent versions of the MTL alleles, dogma that this pathogenic yeast normal ‘white’ cells of the nor of a homolog of the HO is asexual. MTL of C. albicans organism convert to mating- endonuclease that drives shares striking features with MAT specialized ‘opaque’ cells. How switching. A second striking from S. cerevisiae, also existing in this occurs in vivo was unknown, difference between the two yeasts two opposite alleles: MTLa, which as the opaque cell form is is that the mating-type locus of S. encodes a homolog of the unstable at 37°C, but C. albicans cerevisiae controls not only budding yeast a1protein; and has recently been found to mate mating but also meiosis, whereas MTLα, which encodes homologs with high efficiency on skin, where C. albicans has never been of the yeast α1 and α2 proteins. the temperature is several observed to undergo meiosis or The C. albicans MTL alleles are degrees cooler. Furthermore, the sporulate. The a/a/α/α tetraploid larger than those of budding yeast identification of naturally cells produced by mating in C. (8742 and 8861 bp), and encode occurring a/a and α/α albicans have recently been found three additional genes with homozygous diploid strains to undergo chromosome loss to divergent mating-type specific amongst clinical isolates further produce a/a, a/α and α/α diploid alleles. These additional MAT- supports the idea that mating may progeny, and thus might have specific genes encode homologs indeed occur during commensal evolved an alternative version of of poly(A) polymerase, an or pathogenic growth of this the sexual cycle that involves cell oxysterol binding protein, and the ubiquitous human fungal fusion and ploidy reduction but phosphatidylinositol kinase Pik1, pathogen. not meiosis and sporulation, whose roles in mating, if any, are Although there are striking possibly as a means to benefit currently unknown. parallels in the arrangement of the from mating yet survive Most isolates of C. albicans are mating-type locus in S. cerevisiae continuous assault by the host a/α diploids which, like S. and C. albicans despite the ~200 immune system. cerevisiae diploids, are not million years of evolution capable of mating. But C. albicans separating them from a common Cryptococcus neoformans —a strains engineered to be either ancestor, there are two marked step in the evolution of sex homozygous (a/a or α/α) or differences. First, S. cerevisiae is chromosomes hemizygous (a/∆ or α/∆) at MTL a homothallic yeast that readily Cryptococcus neoformans is a mate at low efficiency both in vitro switches mating-type, whereas C. basidiomycete, and so quite and during infection in an animal albicans is a heterothallic yeast divergent in evolution from S. model. More recent studies have that does not switch mating-type cerevisiae and C. albicans, which shown that mating can occur with — there is no sign in the genome are both ascomycetes. And yet Current Biology Vol 13 No 20 R794
information in a fungal pathogen. Saccharomyces cerevisiae Cryptococcus neoformans The MAT locus of C. neoformans shares features both with the other α α Y a1 1 genes X α known fungal MAT loci and the α SXI1α a genes α2 α2 a genes larger, more complex sex chromosomes of animals and a cell α cell a cell α cell plants. α The products encoded by the X Y SXI1 α2 α2 a genes Haploid- MAT locus of C. neoformans fall Haploid- Z Sxi1α specific genes into three functional categories: a1α2 specific genes Dikaryon/diploid- the mating-type-specific cell Z Sxi1α specific genes identity determinant, elements of Diploid cell Dikaryon or diploid cell a specialized MAP kinase Current Biology cascade that governs mating, and proteins of a variety of different Figure 2. Two fungal mating paradigms: S. cerevisiae and C. neoformans. In the budding yeast S. cerevisiae, cells express either of two homeodomain proteins, a1 functional groups which may or or α2, depending on which cassette resides at the active MAT locus. In α cells, an α2–α2 may not be involved in mating. In homodimer represses a-specific genes, while another MATα-encoded product (α1) acti- addition to encoding pheromones vates α-specific genes. Upon cell fusion, haploid-specific genes (including α1) are and pheromone receptors repressed by the a1–α2 heterodimer and a-specific genes continue to be repressed by involved in the earliest steps in α α the 2– 2 homodimer. In contrast, in the basidiomycete C. neoformans, the cell-identity mate recognition — a feature factor Sxi1α is present at low levels in haploid cells. SXI1α expression is activated fol- lowing cell fusion by factors that remain to be identified (X and Y), allowing it to partici- shared with the MAT loci of pate with a partner from a-cells (depicted here as Z) in repression of haploid-specific several other basidiomycetes — genes and activation of unknown targets involved in dikaryon and diploid cell functions. the C. neoformans MAT locus also encodes several elements of the like C. albicans, C. neoformans is strains, and are endowed with the pheromone-activated MAP kinase a ubiquitous human fungal unusual ability to differentiate by a cascade itself. These include pathogen to which we are all process known as haploid fruiting homologs of the Ste20 PAK exposed early in life, as it occurs which involves filamentation and kinase, the Ste11 MEK kinase, worldwide in association with the production of spores of only and the transcription factor Ste12. pigeon guano and certain tree the α mating-type. Thus a and α cells have divergent species. The organism can reside The entire a and α alleles of the alleles of these pathway elements, in a dormant latent state in mating-type locus have recently and so employ two somewhat granulomas and later emerge to been cloned and sequenced from different signaling cascades, cause life-threatening infections two divergent varieties of C. which may contribute to the of the central nervous system. neoformans. The MAT locus spans unique cell-type specific The sexual cycle of C. >100 kb and contains more than 20 specializations involving mating, neoformans involves a bipolar genes, some of which function in haploid fruiting and pathogenesis. mating-type system with haploid a differentiation and virulence, others The MAT locus also encodes a and α cells that do not switch having no clear link to either. The large number of other gene mating-type. In contrast to S. one gene unique to the α allele of products, some essential. cerevisiae, where cell fusion is the MAT locus encodes a Whether these genes play mating- immediately followed by nuclear homeodomain factor, Sxi1α, which and virulence-specific roles, or fusion to establish the diploid functional studies have shown is a have simply been entrapped in the state, nuclear fusion is delayed in key cell fate determinant that MAT locus during its expansion, C. neoformans and other establishes a/α cell identity (Figure remains to be determined. basidiomycetes, and the fusion of 2). Sxi1α is related to other fungal There is a striking contrast yeast-like a and α cells that homeodomain proteins, including between the compact mating-type occurs in response to nutrient a1 and α2 of S. cerevisiae and C. locus of S. cerevisiae, which spans limitation produces a dikaryon, albicans. Remarkably, the a and α only ~700 bp and encodes only which grows filamentously. The alleles of the C. neoformans MAT one or two key cell fate filament tips ultimately produce a locus are otherwise composed of determinants, and the C. fruiting structure, the basidium, divergent sets of the same genes neoformans mating-type locus, where nuclear fusion, meiosis and and so evolved from a common which spans more than 100 kb and sporulation occur. Interest in the ancestral DNA region, which has includes more than 20 genes. And mating-type locus of this been extensively remodeled. while S. cerevisiae is a homothallic pathogenic fungus was fueled by Comparison of the divergent MAT yeast that can switch mating-type, early studies which revealed that alleles has suggested molecular C. neoformans is heterothallic (like mating-type is linked to mechanisms by which the locus C. albicans) and does not switch physiology and virulence in C. might expand or contract by mating-type. There are no silent neoformans. Strains of α mating- transposition and inversions. mating-type cassettes or HO type are more common in nature These studies have established a endonuclease in C. neoformans, and the clinical setting, can be novel paradigm for the and it would require a large more virulent than congenic a arrangement of mating-type amount of genetic coding capacity Current Biology Vol 13 No 20 R795 to accommodate mating-type the initial stages of mate specializations, is a poignant switching in this pathogenic recognition and cell fusion. example of the common theme of basidiomycete. Moreover, because Another unique feature of fungi evolution and diversification that C. neoformans occurs with tetrapolar systems is that the has occurred in biological systems predominantly as a haploid a and b regions of the MAT locus as they evolve to exploit different organism in which the diploid state are most commonly multi- rather environmental niches. The roles of is unstable and transient, it does than bi-allelic, giving rise to the MAT locus in directing the not require a mating-type hundreds or even thousands of establishment of cell-type identity switching mechanism to rapidly different mating-types. In the and microbial virulence, and in the reenter the diploid state. The spore most extreme examples, such as evolution of unique and chains produced by C. neoformans the model mushroom fungi specialized modes of sexual are also mixed, providing ready Schizophyllum commune and recombination, will likely continue opportunity for siblings of opposite Coprinus cinereus, the control of to capture the imagination of mating-type to again engage in cell–cell fusion by pheromones biologists for many years to come. mating. In summary, divergence has been dispensed with: each into the homothallic and encounter of two different strains Acknowledgments heterothallic lifestyles may have results in promiscuous cell–cell Our studies are supported by contributed to the evolution of two fusion, and post-fusion steps in grants from the NIH/NIAID, the very different types of cell-identity- the sexual cycle require two nuclei Burroughs Wellcome Fund, and determining systems in these of different mating-types. This the Howard Hughes Medical fungi. An interesting similarity unique association of pheromones Institute. between the MAT loci of C. and pheromone receptor genes albicans and C. neoformans is the with the MAT locus likely arose in Further reading inclusion of additional and the basidiomycete lineage, and in Herskowitz, I. (1989). A regulatory essential genes, which likely affect organisms with a bipolar mating hierarchy for cell specialization in yeast. Nature 342, 749–757. both the evolution of this unique system, such as C. neoformans, it Hull, C.M., Davidson, R.C., and region of the genome and its is linked to the homeodomain Heitman, J. (2002). Cell identity and biological functions. region, whereas in those with a sexual development in tetrapolar system, such as U. Cryptococcus neoformans are controlled by the mating-type- Ustilago maydis — a tetrapolar maydis, it is not. specific homeodomain protein paradigm Sxi1α. Genes Dev. 16, 3046–3060. In contrast to the ascomycetes Conclusions and outlook Hull, C.M., and Heitman, J. (2002). and the pathogen C. neoformans, We now understand at a molecular Genetics of Cryptococcus neoformans. Annu. Rev. Genet. 36, many basidiomycetous fungi level how mating-type loci govern 557–615. arrange their mating-type the establishment of bipolar and Hull, C.M., and Johnson, A.D. (1999). information in a distinctly different tetrapolar mating-type systems, Identification of a mating type-like fashion. One example is the maize and how unique adaptations have locus in the asexual pathogenic pathogen U. maydis, in which the occurred in the budding yeast to yeast Candida albicans. Science 285, 1271–1275. filamentous dikaryon produced by enable it to switch mating-type. Kahmann, R., and Bölker, M. (1996). mating is the infectious form of the One conserved general feature of Self/nonself recognition in fungi: organism. U. maydis arranges its fungal MAT loci is the presence of old mysteries and simple solutions. mating-type information into two genes encoding transcription Cell 85, 145–148. Kronstad, J.W., and Staben, C. (1997). defined regions that lie on different factors which govern the Mating type in filamentous fungi. chromosomes, giving rise to a establishment of cell type identity, Annu. Rev. Genet. 31, 245–276. tetrapolar mating system. The and it is striking how the Lengeler, K.B., Fox, D.S., Fraser, J.A., region of MAT known as the b homeodomain proteins have been Allen, A., Forrester, K., Dietrich, F.S., and Heitman, J. (2002). locus contains a pair of divergently conserved in both ascomycetes Mating-type locus of Cryptococcus transcribed homeodomain and basidiomycetes. And yet in neoformans: a step in the evolution encoding genes, bE and bW, with some examples, this role has been of sex chromosomes. Eukaryot Cell different alleles encoding apparently supplanted by HMG 1, 704–718. Miller, M.G., and Johnson, A.D. (2002). alternative versions of the factors box proteins. Thus far, the mating- White-opaque switching in Candida which can heterodimerize to type locus of C. neoformans is one albicans is controlled by mating- govern the establishment of cell- of only a few examples in which type locus homeodomain proteins type identity and completion of the the typically compact mating-type and allows efficient mating. Cell sexual cycle. This region of the locus has been dramatically 110, 293–302. Soll, D.R., Lockhart, S.R., and Zhao, R. MAT locus shares features with expanded, likely by chromosomal (2003). Relationship between the MAT loci of S. cerevisiae and translocation and rearrangements. switching and mating in Candida C. albicans, and the SXI1α gene of And while this MAT locus shares albicans. Eukaryot Cell 2, 390–397. C. neoformans. some features with those of other In U. maydis, mating-type is fungi that lie within the same Departments of Molecular Genetics and also governed by a second region phylum, it clearly establishes a Microbiology, Medicine, and Pharmacology and Cancer Biology, of the genome, the a locus, which novel paradigm. The diversity of Howard Hughes Medical Institute, Duke encodes pheromones and these fungal mating-type loci, with University Medical Center, Durham, NC pheromone receptors that direct both shared features and novel 27710, USA. E-mail: [email protected]