Relationship between monokaryotic growth rate and mating type in the edible basidiomycete Pleurotus ostreatus

The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBalpha and matBbeta) and to correlate the presence of specific matBalpha and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.     

A New Mating Compatibility Locus in PHYSARUM POLYCEPHALUM

The rate and extent of plasmodium formation were studied in mating tests involving pairs of largely isogenic amoebal strains compatible for mating-type (mt) alleles. A systematic variability was observed: plasmodia formed either rapidly and extensively or slowly and inefficiently. Plasmodium formation was found to be 10³- to 10⁴-fold more extensive in "rapid" crosses than in "slow" crosses. A genetic analysis revealed that the variability reflects the influence of a multiallelic compatibility locus that determines mating efficiency. This compatibility locus (designated matB), together with the original mating type locus, mt (in this work designated matA), constitute a tetrapolar mating specificity system in Physarum polycephalum.     

Diploids in the Cryptococcus neoformans Serotype A Population Homozygous for the α Mating Type Originate via Unisexual Mating

The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the α mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual α population is unclear. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions generating both diploid intermediates and haploid recombinant progeny. Same-sex mating (α-α) also occurs in nature as evidenced by the existence of natural diploid αADα hybrids that arose by fusion between two α cells of different serotypes (A and D). How significantly this novel sexual style contributes to genetic diversity of the Cryptococcus population was unknown. In this study, ∼500 natural C. neoformans isolates were tested for ploidy and close to 8% were found to be diploid by fluorescence flow cytometry analysis. The majority of these diploids were serotype A isolates with two copies of the α MAT locus allele. Among those, several are intra-varietal allodiploid hybrids produced by fusion of two genetically distinct α cells through same-sex mating. The majority, however, are autodiploids that harbor two seemingly identical copies of the genome and arose via either endoreplication or clonal mating. The diploids identified were isolated from different geographic locations and varied genotypically and phenotypically, indicating independent non-clonal origins. The present study demonstrates that unisexual mating produces diploid isolates of C. neoformans in nature, giving rise to populations of hybrids and mixed ploidy. Our findings underscore the importance of same-sex mating in shaping the current population structure of this important human pathogenic fungus, with implications for mechanisms of selfing and inbreeding in other microbial pathogens.     

Nonsex Genes in the Mating Type Locus of Candida albicans Play Roles in a/α Biofilm Formation,Including Impermeability and Fluconazole Resistance

The mating type locus (MTL) of Candida albicans contains the mating type genes and has, therefore, been assumed to play an exclusive role in the mating process. In mating-incompetent a/α cells, two of the mating type genes, MTL a1 and MTLα2, encode components of the a1-α2 corepressor that suppresses mating and switching. But the MTL locus of C. albicans also contains three apparently unrelated “nonsex” genes (NSGs), PIK, PAP and OBP, the first two essential for growth. Since it had been previously demonstrated that deleting either the a/α copy of the entire MTL locus, or either MTLa1 or MTLα2, affected virulence, we hypothesized that the NSGs in the MTL locus may also play a role in pathogenesis. Here by mutational analysis, it is demonstrated that both the mating type and nonsex genes in the MTL locus play roles in a/α biofilm formation, and that OBP is essential for impermeability and fluconazole resistance.     

Molecular and genetic evidence for a tetrapolar mating system in Sparassis latifolia

Sparassis latifolia is a valuable edible fungus cultivated in East Asia that is rich in β-glucans. Understanding the mating system and sexual life cycle is important not only for breeding programs to improve strains but also for studies on speciation and population structures. In the present study, mating experiments using monokaryons derived from two different parental strains were performed. Chi-squared test indicated satisfied Mendel segregation, which supported a tetrapolar mating system. A search in the genome for homologs to the well-defined homeodomain and pheromone/receptors, as well as frequently found flanking genes, resulted in the identification of known mating-type loci previously identified in tetrapolar basidiomycetes, each represented by two idiomorphic alleles on separate contigs. Deficiency of the β-flanking protein in S. latifolia and S. crispa around the MAT-A locus may be explained by the locus being rich in transposable elements adjacent to HD genes. Monokaryotic mycelia are characterized by a slower growth rate and a relative lack of aerial mycelia compared with the parental strain. Chlamydospores can be produced in both monokaryotic and dikaryotic mycelial stages. We provide genetic and molecular evidence for the mating system of S. latifolia, a finding that will be helpful for the cross-breeding of this mushroom.     

Genetic Mapping and Non-Mendelian Segregation of Mating Type Loci in the Oomycete, Phytophthora Infestans

DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in this normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses as RAPD markers, restriction fragment length polymorphisms (RFLPs), single-strand conformational polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balanced lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude from progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.     

Interconversion of Yeast Mating Types II. Restoration of Mating Ability to Sterile Mutants in Homothallic and Heterothallic Strains

The two mating types of the yeast Saccharomyces cerevisiae can be interconverted in both homothallic and heterothallic strains. Previous work indicates that all yeast cells contain the information to be both a and α and that the HO gene (in homothallic strains) promotes a change in mating type by causing a change at the mating type locus itself. In both heterothallic and homothallic strains, a defective α mating type locus can be converted to a functional a locus and subsequently to a functional α locus. In contrast, action of the HO gene does not restore mating ability to a strain defective in another gene for mating which is not at the mating type locus. These observations indicate that a yeast cell contains an additional copy (or copies) of α information, and lead to the "cassette" model for mating type interconversion. In this model, HMa and hmα loci are blocs of unexpressed α regulatory information, and HMα and hma loci are blocs of unexpressed a regulatory information. These blocs are silent because they lack an essential site for expression, and become active upon insertion of this information (or a copy of the information) into the mating type locus by action of the HO gene.     

Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, α and a. The α and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The α and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.     

Regulation of Mating and Meiosis in Yeast by the Mating-Type Region

A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rme enables the diploids a*/α, a/a*, and α/α to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains.     

Interconversion of Yeast Mating Types III. Action of the Homothallism (HO) Gene in Cells Homozygous for the Mating Type Locus

Mating type interconversion in homothallic Saccharomyces cerevisiae has been studied in diploids homozygous for the mating type locus produced by sporulation of a/a/a/α and a/a/α/α tetraploid strains. Mating type switches have been analyzed by techniques including direct observation of cells for changes in α-factor sensitivity. Another method of following mating type switching exploits the observation that a/α cells exhibit polar budding and a/a and α/α cells exhibit medial budding.—These studies indicate the following: (1) The allele conferring the homothallic life cycle (HO) is dominant to the allele conferring the heterothallic life cycle (ho). (2) The action of the HO gene is controlled by the mating type locus—active in a/a and α/α cells but not in a/α cells. (3) The HO (or HO-controlled) gene product can act independently on two mating type alleles located on separate chromosomes in the same nucleus. (4) A switch in mating type is observed in pairs of cells, each of which has the same change.     

Discovery and Functional Study of a Novel Genomic Locus Homologous to Bα-Mating-Type Sublocus of Lentinula edodes

The interaction of mating pheromone and pheromone receptor from the B mating-type locus is the first step in the activation of the mushroom mating signal transduction pathway. The B mating-type locus of Lentinula edodes is composed of Bα and Bβ subloci, each of which contains genes for mating pheromone and pheromone receptor. Allelic variations in both subloci generate multiple B mating-types through which L. edodes maintains genetic diversity. In addition to the B mating-type locus, our genomic sequence analysis revealed the presence of a novel chromosomal locus 43.3 kb away from the B mating-type locus, containing genes for a pair of mating pheromones (PHBN1 and PHBN2) and a pheromone receptor (RCBN). The new locus (Bα-N) was homologous to the Bα sublocus, but unlike the multiallelic Bα sublocus, it was highly conserved across the wild and cultivated strains. The interactions of RcbN with various mating pheromones from the B and Bα-N mating-type loci were investigated using yeast model that replaced endogenous yeast mating pheromone receptor STE2 with RCBN. The yeast mating signal transduction pathway was only activated in the presence of PHBN1 or PHBN2 in the RcbN producing yeast, indicating that RcbN interacts with self-pheromones (PHBN1 and PHBN2), not with pheromones from the B mating-type locus. The biological function of the Bα-N locus was suggested to control the expression of A mating-type genes, as evidenced by the increased expression of two A-genes HD1 and HD2 upon the treatment of synthetic PHBN1 and PHBN2 peptides to the monokaryotic strain of L. edodes.     

Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle involving fusion of haploid MATα and MATa cells. Virulence has been linked to the mating type, and MATα cells are more virulent than congenic MATa cells. To study the link between the mating type and virulence, we functionally analyzed three genes encoding homologs of the p21-activated protein kinase family: STE20α, STE20a, and PAK1. In contrast to the STE20 genes that were previously shown to be in the mating-type locus, the PAK1 gene is unlinked to the mating type. The STE20α, STE20a, and PAK1 genes were disrupted in serotype A and D strains of C. neoformans, revealing central but distinct roles in mating, differentiation, cytokinesis, and virulence. ste20α pak1 and ste20a pak1 double mutants were synthetically lethal, indicating that these related kinases share an essential function. In summary, our studies identify an association between the STE20α gene, the MATα locus, and virulence in a serotype A clinical isolate and provide evidence that PAK kinases function in a MAP kinase signaling cascade controlling the mating, differentiation, and virulence of this fungal pathogen.     

Inbreeding depression in self-incompatible North-American Arabidopsis lyrata: disentangling genomic and S-locus-specific genetic load

Newly formed selfing lineages may express recessive genetic load and suffer inbreeding depression. This can have a genome-wide genetic basis, or be due to loci linked to genes under balancing selection. Understanding the genetic architecture of inbreeding depression is important in the context of the maintenance of self-incompatibility and understanding the evolutionary dynamics of S-alleles. We addressed this using North-American subspecies of Arabidopsis lyrata. This species is normally self-incompatible and outcrossing, but some populations have undergone a transition to selfing. The goals of this study were to: (1) quantify the strength of inbreeding depression in North-American populations of A. lyrata; and (2) disentangle the relative contribution of S-linked genetic load compared with overall inbreeding depression. We enforced selfing in self-incompatible plants with known S-locus genotype by treatment with CO₂, and compared the performance of selfed vs outcrossed progeny. We found significant inbreeding depression for germination rate (δ=0.33), survival rate to 4 weeks (δ=0.45) and early growth (δ=0.07), but not for flowering rate. For two out of four S-alleles in our design, we detected significant S-linked load reflected by an under-representation of S-locus homozygotes in selfed progeny. The presence or absence of S-linked load could not be explained by the dominance level of S-alleles. Instead, the random nature of the mutation process may explain differences in the recessive deleterious load among lineages.     

Maternal inheritance of mitochondria: multipolarity, multiallelism and hierarchical transmission of mitochondrial DNA in the true slime mold Physarum polycephalum

Direct evidence of digestion of paternal mitochondrial DNA (mtDNA) has been found in the true slime mold Physarum polycephalum. This is the first report on the selective digestion of mtDNA inside the zygote, and is striking evidence for the mechanism of maternal inheritance of mitochondria. Moreover, two mitochondrial nuclease activities were detected in this organism as candidates for the nucleases responsible for selective digestion of mtDNA. In the true slime mold, there is an additional feature of the uniparental inheritance of mitochondria. Although mitochondria are believed to be inherited from the maternal lineage in nearly all eukaryotes, the mating types of the true slime mold P. polycephalum is not restricted to two: there are three mating loci—matA, matB, and matC—and these loci have 16, 15, and 3 alleles, respectively. Interestingly, the transmission patterns of mtDNA are determined by the matA locus, in a hierarchical fashion (matA hierarchy) as follows: matA7 > matA2 > matA11 > matA12 > matA15/matA16 > matA1 > matA6. The strain possessing the higher status of matA would be the mtDNA donor in crosses. Furthermore, we have found that some crosses showed biparental inheritance of mitochondria. This review describes the phenomenon of hierarchical transmission of mtDNA in true slime molds, and discusses the presumed molecular mechanism of maternal and biparental inheritance.     

Mating Type and Sporulation in Yeast. II. Meiosis, Recombination, and Radiation Sensitivity in an α_entitystart_#945_entit Diploid with Altered Sporulation Control

In wild-type S. cerevisiae, diploid cells must be heterozygous at the mating-type locus in order to sporulate. In the preceding paper, we described a number of mutants (CSP mutants), isolated from nonsporulating aa and αα parent strains, in which sporulation appeared to be uncoupled from control by mating type. The characterization of one of these mutants (CSP1) is now extended to other processes controlled by mating type. This mutant is indistinguishable from αα cells and unlike aα cells for mating factor production and response, zygote formation, intragenic mitotic recombination, and for X-ray sensitivity. The mutant apparently undergoes a full round of DNA synthesis in sporulation medium, but with delayed kinetics. Only 20% of the cells complete sporulation. Among spores in completed asci, the frequency of both intra- and intergenic recombination is the same as it is for spores produced by aα cells. However, experiments in which cells were shifted from sporulation medium back to minimal growth medium gave a frequency of meiotic recombination between ade2 or leu2 heteroalleles only 25% to 29% as high for CSP1 αα diploid or CSP1 aa disomic cells as for aα diploid or disomic cells. Because the latter result, indicating recombination defectiveness, measured recombinant production in the entire cell population, whereas the result indicating normal recombination sampled only completed spores, we infer that all meiotic recombination events occurring in the population of CSP1 αα cells are concentrated in those few cells which complete sporulation. This high degree of correlation between meiotic recombination and the completion of meiosis and sporulation suggests that recombination may be required for proper meiotic chromosome segregation in yeast just as it appears to be in maize and in Drosophila     

MFalpha1, the gene encoding the alpha mating pheromone of Candida albicans

Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the α mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the α pheromone gene. Here we report the isolation and characterization of the gene MFα1, which codes for the precursor of the α mating pheromone in C. albicans. Two active α-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mfα1 null mutant of C. albicans. The mfα1 null mutant fails to mate as MTLα, while MTLa mfα1 cells are still mating competent. Experiments performed with the synthetic α-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTLα kex2 mutant strain when added exogenously, thereby confirming their roles as α mating pheromones.     

Regulation of Nuclear Positioning and Dynamics of the Silent Mating Type Loci by the Yeast Ku70/Ku80 Complex

We have examined the hypothesis that the highly selective recombination of an active mating type locus (MAT) with either HMLα or HMRa is facilitated by the spatial positioning of relevant sequences within the budding yeast (Saccharomyces cerevisiae) nucleus. However, both position relative to the nuclear envelope (NE) and the subnuclear mobility of fluorescently tagged MAT, HML, or HMR loci are largely identical in haploid a and α cells. Irrespective of mating type, the expressed MAT locus is highly mobile within the nuclear lumen, while silent loci move less and are found preferentially near the NE. The perinuclear positions of HMR and HML are strongly compromised in strains lacking the Silent information regulator, Sir4. However, HMLα, unlike HMRa and most telomeres, shows increased NE association in a strain lacking yeast Ku70 (yKu70). Intriguingly, we find that the yKu complex is associated with HML and HMR sequences in a mating-type-specific manner. Its abundance decreases at the HMLα donor locus and increases transiently at MATa following DSB induction. Our data suggest that mating-type-specific binding of yKu to HMLα creates a local chromatin structure competent for recombination, which cooperates with the recombination enhancer to direct donor choice for gene conversion of the MATa locus.     

αADα Hybrids of Cryptococcus neoformans: Evidence of Same-Sex Mating in Nature and Hybrid Fitness

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, α and a. However, the overwhelming predominance of mating type (MAT) α over a in C. neoformans populations limits α–a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between α isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural αADα hybrids that arose by fusion between two α cells of different serotypes (A and D) were identified and characterized, providing definitive evidence that same-sex mating occurs naturally. A novel truncated allele of the mating-type-specific cell identity determinant SXI1α was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed αADα strains exhibited hybrid vigor both in vitro and in vivo, providing a plausible explanation for their relative abundance in nature despite the fact that AD hybrids are inefficient in meiosis/sporulation and are trapped in the diploid state. These findings provide insights on the origins, genetic mechanisms, and fitness impact of unisexual hybridization in the Cryptococcus population.     

Genetic recombination events which position the friedreich ataxia locus proximal to the D9S15/D9S5 linkage group on chromosome 9q

The absence of recombination between the mutation causing Friedreich ataxia and the two loci which originally assigned the disease locus to chromosome 9 has slowed attempts to isolate and characterize the genetic defect underlying this neurodegenerative disorder. A proximity of less than 1 cM to the linkage group has been proved by the generation of high maximal lod score (Z) to each of the two tightly linked markers D9S15 (Z = 96.69; recombination fraction [θ] = .01) and D9S5 (Z = 98.22; θ = .01). We report here recombination events which indicate that the FRDA locus is located centromeric to the D9S15/D9S5 linkage group, with the most probable order being cen–FRDA–D9S5–D9S15–qter. However, orientation of the markers with respect to the centromere, critical to the positional cloning strategy, remains to be resolved definitively.     

Development of SCAR Markers to Determine the Mating Types of Lepista nuda Protoplast Monokaryons

Lepista nuda (Bull. ex Fr.) Cooke belongs to Tricholomataceae and is an edible fungus with both economic and medical value. Mycelia were isolated from the fruiting bodies of L. nuda and were used to prepare the protoplast monokaryons. One hundred and fifteen monokaryons were obtained and their mating types were determined using somatic incompatibility tests. Protoplast monokaryons segregated into either the A₁B₁ or the A₂B₂ mating types. Inter-simple sequence repeats and sequence-related amplified polymorphism fingerprinting were used to analyse the mating types of these protoplast monokaryons and 16 sequence-characterised amplified region primers were developed to efficiently differentiate between the monokaryon mating types. Multiplex PCR analyses were also established. The data presented here outline a method for the precise and rapid identification of protoplast monokaryon mating types, which has the promise to shorten the period required for conventional crossbreeding.