Discovery of a modified tetrapolar sexual cycle in Cryptococcus amylolentus and the evolution of MAT in the Cryptococcus species complex

Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The human fungal pathogenic and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system (a, α) in which the MAT locus is unusually large (>100 kb) and encodes >20 genes including homeodomain (HD) and pheromone/receptor (P/R) genes. To understand how this unique bipolar mating system evolved, we investigated MAT in the closely related species Tsuchiyaea wingfieldii and Cryptococcus amylolentus and discovered two physically unlinked loci encoding the HD and P/R genes. Interestingly, the HD (B) locus sex-specific region is restricted (～2 kb) and encodes two linked and divergently oriented homeodomain genes in contrast to the solo HD genes (SXI1α, SXI2a) of C. neoformans and Cryptococcus gattii. The P/R (A) locus contains the pheromone and pheromone receptor genes but has expanded considerably compared to other outgroup species (Cryptococcus heveanensis) and is linked to many of the genes also found in the MAT locus of the pathogenic Cryptococcus species. Our discovery of a heterothallic sexual cycle for C. amylolentus allowed us to establish the biological roles of the sex-determining regions. Matings between two strains of opposite mating-types (A1B1×A2B2) produced dikaryotic hyphae with fused clamp connections, basidia, and basidiospores. Genotyping progeny using markers linked and unlinked to MAT revealed that meiosis and uniparental mitochondrial inheritance occur during the sexual cycle of C. amylolentus. The sexual cycle is tetrapolar and produces fertile progeny of four mating-types (A1B1, A1B2, A2B1, and A2B2), but a high proportion of progeny are infertile, and fertility is biased towards one parental mating-type (A1B1). Our studies reveal insights into the plasticity and transitions in both mechanisms of sex determination (bipolar versus tetrapolar) and sexual reproduction (outcrossing versus inbreeding) with implications for similar evolutionary transitions and processes in fungi, plants, and animals.     

Orchestration of sexual reproduction and virulence by the fungal mating-type locus

The mating-type locus (MAT) orchestrates sexual reproduction in fungi. Sexual reproduction is related not only to fitness of an organism, but also correlated with virulence in certain pathogens. In the dandruff-associated fungus Malassesia globosa, although the sexual cycle remains to be discovered, whole genome analysis has led to the hypothesis that mating may occur on host skin. Furthermore, the MAT locus of M. globosa and U. hordei provides evidence that transitions between tetrapolar and bipolar systems have independently occurred. These results, together with studies recapitulating the ancestral tetrapolar mating system in Cryptococcus and the structure of MAT in related smut fungi, have furthered understanding on transitions between different mating systems and the evolution of MAT in the Basidiomycota.     

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.     

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.     

Mating factor linkage and genome evolution in basidiomycetous pathogens of cereals

Sex in basidiomycete fungi is controlled by tetrapolar mating systems in which two unlinked gene complexes determine up to thousands of mating specificities, or by bipolar systems in which a single locus (MAT) specifies different sexes. The genus Ustilago contains bipolar (Ustilago hordei) and tetrapolar (Ustilago maydis) species and sexual development is associated with infection of cereal hosts. The U. hordei MAT-1 locus is unusually large (approximately 500 kb) and recombination is suppressed in this region. We mapped the genome of U. hordei and sequenced the MAT-1 region to allow a comparison with mating-type regions in U. maydis. Additionally the rDNA cluster in the U. hordei genome was identified and characterized. At MAT-1, we found 47 genes along with a striking accumulation of retrotransposons and repetitive DNA; the latter features were notably absent from the corresponding U. maydis regions. The tetrapolar mating system may be ancestral and differences in pathogenic life style and potential for inbreeding may have contributed to genome evolution.     

Primers for mating-type diagnosis in Diaporthe and Phomopsis: their use in teleomorph induction in vitro and biological species definition

Sexual reproduction in ascomycete fungi is governed by the mating-type (MAT) locus. The MAT loci of Diaporthe and its Phomopsis anamorphs differ in only one gene: MAT1-1-1 in mating-type MAT1-1 and MAT1-2-1 in mating-type MAT1-2. In order to diagnose mating-types in Diaporthe and Phomopsis and evaluate their usefulness in teleomorph induction in vitro and biological species delimitation, we designed primers that amplify part of the MAT1-1-1 and MAT1-2-1 genes. MAT phylogenies were generated and compared with ITS and EF1-α phylograms. Species recognised in the EF1-α phylogeny corresponded directly with those determined in the MAT phylogenies. ITS was shown to be highly variable resulting in a large number of phylogenetic species that were discordant with MAT and EF1-α species. Mating experiments were conducted to evaluate the existence of reproductive barriers between some isolates, and their anamorphic morphologies were compared. The primers proved to be useful in the mating-type diagnosis of isolates, selection of compatible mating pairs, and in the assessment of biological species boundaries.     

Glomerella truncata: another Glomerella species with an atypical mating system

In the genus Glomerella all species studied to date do not fit the usual mating system of heterothallic ascomycetes. This study investigated the mating system of G. truncata (anamorph Colletotrichum truncatum), a pathogen responsible for lentil anthracnose. Twenty-two field isolates from the Canadian prairies were crossed in all possible combinations, including selfings. All isolates also were screened for the presence of the MAT1-1 and MAT1-2 idiomorphs by targeting small conserved areas of the MAT genes (the alpha domain and the high mobility group HMG box) with degenerate primers, and a pair of G. truncata-specific HMG primers (CT21HMG) were designed. The results of the classical mating study suggested that G. truncata is heterothallic. Isolates fell into two incompatibility groups, which is consistent with a bipolar mating system but different from what has been described in other Glomerella species. Molecular screening showed that the HMG box used as a marker for the MAT1-2 idiomorph was present in both partners of fertile crosses in G. truncata, unlike in the typical ascomycete system, but as previously described for two other Glomerella species. G. truncata therefore appears to share unusual mating system characteristics with the other Glomerella species studied to date.     

The a and b loci of Ustilago maydis hybridize with DNA sequences from other smut fungi.

The smut fungi are obligately parasitic during the sexual phase of their life cycle, and the mating-type genes of these fungi play key roles in both sexual development and pathogenicity. Among species of smut fungi it is common to find a bipolar mating system in which one locus with two alternate alleles is believed to control cell fusion and establishment of the infectious cell type. Alternatively, several species have a tetrapolar mating system in which two different genetic loci, one of which has multiple alleles, control fusion and subsequent development of the infection hyphae. Cloned sequences from the a and b mating-type loci of the tetrapolar smut fungus Ustilago maydis were used as hybridization probes to DNAs from 23 different fungal strains, including smut fungi with both tetrapolar and bipolar mating systems. In general, all of the smut fungi hybridized with the mating-type genes from U. maydis, suggesting conservation of the sequences involved in mating interactions. A selection of DNAs from other ascomycete and basidiomycete fungi failed to hybridize with the U. maydis mating-type sequences. Exceptions to this finding include hybridization of DNA from the a1 idiomorph of U. maydis to DNA from one strain of U. violacea and hybridization of both a idiomorphs to DNA from Saccharomyces cerevisiae.     

Modification changes of the genetic material in Saccharomyces yeasts

The problem of mating-type switches in heterothallic yeast cells was investigated. In selective system for cytoduction in alpha x alpha crosses alpha-cytoductants were predominantly obtained. Thus matings in alpha x alpha crosses can proceed through non-heritable changes (modifications) of the mating type alpha----a. The frequency of alpha-cytoductants after UV-irradiation of the recipient cells exceeded the control value 50-90 times. The extra copy of MAT alpha dramatically decreased the frequency of cytoductants in alpha x alpha crosses, either spontaneously or after UV-irradiation. The rad18 recipient defective in postreplication repair had 70-times increased level of mating-type modifications, as compared with isogenic Rad+ strain. An explanation consistent with these data is that mating-type modifications are due to phenotypic expression of primary lesions of MAT alpha locus. Such lesions might be expressed as transient a-mating type. After the mating event, these lesions can be repaired or turned to true mutations within the MAT locus. In fact, approximately half of non-mating cytoductants from alpha x alpha crosses had the phenotype of mat alpha 2 mutants.     

担子菌交配型基因的克隆及功能研究进展

担子菌中已有２０多个交配型基因被克隆,Ａ、Ｂ位点的交配型基因具有明显不同的结构特征。Ａ位点的交配型基因编码两类含相似同源结构域的蛋白质,两种蛋白质形成异源二聚体,调节依赖Ａ因子的发育过程;而Ｂ位点的交配型基因编码信息素及其受体。交配型基因编码的同源结构域蛋白质在植物、动物和菌类中都具有广泛的保守性。