MAT Locus Research Articles

Molecular analysis of mating type loci from the mycophenolic acid producer Penicillium brevicompactum: Phylogeny and MAT protein characterization suggest a cryptic sexual life cycle

The mycophenolic acid producing ascomycete Penicillium brevicompactum is considered to be an anamorphic (asexual) species, for which a sexual cycle was never observed. However, since recent reports of otherwise asexually propagating filamentous fungi have demonstrated a sexual cycle controlled by mating type loci, we carried out a molecular analysis of mating type loci from P. brevicompactum. Using data from extensive DNA sequencing analysis, we determined the mating type loci from 22 strains derived from various type culture collections. We found 8 strains carrying a MAT1-1 locus encoding a 362 amino acid alpha domain transcription factor. The other 14 possessed a MAT1-2 locus encoding a 298 amino acid HMG domain transcription factor. cDNA analysis confirmed that both mating type loci are transcriptionally expressed. The karyotype of six selected strains, determined using contour-clamped homogeneous electric field (CHEF) electrophoresis, demonstrated distinct differences in size and numbers of chromosomes between the strains investigated. Interestingly, our phylogenetic survey of 72 strains from 11 different Penicillium species revealed that MAT genes serve as excellent molecular markers to determine phylogenetic relationships among species closely related to P. brevicompactum. Based on our sequencing results, we constructed transformation vectors for site-specific deletion of mating type loci from two selected strains of opposite mating type. Complementation strains were constructed containing both the mating type locus deletion cassette and a MAT-egfp fusion gene. These strains were used for comparative phenotypic analyses between strains containing or lacking the mating type gene. Whereas all MAT1-2 strains were indistinguishable, the MAT1-1 and MAT1-1-1 deletion strains differed distinctly. The MAT1-1-1 deletion strain produced more conidiospores on solid media, but smaller pellets in liquid media. This is probably the consequence of fewer conidial germ tubes than with the wild type mating type strain. Finally, we showed that the MAT-EGPF fusion protein is localized to the nuclei and detectable in protein samples by Western analysis. Together, our results suggest that the asexually propagating fungus P. brevicompactum might be a heterothallic species with a cryptic sexual life cycle.

Molecular Genetic Features of Biological Species of the Genus Saccharomyces

Molecular genetic study of Saccharomyces yeasts of different species was carried out using molecular karyotyping and comparative analysis of a number of nuclear and mitochondrial genes. Multigene phylogenetic analysis showed that the recently described species S. jurei was phylogenetically closest to S. mikatae, while S. bayanus and S. arboricola were the most divergent species within the genus Saccharomyces. Comparative analysis of amino acid sequences of the SUC β-fructosidase genes also indicated a close genetic relationship between the species S. jurei and S. mikatae. The biological species S. cerevisiae, S. kudriavzevii, and S. paradoxus possessed collinear karyotypes. Two reciprocal translocations were found in the S. jurei karyotype, one of which was unique (between chromosomes I and XIII), and the second was common with S. mikatae: VI/VII. Reciprocal translocations in the karyotypes of S. arboricola, S. bayanus, and S. cariocanus were species-specific. It was established that only chromosome III carrying the mating type locus MAT was of approximately the same size in all eight species of the genus Saccharomyces. The adaptive role of reciprocal translocations of chromosomes is discussed.

MAT heterozygosity and the second sterility barrier in the reproductive isolation of Saccharomyces species

The genetic analysis of large numbers of Saccharomyces cerevisiae × S. uvarum (“cevarum”) and S. kudriavzevii × S. uvarum (“kudvarum”) hybrids in our previous studies revealed that these species are isolated by a postzygotic double-sterility barrier. We proposed a model in which the first barrier is due to the abruption of the meiotic process by the failure of the chromosomes of the subgenomes to pair (and recombine) in meiosis and the second barrier is assumed to be the result of the suppression of mating by allospecific MAT heterozygosity. While the former is analogous to the major mechanism of postzygotic reproductive isolation in plants and animals, the latter seems to be Saccharomyces specific. To bolster the assumed involvement of MAT in the second sterility barrier, we produced synthetic alloploid two-species cevarum and kudvarum hybrids with homo- and heterothallic backgrounds as well as three-species S. cerevisiae × S. kudvarum × S. uvarum (“cekudvarum”) hybrids by mass-mating and examined their MAT loci using species- and cassette-specific primer pairs. We found that the allospecific MAT heterozygosity repressed MAT switching and mating in the hybrids and in the viable but sterile spores produced by the cevarum hybrids that had increased (allotetraploid) genomes. The loss of heterozygosity by meiotic malsegregation of MAT-carrying chromosomes in the latter hybrids broke down the sterility barrier. The resulting spores nullisomic for the S. uvarum chromosome produced vegetative cells capable of MAT switching and conjugation, opening the way for GARMe (Genome Autoreduction in Meiosis), the process that leads to chimeric genomes.

Mating genes in Calonectria and evidence for a heterothallic ancestral state.

The genus Calonectria includes many important plant pathogens with a wide global distribution. In order to better understand the reproductive biology of these fungi, we characterised the structure of the mating type locus and flanking genes using the genome sequences for seven Calonectria species. Primers to amplify the mating type genes in other species were also developed. PCR amplification of the mating type genes and multi-gene phylogenetic analyses were used to investigate the mating strategies and evolution of mating type in a collection of 70 Calonectria species residing in 10 Calonectria species complexes. Results showed that the organisation of the MAT locus and flanking genes is conserved. In heterothallic species, a novel MAT gene, MAT1-2-12 was identified in the MAT1-2 idiomorph; the MAT1-1 idiomorph, in most cases, contained the MAT1-1-3 gene. Neither MAT1-1-3 nor MAT1-2-12 was found in homothallic Calonectria (Ca.) hongkongensis, Ca. lateralis, Ca. pseudoturangicola and Ca. turangicola. Four different homothallic MAT locus gene arrangements were observed. Ancestral state reconstruction analysis provided evidence that the homothallic state was basal in Calonectria and this evolved from a heterothallic ancestor.

Differential Contribution of the Parental Genomes to a S. cerevisiae × S. uvarum Hybrid, Inferred by Phenomic, Genomic, and Transcriptomic Analyses, at Different Industrial Stress Conditions.

In European regions of cold climate, S. uvarum can replace S. cerevisiae in wine fermentations performed at low temperatures. S. uvarum is a cryotolerant yeast that produces more glycerol, less acetic acid and exhibits a better aroma profile. However, this species exhibits a poor ethanol tolerance compared with S. cerevisiae. In the present study, we obtained by rare mating (non-GMO strategy), and a subsequent sporulation, an interspecific S. cerevisiae × S. uvarum spore-derivative hybrid that improves or maintains a combination of parental traits of interest for the wine industry, such as good fermentation performance, increased ethanol tolerance, and high glycerol and aroma productions. Genomic sequencing analysis showed that the artificial spore-derivative hybrid is an allotriploid, which is very common among natural hybrids. Its genome contains one genome copy from the S. uvarum parental genome and two heterozygous copies of the S. cerevisiae parental genome, with the exception of a monosomic S. cerevisiae chromosome III, where the sex-determining MAT locus is located. This genome constitution supports that the original hybrid from which the spore was obtained likely originated by a rare-mating event between a mating-competent S. cerevisiae diploid cell and either a diploid or a haploid S. uvarum cell of the opposite mating type. Moreover, a comparative transcriptomic analysis reveals that each spore-derivative hybrid subgenome is regulating different processes during the fermentation, in which each parental species has demonstrated to be more efficient. Therefore, interactions between the two subgenomes in the spore-derivative hybrid improve those differential species-specific adaptations to the wine fermentation environments, already present in the parental species.

The Pheromone and Pheromone Receptor Mating-Type Locus Is Involved in Controlling Uniparental Mitochondrial Inheritance in Cryptococcus.

Mitochondria are inherited uniparentally during sexual reproduction in the majority of eukaryotic species studied, including humans, mice, and nematodes, as well as many fungal species. Mitochondrial uniparental inheritance (mito-UPI) could be beneficial in that it avoids possible genetic conflicts between organelles with different genetic backgrounds, as recently shown in mice, and it could prevent the spread of selfish genetic elements in the mitochondrial genome. Despite the prevalence of observed mito-UPI, the underlying mechanisms and the genes involved in controlling this non-Mendelian inheritance are poorly understood in many species. In Cryptococcus neoformans, a human pathogenic basidiomyceteous fungus, mating types (MATα and MATa) are defined by alternate alleles at the single MAT locus that evolved from fusion of the two MAT loci (P/R encoding pheromones and pheromone receptors, and HD encoding homeodomain transcription factors) that are the ancestral state in the basidiomycota. Mitochondria are inherited uniparentally from the MATa parent in C. neoformans, and this requires the SXI1α and SXI2a HD factors encoded by MAT However, there is evidence that additional genes contribute to the control of mito-UPI in Cryptococcus Here, we show that in C. amylolentus, a sibling species of C. neoformans with unlinked P/R and HD MAT loci, mito-UPI is controlled by the P/R locus and is independent of the HD locus. Consistently, by replacing the MATα alleles of the pheromones (MF) and pheromone receptor (STE3) with the MATa alleles, we show that these P/R locus-defining genes indeed affect mito-UPI in C. neoformans during sexual reproduction. Additionally, we show that during early stages of C. neoformans sexual reproduction, conjugation tubes are always produced by the MATα cells, resulting in unidirectional migration of the MATα nucleus into the MATa cell during zygote formation. This process is controlled by the P/R locus and could serve to physically restrict movement of MATα mitochondria in the zygotes, and thereby contribute to mito-UPI. We propose a model in which both physical and genetic mechanisms function in concert to prevent the coexistence of mitochondria from the two parents in the zygote, and subsequently in the meiotic progeny, thus ensuring mito-UPI in pathogenic Cryptococcus, as well as in closely related nonpathogenic species. The implications of these findings are discussed in the context of the evolution of mito-UPI in fungi and other more diverse eukaryotes.

Mating-Type-Specific Ribosomal Proteins Control Aspects of Sexual Reproduction in Cryptococcus neoformans.

The MAT locus of Cryptococcus neoformans has a bipolar organization characterized by an unusually large structure, spanning over 100kb. MAT genes have been characterized by functional genetics as being involved in sexual reproduction and virulence. However, classical gene replacement failed to achieve mutants for five MAT genes (RPL22, RPO41, MYO2, PRT1, and RPL39), indicating that they are likely essential. In the present study, targeted gene replacement was performed in a diploid strain for both the α and a alleles of the ribosomal genes RPL22 and RPL39 Mendelian analysis of the progeny confirmed that both RPL22 and RPL39 are essential for viability. Ectopic integration of the RPL22 allele of opposite MAT identity in the heterozygous RPL22a/rpl22αΔ or RPL22α/rpl22aΔ mutant strains failed to complement their essential phenotype. Evidence suggests that this is due to differential expression of the RPL22 genes, and an RNAi-dependent mechanism that contributes to control RPL22a expression. Furthermore, via CRISPR/Cas9 technology, the RPL22 alleles were exchanged in haploid MATα and MATa strains of C. neoformans These RPL22 exchange strains displayed morphological and genetic defects during bilateral mating. These results contribute to elucidating functions of C. neoformans essential mating type genes that may constitute a type of imprinting system to promote inheritance of nuclei of both mating types.

The signaling mechanisms involved in the dimorphic phenomenon of the Basidiomycota fungus Ustilago maydis.

In the present manuscript, we describe the mechanisms involved in the yeast-to-hypha dimorphic transition of the plant pathogenic Basidiomycota fungus Ustilago maydis. During its life cycle, U. maydis presents two stages: one in the form of haploid saprophytic yeasts that divide by budding and the other that is the product of the mating of sexually compatible yeast cells (sporidia), in the form of mycelial dikaryons that invade the plant host. The occurrence of the involved dimorphic transition is controlled by the two mating loci a and b. In addition, the dimorphic event can be obtained in vitro by different stimuli: change in the pH of the growth medium, use of different carbon sources, and by nitrogen depletion. The presence of other factors and mechanisms may affect this phenomenon; among these, we may cite the PKA and MAPK signal transduction pathways, polyamines, and factors that affect the structure of the nucleosomes. Some of these factors and conditions may affect all these dimorphic events, or they may be specific for only one or more but not all the processes involved. The conclusion reached by these experiments is that U. maydis has constituted a useful model for the analysis of the mechanisms involved in cell differentiation of fungi in general.

Integrative Activity of Mating Loci, Environmentally Responsive Genes, and Secondary Metabolism Pathways during Sexual Development of Chaetomium globosum.

The origins and maintenance of the rich fungal diversity have been longstanding issues in evolutionary biology. To investigate how differences in expression regulation contribute to divergences in development and ecology among closely related species, transcriptomes were compared between Chaetomium globosum, a homothallic pathogenic fungus thriving in highly humid ecologies, and Neurospora crassa, a heterothallic postfire saprotroph. Gene expression was quantified in perithecia at nine distinct morphological stages during nearly synchronous sexual development. Unlike N. crassa, expression of all mating loci in C. globosum was highly correlated. Key regulators of the initiation of sexual development in response to light stimuli-including orthologs of N. crassasub-1, sub-1-dependent gene NCU00309, and asl-1-showed regulatory dynamics matching between C. globosum and N. crassa Among 24 secondary metabolism gene clusters in C. globosum, 11-including the cochliodones biosynthesis cluster-exhibited highly coordinated expression across perithecial development. C. globosum exhibited coordinately upregulated expression of histidine kinases in hyperosmotic response pathways-consistent with gene expression responses to high humidity we identified in fellow pathogen Fusarium graminearum Bayesian networks indicated that gene interactions during sexual development have diverged in concert with the capacities both to reproduce asexually and to live a self-compatible versus self-incompatible life cycle, shifting the hierarchical roles of genes associated with conidiation and heterokaryon incompatibility in N. crassa and C. globosum This divergence supports an evolutionary history of loss of conidiation due to unfavorable combinations of heterokaryon incompatibility in homothallic species.IMPORTANCE Fungal diversity has amazed evolutionary biologists for decades. One societally important aspect of this diversity manifests in traits that enable pathogenicity. The opportunistic pathogen Chaetomium globosum is well adapted to a high-humidity environment and produces numerous secondary metabolites that defend it from predation. Many of these chemicals can threaten human health. Understanding the phases of the C. globosum life cycle in which these products are made enables better control and even utilization of this fungus. Among its intriguing traits is that it both is self-fertile and lacks any means of propagule-based asexual reproduction. By profiling genome-wide gene expression across the process of sexual reproduction in C. globosum and comparing it to genome-wide gene expression in the model filamentous fungus N. crassa and other closely related fungi, we revealed associations among mating-type genes, sexual developmental genes, sexual incompatibility regulators, environmentally responsive genes, and secondary metabolic pathways.

Mating-type switching and mating-type gene array expression in the methylotrophic yeast Ogataea thermomethanolica TBRC656

The methylotrophic yeast, Ogataea thermomethanolica TBRC656, is an attractive host organism for heterologous protein production owing to the availability of protein expression vectors and a genome-editing tool. In this study, we focused on mating-type switching and gene expression in order to elucidate its sexual life cycle and establish genetic approaches applicable for the strain. A putative mating-type gene cluster was identified in TBRC656 that is syntenic to the cluster in Ogataea parapolymorpha DL-1 (previously named Hansenula polymorpha). Like DL-1, TBRC656 possesses two mating loci, namely MATa and MATα, and also shows flip-flop mating-type switching. Interestingly, unlike any other methylotrophic yeast, TBRC656 robustly switched mating type during late growth in rich medium (YPD). Under nutrient depletion, mating-type switching was observed within one hour. Transcription from both MATa and MATα mating loci was detected during growth in YPD, and possibly induced upon nitrogen depletion. Gene expression from MATα was detected as a single co-transcript from a three-gene array (α2-α1-a1S). Deletion of a putative a1S ORF at the MATα locus had no observed effect on mating-type switching but demonstrated significant effect on mating-type gene expression at both MATa and MATα loci.

Frequency of the Mating-Type (MAT1) in Histoplasma capsulatum Isolates from Buenos Aires, Argentina.

Sex is genetically determined in Histoplasma capsulatum, governed by a sex-specific region in the genome called the mating-type locus (MAT1). We investigate the distribution of isolates of two H. capsulatum mating types in the clades circulating in Buenos Aires, Argentina. Forty-nine H. capsulatum isolates were obtained from the culture collection of the Mycology Center. The MAT1 locus was identified by PCR from the yeast suspension. The analysis of forty-eight isolates from clinical samples exhibited a ratio of 1.7 (MAT1-1:MAT1-2) and the only isolate from soil was MAT1-1. Forty-five H. capsulatum isolates belonged to the LAm B clade (H. capsulatum from Latin American group B clade) and showed a ratio of 1.8 (MAT1-1:MAT1-2). These results suggest an association between the mating types in isolates belonging to the LAm B clade. It remains to be defined whether a greater virulence should be attributed to the differences between the strains of the opposite mating type of the LAm B clade.

Whole Genome Sequence of the Commercially Relevant Mushroom Strain Agaricus bisporus var. bisporus ARP23.

Agaricus bisporus is an extensively cultivated edible mushroom. Demand for cultivation is continuously growing and difficulties associated with breeding programs now means strains are effectively considered monoculture. While commercial growing practices are highly efficient and tightly controlled, the over-use of a single strain has led to a variety of disease outbreaks from a range of pathogens including bacteria, fungi and viruses. To address this, the Agaricus Resource Program (ARP) was set up to collect wild isolates from diverse geographical locations through a bounty-driven scheme to create a repository of wild Agaricus germplasm. One of the strains collected, Agaricus bisporus var. bisporus ARP23, has been crossed extensively with white commercial varieties leading to the generation of a novel hybrid with a dark brown pileus commonly referred to as ‘Heirloom’. Heirloom has been successfully implemented into commercial mushroom cultivation. In this study the whole genome of Agaricus bisporus var. bisporus ARP23 was sequenced and assembled with Illumina and PacBio sequencing technology. The final genome was found to be 33.49 Mb in length and have significant levels of synteny to other sequenced Agaricus bisporus strains. Overall, 13,030 putative protein coding genes were located and annotated. Relative to the other A. bisporus genomes that are currently available, Agaricus bisporus var. bisporus ARP23 is the largest A. bisporus strain in terms of gene number and genetic content sequenced to date. Comparative genomic analysis shows that the A. bisporus mating loci in unifactorial and unsurprisingly highly conserved between strains. The lignocellulolytic gene content of all A. bisporus strains compared is also very similar. Our results show that the pangenome structure of A. bisporus is quite diverse with between 60–70% of the total protein coding genes per strain considered as being orthologous and syntenically conserved. These analyses and the genome sequence described herein are the starting point for more detailed molecular analyses into the growth and phenotypical responses of Agaricus bisporus var. bisporus ARP23 when challenged with economically important mycoviruses.

Characterization of fungal species associated with cladode brown spot on Nopalea cochenillifera in Brazil

Cladode brown spot (CBS) is an important disease of Nopalea cochenillifera in the semiarid region of Northeastern Brazil. It has been reported in several countries, but its etiology is controversial, attributed to a complex of pathogens. Fifty fungal isolates were obtained and identified based on morphology and phylogeny, through analysis of the ribosomal DNA internal transcribed spacers (ITS), translation elongation factor 1-alpha (TEF1-alpha), β-tubulin (TUB-2), second largest subunit of RNA polymerase (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the mating type locus MAT1–2 (ApMAT) partial gene sequences. Seven fungal general and twenty species were identified – Alternaria sp., A. longipes and A. tenuissima; Colletotrichum fructicola and C. siamense; Fusarium lunatum, F. incarnatum, and F. verticillioides; Lasiodiplodia euphorbicola, L. iraniensis, L. pseutheobromae, L. theobromae and Lasiodiplodia sp.; Neofusicoccum batangarum; Neopestalotiopsis australis, N. protearum and Neopestalotiopsis sp.; and Nigrospora sphaerica, N. hainanensis and Nigrospora sp. Four of these species had already been reported on Cactaceae of the genus Opuntia, but only N. batangarum, C. fructicola and C. siamense have been reported to date on the genus Nopalea. All isolates were pathogenic to detached cladodes of N. cochenillifera ‘Miuda’. Differences in aggressiveness were observed among the species, with L. iraniensis and F. lunatum characterized as the most aggressive species, whilst F. verticillioides as the least aggressive. This study provide important information on the fungi associated with cladode brown spot and improve the strategies for the management of disease on N. cochenillifera.

Design and validation of a robust multiplex polymerase chain reaction assay for MAT idiomorph within the Fusarium fujikuroi species complex

ABSTRACTWe discovered that published polymerase chain reaction (PCR) assays for determining mating type (MAT) idiomorph failed to genotype some of the Fusarium fujikuroi species complex (FFSC) isolates recovered from Mangifera indica (mango), Swietenia macrophylla (big-leaf mahogany), Annona muricata (soursop), Bursera sp., and Tabebuia sp. in Mexico. Thus, the primary objective of this study was to design and validate a robust multiplex PCR–based diagnostic for typing MAT within the FFSC. To accomplish this objective, we mined the MAT1-1 or MAT1-2 locus from the genomes of 60 FFSC isolates, representing 56 phylospecies, and from four species in its sister group, the F. nisikadoi species complex (FNSC). Bioinformatic searches were facilitated by targeting DNA lyase (SLA2) and apurinic endonuclease (APN1), the genes that flank the MAT locus in Fusarium. As expected, three genes were identified within MAT1-1 (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and two in MAT1-2 (MAT1-2-1 and MAT1-2-9), using the ab initio prediction tool AUGUSTUS. Of the three multiplex PCR assays we designed and tested, the one targeting MAT1-1-2 and MAT1-2-1 successfully genotyped the entire 71-isolate validation panel, which included 56 FFSC and 4 FNSC phylospecies. By contrast, the published PCR assays we tested produced positive genotypes for only 46.5–59% of the 71-isolate validation panel, but only when they were run as a uniplex assay. Although only one-fifth of the FFSC/FNSC are known to reproduce sexually, our results suggest that if they possess a sexual cycle, it is heterothallic (self-sterile).

Cohesin Impedes Heterochromatin Assembly in Fission Yeast Cells Lacking Pds5.

The fission yeast Schizosaccharomyces pombe is a powerful genetic model system for uncovering fundamental principles of heterochromatin assembly and epigenetic inheritance of chromatin states. Heterochromatin defined by histone H3 lysine 9 methylation and HP1 proteins coats large chromosomal domains at centromeres, telomeres, and the mating-type (mat) locus. Although genetic and biochemical studies have provided valuable insights into heterochromatin assembly, many key mechanistic details remain unclear. Here, we use a sensitized reporter system at the mat locus to screen for factors affecting heterochromatic silencing. In addition to known components of heterochromatin assembly pathways, our screen identified eight new factors including the cohesin-associated protein Pds5. We find that Pds5 enriched throughout heterochromatin domains is required for proper maintenance of heterochromatin. This function of Pds5 requires its associated Eso1 acetyltransferase, which is implicated in the acetylation of cohesin. Indeed, introducing an acetylation-mimicking mutation in a cohesin subunit suppresses defects in heterochromatin assembly in pds5∆ and eso1∆ cells. Our results show that in cells lacking Pds5, cohesin interferes with heterochromatin assembly. Supporting this, eliminating cohesin from the mat locus in the pds5∆ mutant restores both heterochromatin assembly and gene silencing. These analyses highlight an unexpected requirement for Pds5 in ensuring proper coordination between cohesin and heterochromatin factors to effectively maintain gene silencing.

A Sir2-regulated locus control region in the recombination enhancer of Saccharomyces cerevisiae specifies chromosome III structure.

The NAD+-dependent histone deacetylase Sir2 was originally identified in Saccharomyces cerevisiae as a silencing factor for HML and HMR, the heterochromatic cassettes utilized as donor templates during mating-type switching. MATa cells preferentially switch to MATα using HML as the donor, which is driven by an adjacent cis-acting element called the recombination enhancer (RE). In this study we demonstrate that Sir2 and the condensin complex are recruited to the RE exclusively in MATa cells, specifically to the promoter of a small gene within the right half of the RE known as RDT1. We also provide evidence that the RDT1 promoter functions as a locus control region (LCR) that regulates both transcription and long-range chromatin interactions. Sir2 represses RDT1 transcription until it is removed from the promoter in response to a dsDNA break at the MAT locus induced by HO endonuclease during mating-type switching. Condensin is also recruited to the RDT1 promoter and is displaced upon HO induction, but does not significantly repress RDT1 transcription. Instead condensin appears to promote mating-type donor preference by maintaining proper chromosome III architecture, which is defined by the interaction of HML with the right arm of chromosome III, including MATa and HMR. Remarkably, eliminating Sir2 and condensin recruitment to the RDT1 promoter disrupts this structure and reveals an aberrant interaction between MATa and HMR, consistent with the partially defective donor preference for this mutant. Global condensin subunit depletion also impairs mating-type switching efficiency and donor preference, suggesting that modulation of chromosome architecture plays a significant role in controlling mating-type switching, thus providing a novel model for dissecting condensin function in vivo.

Structural variation and phylogenetic analysis of the mating-type locus in the genus Morchella

ABSTRACTTrue morels (Morchella spp.) are a group of edible fungi that are in high demand worldwide. However, this group of fungi remains poorly understood in terms of its genetic structure, life cycle, and mating system. In the present study, we cloned the MAT locus in Morchella sp. Mes-20 using long-range polymerase chain reaction (PCR) amplification. Our results showed that the MAT1-2 idiomorph in the single-ascospore isolate YAASMCB-3 was 7.5 kb in length, harboring a single MAT1-2-1 gene, whereas the MAT1-1 idiomorph in the isolate YAASMCB-15 was 7.8 kb, carrying both MAT1-1-1 and MAT1-1-10 genes. Mating-type diagnostic assays of single-ascospore populations confirmed that Mes-20 is heterothallic. In addition, 42 collections belonging to 17 morel species (7 black morel species [Elata clade], 9 yellow morel species [Esculenta clade], and 1 species from the Rufobrunnea clade) were used to clone and characterize the MAT1-1-1 and MAT1-2-1 genes for development of the mating gene genealogies. In concordance with the multilocus phylogenetic trees, collections for the respective species were readily distinguished by well-supported lineages in mating gene genealogies. The topologies were consistent within the Elata clade, whereas the branching order and sister-group relationships slightly varied within the Esculenta clade. Our results show that species in the genus Morchella could be delimited by mating gene genealogies.

Global distribution of mating types shows limited opportunities for mating across populations of fungi causing boxwood blight disease

Boxwood blight is a disease threat to natural and managed landscapes worldwide. To determine mating potential of the fungi responsible for the disease, Calonectria pseudonaviculata and C. henricotiae, we characterized their mating-type (MAT) loci. Genomes of C. henricotiae, C. pseudonaviculata and two other Calonectria species (C. leucothoes, C. naviculata) were sequenced and used to design PCR tests for mating-type from 268 isolates collected from four continents. All four Calonectria species have a MAT locus that is structurally consistent with the organization found in heterothallic ascomycetes, with just one idiomorph per individual isolate. Mating type was subdivided by species: all C. henricotiae isolates possessed the MAT1-1 idiomorph, whereas all C. pseudonaviculata isolates possessed the MAT1-2 idiomorph. To determine the potential for divergence at the MAT1 locus to present a barrier to interspecific hybridization, evolutionary analysis was conducted. Phylogenomic estimates showed that C. henricotiae and C. pseudonaviculata diverged approximately 2.1 Mya. However, syntenic comparisons, phylogenetic analyses, and estimates of nucleotide divergence across the MAT1 locus and proximal genes identified minimal divergence in this region of the genome. These results show that in North America and parts of Europe, where only C. pseudonaviculata resides, mating is constrained by the absence of MAT1-1. In regions of Europe where C. henricotiae and C. pseudonaviculata currently share the same host and geographic range, it remains to be determined whether or not these two recently diverged species are able to overcome species barriers to mate.

Fifty Aureobasidium pullulans genomes reveal a recombining polyextremotolerant generalist.

SummaryThe black yeast Aureobasidium pullulans is a textbook example of a generalistic and ubiquitous fungus thriving in a wide variety of environments. To investigate whether A. pullulans is a true generalist, or alternatively, whether part of its versatility can be attributed to intraspecific specialization masked by cryptic diversification undetectable by traditional phylogenetic analyses, we sequenced and analysed the genomes of 50 strains of A. pullulans from different habitats and geographic locations. No population structure was observed in the sequenced strains. Decay of linkage disequilibrium over shorter physical distances (<100 bp) than in many sexually reproducing fungi indicates a high level of recombination in the species. A homothallic mating locus was found in all of the sequenced genomes. Aureobasidium pullulans appears to have a homogeneous population genetics structure, which is best explained by good dispersal and high levels of recombination. This means that A. pullulans is a true generalist that can inhabit different habitats without substantial specialization to any of these habitats at the genomic level. Furthermore, in the future, the high level of A. pullulans recombination can be exploited for the identification of genomic loci that are involved in the many biotechnologically useful traits of this black yeast.

RNA insertion in DNA as the imprint moiety: the fission yeast paradigm.

This review elaborates on the findings of a new report which possibly resolves the biochemical nature of a novel type of DNA imprint as ribonucleotide and the mechanism of its formation during cell differentiation in fission yeast. The process of mating-type switching in fission yeast, Schizosaccharomyces pombe, displays characteristics of a typical mammalian stem cell lineage, wherein a cell divides to produce an identical cell and a differentiated cell after every two cell divisions. This developmental asymmetry has been ascribed to play a role in generation of a DNA strand-specific imprint at the mat1 locus during lagging strand synthesis and its segregation to one of the two daughter cells by the process of asymmetric, semi-conservative DNA replication. The nature of this imprint and mechanisms of its generation have been a subject of research and debate. A recent report by Singh et al. (Nucleic Acids Res 47:3422-3433. https://doi.org/10.1093/nar/gkz092 , 2019) provides compelling evidence in support of a ribonucleotide as the imprint moiety within the mat1 DNA and demonstrates the role of Mcm10/Cdc23, an important, evolutionarily conserved component of DNA replication machinery in eukaryotes, in installing the imprint through a non-canonical primase activity and interaction with DNA Polα and Swi1. The high degree of conservation of DNA replication machinery, especially the presence of the T7 gene 4 helicase/primase domain in the mammalian orthologs of Mcm10 suggests that similar mechanisms of DNA imprinting may play a role during cell differentiation in metazoans.