Mapping the mating type locus of Tetrahymena thermophila: Meiotic linkage of mat to the ribosomal RNA gene

Tetrahymena thermophila has a multiple mating type system. While a sexually mature cell usually expresses only one mating type, its germline (micronucleus) carries the genetic potential for 5 to 7 mating types. The set of allowed mating types is specified by the mat locus. The choice of which particular mating type is expressed by a cell reflects a somatically inherited, developmentally programmed differentiation of the somatic nucleus (macronucleus). In this work we report that the mat locus maps to the left arm of chromosome 2, as determined by nullisomic deletion mapping. We also report a distance of 29 cM between the mat locus and the ribosomal RNA gene, previously mapped to chromosome 2L. This represents another (rare) case of meiotic linkage in Tetrahymena. © 1992 Wiley-Liss, Inc.     

Ecological Genetics of Tetrahymena thermophila: Mating Types, i-Antigens, Multiple Alleles and Epistasis

Until recently, Tetrahymena thermophila has rarely been isolated from nature. With improved sampling procedures, T. thermophila has been found in ponds in many northeastern states. The availability of resident populations makes possible both population and ecological genetic studies. All seven known mating types have been recovered; no eighth mating type has been found. Crosses among whole-genome homozygotes derived from Pennsylvania isolates reveal a spectrum genotypes with mating type alleles resembling traditional A (IV- and VII-) and B(I-) categories. The genotypes differ significantly with respect to mating type frequency, both among themselves and from previously described genotypes. One A-category genotype appears to lack mating type II, while one A-category and all B-category genotypes have low frequencies of mating type III, thus accounting for the low frequency of III in the pond. The low frequency of III in all five B-category genotypes examined suggests that the founding allele in this region was low for III. These and other differences are discussed both in terms of mating type frequencies in the pond and in terms of the possible molecular structure of mat alleles. By contrast, numerous variants of the cell surface immobilization antigen are found in addition to the previously described i-antigens. Variants of the known SerH alleles include those with restriction fragment length polymorphisms and temperature sensitivity as well as alleles with new antigenic specificity. Multiple alleles are present in single ponds. Genes exhibiting serially dominant epistasis over SerH genes also are found. In two instances (K and C), families of antigenically similar polypeptides are expressed in place of H i-antigen. Molecular weight differences suggest that these paralogous i-antigen genes evolve by gene duplication and unequal crossing over within central repeats. The existence of complex patterns of epistasis together with seasonal changes in i-ag frequencies suggest that i-ag play an important, but as yet unknown, ecological role related to the occurrence of frequent conjugation.     

Genome analysis of sphingolipid metabolism-related genes in Tetrahymena thermophila and identification of a fatty acid 2-hydroxylase involved in the sexual stage of conjugation

Sphingolipids are bioactive lipids present in all eukaryotes. Tetrahymena thermophila is a ciliate that displays remarkable sphingolipid moieties, that is, the unusual phosphonate-linked headgroup ceramides, present in membranes. To date, no identification has been made in this organism of the functions or related genes implicated in sphingolipid metabolism. By gathering information from the T. thermophila genome database together with sphingolipid moieties and enzymatic activities reported in other Tetrahymena species, we were able to reconstruct the putative de novo sphingolipid metabolic pathway in T. thermophila. Orthologous genes of 11 enzymatic steps involved in the biosynthesis and degradation pathways were retrieved. No genes related to glycosphingolipid or phosphonosphingolipid headgroup transfer were found, suggesting that both conserved and innovative mechanisms are used in ciliate. The knockout of gene TTHERM_00463850 allowed to identify the gene encoding a putative fatty acid 2-hydroxylase, which is involved in the biosynthesis pathway. Knockout cells have shown several impairments in the sexual stage of conjugation since different mating types of knockout strains failed to form cell pairs and complete the conjugation process. This fatty acid 2-hydroxylase gene is the first gene of a sphingolipid metabolic pathway to be identified in ciliates and have a critical role in their sexual stage.     

Phylogenetic Analysis of Phaeosphaeria Species Using Mating Type Genes and Distribution of Mating Types in Iran

Phaeosphaeria species are pathogenic on wheat, barley and a wide range of wild grasses. To analyze mating type loci of the Phaeosphaeria species and investigate mating type distribution in Iran, we sequenced mating type loci of 273 Phaeosphaeria isolates including 67 isolates obtained from symptomatic leaves and ears of wheat, barley, and wild grasses from two wheat-growing region in Iran as well as 206 isolates from our collection from other regions in Iran which were isolated in our previous studies. Mating type genes phylogeny was successfully used to determine the species identity and relationships among isolates within the Phaeosphaeria spp. complex. In this study, we reported seven new host records for Phaeosphaeria species and the Phaeosphaeria avenaria f. sp. tritici 3 group was first reported from Iran in this study. Mating type distribution among Phaeosphaeria species was determined. Both mating types were present in all sampling regions from Iran. We observed skewed distribution of mating types in one region (Kohgiluyeh va Boyer-Ahmad) and equal distribution in the other region (Bushehr). However, when considering our entire dataset of 273 Iranian Phaeosphaeria isolates, the ratio of mating types was not deviated significantly from 1:1 suggesting possibilities for isolates of opposite mating type to interact and reproduce sexually, although the sexual cycle may infrequently occur in some regions especially when the climatic conditions are unfavorable for teleomorph development.     

Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation

The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat–, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat+-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat– information. The transformants behave in crosses as do the reference mat+ or mat– strains, thus indicating that the transgenic mat+ and mat– are fully functional even when they have integrated at ectopic sites.     

Microarray and real-time PCR analyses reveal mating type-dependent gene expression in a homothallic fungus

Sordaria macrospora is a homothallic ascomycete which is able to form fertile fruiting bodies without a mating partner. To analyze the molecular basis of homothallism and the role of mating products during fruiting body development, we have deleted the mating type gene Smta-1 encoding a high-mobility group domain (HMG) protein. The ΔSmta-1 deletion strain is morphologically wild type during vegetative growth, but it is unable to produce perithecia or ascospores. To identify genes expressed under control of Smta-1, we performed a cross-species microarray analysis using Neurospora crassa cDNA microarrays hybridized with S. macrospora targets. We identified 107 genes that are more than twofold up- or down-regulated in the mutant. Functional classification revealed that 81 genes have homologues with known or putative functions. Comparison of array data from ΔSmta-1 with those from three phenotypically similar mutants revealed that only a limited set of ten genes is deregulated in all mutants. Remarkably, the ppg2 gene encoding a putative lipopeptide pheromone is 500-fold down-regulated in the ΔSmta-1 mutant while in all other sterile mutants this gene is up-regulated. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Comparative genomics of the mating-type loci of the mushroom Flammulina velutipes reveals widespread synteny and recent inversions

Background

Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs.

Methodology/Principal Findings

We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters.

Conclusions/Significance

In our study we determined that the Agaricales have very large scale synteny at matA (∼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.     

Mating-Type Polymorphic Variation in Euplotes minuta (Ciliophora: Hypotrichida)1

An investigation was carried out to probe into the mating-type structure of a local population of the marine ciliate, Euplotes minuta. From this population, nine different mating types belonging to a unique set were isolated. The nine type-representative wild stocks analyzed were found to be heterozygous at the mating-type (mat) locus and provided, together with their sexual progeny, a total of 15 pure mating types. In E. minuta, the high-multiple nature of the basic mating system controlled by a series of peck-order alleles at a single locus should be considered a virtual certainty. The relationships among the genetic economies of the similar bottom-dwelling marine ciliates of the genus Euplotes, the E. vannus-crassus-minuta group, are discussed.     

Sex-linked transcriptional divergence in the hermaphrodite fungus Neurospora tetrasperma

In the filamentous ascomycete Neurospora tetrasperma, a large (approx. 7 Mbp) region of suppressed recombination surrounds the mating-type (mat) locus. While the remainder of the genome is largely homoallelic, this region of recombinational suppression, extending over 1500 genes, is associated with sequence divergence. Here, we used microarrays to examine how the molecular phenotype of gene expression level is linked to this divergent region, and thus to the mating type. Culturing N. tetrasperma on agar media that induce sexual/female or vegetative/male tissue, we found 196 genes significantly differentially expressed between mat A and mat a mating types. Our data show that the genes exhibiting mat-linked expression are enriched in the region genetically linked to mating type, and sequence and expression divergence are positively correlated. Our results indicate that the phenotype of mat A strains is optimized for traits promoting sexual/female development and the phenotype of mat a strains for vegetative/male development. This discovery of differentially expressed genes associated with mating type provides a link between genotypic and phenotypic divergence in this taxon and illustrates a fungal analogue to sexual dimorphism found among animals and plants.     

What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina

In the heterothallic ascomycete Podospora anserina, the mating-type locus is occupied by two mutually exclusive sequences termed mat+ and mat–. The mat+ sequence contains only one gene, FPR1, while the mat– sequence contains three genes: FMR1, SMR1 and SMR2. Previous studies have demonstrated that FPR1 and FMR1 are required for fertilization. Further analyses have led to the hypothesis that mat+ and mat– genes establish a mat+ and mat– nuclear identity, allowing recognition between nuclei of opposite mating type within the syncytial cells formed after fertilization. This hypothesis was based on the phenotypes of strains bearing mutations in ectopic mat genes. Here we present an analysis of mutations in resident mat– genes which suggests that, unlike FMR1 and SMR2, SMR1 is not involved in establishing nuclear identity. In fact, mutations in these two genes impair nuclear recognition, leading to uniparental progeny, while mutations in SMR1 block the sexual process, probably at a step after nuclear recognition. The nuclear identity hypothesis has also been tested through internuclear complementation tests. In these experiments, the mat– mutants were crossed with a mat+ strain carrying the wild-type mat– genes. Our rationale was that internuclear complementation should not be possible for nuclear identity genes: the relevant genes should show nucleus-restricted expression, and diffusion of their products to other nuclei should not occur. This test confirmed that SMR1 is not a bona fide mat gene since it can fulfill its function whatever its location, in either a mat− or a mat+ nucleus, and even when present in both nuclei. SMR2, but not FMR1, behaves like a nuclear identity gene with respect to internuclear complementation tests. A model is proposed that tentatively explains the ambiguous behaviour of the FMR1 gene and clarifies the respective functions of the three mat– proteins. Received: 15 October 1996 / Accepted: 25 April 1997     

Hybridization between Japanese and North American Chlamydomonas reinhardtii (Volvocales,Chlorophyceae)

The microalga Chlamydomonas reinhardtii is a model organism whose whole genome has been sequenced. Although considered a cosmopolitan species, only eastern North American isolates of C. reinhardtii were available before 2010, when new Japanese isolates were reported. In the study describing the new Japanese isolates, zygote formation between Japanese and North American strains was shown, but germination was not demonstrated. In this study, the germination of intercontinental hybrid zygotes was examined using wild‐type Japanese strains and mutant American strains that cannot utilize nitrate. Several clonal progeny strains were established, and the progeny strains were screened based on mating type and nitrate utilization to confirm their hybrid nature. The establishment of four intercontinental hybrid strains with different phenotypic combinations was confirmed by sequencing mating type‐specific and nitrate reductase‐related genes. The potential for hybrid formation between Japanese and North American strains suggests the existence of a worldwide mating population of C. reinhardtii.     

Control of yeast cell type by the mating type locus: II. Genetic interactions between MATα and unlinked α-specific STE genes

The alleles of the yeast mating type locus, MATα and MATa, determine the yeast cell types, a,α, and a/α. It has been proposed that the MATα2 product negatively regulates expression of unlinked a-specific genes, and that the MATα1 product positively regulates expression of unlinked α-specific genes. The behavior of mutants defective in MATα2, which are deficient in mating and in production of α-factor, can thus be attributed to antagonism between a-specific and α-specific functions expressed simultaneously in matα2^? strains. If this view is correct, then elimination by mutation of the specific functions required to mate as α may allow matα2 mutants to mate as a. In order to test this possibility, we examined the interactions between matα2 mutations and various unlinked mutations that cause α cells but not a cells to be mating defective (α-specific STE mutations). Three α-specific mutations (ste3, ste13 and kex2) were found to be non-allelic. Furthermore, although matα2 mutants mate weakly as a, matα2, ste3 double mutants, but not matα2 ste13 or matα2 kex2 double mutants, mate efficiently as a. The ability of matα2 ste3 strains to mate as a supports the view that matα2 mutants express a-specific mating functions, and suggests that a mating functions are expressed constitutively in MATa cells. The mating behaviour of the matα2 ste3 double mutant is consistent with the proposal that STE3 is positively regulated by the MATα1 product.     

Identification of Races and Mating Types of Cochliobolus carbonum from Corn in the Yunnan Province in China

Northern corn leaf spot, a foliar disease caused by Cochliobolus carbonum, has become prevalent in southwestern China, especially in the Yunnan Province. Races and mating types were identified for 169 isolates collected from 13 prefectures of Yunnan by artificial inoculation using six hybrid corns as differential hosts and by crossing with three standard mating strains: CC092 (MAT1‐2), CC120 (MAT1‐1) and CC026 (MAT1‐1). Results showed the existence of three races: CCR1 (one isolate), CCR2 (43 isolates) and CCR3 (125 isolates). Most isolates were moderately or weakly virulent with only five being highly virulent. CCR3 was widely distributed and significantly more virulent than CCR2 that coexisted with CCR3 in many locations. On Sach's nutrient agar, 20.71% of the Yunnan isolates self‐mated, forming sterile perithecia. Fully developed perithecia could be formed between isolates of different geographic origins, but only 15.98% strains mated successfully with CC092 and 5.33% formed mature perithecia with 4–6 ascospores per asus. Similar results were obtained in crossing with CC026 or CC120. Mating could also occur between CCR3 and CCR2. Both mating types were found in Yunnan with 84 MAT1‐1 strains (one CCR1, 10 CCR2 and 73 CCR3) and 85 MAT1‐2 strains (33 CCR2 and 52 CCR3) and they coexisted in most areas. To identify the mating type rapidly, three specific primers were successfully developed and employed to amplify the mating‐type genes, with stable patterns of 1627 and 876 bp fragments obtained from MAT1‐1 and MAT1‐2 isolates, respectively. The ratio between MAT1‐1 and MAT1‐2 was 1 : 1, indicating that the mating‐type genes segregated randomly in the field naturally.     

Vampirovibrio chlorellavorus draft genome sequence,annotation, and preliminary characterization of pathogenicity determinants

Vampirovibrio chlorellavorus is recognized as a pathogen of commercially‐relevant Chlorella species. Algal infection and total loss of productivity (biomass) often occurs when susceptible algal hosts are cultivated in outdoor open pond systems. The pathogenic life cycle of this bacterium has been inferred from laboratory and field observations, and corroborated in part by the genomic analyses for two Arizona isolates recovered from an open algal reactor. V. chlorellavorus predation has been reported to occur in geographically‐ and environmentally‐diverse conditions. Genomic analyses of these and additional field isolates is expected to reveal new information about the extent of ecological diversity and genes involved in host‐pathogen interactions. The draft genome sequences for two isolates of the predatory V. chlorellavorus (Cyanobacteria; Ca. Melainabacteria) from an outdoor cultivation system located in the Arizona Sonoran Desert were assembled and annotated. The genomes were sequenced and analyzed to identify genes (proteins) with predicted involvement in predation, infection, and cell death of Chlorella host species prioritized for biofuel production at sites identified as highly suitable for algal production in the southwestern USA. Genomic analyses identified several predicted genes encoding secreted proteins that are potentially involved in pathogenicity, and at least three apparently complete sets of virulence (Vir) genes, characteristic of the VirB‐VirD type system encoding the canonical VirB1‐11 and VirD4 proteins, respectively. Additional protein functions were predicted suggesting their involvement in quorum sensing and motility. The genomes of two previously uncharacterized V. chlorellavorus isolates reveal nucleotide and protein level divergence between each other, and a previously sequenced V. chlorellavorus genome. This new knowledge will enhance the fundamental understanding of trans‐kingdom interactions between a unique cosmopolitan cyanobacterial pathogen and its green microalgal host, of broad interest as a source of harvestable biomass for biofuels or bioproducts.     

Gibberella fujikuroi mating population E is associated with maize and teosinte

Isolates of Fusarium subglutinans mating population E are usually found on maize. This fungus forms part of the so-called Gibberella fujikuroi species complex. Previously, F. subglutinans has been associated with two additional mating populations (B and H) and a variety of plant hosts. This was mainly due to a lack of diagnostic morphological characters, but the use of DNA sequence information showed that the strains making up mating populations B, E and H, as well as those associated with the different plant hosts, represent separate species. Recently, another putative mating population has been reported on the wild teosinte relatives of maize. Based on sexual compatibility studies, these isolates were apparently closely related to the pitch canker fungus, F. subglutinans f. sp. pini (= F. circinatum;G. fujikuroi mating population H). The aim of the current study was to determine whether the population of F. subglutinans from teosinte constitutes a new or an existing lineage within the G. fujikuroi complex. For this purpose, portions of the mitochondrial small subunit ribosomal DNA, calmodulin and β-tubulin genes from the fungi were sequenced. Phylogenetic analyses and comparison with sequences from public domain databases indicated that the F. subglutinans isolates from teosinte are most closely related to strains of G. fujikuroi mating population E. These results were confirmed using sexual compatibility studies. The putative mating population from the wild relatives of maize therefore forms part of the existing E-mating population and does not constitute a new lineage in the G. fujikuroi species complex.     

Distribution of Mating Types and Genetic Diversity of Ascochyta rabiei Populations in Tunisia Revealed by Mating-type-specific PCR and Random Amplified Polymorphic DNA Markers

The distribution of mating types of Ascochyta rabiei (teleomorph: Didymella rabiei) was determined in Tunisia using a MAT‐specific PCR assay. Among 123 isolates tested, 80% were MAT1‐1 and 20%MAT1‐2. Only MAT1‐1 isolates were present in the Beja and Bizerte regions of Tunisia, whereas both mating types were present in Nabeul, Kef and Jendouba. In the latter three regions, the hypothesis of random mating could not be rejected based on chi‐squared tests of mating‐type ratios (P > 0.05). The lower frequency of the MAT1‐2 coupled with the restricted distribution of this mating type in Tunisia may indicate a recent introduction of MAT1‐2 in Tunisia. This speculation is consistent with the recent (2001) observation of D. rabiei pseudothecia on chickpea debris in Tunisia. Forty isolates representative of the five regions were genetically analysed using 10 random amplified polymorphic DNA (RAPD) primers to provide a preliminary estimate of genetic diversity of the pathogen in Tunisia. Among 129 putative RAPD loci amplified, 81% were polymorphic and 32 unique RAPD fingerprints were detected. A high level of genetic differentiation was detected among subpopulations (G_ST = 0.33). Cluster analyses revealed that isolates from Bizerte, Beja and Jendouba were genetically similar and distinct from isolates sampled in Nabeul and Kef. MAT1‐1 isolates were clustered separately from MAT1‐2 isolates in Jendouba and Nabeul suggesting that recombination may not yet be occurring in these regions despite the occurrence of both mating types in equal frequency in these regions. This lack of recombination between MAT1‐1 and MAT1‐2 also supports the hypothesis of a recent introduction of MAT1‐2 into Tunisia.     

Mating type‐dependent partner sensing as mediated by VEL1 in Trichoderma reesei

Sexual development in the filamentous model ascomycete Trichoderma reesei (syn. Hypocrea jecorina) was described only a few years ago. In this study, we show a novel role for VELVET in fungi, which links light response, development and secondary metabolism. Vel1 is required for mating in darkness, normal growth and conidiation. In light, vel1 was dispensable for male fertility but essential for female fertility in both mating types. VEL1 impacted regulation of the pheromone system (hpr1, hpr2, hpp1, ppg1) in a mating type‐dependent manner and depending on the mating partner of a given strain. These partner effects only occurred for hpp1 and hpr2, the pheromone precursor and receptor genes associated with the MAT1‐2 mating type and for the mating type gene mat1‐2‐1. Analysis of secondary metabolite patterns secreted by wild type and mutants under asexual and sexual conditions revealed that even in the wild type, the patterns change upon encounter of a mating partner, with again distinct differences for wild type and vel1 mutants. Hence, T. reesei applies a language of pheromones and secondary metabolites to communicate with mating partners and that this communication is at least in part mediated by VEL1.