Mating type gene analyses in the genus Diplodia: From cryptic sex to cryptic species

Cryptic species are common in Diplodia, a genus that includes some well-known and economically important plant pathogens. Thus, species delimitation has been based on the phylogenetic species recognition approach using multigene genealogies. We assessed the potential of mating type (MAT) genes sequences as phylogenetic markers for species delimitation in the genus Diplodia. A PCR-based mating type diagnostic assay was developed that allowed amplification and sequencing of the MAT1-1-1 and MAT1-2-1 genes, and determination of the mating strategies used by different species. All species tested were shown to be heterothallic. Phylogenetic analyses were performed on both MAT genes and also, for comparative purposes, on concatenated sequences of the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1-α) and beta-tubulin (tub2). Individual phylogenies based on MAT genes clearly differentiated all species analysed and agree with the results obtained with the commonly used multilocus phylogenetic analysis approach. However, MAT genes genealogies were superior to multigene genealogies in resolving closely related cryptic species. The phylogenetic informativeness of each locus was evaluated revealing that MAT genes were the most informative loci followed by tef1-α. Hence, MAT genes can be successfully used to establish species boundaries in the genus Diplodia.     

Sexual Reproduction as the Cause of Heat Resistance in the Food Spoilage Fungus Byssochlamys spectabilis (Anamorph Paecilomyces variotii)

Paecilomyces variotii is a common cosmopolitan species that is able to spoil various food- and feedstuffs and is frequently encountered in heat-treated products. However, isolates from heat-treated products rarely form ascospores. In this study we examined by using molecular techniques and mating tests whether this species can undergo a sexual cycle and form ascospores. The population structure of this species was examined by analyzing the nuclear ribosomal internal transcribed spacer 1 (ITS1) and ITS2 and the 5.8S rRNA gene, as well as partial β-tubulin, actin, and calmodulin gene sequences. Phylogenetic analyses revealed that P. variotii is a highly variable species. Partition homogeneity tests revealed that P. variotii has a recombining population structure. In addition to sequence analyses, mating experiments indicated that P. variotii is able to form ascomata and ascospores in culture in a heterothallic manner. The distribution of MAT1-1 and MAT1-2 genes showed a 1:1 ratio in the progeny of the mating experiments. From the sequence analyses and mating data we conclude that P. variotii is the anamorph of Talaromyces spectabilis and that it has a biallelic heterothallic mating system. Since Paecilomyces sensu stricto anamorphs group within Byssochlamys, a new combination Byssochlamys spectabilis is proposed.     

Isolation of the MAT1-1 mating type idiomorph and evidence for selfing in the Chinese medicinal fungus Ophiocordyceps sinensis

Ophiocordyceps sinensis is one of the most valued medicinal fungi in China. Research on the mating system and sexual development is vitally important to this endangered species. Previous efforts devoted to investigate the mating type (MAT) locus of O. sinensis, however, resulted in an incomplete understanding. In this study, the MAT1-1 locus of O. sinensis was investigated. The conserved α-box and HMG-box regions of the MAT1-1-1 and MAT1-1-3 genes, respectively, and a conserved region of the DNA lyase gene were successfully amplified using degenerate PCR. A combination of TAIL-PCR and long-range PCR were used to connect these genes and obtain the sequence of the MAT1-1 locus. Screening of 22 single spore isolates by PCR demonstrated that both the MAT1-1-1 and MAT1-2-1 genes cooccurred within the same isolate. Additionally, both MAT1-1-1 and MAT1-2-1 are expressed in vegetative mycelia, providing evidence that O. sinensis is likely capable of selfing. DAPI (4,6-diamidino-2-phenylindole) staining of ascospores and hyphae showed that a majority of hyphal compartments are binucleate, suggesting that O. sinensis may be pseudohomothallic. Analyses of sequence diversity showed lower levels of genetic diversity in MAT1-1-1 compared to MAT1-2-1, indicating the possibility that different selective pressures act on the two MAT idiomorphs. The MAT1-1-1 sequences of O. sinensis and Tolypocladium inflatum cluster as a monophyletic group consistent with phylogenetic classification of Ophiocordycipitaceae. Comparison of the structure of the MAT1-1 locus across hypocrealean taxa showed that O. sinensis contains all three mating type genes (MAT1-1-1, MAT1-1-2, and MAT1-1-3) and supported previous observations that of the four families in Hypocreales, MAT1-1-3 has undergone a lineage specific loss only in some members of the Cordycipitaceae.     

Deletion and Complementation of the Mating Type (MAT) Locus of the Wheat Head Blight Pathogen Gibberella zeae

Gibberella zeae, a self-fertile, haploid filamentous ascomycete, causes serious epidemics of wheat (Triticum aestivum) head blight worldwide and contaminates grain with trichothecene mycotoxins. Anecdotal evidence dating back to the late 19th century indicates that G. zeae ascospores (sexual spores) are a more important inoculum source than are macroconidia (asexual spores), although the fungus can produce both during wheat head blight epidemics. To develop fungal strains to test this hypothesis, the entire mating type (MAT1) locus was deleted from a self-fertile (MAT1-1/MAT1-2), virulent, trichothecene-producing wild-type strain of G. zeae. The resulting MAT deletion (mat1-1/mat1-2) strains were unable to produce perithecia or ascospores and appeared to be unable to mate with the fertile strain from which they were derived. Complementation of a MAT deletion strain by transformation with a copy of the entire MAT locus resulted in recovery of production of perithecia and ascospores. MAT deletion strains and MAT-complemented strains retained the ability to produce macroconidia that could cause head blight, as assessed by direct injection into wheat heads in greenhouse tests. Availability of MAT-null and MAT-complemented strains provides a means to determine the importance of ascospores in the biology of G. zeae and perhaps to identify novel approaches to control wheat head blight.     

Cloning and analysis of the mating type genes from Cochliobolus heterostrophus

Cochliobolus heterostrophus, a heterothallic Ascomycete, has a single mating type locus with two alternate forms called MAT-1 and MAT-2. MAT-1 was cloned by complementing a MAT-2 strain using a cosmid library from a MAT-1 strain and screening for a homothallic transformant. The cosmid recovered from this transformant was able to re-transform a MAT-2 strain to homothallism and MAT identity was proven by restriction fragment length polymorphism and conventional genetic mapping. All homothallic transformants could mate with either MAT-1 or MAT-2 strains, although the number of ascospores produced by self matings or crosses to MAT-2 strains was low. Progeny of selfed homothallic transformants were themselves homothallic. MAT-2 was cloned by probing a cosmid library from a MAT-2 strain with a fragment of insert DNA from the MAT-1 cosmid. A 1.5 kb subclone of either MAT-containing cosmid was sufficient to confer mating function in transformants. Examination of the DNA sequence of these subclones revealed that MAT-1 and MAT-2 contain 1297 by and 1171 bp, respectively, of completely dissimilar DNA flanked by DNA common to both mating types. Putative introns were found (one in each MAT gene) which, when spliced out, would yield open reading frames (ORFs) that occupied approximately 90% of the dissimilar DNA sequences. Translation of the MAT-1 ORF revealed similarity to the Neurospora crassa MATA, Podospora anserina mat?, and Saccharomyces cerevisiae MATα1 proteins; translation of the MAT-2 ORF revealed similarity to the N. crassa MATa, P. anserina mat+, and Schizosaccharomyces pombe mat-Mc proteins. These gene products are all proven or proposed DNA binding proteins. Those with similarity to MAT-2 are members of the high mobility group.     

Characterization of the mating-type genes in Leptographium procerum and Leptographium profanum

Leptographium procerum and the closely related species Leptographium profanum, are ascomycetes associated with root-infesting beetles on pines and hardwood trees, respectively. Both species occur in North America where they are apparently native. L. procerum has also been found in Europe, China New Zealand, and South Africa where it has most probably been introduced. As is true for many other Leptographium species, sexual states have never been observed in L. procerum or L. profanum. The objectives of this study were to clone and characterize the mating type loci of these fungi, and to develop markers to determine the mating types of individual isolates. To achieve this, a partial sequence of MAT1-2-1 was amplified using degenerate primers targeting the high mobility group (HMG) sequence. A complete MAT1-2 idiomorph of L. profanum was subsequently obtained by screening a genomic library using the HMG sequence as a probe. Long range PCR was used to amplify the complete MAT1-1 idiomorph of L. profanum and both the MAT1-1 and MAT1-2 idiomorphs of L. procerum. Characterization of the MAT idiomorphs suggests that the MAT genes are fully functional and that individuals of both these species are self-sterile in nature with a heterothallic mating system. Mating type markers were developed and tested on a population of L. procerum isolates from the USA, the assumed center of origin for this species. The results suggest that cryptic sexual reproduction is occurring or has recently taken place within this population.     

Molecular organization of the mating type (Mat) locus of Exserohilum monoceras (Setosphaeria monoceras), a bioherbicide agent for Echinochloa weeds

Mating-type genes were cloned and sequenced in the heterothallic phytopathogenic fungus Exserohilum monoceras, a candidate bioherbicide for the control of Echinochloa weeds in rice fields. Using PCR-based methods, we determined both idiomorphs and flanking regions of these genes. The structural organization of the E. monoceras Mat locus was similar to that of other heterothallic ascomycetes. The MAT1-1-1 gene was 1191 bp and encoded a predicted protein of 379 amino acids with an α box domain. The MAT1-2-1 gene was 1093 bp and encoded a predicted protein of 345 amino acids with a high mobility group box domain. Expression of both MAT genes was detected under vegetative and invasive growth conditions. MAT-specific primers were developed to assess the mating-type frequency of E. monoceras field isolates. Both mating types were observed in Japanese field isolates. Analysis of mating types and sexual hybridization of E. monoceras could provide useful approaches for the conventional genetic manipulation of this fungus to produce a more efficient bioherbicide.     

Heterothallism revealed in the root rot fungi Berkeleyomyces basicola and B. rouxiae

Berkeleyomyces basicola and Berkeleyomyces rouxiae, two sister species previously treated collectively as Thielaviopsis basicola, reside in the Ceratocystidaceae (Microascales, Ascomycota). Both species are important root pathogens of many important agricultural crops and ornamental plants. Although T. basicola has been known for more than 150y, a sexual state has never been found and it has been assumed to be an asexual pathogen. The aim of this study was to determine the mating strategy of the two Berkeleyomyces species. Investigation of the genome sequences of two B. basicola isolates allowed for the complete characterization of the MATlocus, revealing that it has a typical heterothallic mating system with the MAT1-1andMAT1-2 idiomorphs occurring in different isolates. PCR amplification using mating type primers developed in this study, showed that the MAT1-1-1andMAT1-2-1 genes were also present in different isolates of B. rouxiae. Pairing of isolates representing the two mating types of both species,using a variety of techniques failed to produce sexual structures. Although we have found no direct evidence that they reproduce sexually, these fungi are clearly heterothallic with both mating types occurring in some countries suggesting that a cryptic sexual cycle could exist for them.     

Analysis of the mating-type loci of co-occurring and phylogenetically related species of Ascochyta and Phoma

Ascochyta and Phoma are fungal genera containing several important plant pathogenic species. These genera are morphologically similar, and recent molecular studies performed to unravel their phylogeny have resulted in the establishment of several new genera within the newly erected Didymellaceae family. An analysis of the structure of fungal mating-type genes can contribute to a better understanding of the taxonomic relationships of these plant pathogens, and may shed some light on their evolution and on differences in sexual strategy and pathogenicity. We analysed the mating-type loci of phylogenetically closely related Ascochyta and Phoma species (Phoma clematidina, Didymella vitalbina, Didymella clematidis, Peyronellaea pinodes and Peyronellaea pinodella) that co-occur on the same hosts, either on Clematis or Pisum. The results confirm that the mating-type genes provide the information to distinguish between the homothallic Pey. pinodes (formerly Ascochyta pinodes) and the heterothallic Pey. pinodella (formerly Phoma pinodella), and indicate the close phylogenetic relationship between these two species that are part of the disease complex responsible for Ascochyta blight on pea. Furthermore, our analysis of the mating-type genes of the fungal species responsible for causing wilt of Clematis sp. revealed that the heterothallic D. vitalbina (Phoma anamorph) is more closely related to the homothallic D. clematidis (Ascochyta anamorph) than to the heterothallic P. clematidina. Finally, our results indicate that homothallism in D. clematidis resulted from a single crossover between MAT1-1 and MAT1-2 sequences of heterothallic ancestors, whereas a single crossover event followed by an inversion of a fused MAT1/2 locus resulted in homothallism in Pey. pinodes.     

Ceratocystidaceae exhibit high levels of recombination at the mating-type (MAT) locus

Mating is central to many fungal life cycles and is controlled by genes at the mating-type (MAT) locus. These genes determine whether the fungus will be self-sterile (heterothallic) or self-fertile (homothallic). Species in the ascomycete family Ceratocystidaceae have different mating strategies, making them interesting to consider with regards to their MAT loci. The aim of this study was to compare the composition of the MAT locus flanking regions in 11 species of Ceratocystidaceae representing four genera. Genome assemblies for each species were examined to identify the MAT locus and determine the structure of the flanking regions. Large contigs containing the MAT locus were then functionally annotated and analysed for the presence of transposable elements. Genes typically flanking the MAT locus in sordariomycetes were found to be highly conserved in the Ceratocystidaceae. The different genera in the Ceratocystidaceae displayed little synteny outside of the immediate MAT locus flanking genes. Even though species ofCeratocystis did not show much synteny outside of the immediate MAT locus flanking genes, species of Huntiella and Endoconidiophora were comparatively syntenic. Due to the high number of transposable elements present in Ceratocystis MAT flanking regions, we hypothesise that Ceratocystis species may have undergone recombination in this region.     

Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae

Fungi capable of sexual reproduction use heterothallic (self-sterile) or homothallic (self-fertile) mating strategies. In most ascomycetes, a single mating type locus, MAT, with two alternative forms (MAT1-1 and MAT1-2) called idiomorphs, controls mating ability. In heterothallic ascomycetes, these alternative idiomorphs reside in different nuclei. In contrast, most homothallic ascomycetes carry both MAT1-1 and MAT1-2 in a single nucleus, usually closely linked. An example of the latter is Gibberella zeae, a species that is capable of both selfing and outcrossing. G. zeae is a devastating cereal pathogen of ubiquitous geographical distribution, and also a producer of mycotoxins that threaten human and animal health. We asked whether G. zeae could be made strictly heterothallic by manipulation of MAT. Targeted gene replacement was used to differentially delete MAT1-1 or MAT1-2 from a wild-type haploid MAT1-1; MAT1-2 strain, resulting in MAT1-1; mat1-2, mat1-1; MAT1-2 strains that were self-sterile, yet able to cross to wild-type testers and, more importantly, to each other. These results indicated that differential deletion of MAT idiomorphs eliminates selfing ability of G. zeae, but the ability to outcross is retained. They also indicated that both MAT idiomorphs are required for self-fertility. To our knowledge, this is the first report of complete conversion of fungal reproductive strategy from homothallic to heterothallic by targeted manipulation of MAT. Practically, this approach opens the door to simple and efficient procedures for obtaining sexual recombinants of G. zeae that will be useful for genetic analyses of pathogenicity and other traits, such as the ability to produce mycotoxins.     

Mutation of a Heterothallic Strain to Homothallism

Upon mutagenesis, a heterothallic αα diploid strain mutated to homothallism. The gene confering homothallism is nuclear, recessive, and unlinked to mating type. This gene is not allelic to the HO gene, which is responsible for previously described instances of homothallism in yeast. We have designated this new gene for homothallism as cmt (change of mating type).