Isolation and characterization of the mating-type idiomorphs from the wheat septoria leaf blotch fungus Mycosphaerella graminicola

Both mating-type loci from the wheat septoria leaf blotch pathogen Mycosphaerella graminicola have been cloned and sequenced. The MAT1-2 gene was identified by screening a genomic library from the MAT1-2 isolate IPO94269 with a heterologous probe from Tapesia yallundae. The MAT1-2 idiomorph is 2772 bp and contains a single gene encoding a putative high-mobility-group protein of 394 amino acids. The opposite idiomorph was obtained from isolate IPO323, which has the complementary mating type, by long-range PCR using primers derived from sequences flanking the MAT1-2 idiomorph. The MAT1-1 locus is 2839 bp in size and contains a single open reading frame encoding a putative alpha1-domain protein of 297 amino acids. Within the nonidiomorphic sequences, homology was found with palI, encoding a membrane receptor from Aspergillus nidulans, and a gene encoding a putative component of the anaphase-promoting complex from Schizosaccharomyces pombe and a DNA-(apurinic or apyrimidinic) lyase from S. pombe. For each of the MAT genes specific primers were designed and tested on an F1 mapping population that was generated from a cross between IPO323 and IPO94269. An absolute correlation was found between the amplified allele-specific fragments and the mating type as determined by backcrosses of each F1 progeny isolate to the parental isolates. The primers were also used to screen a collection of field isolates in a multiplex PCR. An equal distribution of MAT1-1 and MAT1-2 alleles was found for most geographic origins examined.     

Mating type idiomorphs from a French population of the wheat pathogen Mycosphaerella graminicola: widespread equal distribution and low but distinct levels of molecular polymorphism

Septoria tritici blotch caused by the heterothallic ascomycete Mycosphaerella graminicola is currently the most frequent and the most economically damaging disease on wheat worldwide. Five hundred and ten strains of this fungus were sampled from 16 geographical locations representing the major wheat producing areas in France. Multiplex PCR amplification, PCR-RFLP-SSCP screening and sequencing of parts of mating type encoding sequences were performed in order to assess the distribution and molecular polymorphism of the mating type idiomorphs. The two idiomorphs were scored at similar frequencies within all sampled locations. Both mating types were also identified at the leaf spatial scale, on 42% of leaves from which two or three strains were isolated. No correlation was found between distribution of mating types and either host cultivars from which the sampling was carried out or in vitro colony phenotypes observed during the culture of strains on potato dextrose agar (PDA) medium. PCR-RFLP-SSCP assay highlighted only one MAT1-1 strain exhibiting a profile distinct from all other MAT1-1 strains, whereas ten MAT1-2 strains (among which two and four with same profiles, respectively) showed profiles differing from the other MAT1-2 strains. Sequencing revealed that all polymorphisms corresponded to single nucleotide variations and all strains displaying the same single strand conformation polymorphism (SSCP) profiles showed identical nucleotide sequences, thereby confirming the high sensitivity of SSCP. Only two out of the disclosed nucleotide variations were nonsynonymous. This study strongly suggests a large potential for sexual reproduction in the French population of M. graminicola and reports a high conservation of mating type sequences in the fungus at both nucleotide and population levels, with a great difference in molecular variability between the two idiomorphs.     

A combined amplified fragment length polymorphism and randomly amplified polymorphism DNA genetic kinkage map of Mycosphaerella graminicola,the septoria tritici leaf blotch pathogen of wheat

An F(1) mapping population of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, was generated by crossing the two Dutch field isolates IPO323 and IPO94269. AFLP and RAPD marker data sets were combined to produce a high-density genetic linkage map. The final map contained 223 AFLP and 57 RAPD markers, plus the biological traits mating type and avirulence, in 23 linkage groups spanning 1216 cM. Many AFLPs and some RAPD markers were clustered. When markers were reduced to 1 per cluster, 229 unique positions were mapped, with an average distance of 5.3 cM between markers. Because M. graminicola probably has 17 or 18 chromosomes, at least 5 of the 23 linkage groups probably will need to be combined with others once additional markers are added to the map. This was confirmed by pulsed-field gel analysis; probes derived from 2 of the smallest linkage groups hybridized to two of the largest chromosome-sized bands, revealing a discrepancy between physical and genetic distance. The utility of the map was demonstrated by identifying molecular markers tightly linked to two genes of biological interest, mating type and avirulence. Bulked segregant analysis was used to identify additional molecular markers closely linked to these traits. This is the first genetic linkage map for any species in the genus Mycosphaerella or the family Mycosphaerellaceae.     

Avirulence in the wheat septoria tritici leaf blotch fungus Mycosphaerella graminicola is controlled by a single locus

Segregation of avirulence in Mycosphaerella graminicola, a heterothallic ascomycete that causes wheat septoria tritici leaf blotch, was studied in F1, BC1, and F2 populations by inoculation assays on five wheat cultivars in the seedling stage and by amplified fragment length polymorphism and random amplified polymorphic DNA analyses. F1 was generated by crossing isolates IPO323 (avirulent) and IPO94269 (virulent). All F1, BC1, and F2 progeny isolates were virulent on the susceptible check cultivar Taichung 29 and were avirulent on the resistant check cultivar Kavkav-K4500. Avirulence segregation was observed in F1 and in several BC1 and F2 generations on the differential cultivars Shafir, Kavkaz, and Veranopolis at a 1:1 ratio. Avirulence for the three differential cultivars always cosegregated. We conclude that avirulence in isolate IPO323 is controlled by a single, seemingly complex locus.     

Both mating types of the wheat pathogen Mycosphaerella graminicola are present in Morocco

Septoria tritici blotch caused by the heterothallic ascomycete Mycosphaerella graminicola is one of the most currently damaging diseases on wheat crops worldwide. So far, no information was reported about the status of sexual reproduction of this pathogen under Moroccan conditions. We investigated here for the first time the occurrence of the two mating types (MAT1-1 and MAT1-2) of M. graminicola in Morocco by sampling 141 single-conidial isolates from 4 important wheat producing regions (Gharb, Sa?s, Chaouia and Tadla). The mating type of each isolate was determined by amplification with multiplex PCR of a partial sequence from the corresponding idiomorph. Overall, 43% out of the assessed isolates were MAT1-1 and 57 % were MAT1-2. Both mating types were identified within the 3 sampled regions Gharb, Sa?s and Chaouia, but not in Tadla, where only MAT1-2 isolates were found. The presence of the two mating types highlighted here offers a suitable genetic condition for M. graminicola to occur sexual reproduction in Morocco. The potential of sexual recombination will be examined by the study of mating type frequencies using a large sample size as well as by searching and quantification of pseudothecia in the field.     

A Suppressor of Mating-Type Locus Mutations in SACCHAROMYCES CEREVISIAE: Evidence for and Identification of Cryptic Mating-Type Loci

A mutation has been identified that suppresses the mating and sporulation defects of all mutations in the mating-type loci of S. cerevisiae. This suppressor, sir1-1, restores mating ability to mat alpha 1 and mat alpha 2 mutants and restores sporulation ability to mat alpha 2 and mata1 mutants. MATa sir1-1 strains exhibit a polar budding pattern and have reduced sensitivity to alpha-factor, both properties of a/alpha diploids. Furthermore, sir1-1 allows MATa/MATa, mat alpha 1/mat alpha/, and MAT alpha/MAT alpha strains to sporulate efficiently. All actions of sir1-1 are recessive to SIR1. The ability of sir1-1 to supply all functions necessary for mating and sporulation and its effects in a cells are explained by proposing that sir1-1 allows expression of mating type loci which are ordinarily not expressed. The ability of sir1-1 to suppress the mat alpha 1-5 mutation is dependent on the HMa gene, previously identified as required for switching of mating types from a to alpha. Thus, as predicted by the cassette model, HMa is functionally equivalent to MAT alpha since it supplies functions of MAT alpha. We propose that sir1-1 is defective in a function. Sir ("Silent-information regulator"), whose role may be to regulate expression of HMa and HM alpha.     

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.     

Significant difference in pathogenicity between MAT1-1 and MAT1-2 isolates in the wheat pathogen Mycosphaerella graminicola

Five Mycosphaerella graminicola populations from four geographic regions (Australia, Israel, Switzerland, and the USA) were assayed for neutral RFLP markers and mating type idiomorphs. On average, 25-30 genetically distinct isolates were selected from each population and their pathogenicity was measured on two wheat cultivars in a common garden experiment conducted in a greenhouse. A significant difference in pathogenicity was found between MAT1-1 and MAT1-2 isolates. On average, MAT1-1 isolates had 14-22% greater pathogenicity than MAT1-2 isolates. The pattern of higher pathogenicity in MAT1-1 isolates was consistent across four geographical populations and on two wheat cultivars. A uniform and continuous variation in pathogenicity was found among isolates within each mating type, but no genetic differentiation in selectively neutral RFLP loci was found between mating types, consistent with the hypothesis that differences in pathogenicity were not due to the effects of specific pathogenicity genes or non-random genetic backgrounds.     

MgSlt2, a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth

Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1242-bp open reading frame, and encodes a 414-amino-acid protein. We designated this homolog MgSlt2, generated MgSlt2 knockout strains in M. graminicola isolate IPO323, and found several altered phenotypes in vitro as well as in planta. In yeast glucose broth, MgSlt2 disruptants showed a defective polarized growth in the tip cells upon aging, causing substantial local enlargements culminating in large swollen cells containing two to four nuclei. The MgSlt2 disruptants showed a significantly increased sensitivity to several fungicides, including miconazole (2x), bifonazole (>4x), imazalil (5x), and cyproconazole (10x), and were hypersensitive to glucanase. Unlike the wild type, MgSlt2 disruptants did not produce aerial mycelia and did not melanize on potato dextrose agar. Although cytological analysis in planta showed normal penetration of wheat stomata by the germ tubes of the MgSlt2 disruptants, subsequently formed hyphal filaments frequently were unable to branch out and establish invasive growth resulting in highly reduced virulence, and prevented pycnidia formation. Therefore, we conclude that MgSlt2 is a new pathogenicity factor in M. graminicola.     

江苏省稻瘟病菌有性态的研究*

用标准菌株对１９９７～１９９９年在江苏省吴江市,宜兴市、通州市、高邮市和赣榆县采集的３２５个猪瘟病菌单孢分离菌株的可育性和交配型进行了测定,结果表明江苏省稻瘟病菌菌株的育性较低,可交配率为２２．７７％,可育率仅为７．０８％。不同年份、不同地区采集的猪瘟病菌茵株的性亲和力和交配型有较大的差异,三年的交配率分别为２６．６１％、８．２６％和３３．６４％;温州地区和赣榆地区菌株的交配率相对较高,分别为２６．１５％和２５．４２％,宜兴地区菌株的交配率较低,只有１５．３８％。江苏省稻瘟病菌菌株的交配型在不同年份亦出现很大差别,１９９７年２９个可交配菌株中有２１个菌株表现为ＭＡＴ１－２ 交配型,而１９９９年３６个可交配茵株均为ＭＡＴ１－１交配型。用江苏省稻瘟病菌的可育菌株进行互交,２５个组合中只有６个组合能产生子囊壳和子囊,但均不产生子囊孢子,提示江苏省稻瘟病菌在田间产生健康有性后代的几率不大。对杂交后代的遗传学分析表明,菌株的交配型是受单基因控制的。     

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.     

Evaluation of the potential for sexual reproduction in field populations of Cercospora beticola from USA

Cercospora leaf spot, caused by the hemibiotrophic fungal pathogen Cercospora beticola, is the most economically damaging foliar disease of sugarbeet worldwide. Although most C. beticola populations display characteristics reminiscent of sexual recombination, no teleomorph has been described. To assess whether populations in northern United States have characteristics consistent with sexual reproduction, 1024 isolates collected over a 3-y period were analyzed for frequency and distribution of mating type genes. After clone correction, an approximately equal distribution of mating types was found for each sampling year. Mating type frequency was also assessed in individual lesions. Lesions always consisted of isolates with a single mating type and microsatellite haplotype, but both mating types and up to five microsatellite haplotypes could be found on an individual leaf. The MAT1-1-1 and MAT1-2-1 genes were sequenced from 28 MAT1-1 and 28 MAT1-2 isolates, respectively. Three MAT1-1-1 nucleotide haplotypes were identified that encoded a single amino acid sequence. For MAT1-2-1, five nucleotide haplotypes were identified that encoded four protein variants. MAT1-1-1 and MAT1-2-1 gene expression analyses were conducted on plants inoculated with either or both mating types. MAT1-1-1 expression remained low, but MAT1-2-1 spiked during late stages of colonization. A segment of the MAT1-2-1 coding sequence was also found in MAT1-1 isolates. Taken together, these results suggest that C. beticola has the potential for sexual reproduction.     

Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.     

Frequency and Genetic Diversity of the MAT1 Locus of Histoplasma capsulatum Isolates in Mexico and Brazil

The MAT1-1 and MAT1-2 idiomorphs associated with the MAT1 locus of Histoplasma capsulatum were identified by PCR. A total of 28 fungal isolates, 6 isolates from human clinical samples and 22 isolates from environmental (infected bat and contaminated soil) samples, were studied. Among the 14 isolates from Mexico, 71.4% (95% confidence interval [95% CI], 48.3% to 94.5%) were of the MAT1-2 genotype, whereas 100% of the isolates from Brazil were of the MAT1-1 genotype. Each MAT1 idiomorphic region was sequenced and aligned, using the sequences of the G-217B (+ mating type) and G-186AR (− mating type) strains as references. BLASTn analyses of the MAT1-1 and MAT1-2 sequences studied correlated with their respective + and − mating type genotypes. Trees were generated by the maximum likelihood (ML) method to search for similarity among isolates of each MAT1 idiomorph. All MAT1-1 isolates originated from Brazilian bats formed a well-defined group; three isolates from Mexico, the G-217B strain, and a subgroup encompassing all soil-derived isolates and two clinical isolates from Brazil formed a second group; last, one isolate (EH-696P) from a migratory bat captured in Mexico formed a third group of the MAT1-1 genotype. The MAT1-2 idiomorph formed two groups, one of which included two H. capsulatum isolates from infected bats that were closely related to the G-186AR strain. The other group was formed by two human isolates and six isolates from infected bats. Concatenated ML trees, with internal transcribed spacer 1 (ITS1) -5.8S-ITS2 and MAT1-1 or MAT1-2 sequences, support the relatedness of MAT1-1 or MAT1-2 isolates. H. capsulatum mating types were associated with the geographical origin of the isolates, and all isolates from Brazil correlated with their environmental sources.     

Relation between the efficiency of homothallic switching of yeast mating type genes and the distribution of cell types.

Homothallic switching of yeast mating type genes occurs as often as each cell division, so that a colony derived from a single haploid spore soon contains an equal number of MATa and MAT alpha cells. Cells of opposite mating types conjugate, and eventually the colony contains only nonmating MATa/MAT alpha diploids. Mutations that reduce the efficiency of homothallic MAT conversions yield colonies that still contain many haploid cells of the original spore mating type plus a few recently generated cells of the opposite mating type. These (a greater than alpha)- or (alpha greater than a)-mating colonies also contain some nonmating diploid cells. As an alternative to microscopic pedigree analysis to determine the frequency of mating type conversions in a variety of mutant homothallic strains, we analyzed the proportions of MATa, MAT alpha, and MATa/MAT alpha cells in a colony by examining the mating phenotypes of subclones. We developed a mathematical model that described the proportion of cell types in a slow-switching colony. This model predicted that the proportion of nonmating cells would continually increase with the size (age) of a colony derived from a single cell. This prediction was confirmed by determining the proportion of cell types in colonies of an HO swi1 strain that was grown for different numbers of cell divisions. Data from subcloning (a greater than alpha) and (alpha greater than a) colonies from a variety of slow-switching mutations and chromosomal rearrangements were used to calculate the frequency of MAT conversions in these strains.     

Genomic and population analyses of the mating type loci in Coccidioides species reveal evidence for sexual reproduction and gene acquisition

Coccidioides species, the fungi responsible for the valley fever disease, are known to reproduce asexually through the production of arthroconidia that are the infectious propagules. The possible role of sexual reproduction in the survival and dispersal of these pathogens is unexplored. To determine the potential for mating of Coccidioides, we analyzed genome sequences and identified mating type loci characteristic of heterothallic ascomycetes. Coccidioides strains contain either a MAT1-1 or a MAT1-2 idiomorph, which is 8.1 or 9 kb in length, respectively, the longest reported for any ascomycete species. These idiomorphs contain four or five genes, respectively, more than are present in the MAT loci of most ascomycetes. Along with their cDNA structures, we determined that all genes in the MAT loci are transcribed. Two genes frequently found in common sequences flanking MAT idiomorphs, APN2 and COX13, are within the MAT loci in Coccidioides, but the MAT1-1 and MAT1-2 copies have diverged dramatically from each other. Data indicate that the acquisition of these genes in the MAT loci occurred prior to the separation of Coccidioides from Uncinocarpus reesii. An analysis of 436 Coccidioides isolates from patients and the environment indicates that in both Coccidioides immitis and C. posadasii, there is a 1:1 distribution of MAT loci, as would be expected for sexually reproducing species. In addition, an analysis of isolates obtained from 11 soil samples demonstrated that at three sampling sites, strains of both mating types were present, indicating that compatible strains were in close proximity in the environment.     

Homothallic switching of Saccharomyces cerevisiae mating type genes by using a donor containing a large internal deletion.

Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR. The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching. We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event. Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient. However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.