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PubMed2006

A MADS box protein interacts with a mating-type protein and is required for fruiting body development in the homothallic ascomycete Sordaria macrospora.

MADS box transcription factors control diverse developmental processes in plants, metazoans, and fungi. To analyze the involvement of MADS box proteins in fruiting body development of filamentous ascomycetes, we isolated the mcm1 gene from the homothallic ascomycete Sordaria macrospora, which encodes a putative homologue of the Saccharomyces cerevisiae MADS box protein Mcm1p. Deletion of the S. macrospora mcm1 gene resulted in reduced biomass, increased hyphal branching, and reduced hyphal compartment length during vegetative growth. Furthermore, the S. macrospora Deltamcm1 strain was unable to produce fruiting bodies or ascospores during sexual development. A yeast two-hybrid analysis in conjugation with in vitro analyses demonstrated that the S. macrospora MCM1 protein can interact with the putative transcription factor SMTA-1, encoded by the S. macrospora mating-type locus. These results suggest that the S. macrospora MCM1 protein is involved in the transcriptional regulation of mating-type-specific genes as well as in fruiting body development.

Amino Acid Sequence↗

PubMed2001

Characterization of an opsin gene from the ascomycete Leptosphaeria maculans.

An opsin gene (ops) has been characterized from Leptosphaeria maculans, the ascomycete that causes black-leg disease of Brassica species. This is the second opsin identified outside the archaeal and animal kingdoms. The gene encodes a predicted protein with high similarity (70.3%) and identity (53.3%) to the nop-1 opsin of another ascomycete Neurospora crassa. The L. maculans opsin also has identical amino acid residues in 20 of the 22 residues in the retinal-binding pocket of archaeal opsins. Opsin, on the fourth largest chromosome of L. maculans and 22 cM from the mating type locus, is the first cloned gene to be mapped in L. maculans. Opsin is transcribed at high levels in mycelia grown in the presence and absence of light; this pattern is in contrast with that of the N. crassa opsin, which is transcribed only in the light.

Amino Acid Sequence↗

PubMed2002

Comparative degradation of oomycete, ascomycete, and basidiomycete cell walls by mycoparasitic and biocontrol fungi.

Fourteen fungi (primarily representing mycoparasitic and biocontrol fungi) were tested for their ability to grow on and degrade cell walls (CWs) of an oomycete (Pythium ultimum), ascomycete (Fusarium equisetii), and basidiomycete (Rhizoctonia solani), and their hydrolytic enzymes were characterized. Protein was detected in the cultural medium of eleven of the test isolates, and these fungi significantly degraded CWs over the 14-day duration of the experiment. In general, a greater level of CW degradation occurred for F. equisetii and P. ultimum than for R. solani. Fungi that degraded F. equisetii CWs were Coniothyrium minitans, Gliocladium roseum, Myrothecium verrucaria, Talaromyces flavus, and Trichoderma harzianum. Taxa degrading P ultimum CWs included Chaetomium globosum, Coniothyrium minitans, M. verrucaria, Seimatosporium sp., Talaromyces flavus, Trichoderma hamatum, Trichoderma harzianum, and Trichoderma viride. Production of extracellular protein was highly correlated with CW degradation. Considerable variation in the molecular weights of CW-degrading enzymes were detected among the test fungi and the CW substrates in zymogram electrophoresis. Multivariate analysis between CW degradation and hydrolysis of barley beta-glucan (beta1,3- and beta1,4-glucanases), laminarin (beta1,3- and beta1,6-glucanases), carboxymethyl cellulose (endo-beta1,4-glucanases), colloidal chitin (chitinases), and chitosan (chitosanases) was conducted. For F. equisetii CWs, the regression model accounted for 80% of the variability, and carboxymethyl cellulases acting together with beta-glucanases contributed an R2 of 0.52, whereas chitinases and beta-glucanases alone contributed an R2 of 0.11 and 0.12, respectively. Only 61% of the variability observed in the degradation of P. ultimum CWs was explained by the enzyme classes tested, and primarily beta-glucanases (R2 of 0.53) and carboxymethyl cellulases (R2 of 0.08) alone contributed to CW break down. Too few of the test fungi degraded R. solani CWs to perform multivariate analysis effectively. This study identified several fungi that degraded ascomyceteous and oomyceteous, and to a lesser extent, basidiomycetous CWs. An array of enzymes were implicated in CW degradation.

Carbohydrate Metabolism↗

PubMed2001

A lectin from an ascomycete mushroom, Melastiza chateri: no synthesis of the lectin in mycelial isolate.

Using an affinity adsorbent prepared from L-fucose and starch, a lectin was isolated from fruit bodies of an ascomycete mushroom, Melastiza chateri. The lectin was found to cross-react with antiserum against Aleuria aurantia lectin (AAL), that had been obtained from another ascomycete mushroom. The N-terminal amino acid sequence was analyzed, and among 20 residues 12 were the same as AAL. The molecular mass of the lectin estimated by SDS-PAGE was approximately 40 kDa, which is larger than that of AAL. Mycelial isolate was obtained from M. chateri by germinating ascospores, and identified by analyzing restriction fragment length polymorphisms (RFLP) of DNA. The isolate from M. chateri did not synthesize the lectin, although the isolate from A. aurantia had been known to synthesize AAL as much as the fruit body.

Amino Acid Sequence↗

PubMed2004

Conservation and evolution of cis-regulatory systems in ascomycete fungi.

Relatively little is known about the mechanisms through which gene expression regulation evolves. To investigate this, we systematically explored the conservation of regulatory networks in fungi by examining the cis-regulatory elements that govern the expression of coregulated genes. We first identified groups of coregulated Saccharomyces cerevisiae genes enriched for genes with known upstream or downstream cis-regulatory sequences. Reasoning that many of these gene groups are coregulated in related species as well, we performed similar analyses on orthologs of coregulated S. cerevisiae genes in 13 other ascomycete species. We find that many species-specific gene groups are enriched for the same flanking regulatory sequences as those found in the orthologous gene groups fromS. cerevisiae, indicating that those regulatory systems have been conserved in multiple ascomycete species. In addition to these clear cases of regulatory conservation, we find examples of cis-element evolution that suggest multiple modes of regulatory diversification, including alterations in transcription factor-binding specificity, incorporation of new gene targets into an existing regulatory system, and cooption of regulatory systems to control a different set of genes. We investigated one example in greater detail by measuring the in vitro activity of the S. cerevisiae transcription factor Rpn4p and its orthologs from Candida albicans and Neurospora crassa. Our results suggest that the DNA binding specificity of these proteins has coevolved with the sequences found upstream of the Rpn4p target genes and suggest that Rpn4p has a different function in N. crassa.

Amino Acid Motifs↗

PubMedDate not supplied

Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes.

The green fluorescent protein (GFP) has been established as the premier in vivo reporter for investigations of gene expression, protein localization, and cell and organism dynamics. The fungal transformation vector pCT74, with sGFP under the control of the ToxA promoter from Pyrenophora tritici-repentis, effectively expresses GFP in a diverse group of filamentous ascomycetes. Due to the versatility of ToxA promoter-driven expression of GFP, we constructed an additional set of fluorescent protein expression vectors to expand the color palette of fluorescent markers for use in filamentous fungi. EYFP, ECFP and mRFP1 were successfully expressed from the ToxA promoter in its fungus of origin, P. tritici-repentis, and a distant relative, Verticillium dahliae. Additionally the ToxB promoter from P. tritici-repentis drove expression of sGFP in V. dahliae, suggesting a similar potential to the ToxA promoter for heterologous expression in ascomycetes. The suite of fungal transformation vectors presented here promise to be useful for a variety of fungal research applications.

Ascomycota↗

PubMed2000

Three new ascomycetous yeasts from insect-associated arboreal habitats.

A new species of Pichia and two new species of Candida are described and were determined to be genetically isolated from all other currently recognized ascomycetous yeasts from their sequence divergence in the species-variable D1/D2 domain of large subunit (26S) ribosomal DNA. The three species were primarily isolated from the frass of wood-boring insects living in pine and spruce trees. The new species and their type strains are the following: Pichia ramenticola NRRL YB-1985 (CBS 8699), mating type alpha (NRRL YB-3835, CBS 8700, mating type a), Candida piceae NRRL YB-2107 (CBS 8701), and Candida wyomingensis NRRL YB-2152 (CBS 8703). Pichia ramenticola and C. piceae assimilate methanol as a carbon source; P. ramenticola is the first known heterothallic ascomycetous yeast to utilize this compound.

Animals↗

PubMed2001

Aliquandostipitaceae, a new family for two new tropical ascomycetes with unusually wide hyphae and dimorphic ascomata.

In two short surveys of lignicolous, fruitbody-forming ascomycetes in Thailand and southern China, six species were found, of which five were new to science. Two fungi with affinity to the Dothideomycetes, one from Thailand and one from China, are described here in the new genus ALIQUANDOSTIPITE: and included in the new family Aliquandostipitaceae. Aliquandostipite khaoyaiensis was found in a tropical rain forest in Thailand and A. sunyatsenii in a small stream in southern China. Both new species are closely related based on morphological and molecular characteristics and with uncertain affinity to other taxa of the Euascomycetes based on phylogenetic analyses of SSU rDNA sequences. The distinguishing features of the new species are the presence of both sessile and stalked ascomata side by side on the substratum and the widest hyphae known from ascomycetes.

Journal Article↗

PubMed2002

Vegetative incompatibility in the ascomycete Rosellinia necatrix studied by fluorescence microscopy.

We describe our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous ascomycetes, Rosellinia necatrix, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei. Hyphal anastomosis between incompatible strains, which were field and single ascospore isolates, were observed with cell death showing fused hyphae, and nuclei debris which were intensified by staining with ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide but were intensified by staining with acridine orange. A strain was found which did not form a barrier reaction, but which could be shown to undergo cell death and therefore showed a positive vegetative incompatibility reaction. We also examined the vegetative incompatibility among five single ascospore isolates and the putative parent strain from the same perithecium; all strains were incompatible. These results strongly suggest that vegetative incompatibility in R. necatrix is regulated by many loci.

Acridine Orange↗

PubMed2005

Absolute stereochemistry of unusual biopolymers from Ascomycete culture LL-W1278: examples that derivatives of (S)-6-hydroxymellein are also natural fungal metabolites.

The new linear polyesters, W1278-A, B, and C, were isolated from culture extracts of a freshwater Ascomycete fungus. Their structures were elucidated on the basis of spectroscopic analyses and chemical transformations, with the absolute configuration established by the circular dichroism (CD) method using (R)-(-)-6-hydroxymellein as reference. Unexpectedly, W1278-A, B, and C contain (S)-6-hydroxymellein residues as concluded from the display of the opposite ellipticity than that of known (3R)-(-)-3,4-dihydro-6,8-dihydroxy-3-methylisocoumarin as revealed by CD.

Ascomycota↗

PubMed1983

[Protoplast liberation and regeneration in the ascomycete Hypomyces ochraceus (Pers.) Tul].

A rapid and convenient method for producing protoplasts from 3 d old mycelium of the ascomycete Hypomyces ochraceus is described. The procedure involves a Helix pomatia enzyme preparation and sucrose (20%) for stabilization. Pretreatment with disulfide bond reducing agents reduced the amount of viable protoplasts. Formation of protoplasts and different stages of regeneration were observed by phase contrast microscopy. There was only one type of true regeneration from protoplasts to hyphae in 15-30% gelatine medium by direct forming a germ tube from the original protoplast. Cytological events and physiological conditions are discussed.

Animals↗

PubMed1997

Multicellular ascomycetous fungal genomes contain more than 8000 genes.

Fungi comprise a large monophyletic group of uni- and multicellular eukaryotic organisms in which many species are of economic or medical importance. Fungal genomes are variable in size (13-42 Mb), and multicellular species support true spatial and temporal cell-type-specific regulation of gene expression. In a 38.8-kb Aspergillus nidulans contiguous genomic DNA region, a transposable element and 12 potential genes were identified, 7 similar to genes in other organisms. This observation is consistent with the prediction that multicellular ascomycetous fungi harbor 8000-9000 genes in a 36-Mb average genome. Thus, the genomic DNA sequence of filamentous fungi will provide substantial amounts of genetic and functional information that is not available in yeast, for the human and other metazoan minimal gene complement.

Animals↗

PubMed1999

Pyruvate decarboxylase filaments are associated with the cortical cytoskeleton of asci and spores over the sexual cycle of filamentous ascomycetes.

We show that pyruvate decarboxylase (PDC) 8- to 10-nm-diameter filaments, first described in vegetative cells of Neurospora crassa, are ubiquitously present in filamentous fungi. The cellular arrangement of these structures was examined over the entire sexual cycle of the ascomycetes N. crassa, N. tetraesperma, Podospora anserina, and Sordaria macrospora. PDC-filaments were found associated with the cortical microtubule array of asci and ascospores and absent from the vicinity of spindles and spindle pole bodies. Nocodazole-induced depolymerization of the cortical microtubules results in the loss of PDC-filaments. Moreover, a S. macrospora mutant defective in cortical MT distribution shows abnormal PDC organization. Neurospora asci generated on various metabolic conditions, which modify the presence and relative abundance of PDC-filaments in vegetative cells, have identical patterns of subcellular distribution of these structures. A N. crassa mutant (snowflake) that accumulates giant bundles of PDC-filaments during vegetative growth, shows normal distribution of the filaments during ascogenesis. Thus, the regulation conditioning the presence and supramolecular assembly of PDC-filaments in Neurospora differs between vegetative and sexual cells. Taken together, these results suggest that PDC in filamentous fungi may perform two functions, intervening as an enzyme in vegetative metabolism and as a structural protein associated with the cytoskeleton during sexual development.

Actins↗

PubMed1999

Phylogenetic analysis of a dataset of fungal 5.8S rDNA sequences shows that highly divergent copies of internal transcribed spacers reported from Scutellospora castanea are of ascomycete origin.

Using a dataset comprising 5.8S rDNA sequences from a wide range of fungi, we show that some sequences reported recently from the arbuscular mycorrhizal (AM) fungus Scutellospora castanea most likely originate from Ascomycetes. Other ITS and 5.8S sequences which were previously reported are confirmed as being clearly of mycorrhizal origin and are variable within one isolate of S. castanea. However, these results mean that previous conclusions which were drawn regarding the heterokaryotic status of AM fungal spores remain unproven. We provide an enlarged 5.8S rDNA dataset that can be used to check ITS sequences for conflicts with well-established phylogenies of the organisms that they were obtained from.

Amino Acid Sequence↗

PubMed2000

Interfacial self-assembly of fungal hydrophobins of the lichen-forming ascomycetes Xanthoria parietina and X. ectaneoides.

In the symbiotic phenotype of the lichen-forming ascomycetes Xanthoria parietina and X. ectaneoides, a conglutinate, hydrophilic cortex surrounds a system of aerial hyphae with hydrophobic wall surfaces. In X. parietina freeze-fracture electron microscopy showed that a rodlet layer covers the fungal and algal wall surfaces. Extracts of hot SDS-insoluble wall residues isolated from both species contained a protein that revealed a rodlet layer upon interfacial self-assembly. The N-terminal sequence of the 10-kDa protein of X. ectaneoides served to clone cDNA fragments of XEH1 (H1 of X. ectaneoides) and XPH1 (H1 of X. parietina) by RT-PCR. Genomic DNA blot analysis with both lichenized species and the aposymbiotically cultured symbionts of X. parietina showed that XPH1 and XEH1 are fungal single copy genes. The deduced amino acid sequences of the two encoded proteins were 96% identical and showed the characteristics of class I hydrophobins.

Amino Acid Sequence↗

PubMed1991

Integrative transformation of the ascomycete Podospora anserina: identification of the mating-type locus on chromosome VII of electrophoretically separated chromosomes.

Protoplasts of wild-type strain s and a long-lived extrachromosomal mutant (AL2) of the ascomycete Podospora anserina were transformed using a plasmid (pAN7-1) which contains the hygromycin B phosphotransferase gene (hph) of Escherichia coli under the control of Aspergillus nidulans regulatory sequences. After optimizing the transformation procedure, transformation efficiencies of 15-21 transformants/micrograms plasmid DNA were obtained. Using a second selectable vector (pBT3), which contains the beta-tubuline gene of a benomyl-resistant Neurospora crassa mutant, the co-transformation rate was determined. Southern blot hybridization experiments revealed that the transforming plasmid became integrated into the genome of the recipient either as a single copy or as multiple copies. In addition, the data from molecular as well as from classical genetic analyses indicated that in independent transformants vector integration occurred at different positions. The mitotic and meiotic stability of transformants proved to be dependent on the number of integrated plasmid copies. Genetic analyses revealed a transformant in which the integrated vector is closely linked to the mating-type locus. Fractionation of whole chromosomes by pulsed field gel electrophoresis and subsequent hybridization of the immobilized DNAs against radiolabelled vector sequences indicated the largest of seven chromosomes as the chromosome containing the integrated vector and thus the mating-type locus.

Ascomycota↗

PubMed1979

Chromosomal and extrachromosomal control of senescence in the ascomycete Podospora anserina.

In Podospora anserina senescence leading to cellular death occurs regularly after prolonged vegetative propagation. However, the life span of this ascomycete may be extended by various means: 1. Mutations in a least 8 morphogenetic genes belonging to 4 linkage groups postpone drastically or even prevent in certain pairwise combinations (e.g. i viv) the onset of senescence. 2. Inhibitors of mt DNA and of mitochondrial protein synthesis show a life prolonging effect when added in low concentrations to the growth medium. 3. A similar effect was found when mycelia were fed exclusively on non repressive carbon sources. Whereas the anti-aging effect of specific mutated genes is rather permanent, the life prolonging action of the inhibitors and carbon sources is restricted and temporary. These substances have no long lasting effect, since after their removal from the medium aging proceeds. Physiological experiments have further shown the existence of three phases in the life span of Podospora anserina. During the juvenile phase aging is prevented by all of these compounds; during the presenescent phase aging is prevented by inhibitors of mt DNA only, and during the senescent phase aging is irreversible. Senescence may be induced in juvenile protoplasts by DNA extracted from senescent mycelia. This, together with the well known fact that senescence is extrachromosomically inherited, points to extrachromosomal DNA as the causative agent of senescence. This kind of DNA may be connected with or perhaps located in the mitochondria. Collectively, the data are consistent in showing that the syndrome of senescence in Podospora anserina is controlled by a chromosomal-extrachromosomal interaction. In this system, extrachromosomal DNA, perhaps a mt DNA, is identical with the infectious principle initiating the decay of the cell, and nuclear genes supervise its expression.

Aging↗

PubMed1995

Inheritance of chromosomal length polymorphisms in the ascomycete Leptosphaeria maculans.

Pulsed field gel electrophoresis experiments show that chromosomal length polymorphisms are produced during meiosis in the ascomycetous plant pathogen Leptosphaeria maculans. Homologues in tetrads of L. maculans were identified on the basis of their binding to chromosome-specific probes that included beta-tubulin, nitrate reductase, 18S ribosomal DNA and two Random Amplified Polymorphic DNA (RAPD) markers. Changes in size of homologues were followed during meiosis. Significant karyotype variation was evident due to the random assortment of parental homologues of different sizes. In most cases, the progeny had the same-sized homologues as the parents; however, in some instances novel-sized homologues were detected that varied in size from those of the parents by up to 50 kb. Our results are consistent with the hypothesis that these novel chromosomal length polymorphisms are produced by reciprocal recombination between parental homologous chromosomes of unequal sizes.

Ascomycota↗