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Identification of elements in the PDA1 promoter of Nectria haematococca necessary for a high level of transcription in vitro.

Expression of the PDA1 gene in the ascomycete Nectria haematococca MPVI (anamorph: Fusarium solani) is induced by exposure of mycelium to pisatin, an isoflavonoid phytoalexin produced by its host plant, garden pea. The PDA1 gene encodes a cytochrome P-450 monooxygenase which detoxifies pisatin. Regulatory elements controlling transcription from the PDA1 promoter were identified using a homologous Nectria in vitro transcription system through analysis of 5' deletions, specific oligonucleotide competition, and fusion of upstream segments to a heterologous promoter. A promoter-distal element which provided transcriptional activation was localized to a 35-bp region positioned -514 to -483 upstream of the transcriptional start site. This 35-bp region binds a previously characterized pisatin-responsive DNA-binding factor (PRF) and thus may provide pisatin-responsive control of transcription. A second promoter-proximal positive-acting region was found to be necessary for promoter transcription in both homologous and heterologous extracts, and so is likely to bind less genespecific transcription activator(s). A negative-acting element located between these two positive regions may act to make the positive-acting elements interdependent. The identification of an activator responding to pisatin provides a model for the control of a number of genes and processes controlled by host-specific signals, particularly the flavonoids.

Base Sequence↗

Dimorphic behaviour and taxonomy of Trichosporiella sporotrichoides.

Three strains of Trichosporiella sporotrichoides van Oorschot showed divergent reactions to suspension plating. A comparison was made with other yeast-like fungi and some descriptive terms were redefined or introduced. Strain CBS 671.74 developed the same hyphae with lateral conidia as in streak cultures. Strain CBS 577.77 showed initial-stage budding eventually giving rise to hyphae with lateral conidia. CBS 152.76 showed phenotypic segregation with suspension plating and an additional pseudomycelial form in liquid culture. Further differences between the strains were found in carbohydrate composition of cells, ultrastructure of cell walls and in growth patterns on various sugars. Two strains with basidiomycetous affinities are reclassified in the genus Trichosporon. A third strain with ascomycetous affinities is redescribed as Trichosporiella ornithopoda spec. nov.

Culture Media↗

Geosiphon pyriforme, a fungus forming endocytobiosis with Nostoc (cyanobacteria), is an ancestral member of the Glomales: evidence by SSU rRNA analysis.

Geosiphon pyriforme inhabiting the surface of humid soils represents the only known example of endocytobiosis between a fungus (Zygomycotina; macrosymbiont) and cyanobacteria (Nostoc; endosymbiont). In order to elucidate the taxonomical and evolutionary relationship of Geosiphon pyriforme to fungi forming arbuscular mycorrhiza (AM fungi), the small-subunit (SSU) ribosomal RNA genes of Geosiphon pyriforme and Glomus versiforme (Glomales; a typical AM fungus) were analyzed and aligned with SSU rRNA sequences of several Basidiomycetes, Ascomycetes, Chytridiomycetes, and Zygomycetes, together with all AM-fungal (Glomales) sequences published yet. The distinct group of the order Glomales, which includes Geosiphon, does not form a clade with any other group of Zygomycetes. Within the Glomales, two main lineages exist. One includes the families Gigasporaceae and Acaulosporaceae; the other one is represented by the genus Glomus, the members of which are very divergent. Glomus etunicatum and Geosiphon pyriforme both form independent lineages ancestral to the Glomales. The data provided by the present paper confirm clearly that Geosiphon represents a fungus belonging to the Glomales. The question remains still open as to whether or not Geosiphon is to be placed within or outside the genus Glomus, since this genus is probably polyphyletic and not well defined yet. Geosiphon shows the ability of a Glomus-like fungus to form a "primitive" symbiosis with a unicellular photoautotrophic organism, in this case a cyanobacterium, leading to the conclusion that a hypothetical association of a Glomus-like fungus with a green alga as a step during the evolution of the land plants appears probable.

Base Sequence↗

A small insertion in the SSU rDNA of the lichen fungus Arthonia lapidicola is a degenerate group-I intron.

Insertions of less than 100 nt occurring in highly conserved regions of the small subunit ribosomal DNA (SSU rDNA) may represent degenerate forms of the group-I introns observed at the same positions in other organisms. A 63-nt insertion at SSU rDNA position 1512 (relative to the Escherichia coli SSU rDNA) of the lichen-forming fungus Arthonia lapidicola can be folded into a secondary structure with two stem loops and a pairing of the insertion and flanking sequences. The two stem loops may correspond to the P1 and P2, and the insertion-flanking pairing to the P10, of a group-I intron. Considering these small insertions as degenerate introns provides important clues to the evolution and catalytic function of group-I introns. Keywords Ribosomal DNA middle dot Small subunit middle dot 18s middle dot Degenerate introns middle dot Ascomycetes

Ascomycota↗

Yeast species utilizing uric acid, adenine, n-alkylamines or diamines as sole source of carbon and energy.

Yeast strains utilizing uric acid, adenine, monoamines or diamines as sole source of carbon and energy were isolated from several soil samples by the enrichment culture method. The most common species was Trichosporon cutaneum. Strains of Candida catenulata, C. famata, C. parapsilosis, C. rugosa, Cryptococcus laurentii, Stephanoascus ciferrii and Tr. adeninovorans were also isolated. All strains utilizing uric acid as sole carbon source utilized some primary n-alkyl-l-amines, hydroxyamines or diamines as well. The ascomycetous yeast strains showing these characteristics all belonged to species known to assimilate hydrocarbons. Type strains of hydrocarbon-positive yeast species which were not found in the enrichment cultures generally assimilated putrescine, some type strains also butylamine or pentylamine, but none assimilated uric acid. Methanol-positive species were not isolated. Type strains of methanol-positive and of hydrocarbon-negative species did not assimilate uric acid, butylamine or putrescine. Assimilation of putrescine as sole source of carbon and energy may be a valuable diagnostic criterion in yeast taxonomy.

Adenine↗

Trichosporon terrestre sp. nov.

A single strain representative of an undescribed Trichosporon species, was recovered from soil. The cell wall of the strain corresponds in structure to cell walls of the ascomycetous yeasts. On the ground of X-ray inactivation data the strain is assumed to be diploid. A description of the new species is given.

Ascomycota↗

Genetic and molecular characterisation of purine permease genes of Aspergillus nidulans reveals a novel family of transporters conserved in prokaryotes and eukaryotes.

The ascomycete fungus Aspergillus nidulans can utilize purines (adenine, guanine, hypoxanthine, xanthine, and uric acid) as sole nitrogen sources [1]. The expression of most structural genes involved in the pathway of purine uptake and catabolism is subject to uric acid induction, mediated by the product of the positive regulatory gene uaY, and to nitrogen metabolite repression, mediated by the product of the general, positive-acting, GATA-like transcription factor, encoded by the areA gene [1].

Aspergillus nidulans↗

Effect of rice--glycerol complex medium on the production of Lovastatin by Monascus ruber.

Response surface methodology (RSM) was employed to study the effect of the composition of the rice-glycerol complex medium on the production of lovastatin (Lvs) by the ascomycete Monascus ruber in mixed solid-liquid (or submerged) cultures at 25 degrees C. Four components (rice powder, peptone, glycerol, glucose) were studied to evaluate the approximate polynomial for all dependent variables, explaining their effects on the production of Lvs. The best composition derived from RSM regression was (in g/L) rice powder 34.4, peptone 10.8,, glucose 129, KNO3 8.0, MgSO4.7H2O 4.0 and glycerol 36.4 mL/L. With this composition, the Lvs production was 157 mg/L after 10 d of cultivation. In comparison with glycerol and glucose, the rice powder becomes a more suitable carbon source and represents a great potential for the production of Lvs.

Glucose↗

Activity of the AIB factor observed in prokaryotic and eukaryotic microorganisms.

Streptomyces cinnamonensis produces a new substance named AIB (for anti-isobutyrate) factor which, on a solid medium, efficiently counteracts toxic concentrations not only of isobutyrate but also of other salts of short-chain monocarboxylic acids. In the present study we demonstrate that the AIB factor activity is widely spread because this effect was positively detected in 25 of 31 randomly chosen microorganisms (streptomycetes, ascomycetes, zygomycetes and basidiomycetes). The AIB factor produced by the tested microorganisms on an agar media allows for germination, growth, and sporulation of the testing Streptomyces coelicolor on an agar medium containing 20 mmol/L acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and 2-methylbutyrate. The activity of the AIB factor from different sources towards these substances differs.

Ascomycota↗

Production of beta-glucosidase, exo-beta-1,4-glucanase and endo-beta-1,4-glucanase by selected microscopic fungi.

Production of beta-glucosidase, exo-beta-1,4-glucanase and endo-beta-1,4-glucanase was screened in 58 species of imperfect fungi, mucoral fungi and some ascomycetes. beta-Glucosidase activity was found in all of the tested microorganisms, exo-beta-1,4-glucanase activity in 23, and endo-beta-1,4=glucanase activity in 38 microorganisms. Growth on cellobiose was found in all tested microorganisms, growth on carboxymethylcellulose in in 38 tested strains.

Ascomycota↗

Production of alpha-amylase by microscopic fungi.

The amylolytic activity and especially the production of alpha-amylase (EC 3.2.1.1) and alpha-glucosidase (EC 3.2.1.20) was screened in imperfect fungi, mucoral fungi and some ascomycetes. The character of the polysaccharide system, which is responsible for the utilization of alpha (1 to 4) glucan, was specified with a concomitant screening of growth on soluble starch. The amylolytic activity was found in 29 strains out of the 49 tested.

Amylases↗

Secretion of acid phosphatase in Claviceps purpurea--an ultracytochemical study.

The lead phosphate precipitation method showed the reaction product of acid phosphatase (which reflects the presence of the enzyme glycoprotein) in peripheral cytoplasmic vesicles in the ascomycetous fungus Claviceps purpurea. The product appeared to diffuse from these vesicles (diameter 100-200 nm) towards the cell wall, usually to its sites covered by the capsular fibres exhibiting also acid phosphatase activity. This observation of diffusion of secretory glycoprotein in the cytoplasmic matrix and its orientation to the plasmalemma and capsular fibrils suggests an alternative to the well-described secretory mechanism of transport and exocytosis of glycoproteins via membrane-bound transport conveyors fusing with the cell membrane. It confirms and enlarges our previous finding of the reaction product of acid phosphatase performed by ultrastructural cytochemistry in vacuoles (lysosomes), in the growing cell septum, in cytoplasmic vesicles and in the fibres of the external capsule.

Acid Phosphatase↗

Effect of Chalara longipes on decomposition of humic acids from Picea abies needle litter.

The effect of the saprotrophic ascomycete Chalara longipes on the decomposition of humic acids was determined in a cultivation experiment. The fungus was incubated in liquid cultures in a full-strength (F system) and an organic nitrogen-free medium (F-N system), both amended with pure humic acids isolated from spruce forest litter. Fungal biomass production was highest in the F system with humic acids and lowest in the F-N system, the effect of organic nitrogen and humic acids being significant (p < 0.05). The presence of organic nitrogen seems to be essential for growth. The fungus utilized humic acids; molar mass distribution in media obtained by gel-permeation chromatography showed decrease in the humic acids fraction. Decolorization of the media reached 75 and 64 % in the F and F-N systems, respectively. The molar mass of humic acids probably also decreased as estimated from the increase in the absorbance A465/A665 (A4/A6) ratio. It is assumed that the utilization of humic acids may be mediated by the production of organic acids (as their components) and production of some oxidative enzymes.

Ascomycota↗

A group I intron in the 18S ribosomal DNA from the parasitic fungus Isaria japonica.

The nucleotide sequence of the 18S rDNA coding gene in the ascomycetes parasitic fungus Isaria japonica contains a group I intron with a length of 379 nucleotides. The identification of the DNA sequence as a group I intron is based on its sequence homology to other fungal group I introns. Its group I intron contained the highly conserved sequence elements P, Q, R, and S found in other group I introns. Surprisingly, the intron sequence of I. japonica is more similar to that of Ustilago maydis than to the one found in Sclerotinia sclerotiorum. This is in contrast to the sequence identity found on the neighboring rDNA. This is an interesting finding and suggests a horizontal transfer of group I intron sequences.

Ascomycota↗

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.

Alleles↗

Transcription of a nitrate reductase gene isolated from the symbiotic basidiomycete fungus Hebeloma cylindrosporum does not require induction by nitrate.

Ectomycorrhizal fungi contribute to the nitrogen nutrition of their host plants, but no information is available on the molecular control of their nitrogen metabolism. The cloning and pattern of transcriptional regulation of two nitrite reductase genes of the symbiotic basidiomycete Hebeloma cylindrosporum are presented. The genomic copy of one of these genes (nar1) was entirely sequenced; the coding region is interrupted by 12 introns. The nar1 gene, which is transcribed and codes for a putative 908-amino acid polypeptide complemented nitrate reductase-deficient mutants of H. cylindrosporum upon transformation, thus demonstrating that the gene is functional. The second gene (nar2), for which no mRNA transcripts were detected, is considered to be an ancestral, non-functional duplication of nar1. In a 462-nt partial sequence of nar2 two introns were identified at positions identical to those of introns 8 and 9 of nar1, although their respective nucleotide sequences were highly divergent; the exon sequences were much more conserved. In wild-type strains, transcription of nar1 is repressed in the presence of a high concentration of ammonium. High levels of transcription are observed in the presence of either very low nitrogen concentrations or high concentrations of nitrate or organic N sources such as urea, glycine or serine. This indicates that in H. cylindrosporum, in contrast to all nitrophilous organisms studied so far, an exogenous supply of nitrate is not required to induce transcription of a nitrate reductase gene. In contrast, repression by ammonium suggests the existence of a wide-domain regulatory gene, as already characterized in ascomycete species.

Agaricales↗

Molecular mapping of Stb1, a potentially durable gene for resistance to septoria tritici blotch in wheat.

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola (anamorph Septoria tritici), was the most destructive disease of wheat in Indiana and adjacent states before deployment of the resistance gene Stb1 during the early 1970s. Since then, Stb1 has provided durable protection against STB in widely grown wheat cultivars. However, its chromosomal location and allelic relationships to most other STB genes are not known, so the molecular mapping of Stb1 is of great interest. Genetic analyses and molecular mapping were performed for two mapping populations. A total of 148 F1 plants (mapping population I) were derived from a three-way cross between the resistant line P881072-75-1 and the susceptible lines P881072-75-2 and Monon, and 106 F6 recombinant-inbred lines (mapping population II) were developed from a cross between the resistant line 72626E2-12-9-1 and the susceptible cultivar Arthur. Bulked-segregant analysis with random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and microsatellite or simple-sequence repeat (SSR) markers was conducted to identify those that were putatively linked to the Stb1 gene. Segregation analyses confirmed that a single dominant gene controls the resistance to M. graminicola in each mapping population. Two RAPD markers, G7(1200) and H19(520), were tightly linked to Stb1 in wheat line P881072-75-1 at distances of less than 0.68 cM and 1.4 cM, respectively. In mapping population II, the most closely linked marker was SSR Xbarc74, which was 2.8 cM proximal to Stb1 on chromosome 5BL. Microsatellite loci Xgwm335 and Xgwm213 also were proximal to Stb1 at distances of 7.4 cM and 8.3 cM, respectively. The flanking AFLP marker, EcoRI-AGC/ MseI-CTA-1, was 8.4 cM distal to Stb1. The two RAPD markers, G7(1200) and H19(520), and AFLP EcoRI-AGC/ MseI-CTA-1, were cloned and sequenced for conversion into sequence-characterized amplified region (SCAR) markers. Only RAPD allele H19(520) could be converted successfully, and none of the SCAR markers was diagnostic for the Stb1 locus. Analysis of SSR and the original RAPD primers on several 5BL deletion stocks positioned the Stb1 locus in the region delineated by chromosome breakpoints at fraction lengths 0.59 and 0.75. The molecular markers tightly linked to Stb1 could be useful for marker-assisted selection and for pyramiding of Stb1 with other genes for resistance to M. graminicola in wheat.

Ascomycota↗