Properties of a hydrophobin isolated from the mycoparasitic fungus Verticillium fungicola

Verticillium fungicola, isolated from Agaricus bisporus (commercial mushroom), produced significant extracellular hydrophobin when grown for 7 days in a static liquid culture of synthetic minimal medium. The hydrophobin was purified by precipitation with ammonium sulphate (80% saturation), Sephadex G-100 gel filtration, and hydroxyapatite column chromatography. The purified protein yielded a single band in polyacrylamide gel electrophoresis under native conditions, with an apparent molecular mass of 70 +/- 4 kDa, and also another single band in SDS-PAGE, with a molecular mass of 7 +/- 3 kDa. Molecular mass determined with matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) resulted in 7563.9 m/z. The same protein was extracted from the V. fungicola mycelium. Analysis of the amino acid composition revealed the presence of about 50% hydrophobic residues, detecting at least six cysteines, evaluated as cystines, and no free sulfhydryl groups. The protein did not show any glycosylation. On the basis of similarities in hydropathy patterns and solubility characteristics, V. fungicola hydrophobin can be included as a new member of Class II hydrophobins.     

Chemical components and their locations in the Verticillium fungicola cell wall

The chemical structure of cell walls and fractions of Verticillium fungicola, a pathogen of Agaricus bisporus, as well as their corresponding ultrastructures were studied. There are at least three chemically distinct types of carbohydrate polymers: one yielding mannose with lower amounts of galactose and glucose (glucogalactomannan), another one composed mainly of glucose (glucan), and a third one containing only N-acetylglucosamine (chitin). Attempts were made to locate these materials in situ by comparing electron micrographs of shadowed and sectioned cell walls, and also by indirect immunofluorescence. It was shown that none of these polymers constituted a completely physically distinct layer, but there seem to be different solubility properties in the outer, inner, and intermediate layers. It was also shown that fibrillar material (chitin) embedded in cementing glucan constituted the residual inner fraction of the original wall material. Indirect immunofluorescence showed the location of a significant amount of glucogalactomannan on the surface of the walls in which rodlet structures were visualized by electron microscopy.     

Investigating the role of a Verticillium fungicola beta-1,6-glucanase during infection of Agaricus bisporus using targeted gene disruption

Studies on the mycopathogen Verticillium fungicola have shown the up-regulation of beta-1,6-glucanases when grown in the presence of host cell walls and host cell wall components including chitin. These cell-wall-degrading enzymes are hypothesized to contribute to the pathogenic ability of mycopathogens. A beta-1,6-glucanase gene, VfGlu1, showing high similarity to beta-1,6-glucanase genes from Hypocrea virens, Neotyphodium sp., and Trichoderma harzianum, was isolated using degenerate PCR from V. fungicola, a serious mycopathogen of the cultivated mushroom Agaricus bisporus. Agrobacterium-mediated transformation of V. fungicola using homologous DNA from VfGlu1 resulted in homologous integration at the VfGlu1 locus in 75% of transformants, generating mutants disrupted in the VfGlu1 gene. VfGlu1 mutants displayed reduced virulence and diminished ability to utilize chitin as a carbon source, implicating VfGlu1 in the disease process. Agrobacterium-mediated transformation affords an efficient technique for the disruption of genes associated with disease symptom development in the complex V. fungicola-A. bisporus interaction.     

Comparison of partial sequence of the cap binding protein (eIF4E) isolated from Agaricus bisporus and its pathogen Verticillium fungicola

The 3' regions of the gene encoding the cap binding protein eIF4E were successfully isolated from Agaricus bisporus and Verticillium fungicola using a degenerate primer within the eIF4E gene and an anchored oligo d(T) primer. The deduced amino acid sequences contained 173 residues for A. bisporus and 171 residues V. fungicola. Analysis of these sequences shows that despite conserved regions of homology, centering around tryptophan residues, A. bisporus and V. fungicola are very diverse at the amino acid and DNA level. Percentage homology between the two fungi is low at the nucleotide, 35%, and amino acid level, 29%. The highest degree of similarity between the A. bisporus sequence and other published sequences is with the Homo sapiens eIF4E sequence (32%). V. fungicola exhibited highest homology with the eIF4E sequence from Caenorhabditis elegans (34%). Southern analysis of genomic DNA indicated a single copy of the gene within the A. bisporus genome.     

Hydrogen peroxide concentrations detected in Agaricus bisporus sporocarps and relation with their susceptibility to the pathogen Verticillium fungicola

A dry bubble is an undifferentiated structure that forms in place of mushrooms when cultures of Agaricus bisporus are contaminated by Verticillium fungicola. Hydrogen peroxide concentrations were measured in lyophilised samples of bubbles and healthy sporocarps from cultures of genetically related strains of A. bisporus. The strains the more resistant to the pathogen had the higher levels of H2O2 concentration measured in the bubbles, but the differences in the healthy sporocarps were not significant. That is an indication of a higher reaction to the pathogen in the forming sporocarps of A. bisporus strains associated with their partial resistance to V. fungicola.     

Isolation and Characterization of Differentially Expressed Genes in the Mycelium and Fruit Body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-β-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

Isolation and characterization of differentially expressed genes in the mycelium and fruit body of Tuber borchii

The transition from vegetative mycelium to fruit body in truffles requires differentiation processes which lead to edible fruit bodies (ascomata) consisting of different cell and tissue types. The identification of genes differentially expressed during these developmental processes can contribute greatly to a better understanding of truffle morphogenesis. A cDNA library was constructed from vegetative mycelium RNAs of the white truffle Tuber borchii, and 214 cDNAs were sequenced. Up to 58% of the expressed sequence tags corresponded to known genes. The majority of the identified sequences represented housekeeping proteins, i.e., proteins involved in gene or protein expression, cell wall formation, primary and secondary metabolism, and signaling pathways. We screened 171 arrayed cDNAs by using cDNA probes constructed from mRNAs of vegetative mycelium and ascomata to identify fruit body-regulated genes. Comparisons of signals from vegetative mycelium and fruit bodies bearing 15 or 70% mature spores revealed significant differences in the expression levels for up to 33% of the investigated genes. The expression levels for six highly regulated genes were confirmed by RNA blot analyses. The expression of glutamine synthetase, 5-aminolevulinic acid synthetase, isocitrate lyase, thioredoxin, glucan 1,3-beta-glucosidase, and UDP-glucose:sterol glucosyl transferase was highly up-regulated, suggesting that amino acid biosynthesis, the glyoxylate cycle pathway, and cell wall synthesis are strikingly altered during morphogenesis.     

A polymerase chain reaction-based test for Verticillium fungicola causing dry bubble disease on the cultivated mushroom,Agaricus bisporus

A polymerase chain reaction (PCR)-based test is described for the specific detection of Verticillium fungicola var. aleophilum (Vfa), the fungal pathogen causing dry bubble disease on the cultivated button mushroom, Agaricus bisporus. PCR primers were tailored to target a 162-bp arbitrary sequence in the Vfa genome. In PCR amplifications using the primer pair, all of 20 isolates of Vfa that had been collected during a 29-year period at commercial mushroom operations located primarily in North America were found to generate the diagnostic 162-bp DNA product. Conversely, the primers failed to produce the specific amplicon with DNA from isolates representing 5 other species of Verticillium, the pathogenic subspecies V. fungicola var. fungicola from Europe, and 12 other fungal species commonly inhabiting mushroom compost. A protocol was designed enabling a confirmed diagnosis of dry bubble disease in less than 3 h. The PCR-based test should find application for the rapid diagnosis and detection of the fungal pathogen in disease management programs and, potentially, in screening for on-the-farm sources of infection.     

Comparative effect of the fungicide Prochloraz-Mn on Agaricus bisporus vegetative-mycelium and fruit-body cell walls.

Fungicides to control mycopathogens of commercial Agaricus bisporus, a mushroom cultivated for human consumption, are a major field of study, since these chemicals are toxic to both the host and its fungal parasites. The fungicide Prochloraz-Mn, used at its LD50 for A. bisporus, partially inhibited protein biosynthesis in the vegetative mycelial cell walls of this mushroom and caused significant changes in cell-wall polysaccharide structure, as deduced by methylation analysis and gas liquid chromatography-mass spectrometry (GLC-MS). Furthermore, the aggregated mycelial walls showed distinct alterations in their overall chemical composition following the administration of Prochloraz-Mn at the LD50 and the LD50 x1000. As expected, GLC-MS studies indicated that the latter dose caused more appreciable differences in polysaccharide structure. The decrease in mushroom crop yields obtained from industrial cultures treated with Prochloraz-Mn to control V. fungicola infection depended on the dose of the fungicide employed, whereas fruit-body morphology was only slightly affected at the highest Prochloraz-Mn concentration used.     

Zinc and lead uptake by mycelium and regenerating protoplasts of Verticillium marquandii

The ability of the filamentous fungus Verticillium marquandii for Zn²⁺ and Pb²⁺ uptake from aqueous solution was studied. The 24-h-old living mycelium bound Zn²⁺ and Pb²⁺ (206.2 and 324.5 mg/g dry weight, respectively) effectively, in contrast to a very low Zn²⁺ uptake by autoclaved mycelium (20.2 mg/g). The most effective results were noted when the metals were introduced as acetates and incubated with mycelium for 24 h in case of Zn²⁺ while Pb²⁺ achieved the maximum level of metal binding after as early as 3 h. The cell wall was the main site of effective Zn²⁺ and Pb²⁺ binding by V. marquandii mycelium (91.0–93.6% of metals were located in cell wall after 24 h of exposure). The metabolic inhibitors: antimycin A and sodium azide had a strong limitation effect on Zn²⁺ uptake by a 24-h-old living mycelium, whereas Pb²⁺ binding did not decrease to a large extent. The freshly obtained protoplasts accumulated Zn²⁺ and Pb²⁺ on a low level in comparison with cells at different stages of cell wall regeneration. The use of regenerating protoplasts showed that resynthesis of cell wall was necessary for high binding of Zn²⁺, whereas Pb²⁺ uptake on the significant level took place during cell wall regeneration. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of the fungicide Prochloraz-Mn on the cell wall structure of <Emphasis Type="Italic">Verticillium fungicola</Emphasis>

The chemical structure of the cell wall of two isolates of Verticillium fungicola collected from diseased fruit bodies of the commercial mushroom Agaricus bisporus treated with the fungicide Prochloraz-Mn was analyzed. The isolates were obtained during different periods of time and grown in the absence and presence of the LD₅₀ values of the fungicide for V. fungicola. In addition, another V. fungicola isolate collected previous to the routine utilization of Prochloraz-Mn but grown under the same conditions was also analyzed. The overall chemical composition of the cell wall from the three isolates showed detectable differences in their basic components, with a significant decrease in the protein content in fungicide-treated cells. This inhibitory effect was partially compensated by an increase in neutral and/or aminated carbohydrates and was accompanied by appreciable modifications of polysaccharide structure, as deduced after methylation analysis and gas-liquid chromatography-mass spectrometry (GLC-MS). Moreover, differences in hyphal morphology caused by the fungicide were observed by transmission electron microscopy (TEM). Accepted 2 May 2002 Electronic Publication     

Simple staining detects ultrastructural and biochemical differentiation of vegetative hyphae and fruit body initials in colonies of Pleurotus pulmonarius

AIMS: To know the ultrastructural and biochemical differences of vegetative hyphae and fruit body initials in colonies of Pleurotus pulmonarius. METHODS AND RESULTS: Feulgen reagent was used to detects differentiation of hyphae. The intracellular laccases, proteases and beta-1,3-glucanases activity, content of cytoplasmic protein, glycogen and glucans in the cell wall were evaluated in hyphae of fruit body initials and in vegetative hyphae. The thickness of hyphal walls of the vegetative hyphae was also evaluated. Substantial biochemical changes were observed in hyphae of different zones of the fruiting colony. Hyphae at the periphery had thinner walls than in the centre of the colony. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Staining correlated with the enzymatic activity, protein, glycogen and glucans, in mycelium and in fruit body initials. The implications are that hyphal maturity in P. pulmonarius involves storage of glucans, in part at least, in the form of a thickened hyphal wall.     

Quantitative genetics to dissect the fungal-fungal interaction between Lecanicillium verticillium and the white button mushroom Agaricus bisporus

Lecanicillium fungicola (formerly Verticillium fungicola) is responsible for dry bubble disease in the white button mushroom Agaricus bisporus. Selection for resistance to this pathogen raises an important challenge for mushroom breeders. We have investigated the inheritance of resistance to dry bubble under artificial inoculation in three independent experiments, using a progeny of 89 hybrids derived from an intervarietal A. bisporus var. bisporus×A. bisporus var. burnettii cross. Overall, phenotypic correlations were highly significant between the different experiments. Principal component analysis, together with analysis of variance results stated that the disease reactions were accurately assessed using the percentage of bubbles (PB) and the percentage of spotty cap mushrooms (PS) separately rather than with the combination of both. An original contribution of this study lies in the effective use of area under the disease-progress curve (AUDPC) to describe the dry bubble resistance. The continuous phenotypic distribution observed for the resistance traits suggested that tolerance to dry bubble was under polygenic control. Heritability estimates for either PB or AUDPC were high (0.67-0.86) while it was inconsistent for PS (0.33-0.68) suggesting a strong impact of the environment on this latter trait. Earliness and latent period were found highly correlated with disease incidence. The earliest strains appeared to be the most resistant ones. These results contribute to disentangle the complex fungal-fungal A. bisporus / L. fungicola interaction and to provide genetic basis as a prerequisite for mushroom breeding program.     

2-amino-2-deoxy-D-erythrose in Agaricus bisporus

2-Amino-2-deoxy-D-erythrose was isolated from the cell wall of the fruit body of Agaricus bisporus. The structure of the amino sugar was determined by mass spectrography and 1H-NMR spectrography of its acetylated derivative and by paper chromatographic comparisons with authentic 2-amino-2-deoxy-D-erythrose. This amino sugar is a component of the glycoprotein fraction from the cell wall. Its content in the glycoprotein increased markedly, especially during the ripening stage of the fruit body.     

Chemical Analysis of the Lamella Walls of Agaricus bisporus Fruit Bodies

Purified lamella wall fragments of Agaricus bisporus fruit bodies were analyzed and shown to consist of neutral sugars (46.5%), hexosamines (31.7%), proteins (9.5%), some lipid material (10.0%), and ash (1.4%). The cell walls were fractionated on the basis of their polysaccharide solubility in water and alkaline solutions. The isolated fractions, using methylation analysis, exhibited striking chemical structural differences compared with the same fractions obtained from the corresponding vegetative cells and fruit bodies (stipe and pileus) walls. The structural differences detected in the wall seem to correspond to the ultimate differentiation of the mycelium inside the fruit body of A. bisporus. Received: 30 November 1998 / Accepted: 29 January 1999     

A novel developmental stage-specific lectin of the basidiomycete Pleurotus cornucopiae.

A novel lectin was isolated from mycelia of the basidiomycete Pleurotus cornucopiae grown on solid medium. The lectin was purified to homogeneity by mucin-Sepharose affinity chromatography. The molecular mass of the lectin was 40 kDa under reducing conditions, but the subunits were polymerized through disulfide bridges under physiological conditions. Hemagglutinating activity of this lectin was completely inhibited by 2-mercaptoethanol, indicating that the multimer is active. The activity was also inhibited by EDTA, and restored by CaCl2. N-Acetyl-D-galactosamine was the most potent hapten inhibitor. N-terminal amino acid sequence analysis revealed that the mycelial lectin was different from the fruit body lectin of this organism. The mycelial lectin appeared prior to fruit body formation and disappeared during the formation of fruit bodies. The lectin was localized on the surface of solid-medium-grown mycelia, and only dikaryotic, and not monokaryotic, mycelia produced the lectin. These results suggest that the appearance of this lectin is associated with fruit body formation.     

Purification and characterization of a surface lectin from the nematode-trapping fungus Arthrobotrys oligospora.

Several studies have indicated that the capture of nematodes by the nematophagous fungus Arthrobotrys oligospora is mediated by a lectin on the fungal surface. One of the major surface proteins of this fungus showed haemagglutinating activity and was isolated by affinity chromatography using a mucin Sepharose column. Biochemical analysis showed that the protein was a dimeric glycoprotein with a molecular mass of 36 kDa and an isoelectric point of pH 6.5, and contained no sulphur amino acids. The protein was N-terminally blocked; four internal peptides were sequenced, and showed no significant similarity to sequences in the Swiss-Prot or PIR databases. The haemagglutinating activity of the isolated protein was not inhibited by any of the mono- or disaccharides tested, but it was inhibited by the glycoproteins fetuin and mucin. The haemagglutinating activity changed after incubating the protein in buffers of different pH, with maximal activity at pH 11.0 and no activity at pH 2.8. The lectin was tested for different enzymic activities but none were detected. Analysis of the haemagglutinating activity in various cell fractions indicated that the protein was associated with extracellular polymer layers and with the cell wall of the fungus. About the same amount of the haemagglutinating protein was recovered from samples of vegetative mycelium and of mycelium containing nematode-trapping cells.     

Anti-tumor factor in insects' hemolymph

We have purified a lectin from the hemolymph of Sarcophaga peregrina larvae, obtained after injury of their body wall. This lectin recognizes galactose residues and suggested to be involved in the defense mechanism of this insect. We also demonstrated that this lectin induced cytotoxic effects on tumor cells in the presence of murine macrophages. It was found that the murine macrophages had Sarcophaga lectin binding proteins. Using pupal hemocytes and fatbody of this insect, we established an in vitro system that mimics dissociation of the fatbody in vivo. New membrane protein, induced on the surface of the hemocytes at pupation, suggested to participate in the recognition of the fatbody.     

An endogenous lectin "CSL" interacts with glycoprotein components in peripheral nervous system myelin

An endogenous mannose binding lectin isolated from the rat cerebellum, CSL, was localized using immunocytochemical techniques in adult and in developing rat sciatic nerve. The lectin is present in Schwann cell cytoplasm and in compact myelin. It is present very early in Schwann cells and persists throughout postnatal sciatic nerve development. Endogenous ligands for the lectin were detected using iodinated-CSL binding to proteins blotted after polyacrylamide gel electrophoresis. Probably PO and MAG glycoproteins are specifically bound by CSL in contrast with numerous other Concanavalin A binding glycoproteins. A 31 kDa glycoprotein identified in purified preparations of axons of young rats also reacts with CSL. Based on the present developmental biochemical and immunochemical studies, an hypothetical scheme is proposed for the molecular basis of axon-Schwann cell interactions and of stabilization of compact myelin.