Verticillium disease or "dry bubble" of cultivated mushrooms: the Agaricus bisporus lectin recognizes and binds the Verticillium fungicola cell wall glucogalactomannan

The step of recognition and (or) binding for the development of the disease of the cultivated mushroom Agaricus bisporus by the mycoparasite Verticillium fungicola was studied by several approaches: agglutination of V. fungicola germinated spores by an A. bisporus extract from fruit body cell walls, immunofluorescence microscopy of A. bisporus hyphae from fruit bodies and vegetative mycelia pretreated with purified V. fungicola cell wall glucogalactomannan, and finally, by hemagglutination experiments carried out with an A. bisporus fruit body lectin in the presence and absence of the same glucogalactomannan. Hemagglutinating activity of the purified A. bisporus fruit body lectin was clearly inhibited by the V. fungicola glucogalactomannan, whereas in the A. bisporus vegetative mycelium such lectin was not encountered. All the results obtained make evident the recognition and binding of the A. bisporus fruit body lectin to the V. fungicola cell wall glucogalactomannan, clarifying why the mushrooms, but not the vegetative mycelium, become diseased.     

Tvbgn3, a beta-1,6-glucanase from the biocontrol fungus Trichoderma virens, is involved in mycoparasitism and control of Pythium ultimum

Even though beta-1,6-glucanases have been purified from several filamentous fungi, the physiological function has not been conclusively established for any species. In the present study, the role of Tvbgn3, a beta-1,6-glucanase from Trichoderma virens, was examined by comparison of wild-type (WT) and transformant strains in which Tvbgn3 was disrupted (GKO) or constitutively overexpressed (GOE). Gene expression analysis revealed induction of Tvbgn3 in the presence of host fungal cell walls, indicating regulation during mycoparasitism. Indeed, while deletion or overexpression of Tvbgn3 had no evident effect on growth and development, GOE and GKO strains showed an enhanced or reduced ability, respectively, to inhibit the growth of the plant pathogen Pythium ultimum compared to results with the WT. The relevance of this activity in the biocontrol ability of T. virens was confirmed in plant bioassays. Deletion of the gene resulted in levels of disease protection that were significantly reduced from WT levels, while GOE strains showed a significantly increased biocontrol capability. These results demonstrate the involvement of beta-1,6-glucanase in mycoparasitism and its relevance in the biocontrol activity of T. virens, opening a new avenue for biotechnological applications.     

PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of beta-1,3- and beta-1,6-glucans

PHR1 and PHR2 encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen Candida albicans. These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo. Deletion of either gene results in a pH-conditional defect in cell morphology and virulence. Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and beta-galactosidases. Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity. By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH. The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution. The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between beta-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in beta-1,3-glucan-associated beta-1, 6-glucans and, within several generations, a fivefold increase in the chitin content of the walls. The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis. Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between beta-1,6- and beta-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process beta-1,3-glucans and make available acceptor sites for the attachment of beta-1,6-glucans.     

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.     

Endophytic fungal beta-1,6-glucanase expression in the infected host grass

Mutualistic fungal endophytes infect many grass species and often confer benefits to the hosts such as reduced herbivory by insects and animals. The physiological interactions between the endophytes and their hosts have not been well characterized. Fungal-secreted proteins are likely to be important components of the interaction. In the interaction between Poa ampla and the endophyte Neotyphodium sp., a fungal beta-1,6-glucanase is secreted into the apoplast, and activity of the enzyme is detectable in endophyte-infected plants. Sequence analysis indicates the beta-1,6-glucanase is homologous to enzymes secreted by the mycoparasitic fungi Trichoderma harzianum and Trichoderma virens. DNA gel-blot analysis indicated the beta-1,6-glucanase was encoded by a single gene. As a secreted protein, the beta-1,6-glucanase may have a nutritional role for the fungus. In culture, beta-1,6-glucanase activity was induced in the presence of beta-1,6-glucans. From RNA gel blots, similar beta-1,6-glucanases were expressed in tall fescue (Festuca arundinacea Schreb.) and Chewings fescue (Festuca rubra L. subsp. fallax [Thuill] Nyman) infected with the endophyte species Neotyphodium coenophialum and Epichlo? festucae, respectively.     

A refined method for the determination of Saccharomyces cerevisiae cell wall composition and beta-1,6-glucan fine structure.

In yeast and other fungi, cell division, cell shape, and growth depend on the coordinated synthesis and degradation of cell wall polymers. We have developed a reliable and efficient micro method to determine Saccharomyces cerevisiae cell wall composition that distinguishes between beta1,3- and beta1,6-glucan. The method is based on the sequential treatment of cell walls with specific hydrolytic enzymes followed by dialysis. The low molecular weight (MW) products thus separated account for each particular cell wall polymer. The method can be applied to as little as 50-100 mg (wet wt) of radioactively labeled cells. A combination of chitinase and recombinant beta-1,3-glucanase is initially used, releasing all of the chitin and 60-65% of the beta1,3-glucan from the cell walls. Next, recombinant endo-beta-1,6-glucanase from Trichoderma harzianum is utilized to release all the beta-1,6-glucan present in the wall. The chromatographic pattern of endoglucanase digested beta-1,6-glucan provides a characteristic "fingerprint" of beta-1,6-glucan and the fine structure of the oligosaccharides in this pattern was determined by 1H NMR and electrospray ionization mass spectroscopy. The final enzymatic step uses laminarinase and beta-glucosidase to release the remaining beta-1,3-glucan. The cell wall mannan remains as a high MW fraction at the end of the fractionation procedure. Good sensitivity and correlation with cell wall composition determined by traditional methods were observed for wild-type and several cell wall mutants.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

Enzyme Production by the Mycoparasite Verticillium biguttatum against Rhizoctonia solani

Verticillium biguttatum, a mycoparasite of the ubiquitous soil-borne plant pathogen Rhizoctonia solani, excreted chitinase and beta-1,3-glucanase into liquid medium when grown on laminarin and chitin, respectively. Neither chitinase nor beta-1,3-glucanase was produced by the mycoparasite when grown on cell walls of two isolates of R. solani representing anastomosis groups (AG)-3 and AG-8. Extracellular protease was induced by growth on cell walls of the pathogen, whereas beta-1,3-glucanase and chitinase were produced bound to the cell wall of V. biguttatum. This is the first report of chitinase, beta-1,3-glucanase and protease production by V. biguttatum. These enzymes may play a previously unforeseen role in dissolving and penetrating the cell walls of R. solani.     

Purification and properties of a β-1,6-glucanase from Streptomyces sp. EF-14, an actinomycete antagonistic to Phytophthora spp.

Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism. Streptomyces sp. EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp. A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source. The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase. The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5. It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products. Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links. No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme. This is the first beta-1,6-glucanase characterized from an actinomycete.     

Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds

Chitin, a beta-1,4-linked polysaccharide of N-acetylglucosamine, is a major structural component of fungal cell walls. Fungi have multiple classes of chitin synthases that catalyse N-acetylglucosamine polymerization. Here, we demonstrate the requirement for a class V chitin synthase during host infection by the vascular wilt pathogen Fusarium oxysporum. The chsV gene was identified in an insertional mutagenesis screen for pathogenicity mutants. ChsV has a putative myosin motor and a chitin synthase domain characteristic of class V chitin synthases. The chsV insertional mutant and a gene replacement mutant of F. oxysporum display morphological abnormalities such as hyphal swellings that are indicative of alterations in cell wall structure and can be partially restored by osmotic stabilizer. The mutants are unable to infect and colonize tomato plants or to grow invasively on tomato fruit tissue. They are also hypersensitive to plant antimicrobial defence compounds such as the tomato phytoanticipin alpha-tomatine or H2O2. Reintroduction of a functional chsV copy into the mutant restored the growth phenotype of the wild-type strain. These data suggest that F. oxysporum requires a specific class V chitin synthase for pathogenesis, most probably to protect itself against plant defence mechanisms.     

Detection of beta-1,6-glucanase isozymes from Trichoderma strains in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectrofocusing gels.

The filamentous fungus Trichoderma produces, under specific growth conditions, several extracellular fungal cell wall degrading enzymes, amongst them beta-1,6-glucanases. These enzymes seem to play an important role in the antagonistic action of Trichoderma against a wide range of fungal plant pathogens. In this report we describe two different methods for the specific detection of the activity of beta-1,6-glucanase isozymes in gels. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, beta-1,6-glucanase activity can be assayed in the gel by renaturation of the enzyme, incubation with an overlay agarose gel containing solubilized pustulan (a commercially available beta-1,6-glucan), followed by the staining of the agarose gel with Congo Red. In native isoelectrofocusing gels, as little as 1 mU can be detected after incubation with solubilized pustulan followed by a detection reaction of the released reducing sugars with 2,3,5-triphenyltetrazolium chloride. The latter technique has been successfully applied to the screening of beta-1,6-glucanase isozymes from different Trichoderma strains under different growth conditions.     

KRE5 gene null mutant strains of Candida albicans are avirulent and have altered cell wall composition and hypha formation properties

The UDP-glucose:glycoprotein glucosyltransferase (UGGT) is an endoplasmic reticulum sensor for quality control of glycoprotein folding. Saccharomyces cerevisiae is the only eukaryotic organism so far described lacking UGGT-mediated transient reglucosylation of N-linked oligosaccharides. The only gene in S. cerevisiae with similarity to those encoding UGGTs is KRE5. S. cerevisiae KRE5 deletion strains show severely reduced levels of cell wall beta-1,6-glucan polymer, aberrant morphology, and extremely compromised growth or lethality, depending on the strain background. Deletion of both alleles of the Candida albicans KRE5 gene gives rise to viable cells that are larger than those of the wild type (WT), tend to aggregate, have enlarged vacuoles, and show major cell wall defects. C. albicans kre5/kre5 mutants have significantly reduced levels of beta-1,6-glucan and more chitin and beta-1,3-glucan and less mannoprotein than the WT. The remaining beta-1,6-glucan, about 20% of WT levels, exhibits a beta-1,6-endoglucanase digestion pattern, including a branch point-to-linear stretch ratio identical to that of WT strains, suggesting that Kre5p is not a beta-1,6-glucan synthase. C. albicans KRE5 is a functional homologue of S. cerevisiae KRE5; it partially complements both the growth defect and reduced cell wall beta-1,6-glucan content of S. cerevisiae kre5 viable mutants. C. albicans kre5/kre5 homozygous mutant strains are unable to form hyphae in several solid and liquid media, even in the presence of serum, a potent inducer of the dimorphic transition. Surprisingly the mutants do form hyphae in the presence of N-acetylglucosamine. Finally, C. albicans KRE5 homozygous mutant strains exhibit a 50% reduction in adhesion to human epithelial cells and are completely avirulent in a mouse model of systemic infection.     

Cloning and characterization of bgn16.3, coding for a beta-1,6-glucanase expressed during Trichoderma harzianum mycoparasitism

AIMS: To clone and characterize the gene coding for BGN16.3, a beta-1,6-glucanase putatively implicated in mycoparasitism by Trichoderma harzianum, a biocontrol agent used against plant pathogenic fungi. METHODS AND RESULTS: Using degenerate primed PCR and cDNA library screening, we have cloned the cDNA coding BGN16.3. bgn16.3 showed a significant sequence identity (50%) to bgn16.1; however, they both have low identity to the previously cloned bgn16.2, allowing the identification of amino acid sequences putatively involved in the common catalytic activity of the three proteins. bgn16.3 is a single-copy gene and highly homologous sequences are present in all tested Trichoderma species. bgn16.3 expression pattern is analysed by Northern blot, finding that it is expressed during the interaction of T. harzianum CECT 2413 with Botrytis cinerea, supporting the implication of the enzyme in the mycoparasitic process. CONCLUSIONS: The cloned bgn16.3 completes the knowledge on the beta-1,6-glucanase isozyme system from T. harzianum CECT 2413. A highly homologous gene is present in all analysed Trichoderma strains. bgn16.3 is expressed under few specific conditions, including the mycoparasitic process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the knowledge of beta-1,6-glucanases. It implicates this group of enzymes in the mycoparasitism by some biocontrol agents such as T. harzianum.     

Characterization of Aspergillus nidulans mutants deficient in cell wall chitin or glucan.

By screening for the osmotically remediable phenotype, mutations in two genes (orlA and orlB) affecting the cell wall chitin content of Aspergillus nidulans were identified. Strains carrying temperature-sensitive alleles of these genes produce conidia which swell excessively and lyse when germinated at restrictive temperatures. Growth under these conditions is remedied by osmotic stabilizers and by N-acetylglucosamine (GlcNAc). Remediation by GlcNAc suggests that the mutations affect early steps in the synthesis of chitin. Temperature and medium shift experiments indicate that the phenotype is the result of decreased synthesis rather than increased chitin degradation and that osmotic stabilizers act to stabilize a defective wall rather than to stabilize the gene product. Two genes, orlC and orlD, which affect cell wall beta-1,3-glucan content were also identified. Walls from strains carrying mutations in these genes exhibit normal amounts of alpha-1,3-glucan and chitin but reduced amounts of beta-1,3-glucan. As for the chitin-deficient mutants, orlC and orlD mutants spontaneously lyse on conventional media but are remedied by osmotic stabilizers. These results indicate that both chitin and beta-1,3-glucan are likely to contribute to the structural rigidity of the cell wall.     

Production and catabolite repression of Penicillium italicum beta-glucanases.

The filamentous fungus Penicillium italicum, grown in a defined liquid medium, produced beta-1,3-glucanase, which remained essentially bound to the cells, and beta-1,6-glucanase, an essentially extracellular enzyme. When glucose was depleted from the medium, when a limited concentration of glucose (0.2%) was maintained, or when the carbon source was galactose (3%) or lactose (3%), a significant increase in the specific activity of beta-1,3-glucanase, in cell extracts, took place. This was paralleled by a very slow rate of growth, and under glucose limitation, the appearance of beta-1,3-glucanase in the medium was also observed. On the other hand, when an excess of glucose, fructose, or sucrose was present, the specific activity remained constant and active growth was promoted. Laminarin, cellobiose, gentiobiose, and isolated Penicillium italicum walls were not capable of significantly inducing beta-1,3-glucanase synthesis to a level beyond that attained by glucose limitation. A similar behavior was observed for beta-1,6-glucanase. beta-1,3-Glucanase and beta-1,6-glucanase are therefore constitutive enzymes subjected to catabolite repression. The results are discussed in the context of the possible functions that have been suggested for glucanases and related enzymes.     

Two homologous genes,DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae

The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)-anchored proteins and are then transferred to cell wall beta-1,6-glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI-anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cDelta was hypersensitive to a cell wall-digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope-tagged Dcw1p is an N-glycosylated, GPI-anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI-anchored membrane proteins and are required for normal biosynthesis of the cell wall.     

beta-1,6-Glucan synthesis in Saccharomyces cerevisiae

beta-1,6-Glucan is an essential fungal-specific component of the Saccharomyces cerevisiae cell wall that interconnects all other wall components into a lattice. Considerable biochemical and genetic effort has been directed at the identification and characterization of the steps involved in its biosynthesis. Structural studies show that the polymer plays a central role in wall structure, attaching mannoproteins via their glycosylphosphatidylinositol (GPI) glycan remnant to beta-1,3-glucan and chitin. Genetic approaches have identified genes that upon disruption result in beta-1,6-glucan defects of varying severity, often with reduced growth or lethality. These gene products have been localized throughout the secretory pathway and at the cell surface, suggesting a possible biosynthetic route. Current structural and genetic data have therefore allowed the development of models to predict biosynthetic events. Based on knowledge of beta-1,3-glucan and chitin synthesis, it is likely that the bulk of beta-1,6-glucan polymer synthesis occurs at the cell surface, but requires key prior intracellular events. However, the activity of most of the identified gene products remain unknown, making it unclear to what extent and how directly they contribute to the synthesis of this polymer. With the recent availability of new tools, reagents and methods (including genomics), the field is poised for a convergence of biochemical and genetic methods to identify and characterize the biochemical steps in the synthesis of this polymer.     

BGN16.3, a novel acidic beta-1,6-glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413

A new component of the beta-1,6-glucanase (EC 3.2.1.75) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo-beta-1,6-glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic beta-1,6-glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to beta-1,6-glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS/PAGE and a pI of 4.5. The enzyme only showed activity against substrates with beta-1,6-glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement beta-1,6-glucanases in this process is discussed.