Molecular characterization of two ammonium transporters from the ectomycorrhizal fungus Hebeloma cylindrosporum.

Heterologous expression of the yeast triple Mep mutant has enabled the first molecular characterization of AMT/MEP family members in an ectomycorrhizal fungus. External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structure of the ectomycorrhizal symbiosis and therefore molecular studies on ammonium transport in hyphae are urgently needed. The kinetic properties of AMT2 and AMT3 from Hebeloma cylindrosporum were studied in Saccharomyces cerevisiae. Expression of HcAmts in the yeast triple Mep mutant restored ammonium retention within cells. The HcAmts did not complement the ammonium sensing defect phenotype of Mep2Delta cells during pseudohyphal differentiation. Northern blot analysis in H. cylindrosporum showed that the HcAMTs were up-regulated upon nitrogen deprivation and down-regulated by ammonium.

Molecular cloning, characterization and regulation by cadmium of a superoxide dismutase from the ectomycorrhizal fungus Paxillus involutus.

The gene encoding a superoxide dismutase (PiSOD) was cloned by suppressive subtractive hybridization from cDNA library of the ectomycorrhizal fungus, Paxillus involutus, grown under cadmium-stress conditions. The encoded protein was presumed to be localized in the peroxisomes because it contained a C-terminal peroxisomal localization peptide (SKL) and lacked an N-terminal mitochondrial transit peptide. Complementation of an Escherichia coli SOD null strain that is unable to grow in the presence of paraquat or cadmium indicated that cloned Pisod encoded a functional superoxide dismutase. Sensitivity of PiSOD activity to H2O2 but not KCN, and sequence homologies to other SODs strongly suggest that it is a manganese-containing superoxide dismutase. Monitoring PiSOD transcript, immunoreactive polypeptide and superoxide dismutase activity following cadmium stress suggests that the principal level of control is post-translational. This is, to our knowledge, the first insight in the characterization of molecular events that take place in an ectomycorrhizal fungus during exposure to heavy metals.

Effects of heavy metals on nitrogen uptake by Paxillus involutus and mycorrhizal birch seedlings.

The effects of the heavy metals Cu, Cd, Ni, Pb and Zn on [(14)C]methylamine and [(14)C]aminoisobutyric acid uptake were studied in the free-living fungus Paxillus involutus and in mycorrhizal and non-mycorrhizal birch roots. The uptake of both N sources by P. involutus was inhibited by the five metals tested. However, Cu(2+) and Pb(2+) had a greater inhibitory effect. Non-competitive inhibitions were determined between heavy metals and [(14)C]methylamine uptake. [(14)C]Methylamine uptake was reduced by one third by 2 µM Cd(2+) and Cu(2+) in non-mycorrhizal roots, whereas that of mycorrhizal roots was not affected. However, it was reduced by 30 to 80% by 200 µM Cd(2+) and Cu(2+) irrespective of the mycorrhizal status. [(14)C]Aminoisobutyric acid uptake in mycorrhizal roots was not significantly affected by Cd(2+) and Cu(2+), whereas that of non-mycorrhizal roots was decreased by 77% at 200 µM Cu(2+). [(14)C]Aminoisobutyric acid uptake was 4.5 to 6 fold higher in mycorrhizal roots, compared with non-mycorrhizal roots, even under metal exposure. The high efficiency of N acquisition by mycorrhizal birch seedlings under metal exposure might be regarded as a mechanism of stress avoidance.

Ammonium and methylamine transport by the ectomycorrhizal fungus Paxillus involutus and ectomycorrhizas.

Using [(14)C]methylamine as an analogue of ammonium, the kinetics and the energetics of NH(4)(+) transport were studied in the ectomycorrhizal fungus, Paxillus involutus (Batsch) Fr. The apparent half-saturation constant (K(m)) and the maximum uptake rate (V(max)) for the carrier-mediated transport derived from the Eadie-Hofstee transformation were 180 µM and 380 nmol (mg dry wt)(-1) min(-1,) respectively. Both pH dependence and inhibition by protonophores indicate that methylamine transport in P. involutus was dependent on the electrochemical H(+) gradient. Both long-term and short-term uptake experiments were consistent with regulation of ammonium/methylamine transport processes by the presence of an organic nitrogen source. Analysis of methylamine uptake by different P. involutus isolates revealed no obvious trend in the uptake capacities in relation to N deposition at the collection site. Kinetic parameters were determined in P. involutus/Betula pendula (Roth.) axenic association and in detached mycorrhizal roots isolated from forest sites. Enhanced methylamine uptake in the presence of the fungal symbiont was demonstrated. Homogeneous V(max) values were found for axenic and detached mycorrhizas, whereas K(m) values showed greater variations.

Purification and characterization of an anti-(A+B) specific lectin from the mushroom Hygrophorus hypothejus.

A lectin (HHL) was isolated from the fruiting body of the mushroom Hygrophorus hypothejus by a combination of affinity chromatography on stromas of group B erythrocytes embedded in polyacrylamide gel, and DEAE-trisacryl and gel filtration chromatography. Its molecular mass, as determined by gel filtration, is estimated to be 68000 kDa and its structure is tetrameric with four identical subunits assembled with non-covalent bonds. HHL agglutinates specifically A and B blood group erythrocytes and in hemagglutination inhibition assays, exhibits sugar-binding specificity toward lactose, the anomeric alpha form being more effective than the beta form.

Effects of nitrogen source on growth and activity of nitrogen-assimilating enzymes in Douglas-fir seedlings.

Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings were grown for 68 days in a growth chamber in nutrient solutions with ammonium, nitrate or ammonium nitrate as the nitrogen source. Among the nitrogen sources tested, whole-seedling biomass, relative growth rate (RGR), root and shoot elongation, and number of lateral roots, were greatest in seedlings grown with ammonium. In the absence of nitrogen, plant growth and formation of lateral roots were poor. Initially, glutamine synthetase, NAD-glutamate dehydrogenase and aspartate aminotransferase activities were high in young roots and shoots, but all three enzymatic activities decreased after one month of culture. In root apices, glutamine synthetase and aspartate aminotransferase activities were higher than NAD-glutamate dehydrogenase activity. Enzymatic activities were often higher in ammonium-fed seedlings than in seedings supplied with the other forms of nitrogen. Activities of all three enzymes were significantly reduced in seedlings grown in the absence of nitrogen. The beneficial effect of ammonium is discussed on the basis of its involvement in the assimilation pathways of Douglas-fir.

Cytolocalization of glycogen, starch, and other insoluble polysaccharides during ontogeny of Paxillus involutus-Betula pendula ectomycorrhizas.

The Paxillus involutus (Fries) Karsten-Betula pendula Roth association was studied during the early stages of formation. Cytological studies revealed fungal colonization behind the root cap and gradually around the entire root apex. Ultrastructural investigations were carried out and insoluble polysaccharide distribution was followed. The density of starch grains increased in plant cells especially after 4 d of contact between the two partners, but later on decreased strongly in the root cap. Large amounts of glycogen were revealed in the hyphae in certain mycorrhizal regions after 6 d of contact: in the Hartig net, in the inner sheath but only near the net, and all along the outer sheath surrounding the mycorrhiza. Thickenings of the epidermal cell walls were detected as early as 2 d after contact and then varied according to the distance from the root tip. Such polysaccharide distributions are assumed to show a transfer of carbohydrates from the root to the fungus and are discussed in terms of carbon requirements for both partners.

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press

DNA polymorphism in morels: complete sequences of the internal transcribed spacer of genes coding for rRNA in Morchella esculenta (yellow morel) and Morchella conica (black morel).

The internal transcribed spacer (ITS) of the gene coding for rRNA was sequenced in both directions with the gene walking technique in a black morel (Morchella conica) and a yellow morel (M. esculenta) to elucidate the ITS length discrepancy between the two species groups (750-bp ITS in black morels and 1,150-bp ITS in yellow morels.

Polymorphism in morels: isozyme electrophoretic analysis.

The aim of this study was to assess whether isozyme polymorphism in different members of the Morchellaceae could be used to improve the systematics in this fungal group and to characterize intraspecific crossings between monosporal strains in Morchella esculenta. For this purpose, isozyme electrophoretic analysis of the following enzymes was performed: glutamine synthetase, NAD-glutamate dehydrogenase, NADP-glutamate dehydrogenase, aspartate aminotransferase, malate dehydrogenase, NAD-glyceraldehyde phosphate dehydrogenase, glucose phosphate isomerase, and superoxide dismutase. The analyses allowed discrimination at the inter- or intra-specific levels and could help to establish a method of identification for strains in the Morchellaceae. To a certain extent they appeared to be suitable to analyze interactions of monosporal strains of Morchella esculenta in pairing experiments. The polymorphism shown in this study was consistent with the phylogenetic relationships between the investigated strains only at the genus level.Key words: isozyme analysis, electrophoresis, Morchella sp., polymorphism.

Isolation and characterization of an N-acetyllactosamine-binding lectin from the mushroom Laetiporus sulfureus.

A hemagglutinating and hemolytic lectin (PSL) has been isolated from carpophores of the parasitic mushroom Laetiporus sulfureus by affinity chromatography on Sepharose. Its molecular weight, as determined both by gel filtration and by electrophoresis in non-denaturing conditions, is about 190,000 and its structure is tetrameric, with two distinct types of subunits (about 60,000 and 36,000). It appeared homogeneous on HPLC gel filtration but exhibited microheterogeneity on isoelectric focusing. Hapten inhibition assay indicated that the Laetiporus lectin is specific for N-acetyllactosamine residues and that hemagglutinating and hemolysis activities are supported by the same site.

Discrimination of some African Armillaria species by isozyme electrophoretic analysis.

Ten Armillaria isolates, collected from various host plants and widespread geographical origins in tropical Africa, were cultivated on orange fragments in the presence of water and ran different culture media in order to optimize enzyme and mycelial cord production. Seven enzymes involved in the primary metabolism of nitrogen and carbon (glutamate dehydrogenases, aspartate aminotransferase, malate dehydrogenase, glucose n-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and isocitrate dehydrogenase) were extracted from the mycelial cords and analyzed by polyacry lamide gel electrophoresis. Cluster analysis based on calculated similarity values derived from isozyme banding patterns separated the isolates into five groups. Two isolates considered as belonging to A. mellea ssp. africana (an African species closely related to European A. mellea) were present in a clearly Separated cluster when compared to the other isolate groups. Two Kenyan isolates, belonging to an as yet unnamed biological species, which were characterized by the production of few slow growing mycelial cords, were also found in a separate cluster with slightly greater similarity coefficients to the other isolates. The six other isolates, referred to as isolates of A. heimii (a highly variable species with different sexual systems) fell into three sub-clusters of variable homology. The two homothallic heimii isolates from Tanzania and Malawi, which were very closely related, displaying 100% isozyme similarity, exhibited a higher degree of similarity with the two other homothallic heimii isolates from Zimbabwe and Congo, than with the two heterothallic unifactorial heimii isolates from Cameroon and Gabon. The value of isozymes in the classification of African Armillaria spp. is discussed.

Purification and Characterization of Glutamine Synthetase and NADP-Glutamate Dehydrogenase from the Ectomycorrhizal Fungus Laccaria laccata.

Glutamine synthetase (GS) and NADP-dependent glutamate dehydrogenase (NADP-GDH) play a key role in nitrogen assimilation in the ectomycorrhizal fungus Laccaria laccata (Scop. ex Fr. Cke) strain S 238. The two enzymes were purified to apparent electrophoretic homogeneity by a three-step procedure involving diethylaminoethyl (DEAE)-Trisacryl and affinity chromatography, and DEAE-5PW fast protein liquid chromatography. This purification scheme resulted in a 23 and 62% recovery of the initial activity for GS and NADP-GDH, respectively. Purified GS had a specific activity of 713 nanomoles per second per milligram protein and a pH optimum of 7.2. Michaelis constants (millimolar) for the substrates were NH(4) (+) (0.024), glutamate (3.2), glutamine (30), ATP (0.18), and ADP (0.002). The molecular weight (M(r)) of native GS was approximately 380,000; it was composed of eight identical subunits of M(r) 42,000. Purified NADP-GDH had a specific activity of 4130 nanomoles per second per milligram protein and a pH optimum of 7.2 (amination reaction). Michaelis constants (millimolar) for the substrates were NH(4) (+) (5), 2-oxoglutarate (1), glutamate (26), NADPH (0.01), and NADP (0.03). Native NADP-GDH was a hexamer with a M(r) of about 298,000 composed of identical subunits with M(r) 47,000. Polyclonal antibodies were produced against purified GS and NADP-GDH. Immunoprecipitation tests and immunoblot analysis showed the high reactivity and specificity of the immune sera against the purified enzymes.

Glutamate dehydrogenases in ectomycorrhizas of spruce (Picea excelsa L.) and beech (Fagus sylvatica L.).

The ammonia assimilation enzyme glutamate dehydrogenase as studied in extracts of spruce (Picea excelsa L.) roots, mycelium of a mycorrhizal fungus (Hebeloma sp.) and associated ectomycorrhizas. Evidence from enzyme reactions in crude extracts, electrophoretic patterns and immunological tests Using antibodies raised against purified NADP-GDH of Cenococcum geophilum Fr. consistently showed that Hebeloma NADP-dependent GDH was active in spruce ectomycorrhizas. Histochemical studies associated some NADP-GDH activity with the Hartig net. By contrast, the NADP-GDH fungal pathway was strongly suppressed in beech (Fagus sylvatica L.) associations with Hebeloma crustuliniforme (Bull. ex St Amans) Quél. and Paxillus involutus (Batsch ex Fr.) Fr.

Enzyme-Linked Immunosorbent Assay of Fungal NADP-Glutamate Dehydrogenase.

A sensitive and reliable method has been developed for the quantitation of NADP(+)-glutamate dehydrogenase from the phytopathogenic Ascomycete Sphaerostilbe repens using a two-step competitive enzyme-linked immunosorbent assay. Purified enzyme was adsorbed noncovalently to polystyrene wells and rabbit immunserum was allowed to bind to antigensensitized wells. Bound specific antibody was visualized by goat antirabbit immunoglobulin covalently linked to alkaline phosphatase using paranitrophenylphosphate as the substrate. Increasing amounts of purified enzyme or crude fungal extracts were quantitated by their ability to inhibit specific antibody adsorption to antigen-coated polystyrene wells. This system proves to be useful in the range of 10 to 80 nanograms of enzyme level. Using this assay, identical amounts of NADP(+)-glutamate dehydrogenase were found in mycelia grown on nitrate and ammonia sources.

Influence of calcium on the differentiation and growth of aggregated organs in Sphaerostilbe repens.

In the ascomycete Sphaerostilbe repens the vegetative mycelium aggregated into structures composed of aerial coremia and rhizomorphs immersed in the culture medium. The morphology and growth of the cultures were examined on a defined medium with and without calcium supplementation. Initiation and growth of aggregated organs occurred only if calcium was present continuously in the culture medium. On calcium-deficient media the fungus produced only vegetative mycelium which retained its ability to aggregate in the presence of the cation. Small concentrations of calcium (less than 1 ppm) stimulated aggregation of the mycelium, but higher concentrations (about 200 ppm) were necessary to obtain optimum development of the aggregated organs. Strontium partially replaced calcium, but lithium did not, as a stimulator of aggregation.