Cadmium-responsive thiols in the ectomycorrhizal fungus Paxillus involutus

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (gamma-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and gamma-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.     

Polyamine biosynthesis in the ectomycorrhizal fungus Paxillus involutus exposed to zinc

Following exposure of the ectomycorrhizal fungus Paxillus involutus to 3·75 μg ml^-1 or 15 μg ml^-1 zinc for 2 weeks, growth was unaffected, putrescine and spermidine concentrations remained unaltered, while spermine concentration increased substantially. This was accompanied by small changes in the activities of ornithine decarboxylase and S -adenosylmethionine decarboxylase. There was, however, reduced synthesis of aminopropylcadaverine and N,N' bis (3-aminopropyl)cadaverine at the higher zinc concentration. It is possible that the increased spermine concentration may have resulted from displacement of the amine from the cell wall by the metal ion.     

Screening for rapidly evolving genes in the ectomycorrhizal fungus Paxillus involutus using cDNA microarrays

We have examined the variations in gene content and sequence divergence that could be associated with symbiotic adaptations in the ectomycorrhizal fungus Paxillus involutus and the closely related species Paxillus filamentosus. Strains with various abilities to form mycorrhizae were analysed by comparative genomic hybridizations using a cDNA microarray containing 1076 putative unique genes of P. involutus. To screen for genes diverging at an enhanced and presumably non-neutral rate, we implemented a simple rate test using information from both the variations in hybridizations signal and data on sequence divergence of the arrayed genes relative to the genome of Coprinus cinereus. C. cinereus is a free-living saprophyte and is the closest evolutionary relative to P. involutus that has been fully sequenced. Approximately 17% of the genes investigated were detected as rapidly diverging within Paxillus. Furthermore, 6% of the genes varied in copy numbers between the analysed strains. Genome rearrangements associated with this variation including duplications and deletions may also play a role in adaptive evolution. The cohort of divergent and duplicated genes showed an over-representation of either orphans, genes whose products are located at membranes, or genes encoding for components of stress/defence reactions. Some of the identified genomic changes may be associated with the variation in host specificity of ectomycorrhizal fungi. The proposed procedure could be generally applicable to screen for rapidly evolving genes in closely related strains or species where at least one has been sequenced or characterized by expressed sequence tag analysis.     

Endogenous polyamine content and metabolism in the ectomycorrhizal fungus Paxillus involutus

Endogenous polyamine content of the ectomycorrhizal fungus Paxillus involutus , as well as the activity of its biosynthetic enzymes in relation to mycelia ageing were investigated in this work. Polyamines in free, PCA-soluble and insoluble conjugated forms, are present in Paxillus involutus mycelia in relatively high amounts and the ratio of putrescine to spermidine is age-dependent. Both arginine- and ornithine-decarboxylases are present, but putrescine biosynthesis proceeds mostly via ornithine decarboxylase and decreases with the age of mycelia. There was a large release of free polyamines from mycelia which showed age-dependent features. Clear polyamine uptake was observed in 2-wk-old mycelia and no competition between putrescine and cadaverine was detected. Putrescine uptake seems to reduce ornithine decarboxylase activity, but does not affect arginine decarboxylase.     

The effect of the polyamine biosynthesis inhibitor DFMO on the ectomycorrhizal fungus Paxillus involutus

DFMO reduced mycelial growth of the ectomycorrhizal fungus Paxillus involutus . This was accompanied by reduced activities of ornithine decarboxylase, S-adenosylmethionine decarboxylase and diamine oxidase, and unchanged polyamine oxidase activity. Although DFMO treatment did not alter putrescine or spermidine concentrations significantly, spermine concentration was substantially reduced.     

Evolution of nucleotide sequences and expression patterns of hydrophobin genes in the ectomycorrhizal fungus Paxillus involutus

Hydrophobins are small, secreted proteins that play important roles in the development of pathogenic and symbiotic fungi. Evolutionary mechanisms generating sequence and expression divergence among members in hydrophobin gene families are largely unknown. Seven hydrophobin (hyd) genes and one hyd pseudogene were isolated from strains of the ectomycorrhizal fungus Paxillus involutus. Sequences were analysed using phylogenetic methods. Expression profiles were inferred from microarray experiments. The hyd genes included both young (recently diverged) and old duplicates. Some young hyd genes exhibited an initial phase of enhanced sequence evolution owing to relaxed or positive selection. There was no significant association between sequence divergence and variation in expression levels. However, three hyd genes displayed a shift in the expression levels or an altered tissue specificity following duplication. The Paxillus hyd genes evolve according to the so-called birth-and-death model in which some duplicates are maintained for a long time, whereas others are inactivated through mutations. The role of subfunctionalization and/or neofunctionalization for preserving the hyd duplicates in the genome is discussed.     

Effect of nitrogen level on acid phosphatase activity of eight isolates of ectomycorrhizal fungus Paxillus involutus cultured in vitro

The higher levels of nitrogen in ammonium form stimulated the growth of mycelia and increased the accessible as well as the total acid phosphatase activity of Paxillus involutus (Batsch) Fr. isolates grown in pure culture. Rates of mycelia growth and acid phosphatase activities varied widely from one isolate to another. Scots pine (Pinus sylvestris L.) seedlings were inoculated with different P. involutus isolates in axenic conditions. Shoots of pine seedlings with mycorrhizae contained more phosphorus than shoots of non-mycorrhizal seedlings. The relations between growth and phosphatase activity of P. involutus isolates and their efficiency in supplying the host plant with phosphate are discussed.     

Characterization and regulation of PiDur3, a permease involved in the acquisition of urea by the ectomycorrhizal fungus Paxillus involutus

Urea, which is known to be a source of nitrogen for the growth of many organisms, represents an important fertilizer in forest soils. Since most trees form symbiotic associations with ectomycorrhizal fungi, the capacities of these symbionts to take up and assimilate urea would determine the efficiency of urea nitrogen salvaging by plants. We showed that Paxillusinvolutus, an ectomycorrhizal basidiomycete, is capable of using urea as sole nitrogen source. We report the molecular characterization of an active urea transporter (PiDur3) isolated from this fungus. We demonstrated that the import of urea is a minor event on ammonium condition, since the expression of PiDUR3 is repressed by the high intracellular glutamine pool. Interestingly, on urea nutritive condition, the uptake of urea is rather mediated by the intracellular urea pool and particularly by urease efficiency.     

Characterization and regulation of PiDur3, a permease involved in the acquisition of urea by the ectomycorrhizal fungus Paxillus involutus.

Urea, which is known to be a source of nitrogen for the growth of many organisms, represents an important fertilizer in forest soils. Since most trees form symbiotic associations with ectomycorrhizal fungi, the capacities of these symbionts to take up and assimilate urea would determine the efficiency of urea nitrogen salvaging by plants. We showed that Paxillusinvolutus, an ectomycorrhizal basidiomycete, is capable of using urea as sole nitrogen source. We report the molecular characterization of an active urea transporter (PiDur3) isolated from this fungus. We demonstrated that the import of urea is a minor event on ammonium condition, since the expression of PiDUR3 is repressed by the high intracellular glutamine pool. Interestingly, on urea nutritive condition, the uptake of urea is rather mediated by the intracellular urea pool and particularly by urease efficiency.     

Variation in host specificity and gene content in strains from genetically isolated lineages of the ectomycorrhizal fungus Paxillus involutus s. lat.

Ectomycorrhizal fungi are known to vary in host range. Some fungi can enter into symbiosis with multiple plant species, while others have restricted host ranges. The aim of this study was to examine variation in host specificity among strains from the basidiomycete Paxillus involutus s. lat. Recent studies have shown that this fungus consists of at least four genetically isolated lineages, phylogenetic species (PS) I (which corresponds to the morphological species Paxillus obscurosporus), PS II (P. involutus s. str.), PS III (Paxillus validus), and PS IV (not yet supported by any reference material). Thirty-five Paxillus strains of PS I to IV were examined in microcosms for their capacity to infect birch (Betula pendula) and spruce (Picea abies). Seventeen strains were compatible and formed mycorrhizae with both tree species. Seven strains were incompatible with both birch and spruce. The gene content in three pairs of incompatible and compatible strains PS I, II, and III were compared using microarray-based comparative genomic hybridizations. Of 4,113 P. involutus gene representatives analyzed, 390 varied in copy numbers in at least one of the three pairwise comparisons. Only three reporters showed significant changes in all three pairwise comparisons, and none of these were changed in a similar way in three comparisons. Our data indicate that changes in host range have occurred frequently and independently among strains in P. obscurosporus, P. involutus s. str., and P. validus. No evidence was obtained demonstrating that these changes have been associated with the gain or loss of similar genes in these three species.     

Cross-colonization of Scots pine (Pinus sylvestris) seedlings by the ectomycorrhizal fungus Paxillus involutus in the presence of inhibitory levels of Cd and Zn

The effects of Cd and Zn on cross-colonization by Paxillus involutus of Scots pine seedlings was examined by using pairs of ectomycorrhizal (ECM) and non-mycorrhizal (NM) seedlings grown in the same vessel. This was done to assess, first, the ability of P. involutus to colonize NM Scots pine seedlings by growth from colonized roots of other Scots pine seedlings in the presence of Cd or Zn, and, second whether ECM colonization of Scots pine by P. involutus provided a competitive advantage over NM seedlings. Ectomycorrhizal colonization of Scots pine was shown to be more sensitive than Scots pine itself to Cd and Zn, but prior colonization did provide a competitive advantage with respect to biomass production. This beneficial effect over NM seedlings was, however, equal in the control, Cd and Zn treatments, and was due simply to growth stimulation in the presence of ECM colonization. Cross-colonization from an ECM to a NM seedling was reduced but not prevented by Cd and Zn. Cd had a more negative effect on cross-colonization than on initial colonization of seedlings, whereas Zn had an equally inhibitory effect on both parameters. These results have important implications for plant establishment on metal-contaminated sites. If cross-colonization between plants is reduced by toxic metals, plant establishment on contaminated sites might be retarded.     

Changes in carbon allocation and expression of carbon transporter genes in Betula pendula Roth. colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr.

Comparative analyses of aspects of the carbon (C) physiology and the expression of C transporter genes in birch (Betula pendula Roth.) colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. were performed using mycorrhizal (M) and non‐mycorrhizal (NM) plants of similar foliar nutrient status. After six months of growth, the biomass of M plants was significantly lower than that of NM plants. Diurnal C budgets of both sets of plants revealed that M plants exhibited higher rates of photosynthesis and root respiration expressed per unit dry weight. However, the diurnal net C gain of M and NM plants remained similar. Ectomycorrhizal roots contained higher soluble carbohydrate pools and increased activity of cell wall invertase, suggesting that additional C was allocated to these roots and their ectomycorrhizal fungi consistent with an increased sink demand for C due to the presence of the mycobiont. In M roots, the expression of two hexose and one sucrose transporter genes of birch were reduced to less than one‐third of the expression level observed in NM roots. Analysis using a probe against the birch ribosomal internal transcribed spacer region revealed that M roots contained 22% less plant RNA than NM roots. As the expression of birch hexose and sucrose transporter genes was reduced to a much greater extent, this suggests that these specific genes were down‐regulated in response to alterations in C metabolism within M roots.     

Decomposition of cellulose,soil organic matter and plant litter in a temperate grassland soil

The formation of mor humus in an experimental grassland plot, which has been acidified by long-term fertiliser treatment, has been studied by comparing the rates of cellulose, soil organic matter and plant litter decay with those in an adjacent plot with near-neutral pH and mull humus. The decomposition of cellulose filter paper in litter bags of 5 mm, 1-mm and 45-μm mesh size buried at 3 to 4 cm depth the plots was followed by measuring the weight loss and changes in glucose content over a 6 month period. Soil pH was either 5.3 or 4.3. Decomposition of native soil organic matter and plant litter in soil from the same plots were followed using CO₂ evolution in laboratory microcosms. Cellulose weight loss at pH 5.3 was greatest from the 5-mm mesh bags and least from the 45-um mesh bags. At pH 4.3 there was little weight loss from bags and no significant differences in weight loss between bags with different sized mesh. There was, however, a reduction in the glucose content of the hydrolysed and derivatised filter paper with time. The decomposition rate of native soil organic matter in the low pH soil was increased to that observed in the less acid soil when the pH of the former was increased from 4.3 to 5.3. The increase in decomposition rate of added plant litter in the more acid soil as a result of CA(OH)₂ addition was only 60% of that observed in the soil with pH 5.3. These data support the hypothesis that the absence of soil animals and the restricted microbial decomposition in the acidic soil was responsible for mor humus formation.     

Spatial patterns of gene expression in the extramatrical mycelium and mycorrhizal root tips formed by the ectomycorrhizal fungus Paxillus involutus in association with birch (Betula pendula) seedlings in soil microcosms

Functional compartmentation of the extramatrical mycelium of ectomycorrhizal (ECM) fungi is considered important for the operation of ECM associations, although the molecular basis is poorly characterized. Global gene expression profiles of mycelium colonizing an ammonium sulphate ((NH4)2SO4) nutrient patch, rhizomorphs and ECM root tips of the Betula pendula-Paxillus involutus association were compared by cDNA microarray analysis. The expression profiles of rhizomorphs and nutrient patch mycelium were similar to each other but distinctly different from that of mycorrhizal tips. Statistical analyses revealed 337 of 1075 fungal genes differentially regulated among these three tissues. Clusters of genes exhibiting distinct expression patterns within specific tissues were identified. Genes implicated in the glutamine synthetase/glutamate synthase (GS/GOGAT) and urea cycles, and the provision of carbon skeletons for ammonium assimilation via beta-oxidation and the glyoxylate cycle, were highly expressed in rhizomorph and nutrient patch mycelium. Genes implicated in vesicular transport, cytoskeleton organization and morphogenesis and protein degradation were also differentially expressed. Differential expression of genes among the extramatrical mycelium and mycorrhizal tips indicates functional specialization of tissues forming ECM associations.