Expression of the nitrate transporter nrt2 gene from the symbiotic basidiomycete Hebeloma cylindrosporum is affected by host plant and carbon sources.

Although the function of the extramatrical mycelium of ectomycorrhizal fungi is considered essential for the acquisition of nitrogen by forest trees, gene regulation in this fungal compartment is poorly characterized. In this study, the expression of the nitrate transporter gene nrt2 from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum was shown to be regulated by plant host and carbon sources. In the presence of a low fructose concentration, nrt2 expression could not be detected in the free-living mycelium but was high in the extramatrical symbiotic mycelium associated to the host plant Pinus pinaster. In the absence of nitrogen or in the presence of nitrate, high sugar concentrations in the medium were able to enhance nrt2 expression. Nevertheless, in the presence of high fructose concentration, high ammonium concentration still completely repressed nrt2 expression indicating that the nitrogen repression overrides sugar stimulation. This is the first report revealing an effect of host plant and of carbon sources on the expression of a fungal nitrate transporter-encoding gene.

Soil eukaryotic functional diversity, a metatranscriptomic approach.

To appreciate the functional diversity of communities of soil eukaryotic micro-organisms we evaluated an experimental approach based on the construction and screening of a cDNA library using polyadenylated mRNA extracted from a forest soil. Such a library contains genes that are expressed by each of the different organisms forming the community and represents its metatranscriptome. The diversity of the organisms that contributed to this library was evaluated by sequencing a portion of the 18S rDNA gene amplified from either soil DNA or reverse-transcribed RNA. More than 70% of the sequences were from fungi and unicellular eukaryotes (protists) while the other most represented group was the metazoa. Calculation of richness estimators suggested that more than 180 species could be present in the soil samples studied. Sequencing of 119 cDNA identified genes with no homologues in databases (32%) and genes coding proteins involved in different biochemical and cellular processes. Surprisingly, the taxonomic distribution of the cDNA and of the 18S rDNA genes did not coincide, with a marked under-representation of the protists among the cDNA. Specific genes from such an environmental cDNA library could be isolated by expression in a heterologous microbial host, Saccharomyces cerevisiae. This is illustrated by the functional complementation of a histidine auxotrophic yeast mutant by two cDNA originating possibly from an ascomycete and a basidiomycete fungal species. Study of the metatranscriptome has the potential to uncover adaptations of whole microbial communities to local environmental conditions. It also gives access to an abundant source of genes of biotechnological interest.

How does a symbiotic fungus modulate expression of its host-plant nitrite reductase?

* In the mycorrhizal association, changes in the metabolic activities expressed by the plant and fungal partners could result from modulations in the quantity and nature of nutrients available at the plant-fungus interface. This hypothesis was tested for the nitrite reductase gene in the association Hebeloma cylindrosporumxPinus pinaster. * Transcripts from plant and fungal nitrite reductases and a fungal ammonium transporter were quantified in control uninoculated roots, extraradical mycelia and mycorrhizas formed by either wild-type or nitrate reductase deficient fungal strains. * The fungal genes were downregulated in mycorrhizas compared with extraradical hyphae. The plant nitrite reductase was induced only transiently by NO(3)(-) in the association with a wild-type strain, but permanently expressed at a high level in mycorrhizas formed by the deficient mutant. * These results suggest that reduced nitrogen compounds transferred from the fungus to the root cortical cells repress the plant nitrite reductase, thus highlighting a plant gene regulation by the nutrients available in the Hartig net.

Green fluorescent protein expression in the symbiotic basidiomycete fungus Hebeloma cylindrosporum.

The symbiotic basidiomycete Hebeloma cylindrosporum is a model fungal species used to study ectomycorrhizal symbiosis at the molecular level. In order to have a vital marker, we developed a green fluorescent protein (GFP) reporter system efficiently expressed in H. cylindrosporum using the sgfp coding region bordered by two introns fused to the saprophytic basidiomycete Coprinopsis cinerea cgl1 promoter. Expression of this reporter system was tested under different environmental conditions in two transformants, and glucose was shown to repress gfp expression. Such a reporter system will be used in plant-fungus interaction to evaluate sugar supply by the plant to the compatible mycorrhizal symbiont and to compare the expression of various genes of interest in the free-living mycelia, in the symbiotic (mycorrhizas) and the reproductive (fruit bodies) structures formed by H. cylindrosporum.

Intraspecific variation in use of different organic nitrogen sources by the ectomycorrhizal fungus Hebeloma cylindrosporum.

The ectomycorrhizal (ECM) fungus Hebeloma cylindrosporum is an appropriate model to study the intraspecific functional diversity of ECM fungi in forest ecosystems. Numerous metabolic genes, specifically genes related to nitrogen assimilation, have been characterised for this species and the spatial and temporal structures of its natural populations have been extensively worked out. In this paper, we reveal the extent to which intraspecific variation exists within this fungus for the ability to use organic nitrogen, an important functional characteristic of ECM fungi. In addition to ammonium and nitrate, H. cylindrosporum can use at least 13 different amino acids out of 21 tested as sole nitrogen source, as well as urea and proteins. By screening 22 genetically different wild type haploid strains we identified obvious differences in use of six nitrogen sources: alanine, glycine, phenylalanine, serine, bovine serum albumin and gelatine. Of the 22 haploid strains, 11 could not use at least one of these six nitrogen sources. The inability of some haploid strains to use a nitrogen source was found to be a recessive character. Nevertheless, obvious differences in use of the four amino acids tested were also measured between wild type dikaryons colonising a common Pinus pinaster root system. This study constitutes the basis for future experiments that will address the consequences of the functional diversity of an ECM fungus on the functioning of the ECM symbiosis under natural conditions.

Below-ground distribution and persistence of an ectomycorrhizal fungus.

• In most studies, the distribution of the mycelia of ectomycorrhizal fungi in forest soils has been inferred from areas occupied by fruit bodies. Here, we investigated the below-ground spatio-temporal distribution of Hebeloma cylindrosporum by polymerase chain reaction quantification of a specific DNA sequence present in DNA extracted from soil. • Soil samples were collected in a Pinus pinaster stand located in a campsite where fruit bodies of H. cylindrosporum had been mapped from 1990 to 2000. • In samples collected underneath fruit bodies, DNA of H. cylindrosporum was always detected in large amounts. However, this DNA was often undetectable in the absence of fruit body even 1 yr after their disappearance. This result was supported by the failure to identify mycorrhizas of this species on seedlings planted in soil samples collected in places where fruit bodies were present 1 yr before sampling. • This pattern suggests a recolonization of the site each year by H. cylindrosporum basidiospores in 'receptive' patches of ground created either by the local elimination of competitors or by local nutrient enrichments, which could frequently occur in a campsite. Our results demonstrate that an ectomycorrhizal species can be completely eliminated from the roots within 1 yr and does not necessarily contribute to the next generation of mycorrhizas.

Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum.

Symbiotic ectomycorrhizal fungi contribute to the nitrogen nutrition of their host-plants but little information is available on the molecular control of their nitrogen metabolism. We cloned and characterised genes encoding a nitrite reductase and a nitrate transporter in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. These two genes are divergently transcribed and linked to a previously cloned nitrate reductase gene, thus demonstrating that nitrate assimilation gene clusters occur in homobasidiomycetes. The nitrate transporter polypeptide (NRT2) is characterised by 12 transmembrane domains and presents both a long putative intracellular loop and a short C-terminal tail, two structural features which distinguish fungal high-affinity transporters from their plant homologues. In different wild-type genetic backgrounds, transcription of the two genes was repressed by ammonium and was strongly stimulated not only in the presence of nitrate but also in the presence of organic nitrogen sources or under nitrogen deficiency.

Spatial distribution of the below-ground mycelia of an ectomycorrhizal fungus inferred from specific quantification of its DNA in soil samples.

In natural forest ecosystems several ectomycorrhizal fungal species cohabit on host plant root systems. To evaluate the ecological and functional impact of each species, it is necessary to appreciate the distribution and abundance of its mycelia in the soil. We developed a competitive PCR (cPCR) method for the basidiomycete Hebeloma cylindrosporum that allows quantification of its DNA in complex DNA mixtures extracted directly from soil samples. The target sequence chosen for the cPCR analysis was a 533-bp fragment of the nuclear ribosomal intergenic spacer, amplified using two species-specific primers. The detection threshold of the cPCR protocol developed was 0.03 pg of genomic DNA. This method was applied to soil samples collected from beneath and at various distances from a group of fruit bodies in a Pinus pinaster forest stand. The results revealed that H. cylindrosporum below-ground biomass was concentrated directly underneath the fruit bodies or very close to them, while no DNA of this species could be detected in soil samples collected at more than 50 cm away. In the vicinity of fruit bodies, H. cylindrosporum soil DNA concentration varied considerably (between 10 and 0.07 ng g soil(-1)) and decreased sharply with increased distance from the fruit bodies. This work demonstrates the potential of competitive quantitative PCR for the study of the distribution, abundance and persistence of the mycelia of an ectomycorrhizal fungal species in soil.

Isolation and characterization of nitrate reductase deficient mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum.

To clarify the role of the fungal nitrate assimilation pathway in nitrate reduction by mycorrhizal plants, nitrate reductase (NR)-deficient (NR- ) mutants of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum Romagnesi have been selected. These mutants were produced by u.v. mutagenesis on protoplasts originating from homokaryotic mycelia belonging to complementary mating types of this heterothallic tetrapolar species. Chlorate-resistant mutants were first selected in the presence of different nitrogen (N) sources in the culture medium. Among 1495 chlorate resistant mycelia, 30 failed to grow on nitrate and lacked a detectable NR activity. Growth tests on different N sources suggested that the NR activity of all the different mutants is specifically impaired as a result of mutations in either the gene coding for NR apoprotein or genes controlling the synthesis of the molybdenum cofactor. Furthermore, restoration of NR activity in some of the dikaryons obtained after crosses between the different mutant mycelia suggested that not all the selected mutations mapped in the same gene. Utilization of N on a NH4 15 NO3 medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas 15 N enrichment values indicated that 13-27% of N present in 13-d-old wild-type mycelia originated from nitrate. Apparently, the mutant mycelia do not compensate their inability to use nitrate by a more efficient use of ammonium. These different NR mutants still form mycorrhizas with the habitual host plant, Pinus pinaster (Ait.), making them suitable for study of the contribution of the fungal nitrate assimilation pathway to nitrate assimilation by mycorrhizal plants.

The ectomycorrhizal basidiomycete Hebeloma circinans harbors a linear plasmid encoding a DNA- and RNA polymerase.

Bulk DNA isolated from the ectomycorrhizal basidiomycete Hebeloma circinans was treated with proteinase K and submitted to agarose gel electrophoresis. In addition to high molecular weight genomic DNA, three minor bands were detected. The band with the highest electrophoretic mobility (2.2 kbp) corresponds to double-stranded RNA. The two other bands, termed pHC1 and pHC2, were shown to be dsDNA molecules of 10.3 and 9.1 kbp, respectively. Treatment of the pHC elements with 3'- and 5'-specific exonucleases revealed a linear structure and proved that the 5' ends are protected from digestion; for pHC2, linearity was confirmed by restriction mapping. A 3.2 kbp HindIII fragment of pHC2 was cloned and sequenced; it contains two open reading frames encoding putative viral B type DNA and RNA polymerases. Thus, the fungus harbors a typical linear plasmid, up to now, rarely described for basidiomycetes and hitherto unknown for mycorrhizal species.