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.     

Ammonium-assimilating enzymes and their regulation in wild and NADP-glutamate dehydrogenase-deficient strains of the ectomycorrhizal fungus Hebeloma cylindrosporum

Hebeloma cylindrosporum strain h 17 was grown on media containing either glutamate or ammonium as nitrogen source. Growth tests and in vitro activity measurements revealed that both glutamine synthetase (GS. EC 6.3.1.2) and NADP-specific glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) are fully functional in wild type mycelia grown on glutamate or ammonium as sole nitrogen source. However, NADP-GDH appeared to be more active than GS in stationary growing mycelia. NADP-GDH is also able to sustain adequate ammonium assimilation in methionine sulfoximine (MSX)-treated mycelia since they grew as well as mycelia fed with ammonium alone. The NADP-GDH also appeared to be L-glutamate inducible whereas GS was repressed by ammonium. The NADP-GDH deficient strain, when transferred from a glutamate containing medium to an ammonium containing medium, exhibited a derepressed GS, although this enzyme did not fully substitute for the deficiency of NADP-GDH in ammonium assimilation. The low NADP-GDH activity of the mutant strain exhibited a reduced mobility on a 6% constant polyacrylamide gel. By contrast, the two enzymes had identical molecular weights, estimated to be ca 295 kDa on gradient polyacrylamide gel. The involvement of NADP-GDH and GS enzymes in nitrogen assimilation is discussed.     

Peptide uptake in the ectomycorrhizal fungus Hebeloma cylindrosporum: characterization of two di- and tripeptide transporters (HcPTR2A and B)

Constraints on plant growth imposed by low availability of nitrogen are a characteristic feature of ecosystems dominated by ectomycorrhizal plants. Ectomycorrhizal fungi play a key role in the N nutrition of plants, allowing their host plants to access decomposition products of dead plant and animal materials. Ectomycorrhizal plants are thus able to compensate for the low availability of inorganic N in forest ecosystems. The capacity to take up peptides, as well as the transport mechanisms involved, were analysed in the ectomycorrhizal fungus Hebeloma cylindrosporum. The present study demonstrated that H. cylindrosporum mycelium was able to take up di- and tripeptides and use them as sole N source. Two peptide transporters (HcPTR2A and B) were isolated by yeast functional complementation using an H. cylindrosporum cDNA library, and were shown to mediate dipeptide uptake. Uptake capacities and expression regulation of both genes were analysed, indicating that HcPTR2A was involved in the high-efficiency peptide uptake under conditions of limited N availability, whereas HcPTR2B was expressed constitutively.     

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 NH₄¹⁵NO₃ medium was studied for two mutant strains and their corresponding wild-type homokaryons. None of the mutants could use nitrate whereas ¹⁵N 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.     

Microsatellite markers for Hebeloma species developed from expressed sequence tags in the ectomycorrhizal fungus Hebeloma cylindrosporum

The increasing number of expressed sequence tag (EST) projects dedicated to ectomycorrhizal fungi is translating into the release of large sets of ESTs. The aim of this study was to develop and test simple sequence repeat (SSR) markers from EST databases of the model ectomycorrhizal fungus Hebeloma cylindrosporum. Six SSR markers were found to be both unambiguously scorable and polymorphic among 12 H. cylindrosporum isolates. Two SSR markers were transferable to other Hebeloma species and one marker was interestingly found to be polymorphic among seven H. crustuliniforme isolates.     

Molecular and functional characterization of a Na(+)-K(+) transporter from the Trk family in the ectomycorrhizal fungus Hebeloma cylindrosporum

Ectomycorrhizal symbiosis between fungi and woody plants strongly improves plant mineral nutrition and constitutes a major biological process in natural ecosystems. Molecular identification and functional characterization of fungal transport systems involved in nutrient uptake are crucial steps toward understanding the improvement of plant nutrition and the symbiotic relationship itself. In the present report a transporter belonging to the Trk family is identified in the model ectomycorrhizal fungus Hebeloma cylindrosporum and named HcTrk1. The Trk family is still poorly characterized, although it plays crucial roles in K(+) transport in yeasts and filamentous fungi. In Saccharomyces cerevisiae K(+) uptake is mainly dependent on the activity of Trk transporters thought to mediate H(+):K(+) symport. The ectomycorrhizal HcTrk1 transporter was functional when expressed in Xenopus oocytes, enabling the first electrophysiological characterization of a transporter from the Trk family. HcTrk1 mediates instantaneously activating inwardly rectifying currents, is permeable to both K(+) and Na(+), and displays channel-like functional properties. The whole set of data and particularly a phenomenon reminiscent of the anomalous mole fraction effect suggest that the transport does not occur according to the classical alternating access model. Permeation appears to occur through a single-file pore, where interactions between Na(+) and K(+) might result in Na(+):K(+) co-transport activity. HcTrk1 is expressed in external hyphae that explore the soil when the fungus grows in symbiotic condition. Thus, it could play a major role in both the K(+) and Na(+) nutrition of the fungus (and of the plant) in nutrient-poor soils.     

Functional expression of the green fluorescent protein in the ectomycorrhizal model fungus Hebeloma cylindrosporum

Hebeloma cylindrosporum is a model fungus for mycorrhizal studies because of its fast growth rate, simple nutritional requirements, and completion of its life cycle in vitro, and because it is amenable to transformation. To advance cell biological research during establishment of symbiosis, a tool that would enable the direct visualisation of fusion proteins in the different symbiotic tissues [namely, the expression of reporter genes such as Green Fluorescent Protein (GFP)] was still a missing tool. In the present study, H. cylindrosporum was transformed using Agrobacterium carrying the binary plasmid pBGgHg containing the Escherichia coli hygromycin B phosphotransferase (hph) and the EGFP genes, both under the control of the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter. EGFP expression was successfully detected in transformants. The fluorescence was uniformly distributed in the hyphae, while no significant background signal was detected in control hyphae. The suitability of EGFP for reporter gene studies in Hebeloma cylindrosporum was demonstrated opening up new perspectives in the Hebeloma genetics.Tobias Müller and Mariam Benjdia contributed equally to this work.     

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.     

Genetical variability of glutamate dehydrogenase activity in monokaryotic and dikaryotic mycelia of the ectomycorrhizal fungus Hebeloma cylindrosporum

Summary Glutamate dehydrogenase (GDH) is the key enzyme of ammonium assimilation by ectomycorrhizal fungi. Its activity might be use as a criterion to select mycelia capable of enhancing the nitrogen nutrition of the host plants. Genetical variability of the GDH activity of the ectomycorrhizal fungus Hebeloma cylindrosporum Romagnési was studied in an attempt to determine if this enzyme activity could be improved by way of chromosomal genetics. The activity of 11 wild strains was compared with that of 70 mycelia obtained as the progeny of a laboratory fruiting strain HC1. These 70 mycelia were 20 monokaryons (5 for each mating type) and the 50 synthesized dikaryons obtained from all the compatible fusions between these monokaryons. The specific GDH activity of the 11 wild strains ranged from 1.5 to 11.6 nkat mg^-1 fungal protein. The activity of the monokaryotic progeny of the HC1 strain was, on average, three times lower (2.85 n kat mg^-1 fungal protein) than that of the parental dikaryon. In contrast, synthesized dikaryons originating from these monokaryons were very variable and had an average values similar to that of the parental dikaryon (9.1 nkat mg^-1 fungal protein). Eighteen of these synthesized dikaryons contained an activity higher than that of the original HC1 strain. The variation of the GDH activity of these dikaryons involves additive and non additive (interactive) components, each of them accounting for ca. 50% of the genetical variation. The non additive variation could not be explained by a model involving only dominance. These results are discussed with reference to the genetical improvement of mycorrhizal fungi in order to enhance nitrogen nutrition of the host plants.Abbreviations GDH glutamate dehydrogenase - IAA indole-3-acetic acid - NADP nicotimamide adenine dinucleotide phosphate     

Fine-scale structure of populations of the ectomycorrhizal fungus Hebeloma cylindrosporum in coastal sand dune forest ecosystems

The basidiomycete mushroom Hebeloma cylindrosporum is a frequently found pioneer ectomycorrhizal species naturally associated with Pinus pinaster trees growing in coastal sand dune ecosystems along the Atlantic south-west coast of France. The genotypic diversity and spatial structure of three populations of this fungal species have been studied. At each site the basidiocarps were mapped, sampled and propagated as pure mycelial cultures. For each of the isolates, we have studied polymorphisms in the mitochondrial genome, polymorphisms at two different nuclear loci and also fingerprints produced with a multicopy DNA probe. The comparison of the different polymorphisms obtained, with each of the four molecular methods used, allowed the identification of several of the different genets present in each site. In two of the studied sites most of the basidiocarps, which often occurred as dense patches of 10–30 in 1 m² or less, were of a unique genotype, suggesting the below-ground mycelia to be of a small size (from 50 cm² to approx. 7 m² for the larger mycelia) and that the root system of a single Pinus tree can host several genets of the same symbiotic fungus. In the two sites, which were studied again after a 3-year interval, none of the genotypes identified in the first year of sampling was re-identified 3 years later. These results contrast with those reported for other species of soilborne homobasidiomycete species, either ectomycorrhizal, parasitic or saprophytic, showing mostly large clones resulting from the vegetative growth and from persistence of below-ground mycelia. Sexual reproduction through meiospore dispersal seems to play a key role in the structuring of the populations of H. cylindrosporum. Mycelia associated with the root systems seem to be replaced after 1 or a few years, during which basidiocarp differentiation takes place. As opposed to the few other studied ectomycorrhizal species, H. cylindrosporum has the characteristics of ruderal species, with a short life-span adapted to pioneer situations, e.g. to nutrient-poor and unstable sandy soils of coastal sand dunes.     

Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum

We transformed haploid mycelium of Hebeloma cylindrosporum via Agrobacterium tumefaciens and optimised the procedure to develop a new tool for insertional mutagenesis in this fungus. Southern blot analysis of 83 randomly selected transformants showed that they all contained plasmid inserts. Each of them showed a unique hybridisation pattern, suggesting that integration was random in the fungal genome. Sixty percent of transformants obtained in the presence of bacteria pre-treated with acetosyringone integrated a single transferred DNA copy. Thermal asymmetric interlaced polymerase chain reaction allowed us to recover the left border and the right border junctions in 85% and 15% of transformants analysed, respectively. Results show that A. tumefaciens-mediated transformation may be a powerful tool for insertional mutagenesis in H. cylindrosporum.     

Correspondence between genet diversity and spatial distribution of above- and below-ground populations of the ectomycorrhizal fungus Hebeloma cylindrosporum

Population studies of ectomycorrhizal fungal species have largely relied upon fruit body (the reproductive organ) sampling. Analysis of the fruit bodies alone supposes that they reflect the present and spatial organization of all below-ground genets (mycorrhizas and extramatrical mycelia). The relation between fruit bodies and ectomycorrhizas was investigated for the basidiomycete agaric Hebeloma cylindrosporum in four Pinus pinaster stands in south-west France. Genet identification was based on the comparison of polymorphisms within a hypervariable segment of the ribosomal intergenic spacer amplified by polymerase chain reaction (PCR) using a H. cylindrosporum species-specific primer. Mycorrhizas were sorted from soil samples collected underneath patches of fruit bodies or patches where fruit bodies had or had not been observed during the years prior to mycorrhiza collection. On average 65% of the 1026 mycorrhizas collected underneath fruit bodies were formed by H. cylindrosporum, whereas only 2% of the 954 collected in places from where fruit bodies were absent were formed by this species. All genotypes identified above ground were also identified below ground. In patches where one genotype formed all or more than 90% of the fruit bodies, the same genotype formed all or a large majority of the mycorrhizas. In patches occupied by several different fruiting genotypes, additional nonfruiting ones could be present on the root systems. In all cases, the mycorrhizas of one genotype were found no more than 10-20 cm away from its corresponding fruit bodies, and fruit body disappearance at a given place was associated with the disappearance of the corresponding mycorrhizas within 1 year. Although there was not a strict coincidence between the total numbers of genets present below ground and of those forming fruit bodies, fruit body analysis for H. cylindrosporum appears to reflect both the genetic diversity and the spatial structure of its below-ground populations. The results obtained also illustrate the rapid turnover of ectomycorrhizal fungal species on the root systems in the absence of any obvious major disturbance of the ecosystem.     

Rooting and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris are enhanced by the ectomycorrhizal fungus Hebeloma cylindrosporum

The effect of different strains of the ectomycorrhizal fungus Hebeloma cylindrosporum on rooting in vitro and acclimatization of micropropagated cuttings of Pinus pinaster and Pinus sylvestris was studied. Two clones of P. pinaster and one of P. sylvestris were unable to root in the absence of auxin, but were induced to root on a medium devoid of auxin by all the fungal strains. Wild-type and indoleacetic acid (IAA)-overproducing mutant strains of the fungus stimulated rooting of clones showing a good reactivity to auxin to the same extent. In contrast, with a clone of P. sylvestris that showed low reactivity to auxin, IAA-overproduction by the fungus was advantageous for the induction of rooting of cuttings. Adventitious roots formed in the presence of a fungal strain were completely surrounded by a loosely packed network of hyphae which formed mycorrhizas as soon as roots grew outside the agar medium. During acclimatization, fungal inoculation improved the survival of rooted cuttings. At the end of acclimatization, fungal mycelia could be easily detected in the culture substrate of cuttings inoculated with dikaryotic strains and most of the pines' short roots were mycorrhizal. Monokaryotic mycelia, which have a lower growth rate and a lower infectivity, displayed poor ability to colonize the substrate and to form mycorrhizas. Two months after the end of acclimatization, fungal inoculation frequently depressed the growth of acclimatized cuttings of the clone J of P. pinaster . No depressive effect was observed with clone 78 and growth stimulation could even be observed with the infective dikaryon D1 which formed numerous mycorrhizas. From these studies, it was concluded that ectomycorrhizal fungi could be a suitable tool for improving rooting in vitro and survival at acclimatization of micropropagated conifer cuttings.