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.     

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.     

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.     

The evolution of reproductive isolation in the ectomycorrhizal Hebeloma crustuliniforme aggregate (Basidiomycetes) in northwestern Europe: a phylogenetic approach

Abstract. To reconstruct the evolution of reproductive isolation in the ectomycorrhizal Hebeloma crustuliniforme aggregate (Basidiomycetes), phylogenetic relationships were determined between strains that belong to a clade consisting of nine intercompatibility groups (ICGs, biological species). Four of these nine ICGs are partially compatible and belong to the H. crustuliniforme aggregate. Different levels of partial compatibility have been found between these four ICGs. Between ICGs 3 and 4, 15% of the combinations were compatible. One strain was compatible with all isolates of both ICGs 3 and 4 and also with one isolate of ICG 2. Both a nuclear phylogeny, based on ribosomal IGS sequence data, and a mitochondrial phylogeny, based on a group-I intron located in the large subunit ribosomal RNA gene (LrRNA), were reconstructed. The level of incompatibility was compared with the phylogenetic history of individuals belonging to this clade.
Different relationships were found between the level of compatibility and the relative age of the most recent common ancestor (MRCA) for different ICGs. On the one hand, the evolution of incompatibility between ICGs 2 and 3/4 is most consistent with the class of "divergence- first" models because a positive correlation was found between the relative age of the MRCA and the level of incompatibility for ICG 2 versus 3/4. On the other hand, the lack of such a correlation for ICGs 3 and 4 shows that (partial) incompatibility between these ICGs has arisen without strong divergence. The ecological (and to a lesser extent geographical) differences found between ICGs 3 and 4 suggest that selection for incompatibility, associated with host tree preference, has been important in the evolution of incompatibility between these two ICGs. The incongruence between the nuclear and mitochondrial trees for ICG 1 could be explained by a hybrid origin of this ICG, with different donors of the mitochondrial and nuclear sequences.     

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.     

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.     

Effect of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of arbuscular mycorrhiza-forming Prunus avium and Prunus cerasus

 The effect of different genotypes of the ectomycorrhizal fungus Hebeloma cylindrosporum on in vitro rooting of micropropagated cuttings of Prunus avium and P. cerasus was studied in an attempt to determine whether ectomycorrhizal fungi could enhance in vitro adventitious root formation in plants which form arbuscular endomycorrhizas. The rooting percentage of P. avium cuttings was approximately 16% in the absence of hormonal treatment; it increased up to 30% in the presence of 5.7 μM IAA which was the most favourable auxin concentration. The rooting percentage of cuttings cultivated in the absence of IAA was enhanced by all the studied strains of H. cylindrosporum. It ranged from 50 to 60% with the IAA-overproducing mutant D 111 or the wild-type dikaryon D1, to 100% in the presence of the mutants 331 or D 117. The cuttings of P. cerasus showed a higher rooting ability than those of P. avium since approximately 40% of them were able to root in the absence of hormonal treatment. Except for the mutant D117, their rooting percentage was not significantly improved by H. cylindrosporum. Fungal inoculation also affected the survival of cuttings at acclimatization: 50% of the uninoculated P. avium cuttings survived whereas the survival percentage of inoculated cuttings ranged from 30 to 100% depending on the fungal genotype. With P. cerasus, the percentage of survival of uninoculated cuttings ranged from 85 to 100% and fungi either did not significantly improve it or lowered it. At acclimatization fungal hyphae could be observed in close contact with adventitious roots, but they did not establish mycorrhizal association. The shoot height of P. avium plantlets obtained from inoculated cuttings was not significantly different from that of plantlets originating from uninoculated ones. By contrast, fungal inoculation generally depressed the growth of acclimatized P. cerasus plantlets. The possibility of using ectomycorrhizal fungi as a tool to enhance rooting of micropropagated cuttings of plants which do not form ectomycorrhizas is discussed. Received: 25 November 1996 / Accepted: 2 June 1997     

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.     

Viability of ectomycorrhizal fungus mycelium entrapped in calcium alginate gel

 We studied the viability of fragmented mycelium of Pisolithus tinctorius and Paxillus involutus entrapped in calcium alginate gel to determine the efficacy of this method of producing ectomycorrhizal fungus inoculum. Fungi were grown in MMN solution at 28  °C before being fragmented in a blender and subsequently entrapped in calcium alginate. We tested different ratios of alginate and mycelium suspension to 0.7 M CaCl₂. The ratio 8 : 10 resulted in well-formed beads of the highest viability for Paxillus involutus (99%) and for Pisolithus tinctorius (75%). Paxillus involutus mycelium was more than 90% viable when entrapped mycelium was 10 to 50 days old, and Pisolithus tinctorius attained its highest viability (55%) for 20- to 40-day-old mycelium. Gel entrapped Paxillus involutus mycelium grew well at all temperatures after 30 days of storage, but viability significantly decreased after 60 days storage at 6  °C on dry filter paper. For gel-entrapped Pisolithus tinctorius mycelium, viability was highest when stored at 25  °C in 0.7 M CaCl₂. Entrapment of Paxillus involutus fragmented mycelium in calcium alginate beads under the conditions that we propose can be used successfully to produce inoculum. Accepted: 11 October 1998     

Extracellular complexation of Cd in the Hartig net and cytosolic Zn sequestration in the fungal mantle of Picea abies – Hebeloma crustuliniforme ectomycorrhizas

Compartmentation of heavy metals on or within mycorrhizal fungi may serve as a protective function for the roots of forest trees growing in soils containing elevated concentrations of metals such as Cd and Zn. In this paper we present the first quantitative measurements by X‐ray microanalysis of heavy metals in high‐pressure frozen and cryosectioned ectomycorrhizal fungal hyphae. We used this technique to analyse the main sites of Cd and Zn in fungal cells of mantle and Hartig net hyphae and in cortical root cells of symbiotic Picea abies – Hebeloma crustuliniforme associations to gain new insights into the mechanisms of detoxification of these two metals in Norway spruce seedlings. The mycorrhizal seedlings were exposed in growth pouches to either 1 mM Cd or 2 mM Zn for 5 weeks. The microanalytical data revealed that two distinct Cd‐ and Zn‐binding mechanisms are involved in cellular compartmentation of Cd and Zn in the mycobiont. Whereas extracellular complexation of Cd occurred predominantly in the Hartig net hyphae, both extracellular complexation and cytosolic sequestration of Zn occurred in the fungal tissue. The vacuoles were presumed not to be a significant pool for Cd and Zn storage. Cadmium was almost exclusively localized in the cell walls of the Hartig net (up to 161 mmol kg ^{? 1} DW) compared with significantly lower concentrations in the cell walls of mantle hyphae (22 mmol kg ^{? 1} DW) and in the cell walls of cortical cells (15 mmol kg ^{? 1} DW). This suggests that the apoplast of the Hartig net is a primary accumulation site for Cd. Zinc accumulated mainly in the cell walls of the mantle hyphae (111 mmol kg ^{? 1} DW), the Hartig net hyphae (130 mmol kg ^{? 1} DW) and the cortical cells (152 mmol kg ^{? 1} DW). In addition, Zn occurred in high concentrations in the cytoplasm of the fungal mantle hyphae (up to 164 mmol kg ^{? 1} DW) suggesting that both the cell walls and the cytoplasm of fungal tissue are the main accumulation sites for Zn in P. abies resulting in decreased Zn transfer from the fungus to the root.     

Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes

Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of Cl^– into the medium, followed by a K⁺ efflux; (ii) an influx of Ca²⁺ (measured as accumulation of ⁴⁵Ca²⁺ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H₂O₂. The removal of extracellular Ca²⁺ by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca²⁺ release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions.Abbreviations DW dry weight - FW fresh weight - TMB-8 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octyl ester We thank Prof. M. Zenk (Universität München, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universität Tübingen, Germany) for isolates of Hebeloma crustuliniforme Tü 704. We are also thankful to Dr. W. Mayer (Universität Tübingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by Graduiertenkolleg Interaktion in Waldökosystemen (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenförderungsgesetz.