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Keywords:

  • cell wall;
  • ectomycorrhiza;
  • hydrophobin;
  • mycelium;
  • symbiosis;
  • tree

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The immunolocalization of one of the hydrophobins of Pisolithustinctorius (HYDPt-1) is reported. Hydrophobin proteins play key roles in adhesion and aggregation of fungal hyphae, and it is already known that formation of ectomycorrhizas on eucalypt roots enhances the accumulation of hydrophobin mRNAs in the mycelium of Pisolithus tinctorius.
  • • 
    Purification of SDS-insoluble proteins from the mycelium of P. tinctorius showed the presence of a 13 kDa polypeptide with properties of class I hydrophobin.
  • • 
    Polyconal antibodies were raised against a recombinant HYDPt-1 polypeptide, and these were used for immunofluorescence-coupled transmission electron microscopy.
  • • 
    HYDPt-1 is a cell wall protein located at the surface of the hyphae with no preferential accumulation in the fungal cells of the different tissues of the ectomycorrhiza (i.e. extraradical hyphae, mantle or Hartig net).

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Ectomycorrhizal basidiomycetes form a symbiotic association with roots of many tree species. This symbiosis is widespread in temperate and boreal forests, and can be used to increase forest production (Smith & Read, 1997). Ectomycorrhizal fungi have a low capacity for saprophytic colonization of soil: they need to be connected to a host tree in order to develop an abundant mycelium in the soil and to complete their sexual cycle. Thus, the ability of ectomycorrhizal hyphae to bind the surface of a host root is essential for the dissemination and the survival of the fungus, and for the success of mycorrhiza formation. However, the cellular and molecular events triggering the attachment of the hyphae to the root surface or the differentiation of the organized symbiotic tissues are still poorly understood (Martin et al., 1997; Barker et al., 1998; Martin et al., 1999). In ectomycorrhizas various cell wall components are probably essential for: the mycobiont –host interactions; the aggregation of hyphae forming the mantle ensheathing the root, and the penetration of the plant apoplast by fungal hyphae (Peterson & Bonfante, 1994; Bonfante, 2000).

Cell walls are fundamental cellular structures involved in sensing the environment by changing cell surface composition and topology of receptor-like molecules. The protein complement of the cell wall plays an essential role in cell-to-cell communication and adhesion. To understand the cellular and molecular communication between the two partners of the ectomycorrhizal symbiosis, we have identified several fungal cell wall proteins and genes which are thought to play key roles in the early steps of mycorrhiza formation (Tagu & Martin, 1996; Martin et al., 1999). The synthesis of acidic polypeptides in the ectomycorrhizal basidiomycete Pisolithus tinctorius is strikingly increased during eucalypt root colonization and the members of these symbiosis-regulated acidic polypeptides (SRAPs) share structural features with adhesins (Laurent et al., 1999). Three other mRNAs –hydPt-1, hydPt-2 and hydPt-3– are also highly accumulated during the establishment of the symbiosis. They code for three different polypeptides (Tagu et al., 1996, 1998) all probably belonging to class I hydrophobins as defined by Wessels (1997). Hydrophobins are small polypeptides, moderately hydrophobic with a conserved distribution of eight cysteine residues. They are cell wall or excreted fungal morphogenetic proteins involved in many aspects of fungal biology where adhesion occurs (Wessels, 1997; Kershaw & Talbot, 1998). Their roles are related to the amphipathic structure of these proteins, and their ability to self-assemble at an hydrophobic/hydrophilic (air/liquid) interface (Wösten et al., 1993). Hydrophobins accumulate at the surface of aerial hyphae or spores and direct their hydrophobic domains towards the air. This hydrophobic layer can be involved in aerial growth and aggregation of hyphae (fruiting bodies) or spore adhesion to hydrophobic host surfaces (cuticle), and may have a protective role in resistance to desiccation (Temple et al., 1997; Wessels, 1997; Kershaw & Talbot, 1998; Wösten et al., 1999).

The roles of hydrophobins in the formation of ectomycorrhiza are still under debate and the subcellular localization of these proteins remains unknown. As a first step to the understanding of the function of hydrophobins in the ectomycorrhizal symbiosis, a recombinant HYDPt-1 polypeptide was obtained in Escherichia coli and used for polyclonal antibody production. Here we report the localization of HYDPt-1 polypeptides in the Eucalyptus globulusPisolithus tinctorius mycorrhiza.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fungal isolate, bacterial strain, and culture conditions

The dikaryotic strain 441 of P. tinctorius[(Pers.) Coker & Couch, Synonym = P. arhizus (Scop. Pers.)] used in this study was kept in the Collection of Ectomycorrhizal Fungi of the Forest Microbiology Laboratory (INRA-Nancy, France) and grown as previously described (Nehls et al., 1998) on cellophane covering an agar medium containing Pachlewski medium at low concentration of glucose (5 mM). Seeds of Eucalyptus globulus ssp. bicostata Kirkp. were pretreated and germinated, and seedlings grown as previously described (Nehls et al., 1998). Lateral roots of E.globulus were inoculated with P. tinctorius mycelium in Petri dishes and harvested after a few days of contact corresponding to early stages of ectomycorrhiza formation (Burgess et al., 1996). For the production of aerial mycelium of P. tinctorius, colonies were incubated on Pachlewski medium at high concentration of glucose (100 mM) directly on the agar medium without the cellophane membrane. After 3 weeks in the dark at 25°C, plates were frozen at −80°C and aerial hyphae were collected in liquid nitrogen by scraping the plates. The Escherichia coli strain recipient for the gene fusion construct was M15[pREP4] (Qiagen, Hilden, Germany), containing the low-copy pREP4 plasmid which confers resistance to kanamycine, and which constitutively expresses the lac repressor protein encoded by the lacIq gene.

Isolation of hydrophobins from aerial hyphae

Hydrophobins were extracted from aerial hyphae of P. tinctorius as described by de Vries et al. (1993). Briefly, cell walls were obtained by fragmentation using an X-press and by incubation in a hot-SDS buffer (Wessels et al., 1991). Cell walls were dried, resuspended in trifluoroacetic acid (TFA) and sonicated in order to disassemble hydrophobins. After centrifugation, the TFA supernatant was transferred into a new tube and evaporated. Performic acid treatment of the TFA-extracted material was done as described by Wessels et al. (1991). The extracted residues were resuspended in loading buffer [0.1 M Tris-HCl pH 6.8, SDS 2% (w/v), 20% (v/v) glycerol, 0.02% (w/v) bromophenol blue]. SDS-PAGE of the extracts was performed using 15% (w/v) acrylamide gel. Proteins were stained with colloidal G250 Coomassie brilliant blue (Neuhof et al., 1988).

Construction of the Histidine-tagged HYDPt-1 fusion protein

The fragment containing the hydrophobin HYDPt-1 coding sequence was amplified by PCR from the hydPt-1 cDNA (GenBank accession number: U29605) and cloned into pBS (Tagu et al., 1996). For the construction of the Histidine-tagged (His-tagged) fusion protein, two primers were designed: the first (5′-cgcGGATCCGAGACCAATGCTCAGCGTATGG-3′) contained a BamHI recognition site and matches the codons of the first amino acids after the putative signal peptide (Tagu et al., 1998). The second primer (5′-gggAAGCTTAACGTGCTGGACACGTCG-3′) matched the last amino acids of the HYDPt-1 sequence and harboured a restriction site for HindIII. Three bases (lower case) were added upstream of the two primers in order to help restriction of the fragment. The expected size of the amplified fragment was 462 bp. For PCR amplification, 5 ng of purified pBS-hydPt-1 was mixed with 1.5 µM of each primer, 1.25 mM of dNTP, and 1.25 units of Taq polymerase (provided with its buffer by Quantum-Appligene-Oncor, Illkirch, France), in a 25-µL final reaction volume. Amplification was done in a Perkin Elmer GeneAmp PCR System 9600, with an initial denaturation step at 94°C for 1 min, followed by 30 cycles of 94°C for 0.5 min, 55°C for 0.5 min and 72°C for 1 min. A final elongation step was performed at 72°C for 10 min and the reaction was stopped at 4°C until gel analysis. The DNA fragments were analysed by agarose gel electrophoresis and purified by the QIAquick Gel Extraction Kit from Qiagen. Amplified DNA fragments were digested with BamHI and HindIII before cloning in pQE30 (Qiagen, Hilden, Germany) containing a 6 × His-tag coding sequence upstream of the cloning sites. E. coli M15[pREP4] competent cells were transformed with the ligation mixture and selection was performed on kanamycine (pREP4) and ampicilline (pQE30). Four resistant colonies were randomly picked and the presence of the recombinant plasmid was checked by PCR and sequencing. Small scale extractions were performed under denaturing conditions (see next paragraph) in order to select the strain which produced the largest amount of recombinant protein.

Isolation of the His-tagged HYDPt-1 fusion protein

Growth of bacteria was performed following the manufacturer’s instructions: recombinant E. coli M15[pREP4] was grown in 0.5 L Luria-Bertani medium (Sambrook et al., 1989) supplemented with 0.2% (w/v) glucose and antibiotics. Recombinant protein expression was induced by 1 mM isopropylthiogalactoside (IPTG) for 5 h, bacteria were harvested by centrifugation, and the pellet kept at −80°C pending the protein extraction. The pellet was thawed for 15 min on ice and cells were lysed by the addition of 5 ml of 6 M guanidium hydrochloride buffered in 0.01 M Tris-HCl pH 8. Lysis was enhanced by a 10 minute sonication in a water bath. After centrifugation, the lysate was mixed with 1 ml of 50% (w/v) Ni-NTA (nickel-nitrilotriacetic acid) slurry resins for 1 h. The lysate-resin mixture was loaded into a column and washed by 8 ml of buffer C (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-HCl pH 6.3). The recombinant protein was eluted with 4 × 0.5 ml of buffer D (same as buffer C but pH 5.9) and 5 × 1 ml of buffer E (same as buffer C but pH 4.5). Aliquots of fractions D and E were analysed by SDS-PAGE for the presence of the recombinant protein using 15% (w/v) acrylamide gels which were silver stained.

HPLC purification of hydrophobins

The most abundant protein present in fraction E was purified by reverse phase HPLC (RP-HPLC) using a 5-µm Supelcosil LC 318 column (5 cm × 4.6 mm ID) protected by a 5-µm Supelcosil LC 318 guard column (2 cm × 4.6 mm ID) (Supelco, Bellefonte, Pensylvania, USA). The injection volume was 200 µL. The mobile phase used for the separation of the proteins consisted of 0.1% (v/v) trifluoracetic acid (TFA) (solvent A) and 0.1% (v/v) TFA in acetonitrile (solvent B). The protein samples were eluted by means of the following gradient of acetonitrile: 0–40% in 15 min, 40–80% in 20 min, 80–90% in 2 min and 90% in 3 min. The gradient was then immediately returned to 0%. The flow rate was 0.8 ml min−1 and detection was performed at 214 nm. The peaks were collected, vacuum dried and analysed by SDS-PAGE.

The N-terminal sequence of the recombinant protein (transferred onto PVDF membrane) was determined using an automatic LF 3000 Protein Sequencer (Beckman, Berkeley, CA, USA) equipped with an online Gold HPLC system for detection of the phenylthiohydantoin amino acids (De Bellis et al., 1998).

Antibody production

Polyclonal antibodies were raised against the recombinant HPLC-purified HYDPt-1 polypeptide (100 µg) by immunizing a rabbit. Initial immunization was with Freund’s complete adjuvant and subsequent boosts after 10, 20 and 30 d were with Freund’s incomplete adjuvants. Blood samples were collected from the ear vein.

Immunofluorescence, transmission electron microscopy and immunolabelling

Immunofluorescence experiments were performed on E. globulus roots colonized by P. tinctorius hyphae according to two protocols. In the first procedure, unfixed segments were embedded in 10% (w/v) low melting point agarose. Sections (100 µm) were prepared using a Balzer Vibratome series 1.000 apparatus. In the second procedure, the segments were fixed in 2.5% (v/v) glutaraldehyde, postfixed in 1% (w/v) osmium tetroxide, embedded in LR White resin (as described in the next paragraph) and semithin sections was obtained with a Reichert-Jung Ultracut. Sections from both the samples were incubated overnight at 4°C with the polyclonal antibody anti-HYDPt-1 (dilution 1 : 200–1 : 500) in phosphate buffer containing 1% (w/v) BSA. Sections were washed 3 times for 15 min in phosphate buffer, saturated for 30 min with 1% (w/v) BSA in phosphate buffer, and incubated at room temperature in the dark for 3 h with a goat antirabbit IgG conjugated to fluorescein isothiocyanate (FITC) (dilution 1 : 80). The sections were washed as before, mounted, and observed using a scanning microscope (Optiphot-2 View Scan DVC-250, Nikon) at 494 nm. Labelling specificity was determined by replacing the primary antibody with (a) the buffer and (b) the preimmune serum.

For transmission electron microscopy (TEM), mycelium of P. tinctorius and inoculated roots of E. globulus were fixed in 2.5% (v/v) glutaraldehyde in 10 mM Na-phosphate buffer (pH 7.2) overnight at 4°C. After washing in the same buffer, they were postfixed in 1% (w/v) osmium tetroxide in water for 1 h, washed three times with water, and dehydrated in an ethanol series (30, 50, 70, 90, 100% (v/v); 15 min each step) at room temperature. The samples were infiltrated in 2 : 1 (v/v) ethanol : LR White (Polysciences, Warrington, PE, USA) for 1 h, 1 : 2 (v/v) ethanol : LR White resin for 2 h, 100% LR White overnight at 4°C and embedded in LR White resin, according to Moore et al. (1991). Semi-thin sections (1 µm) were stained with 1% (w/v) toluidine blue for morphological observations. Immunogold labelling (dilution 1 : 500) was performed on thin sections as described by Balestrini et al. (1996) and observed with a Philips CM10 transmission electron microscope. Labelling specificity was determined by replacing the primary antibody with the preimmune serum.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Extraction of hydrophobins from Pisolithus tinctorius aerial hyphae

In order to characterize the native hydrophobins from P. tinctorius, their purification was initiated. The high accumulation of hydrophobin transcripts in aerial hyphae of P. tinctorius (Tagu et al., 1996) suggests the presence of self-assembled hydrophobin polypeptides at the surface of the cell wall. Cell walls were, thus, extracted from the mycelium of P. tinctorius and SDS soluble polypeptides were removed. The SDS insoluble fraction (probably containing a complex of self-assembled hydrophobin) was treated with TFA in order to release water soluble monomers of hydrophobins. SDS-PAGE analysis (Fig. 1) showed the presence of a major 13 kDa-polypeptide in this sample, suggesting that class I hydrophobins were extracted from P. tinctorius mycelium. An oxidation with performic acid caused a shift in the apparent molecular weight of the 13 kDa polypeptide to 17 kDa position (Fig. 1). Despite several trials, no sequence was obtained.

image

Figure 1. Detection of class I hydrophobin in P. tinctorius. Hot-SDS extracted cell walls of aerial hyphae were extracted with trifluoroacetic acid (TFA) and the extract was analysed by SDS-PAGE [15%(w/v) acrylamide] before (lane TFA) and after performic acid oxidation (lane PA). The gel was stained with colloidal Coomassie brilliant blue. The amount of material loaded per lane was derived from 3 mg cell walls (dry wt).

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Purification of the His-tagged HYDPt-1 fusion protein

In order to raise polyclonal antibodies against a hydrophobin of P. tinctorius, a recombinant His-tagged HYDPt-1 polypeptide was expressed in E. coli. The two primers used for the construction were designed to amplify a sequence lacking the signal peptide. Highly denaturing extraction conditions (6 M guanidinium hydrochloride) were required to solubilize the polypeptide. This suggests that the recombinant hydrophobin probably formed inclusion bodies. Elution of the solubilized recombinant protein from the Ni-NTA affinity column was performed using a pH gradient; a major band of about 17 kDa was eluted at pH 4.5 (Fig. 2a, lane 1). The yield ranged from 1 to 2 mg of recombinant polypeptide per litre of bacterial cell suspensions. Other polypeptides from the E. coli lysate coeluted with the recombinant protein (Fig. 2a, lane 1) and a RP-HPLC purification was carried out to further purify the putative His-tagged HYDPt-1.

image

Figure 2. Purification by RP-HPLC of the His-tagged HYDPt-1 fusion protein. (a) Bacterial extracts prepurified by IMAC were loaded onto a LC318 column and analysed by RP-HPLC at 214 nm through a gradient of acetonitrile. The arrow indicates the peak eluted at 43% (v/v) acetonitrile which corresponds to the purified recombinant HYDPt-1 (lane 2 of the inset), compared to the IMAC purified polypeptide before RP-HPLC purification (lane 1 of the inset). SDS-PAGE was performed on 15% (w/v) acrylamide gels and the polypeptides were silver stained. (b) Comparison of the sequences of the recombinant HYDPt-1 polypeptide deduced from the cDNA, and obtained after sequencing of the 17 kDa RP-HPLC purified band.

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The 17 kDa protein was purified by injection of the pH 4.5 eluted fraction using RP-HPLC. Three major peaks were observed after elution through an acetonitrile gradient (Fig. 2a). A SDS-PAGE analysis of these three different peaks identified the presence of the 17 kDa protein in the fraction eluted at 43% acetonitrile (Fig. 2a, arrow and lane 2). The RP-HPLC purified 17 kDa-polypeptide was partially sequenced (Fig. 2b) and the sequence matched perfectly to the 6xHis tag and the first nine N-terminal amino acids of the HYDPt-1 sequence deduced from its cDNA.

Localization of antigens recognized by anti-HYDPt-1 antibodies

Polyclonal antibodies raised against the purified recombinant polypeptide were used for the localization of HYDPt-1 in P. tinctorius hyphae colonizing E. globulus roots. Indirect immunofluoresence (IIF) performed on mycorrhizal roots led to an intense labelling of extramatrical hyphae, hyphae constituting the mantle and penetrating the root on both Vibratome (Fig. 3a–c) and semithin sections (Fig. 3d–f). In a young mycorrhizal root, hyphae developing at the root surface and between the grooves of the epidermal cells (Fig. 3a) were easily identified. When the mantle is fully developed (Fig. 3f), the labelling was detected at the surface of all the hyphae (Fig. 3c,d). In the control sections, where the primary antibody was omitted (Fig. 3b) or the preimmune serum was used instead of the anti-HYDPt-1 (Fig. 3e), a weak brown-yellow autofluorescence of the root epidermal cell walls and some mantle hyphae was the only signal to be seen.

image

Figure 3. Immunofluorescence labelling of P. tinctorius hyphae on E. globulus roots with anti-HYDPt-1 antibodies. Vibratome sections (a,b,c) and LRWhite semithin sections (d,e,f) from mycorrhizal roots are used. (a) A specific green labelling is detected at the surface of the hyphae following the outline of the epidermal cells (arrows) in a young ectomycorrhizal root. (b) Control section where the anti-HYDPt-1 antibody is omitted. The FITC-labelled secondary antibody does not lead to any signal on the fungal walls, while epidermal cell walls show a weak brown-yellow autofluorescence. (c) After treatment with the anti-HYDPt-1 antibody, a strong green signal is evident at the surface of all the mantle hyphae in a mature ectomycorrhiza (M). Host walls of the epidermal cells and root hairs show a weak brown-yellow signal due to autofluorescence. (d) After treatment with the anti-HYDPt-1 antibody, an intense green fluorescence is present at the surface of all the mantle hyphae on a semithin section. (e) No specific labelling is present in a control section treated with the preimmune serum and with a secondary antibody conjugated to FITC. Epidermal cell walls and some mantle hyphae show a weak brown-yellow autofluorescence. (f) Semithin section of a mature mycorrhizal root are observed under a light microscope after staining with Toluidine blue. The loose external and the compact inner mantles ensheathing the root are seen. (M). E, epidermal cell. In a, b, c, f bars correspond to 9 µm; in d, e bars correspond to 10 µm.

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Gold-labelling was located on the cell wall of free-living hyphae of P. tinctorius (Fig. 4), and hyphae colonizing the root (Fig. 5). Only traces of immunogold labelling was detected at the surface of longitudinally structured-sectioned hyphae (Fig. 4a): gold particles occurred at the surface of the fungal cells and were better detected on tangential sections (Fig. 4b). Hyphae in contact with E. globulus roots were regularly immuno-labelled. The distribution pattern was comparable in hyphae contacting the outer surface of epidermal cells (Fig. 5a), and in hyphae which actively grow around the root (Fig. 5b). Here, the labelling was limited to the fungal wall, mostly present inside an electron transparent layer. Labelling did not occur on the material observed at the hyphal surface (Fig. 5a) or in the loose electron-opaque material separating hyphae (Fig. 5b,c). The fungal walls in contact with the root epidermal cells or with adjacent hyphae showed similar labelling as the hyphal surfaces in contact with the air (Fig. 5a). Proteins recognized by HYDPt-1 antibodies were clearly detected on the cell walls of hyphae forming the mantle (Fig. 5c,d) or penetrating between two epidermal cells to form the Hartig net (Fig. 5e). No labelling was present in the plant cell wall (Fig. 5a,c) and limited labelling was seen on control sections where the preimmune serum was used instead of HYDPt-1 antibodies (Fig. 5f) or where the first antibody was omitted (not shown).

image

Figure 4. Immunogold localization of HYDPt-1 hydrophobins on thin sections from mycelium of P. tinctorius grown in pure culture. (a) In growing hyphae, hyphal walls are labelled after treatment with the antibody. (b) Gold particules (arrow heads) are present at the hyphal surface as seen in a tangential section. h: hypha. Bars, 0.2 µm.

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image

Figure 5. Immunogold localization of HYDPt-1 on thin sections from E. globulus roots colonized by P. tinctorius. (a) Hyphae contact an epidermal cell. The fungal walls are labelled after treatment with anti-HYDPt-1 antibodies (arrow heads). No labelling is detected on the host wall. (b) Gold granules (arrow heads) are regularly present on electron transparent layer of the walls of actively growing extramatrical hyphae. Labelling is not detected on the fibrillar material at the surface of hyphae nor in the loose electron-dense material among hyphae. (c) Mantle region: fungal walls are regularly labelled. (d) Magnification of the mantle hyphae. Labelling is evenly distributed over the fungal cell walls (arrow heads). (e) Cell wall labelling of hyphae progressing between two epidermal cells during the establishment of the Hartig net. Gold granules are present on the fungal wall (arrow heads), while no labelling occurs on the host wall. (f) Limited labelling is found in the control section where the preimmune serum alone was used. h: hypha, E: epidermal cells, w: host wall, n: fungal nucleus. Bars, 0.3 µm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Colonization of roots by ectomycorrhizal fungi is a key step for a successful development of the symbiosis. Root and fungal cell surfaces are, thus, important cellular structures involved in the earliest steps of mycorrhiza development. Many studies have shown that cell walls of the partners were altered in their ultrastructure and composition (Peterson & Bonfante, 1994). Cell wall proteins – like adhesins, integrins, lectins or arabinogalactan proteins – are known to be involved in adhesive processes and cell-to-cell communication (Knox, 1994; Patti & Hook, 1994; Pemberton, 1994; Shyy & Chien, 1997). Hydrophobins, which have been widely found among filamentous fungi, are probably involved in aggregation (Wessels, 1997; Kershaw & Talbot, 1998) and adhesion (Wösten et al., 1994b), but could also be involved in tolerance to desiccation (Temple et al., 1997).

The role of the different hydrophobins in a given fungus is not well documented, except for Schizophyllum commune (Wessels, 1999). It is not known whether these hydrophobins have redundant or specific functions. Complementation of the mpg1 mutant phenotype of Magnaporthe grisea was partially successful after over-expression of several types of hydrophobin genes from different fungal species. This indicates that the various hydrophobins are functionally related (Kershaw et al., 1998). The temporal expression of the three P. tinctorius hydrophobin genes in ectomycorrhizas is similar (Tagu et al., 1996, 1998) and thus cannot give any clues on the specific role of each of these polypeptides. An analysis of the spatial distribution of these proteins is, thus, necessary in order to know whether particular hydrophobins are involved in: the adhesion of hyphae to the root surface; the aggregation of hyphae in the mantle, and/or the penetration of the mycelium by reinforcement of the cell wall.

Purification of SDS-insoluble proteins from the mycelium of P. tinctorius showed the presence of a 13-kDa polypeptide with properties of class I hydrophobin. The fact that oxidation of the extract by performic acid was responsible for a change in the electrophoretic mobility of this protein further suggests that the identified polypeptide corresponds to an hydrophobin (de Vries et al., 1993). However, we were unable to identify this polypeptide by sequencing, probably because the amount available was too small. Antibodies raised against the recombinant HYDPt-1 protein did not cross-react with this 13 kDa protein on western blot (data not shown). That a highly accumulated mRNA gives rise to only a small amount of the corresponding polypeptides has already been observed for hydrophobins (Talbot et al., 1996), because of the strong anchoring of self-assembled hydrophobins in the cell wall. In order to obtain more material for the preparation of antibodies, a recombinant polypeptide HYDPt-1 was produced in E. coli. A 17-kDa polypeptide was identified and eluted at pH 4.5. Its apparent molecular weight was slightly higher than the expected size (14.5 kDa) probably because of changes in gel mobility due to hydrophobic domains. Formation of intramolecular disulphide bridges between the different cysteines of the polypeptide could also partly explained this discrepancy. A further purification by RP-HPLC was needed to eliminate contaminants from the recombinant fusion polypeptide. The elution of the recombinant protein was effective at c. 43% acetonitrile, as for the polypeptide previously identified in P. tinctorius mycelium (data not shown). A similar result was described for the EAS hydrophobin from the rodlet layer of spores of Neurospora crassa (Templeton et al., 1995). This confirms the relative hydrophibicity of the monomeric recombinant HYDPt-1. The yield of production was low (1 mg l−1 of bacterial culture) and comparable to the one described by Peñas et al. (1998) for the hydrophobin FbhA from Pleurotus ostreatus. The codon usage of the hydPt-1 cDNA cannot by itself explain this low production. Low expression levels could be related to the toxicity or the instability of the recombinant protein. It is known that hydrophobic regions – as in the central part of HYDPt-1 (Tagu & Martin, 1996) – often have a toxic effect on bacterial cells (The QiAexpressionist, Qiagen, Hilden, Germany).

The aim of this study was to localize HYDPt-1 on sections of ectomycorrhizas. A previous report indicated that the synthesis of hydPt-1 mRNAs was up-regulated during the early stages of E. globulus – P. tinctorius ectomycorrhiza formation (Tagu et al., 1996). Thus, in this work, sections of eucalypt roots colonized by the P. tinctorius mycelium were used at the time when hyphae anchored on the root surfaces, began to aggregate to form the mantle, and initiated root cell penetration. The tissue distribution of HYDPt-1 in P. tinctorius mycelium was obtained by IIF and clearly indicated that all the different hyphae forming the ectomycorrhiza (extramatrical, forming the mantle or penetrating the root) were immuno-labelled. TEM showed that HYDPt-1 antibodies were located at the surface of P. tinctorius cell walls. This observation is in agreement with the presence of a putative signal peptide in the nucleotidic sequence of the cDNA. During the saprotrophic and symbiotic growth, the differentiation of the symbiotic interfaces, namely hyphae/root surface, hyphae/hyphae, hyphae/root apoplasm, did not seem to modify this location pattern. In aerial hyphae of S. commune, the hydrophobin SC3p is located at the outer surface of the cell walls that are in contact with air or with a synthetic hydrophobic surface, like Teflon (Wösten et al., 1993, 1994a, 1994b). However, when hyphae are sticked, SC3p is absent from the hyphal/hyphal interface (Wösten et al., 1994a). In fruiting bodies of S. commune the SC4p protein is also located at the surface of hyphae of the plectenchyma, and at the interface with air cavities (Wessels et al., 1995; Lugones et al., 1999). This is different from our observation which described an homogenous labelling on the cell walls of P. tinctorius both at hyphae/hyphae and hyphae/root surface interfaces. In Pleurotus ostreatus, hydrophobins are also distributed at the surface of the cell wall of aerial hyphae (Ásgeirsdóttir et al., 1998) in a similar manner as for P. tinctorius ectomycorrhizas.

The presence of HYPt-1 related molecules at the surface of P. tinctorius hyphae growing on agar media suggests that hydrophobins are proteins being accumulated also in nonspecialized hyphae. This observation fits well with previous data indicating the accumulation of hydPt-1 mRNA extracted from aerial hyphae (Tagu et al., 1996). In the present study, the labelling was particularly intense on the electron-transparent layer where glucans and chitin were previously located (Martin et al., 1999). This could suggest a structural interaction between hydrophobins and other cell wall polymers. HYDPt-1 showed a distribution similar to another family of symbiosis-regulated cell wall proteins from P. tinctorius: the 32 kDa-SRAPs (Laurent et al., 1999). As hydrophobins, 32 kDa-SRAPs are abundant up-regulated cell wall proteins in P. tinctorius. This suggests that hydrophobins and SRAPs belong to a complex array of extracellular polypeptides probably associated to other cell wall polymers and produced during the symbiotic interaction. Co-localization of the different hydrophobins and members of 32 kDa-SRAPs will be performed to test this hypothesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors would like to emphasise the constant support of Prof. J.G.H. Wessels (University of Groningen), who was always willing to help us in our research. We acknowledge Dr F. Lapeyrie (INRA-Nancy), Dr H.A.B. Wösten (University of Groningen) and Luciana Vallorani (University of Urbino) for constructive advice, Dr Luis Lugones (University of Groningen) for his help with the hydrophobin extraction, and Béatrice Palin and Mickaël Scarpetta for their technical assistance. A 6 months sabbatical stay by DT at the University of Urbino (Italy) was granted by INRA.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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