• Metallothioneins are small polypeptides involved in metal tolerance of many eukaryotes. Here we characterized the Pimt1 gene, coding for a metallothionein from the ectomycorrhizal fungus Paxillus involutus.
• Expression of Pimt1 in P. involutus under metal stress conditions was measured by northern blot and RT-PCR analyses. The full-length cDNA was used to perform functional complementation in yeast mutant strains and agrotransformation of Hebeloma cylindrosporum.
• Heterologous expression in yeast showed that PiMT1 was able to complement the hypersensitivity of mutant strains to cadmium (Cd) and copper (Cu), but not to zinc (Zn). Transcripts were almost undetectable under control conditions, whereas Cu and Cd, but not Zn, strongly induced Pimt1 expression in P. involutus. Constitutive overexpression of Pimt1 in H. cylindrosporum conferred a higher copper tolerance.
• The present study identified PiMT1 as a potential determinant in the response of mycorrhizal fungi to Cu and Cd stress. Additionally, we demonstrated the usefulness of mycorrhizal fungi transformation using Agrobacterium technology to approach gene function.
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Like most other organisms, ectomycorrhizal fungi possess a range of molecular mechanisms at the cellular level to tolerate heavy metals (reviewed in Bellion et al., 2006). Cysteine-rich heavy metal-binding peptides, metallothioneins (MTs) and phytochelatins (PCs), are important chelators of metal ions in the cytosol (Cobbett & Goldsbrough, 2002). Unlike PCs, which are enzymatically synthesized polypeptides, MTs are genome-encoded peptides. They are spread over pro- and eukaryotic kingdoms, but their precise physiological role has not yet been elucidated. Proposed functions of MTs in eukaryotic cells include metal homeostasis and cellular detoxification of metals. This was demonstrated by overexpression experiments of MTs in various organisms: yeast CUP1 gene transferred to cauliflower resulted in a 16-fold higher Cd tolerance and accumulation (Hasegawa et al., 1997). Transfer of mouse MT1 or human MT2 to Nicotiana tabacum and Brassica napus (Misra & Gedamu, 1989; Pan et al., 1994) resulted in a constitutively enhanced Cd tolerance in these plants.
Fungal MTs show highly distinct patterns of constitutive or metal-inducible gene expression. The Saccharomyces cerevisiae cup1 is specifically regulated by copper, while zinc, cadmium and gold are unable to induce cup1 (Butt et al., 1984; Okuyama et al., 1999). Induction of MT synthesis led to a binding of excess free Cu ions in the cytosol, and conferred resistance to Cu (Welch et al., 1983; Fogel et al., 1988).
The response of mycorrhizal fungi to toxic metals is of interest in view of their importance in the reclamation of polluted sites and their role in tree growth. As we reported earlier, the ectomycorrhizal fungus Paxillus involutus is particularly tolerant to metal (Blaudez et al., 2000b). We have also demonstrated that P. involutus could tightly bind cadmium to cell walls, accumulate Cd within the cytoplasm or in the vacuolar compartment (Blaudez et al., 2000a). We further hypothesized that Cd could induce oxidative stress (Jacob et al., 2001), as already demonstrated for S. cerevisiae. There are few reports on MT-like sequences in mycorrhizal fungi, obtained in the course of expressed sequence tag projects. Genes coding for putative MTs have been isolated in the ectomycorrhizal basidiomycete Pisolithus tinctorius (Voiblet et al., 2001) and in the arbuscular mycorrhizal (AM) fungus Gigaspora rosea (Stommel et al., 2001). [Correction added after online publication 7 February 2007: in the preceding sentence Glomus intraradices was corrected to Gigaspora rosea]. More recently, the existence of a MT-encoding gene from the AM fungus Gigaspora margarita was reported (Lanfranco et al., 2002).
We screened 2040 arrayed cDNAs of Paxillus involutus to identify Cd-responsive genes using differential hybridization. Our results suggest that Cd complexation by phenolic compounds or by complexing peptides such as MTs is probably a key determinant of the cellular response to Cd in P. involutus (Jacob et al., 2001). In addition, using an improved HPLC method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (γ-glutamylcysteine, glutathione) to higher molecular mass (PCs), we found that glutathione and γ-glutamylcysteine contents increased when Paxillus involutus was exposed to Cd. The content of an additional compound with a high molecular mass, most probably related to a MT, increased drastically in mycelia exposed to Cd (Courbot et al., 2004).
Here we report the cloning and characterization of the Pimt1 gene coding for a MT in P. involutus. The full-length cDNA was able to complement the growth deficiency of the S. cerevisiae mutant Δyap1 on Cd as well as the growth deficiency of Δcup2 on Cu. Abundance of the Pimt1 transcript increased dramatically during induction by Cu and Cd, but not by Zn. Moreover, using agrotransformation, we were able to generate transgenic strains of the ectomycorrhizal fungus Hebeloma cylindrosporum overexpressing the Pimt1 gene, conferring increased Cu tolerance.
Materials and Methods
Organisms and culture media
The origin and the growth conditions of the ectomycorrhizal fungi P. involutus (Batsch) Fr. (ATCC 200175) and H. cylindrosporum (Romagnesi) (strain h1) have been described by Jacob et al. (2001) and Javelle et al. (2001), respectively. Classical procedures for manipulating E. coli have been described previously (Sambrook & Russell, 2001). For transcriptional induction experiments, P. involutus colonies were grown on cellophane-covered agar medium for 10 d and transferred to liquid medium for 24 h (adaptation period). Colonies were further transferred to liquid medium containing various metal concentrations for various lengths of time (see the legend in Fig. 3).
The yeast strains used for heterologous expression of Pimt1 were cup2::kanMX4 (Δcup2) and zrc1::kanMX4 (Δzrc1), derived from the wild-type strain BY4741 (MATa his3 leu2 met15 ura3), and the strain yap1::TRP1 (Δyap1), derived from the wild-type strain W303B (MATa his3 can1-100 ade2 leu2 trp1 ura3). Growth was on SD medium (yeast nitrogen-based (YNB) medium without amino acids, with 2% (w/v) galactose or glucose as carbon source and 0.192% yeast synthetic drop-out medium without uracil). Yeast cultures were adjusted to OD = 1.0, and 5 µl of serial dilutions were spotted on SD medium without extra metal or supplemented with 40 µm CdCl2, 150 µm CuSO4 or 10 mm ZnCl2. Plates were incubated for 4 d at 30°C.
Pimt1 cloning and functional expression in yeast
The Pimt1 cDNA (accession number AY525379) was cloned from a cDNA array experiment (Courbot et al., 2004) and subcloned from the pGEM®-T clone into the pYES2 plasmid (Invitrogen, Cergy Pontoise, France), using MT1 (5′-CCCCCAAGCTTATGAACACCATCACCTCTG-3′) and MT2 (5′-CTGCAGAATTCTTAGCACTTGCATTCACCAGG-3′) primers, introducing HindIII and EcoRI sites (sites underlined). The pYES2-Pimt1 construct and the empty vector pYES2 were used to transform the Δyap1, Δcup2 and Δzrc1 strains as described previously (Gietz et al., 1992). The various transformants were selected on SD medium without ura. They were further cultured in liquid SD medium before being transferred for 4 d at 30°C onto SD-agar plates supplemented (or not) with CdCl2, CuSO4 or ZnCl2.
The adjacent DNA sequence upstream from Pimt1 in the P. involutus genome was investigated with the Clontech Universal GenomeWalker kit (Clontech Laboratories, Inc., Heidelberg, Germany). Briefly, P. involutus genomic DNA was digested with one of four different blunt-end restriction enzymes (provided by the manufacturer). A Pimt1-specific primer (MTgw; 5′-TTAGCACTTGCATTCACCAGGCTTG-3′) was designed and used in combination with the adaptor-specific primer AP1 (and AP2 in a second step) to amplify large genomic segments adjacent to Pimt1. PCR products were gel-purified with the Qiagen QIAquick gel extraction kit (Qiagen, Hilden, Germany) for sequencing.
DNA and RNA isolation and northern blot analysis
Fungal colonies were fixed in liquid nitrogen. Tissues were ground (Blender RETSCH model MM 300; Qiagen) and total RNA isolation was performed with the RNeasy Plant Mini kit (Qiagen) from approx. 100 mg of frozen mycelia. According to the manufacturer's recommendations, a buffer containing guanidium hydrochloride was used instead of one containing guanidium isothiocyanate, to avoid solidification of samples as a result of secondary metabolites in mycelia of filamentous fungi. DNA removal was achieved by incubating samples with RNase-free DNase (Qiagen) as described by the manufacturer. An average of 800 ng total RNA per mg frozen material was isolated and stored in DEPC-treated water at −70°C until further use. Genomic DNA was obtained from fungal colonies fixed and ground as described above and extracted with the DNeasy Plant Mini kit (Qiagen) from approx. 100 mg of frozen mycelia, according to the manufacturer's recommendations.
Samples of total RNAs (20 µg) were separated on 1.5% w/v formaldehyde agarose gels, transferred to positive nylon membranes (Appligene-Oncor, Illkirch Graffenstaden, France) by capillary elution according to standard procedures (Sambrook & Russell, 2001), and fixed by UV irradiation for 4 min. Membranes were then prehybridized for 2 h at 37°C, as previously described (Jacob et al., 2001). The radioactive probe [α-32P]dCTP-labeled cDNA Pimt1 was denatured and added to the prehybridization buffer. After 24 h of incubation with the probe, membranes were subjected to three washes in 2 × SSC/0.5% SDS at room temperature for 30 min, followed by one wash in 1 × SSC/0.1% SDS at 65°C for 40 min. Blots were rehybridized with a 5.8 S rRNA probe to monitor RNA amounts and to ensure equal RNA loading. Autoradiographs were analyzed by densitometric scanning using the Molecular Analyst computer software Quantity one (Bio-Rad, Hercules, CA, USA).
Agrotransformation of H. cylindrosporum with Pimt1 cDNA
The Pimt1 cDNA was PCR-amplified using the primers PiMtBamF 5′-CCCGGATCCATGAACACCATCACCTCTGT-3′ and PiMtBamR 5′-CCCGGATCCTTAGCACTTGCATTCACCA-3′ and subcloned from the pGEM®-T clone into the BamHI-digested pSK-GPD plasmid (Fitzgerald et al., 2003). The Pimt1 cDNA was thus under the control of the Aspergillus nidulans gpd-promoter and the Glomerella cingulata gpd-terminator. The promoter/Pimt1/terminator cassette was NotI excised and subcloned into the pFAT3 binary plasmid (Fitzgerald et al., 2003) to obtain the pFAT3-Pimt1 construction, which was further used to transform the Agrobacterium tumefaciens strain EHA101. Transformation of cells with the empty vector (EV) pFAT3 containing the hph gene within the T-DNA region was also performed, to obtain a control transgenic strain, named the EV strain.
A. tumefaciens-mediated transformation of H. cylindrosporum was carried out as described previously (Pardo et al., 2002). Briefly, fungal colonies were grown from 2 mm agar plugs on cellophane membranes on N2P2 medium (Gay, 1990) at 22°C in the dark for 2 d and then transferred to N2P2-induction plates (N2P2 medium with lower sugar content (glucose 2 g l−1 as the sole carbon source) supplemented with 40 mm MES at pH 5.3, 0.5% glycerol and 200 µm acetosyringone). The colonies were then inoculated with 50 ml of an induced A. tumefaciens culture prepared according to Pardo et al. (2002). The cocultivation plates were incubated at 22°C for 3 d in the dark; cellophane membranes containing the fungal colonies were transferred to new plates containing the selection medium (N2P2 medium supplemented by 100 mg l−1 chloramphenicol, 100 mg l−1 gentamycin and 12.5 mg l−1 tetracycline for killing A. tumefaciens cells, and 200 mg l−1 of hygromycin B for selection of H. cylindrosporum transformants) and kept at 22°C for 10 d in the dark. Resistant colonies were individually harvested and isolated for vegetative propagation in N2P2 medium containing 200 mg l−1 hygromycin B.
PCR amplification from H. cylindrosporum colonies
PCR amplifications were done on cDNA obtained through reverse transcription of 500 ng RNA for 60 min at 37°C (Qiagen Omniscript RT Kit), using the primers PiMt-f (5′-ATGAACACCATCACCTCTGTCC-3′) and PiMt-r (5′-TTAGCACTTGCATTCACCAGG-3′) for Pimt1, and hph-f (5′-ATGCCTGAACTCACCGCGACAGTCTGT-3′) and hph-r (5′-CTATTCCTTTGCCCTCGGACGAGT-3′) for the hygromycin resistance gene. The RT products were amplified by PCR under the following conditions: 94°C for 2 min followed by 32 cycles for hph and Pimt1 at 94°C for 30 s, 65°C for 45 s and 72°C for 1 min using an Eppendorf Mastercycler (Eppendorf, Le Pecq, France). For PCR amplification on genomic DNA, similar amplification conditions were used on extracted genomic DNA, except that 40 cycles were used.
Phenotype screening for H. cylindrosporum overexpressors
To screen for a phenotype, experiments were performed in which the transformed clones were grown on solid cellophane covered medium containing different metal concentrations. The concentration ranges of metals used were 0.05–1.0 mm, 0.01–0.6 mm and 0.5–5.5 mm for CuSO4, CdCl2 and ZnCl2, respectively. Radial growth and fresh weight were recorded after 14 d of growth.
Cloning and sequence analysis of Pimt1
The Pimt1 cDNA encodes a 34-amino-acid protein (3.42 kDa) with a predicted pI of 7.6. All but one of the cysteine residues are part of the characteristic MT motif C-X-C and P. involutus MT1 contains only one domain bearing three C-X-C motifs (Fig. 1), as found for other fungal MTs. The size of the polypeptide and the proportion of cysteine (seven conserved cysteine residues, representing 20.7% of the total amino acid content of the peptide) indicate that PiMT1 could be assigned to subfamily f1 of fungal MTs (Binz & Kägi, 1999). However, the sequence pattern (C-G-C-S-x(4)-C-x-C-x(3,4)-C-x-C-S-x-C), which identified the members of this subfamily, does not exactly match the PiMT1 sequence. PiMT1 is most closely related to MT-like polypeptides from Agaricus bisporus, Uromyces fabae and two translated cDNA sequences from Antrodia cinnamomea and Lentinula edodes (Fig. 1). It shares only 21.5% identity with MT1 from the AM fungus G. margarita (Lanfranco et al., 2002), with 13 identical amino acids.
Genome walking experiments allowed us to isolate a fragment of 0.8 kb upstream from the start codon of Pimt1. Interestingly, a MRE (metal responsive element) motif was found at position −695 relative to the first nucleotide of the ORF. The MRE consists of a conserved 7 bp core sequence (5′-TGCRCNC-3′) (with R = A/G; N = any residue) surrounded by semiconserved flanking sequences. MREs are cis-regulatory DNA sequences that specifically bind transcription factors and are essential and sufficient for transcriptional induction upon heavy metal load (Stuart et al., 1985).
Expression of Pimt1 in yeast mutants
To characterize PiMT1 further, the Pimt1 cDNA was expressed in three S. cerevisiae mutant strains, which are unable to grow on high concentrations of various metals, and growth was then monitored on both control and metal-supplemented media (Fig. 2). The yap1 gene encodes a transcription factor related to the mammalian AP-1 complex that positively controls various genes involved in metal tolerance and, more generally, oxidative stress tolerance in yeast (Kuge & Jones, 1994). Its deletion renders the yeast mutant highly sensitive to Cd (Wu et al., 2003). Figure 2 shows that the Δyap1 strain transformed with the empty vector was unable to grow at 40 µm Cd, whereas the Cd-sensitive phenotype of the Δyap1 mutant was fully complemented by Pimt1. The yeast mutant Δcup2 is highly sensitive to Cu because of the complete disruption of the factor CUP2 that regulates cup1 expression (Welch et al., 1983; Buchman et al., 1989). Complementing the results obtained with the Δyap1 mutant, an increased tolerance to Cu (Fig. 2) was conferred to Δcup2 cells by Pimt1. Deletion of the zrc1 gene, which encodes a transporter that sequesters Zn into the vacuole, renders the mutant highly sensitive to Zn (Li & Kaplan, 1998). However, transformation with Pimt1 did not restore growth of Δzrc1 on 10 mm Zn (Fig. 2).
Transcript expression pattern of Pimt1
In order to gain further insight at the molecular level concerning responses towards metals in ectomycorrhizal fungi, we followed the expression of Pimt1 by northern blot analysis. A single mRNA of c. 100 bp was detected (Fig. 3). The basal mRNA expression of Pimt1 remained relatively constant at low levels during transfer of the mycelium on medium without excess metal ions. However, the expression of Pimt1 could be markedly enhanced by addition of Cu (Fig. 3a) or Cd (Fig. 3b). The induction of Pimt1 expression reached a maximum at 0.8 mm Cu and 0.176 mm Cd, after 24 and 48 h incubation, respectively. Conversely, Pimt1 was not induced by Zn (Fig. 3c). Rehybridization with a 5.8 S rRNA probe showed a similar signal in all treatments, which ensured equal RNA loading (data not shown).
Transformation of H. cylindrosporum
Two hundred and thirty-three H. cylindrosporum colonies were cocultivated with Agrobacterium containing the pFAT3-Pimt1 construction, including the Pimt1 cDNA under the control of a promoter for constitutive expression and a hygromycin resistance gene (hph). From this set of 233 colonies, 42 hygromycin-resistant transformants could be isolated. Amplification on genomic DNA demonstrated that pFAT3-Pimt1-transformed H. cylindrosporum colonies had integrated the Pimt1 transgene, which was not detected in wild-type and EV-transformed colonies (Fig. 4a). Analyses of gene expression by RT-PCR showed expression of the hygromycin resistance gene in pFAT3-Pimt1-transformed and EV-transformed but not in wild-type colonies (Fig. 4b). Conversely, expression of Pimt1 was detected only in pFAT3-Pimt1-transformed H. cylindrosporum colonies, but not in wild-type and EV-transformed colonies (Fig. 4c). In a further control, we found that genomic amplification of the spectinomycin resistance gene (present on the binary vector) occurred only in transformed A. tumefaciens cells but not in H. cylindrosporum-transformed colonies, demonstrating that transformants were Agrobacterium-free. All transformants retained resistance to hygromycin after several sequential cultures on nonselective medium. Thus hph appeared to be a stable selection marker for H. cylindrosporum.
The MT7 transformant did show a phenotype of higher Cu tolerance with increased diameter colony already at 0.6 mm Cu (Fig. 5a). The two transformants, MT3 and MT7, showed increased biomass production as compared with EV-transformed colonies on 0.8 mm Cu (Fig. 5b). The phenotype was mostly evidenced on 1.0 mm Cu, where the empty vector control showed no residual growth and a similar pattern was observed for the wild-type colony (data not shown). There was no increased tolerance to Zn, Cd or Ni (data not shown).
Metallothioneins are low-molecular-weight, cysteine-rich metal-binding proteins found in a wide variety of organisms, including bacteria, fungi, plant and animal species. MTs bind essential and nonessential heavy metals. Data on functional characterization of P. involutus MT1 obtained with the three yeast mutant strains further suggest that PiMT1 specifically binds Cd and Cu, but not Zn. We also describe here the MT gene expression patterns. Pimt1 transcript expression was strongly induced by Cu and Cd, but not by Zn. These findings on Cd induction contrast with those on Podospora anserina MT1 transcription, which could be clearly up-regulated by Cu ions but not by Cd, Zn or Mn ions (Averbeck et al., 2001). MT1 from G. margarita also responded to Cu, but not to Cd exposure (Lanfranco et al., 2002). In Candida glabrata, both genes coding MTs were induced by Cu and Ag but not by Cd salts (Mehra et al., 1989). However, in the filamentous cyanobacterium Oscillatoria brevis, a MT gene was markedly increased under Cd and Zn exposure (Liu et al., 2003). However, it seems that the Pimt1 response to Cu was faster and more intense than the response to Cd, because Cd detoxification may also involve other mechanisms, as discussed later. Alternatively, the isolated MT may have a higher binding capacity for Cu than for Cd, which is also reflected by the ability of the PiMT1 overexpression to confer a higher tolerance to Cu but not to Cd in H. cylindrosporum. Many eukaryotic organisms contain a family of MT genes. In P. involutus another distantly related MT was found in the EST library (named Pimt2). However, transcripts corresponding to this partial cDNA did not increase under Cd exposure (M. Courbot, unpublished). This is in agreement with the finding that C. glabrata MT2 dominates in cellular resistance to Cu salts, whereas MT1 does not confer any appreciable Cu tolerance (Thorvaldsen et al., 1995). The differential metal inducibility of MTs could originate from different metal binding domains present in the MT promoters and/or to distinct metal-specific transcription factors involved in the regulation of MTs. In animal cells, MT gene expression is induced by metals via multiple metal-responsive elements (MRE) present in the MT gene 5′-regulatory region (Samson & Gedamu, 1998). Moreover, in S. cerevisiae, cup1 and crs5, the two MT genes counteracting metal cytotoxicity by sequestration of cytosolic copper are also induced at the transcriptional level by ACE1 in response to copper excess (Thiele, 1988; Culotta et al., 1994). The transcription factor ACE1 binds to the cup1 and crs5 promoters, increasing their rate of transcription by as much as 50-fold (Thorvaldsen et al., 1995). Similarly, the crf1 gene isolated from Yarrowia lipolytica codes for a transcription factor that confers resistance to Cu and Cd, with MTs as potential target genes (Garcia et al., 2002). Promoter sequence analysis of the Pimt1 gene revealed the presence of at least one putative metal regulatory sequence, suggesting that, similarly to yeast MTs, Pimt1 may also be transcriptionally activated by metal species through a transcription factor binding to MRE.
The ultimate demonstration that a particular gene encodes a protein with a specific function (e.g. metal chelation) relies on an efficient transformation system, either to knock out or to overexpress the gene of interest. To perform genetic transformation of filamentous fungi, two strategies prevail nowadays: transformation of protoplasts and agrotransformation. Agrobacterium-mediated transformation has proven itself to be a simple and reproducible fungal transformation method in many instances. Unlike protoplast transformation, Agrobacterium-mediated transformation does not rely on cell-wall-degrading enzymes and avoids the widely reported inconsistencies that arise through differences in activity of enzyme batches. DNA transfer from A. tumefaciens has been used for both gene knockout (Zeilinger, 2004) and gene transformation studies (De Groot et al., 1998) in filamentous fungi, including mycorrhizal fungi (Pardo et al., 2002, 2005; Kemppainen et al., 2005) and is being developed as a system for insertional mutagenesis in other filamentous fungi (Combier et al., 2003). This technique is reported to be easier to set up and to give higher efficiency in transformation than PEG-mediated transformation of protoplasts (Fitzgerald et al., 2003). The present work has demonstrated that the overexpression of genes through agrotransformation is also a reliable procedure to study gene function in ectomycorrhizal fungi. The increased tolerance to Cu, but not other metals, in H. cylindrosporum transformants overexpressing the Pimt1 transcript, together with the greatest degree of transcript induction in P. involutus by Cu, point to a primary role for this MT in Cu tolerance. The lack of any phenotype for many of the tested transformed clones could be the result of: (i) promoter weakness, eventually combined with an unfavorable transgene localization in the genome or truncated T-DNA transfer (although the Pimt1 sequence is close to the first transferred right border); (ii) endogenous defense mechanisms overwhelming the effects of the transgene expression.
Our previous studies suggested that metal tolerance in P. involutus is achieved by complex mechanisms involving nonspecific binding to cell walls (Blaudez et al., 2000a), responses to oxidative stress (Jacob et al., 2001) and further chelation of metals within cells by Glutathione (GSH) and complexing cysteine-rich peptides that we hypothesized to be MT (Courbot et al., 2004). In the present study, we improve our understanding of the Cd and Cu tolerance mechanisms in P. involutus by demonstrating the key role of a MT in Cd and Cu tolerance. It was demonstrated that constitutive expression of MT can inhibit the biosynthesis or accumulation of PCs in both C. glabrata and Schizosaccharomyces pombe cultures, and the authors further suggested that if expression of MT occurs, sequestration of metal ions by MTs appears the dominant metal detoxification pathway (Yu et al., 1994). However, we unsuccessfully searched for PC-like compounds in P. involutus using an improved HPLC procedure (Courbot et al., 2004). We might therefore conclude that under the present growth conditions, Cu and Cd sequestration in the cytosol of P. involutus occurs mainly through MT complexation. However, given the high glutathione content in P. involutus (Courbot et al., 2004) and the measured flow of Cd into the vacuoles (Blaudez et al., 2000a), we cannot exclude that Cd may also be transferred into the vacuoles as GSH complexes. Knowledge about metal tolerance mechanisms in ectomycorrhizal fungi will lead on to powerful applications in bioremediation.
We thank K. M. Plummer (University of Auckland, New Zealand) for the gift of the vectors pSK-GPD and pFAT3 and Prof. S. Ottonello (University of Parma, Italy) for the gift of the strains W303B, Δyap1 and EHA 101. We thank Prof. A. G. Pardo (University of Quilmes, Argentina) for helpful discussions within the course of the France-Argentina ECOS-Sud program and Dr S. Mugford (University of Oxford, UK) for carefully reading the manuscript.