Stable genetic transformation of the ectomycorrhizal fungus Pisolithus tinctorius

Journal of Microbiological Methods 63 (2005) 45 – 54 www.elsevier.com/locate/jmicmeth

Stable genetic transformation of the ectomycorrhizal fungus Pisolithus tinctorius Aı´da V. Rodrı´guez-Tovara,d, Roberto Ruiz-Medranoa, Aseneth Herrera-Martı´neza, Blanca E. Barrera-Figueroaa, M. Eugenia Hidalgo-Laraa, Blanca E. Reyes-Ma´rquezb, Jose´ Luis Cabrera-Poncec, Marı´a Valde´sd, Beatriz Xoconostle-Ca´zaresa,T a

Departamento de Biotecnologı´a y Bioingenierı´a, Irapuato, Centro de Investigacio´n y Estudios Avanzados del IPN, Av. IPN. 2508, San Pedro Zacatenco 07360 Me´xico, D.F. b Departamento de Biologı´a Celular, Irapuato, Centro de Investigacio´n y Estudios Avanzados del IPN, Av. IPN. 2508, San Pedro Zacatenco 07360 Me´xico, D.F. c Departamento Ingenierı´a Gene´tica de Plantas, Irapuato, Centro de Investigacio´n y Estudios Avanzados del IPN, Av. IPN. 2508, San Pedro Zacatenco 07360 Me´xico, D.F. d Escuela Nacional de Ciencias Biolo´gicas IPN, Carpio y Plan de Ayala, Me´xico D.F. Received 2 September 2004; received in revised form 17 February 2005; accepted 18 February 2005 Available online 2 June 2005

Abstract In the present work the genetic transformation and the expression of gene markers in transgenic Pisolithus tinctorius are reported. The ectomycorrhizae are facultative symbionts of plant roots, which are capable of affording mineral nutrients to its co-host in exchange of fixed carbon. Given the importance of this association (more than 80% of gymnosperms are associated with these fungi), its study from both basic and applied viewpoints is relevant. We have transformed this fungus with reporter genes and analyzed their expression in its saprophytic state. Genetic transformation was performed by microprojectile bombardment and Agrobacterium-mediated transformation. This last method proved to be the more efficient. Southern analysis of biolistic-transformed fungi revealed the random integration of the transgene into the genome. The accumulation of the transcript of the reporter gene was demonstrated by RT-PCR. The visualization of GFP-associated fluorescence in saprophytic mycelia confirmed the expression of the reporter gene. This is the first report on the stable transformation and expression of GFP in the ectomycorrhizal fungus P. tinctorius. D 2005 Elsevier B.V. All rights reserved. Keywords: Agro-transformation; Microprojectile bombardment; GFP expression

T Corresponding author. Tel.: +52 55 5061 3800x4315; fax: +52 55 5061 3313. E-mail address: [email protected] (B. Xoconostle-Ca´zares). 0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2005.02.016

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1. Introduction Mycorrhiza is a mutualistic symbiotic association between soil fungi and plant root systems. There are seven types of mycorrhizal associations, among which arbuscular mycorrhiza and ectomycorrhiza are the most widely distributed. In nature, 83% of dicotyledonous, 79% of monocotyledonous and all gymnosperms are associated with these fungi (Wilcox, 1991). Mycorrhiza absorbs mineral nutrients from soil and transports them to the root, while the plant provides photoassimilates, which are utilized by the fungus to grow and extend its mycelium to colonize the soil. Ectomycorrhizal fungi are capable of infecting several arboreous species; thus, this symbiosis is widespread in both temperate and boreal forests and has been proposed to enhance significantly forest production (Smith and Read, 1997). Ectomycorrhized plants survive better in adverse environmental conditions such as marginal soils, drought, pathogen attack, extreme pH and temperatures and other types of stress. The basidiomycete Pisolithus tinctorius is an almost ubiquitous ectomycorrhizal fungus, able to establish successful symbiosis with a wide range of plants (Marx, 1977), and it has been used as a model system for the analysis of ectomycorrhizal symbiotic interactions (Martin and Tagu, 1998; Martin et al., 1999). Studies on this symbiosis have focused on the characterization of cDNA clones obtained from ectomycorrhized Eucalyptus globulus (Tagu et al., 1993; Tagu and Martin, 1995, 1996; Voiblet et al., 2001). Also, gene expression profiles in ectomycorrhiza under different conditions during symbiosis has been analyzed in both symbionts (Nehls et al., 1998, 2000; Hebe et al., 1999; Sundaram et al., 2001; Martin et al., 2001). In other symbiosis (such as nitrogen fixing bacteria with legumes; Bladergroen and Spaink, 1998), the use of reporter genes fused to upstream sequences from genes potentially involved in the establishment of the association has shed important insights on the whole process. The use of transgenic (recombinant) mycorrhiza will thus be of value to study the regulation of gene expression during the symbiotic differentiation process. In fact, there is evidence of differentially expressed genes from both symbionts, which are regulated in a coordinated fashion (Kaldorf et al., 1998). Therefore, another important issue will surely be to determine the factors

involved in its regulation. However, the use of transgenic fungi for the analysis of gene expression during the establishment of the symbiosis has not been reported to date, because few transformation systems have been reported, while DNA regulatory sequences have not been described. The knowledge of the molecular mechanisms underlying this plant– microbe interaction will greatly benefit from the use of recombinant fungi harboring reporter genes; the expression of which could be regulated differentially, either directly or indirectly, by plant factors. Several methods for fungal genetic transformation have been reported (for a review, see Mullins and Kang, 2001). The capacity to genetically manipulate ectomycorrhizal fungi has an enormous potential from a basic and applied viewpoint; i.e. the expression of transgenes during the symbiosis would in principle limit the synthesized protein to the root, and would thus avoid potential non-desirable effects in the rest of the plant (Lemke et al., 1998). The genetic transformation of Laccaria laccata (Barrett et al., 1990) and Hebeloma cylindrosporum (Marmeisse et al., 1992) two ectomycorrhizal fungi, has been achieved using protoplasts. Particle gene bombardment is another method that has been used for fungal genetic transformation. In this procedure; microprojectiles covered with DNA are accelerated onto intact cells using high pressure (Sanford et al., 1993). Two ectomycorrhizal fungal species have been transformed by this procedure, Paxillus involutus (Bills et al., 1995) and Laccaria bicolor (Bills et al., 1999). Another attractive molecular tool for fungal genetic transformation is based on the use of Agrobacterium tumefaciens. This phytopathogenic bacterium has been widely used for the introduction of transgenes mostly into dicotyledonous plants. Agro-transformation of Agaricus bisporus has been performed using pieces of the fruiting body, yielding high efficiency (Chen et al., 2000). Three ectomycorrhiza-forming basidiomycetes, Suillus bovinus, H. cylindrosporum and P. involutus have also been transformed using this system. (Pardo et al., 2002). In addition, Agro-transformation has been described as a tool for insertional mutagenesis in H. cylindrosporium (Combier et al., 2003). However, analysis of transgene expression has not been described in these fungi. The present report describes the gene expression pattern of geneticallytransformed ectomycorrhizal fungus P. tinctorius. In

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addition, the transgene stability was analyzed and its expression in the saprophytic state was monitored. Our results suggest that the transgene integrates into the P. tinctorius genome in a stable manner, and heterologous regulatory sequences used for the expression of the transgenes are functional in this fungus, as evidenced by the detection of the mRNA transgene and the visualization of GFP-associated fluorescence.

2. Materials and methods 2.1. Strain and culture media A P. tinctorius strain isolated from Quere´taro, Central Me´xico, described by Valde´s (1985) was used in this study. This fungus was propagated using MNM medium (Marx, 1977). The selective media supplemented with hygromycin (200 Ag ml 1) (SigmaAldrich, St. Louis MO) was used for selection of transformed fungi. Escherichia coli strain DH5a (Invitrogen; Carlsbad, CA) was used for plasmid propagation, and was grown in TB liquid medium (Sambrook et al., 1989). The A. tumefaciens LBA4404 strain bearing plasmid pAF1 (a kind donation from Dr. June Simpson, CINVESTAV—Irapuato, Mexico), was grown in YEB solid medium (Ausubel et al., 1999) supplemented with kanamycin (50 Ag ml 1) (SigmaAldrich). The A. tumefaciens induction medium consisted of YEB medium supplemented with acetosyringone at 140 AM (Sigma-Aldrich). 2.2. Plasmids The A. tumefaciens binary vector pAF1 contains the E. coli hph structural gene under control of the promoter and terminator sequences of the tryptophan synthase C gene of Aspergillus nidulans (dos Reis et al., 2004). A newly-constructed binary vector, named pTUB-Hph/EGFP, harbors the translational fusion of the hph gene with the enhanced green fluorescent protein (EGFP) coding region, driven by Ustilago maydis a-tubulin regulatory sequences. This vector was constructed as follows: the hph-EGFP ORF from the plasmid pHygEGFP (Clontech, Palo Alto CA) was subcloned into the plasmid pUm3, which contains the promoter and terminator sequences of the U. maydis tubulin encoding-gene. Then, the expression unit,

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previously filled-in with the Klenow fragment (New England Biolabs) was subcloned into the filled-in XbaI site of the binary vector pAF1, previously described. 2.3. Transformation by microprojectile bombardment Particle gene bombardment was performed using a Biolistic device (Biorad, Hercules, CA). Tungsten micro carriers of 6 Am in diameter were covered with circular pAF1 plasmid DNA containing CaCl2 and spermidine as described by de la Fuente et al. (1997). Particles were delivered onto fungal mycelium at a pressure of 900 lb/in2. The fungal aerial mycelium was previously dissected with a sterile razor blade into 0.5 cm2 pieces and separately placed on petri dishes containing MNM medium. Since disruption of aerial P. tinctorius hyphae prevented cell regeneration after transformation, pieces of agar containing intact mycelia were used. The calculation of genetic transformation efficiency was expressed as 5 mm2-myceliacontaining agar-pieces with fungal growth in the presence of the selection marker. 2.4. Agro-transformation A. tumefaciens LBA4404 strain harboring either the binary plasmids pAF1 or pTUB-Hyg/EGFP was grown in YEB solid medium supplemented with kanamycin (50 Ag ml 1) at 28 8C for 48 h. A single colony was then inoculated in 50 ml of YEB liquid medium supplemented with kanamycin (50 Ag ml 1), and propagated at 28 8C for 48 h with continuous agitation (200 rpm) until the culture reached an O.D.(600 nm) of 0.4. The culture was then supplemented with acetosyringone (Sigma-Aldrich) at a final concentration of 140 AM. Induced culture was incubated for additional 4 h. Both bacterial and fungal cells were co-cultivated by adding 10 ml of induced A. tumefaciens to petri dishes containing agar-pieces with P. tinctorius mycelia. Cells were incubated overnight at 28 8C after which, the fungus was extensively rinsed with distilled sterile water, transferred to MNM medium containing 150 Ag ml 1 Cefatoxime (Sigma-Aldrich) and incubated for two more days. Finally, potential transformed mycelium was transferred onto MNM solid medium containing 200 Ag ml 1 hygromycin and incubated for 2 weeks at 28 8C.

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2.5. Expression analysis of reporter EGFP in saprophytic fungus Genetically-transformed mycelium was grown on glass slides covered with a thin layer of solid MNM culture media in petri dishes. The slides were carefully removed, and a cover slip was then deposited onto the fresh tissue for microscopic visualization with the fluorescence microscope. EGFP expression was monitored in P. tinctorius aerial mycelium and Pinus greggii colonized roots. Fluorescence emission of the detached mycelium was examined with the use of a confocal laser-scanning microscope (Biorad). The green fluorescence emission associated with GFP was detected using a krypton/argon laser with the following filter settings: 488 nm excitation and 525 nm emission. Images were recorded and processed using Adobe Photoshop 6.0 image software (Sunny Valley, CA). 2.6. PCR assays The identity of P. tinctorius strain was performed by amplifying and sequencing the ITS region comprising the 5.8 ribosomal DNA gene and partial sequences of the 18 S and 28 S ribosomal genes. Primers ITS5 (forward) and ITS4 (reverse), were designed by White et al. (1990). For the detection of the transgenes in P. tinctorius by PCR, total DNA was extracted from 50 mg of frozen aerial mycelium by using a modified CTAB method (Murray and Thompson, 1980; Allers and Lichten, 2000). The reaction was performed on a T-Gradient thermoblock (Biometra, Germany). The following components were mixed to amplify the hph gene from the P. tinctorius genome. The reaction was carried out using 100 ng of P. tinctorius total DNA, 1.5 mM MgCl2, 3 AM of each primer, forward, 5V-ATGAAAAAGCCTGAACTCACCGCGACGT-3V and reverse: 5V-CTATTCCTTTGCCCTCGGACGAGTGCTG-3V, 5 AM dNTPs, 1  PCR buffer, and 1 unit of Taq polymerase (GIBCO, Invitrogen). PCR conditions were as follows: 95 8C, 30 s; 52 8C, 30 s and 72 8C, 1 min, for 30 cycles. The PCR products were resolved by electrophoresis on a 0.8% agarose gel, stained with ethidium bromide and visualized using an EDAS system (Kodak, Rochester, NY). A similar procedure was performed for RT-PCR of the hph transcript. RNA was obtained using a modified protocol described by Logemann et al.

(1987). DNaseI (Ambion) treated-RNA was used as template for RT-PCR. Assays were performed using the Superscript one-step RT-PCR with platinum Taq kit, according to the recommendations of the manufacturer (Invitrogen). PCR primers (forward: 5V-CAACATTTCCGTGTCGCCCTTATT-3V and reverse: 5VCCGATCGTTGTCAGAAGTAAGTTGG-3V) corresponding to a 400 bp region of the h-lactamase gene harbored in pAF1. The reaction was performed as described above. 2.7. Southern blot analysis Purified genomic DNA (10 Ag) of individual transformants was digested with EcoRI, separated on an agarose gel and transferred to positivelycharged nylon membranes as described (Sambrook et al., 1989). The probe was radioactively labeled with [a-32P]-dCTP using a random priming labeling kit (NEN, Dupont, Boston MA). The hybridization was carried out under high stringency conditions using the following solution: 0.5 M NaH2PO4, pH8.0, 7% SDS, 1% BSA, 1 mM EDTA (Ausubel et al., 1999). Posthybridization washes were performed at 65 8C with 2  and 0.1  SSC and the membrane was finally exposed to a Biomax X-ray film at 80 8C (Kodak, Rochester, NY). 2.8. Transgene stability in the P. tinctorius saprophytic state Independent transgenic isolates from Agro-transformed P. tinctorius mycelia were propagated on MNM both solid and liquid medium in absence of hygromycin for 5–10 months. The fungi were transferred to selective media and their growth evaluated. Fungal transformants producing the brown pigment (characteristic of this species) in the presence of hygromycin were considered metabolically active, and thus selected for further analyses. The presence of the transgene was monitored by PCR and Southern blot analyses as described before. 2.9. Root colonization with ectomycorrhiza Forty (four week-old) P. greggii plantlets were inoculated with wild type or genetically-transformed

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P. tinctorius mycelia. The seedlings were grown for three months using previously sterilized sphagnum– vermiculite mixture (1 : 1), in a controlled environment chamber (Conviron, Manitoba, Canada), programmed with the following conditions: 16 h light, 8 h darkness, 27 8C and 70% relative humidity. Pine roots were inoculated with P. tinctorius mycelia propagated in vitro. Fungal colonization was quantified as the number of first- and second-order lateral roots present in each primary roots from colonized and non-colonized roots. Cross-sections of fresh tissue, excised from colonized roots were visualized in order to confirm the presence of fungal sheath.

3. Results 3.1. P. tinctorius is a new strain isolated from central Me´xico The determination of the P. tinctorius strain was performed by amplifying and sequencing the ITS region comprised by the 18 S, 5.8 S and 28 S gene coding region as described in Materials and methods. Sequence analysis showed that this isolate from Quere´taro, Me´xico, deposited in the Genbank with the accession number 647903 is 97% homologous to the described P. tinctorius H237 strain (Martin et al., 2002, Genbank accession number AF374712). 3.2. Generation of genetically-transformed P. tinctorius mycelia using biolistic procedures The ability of disrupted mycelium to form aerial mycelium was impaired after the transformation procedure. For this reason, 0.5 mm2 agar-pieces containing intact aerial mycelium were used for further transformation assays. Microprojectile bombardment yielded several independent transformants when using pAF1 or pTUB-Hph/EGFP, and the calculated transformation efficiency was 10% of mycelium pieces/Ag of circular DNA. Two weeks after transformation, the colonies that grew in the presence of the antibiotic were collected for further analyses. Mycelia bombarded with tungsten particles devoid of DNA failed to produce spontaneous antibiotic-resistant mycelia pieces after two or three weeks. However, for longer incubation periods, production of brown pigment was

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observed in the wild type strain, an indication of metabolic activity of such non-transformed mycelium. In Fig. 1, panel A, the result of a PCR of mycelia transformed with pAF1 using primers specific for a fragment of the E. coli hygromycin phosphotransferase gene is shown. In it, a single product of the expected size (1 kbp) was obtained in the colonies displaying antibiotic resistance. 3.3. A. tumefaciens-mediated transformation A. tumefaciens harboring the pTUB-Hph/EGFP binary plasmid efficiently transferred the T-DNA to hyphal cells as shown by active fungal cell growth on selective culture medium. The procedure yielded hygromycin-resistant mycelia cells when the bacterial culture was induced with acetosyringone. After fifteen days, fungal mycelia were harvested for further analyses. The calculated efficiency was of 60% agarpieces containing mycelia that were able to grow under selective pressure referred to the total number of agarpieces. Evidence of transformation was obtained by PCR, where a band of the expected size (1 kbp) was obtained (Fig. 1, panel B). As shown by control assays, the wild type strain failed to amplify the hph gene and PCR of the h-lactamase gene was similarly negative, thus discarding contamination with bacteria (data not shown). Genetic stability of transgenic P. tinctorius was evaluated by propagating fungi without selective pressure for five to ten months, and then tested for the presence of the hph gene by Southern blot analysis. As shown in Fig. 1, panel C, bands of different sizes were detected in independent transformants, indicating random insertion of the T-DNA into the fungal genome. The detection of the transgene in previously transformed cells failed in only one out of eight tested fungi (Fig. 1C, lane 2), indicating a high mitotic stability. These results strongly suggest that the transgene was stably integrated into the genome. 3.4. Transgenic P. tinctorius displayed similar mycorrhizal colonization capacities than wild type strain The colonization capacity of transgenic fungi was investigated by infecting 4 week-old P. greggii plantlets maintained under controlled conditions. In parallel experiments, both wild type and transgenic fungi were used to infect plant roots. Colonized roots

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with wild type strains displayed in average 1.8 secondary branches/cm of primary roots, while transgenic fungus produced 1.7 branches/cm. Roots of

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non-colonized plants formed 0.3 secondary roots/cm. Thus, transgenic fungi induced secondary roots with the same efficiency than the wild type strains. 6

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probe 0.5 kb Fig. 1. Presence of transgene in transformed fungi. (A) Agarose gel of PCR products obtained from transgenic fungi transformed by biolistics delivery of DNA procedures. Molecular weight marker (lane 1), phsp70-Hyg-GFP DNA (lane 2), total DNA from wild type P. tinctorius (lane 3), total DNA from transgenic P. tinctorius obtained via microprojectile bombardment, harboring phsp70-Hyg-GFP (lanes 4, 5 and 6). (B) Agarose gel of PCR products obtained by Agro-transformation. Molecular weight marker (lane 1), pAF1 (lane 2), total DNA from wild type P. tinctorius (lane 3), PCR product from Agro-transformed cells with pAF1 (lane 4). (C) Southern blot of Agro-transformed fungi, using total DNA digested with EcoRI. DNA from wild type P. tinctorius (lane 1), DNA of independent transformed fungi, grown without selective pressure (lanes 2–9). (D) Diagram of T-DNA inserted into the P. tinctorius genome by Agro-transformation, indicating the position of the probe used in the Southern blot.

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mycorrhized roots was not conclusive because of the high autofluorescence emitted by the plant tissue (data not shown).

Consistent with these observations, microscopic analysis of fresh root preparations showed no differences in colonization, in that the presence of the fungal mantle and Hartig net were observed in both cases (data not shown).

4. Discussion 3.5. Hyg-GFP is efficiently expressed in P. tinctorius We report in the present work the generation of genetically-transformed P. tinctorius using both biolistic microparticle bombardment and Agrobacteriummediated genetic transformation. In terms of efficiency, agro-transformation yielded the highest efficiency (60%), compared with biobalistic (10%), and was quite reproducible. A limitation in the transformation of several species is the availability of functional regulatory sequences and reporter genes, since no regulatory sequences from P. tinctorius have been tested. We chose to use U. maydis tubulin promoter and terminator sequences, based on the fact that both U. maydis (a plant pathogen) and P. tinctorius are basidiomycetes. Thus, in view of our results it is altogether possible that in the particular case of the tubulin gene, it might be regulated in a similar manner in both systems. A second promoter utilized was trpC from A. nidulans, an ascomycete, regulating the expression of hph in pAF1. This promoter has been described to be functional in several evolutionarily distant species, and, indeed, this promoter was also functional in P. tinctorius. The presence of the introduced transgene in the P. tinctorius genome, which was stable in almost 90%

Saprophytic, transgenic fungi bearing the Hygromycin-GFP protein fusion were assayed for detection of Hph-GFP coding mRNA. This was detected by RTPCR assays as described in Materials and methods. As shown in Fig. 2 panel B, a sharp, single band was amplified when DNaseI treated-RNA from transgenic fungus was used as template. No detection of transgene mRNA in wild type fungus was observed. In order to visualize the reporter protein GFP, transgenic fungi obtained by Agro-transformation were analyzed to determine GFP expression and its accumulation in aerial mycelia (Fig. 3). Wild type and transformed three week-old mycelium were examined using confocal microscopy. GFP-associated green fluorescence was confined mostly to cytoplasm, but was also observed in defined regions, probably nuclei (Fig. 3, panel A, B and C), as previously described in other systems (Garcia-Mata et al., 1999). No green fluorescence was detected in wild type P. tinctorius. (Fig. 3, panel D, E and F). On the other hand, no significantly higher GFP accumulation was observed in older tissues, probably because of the slow metabolism of P. tinctorius. GFP detection in

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Fig. 2. The transgene is actively transcribed. (A) Total RNA from wild type fungus and from biolistic-transformed fungus harboring phsp70Hyg-GFP. (B) RT-PCR of hph using as template the RNA previously treated with DNaseI. Arrow in B indicates the band of the expected size (0.4 kbp) of the amplified hph transcript.

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Fig. 3. Green fluorescent protein expression in transgenic fungi. Confocal microscopy images of GFP-associated fluorescence in mycelia from wild type and Agro-transformed P. tinctorius. (A) Mycelia grown on a slide showing green fluorescence due to GFP accumulation. (B) The same field was observed with visible light. (C) Both images were superimposed. (D) No fluorescence was detected in wild type fungi using the same filter settings for GFP detection. (E) The mycelium was observed with visible light. Both images were also superimposed. Bar in A represents 100 Am, and is common to (B-F).

of the transformants after 10 months of continuous growth, strongly supports the notion that the transgene was mitotically stable. The efficient expression of foreign genes in transgenic organisms obviously depends on both transgene transcription and translation. Hph mRNA accumulation was detected in transgenic fungus, indicating the functionality of the gene expression unit. The analysis of antibioticresistant fungus expressing GFP under the hsp70 promoter indirectly demonstrated that it is correctly transcribed and translated in P. tinctorius. GFPassociated fluorescence was observed in saprophytic and extramatrical mycelia. However, no fluorescence was clearly distinguished within root plant tissue, probably due to the presence of lignin and other phenolic compounds present in the root, the emission of which coincides with the GFP spectrum. As for its capacity of interaction with the plant symbiont, transgenic P. tinctorius displayed similar

colonization efficiency when compared to the wild type fungus. This suggests that the presence and/or expression of the transgene did not affect significantly the fitness of the transformed fungus, nor did it interrupt genes potentially involved in the processes of recognition and establishment of the symbiosis. Because of the high efficiency of agro-transformation and stability of the transgenic fungus, this system is proposed as a potentially useful tool for the generation of insertional mutants as well for the study of gene regulation during the development of the ectomycorrhizal symbiosis.

Acknowledgements We wish to thank Dr. Jose´ Ruiz Herrera for critical review of the manuscript. Thanks are also given to the members of our laboratory for useful discussions.

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