Genetic transformation of ectomycorrhizal fungi mediated by Agrobacterium tumefaciens

Mycol. Res. 106 (2) : 132–137 (February 2002).

# The British Mycological Society

132

DOI : 10.1017\S0953756201005378 Printed in the United Kingdom.

Genetic transformation of ectomycorrhizal fungi mediated by Agrobacterium tumefaciens

Alejandro G. PARDO1,2,*†, Mubashir HANIF1, Marjatta RAUDASKOSKI1 and Markus GORFER1,‡ " Department of Biosciences, Division of Plant Physiology, P.O.B. 56, University of Helsinki, Helsinki F-00014, Finland. # Programa de InvestigacioT n en Interacciones BioloT gicas, Centro de Estudios e Investigaciones y Departamento de Ciencia y TecnologıT a, Universidad Nacional de Quilmes, Roque SaT enz Peng a 180, (B1876BXD) Bernal, Provincia de Buenos Aires, Argentina. E-mail : apardo!unq.ed.ar Received 2 February 2001 ; accepted 13 October 2001.

A technique was developed for transforming the ectomycorrhiza-forming basidiomycetes Suillus bovinus, Hebeloma cylindrosporum, and Paxillus involutus based on Agrobacterium tumefaciens-mediated T-DNA transfer. The selection marker employed was the Shble gene conferring resistance to phleomycin under control of the Schizophyllum commune GPD promoter and terminator. Transformants from all three investigated species were shown by PCR to contain the GPDScP-Shble-GPDScT construct, although the fate of the foreign DNA (integrated vs episomal, single-copy vs multi copy) could not be determined. The mycorrhiza formed between S. bovinus Bler transformants and Pinus sylvestris did not reveal any differences from those formed with untransformed Suillus bovinus.

INTRODUCTION Ectomycorrhizas are an ecologically and economically important symbiosis between the fine roots of trees, including most species in boreal and temperate forests, and the soil-borne mycelium of a wide range of fungi belonging mainly to the basidiomycetes. After many decades of careful description of the anatomical, physiological and biochemical features of ectomycorrhiza, recent years have brought new insights at the molecular level. For example, fungal components regulating fine root morphogenesis and plant gene expression have been identified (Nehls et al. 1998a, Ditengou & Lapeyrie 2000), as well as cytoskeletal elements that are involved in the morphological changes that take place when plant roots and fungal mycelium become symbiotic (Tarkka et al. 2000, Gorfer et al. 2001). Alterations in the composition of the fungal cell wall during ectomycorrhizal development are mirrored by differences in gene- and protein-expression levels and undoubtedly point to the special requirements and features of the mutualistic interface (Tagu & Martin * Corresponding author. † Present address : INRA, Centre de Nancy, UMR IaM, 5480 Champenoux, France. ‡ Present address : Ecowork Lab coratoria, La$ ngenfeldgasse 27, 1120 Vienna, Austria.

1996, Laurent et al. 1999). Models explaining equal distribution of plant photosynthates between the two partners in the symbiotic organ arose from studies on sugar transporters (Nehls et al. 1998b). Assignment of functions to genes and their products has however been limited to deduction from sequence homologies, subcellular localisation studies and expression in heterologous hosts since transformation techniques for the vast majority of ectomycorrhizal basidiomycetes have not been readily available. Exceptions are Laccaria laccata (Barret, Dixon & Lemke 1990) and Hebeloma cylindrosporum (Marmeisse et al. 1992), which have been transformed by the protoplast method, and Paxillus involutus (Bills et al. 1995) and Laccaria bicolor (Bills et al. 1999), which have been transformed by particle bombardment. Since the first report on successful genetic transfer from Agrobacterium tumefaciens to the yeast Saccharomyces cerevisiae (Bundock et al. 1995), a number of mainly ascomycetous filamentous fungi have been shown to be amenable to this transformation system (de Groot et al. 1998, Gouka et al. 1999, Abuodeh et al. 2000, Chen et al. 2000). DNA-transfer to fungi apparently functions via the same mechanism as to plants, where acetosyringone (AS) induces virulence (vir) gene expression, resulting in generation of a singlestranded DNA flanked by the direct repeats of the left

A. G. Pardo and others and right border, the so-called T-DNA. This DNA is protected by a proteinaceous coat and is transferred via a transmembrane structure to the host. Upon import of the nucleoprotein filament into the nucleus, the T-DNA integrates into the host genome, preferentially as a single copy Zhu et al. 2000). Whereas the whole machinery for transfer of the T-DNA from Agrobacterium to the plant or fungus seems to be provided by the bacterium, integration of the foreign DNA into the genome depends on host factors. In plants the preferred mode of integration is by illegitimate recombination, since no extensive homology exists between the T-DNA and its site of integration into the plant genome. In S. cerevisiae, on the other hand, TDNA can be targeted to a specific site by inclusion of homologous sequences into it. Homologous recombination can improve transformation efficiency of yeast after cocultivation by a factor of approximately 200 (Bundock & Hooykaas 1996). In the present study we have investigated the applicability of the Agrobacterium system for transformation of the ectomycorrhizal basidiomycetes Suillus bovinus, which is the major object of investigation in our group, and Hebeloma cylindrosporum and Paxillus involutus, two species that were already previously successfully transformed by other methods (Marmeisse et al. 1992, Bills et al. 1995). MATERIALS AND METHODS Bacterial and fungal strains For cloning Escherichia coli XL1-Blue (Stratagene, CA) was used. Agrobacterium tumefaciens LBA1100 from Paul Hooykaas (Leiden University) was used for the transformation of ectomycorrhizal fungi. The ectomycorrhizal homobasidiomycetes used as recipients in cocultivations with A. tumefaciens were : Suillus bovinus isolate no. 096 from David Read (University of Sheffield) ; Hebeloma cylindrosporum dikaryotic strain HC1 ; and Paxillus involutus from Scotland Bush State (Institute of Terrestrial Ecology). Voucher specimens are kept in the Universidad Nacional de Quilmes Fungal and Bacterial Culture Collection, Argentina.

133 H O ; 10 mg MnSO :H O ; 10 mg FeCl :6 H O ; 1 mg # % # $ # ZnSO :7 H O ; 50 mg inositol ; pH" 6 (unadjusted ; % # for pH7n5 adjusted with 1  KOH). Minimal medium for Agrobacterium l−" : 10n5 g K HPO ; 4n5 g KH PO ; # % # % 1 g (NH ) SO ; 0n5 g Na -citrate :2H O ; 0n2 g %# % $ # MgSO :7 H O ; 1 mg thiamine-HCl ; 2 g glucose ; % # 100 µg ml−" kanamycin for selection of transformants. Induction medium : minimal medium plus 40 mM MES and 0n5 % glycerol pH 5n3. Induction agar : induction medium plus 2 % agar. Selection media : malt agar or Moser pH 7n5j100 µg ml−" cefotaxime ; 100 µg ml−" ampicillin ; 125 µg ml−" tetracycline, and the appropriate amount of phleomycin (Cayla, Toulouse), 100 µg ml−" for selection of Suillus bovinus transformants, 300 µg ml−" for Hebeloma cylindrosporum, and 50 µg ml−" for Paxillus involutus. Plasmid construction The phleomycin resistance box (GPDScP-ShbleGPDScT) in pGPhT (Schuren & Wessels 1994) was shortened by taking out a 0n7 kb HindIII\SalI-fragment from the promoter region resulting in plasmid pGFT17. The 1n5 kb phleomycin resistance box was excised as a HindIII\EcoRI fragment and inserted into pBIN19 (Bevan 1984). The vector was electroporated into Agrobacterium tumefaciens LBA1100 by the Gene Pulser (Bio-Rad, Hercules, USA) according to the manufacturer’s protocol, except that modified TB medium l−" (11n8 g casein hydrolysate ; 23n6 g yeast extract ; 9n4 g K HPO ; 2n2 g KH PO ; 1 g glucose) was # % # % used for preparing Agrobacterium competent cells. T-DNA transfer

Pinus sylvestris seeds were obtained from the Haapastensyrja$ Tree Breeding Centre, Finland. The seeds had originally been collected from pine trees belonging to class B3 in Hyryla$ , Southern Finland (Niini 1998). Synthesis of ectomycorrhiza between Suillus bovinus and Pinus sylvestris was carried out according to Timonen et al. (1993).

Fungal colonies were pre-grown on cellulose membranes (CelluSep T3 dialysis membranes with a nominal molecular weight cut-off of 12 000–14 000 ; Membrane Filtration Products Inc., TX) on ME-agar for 7 d at 20 mC and then transferred to induction agar plates with or without 200 µM AS (Aldrich, WI). The colonies were inoculated with 50 µl of Agrobacterium tumefaciens (pBIN19-17) culture prepared as follows : an overnight culture in minimal medium plus kanamycin at 29 m (OD " 0n2) was centrifuged, the supernatant replaced '!! by induction media plus kanamycin and grown for 6 h at 29 m. Kanamycin was omitted from the media when untransformed A. tumefaciens was used for control experiments. The cocultivation plates were incubated at 20 m for 4 d, whereafter membranes with the fungal colonies were transferred to selection plates, kept at 4 m overnight and then shifted to the growing temperature (20 m).

Media composition

Screening of transformants

Malt extract agar (ME-agar) : 1 % malt extract ; 2 % agar. Moser 6 (modified from Moser 1963) l−" : 15 g agar ; 10 g glucose ; 2 g peptone ; 0n2 g yeast extract ; 0n5 g KH PO ; 150 mg MgSO :7 H O ; 75 mg CaCl :2 # % % # #

Mycelium from the putative transformants and wild type strains grown on cellulose membranes on ME-agar for 1 wk was frozen in liquid nitrogen, ground to a fine powder, and DNA was extracted by the CTAB method

Pine seeds and mycorrhiza formation

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(Ausubel et al. 1995). Putative transformants were checked by amplifying 0n8 kb and 0n5 kb fragments corresponding to parts of the Phle construct by PCR. About 100 ng of genomic DNA and " 10 pg of plasmid DNA respectively was routinely used as template. The PCR program for both amplifications was as follows : initial denaturation at 94 m for 5 min, 35 cycles of 94 m for 45 s, 64 m for 30 s and 72 m for 45 s, and final extension at 72 m for 7 min. Several amplified 0n8 kb fragments were cheked by sequencing with a Stretch DNA Sequencer ABI model 373A (University of Maine, DNA Sequencing Facility, ME) after purification from the PCR mix with a WizardR PCR preps DNA purification system (Promega, WI). In order to be sure that the transformed fungal DNA was free of bacterial DNA a control PCR was carried out with specific primers for pBIN19 annealing outside of the T-DNA. These PCR reactions should be negative in truly transformed fungi but positive if Agrobacterium cells were contaminating the mycelium. In this case primers BIN-1 [5h-(4821)-TCGCTGAACGGTTGCGAGAT-(4840)-3h] and BIN-2 [5h-(5574)-GTAGGTTCGAGTCGCGAGAT-(5555)3h] were used. These primers anneal to parts of the tetA gene in pBIN19 (accesion numbers U09365 ; GI520486) outside of the T-DNA region. The PCR program was as follows : initial denaturation at 95 m for 2 min, 35 cycles of 94 m for 45 s, 50 m for 1 min and 72 m for 45 s, and final extension at 72 m for 5 min. In the case of A. tumefaciens as template, an overnight culture in minimal medium without or with kanamycin (A. tumefaciens LBA 1100 and pBIN19-17 respectively) at 29 m (OD " 0n2) was centrifuged, the supernatant '!! replaced by distilled water, boiled for 10 min, and used as template (1 µl) in the PCR reaction. Under the present conditions this PCR proved to be sensible with as less as 1 pg of plasmid DNA. In order to know whether DNA in untransformed fungal cells was able to be amplified a control PCR targeted to the fungal ribosomal internal transcriber spacer (ITS) was carried out. In this case primers ITS1 [5h-TCCGTAGGTGAACCTGCGG-3h] and ITS4 [5hTCCTCCGCTTATTATTGATATGC-3h] constructed for molecular phylogenetic studies of fungi (White et al. 1990) were used. The PCR program was the same as for pBIN19 except that 30 cycles of amplification were carried out.

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Fig. 1. Vector pBIN19-17, which contains the Phle construct (HindIII-GPDScP-Shble-GPDScT-EcoRI) and the Neomarker under control of plant transcriptional elements between the left and right border repeats comprising the socalled T-DNA, and the Kanr-marker for selection in Escherichia coli and Agrobacterium tumefaciens (not drawn to scale).

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Fig. 2. Sequences of primers used for PCR analysis of transformants, their position in the Phle-box and the size of the expected amplification products.

As a marker for selection of transformants the Streptoalloteicus hindustanus phleomycin resistance gene (Shble) was chosen, since : (1) it has already been shown to be efficiently transcribed and translated in other homobasidiomycetes (Schuren & Wessels 1994) ; (2) it was available as a fusion to transcription-control elements from a homobasidiomycete, the promoter and terminator region of the highly and constitutively

expressed GPD gene (glyceraldehyde-3-phosphate dehydrogenase) from Schizophyllum commune (Schuren & Wessels 1994) ; and (3) resistance levels to phleomycin for at least two of the investigated fungi – Paxillus involutus and Suillus bovinus – were in a range allowing efficient selection of transformants. Growth of P. involutus was completely abolished at phleomycin

RESULTS AND DISCUSSION

A. G. Pardo and others

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Fig. 3. Analysis of ectomycorrhizal basidiomycetes Bler transformants by PCR. (A) PCR targeted to the fungal ribosomal ITS : Lane 1, untransformed Suillus bovinus ; Lane 2, untransformed Hebeloma cylindrosporum ; Lane 3, untransformed Paxillus involutus ; Lane 4, negative control without template. The PCR was performed with primers ITS1 and ITS4. (B) Analysis of P. involutus transformants ; Lanes 1–3, individual transformants (100 %, 18 out of 18, scored positive by PCR) ; Lane 4, untransformed Paxillus involutus ; Lane 5, positive control with pGFT-17 as 100 bp template, Lane 6, 100 bp ladder. The PCR was performed with the primers GPDPok and BLEant resulting in an 0n8 kb product. (C) As B, but the PCR performed with the primers GPDPsen and BLEant resulting in an 0n5 kb product. (D) Analysis of H. cylindrosporum Bler transformants by PCR ; Lanes 1–3, individual transformants (86 %, 13 out of 15, scored positive by PCR) ; Lane 4, untransformed H. cylindrosporum ; Lane 5, positive control with pGFT17 as template ; Lane 6, 100 bp ladder. The PCR was performed with the primers GPDPok and BLEant resulting in an 0n8 kb product as indicated by the arrow. (E) As D but the PCR performed with primers GPDPsen and BLEant resulting in an 0n5 kb product as indicated by the arrow. (F) Analysis of S. bovinus Bler transformants by PCR : Lanes 1–3, individual transformants after reisolation from mycorrhizal mantle (see text for details) ; Lane 4, untransformed S. bovinus ; Lane 5, positive control with pGFT-17 as a template ; Lane 6, 100 bp ladder. PCR with the primers GPDPok and BLEant resulting in an 0n8 kb product as indicated by the arrow. (G). As F but the PCR performed with the primers GPDPsen and BLEant resulting in an 0n5 kb product as indicated by the arrow. In S. bovinus 73 % of the transformants (11 out of 15) scored positive by PCR. (H) PCR targeted to parts of the tetA gene in pBIN19 out of the T-DNA : Lane 1, individual transformant of S. bovinus (100 %, 15 out of 15, transformants scored negative in this PCR). Lane 2, individual transformant of H. cylindrosporum (100 %, 15 out of 15, transformants scored negative in this PCR) ; Lane 3, individual transformant P. involutus (100 %, 18 out of 18, transformants scored negative in this PCR) ; Lane 4, A. tumefaciens LBA1100 ; Lane 5, A. tumefaciens (pBIN1917) ; Lane 6, pBIN19-17 (1 pg) ; Lane 7, 100 bp ladder.

concentrations as low as 5 µg ml−" and growth of S. bovinus at 50 µg ml−". Hebeloma cylindrosporum showed occasionally residual growth at concentrations of up to 100 µg ml−" and only at 200 µg ml−" phleomycin was no growth observed. The phleomycin resistance box (GPDScP-Shble-GPDScT) was inserted into the TDNA region of the binary vector pBIN19 (Bevan 1984) and the resulting plasmid with a size of 13n25 kb was named pBIN19-17 (Fig. 1).

on phleomycin plates gave rise to resistant fungal colonies only when transformed A. tumefaciens was used for cocultivation together with AS, indicating that induction of the vir-genes was essential for T-DNA transfer between A. tumefaciens and hyphae of ectomycorrhizal fungi. Up to 80 % of the colonies were found to be transformed to phleomycin resistance after cocultivation, but this varied largely between individual experiments.

Transformation procedure

Analysis of transformants

Since neither spores nor protoplasts are easily available from the majority of ectomycorrhizal fungi, a transformation procedure with direct DNA transfer from Agrobacterium to mycelium was developed. Selection

Following four rounds of selection on phleomycin, mycelium of potential transformants selected at random was transferred to non-selective medium for DNA isolation. PCR-analysis with primers corresponding to

Transforming ectomycorrhizal-forming basidiomycetes

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the Schizophyllum commune GPD-promoter and Shble (Fig. 2) gave the expected bands, which were never detected in DNA from untransformed mycelium of the investigated fungi (Fig. 3). Untransformed fungal DNA was able to be amplified as is shown in Fig. 3A where a control PCR targeted to ribosomal ITS was carried out. In the case of Paxillus involutus all phleomycin resistant (Bler) colonies (18) contained the phleomycin resitance box as deduced from PCR results whereas only 86 % (13 out of 15) of Hebeloma cylindrosporum Bler-colonies and 73 % (11 out of 15) of S. bovinus Blercolonies scored positive by PCR analysis. This most probably reflects the higher sensitivity of P. involutus to phleomycin. Bler-colonies from S. bovinus and H. cylindrosporum may actually represent a heterogeneous mixture of transformed and untransformed hyphae, rather than result from spontaneous mutations to Bler or adaptation to high levels of phleomycin, since Blercolonies were never obtained in control experiments with untransformed Agrobacterium tumefaciens or ASfree induction media. In Bler-colonies that scored negative in PCR the amount of transforming DNA as a proportion of the total DNA may be below the detection limit of the method. Furthermore, transformed fungal DNA was proved to be free of Agrobacterium DNA as these samples were always unable to be amplified by PCR when the primers BIN1 and BIN-2, which anneal to parts of the tetA gene in pBIN19 outside of the T-DNA, were used (Fig. 3H). Besides, two randomly chosen S. bovinus, one H. cylindrosporum and one P. involutus Bler-transformants were used to check whether the 0n8 kb fragment derived by PCR corresponded to the transferred phleomycin resistance box. In all these cases the construct identity was confirmed by sequencing a 0n5 kb fragment using the GPDsen primer (data not shown). Transformants that were shown by PCR to contain the phleomycin resistance box were subjected to Southern analysis. As a probe the 0n8 kb PCR-product containing part of the GPDSc-promoter and most of the Shble-gene was used. Surprisingly, DNA hybridisation signals could not be detected in any of the samples. This probably again reflects the fact that the transformants are composed of a majority of untransformed nuclei and only a minority of actually transformed nuclei, which are, however, able to sustain growth under selective pressure. Re-selection of transformants on increased phleomycin concentrations and increased pH still did not allow detection of positive bands on a Southern blot. Increasing the pH from " 6 on Moser 6 medium to 7n5 lowered the effective phleomycin concentration for untransformed S. bovinus from 50 µg ml−" to 25 µg ml−" without affecting growth under non-selective conditions (Moser medium was used in these experiments instead of ME-agar due to its increased buffering capacity). Three randomly chosen S. bovinus Bler-transformants were used for synthesis of mycorrhiza with Pinus sylvestris. No differences in ectomycorrhiza formation

were seen between transformed and untransformed S. bovinus and vigorously growing phleomycin-resistant mycelium was readily obtained when ectomycorrhizal short roots were dissected and incubated on ME-agar j200 µg ml−" phleomycin, indicating that transformed nuclei took part in establishing the mycorrhizal association. Mycelium obtained by this procedure was subjected to PCR analysis and the Bler-marker could be detected (Figs 3 F–G, lanes 1–3). Nevertheless, in Southern blots they still scored negative, indicating that formation of mycorrhizae did not sufficierly alter the ratio between transformed and untransformed nuclei. CONCLUSIONS Herein we show that ectomycorrhizal basidiomycetes can be transformed by the Agrobacterium system, although the protocol still needs optimization. We assume that the T-DNA is integrated predominantly as a single copy into the genome as already previously shown for a wide range of fungi including another basidiomycete, Agaricus bisporus (Chen et al. 2000). Even though we could in none of the investigated transformants detect the transferred DNA by Southern analysis, generation of phleomycin-resistant transformants was strictly dependent on the presence of pBIN19-17, the plasmid carrying the GPDScP-ShbleGPDScT construct between its T-DNA borders, and AS, which induces the vir-operon in Agrobacterium for efficient T-DNA transfer. Likewise, the GPDScP-ShbleGPDScT construct could be detected by PCR only from transformants and never from the untransformed parental strains. Failure to detect the transferred DNA could be explained by the presence of both transformed and untransformed nuclei in the mycelium of the transformants. A 1 : 1 ratio of transformed to untransformed nuclei is already expected from the dikaryotic nature of the strains used in this study, and this ratio could have been further changed in favour of untransformed nuclei by the methodological restrictions imposed by special features of the investigated fungi. Under laboratory conditions it is difficult to obtain single cells (spores or viable protoplasts), which would be necessary to produce genetically homogeneous mycelium. As the best alternative, only small inocula were taken from the margins of Bler colonies for subculturing, and consistent with the idea that the transformants contained a heterogenous mixture of transformed and untransformed nuclei, often only a minority of subcultured inocula from the same transformant continued to grow on selective medium. Future experiments with different selection markers may help to resolve the problems in the transformation system. Furthermore, fluorescence in situ hybridization (FISH) (Taga & Murata 1994, Trouvelot et al. 1999) may be a useful tool to allow the differentiation of transformed and non transformed nuclei within a single fungal colony. All herein tested fungal species could be transformed via cocultivation indicating that this system

A. G. Pardo and others may prove useful for most if not all ectomycorrhizaforming basidiomycetes currently used for molecular genetic studies. A C K N O W L E D G E M E N TS We thank Paul Hooykaas for providing Agrobacterium tumefaciens strain LBA1100 and vector pBIN19, and Jos Wessels for providing plasmid pGPhT. This work was supported by grants from Universidad Nacional de Quilmes, Argentina and Center for International Mobility, Finland to A.G.P., and the Academy of Finland to M.R. The authors are specially grateful to Marjukka Uuskalio and Minna Kemppainen for their help. A. G. P. is a member of the Scientific Research Career of CONICET (Argentina).

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