Skip to main content
SpringerLink
Log in

The dominant Hc.Sdh R carboxin-resistance gene of the ectomycorrhizal fungus Hebeloma cylindrosporum as a selectable marker for transformation

  • Technical Note
  • Published:
Current Genetics Aims and scope Submit manuscript

Abstract

In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.Sdh R that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe–S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 μg l−1 carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 μg l−1 of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.Sdh R cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0–30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60–95%.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Barret V, Dixon RK, Lemke PA (1990) Genetic transformation of a mycorrhizal fungus. Appl Microbiol Biotechnol 33:313–316

    Article  Google Scholar 

  • Bills SN, Richter DL, Podila GK (1995) Genetic transformation of the ectomycorrhizal fungus Paxillus involutus by particle bombardment. Mycol Res 99:557–561

    Article  Google Scholar 

  • Bills SN, Podila GK, Hiremath ST (1999) Genetic engineering of the ectomycorrhizal fungus Laccaria bicolor for use as a biological control agent. Mycologia 9:237–242

    Article  Google Scholar 

  • Broomfield PLE, Hargreaves JA (1992) A single amino-acid change in the iron-sulphur protein subunit of succinate dehydrogenase confers resistance to carboxin in Ustilago maydis. Curr Genet 22:117–121

    Article  PubMed  CAS  Google Scholar 

  • Bundock P, Hooykaas PJJ (1996) Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination. Proc Natl Acad Sci USA 93:15272–15275

    Article  PubMed  CAS  Google Scholar 

  • Chen X, Stone M, Schlagnhaufer C, Romaine CP (2000) A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ Microbiol 66:4510–4513

    Article  PubMed  CAS  Google Scholar 

  • Combier JP, Melayah D, Raffier C, Gay G, Marmeisse R (2003) Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMS Microbiol Lett 220:141–148

    Article  PubMed  CAS  Google Scholar 

  • Combier JP, Melayah D, Raffier C, Pépin R, Marmeisse R, Gay G (2004) Non mycorrhizal (Myc−) mutants of Hebeloma cylindrosporum obtained through insertional mutagenesis. Mol Plant Microbe Interact 17:1029–1038

    Article  PubMed  CAS  Google Scholar 

  • Debaud JC, Gay G (1987) In vitro fruiting under controlled conditions of the ectomycorrhizal fungus Hebeloma cylindrosporum associated with Pinus pinaster. New Phytol 105:429–435

    Article  Google Scholar 

  • Grimaldi B, de Raaf MA, Filetici P, Ottonello S, Ballario P (2005) Agrobacterium-mediated gene transfer and enhanced green fluorescent protein visualization in the mycorrhizal ascomycete Tuber borchii: a first step towards truffle genetics. Curr Genet 48:7–69

    Article  Google Scholar 

  • Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994

    Article  PubMed  CAS  Google Scholar 

  • Hanif M, Pardo AG, Gorfer M, Raudaskoski M (2002) T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker. Curr Genet 41:183–188

    Article  PubMed  CAS  Google Scholar 

  • Honda Y, Matsuyama T, Irie T, Watanabe T (2000) Carboxin resistance transformation of the basidiomycetous fungus Pleurotus ostreatus. Curr Genet 37:209–212

    Article  PubMed  CAS  Google Scholar 

  • Irie T, Sato T, Saito K, Honda Y, Watanabe T, Kuwahara M, Enei H (2003) Construction of a homologous selectable marker gene for Lentinus elodes transformation. Biosci Biotechnol Biochem 67:2006–2009

    Article  PubMed  CAS  Google Scholar 

  • Ito Y, Muraguchi H, Seshime Y, Oita S, Yanagi SO (2004) Flutolanil and carboxin resistance in Coprinus cinereus conferred by a mutation in the cytochrome b 560 subunit of succinate dehydrogenase complex (Complex II). Mol Genet Genomics 272:328–335

    Article  PubMed  CAS  Google Scholar 

  • Johansson T, Le Quéré A, Ahrén D, Söderström B, Erlandsson R, Lundeberg J, Uhlén M, Tunlid A (2004) Transcriptional responses of Paxillus involutus and Betula pendula during formation of ectomycorrhizal root tissue. Mol Plant Microbe Interact 17:202–215

    Article  PubMed  Google Scholar 

  • Kemppainen M, Circosta A, Tagu D, Martin F, Pardo AG (2005) Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238 N. Mycorrhiza 16:19–22

    Article  PubMed  CAS  Google Scholar 

  • Keon JPR, White GA, Hargreaves JA (1991) Isolation, characterization and sequence of a gene conferring resistance to the systemic fungicide carboxin from the maize smut pathogen, Usitlago maydis. Curr Genet 19:475–481

    Article  PubMed  CAS  Google Scholar 

  • Lazo GR, Stein PA, Ludwig RA (1991) A DNA Transformation-competent Arabidopsis genomic library in Agrobacterium. Nat Biotechnol 9:963–967

    Article  CAS  Google Scholar 

  • Marmeisse R, Gay G, Debaud JC, Casselton LA (1992) Genetic transformation of the symbiotic basidiomycete fungus Hebeloma cylindrosporum. Curr Genet 22:41–45

    Article  PubMed  CAS  Google Scholar 

  • Martin F, Dupessis S, Ditengou F, Lagrange H, Voiblet C, Lapeyrie F (2001) Development cross talking in the ectomycorrhizal symbiosis: signals and communication genes. New Phytol 151:145–154

    Article  CAS  Google Scholar 

  • Martin F, Kohler A, Duplessis S (2007) Living in harmony in the wood underground: ectomycorrhizal genomics. Curr Opin Plant Biol 10:204–210

    Article  PubMed  CAS  Google Scholar 

  • Martin F, Aerts A, Ahrén D, Brun A, Danchin EGJ, Duchaussoy F, Gibon J, Kohler A, Lindquist E, Pereda V, Salamov A, Shapiro HJ, Wuyts J, Blaudez D, Buée M, Brokstein P, Canbäck B, Cohen D, Courty PE, Coutinho PM, Delaruelle C, Detter JC, Deveau A, DiFazio S, Duplessis S, Fraissinet-Tachet L, Lucic E, Frey-Klett P, Fourrey C, Feussner I, Gay G, Grimwood J, Hoegger PJ, Jain P, Kilaru S, Labbé J, Lin YC, Legué V, Le Tacon F, Marmeisse R, Melayah D, Montanini B, Muratet M, Nehls U, Niculita-Hirzel H, Oudot-Le Secq MP, Peter M, Quesneville H, Rajashekar B, Reich M, Rouhier N, Schmutz J, Yin T, Chalot M, Henrissat B, Kües U, Lucas S, Van de Peer Y, Podila GK, Polle A, Pukkila PJ, Richardson PM, Rouzé P, Sanders IR, Stajich JE, Tunlid A, Tuskan G, Grigoriev IV (2008) The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452(7183):88–92

    Article  PubMed  CAS  Google Scholar 

  • Mogensen EG, Challen MP, Strange RN (2006) Reduction in solanapyrone phytotoxin production by Ascochyta rabiei transformed with Agrobacterium tumefaciens. FEMS Microbiol Lett 255:255–261

    Article  PubMed  CAS  Google Scholar 

  • Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsherker N, Smith JC, Markham AF (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17:2503–2516

    Article  PubMed  CAS  Google Scholar 

  • Pardo AG, Hanif M, Raudaskoski M, Gorfer M (2002) Genetic transformation of ectomycorrhizal fungi mediated by Agrobacterium tumefaciens. Mycol Res 106:132–137

    Article  CAS  Google Scholar 

  • Rao PS, Niederpruem DJ (1969) Carbohydrate metabolism during morphogenesis of Coprinus lagopus (sensu Buller). J Bacteriol 100:1222–1228

    PubMed  CAS  Google Scholar 

  • Rouillon R, Gay G, Bernillon J, Favre-Bonvin J, Bruchet G (1986) Analysis by HPLC-mass spectrometry of the indole compounds released by the ectomycorrhizal fungus Hebeloma hiemale Bres. in pure culture. Can J Bot 64:1893–1897

    Article  CAS  Google Scholar 

  • Ruiz-Herrera J, Martinez-Espinoza AD, Alvarez PE, Xoconostle-Cázares B (1999) Carboxin-resistant mutant of Ustilago maydis is impaired in its pathogenicity for Zea mays. Curr Microbiol 39:291–294

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, NY

    Google Scholar 

  • Smith SE, Read S (1997) Mycorrhizal symbiosis, 2nd edn. Academic press, London

    Google Scholar 

  • Topp CN, Ruiz-Herrera J, Martinez-Espinoza AD, Gold SE (2002) Integration of the gene for carboxin resistance does not impact Ustilago maydis-maize interaction. Curr Microbiol 44:67–70

    Article  PubMed  CAS  Google Scholar 

  • Van Kan JA, van den Ackerveken GF, de Wit PJ (1991) Cloning and characterization of cDNA of avirulence gene avr9 of the fungal pathogen Cladosporium fulvum, causal agent of tomato leaf mold. Mol Plant Microbe Interact 4:52–59

    PubMed  Google Scholar 

  • Voiblet C, Duplessis S, Encelot N, Martin F (2001) Identification of symbiosis-regulated genes in Eucalyptus globulus–Pisolithus tinctorius ectomycorrhiza by differential hybridization of arrayed cDNAs. Plant J 25:181–191

    Article  PubMed  CAS  Google Scholar 

  • Wiemken V, Boller T (2002) Ectomycorrhiza: gene expression, metabolism and the wood-wide web. Cur Opin Plant Biol 5:1–7

    Article  Google Scholar 

Download references

Acknowledgments

Authors thank P. Zurcher, A. Azandégbé, A. Perrin and K. Garcia for their valuable assistance and C.P. Romaine (The Pensylvania State University, Pensylvania, USA) for kindly donating the Agrobacterium strain AGL-1.

Author information

Authors and Affiliations

  1. Université de Lyon, 69003, Lyon, France

    Chrisse Ngari, Jean-Philippe Combier, Jeanne Doré, Roland Marmeisse, Gilles Gay & Delphine Melayah

  2. Ecologie Microbienne, IFR 41, Université Lyon 1 CNRS UMR 5557, 69622, Villeurbanne, France

    Chrisse Ngari, Jean-Philippe Combier, Jeanne Doré, Roland Marmeisse, Gilles Gay & Delphine Melayah

Authors
  1. Chrisse Ngari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jean-Philippe Combier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeanne Doré
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Roland Marmeisse
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gilles Gay
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Delphine Melayah
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gilles Gay.

Additional information

Communicated by U. Kues.

The authors Chrisse Ngari and Jean-Philippe Combier contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ngari, C., Combier, JP., Doré, J. et al. The dominant Hc.Sdh R carboxin-resistance gene of the ectomycorrhizal fungus Hebeloma cylindrosporum as a selectable marker for transformation. Curr Genet 55, 223–231 (2009). https://doi.org/10.1007/s00294-009-0231-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00294-009-0231-4

Keywords

Search

Navigation