Abstract
The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barrett V, Dixon RK, Lemke PA (1990) Genetic transformation of a mycorrhizal fungus. Appl Microbiol Biotechnol 33:313–316
Bills S, Podila G, Hiremath S (1999) Genetic engineering of a fungus Laccaria bicolor for use as a biological control agent. Mycologia 9:237–242
Bundock P, Dulk-Ras A, Beijersbergen A, Hooykaas PJJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:3206–3214
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
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
Combier JP, Melayah D, Raffier C, Pépin R, Marmeisse R, Gay G (2004) Nonmycorrhizal (Myc−) mutants of Hebeloma cylindrosporum obtained through insertional mutagenesis. Mol Plant-Microb Interact 17:1029–1038
de Groot MJA, Bundock P, Hooykaas PJJ, Beirjesbrgen AGM (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842
Di Battista C, Selosse MA, Bouchard D, Stenstrøm E, Le Tacon F (1996) Variation in symbiotic efficiency, phenotypic characters and ploidy level among different isolates of the ectomycorrhizal basidiomycete Laccaria bicolor strain S238. Mycol Res 100:1315–1324
Duplessis S, Courty P, Tagu D, Martin F (2005) Transcript patterns associated with ectomycorrhiza development in Eucalyptus globulus and Pisolithus microcarpus. New Phytol 165:599–611
Fitzgerald A, van Kan J, Plummer K (2004) Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inequalis, by expression of RNA with chimeric inverted repeats. Fungal Genet Biol 41:963–971
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
Hellens R, Mullineaux P, Klee H (2000) Technical focus: a guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–451
Johansson T, Le Quéré A, Ahren D, Søderstrøm 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-Microb Interact 17:202–215
Kadotani N, Nakayashiki H, Tosa Y, Mayama S (2003) RNA silencing in the phytopathogenic fungus Magnaporthe oryzae. Mol Plant-Microb Interact 16:769–776
Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci U S A 98:1871–1876
Lammers P, Tuskan G, DiFazio S, Podila G, Martin F (2004) Mycorrhizal symbionts of Populus to be sequenced by the United States Department of Energy's Joint Genome Institute. Mycorrhiza 14:63–64
Laurent P, Voilet C, Tagu D, de Carvalho D, Nehls U, De Bellis R, Ballestrini R, Bauw G, Bonfante P, Martin F (1999) A novel class of ectomycorrhiza-regulated cell wall polypeptides in Pisolithus tinctorius. Mol Plant-Microb Interact 12:862–871
Leclerque A, Wan H, Abschütz A, Chen S, Mitina G, Zimmermann H, Schairer H (2004) Agrobacterium-mediated insertional mutagenesis (AIM) of the entomophatogenic fungus Beauveria bassiana. Curr Genet 45:111–119
Martin F (2001) Frontiers in molecular mycorrhizal research—genes, loci, dots and spins. New Phytol 150:499–505
Martin F, Delaruelle C, Ivory M (1998) Genetic variability in intergenic spacers of ribosomal DNA in Pisolithus isolates associated with pine, Eucalyptus and Afzelia in lowland Kenian forest. New Phytol 139:341–352
Martin F, Tuskan GA, DiFazio SP, Lammers P, Newcombe G, Podila GK (2004) Symbiotic sequencing for the Populus mesocosm. New Phytol 161:330–335
Pardo AG, Hanif M, Raudaskoski M, Gorfer M (2002) Genetic transformation of ectomycorrhizal fungi mediated by Agrobacterium tumefaciens. Mycol Res 106:132–137
Peter M, Courty PE, Kohler A, Delaruelle C, Martin D, Tagu D, Frey-Klett P, Duplessis S, Chalot M, Podila G, Martin F (2003) Analysis of expressed sequence tags from the ectomycorrhizal basidiomycetes Laccaria bicolor and Pisolithus microcarpus. New Phytol 159:117–129
Podila GK, Zheng J, Balasubramanian S, Sundaram S, Hieremath S, Brand J, Hymes M (2002) Molecular interactions in ectomycorrhizas: identification of fungal genes involved in early symbiotic interactions between Laccaria bicolor and red pine. Plant Soil 244:117–128
Tinland B (1996) The integration of T-DNA in plant genomes. Trends Plant Sci 1:178–184
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
Wiemken V, Boller T (2002) Ectomycorrhiza: gene expression, metabolism and the wood-wide web. Curr Opin Plant Biol 5:355–361
Acknowledgements
We are grateful to Peter Romaine and Carl Schlagnhaufer for providing A. tumefaciens strain AGL-1 and plasmid pBGgHg and to Paul Hooykaas for providing A. tumefaciens strain LBA1100. A.G.P. is a member of the Scientific Research Career of CONICET (Argentina). This programme was financed by INRA, Région de Lorraine, PICT-ANPCyT, UNQ and the France–Argentina ECOS-Sud grants.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kemppainen, M., Circosta, A., Tagu, D. et al. Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N. Mycorrhiza 16, 19–22 (2005). https://doi.org/10.1007/s00572-005-0008-7
Received:
Revised:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00572-005-0008-7