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The Aspergillus nidulans amdS gene as a marker for the identification of multicopy T-DNA integration events in Agrobacterium-mediated transformation of Aspergillus awamori

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Abstract

The Aspergillus nidulans amdS selection marker was used for the identification of multicopy T-DNA insertions in Agrobacterium-mediated transformation of Asp. awamori. The selection of transformants on agar plates containing acetamide as sole nitrogen source and hygromycin resulted in a six-fold decrease in the transformation frequency, compared with the transformation frequency obtained after hygromycin selection alone. However, it was found that 47% of the transformants obtained after hygromycin and acetamide double selection contained multiple T-DNA integrations. Furthermore, it was found that the multicopy transformants could easily be identified based on their growth rate on agar plates containing acetamide medium. Based on these data, it can be concluded that the amdS marker can also be used as a selection marker in Agrobacterium-mediated transformation of Asp. awamori and that it is a very useful marker to identify those transformants containing multiple T-DNA integrations.

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References

  • Abuodeh RO, Orbach MJ, Mandel MA, Das A, Galgiani JN (2000) Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens. J Infect Dis 181:2106–2110

    CAS  PubMed  Google Scholar 

  • Amey RC, Athey-Pollard A, Burns C, Mills PR, Bailey A, Foster GD (2002) PEG-mediated and Agrobacterium-mediated transformation in the mycopathogen Verticillium fungicola. Mycol Res 106:4–11

    Article  Google Scholar 

  • Beijersbergen AG, Den Dulk A, Schilperoort RA, Hooykaas PJ (1992) Conjugative transfer by the virulence system of Agrobacterium tumefaciens. Science 256:1324–1327

    CAS  Google Scholar 

  • Bundock P, Dulk-Ras A, Beijersbergen A, Hooykaas PJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14:3206–3214

    CAS  PubMed  Google Scholar 

  • Bundock P, Attikum H van, Dulk-Ras A, Hooykaas PJ (2002) Insertional mutagenesis in yeasts using T-DNA from Agrobacterium tumefaciens. Yeast 19:529–536

    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

    CAS  Google Scholar 

  • Corrick CM, Twomey AP, Hynes MJ (1987) The nucleotide sequence of the amdS gene of Aspergillus nidulans and the molecular characterization of 5′ mutations. Gene 53:63–71

    Article  CAS  PubMed  Google Scholar 

  • Covert SF, Kapoor P, Lee M, Briley A, Nairn CJ (2001) Agrobacterium-mediated transformation of Fusarium circinatum. Mycol Res 105:259–264

    CAS  Google Scholar 

  • Degefu Y, Hanif M (2003) Agrobacterium tumefaciens-mediated transformation of Helminthosporium turcicum, the maize leaf-blight fungus. Arch Microbiol 108:279–284

    Article  Google Scholar 

  • Fitzgerald AM, Mudge AM, Gleave AP, Plummer KM (2003) Agrobacterium and PEG-mediated transformation of the phytopathogen Venturia inaequalis. Mycol Res 107:803–810

    Article  CAS  PubMed  Google Scholar 

  • Gelvin SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu Rev Plant Physiol Plant Mol Biol 51:223–256

    CAS  Google Scholar 

  • Gouka RJ, Gerk C, Hooykaas PJ, Bundock P, Musters W, Verrips CT, Groot MJ de (1999) Transformation of Aspergillus awamoriby Agrobacterium tumefaciens-mediated homologous recombination. Nat Biotechnol 17:598–601

    Article  CAS  PubMed  Google Scholar 

  • Groot MJ de, Bundock P, Hooykaas PJ, Beijersbergen AG (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842

    PubMed  Google Scholar 

  • Hanegraaf PP, Punt PJ, Hondel CA van den, Dekker J, Yap W, Verseveld HW van, Stouthamer AH (1991) Construction and physiological characterization of glyceraldehyde-3-phosphate dehydrogenase overproducing transformants of Aspergillus nidulans. Appl Microbiol Biotechnol 34:765–771

    CAS  PubMed  Google Scholar 

  • Hynes MJ, Pateman JA (1970) The genetic analysis of regulation of amidase synthesis in Aspergillus nidulans. I. Mutants able to utilize acrylamide. Mol Gen Genet 108:97–106

    CAS  PubMed  Google Scholar 

  • Kelly JM, Hynes MJ (1985) Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans. EMBO J 4:475–479

    CAS  PubMed  Google Scholar 

  • Kolar M, Punt PJ, Hondel CA van den, Schwab H (1988) Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene. Gene 62:127–134

    CAS  PubMed  Google Scholar 

  • Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci USA 98:1871–1876

    CAS  PubMed  Google Scholar 

  • Leclerque A, Wan H, Abschutz A, Chen S, Mitina GV, Zimmermann G, Schairer HU (2003) Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana. Curr Genet (in press)

  • Malonek S, Meinhardt F (2001) Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic ascomycete Calonectria morganii. Curr Genet 40:152–155

    CAS  PubMed  Google Scholar 

  • Mattanovich D, Ruker F, Machado AC, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H (1989) Efficient transformation of Agrobacterium spp. by electroporation. Nucleic Acids Res 17:6747

    CAS  PubMed  Google Scholar 

  • Meyer V, Mueller D, Strowig T, Stahl U (2003) Comparison of different transformation methods for Aspergillus giganteus. Curr Genet 43:371–377

    Article  CAS  PubMed  Google Scholar 

  • Michielse CB, Ram AFJ, Hooykaas PJJ, Hondel CAMJJ van den (2004a) Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full length VirD2, VirC2 and VirE2 leads to the insertion of aberrant T-DNA structures. J Bacteriol (in press)

  • Michielse CB, Ram AFJ, Hooykaas PJJ, Hondel CAMJJ van den (2004b) Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori. Fungal Genet Biol (in press)

  • Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S (2001) Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology 91:173–180

    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 

  • Penttila M, Nevalainen H, Ratto M, Salminen E, Knowles J (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164

    PubMed  Google Scholar 

  • Punt PJ, Hondel CA van den (1992) Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. Methods Enzymol 216:447–457

    CAS  PubMed  Google Scholar 

  • Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, Hondel CA van den (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56:117–124

    CAS  PubMed  Google Scholar 

  • Rho HS, Kang S, Lee YH (2001) Agrobacterium tumefaciens-mediated transformation of the plant pathogenic fungus, Magnaporthe grisea. Mol Cells 12:407–411

    CAS  PubMed  Google Scholar 

  • Rodriguez RJ, Yoder OC (1987) Selectable genes for transformation of the fungal plant pathogen Glomerella cingulata f.sp. phaseoli (Colletotrichum lindemuthianum). Gene 54:73–81

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Verdoes JC, Punt PJ, Hondel CAM van den (1995) Molecular genetic strain improvement for the overproduction of fungal proteins by filamentous fungi. Appl Microbiol Biotechnol 43:195–205

    Article  CAS  Google Scholar 

  • Vijn I, Govers F (2003) Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans. Mol Plant Pathol 4:459–467

    Article  CAS  Google Scholar 

  • Zupan JR, Zambryski P (1995) Transfer of T-DNA from Agrobacterium to the plant cell. Plant Physiol 107:1041–1047

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We would like to thank Simon Flitter and Patricia vanKuyk for critically reading this manuscript. This work was supported by Unilever Research, The Netherlands.

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Authors and Affiliations

  1. Institute of Biology, Clusius Laboratory, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands

    C. B. Michielse, A. F. J. Ram & C. A. M. J. J. van den Hondel

  2. Department of Microbiology, TNO Nutrition, Utrechtseweg 48, 3700 AJ, Zeist, The Netherlands

    A. F. J. Ram & C. A. M. J. J. van den Hondel

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  1. C. B. Michielse
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  2. A. F. J. Ram
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  3. C. A. M. J. J. van den Hondel
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Correspondence to C. B. Michielse.

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Communicated by U. Kück

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Michielse, C.B., Ram, A.F.J. & van den Hondel, C.A.M.J.J. The Aspergillus nidulans amdS gene as a marker for the identification of multicopy T-DNA integration events in Agrobacterium-mediated transformation of Aspergillus awamori . Curr Genet 45, 399–403 (2004). https://doi.org/10.1007/s00294-004-0500-1

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  • DOI: https://doi.org/10.1007/s00294-004-0500-1

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