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Agrobacterium tumefaciens-Mediated Transformation

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Book cover Genetic Transformation Systems in Fungi, Volume 1

Part of the book series: Fungal Biology ((FUNGBIO))

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Abstract

The use of Agrobacterium tumefaciens-mediated transformation for achieving genetic transformation of fungi has steadily increased over the last decade, and has proven to be almost universally applicable technique once suitable selection markers have been developed. In recent years the major technical advances has been made within the initial steps of the process, more specifically the efficient construction of plasmids for performing targeted genome modifications. This chapter provides a generic protocol for performing genetic transformation of ascomycetes via A. tumefaciens-mediated transformation (AMT) and guidelines for optimizing the AMT process with new fungal species. The chapter also includes a highly efficient vector construction system based on Uracil Specific Excisions Reagent (USER) cloning and specific PCR generated building blocks, which can be combined ad hoc to create complex plasmids in a single cloning step.

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References

  • Ando A, Sumida Y, Negoro H, Suroto DA, Ogawa J, Sakuradani E, Shimizu S (2009) Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer breeding. Appl Environ Microbiol 75(17):5529–5535

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14(13):3206–3214

    PubMed  CAS  PubMed Central  Google Scholar 

  • Burns C, Leach KM, Elliott TJ, Challen MP, Foster GD, Bailey A (2006) Evaluation of Agrobacterium-mediated transformation of Agaricus bisporus using a range of promoters linked to hygromycin resistance. Mol Biotechnol 32:129–138

    Article  PubMed  CAS  Google Scholar 

  • Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird G-S, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99(12):7877–7882

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Choi J, Park J, Jeon J, Chi MH, Goh J, Yoo SY, Park J, Jung K, Kim H, Park SY, Rho HS, Kim S, Kim BR, Han SS, Kang S, Lee YH (2007) Genome-wide analysis of T-DNA integration into the chromosomes of Magnaporthe oryzae. Mol Microbiol 66:371–382

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Citovsky V, Kozlovsky SV, Lacroix B, Zaltsman A, Dafny-Yelin M, Vyas S, Tovkach A, Tzfira T (2007) Biological systems of the host cell involved in Agrobacterium infection. Cell Microbiol 9(1):9–20

    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(1):141–148

    Article  PubMed  CAS  Google Scholar 

  • Covert SF, Kapoor P, Lee M, Briley A, Nairn CJ (2001) Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. Mycol Res 105(03):259–264

    Article  CAS  Google Scholar 

  • de Groot MJ, Bundock P, Hooykaas PJ, Beijersbergen AG (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16(9):839–842, Erratum in: Nat Biotechnol 1998 16(11):1074

    Article  PubMed  Google Scholar 

  • Flaherty JE, Pirttila AM, Bluhm BH, Woloshuk CP (2003) PAC1, a pH regulatory gene from Fusarium verticillioides. Appl Environ Microbiol 69:5222–5227

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Frandsen RJ (2011) A guide to binary vectors and strategies for targeted genome modification in fungi using Agrobacterium tumefaciens-mediated transformation. J Microbiol Methods 87(3):247–262

    Article  PubMed  CAS  Google Scholar 

  • Frandsen RJ, Andersson JA, Kristensen MB, Giese H (2008) Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi. BMC Mol Biol 9:70

    Article  PubMed  PubMed Central  Google Scholar 

  • Fullner KJ, Nester EW (1996) Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J Bacteriol 178(6):1498–1504

    PubMed  CAS  PubMed Central  Google Scholar 

  • Fulton TR, Ibrahim N, Losada MC, Grzegorski D, Tkacz JS (1999) A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium. Mol Gen Genet 262(4–5):714–720

    Article  PubMed  CAS  Google Scholar 

  • Gauthier GM, Sullivan TD, Gallardo SS, Brandhorst TT, Vanden Wymelenberg AJ, Cuomo CA, Suen G, Currie CR, Klein BS (2010) SREB, a GATA transcription factor that directs disparate fates in Blastomyces dermatitidis including morphogenesis and siderophore biosynthesis. PLoS Pathog 6(4):e1000846

    Article  PubMed  PubMed Central  Google Scholar 

  • Geu-Flores F, Nour-Eldin HH, Nielsen MT, Halkier BA (2007) USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res 35(7):e55

    Article  PubMed  PubMed Central  Google Scholar 

  • Hamilton CM, Frary A, Lewis C, Tanksley SD (1996) Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc Natl Acad Sci U S A 93(18):9975–9979

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Hansen BG, Salomonsen B, Nielsen MT, Nielsen JB, Hansen NB, Nielsen KF, Regueira TB, Nielsen J, Patil KR, Mortensen UH (2011) Versatile enzyme expression and characterization system for Aspergillus nidulans, with the Penicillium brevicompactum polyketide synthase gene from the mycophenolic acid gene cluster as a test case. Appl Environ Microbiol 77(9):3044–3051

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180

    Article  CAS  Google Scholar 

  • Li GH, Zhou ZZ, Liu GF, Zheng FC, He CZ (2007) Characterization of T-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae. Curr Genet 51:233–243

    Article  PubMed  CAS  Google Scholar 

  • Lisby M, Mortensen UH, Rothstein R (2003) Colocalization of multiple DNA double-strand breaks at a single Rad52 repair centre. Nat Cell Biol 5(6):572–577

    Article  PubMed  CAS  Google Scholar 

  • Lugones LG, Scholtmeijer K, Klootwijk R, Wessels JGH (1999) Introns are necessary for mRNA accumulation in Schizophyllum commune. Mol Microbiol 32:681–689

    Article  PubMed  CAS  Google Scholar 

  • McClelland CA, Chang YC, Kwon-Chung KJ (2005) High frequency transformation of Cryptococcus neoformans and Cryptococcus gattii by Agrobacterium tumefaciens. Fungal Genet Biol 42:904–913

    Article  PubMed  CAS  Google Scholar 

  • Michielse CB, Arentshorst M, Ram AFJ, van den Hondel CAMJJ (2005) Agrobacterium mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori. Fungal Genet Biol 42:9–19

    Article  PubMed  CAS  Google Scholar 

  • Nørholm MH (2010) A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnol 10:21

    Article  PubMed  PubMed Central  Google Scholar 

  • Nour-Eldin HH, Hansen BG, Nørholm MHH, Jensen JK, Halkier BA (2006) Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Res 34(18):e122

    Article  PubMed  PubMed Central  Google Scholar 

  • Pall ML, Brunelli JP (1993) A series of six compact fungal transformation vectors containing polylinkers with multiple unique restriction sites. Fungal Genet Newsl 40:59–62

    Google Scholar 

  • Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56(1):117–124

    Article  PubMed  CAS  Google Scholar 

  • Schell J, Van Montagu M (1977) The Ti-plasmid of Agrobacterium tumefaciens, a natural vector for the introduction of nif genes in plants? Basic Life Sci 9:159–179

    PubMed  CAS  Google Scholar 

  • Sørensen LQ, Larsen JE, Khorsand-Jamal P, Nielsen KF, Frandsen RJN (2014) Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system. BMC Mol Biol 15(1):15

    Article  PubMed  PubMed Central  Google Scholar 

  • Takken FLW, van Wijk R, Michielse CB, Houterman PM, Ram AFJ, Cornelissen BJC (2004) A one-step method to convert vectors into binary vectors suited for Agrobacterium-mediated transformation. Curr Genet 45:242–248

    Article  PubMed  CAS  Google Scholar 

  • van Attikum H, Bundock P, Hooykaas PJ (2001) Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration. EMBO J 20(22):6550–6558

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang Y, DiGuistini S, Wang TCT, Bohlmann J, Breuil C (2010) Agrobacterium meditated gene disruption using split-marker in Grosmannia clavigera, a mountain pine beetle associated pathogen. Curr Genet 56:297–307

    Article  PubMed  Google Scholar 

  • Waring RB, May GS, Morris NR (1989) Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin-coding genes. Gene 79(1):119–130

    Article  PubMed  CAS  Google Scholar 

  • Wei DS, Zhang YH, Xing LJ, Li MC (2010) Agrobacterium rhizogenes-mediated transformation of a high oil-producing filamentous fungus Umbelopsis isabellina. J Appl Genet 51(2):225–232

    Article  PubMed  Google Scholar 

  • Yamada M, Yawata K, Orino Y, Ueda S, Isogai Y, Taguchi G, Shimosaka M, Hashimoto S (2009) Agrobacterium-tumefaciens-mediated transformation of antifungal-lipopeptide-producing fungus Coleophoma empetri F-11899. Curr Genet 55(6):623–630

    Article  PubMed  CAS  Google Scholar 

  • Zupan J, Muth TR, Draper O, Zambryski P (2000) The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J 23:11–28

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Systems Biology, Group for Eukaryotic Molecular Cell Biology, Technical University of Denmark, Sǿltofts plads building 223, room 226, Lyngby, DK-2800, Denmark

    Rasmus John Normand Frandsen Ph.D., M.Sc., B.Sc.

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  1. Rasmus John Normand Frandsen Ph.D., M.Sc., B.Sc.
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Correspondence to Rasmus John Normand Frandsen Ph.D., M.Sc., B.Sc. .

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Editors and Affiliations

  1. Applied Biochemistry Department, DSM Biotechnology Center, Delft, The Netherlands

    Marco A. van den Berg

  2. Global Marker Technology Lab (DuPont-Pioneer), E.I.DuPont India Pvt Ltd, DuPont Knowledge Center, Hyderabad, Telangana, India

    Karunakaran Maruthachalam

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Frandsen, R.J.N. (2015). Agrobacterium tumefaciens-Mediated Transformation. In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 1. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10142-2_14

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