Skip to main content
SpringerLink
Log in

Targeted Gene Replacement in Fungal Pathogens via Agrobacterium tumefaciens- Mediated Transformation

  • Protocol
  • First Online:
Plant Fungal Pathogens

Part of the book series: Methods in Molecular Biology ((MIMB,volume 835))

Abstract

Genome sequence data on fungal pathogens provide the opportunity to carry out a reverse genetics approach to uncover gene function. Efficient methods for targeted genome modifications such as knockout and in locus over-expression are in high demand. Here we describe two efficient single-step cloning strategies for construction of vectors for Agrobacterium tumefaciens-mediated transformation (ATMT). Targeted genome modifications require integration by a homologous double crossover event, which is achieved by placing target sequences on either side of a selection marker gene in the vector. Protocols are given for two single-step vector construction techniques. The In-Fusion cloning technique is independent of compatible restriction enzyme sites in the vector and the fragment to be cloned. The method can be directly applied to any vector of choice and it is possible to carry out four fragment cloning without the need for subcloning. The cloning efficiency is not always as high as desired, but it still presents an efficient alternative to restriction enzyme and ligase-based cloning systems. The USER technology offers a higher four fragment cloning efficiency than In-Fusion, but depends on specific structures in the binary vector. The available fungal binary vectors adapted for the USER system are described and protocols are provided for vector design and construction. A general protocol for verification of the resulting gene replacement events in the recipient fungal cells is also given. The cloning systems described above are relevant for all transformation vector constructs, but here we describe their application for ATMT compatible binary vectors. Protocols are provided for ATMT exemplified by Fusarium graminearum. For large-scale reverse genetic projects, the USER technology is recommended combined with ATMT.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Matzke, A.J. and Chilton, M.D. (1981) Site-specific insertion of genes into T-DNA of the Agrobacterium tumor-inducing plasmid: an approach to genetic engineering of higher plant cells. Journal of Molecular and Applied Genetics 1(1), 39–49.

    PubMed  CAS  Google Scholar 

  2. Chan, M.T., Chang, H.H., Ho, S.L., Tong, W.F. and Yu, S.M. (1993) Agrobacterium-mediated production of transgenic rice plants expressing a chimeric alpha-amylase promoter/beta-glucuronidase gene. Plant Molecular Biology 3, 491–506.

    Article  Google Scholar 

  3. Lee, L.-Y. and Gelvin, S.B. (2008) T-DNA Binary Vectors and Systems. Plant Physiology 146, 325–332.

    Article  PubMed  CAS  Google Scholar 

  4. Gelvin, S.B. (2003) Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool. Microbiology and Molecular Biology Reviews 67(1), 16–37.

    Article  PubMed  CAS  Google Scholar 

  5. Pasternak, J.J., Gruber, M.Y., Thompson, J.E. and Glick, B.R. (1983) Development of DNA-mediated transformation systems for plants. Biotechnology Advances 1(1), 1–15.

    Article  PubMed  CAS  Google Scholar 

  6. Bundock, P., Dendulkras, A., Beijersbergen, A. and Hooykaas, P.J. (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO Journal 14(13), 3206–3214.

    PubMed  CAS  Google Scholar 

  7. de Groot, M.J., Bundock, P., Hooykaas, P.J. and Beijersbergen, A.G. (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology 16(9), 839–842.

    Article  PubMed  Google Scholar 

  8. Meyer, V., Mueller, D., Strowig, T. and Stahl, U. (2003) Comparison of different transformation methods for Aspergillus giganteus. Current Genetics 43(5), 371–377.

    Article  PubMed  CAS  Google Scholar 

  9. Walton, F.J., Idnurm, A. and Heitman, J. (2005) Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Molecular Microbiology 57(5), 1381–1396.

    Article  PubMed  CAS  Google Scholar 

  10. Betts, M.F., Tucker, S.L., Galadima, N., Meng, Y., Patel, G., Li, L., Donofrio, N., Floyd, A., Nolin, S., Brown, D., Mandel, M.A., Mitchell, T.K., Xu, J.R., Dean, R.A., Farman, M.L. and Orbach, M.J. (2007) Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae. Fungal Genetics and Biology 44(10), 1035–1049.

    Article  PubMed  CAS  Google Scholar 

  11. Blaise, F., Remy, E., Meyer, M., Zhou, L.G., Narcy, J.P., Roux, J., Balesdent, M.H., Rouxel, T. (2007) A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans. Fungal Genetics and Biology 44(2), 123–138.

    Article  PubMed  CAS  Google Scholar 

  12. Jeon, J., Park, S.Y., Chi, M.H., Choi, J., Park, J., Rho, H.S., Kim, S., Goh, J., Yoo, S., Choi, J., Park, J.Y., Yi, M., Yang, S., Kwon, M.J., Han, S.S., Kim, B.R., Khang, C.H., Park, B., Lim, S.E., Jung, K., Kong, S., Karunakaran, M., Oh, H.S., Kim, H., Kim, S., Park, J., Kang, S., Choi, W.B., Kang, S. and Lee, Y.H. (2007) Genome-wide functional analysis of pathogenicity genes in the rice blast fungus. Nature Genetics 39(4), 561–565.

    Article  PubMed  CAS  Google Scholar 

  13. Malz, S., Grell, M.N., Thrane, C., Maier, F.J., Rosager, P., Felk, A., Albertsen, K.S., Salomon, S., Bohn, L., Schäfer, W. and Giese, H. (2005) Identification of a gene cluster responsible for the biosynthesis of aurofusarin in the Fusarium graminearum species complex. Fungal Genetics and Biology 42(5), 420–433.

    Article  PubMed  CAS  Google Scholar 

  14. Cardoza, R.E., Vizcaino, J.A., Hermosa, M.R., Monte, E. and Gutiérrez, S. (2006) A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast- and Agrobacterium-mediated transformation. The Journal of Microbiology 44(4), 383–395.

    PubMed  CAS  Google Scholar 

  15. Zwiers, L.H. and De Waard, M.A. (2001) Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycos-phaerella graminicola. Current Genetics 39(5–6), 388–393.

    Article  PubMed  CAS  Google Scholar 

  16. Gardiner, D.M. and Howlett, B.J. (2004) Negative selection using thymidine kinase increases the efficiency of recovery of transformants with targeted genes in the filamentous fungus Leptosphaeria maculans. Current Genetics 45(4), 249–255.

    Article  PubMed  CAS  Google Scholar 

  17. Zhang, A., Lu, P., Dahl-Roshak, A.M., Paress, P.S., Kennedy, S., Tkacz, J.S. and An, Z. (2003) Efficient disruption of a polyketide synthase gene ( pks1) required for melanin synthesis through Agrobacterium-mediated transformation of Glarea lozoyensis. Molecular Genetics and Genomics 268(5), 645–655.

    PubMed  CAS  Google Scholar 

  18. Frandsen, R.J., Albertsen, K.S., Stougaard, P., Sørensen, J.L., Nielsen, K.F., Olsson, S. and Giese, H. (2010) Methylenetetrahydrofolate reductase activity is involved in the plasma membrane redox system required for pigment biosynthesis in filamentous fungi. Eukaryotic Cell 9(8), 1225–1235.

    Article  PubMed  CAS  Google Scholar 

  19. Yi, M., Chi, M.H., Khang, C.H., Park, S.Y., Kang, S., Valent, B. and Lee, Y.H. (2009) The ER Chaperone LHS1 Is Involved in Asexual Development and Rice Infection by the Blast Fungus Magnaporthe oryzae. The Plant Cell 21(2), 681–695.

    Article  PubMed  CAS  Google Scholar 

  20. Wang, Y., DiGuistini, S., Wang, T.C., Bohlmann, J. and Breuil, C. (2010) Agrobacterium-meditated gene disruption using split-marker in Grosmannia clavigera, a mountain pine beetle associated pathogen. Current Genetics 56(3), 297–307.

    Article  PubMed  Google Scholar 

  21. Hamilton, M.D., Nuara, A.A., Gammon, D.B., Buller, R.M. and Evans, D.H. (2007) Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase. Nucleic Acids Research 35(1), 143–151.

    Article  PubMed  CAS  Google Scholar 

  22. Clontech Laboratories (2008) In-FusionTM PCR Cloning Kit User Manual PT3650-1

    Google Scholar 

  23. Zhu, B., Cai, G., Hall, E.O. and Freeman, G.J. (2007) In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43(3), 354–359.

    Article  PubMed  CAS  Google Scholar 

  24. Frandsen, R.J., Andersson, J.A., Kristensen, M.B. and Giese, H. (2008) Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi. BMC Molecular Biology 9:70.

    Article  PubMed  Google Scholar 

  25. New England Biolabs (2008) Instruction manual: USER™ Friendly Cloning Kit - A Novel Tool for Cloning PCR Products by Uracil Excision. Version 1.3.

    Google Scholar 

  26. Fulton, T.R., Ibrahim, N., Losada, M.C., Grzegorski, D. and Tkacz, J.S. (1999) A melanin polyketide synthase (PKS) gene from Nodulisporium sp. that shows homology to the pks1 gene of Colletotrichum lagenarium. Molecular Genetics and Genomics 262, 714–720.

    CAS  Google Scholar 

  27. Geu-Flores, F., Nour-Eldin, H.H., Nielsen, M.T. and Halkier, B.A. (2007) USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Research 35(7), e55.

    Article  PubMed  Google Scholar 

  28. van Attikum, H. and Hooykaas, P.J.J. (2003) Genetic requirements for the targeted integration of Agrobacterium T-DNA in Saccharomyces cerevisiae. Nucleic Acids Research 31(3), 826–832.

    Article  PubMed  Google Scholar 

  29. Bashi, Z.D., Khachatourians, G. and Hegedus, D.D. (2010) Isolation of fungal homokaryotic lines from heterokaryotic transformants by sonic disruption of mycelia. Biotechniques 48(1), 41–46.

    Article  PubMed  CAS  Google Scholar 

  30. Frandsen, R.J., Nielsen, N.J., Maolanon, N., Sørensen, J.C., Olsson, S., Nielsen, J. and Giese, H. (2006) The biosynthetic pathway for aurofusarin in Fusarium graminearum reveals a close link between the naphthoquinones and naphthopyrones. Molecular Microbiology 61(4), 1069–1080.

    Article  PubMed  CAS  Google Scholar 

  31. Michielse, C.B., Hooykaas, P.J., van den Hondel, C.A. and Ram, A.F. (2005) Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Current Genetics 48(1), 1–17.

    Article  PubMed  CAS  Google Scholar 

  32. Tendulkar, S.R., Gupta, A. and Chattoo, B.B. (2003) A Simple protocol for isolation of fungal DNA. Biotechnology Letters 25, 1941–1944.

    Article  PubMed  CAS  Google Scholar 

  33. Nielsen, J.B., Nielsen, M.L. and Mortensen, U.H. (2007) Transient disruption of non-homologous end-joining facilitates targeted genome manipulations in the filamentous fungus Aspergillus nidulans. Fungal Genetics and Biology 45(3), 165–170.

    Article  PubMed  Google Scholar 

  34. Nørholm, M.H. (2010) A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnology 10, 21.

    Article  PubMed  Google Scholar 

  35. Fernández-Martín, R., Cerdá-Olmedo, E. and Avalos, J. (2000) Homologous recombination and allele replacement in transformants of Fusarium fujikuroi. Molecular Genetics and Genomics 263(5), 838–845.

    Google Scholar 

  36. Abuodeh, R.O., Orbach, M.J., Mandel, M.A., Das, A. and Galgiani, J.N. (2000) Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens. Journal of Infectious Diseases 181(6), 2106–2110.

    Article  PubMed  CAS  Google Scholar 

  37. Mullins, E.D., Chen, X., Romaine, P., Raina, R., Geiser, D.M. and Kang, S. (2001) Agrobacterium-Mediated Transformation of Fusarium oxysporum: An Efficient Tool for Insertional Mutagenesis and Gene Transfer. Phytopathology 91(2), 173–180.

    Article  PubMed  CAS  Google Scholar 

  38. Frandsen, R.J., Schütt, C., Lund, B.W., Staerk, D., Nielsen, J., Olsson, S. and Giese, H. (2011) Two novel classes of enzymes are required for the biosynthesis of aurofusarin in Fusarium graminearum. Journal of Biological Chemistry 286(12), 10419–10428.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Systems Biology, Center for Microbial Biotechnology, Technical University of Denmark, Lyngby, Denmark

    Rasmus John Normand Frandsen

  2. Bioneer A/S, Hørsholm, Denmark

    Mette Frandsen

  3. Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Ålborg, Denmark

    Henriette Giese

Authors
  1. Rasmus John Normand Frandsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mette Frandsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Henriette Giese
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rasmus John Normand Frandsen .

Editor information

Editors and Affiliations

  1. Northern Crops Science Laboratory, USDA-ARS, North 18th Street 1307, Fargo, 58105-5677, North Dakota, USA

    Melvin D. Bolton

  2. Lab. Phytopathology, Wageningen University, Droevendaalsesteeg 1, Wageningen, 6708 PB, Netherlands

    Bart P.H.J. Thomma

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Frandsen, R.J.N., Frandsen, M., Giese, H. (2012). Targeted Gene Replacement in Fungal Pathogens via Agrobacterium tumefaciens- Mediated Transformation. In: Bolton, M., Thomma, B. (eds) Plant Fungal Pathogens. Methods in Molecular Biology, vol 835. Humana Press. https://doi.org/10.1007/978-1-61779-501-5_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-501-5_2

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-500-8

  • Online ISBN: 978-1-61779-501-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation