Skip to main content
SpringerLink
Log in

Transformation of Fusarium oxysporum f. sp. cubense, causal agent of Fusarium wilt of banana, with the green fluorescent protein (GFP) gene

  • Published:
Australasian Plant Pathology Aims and scope Submit manuscript

Abstract

Fusarium oxysporum f. sp. cubense (Foe) is the causal agent of Fusarium wilt (Panama disease) of bananas in most tropical and subtropical banana-producing regions of the world. The fungus infects through roots, colonises the rhizomes and eventually blocks the vascular system of the pseudostems, resulting in plant death. The green fluorescent protein (GFP) emits green fluorescence when excited by blue light, making it a useful tool to study early stages of fungal infection. The objective of this study was to transform Foc isolates with the GFP gene. Isolates representing ‘subtropical’ race 4 of the fungus were transformed with the sGFP derivative using hygromycin as a selectable marker. Efficiency and transformation of spheroplasts depended on mycelium age, the choice of enzymes and the temperature and duration of incubation. The transformed isolates did not differ markedly from the wild type isolates in growth and morphological characteristics in vitro. Fluorescence microscopy showed expression of the green fluorescent protein in fungal structures. The presence of the GFP DNA in the fungal cells was confirmed by PCR using a GFP-specific primer pair and Southern blot analysis. Pathogenicity tests showed that the transformation process did not alter pathogenicity of Foc isolates. Fungal hyphae within tissues of infected plants could be seen to fluoresce and the transformed fungus was re-isolated from artificially inoculated plants. Transformants of Foc will facilitate future infection studies with this pathogen on banana.

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

Similar content being viewed by others

References

  • Berteaux-Lecellier V, Zickler D, Debuchy R, Panvier-Adoutte A, Thompson-Coffe C, Picard M (1998) A homologue of the yeast SHE4 gene is essential for the transition between the syncytial and cellular stages during sexual reproduction of the fungus Podospora anserina. EM BO Journal 17, 1248–1258.

    Article  CAS  Google Scholar 

  • Booth C (1971) ‘The genus Fusarium.’ (Commonwealth Mycological Institute: Kew, England)

    Google Scholar 

  • Bottin A, Larche L, Villalba F, Gaulin E, Esquerre-Tugaye MT, Rickauer M (1999) Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe—plant interaction in the tobacco pathogen Phytophthora parasitica var nicotianae. FEMS Microbiology Letters 176, 51–56. doi:10.1016/S0378-1097(99)00218-9

    Article  CAS  PubMed  Google Scholar 

  • Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.

    Article  CAS  PubMed  Google Scholar 

  • Chalfie M, Kain S (1998) ‘Green fluorescent protein: properties, applications and protocols.’ (John Wiley & Sons Inc.: New York, USA)

    Google Scholar 

  • Chiu W, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Current Biology 6, 325–330.

    Article  CAS  PubMed  Google Scholar 

  • Churchill ACL, Ciuffetti LM, Hansen DR, Van Etten HD, Van Alfen NK (1990) Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids. Current Genetics 17, 25–31.

    Article  CAS  Google Scholar 

  • Cormack BP, Bertram G, Egerton M, Gow NA, Falkow S, Brown AJ (1997) Yeast-enhanced green fluorescent protein (yEGFP), a reporter of gene expression in Candida albicans. Microbiology 143, 303–311.

    Article  CAS  PubMed  Google Scholar 

  • Cubitt AB, Heim R, Adams SR, Boyd AE, Gross LA, Tsien RY (1995) Understanding, improving and using green fluorescent proteins. Trends in Biochemical Sciences 20, 448–455. doi: 10.1016/S0968-0004(00)89099-4

    Article  CAS  PubMed  Google Scholar 

  • Du W, Huang Z, Flaherty JE, Wells K, Payne GA (1999) Green fluorescent protein as a reporter to monitor gene expression and food colonization by Aspergillus flavus. Applied and Environmental Microbiology 65, 834–836.

    CAS  PubMed  Google Scholar 

  • Dumas B, Centis S, Sarrazin N, Esquerre-Tugaye MT (1999) Use of green fluorescent protein to detect expression of an endopolygalcturonase gene of Colletotrichum lindemuthianum during bean infection. Applied and Environmental Microbiology 65, 1769–1771.

    CAS  PubMed  Google Scholar 

  • Follin JC, Laville E (1966) Variations chez le Fusarium oxysporum. Comportement des lignees issuees des differents organs de multiplication. Fruits 21, 261–268.

    Google Scholar 

  • Freitag M, Ciuffetti LM, Selker EU (2001) Expression and visualization of green fluorescent protein (GFP) in Neurospora crassa. Fungal Genetics Newsletter 48, 15–19.

    Google Scholar 

  • Haseloff J, Amos B (1995) GFP in plants. Trends in Genetics 11, 328–329. doi: 10.1016/0168-9525(95)90186-8

    Article  CAS  PubMed  Google Scholar 

  • Hawksworth DL, Kirk PM, Sutton BC, Pegler DN (1995) ‘Dictionary of the fungi.’ (CAB International: Wallingford, UK)

    Google Scholar 

  • Kershaw MJ, Walkey G, Talbot NJ (1998) Complementation of the Mpg1 mutant phenotype in Magnaporthe grisea reveals functional relationships between fungal hydrophobins. The EMBO Journal 17, 3838–3849. doi: 10.1093/EMBOJ/17.14.3838

    Article  CAS  PubMed  Google Scholar 

  • Lagopodi AL, Ram AFJ, Lamers GEM, Punt PJ, Van den Hondel CAMJJ, Lugtenberg BJJ, Bloemberg GV (2002) Novel aspects of tomato root colonization and infection by Fusarium oxysporum f sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker. Molecular Plant-Microbe Interactions 15, 172–179.

    Article  CAS  PubMed  Google Scholar 

  • Lorang JM, Tuori RP, Martinez JP, Sawyer TL, Redman RS, et al. (2001) Green fluorescent protein is lighting up fungal biology. Applied andEnvironmental Microbiology 67, 1987–1994. doi:10.1128/AEM.67.5.1987-1994.2001

    Article  CAS  Google Scholar 

  • Maor R, Puyesky M, Horwitz BA, Sharon A (1998) Use of green fluorescent protein (GFP) for studyingdevelopment and fungal—plantinteraction in Cochliobolus heterostrophus. Mycological Research 102, 491–496. doi:10.1017/S0953756297005789

    Article  Google Scholar 

  • Nelson PE, Toussoun TA, Marasas WFO (1983) ‘Fusarium Species: an illustrated manual foridentification.’ (Pennsylvania State University Press: University Park, USA)

    Google Scholar 

  • Niedenthal RK, Riles L, Johnston M, Hegemann JH (1996) Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast. Yeast (Chichester, England) 12, 773–786. doi: 10.1002/(SICI)1097-0061(19960630)12:83.3.CO;2-C

    Article  CAS  Google Scholar 

  • Orjeda G (1998) ‘Evaluation of Musa germplasm for resistance to Sigatoka diseases and Fusarium wilt.’ (International Plant Genetic Resources Institute, Rome, Italy (IPGRI), International Network for the Improvement of Banana and Plantain, (INIBAP), Montpellier, France, ACP-EU Technical Centre for Agricultural and Rural Cooperation (CTA), Wageningen, The Netherlands)

    Google Scholar 

  • Pines J (1995) GFP in mammalian cells. Trends in Genetics 11, 326–327. doi: 10.1016/S0168-9525(00)89092-7

    Article  CAS  PubMed  Google Scholar 

  • Prasher DC (1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene 111, 229–233. doi: 10.1016/0378-1119(92)90691-H

    Article  CAS  PubMed  Google Scholar 

  • Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology 1, 17–20.

    Article  CAS  Google Scholar 

  • Sam brook J, Fritsch EF, Maniatis T (1989) ‘Molecular cloning: a laboratory manual.’ (Cold Spring Harbor Press: New York, USA)

    Google Scholar 

  • Sheen J, Hwang S, Niwa Y, Kobayashi H, Galbraith DM (1995) Green fluorescent protein as a new vital marker in plant cells. The Plant Journal 8, 777–784. doi: 10.1046/J.1365-313X.1995.08050777.X

    Article  CAS  PubMed  Google Scholar 

  • Skinner W, Bailey A, Renwick A, Keon J, Gurr S, Hargreaves J (1998) A single amino-acid substitution in the iron-sulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola. Current Genetics 34, 393–398. doi:10.1007/S002940050412

    Article  CAS  PubMed  Google Scholar 

  • Spellig T, Bottin A, Kahmann R (1996) Green fluorescent protein (GFP) as a new vital marker in the phyto pathogenic fungus Ustilago maydis. Molecular & General Genetics 252, 503–509. doi:10.1007/S004380050257

    CAS  Google Scholar 

  • Stover RH (1972) Fusarial wilt (Panama Disease) of bananas and other Musa species. In ‘Banana, plaintain and abaca diseases’. (Commonwealth Mycological Institute: Kew, England)

    Google Scholar 

  • Suelmann R, Fischer R (1997) Nuclear traffic in fungal hyphae: in vivo study of nuclear migration and positioning in Aspergillus nidulans. Molecular Microbiology 25, 757–769. doi: 10.1046/J.l365-2958.1997.5131873.X

    Article  CAS  PubMed  Google Scholar 

  • Suelmann R, Fischer R (2000) Mitochondrial movement and morphology depend on an intact actin cytoskeleton in Aspergillus nidulans. Cell Motility and the Cytoskeleton 45, 42–50. doi:10.1002/(SICI)1097-0169(200001)45:13.3.CO;2-3

    Article  CAS  PubMed  Google Scholar 

  • Tsien RY (1998) The green fluorescent protein. Annual Review of Biochemistry 67, 509–544. doi:10.1146/ANNUREV.BIOCHEM.67.1.509

    Article  CAS  PubMed  Google Scholar 

  • Van West P, Reid B, Campbell TA, Sandrock RW, Fry WE, Kamoun S, Gow NAR (1999) Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora. FEMS Microbiology Letters 178, 71–80. doi: 10.1016/S0378-1097(99)00320-1

    Article  PubMed  Google Scholar 

  • Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes of Neurospora crassa. Proceedings of the National Academy of Sciences of the United States of America 83, 4869–4873.

    Article  CAS  PubMed  Google Scholar 

  • Waite BH, Stover RH (1960) Studies on Fusarium wilt of bananas, VI. Variability and the cultivar concept in Fusarium oxysporum f. cubense. Canadian Journal of Botany 38, 985–994.

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Microbiology, Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, 0002, Pretoria, South Africa

    Marinda Visser, Michael J. Wingfield & Alius Viljoen

  2. Genetics, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, 0002, Pretoria, South Africa

    Brenda D. Wingfield

  3. Department of Plant Pathology, University of California, 95616, Davis, California, USA

    Thomas R. Gordon

Authors
  1. Marinda Visser
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thomas R. Gordon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Brenda D. Wingfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael J. Wingfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alius Viljoen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marinda Visser.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Visser, M., Gordon, T.R., Wingfield, B.D. et al. Transformation of Fusarium oxysporum f. sp. cubense, causal agent of Fusarium wilt of banana, with the green fluorescent protein (GFP) gene. Australasian Plant Pathology 33, 69–75 (2004). https://doi.org/10.1071/AP03084

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1071/AP03084

Keywords

Search

Navigation