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Australasian Plant Pathology

, Volume 33, Issue 1, pp 69–75 | Cite as

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

  • Marinda VisserEmail author
  • Thomas R. Gordon
  • Brenda D. Wingfield
  • Michael J. Wingfield
  • Alius Viljoen
Article

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.

Keywords

Green Fluorescent Protein Fusarium Oxysporum Australasian Plant Pathology Fusarium Wilt Banana Plant 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 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.CrossRefGoogle Scholar
  2. Booth C (1971) ‘The genus Fusarium.’ (Commonwealth Mycological Institute: Kew, England)Google Scholar
  3. 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)00218-9CrossRefPubMedGoogle Scholar
  4. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.CrossRefPubMedGoogle Scholar
  5. Chalfie M, Kain S (1998) ‘Green fluorescent protein: properties, applications and protocols.’ (John Wiley & Sons Inc.: New York, USA)Google Scholar
  6. 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.CrossRefPubMedGoogle Scholar
  7. 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.CrossRefGoogle Scholar
  8. 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.CrossRefPubMedGoogle Scholar
  9. 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)89099-4CrossRefPubMedGoogle Scholar
  10. 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.PubMedGoogle Scholar
  11. 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.PubMedGoogle Scholar
  12. 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
  13. 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
  14. Haseloff J, Amos B (1995) GFP in plants. Trends in Genetics 11, 328–329. doi: 10.1016/0168-9525(95)90186-8CrossRefPubMedGoogle Scholar
  15. Hawksworth DL, Kirk PM, Sutton BC, Pegler DN (1995) ‘Dictionary of the fungi.’ (CAB International: Wallingford, UK)Google Scholar
  16. 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/17.14.3838CrossRefPubMedGoogle Scholar
  17. 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.CrossRefPubMedGoogle Scholar
  18. 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.67.5.1987-1994.2001CrossRefGoogle Scholar
  19. 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/S0953756297005789CrossRefGoogle Scholar
  20. Nelson PE, Toussoun TA, Marasas WFO (1983) ‘Fusarium Species: an illustrated manual foridentification.’ (Pennsylvania State University Press: University Park, USA)Google Scholar
  21. 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-CCrossRefGoogle Scholar
  22. 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
  23. Pines J (1995) GFP in mammalian cells. Trends in Genetics 11, 326–327. doi: 10.1016/S0168-9525(00)89092-7CrossRefPubMedGoogle Scholar
  24. Prasher DC (1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene 111, 229–233. doi: 10.1016/0378-1119(92)90691-HCrossRefPubMedGoogle Scholar
  25. Raeder U, Broda P (1985) Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology 1, 17–20.CrossRefGoogle Scholar
  26. Sam brook J, Fritsch EF, Maniatis T (1989) ‘Molecular cloning: a laboratory manual.’ (Cold Spring Harbor Press: New York, USA)Google Scholar
  27. 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.XCrossRefPubMedGoogle Scholar
  28. 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/S002940050412CrossRefPubMedGoogle Scholar
  29. 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/S004380050257Google Scholar
  30. 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
  31. 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.XCrossRefPubMedGoogle Scholar
  32. 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;2-3CrossRefPubMedGoogle Scholar
  33. Tsien RY (1998) The green fluorescent protein. Annual Review of Biochemistry 67, 509–544. doi:10.1146/ANNUREV.BIOCHEM.67.1.509CrossRefPubMedGoogle Scholar
  34. 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)00320-1CrossRefPubMedGoogle Scholar
  35. 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.CrossRefPubMedGoogle Scholar
  36. 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.CrossRefGoogle Scholar

Copyright information

© Australasian Plant Pathology Society 2004

Authors and Affiliations

  • Marinda Visser
    • 1
    Email author
  • Thomas R. Gordon
    • 3
  • Brenda D. Wingfield
    • 2
  • Michael J. Wingfield
    • 1
  • Alius Viljoen
    • 1
  1. 1.Department of Microbiology, Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI)University of PretoriaPretoriaSouth Africa
  2. 2.Genetics, Forestry and Agricultural Biotechnology Institute (FABI)University of PretoriaPretoriaSouth Africa
  3. 3.Department of Plant PathologyUniversity of CaliforniaDavisUSA

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