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Transformation of Lithium Acetate-treated Neurospora crassa

  • John V. PaiettaEmail author
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Part of the Fungal Biology book series (FUNGBIO)

Abstract

DNA-mediated transformation provides a powerful tool for both genome analysis and gene manipulation. Advances in transformation procedures of filamentous fungi have played a key role in making these organisms tractable on a molecular genetic level. A number of different transformation protocols exist for fungal transformation, each with inherent advantages for different applications. To be described here, lithium acetate has been used with success in preparing Neurospora crassa conidia for transformation. The technique involves the treatment of germinating conidia consecutively with (1) lithium acetate, (2) transforming DNA, and (3) PEG; followed by a brief heat shock before plating. The lithium acetate procedure provides a means for rapid and efficient transformation of N. crassa when moderate transformation frequencies are useful. Further, the technique has general applicability to a variety of fungal species.

Keywords

Neurospora crassa Lithium acetate Transforming DNA PEG Filamentous fungi Exogenous DNA 

Notes

Acknowledgments

This research was supported by a Medical Innovations Grant from the Wright State University Boonshoft School of Medicine. I thank Heather Hostetler for microscopy assistance.

References

  1. Allison DS, Rey MW, Berka RM, Armstrong G, Dunn-Coleman NS (1992) Transformation of the thermophilic fungus Humicola grisea var. thermoidea and overproduction of Humicola glucoamylase. Curr Genet 21:225–229CrossRefGoogle Scholar
  2. Alton NK, Hautala JA, Giles NH, Kushner SR, Vapnek D (1978) Transcription and translation in E. coli of hybrid plasmids containing the catabolic dehydroquinase gene from Neursopora crassa. Gene 4:241–259PubMedCrossRefGoogle Scholar
  3. Bej AK, Perlin MH (1989) A high efficiency transformation system for the basidiomycete Ustilago violacea employing hygromycin resistance and lithium-acetate treatment. Gene 80:171–176PubMedCrossRefGoogle Scholar
  4. Binninger DM, Skrzynia C, Pukkila PJ, Casselton LA (1987) DNA-mediated transformation of the basidomycete Coprinus cinerus. EMBO J 6:835–840PubMedPubMedCentralGoogle Scholar
  5. Butters JA, Hollomon DW (1996) Molecular analysis of azole fungicide resistance in a mutant of Ustilago maydis. Pest Manag Sci 46:277–298CrossRefGoogle Scholar
  6. Case ME, Schweier M, Kushner SR, Giles NH (1979) Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci U S A 76:5259–5263PubMedCrossRefPubMedCentralGoogle Scholar
  7. Danta-Barbosa C, Araujo EF, Moraes LMP, Vainstein MH, Azevedo MO (1998) Genetic transformation of germinated conidia of the thermophilic fungus Humicola grisea var. thermoidea to hygromycin B resistance. FEMS Microbiol Lett 169:185–190CrossRefGoogle Scholar
  8. Davis RH, deSerres FJ (1970) Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol 71A:79–143CrossRefGoogle Scholar
  9. Dhawale SS, Paietta JV, Marzluf GA (1984) A new, rapid and efficient transformation procedure for Neurospora. Curr Genet 8:77–79Google Scholar
  10. Dickman MB (1988) Whole cell transformation of the alfalfa pathogen Colletotrichum trifolii. Curr Genet 14:241–246CrossRefGoogle Scholar
  11. Dickman MB, Partridge JE (1989) Use of molecular markers for monitoring fungi involved in stalk rot of corn. Theor Appl Genet 77:535–539PubMedCrossRefGoogle Scholar
  12. Dickman MB, Podila GK, Kolattukudy PE (1989) Insertion of cutinase gene into a wound pathogen enables it to infect intact host. Nature 342:446–448CrossRefGoogle Scholar
  13. Ebbole D, Sachs MS (1990) A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia. Fungal Genet Newslett 37:17–18Google Scholar
  14. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34PubMedCrossRefGoogle Scholar
  15. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168PubMedPubMedCentralGoogle Scholar
  16. Marek ET, Schardl CL, Smith DA (1989) Molecular transformation of Fusarium solani with an antibiotic resistance marker having no fungal DNA homology. Curr Genet 15:421–428PubMedCrossRefGoogle Scholar
  17. Ninomiya Y, Suzuki K, Ishii C, Inoue H (2004) Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci U S A 101:12248–12253PubMedCrossRefPubMedCentralGoogle Scholar
  18. Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461PubMedPubMedCentralGoogle Scholar
  19. Paietta JV, Marzluf GA (1984) Transformation of lithium acetate-treated Neurospora with mini-preps of plasmid DNA. Fungal Genet Newslett 31:40–41Google Scholar
  20. Paietta JV, Marzluf GA (1985) Gene disruption by transformation in Neurospora crassa. Mol Cell Biol 5:1554–1559PubMedPubMedCentralGoogle Scholar
  21. Soliday CL, Dickman MB, Kolattukudy PE (1989) Structure of the cutinase gene and detection of promoter activity in the 5’-flanking region by fungal transformation. J Bacteriol 171:1942–1951PubMedPubMedCentralGoogle Scholar
  22. Staben C, Jensen B, Singer M, Pollock J, Schectman M, Kinsey J, Selker E (1989) Use of a bacterial Hygromycin B resistance gene as a dominant selectable marker in Neurospora crassa transformation. Fungal Genet Newslett 36:79–81Google Scholar
  23. Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes of Neurospora crassa. Proc Natl Acad Sci U S A 83:4869–4873PubMedCrossRefPubMedCentralGoogle Scholar
  24. Worsham PL, Goldman WE (1990) Development of a genetic transformation system for Histoplasma capsulatum: complementation of uracil auxotrophy. Mol Gen Genet 221:358–362PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyWright State UniversityDaytonUSA

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