Current Genetics

, Volume 48, Issue 4, pp 270–275 | Cite as

A two-step protocol for efficient deletion of genes in the filamentous ascomycete Podospora anserina

  • Andrea Hamann
  • Kristin Krause
  • Alexandra Werner
  • Heinz D. Osiewacz
Technical Note

Abstract

Deletion of genes in Podospora anserina via conventional methods is an inefficient and time-consuming process since homologous recombination occurs normally only at low frequency (about 1%). To improve the efficiency of replacement, we adopted the two-step protocol developed for Aspergillus nidulans (Chaveroche et al. in Nucleic Acids Res 28:E97, 2000). As a prerequisite, a vector was generated containing a blasticidin resistance cassette for selection in the Escherichia coli host strain KS272 (pKOBEG) and a phleomycin resistance cassette for selection in P. anserina. A derivative of this vector, into which short (∼250 bp) PCR-generated sequences flanking the gene to be deleted have been integrated, is introduced into the E. coli host strain which contains a cosmid with the gene of interest and long 5′ and 3′ flanking sequences. Subsequently, a cosmid is reisolated from E. coli in which the gene of interest is replaced by the resistance cassette. This construct is used to transform P. anserina. The long stretches flanking the resistance cassette facilitate recombination with homologous sequences in the fungal genome and increase the efficiency of gene deletion up to 100%. The procedure is not dependent on the availability of specific auxotrophic mutant strains and may be applicable to other fungi.

Keywords

Podospora anserina Knockout Gene replacement Homologous recombination 

Notes

Acknowledgements

We greatly acknowledge Prof. Dr Christophe d’Enfert (Paris, France) for the possibility to use the E. coli strain KS272 (pKOBEG) and Prof. Dr. Axel Brakhage (Jena, Germany) and Prof. Dr. Ulrich Kück (Bochum, Germany) for helpful hints on the development of the knockout procedure. We also greatly acknowledge the P. anserina sequencing consortium coordinated at Paris, France (http://www.genoscope.cns.fr/externe/English/Projets/Projet_GA/GA.html) for making the P. anserina contigs publicly available. The experimental work is supported by a grant of the European Commission (LSHM-CT-2004-512020) to HDO.

References

  1. Averbeck NB, Borghouts C, Hamann A, Specke V, Osiewacz HD (2001) Molecular control of copper homeostasis in filamentous fungi: increased expression of a metallothionein gene during aging of Podospora anserina. Mol Gen Genet 264:604–612CrossRefPubMedGoogle Scholar
  2. Calmels S, Parriche M, Durand H, Tiraby G (1991) High efficiency transformation of Tolypocladium geodes conidiospores to phleomycin resistance. Curr Genet 20:309–314CrossRefPubMedGoogle Scholar
  3. Chaveroche M-K, Ghigo J-M, d’Enfert C (2000) A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res 28:E97CrossRefPubMedGoogle Scholar
  4. Contamine V, Zickler D, Picard M (2004) The Podospora rmp1 gene implicated in nucleus-mitochondria cross-talk encodes an essential protein whose subcellular location is developmentally regulated. Genetics 166:135–150CrossRefPubMedGoogle Scholar
  5. Dementhon K, Saupe SJ, Clavé C (2004) Characterization of IDI-4, a bZIP transcription factor inducing autophagy and cell death in the fungus Podospora anserina. Mol Microbiol 53:1625–1640CrossRefPubMedGoogle Scholar
  6. Dufour E, Boulay J, Rincheval V, Sainsard-Chanet A (2000) A causal link between respiration and senescence in Podospora anserina. Proc Natl Acad Sci USA 97:4138–4143CrossRefPubMedGoogle Scholar
  7. Jekosch K, Kück U (2000) Glucose dependent transcriptional expression of the cre1 gene in Acremonium chrysogenum strains showing different levels of cephalosporin C production. Curr Genet 37:388–395CrossRefPubMedGoogle Scholar
  8. Kooistra R, Hooykaas PJJ, Steensma HY (2004) Efficient gene targeting in Kluyveromyces lactis. Yeast 21:781–792CrossRefPubMedGoogle Scholar
  9. Kück U, Pöggeler S (2004) pZHK2, a bi-functional transformation vector, suitable for two step gene targeting. Fungal Genet Newsl 51:4–6Google Scholar
  10. Langfelder K, Gattung S, Brakhage AA (2002) A novel method used to delete a new Aspergillus fumigatus ABC transporter-encoding gene. Curr Genet 41:268–274CrossRefPubMedGoogle Scholar
  11. Lecellier G, Silar P (1994) Rapid methods for nucleic acids extraction from Petri dish-grown mycelia. Curr Genet 25:122–123CrossRefPubMedGoogle Scholar
  12. Lorin S, Dufour E, Boulay J, Begel O, Marsy S, Sainsard-Chanet A (2001) Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina. Mol Microbiol 42:1259–1267CrossRefPubMedGoogle Scholar
  13. Malagnac F, Lalucque H, Lepère G, Silar P (2004) Two NADPH oxidase isoforms are required for sexual reproduction and ascospore germination in the filamentous fungus Podospora anserina. Fungal Genet Biol 41:982–997CrossRefPubMedGoogle Scholar
  14. 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 USA 101:12248–12253CrossRefPubMedGoogle Scholar
  15. Osiewacz HD (1994) A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes. Curr Genet 26:87–90CrossRefPubMedGoogle Scholar
  16. Pinan-Lucarré B, Paoletti M, Dementhon K, Coulary-Salin B, Clavé C (2003) Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina. Mol Microbiol 47:321–333CrossRefPubMedGoogle Scholar
  17. Rizet G (1952) Les phénomènes de barrages chez Podospora anserina. I Analyse génétique des barrages entre souches S et s. Rev Cytol Biol Veget 13:51–92Google Scholar
  18. Ruprich-Robert G, Zickler D, Berteaux-Lecellier V, Vélot C, Picard M (2002) Lack of mitochondrial citrate synthase discloses a new meiotic checkpoint in a strict aerobe. EMBO J 21:6440–6451CrossRefPubMedGoogle Scholar
  19. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  20. Sellem CH, Lemaire C, Lorin S, Dujardin G, Sainsard-Chanet A (2005) Interaction between the oxa1 and rmp1 genes modulates respiratory complex assembly and life span in Podospora anserina. Genetics 169:1379–1389CrossRefPubMedGoogle Scholar
  21. Silar P, Barreau C, Debuchy R, Kicka S, Turcq B, Sainsard-Chanet A, Sellem CH, Billault A, Cattolico L, Duprat S, Weissenbach J (2003) Characterization of the genomic organization of the region bordering the centromere of chromosome V of Podospora anserina by direct sequencing. Fungal Genet Biol 39:250–263CrossRefPubMedGoogle Scholar
  22. Stumpferl SW, Stephan O, Osiewacz HD (2004) Impact of a disruption of a pathway delivering copper to mitochondria on Podospora anserina metabolism and life span. Eukaryotic Cell 3:200–211CrossRefPubMedGoogle Scholar
  23. Yang L, Ukil L, Osmani A, Nahm F, Davies J, De Souza CPC, Dou X, Perez-Balaguer A, Osmani SA (2004) Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans. Eukaryotic Cell 3:1359–1362CrossRefPubMedGoogle Scholar
  24. Yu J-H, Hamari Z, Han K-H, Seo J-A, Reyes-Domínguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41:973–981CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Andrea Hamann
    • 1
  • Kristin Krause
    • 1
  • Alexandra Werner
    • 1
  • Heinz D. Osiewacz
    • 1
  1. 1.BiozentrumBotanisches InstitutFrankfurtGermany

Personalised recommendations