Abstract
Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. However, this event is rare in organisms like the filamentous fungus Podospora anserina. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. By using the ΔPaKu70 strain, it is now possible (1) to produce deletion mutants with an efficiency of 100%, (2) to achieve allelic exchange by introducing a mutated allele associated with a selection cassette at the locus, (3) to introduce a mutation in a gene without co-insertion of a selectable marker and without any modification of the target locus.
This is a preview of subscription content, log in via an institution to check access.
References
Bergès T, Barreau C (1989) Heat shock at an elevated temperature improves transformation efficiency of protoplasts from Podospora anserina. J Gen Microbiol 135:601–604
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. EMBO J 17:1248–1258
Briand JF, Navarro F, Rematier P, Boschiero C, Labarre S, Werner M, Shpakovski GV, Thuriaux P (2001) Partners of Rpb8p, a small subunit shared by yeast RNA polymerases I, II and III. Mol Cell Biol 21:6056–6065
Chaveroche MK, Ghigo JM, d’Enfert C (2000) A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res 28:E97
Colot HV, Park G, Turner GE, Ringelberg C, Crew CM, Litvinkova L, Weiss RL, Borkovich KA, Dunlap JC (2006) A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci USA 103:10352–10357
Coppin E, Debuchy R (2000) Co-expression of the mating-type genes involved in internuclear recognition is lethal in Podospora anserina. Genetics 155:657–669
da Silva Ferreira ME, Kress MR, Savoldi M, Goldman MH, Hartl A, Heinekamp T, Brakhage AA, Goldman GH (2006) The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell 5:207–211
Debuchy R, Arnaise S, Lecellier G (1993) The mat- allele of Podospora anserina contains three regulatory genes required for the development of fertilized female organs. Mol Gen Genet 241:667–673
Dequard-Chablat M, Rotig A (1997) Homologous and heterologous expression of a ribosomal protein gene in Podospora anserina requires an intron. Mol Gen Genet 253:546–552
Esser K (1974) Podospora anserina. In: King RC (ed) Handbook of genetics. Plenum Press, New York, pp 531–551
Goins CL, Gerik KJ, Lodge JK (2006) Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes. Fungal Genet Biol 43:531–544
Hamann A, Krause K, Werner A, Osiewacz HD (2005) A two-step protocol for efficient deletion of genes in the filamentous ascomycete Podospora anserina. Curr Genet 48:270–275
Kooistra R, Hooykaas PJ, Steensma HY (2004) Efficient gene targeting in Kluyveromyces lactis. Yeast 21:781–792
Krappmann S, Sasse C, Braus GH (2006) Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end-joining-deficient genetic background. Eukaryot Cell 5:212–215
Krugel H, Fiedler G, Smith C, Baumberg S (1993) Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei. Gene 127:127–131
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 42:1259–1267
Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CA, Ram AF (2007) Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol 128:770–775
Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L, Murray SL, Hynes MJ, Osmani SA, Oakley BR (2006) A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics 172:1557–1566
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 101:12248–12253
Poggeler S, Kuck U (2006) Highly efficient generation of signal transduction knockout mutants using a fungal strain deficient in the mammalian ku70 ortholog. Gene 378:1–10
Ridder R, Osiewacz HD (1992) Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency. Curr Genet 21:207–213
Rizet G (1952) Les phénomènes de barrage chez Podospora anserina. I. Analyse génétique des barrages entres les souches S et s. Rev Cytol Biol Veget 13:51–92
Rizet G, Engelmann C (1949) Contribution à l’étude génétique d’un ascomycète tétrasporé: Podospora anserina. Rehm Rev Cytol Biol Veget 11:201–304
Silar P (1995) Two new easy to use vectors for transformation. Fungal Genet Newsl 42:73
Takahashi T, Masuda T, Koyama Y (2006) Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae. Mol Genet Genomics 275:460–470
Turgeon BG, Debuchy R (2007) Cochliobolus and podospora: mechanisms of sex determination and the evolution of reproductive life style. In: Heitman J, Kronstad JW, Taylor JW, Casselton LA (eds) Sex in fungi: molecular determination and evolutionary implications. ASM Press, Washington DC, pp 93–121
Acknowledgments
We thank C. Herbert and L. Sperling for helpful comments on the manuscript. This work was supported by grants from the Association Française contre les Myopathies, grants from the National Research Agency (ANR-05-BLAN-0385-01) and grants from the European Commission via the Integrated Project MiMage (LSHM-CT-2004-512020).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by H. Osiewacz.
Rights and permissions
About this article
Cite this article
El-Khoury, R., Sellem, C.H., Coppin, E. et al. Gene deletion and allelic replacement in the filamentous fungus Podospora anserina . Curr Genet 53, 249–258 (2008). https://doi.org/10.1007/s00294-008-0180-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-008-0180-3