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Recombination and Gene Targeting in Neurospora

  • Keiichiro Suzuki
  • Hirokazu Inoue
Chapter
  • 2.2k Downloads
Part of the Fungal Biology book series (FUNGBIO)

Abstract

Genetic manipulation, especially targeted gene replacement, is a potential powerful tool for gene functional research and industrial engineering in filamentous fungi. However, low frequency of gene targeting in most filamentous fungi has hampered research on the molecular mechanisms of these species. In this chapter, we describe the relationship between exogenous DNA integration events and cellular DNA double-strand repair machinery in one of the model filamentous fungi, Neurospora crassa. Based on the molecular mechanism, it has been proven that the gene-targeting frequency is dramatically increased when nonhomologous end-joining, that promotes chromosomal random integration, was deficient in Neurospora and various other fungi. This technique has opened a new avenue for genetic manipulation in filamentous fungi.

Keywords

Gene targeting Homologous recombination (HR) Nonhomologous end-joining (NHEJ) Homologous integration (HI) Nonhomologous integration (NHI) 

References

  1. Alshahni MM, Yamada T, Takatori K, Sawada T, Makimura K (2011) Insights into a nonhomologous integration pathway in the dermatophyte Trichophyton mentagrophytes: efficient targeted gene disruption by use of mutants lacking ligase IV. Microbiol Immunol 55(1):34–43PubMedCrossRefGoogle Scholar
  2. Bertolini LR, Bertolini M, Maga EA, Madden KR, Murray JD (2009) Increased gene targeting in Ku70 and Xrcc4 transiently deficient human somatic cells. Mol Biotechnol 41(2):106–114PubMedCrossRefGoogle Scholar
  3. Borkovich KA, Alex LA, Yarden O, Freitag M, Turner GE, Read ND, Seiler S, Bell-Pedersen D, Paietta J, Plesofsky N, Plamann M, Goodrich-Tanrikulu M, Schulte U, Mannhaupt G, Nargang FE, Radford A, Selitrennikoff C, Galagan JE, Dunlap JC, Loros JJ, Catcheside D, Inoue H, Aramayo R, Polymenis M, Selker EU, Sachs MS, Marzluf GA, Paulsen I, Davis R, Ebbole DJ, Zelter A, Kalkman ER, O’Rourke R, Bowring F, Yeadon J, Ishii C, Suzuki K, Sakai W, Pratt R (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68(1):1–108PubMedCrossRefPubMedCentralGoogle Scholar
  4. Bugeja HE, Boyce KJ, Weerasinghe H, Beard S, Jeziorowski A, Pasricha S, Payne M, Schreider L, Andrianopoulos A (2012) Tools for high efficiency genetic manipulation of the human pathogen Penicillium marneffei. Fungal Genet Biol 49(10):772–778PubMedCrossRefGoogle Scholar
  5. Chang PK (2008) A highly efficient gene-targeting system for Aspergillus parasiticus. Lett Appl Microbiol 46(5):587–592PubMedCrossRefGoogle Scholar
  6. Chang PK, Scharfenstein LL, Wei Q, Bhatnagar D (2010) Development and refinement of a high-efficiency gene-targeting system for Aspergillus flavus. J Microbiol Methods 81(3):240–246PubMedCrossRefGoogle Scholar
  7. Choquer M, Robin G, Le Pecheur P, Giraud C, Levis C, Viaud M (2008) Ku70 or Ku80 deficiencies in the fungus Botrytis cinerea facilitate targeting of genes that are hard to knock out in a wild-type context. FEMS Microbiol Lett 289(2):225–232PubMedCrossRefGoogle Scholar
  8. 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 U S A 103(27):10352–10357PubMedCrossRefPubMedCentralGoogle Scholar
  9. Critchlow SE, Jackson SP (1998) DNA end-joining: from yeast to man. Trends Biochem Sci 23(10):394–398PubMedCrossRefGoogle Scholar
  10. 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(1):207–211PubMedCrossRefPubMedCentralGoogle Scholar
  11. de Boer P, Bastiaans J, Touw H, Kerkman R, Bronkhof J, van den Berg M, Offringa R (2010) Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in deltalig4 or deltaku70 mutants. Fungal Genet Biol 47(10):839–846PubMedCrossRefGoogle Scholar
  12. de Jong JF, Ohm RA, de Bekker C, Wosten HA, Lugones LG (2010) Inactivation of ku80 in the mushroom-forming fungus Schizophyllum commune increases the relative incidence of homologous recombination. FEMS Microbiol Lett 310(1):91–95PubMedCrossRefGoogle Scholar
  13. Dunlap JC, Borkovich KA, Henn MR, Turner GE, Sachs MS, Glass NL, McCluskey K, Plamann M, Galagan JE, Birren BW, Weiss RL, Townsend JP, Loros JJ, Nelson MA, Lambreghts R, Colot HV, Park G, Collopy P, Ringelberg C, Crew C, Litvinkova L, DeCaprio D, Hood HM, Curilla S, Shi M, Crawford M, Koerhsen M, Montgomery P, Larson L, Pearson M, Kasuga T, Tian C, Basturkmen M, Altamirano L, Xu J (2007) Enabling a community to dissect an organism: overview of the Neurospora functional genomics project. Adv Genet 57:49–96PubMedCrossRefPubMedCentralGoogle Scholar
  14. El-Khoury R, Sellem CH, Coppin E, Boivin A, Maas MF, Debuchy R, Sainsard-Chanet A (2008) Gene deletion and allelic replacement in the filamentous fungus Podospora anserina. Curr Genet 53(4):249–258PubMedCrossRefGoogle Scholar
  15. Fang Z, Zhang Y, Cai M, Zhang J, Zhou X (2012) Improved gene targeting frequency in marine-derived filamentous fungus Aspergillus glaucus by disrupting ligD. J Appl Genet 53(3):355–362PubMedCrossRefGoogle Scholar
  16. Fattah FJ, Lichter NF, Fattah KR, Oh S, Hendrickson EA (2008) Ku70, an essential gene, modulates the frequency of rAAV-mediated gene targeting in human somatic cells. Proc Natl Acad Sci U S A 105(25):8703–8708PubMedCrossRefPubMedCentralGoogle Scholar
  17. Fox BA, Ristuccia JG, Gigley JP, Bzik DJ (2009) Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining. Eukaryot Cell 8(4):520–529PubMedCrossRefPubMedCentralGoogle Scholar
  18. Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D, FitzHugh W, Ma LJ, Smirnov S, Purcell S, Rehman B, Elkins T, Engels R, Wang S, Nielsen CB, Butler J, Endrizzi M, Qui D, Ianakiev P, Bell-Pedersen D, Nelson MA, Werner-Washburne M, Selitrennikoff CP, Kinsey JA, Braun EL, Zelter A, Schulte U, Kothe GO, Jedd G, Mewes W, Staben C, Marcotte E, Greenberg D, Roy A, Foley K, Naylor J, Stange-Thomann N, Barrett R, Gnerre S, Kamal M, Kamvysselis M, Mauceli E, Bielke C, Rudd S, Frishman D, Krystofova S, Rasmussen C, Metzenberg RL, Perkins DD, Kroken S, Cogoni C, Macino G, Catcheside D, Li W, Pratt RJ, Osmani SA, DeSouza CP, Glass L, Orbach MJ, Berglund JA, Voelker R, Yarden O, Plamann M, Seiler S, Dunlap J, Radford A, Aramayo R, Natvig DO, Alex LA, Mannhaupt G, Ebbole DJ, Freitag M, Paulsen I, Sachs MS, Lander ES, Nusbaum C, Birren B (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422(6934):859–868PubMedCrossRefGoogle Scholar
  19. 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(8):531–544PubMedCrossRefGoogle Scholar
  20. Guangtao Z, Hartl L, Schuster A, Polak S, Schmoll M, Wang T, Seidl V, Seiboth B (2009) Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina. J Biotechnol 139(2):146–151PubMedCrossRefGoogle Scholar
  21. Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24(13):2519–2524PubMedCrossRefPubMedCentralGoogle Scholar
  22. Haarmann T, Lorenz N, Tudzynski P (2008) Use of a nonhomologous end joining deficient strain (Deltaku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis. Fungal Genet Biol 45(1):35–44PubMedCrossRefGoogle Scholar
  23. Handa N, Noguchi Y, Sakuraba Y, Ballario P, Macino G, Fujimoto N, Ishii C, Inoue H (2000) Characterization of the Neurospora crassa mus-25 mutant: the gene encodes a protein which is homologous to the Saccharomyces cerevisiae Rad54 protein. Mol Gen Genet 264(1–2):154–163PubMedCrossRefGoogle Scholar
  24. Hatakeyama S, Ishii C, Inoue H (1995) Identification and expression of the Neurospora crassa mei-3 gene which encodes a protein homologous to Rad51 of Saccharomyces cerevisiae. Mol Gen Genet 249(4):439–446PubMedCrossRefGoogle Scholar
  25. He Y, Liu Q, Shao Y, Chen F (2013) Ku70 and ku80 null mutants improve the gene targeting frequency in Monascus ruber M7. Appl Microbiol Biotechnol 97(11):4965–4976PubMedCrossRefGoogle Scholar
  26. Heyer WD, Ehmsen KT, Liu J (2010) Regulation of homologous recombination in eukaryotes. Annu Rev Genet 44:113–139PubMedCrossRefPubMedCentralGoogle Scholar
  27. Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci U S A 75(4):1929–1933PubMedCrossRefPubMedCentralGoogle Scholar
  28. Iiizumi S, Kurosawa A, So S, Ishii Y, Chikaraishi Y, Ishii A, Koyama H, Adachi N (2008) Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting. Nucleic Acids Res 36(19):6333–6342PubMedCrossRefPubMedCentralGoogle Scholar
  29. Ishibashi K, Suzuki K, Ando Y, Takakura C, Inoue H (2006) Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora. Proc Natl Acad Sci U S A 103:14871–14876PubMedCrossRefPubMedCentralGoogle Scholar
  30. Ishidoh KI, Kinoshita H, Ihara F, Nihira T (2014) Efficient and versatile transformation systems in entomopathogenic fungus Lecanicillium species. Curr Genet 60(2):99–108PubMedCrossRefGoogle Scholar
  31. 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(1):212–215PubMedCrossRefPubMedCentralGoogle Scholar
  32. Kuck U, Hoff B (2010) New tools for the genetic manipulation of filamentous fungi. Appl Microbiol Biotechnol 86(1):51–62PubMedCrossRefGoogle Scholar
  33. Lan X, Yao Z, Zhou Y, Shang J, Lin H, Nuss DL, Chen B (2008) Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency. Curr Genet 53(1):59–66PubMedCrossRefGoogle Scholar
  34. Li ZH, Du CM, Zhong YH, Wang TH (2010) Development of a highly efficient gene targeting system allowing rapid genetic manipulations in Penicillium decumbens. Appl Microbiol Biotechnol 87(3):1065–1076PubMedCrossRefGoogle Scholar
  35. Ma JL, Kim EM, Haber JE, Lee SE (2003) Yeast Mre11 and Rad1 proteins define a Ku-independent mechanism to repair double-strand breaks lacking overlapping end sequences. Mol Cell Biol 23(23):8820–8828PubMedCrossRefPubMedCentralGoogle Scholar
  36. 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(4):770–775PubMedCrossRefGoogle Scholar
  37. Mizutani O, Kudo Y, Saito A, Matsuura T, Inoue H, Abe K, Gomi K (2008) A defect of LigD (human Lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae. Fungal Genet Biol 45(6):878–889PubMedCrossRefGoogle Scholar
  38. Nakazawa T, Ando Y, Kitaaki K, Nakahori K, Kamada T (2011) Efficient gene targeting in DeltaCc.ku70 or DeltaCc.lig4 mutants of the agaricomycete Coprinopsis cinerea. Fungal Genet Biol 48(10):939–946PubMedCrossRefGoogle Scholar
  39. 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(3):1557–1566PubMedCrossRefPubMedCentralGoogle Scholar
  40. Nielsen JB, Nielsen ML, Mortensen UH (2008) Transient disruption of non-homologous end-joining facilitates targeted genome manipulations in the filamentous fungus Aspergillus nidulans. Fungal Genet Biol 45(3):165–170PubMedCrossRefGoogle Scholar
  41. 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(33):12248–12253PubMedCrossRefPubMedCentralGoogle Scholar
  42. Nishizawa-Yokoi A, Nonaka S, Saika H, Kwon YI, Osakabe K, Toki S (2012) Suppression of Ku70/80 or Lig4 leads to decreased stable transformation and enhanced homologous recombination in rice. New Phytol 196(4):1048–1059PubMedCrossRefPubMedCentralGoogle Scholar
  43. Orr-Weaver TL, Szostak JW, Rothstein RJ (1981) Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A 78(10):6354–6358PubMedCrossRefPubMedCentralGoogle Scholar
  44. Perkins DD, Barry EG (1977) The cytogenetics of Neurospora. Adv Genet 19:133–285PubMedCrossRefGoogle Scholar
  45. Poggeler S, Kuck U (2006) Highly efficient generation of signal transduction knockout mutants using a fungal strain deficient in the mammalian ku70 ortholog. Gene 378:1–10PubMedCrossRefGoogle Scholar
  46. Sakuraba Y, Schroeder AL, Ishii C, Inoue H (2000) A Neurospora double-strand-break repair gene, mus-11, encodes a RAD52 homologue and is inducible by mutagens. Mol Gen Genet 264(4):392–401PubMedCrossRefGoogle Scholar
  47. Schorsch C, Kohler T, Boles E (2009) Knockout of the DNA ligase IV homolog gene in the sphingoid base producing yeast Pichia ciferrii significantly increases gene targeting efficiency. Curr Genet 55(4):381–389PubMedCrossRefGoogle Scholar
  48. Schroeder AL, Inoue H, Sachs MS (1998) DNA repair in Neurospora. DNA Damage and Repair 1:503–538CrossRefGoogle Scholar
  49. Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271PubMedCrossRefGoogle Scholar
  50. Szewczyk E, Kasuga T, Fan Z (2013) Efficient sequential repetitive gene deletions in Neurospora crassa employing a self-excising beta-recombinase/six cassette. J Microbiol Methods 92(3):236–243PubMedCrossRefGoogle Scholar
  51. 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(5):460–470PubMedCrossRefGoogle Scholar
  52. Tanaka S, Ishii C, Hatakeyama S, Inoue H (2010) High efficient gene targeting on the AGAMOUS gene in an Arabidopsis AtLIG4 mutant. Biochem Biophys Res Commun 396(2):289–293PubMedCrossRefGoogle Scholar
  53. Tani S, Tsuji A, Kunitake E, Sumitani J, Kawaguchi T (2013) Reversible impairment of the ku80 gene by a recyclable marker in Aspergillus aculeatus. AMB Express 3(1):4PubMedCrossRefPubMedCentralGoogle Scholar
  54. Ushimaru T, Terada H, Tsuboi K, Kogou Y, Sakaguchi A, Tsuji G, Kubo Y (2010) Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer. Mol Genet Genomics 284(5):357–371PubMedCrossRefGoogle Scholar
  55. Villalba F, Collemare J, Landraud P, Lambou K, Brozek V, Cirer B, Morin D, Bruel C, Beffa R, Lebrun MH (2008) Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining. Fungal Genet Biol 45(1):68–75PubMedCrossRefGoogle Scholar
  56. Yu X, Gabriel A (2003) Ku-dependent and Ku-independent end-joining pathways lead to chromosomal rearrangements during double-strand break repair in Saccharomyces cerevisiae. Genetics 163(3):843–856PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Keiichiro Suzuki
    • 1
  • Hirokazu Inoue
    • 2
  1. 1.Laboratory of Genetics, Department of Regulation-Biology, Faculty of ScienceSaitama UniversitySaitamaJapan
  2. 2.Regulation Biology, Faculty of ScienceSaitama UniversitySaitamaJapan

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