Abstract
To overcome the challenges met with gene deletion in the plant pathogen Verticillium dahliae, a mutant strain with impaired non-homologous end joining DNA repair was generated to improve targeted gene replacement frequencies. A V. dahliae 991 ΔVdku70 null mutant strain was generated using Agrobacterium tumefaciens-mediated transformation. Despite having impaired non-homologous end joining DNA repair function, the ΔVdku70 strain exhibited normal growth, reproduction capability, and pathogenicity when compared with the wild-type strain. When the ΔVdku70 strain was used to delete 2-oxoglutarate dehydrogenase E2, ferric reductase transmembrane component 3 precursor, and ferric reductase transmembrane component 6 genes, gene replacement frequencies ranged between 22.8 and 34.7 % compared with 0.3 and 0.5 % in the wild-type strain. The ΔVdku70 strain will be a valuable tool to generate deletion strains when studying factors that underlie virulence and pathogenesis in this filamentous fungus.
Similar content being viewed by others
References
Aylon Y, Kupiec M (2004) DSB repair: the yeast paradigm. DNA Repair 3:797–815
Boulton SJ, Jackson SP (1996) Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J 15:5093–5103
Choquer M, Fournier E, Kunz C, Levis C, Pradier JM, Simon A, Viaud M (2007) Botrytis cinerea virulence factors: new insights into a necrotrophic and polyphageous pathogen. FEMS Microbiol Lett 277:1–10
da Silva Ferreira M, Kress M, Savoldi M, Goldman M, Hartl A, Heinekamp T, Brakhage A, Goldman G (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
Dudásová Z, Dudás A, Chovanec M (2004) Non-homologous end-joining factors of Saccharomyces cerevisiae. FEMS Microbiol Rev 28:581–601
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 53:249–258
Frandsen RJ, Frandsen M, Giese H (2012) Targeted gene replacement in fungal pathogens via Agrobacterium tumefaciens-mediated transformation. Plant Fungal Pathog 835:17–45
Gao F, Zhou BJ, Li GY, Jia PS, Li H, Zhao YL, Zhao P, Xia GX, Guo HS (2010) A glutamic acid-rich protein identified in V. dahliae from an insertional mutagenesis affects microsclerotial formation and pathogenicity. PLoS One 5:e15319
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
Haarmann T, Lorenz N, Tudzynski P (2008) Use of a nonhomologous end joining deficient strain (Δku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis. Fungal Genet Biol 45:35–44
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 97:4965–4976
Hefferin ML, Tomkinson AE (2005) Mechanism of DNA double-strand break repair by non-homologous end joining. DNA Repair 4:639–648
Hoff B, Kamerewerd J, Sigl C, Zadra I, Kuck U (2010) Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain DeltaPcku70 show up-regulation of genes from the HOG pathway. Appl Microbiol Biotechnol 4:1081–1094
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 103:14871–14876
Iwabuchi K, Hashimoto M, Matsui T, Kurihara T, Shimizu H, Adachi N, Ishiai M, Yamamoto K, Tauchi H, Takata M, Koyama H, Date T (2006) 53BP1 contributes to survival of cells irradiated with X-ray during G1 without Ku70 or Artemis. Genes Cells 11:935–948
Klose S, Ajwa HA, Fennimore SA, Martin FN, Browne GT, Subbarao KV (2007) Dose response of weed seeds and soilborne pathogens to 1, 3-D and chloropicrin. Crop Prot 26:535–542
Kooistra R, Hooykaas PJ, Steensma HY (2004) Efficient gene targeting in Kluyveromyces lactis. Yeast 9:781–792
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
Lan XW, Yao ZT, Zhou Y, Shang JJ, Lin HY, Nuss DL, Chen BS (2008) Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency. Curr Genet 53:59–66
Lee SE, Moore JK, Holmes A, Umezu K, Kolodner RD, Haber JE (1998) Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94:399–409
Levy M, Erental A, Yarden O (2008) Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum. Mol plant pathol 9:719–725
Maruyama JI, Kitamoto K (2008) Multiple gene disruptions by marker recycling with highly efficient gene-targeting background (ΔligD) in Aspergillus oryzae. Biotechnol Lett 30:1811–1817
Mullins ED, Chen X, Romaine P, Raina R, Geiser DM, Kang S (2001) Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology 91:173–180
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 45:165–170
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 101:248–253
Pöggeler S, Kück U (2006) Highly efficient generation of signal transduction knockout mutants using a fungal strain deficient in the mammalian ku70 ortholog. Gene 378:1–10
Rauyaree P, Ospina-Giraldo MD, Kang S, Bhat RG, Subbarao KV, Grant SJ, Dobinson KF (2005) Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae. Curr Genet 48:109–116
Snoek IS, Van der Krogt ZA, Touw H, Kerkman R, Pronk JT, Bovenberg RA, van den Berg MA, Daran JM (2009) Construction of an hdfA Penicillium chrysogenum strain impaired in non-homologous end-joining and analysis of its potential for functional analysis studies. Fungal Genet Biol 5:418–426
Tachibana A (2004) Genetic and physiological regulation of non-homologous end-joining in mammalian cells. Adv Biophys 38:21–44
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
Takita Y, Takahara M, Nogami S, Anraku Y, Ohya Y (1997) Applications of the long and accurate polymerase chain reaction method in yeast molecular biology: direct sequencing of the amplified DNA and its introduction into yeast. Yeast 13:763–768
Teo SH, Jackson SP (1997) Identification of Saccharomyces cerevisiae DNA ligase IV: involvement in DNA double-strand break repair. EMBO J 16:4788–4795
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 284:357–371
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 45:68–75
Wang HC, Perrault AR, Takeda Y, Qin W, Wang HY, Iliakis G (2003) Biochemical evidence for Ku-independent backup pathways of NHEJ. Nucleic Acids Res 31:5377–5388
Zhang BL, Yang YW, Chen TZ, Yu WG, Liu TL, Li HJ, Fan XH, Ren YZ, Shen DY, Liu L, Dou DL, Chang YH (2012) Island cotton Gbve1 gene encoding a receptor-like protein confers resistance to both defoliating and non-defoliating isolates of Verticillium dahliae. PLoS ONE 7:e51091
Zhong GZ, 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 139:146–151
Zhou BJ, Jia PS, Gao F, Guo HS (2012) Molecular characterization and functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene family from V. dahliae. Mol Plant Microbe Interact 25:964–975
Acknowledgments
This work was supported by a Grant from the Key Project for Breeding Genetic Modified Organisms (2014ZX08005-004). We would like to thank Dr. Latifur Rehman, for reading this manuscript.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Qi, X., Su, X., Guo, H. et al. A ku70 null mutant improves gene targeting frequency in the fungal pathogen Verticillium dahliae . World J Microbiol Biotechnol 31, 1889–1897 (2015). https://doi.org/10.1007/s11274-015-1907-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11274-015-1907-1