title: Efficient gene targeting in ΔCc.ku70 or ΔCc.lig4 mutants of the agaricomycete Coprinopsis cinerea.
abstract: Coprinopsis cinerea is a model for studies of sexual development in agaricomycetes (homobasidiomycetes). Efficient gene targeting should facilitate such studies, especially because increasing genome and transcriptome information is now available in C. cinerea. To estimate the frequency of gene disruption by homologous integration in this fungus, we tried to disrupt Cc.wc-2, which encodes a WC-2 homolog, a partner of the fungal blue-light photoreceptor, WC-1. Disruption of Cc.wc-2 did not occur when recipients (protoplasts) of the disrupting construct were prepared from asexual spores, oidia, from the wild type, 326, while it occurred when protoplasts were prepared from mycelial cells from the same strain, albeit at a low frequency (3%). Double-stranded RNA-mediated silencing of a ku70 homolog, named Cc.ku70, or the lig4 homolog Cc.lig4 more or less increased the frequency of Cc.wc-2 targeting. On the basis of these results, we disrupted Cc.ku70 using a Cc.lig4-silenced strain. We then disrupted Cc.lig4 using the Cc.ku70 disruptant. We found that the disruption of Cc.ku70 or Cc.lig4 greatly enhanced gene targeting. In addition, this study demonstrates that Cc.wc-2 is involved in blue light perception in this fungus.
authors.affiliation: Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Okayama, Japan.
authors.forename: Takehito
authors.initials: T
authors.lastname: Nakazawa
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ando
authors.forename: Kohei
authors.initials: K
authors.lastname: Kitaaki
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Nakahori
authors.forename: Takashi
authors.initials: T
authors.lastname: Kamada
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics*; D003302:Coprinus / Q000235:genetics* / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D020868:Gene Silencing; D018390:Gene Targeting; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009154:Mutation; D025282:Mycelium / Q000235:genetics; D011523:Protoplasts / Q000378:metabolism; D011995:Recombination, Genetic; D013172:Spores, Fungal / Q000235:genetics; D059467:Transcriptome
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D000072200:Ku Autoantigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP