Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing

• Published in: Microbiology (Society for General Microbiology) 2005-11, Vol.151 (11), p.3669-3678
• Main Authors: Namekawa, Satoshi H, Iwabata, Kazuki, Sugawara, Hiroko, Hamada, Fumika N, Koshiyama, Akiyo, Chiku, Hiroyuki, Kamada, Takashi, Sakaguchi, Kengo
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Basidiomycetes; Basidiomycotina; Biological and medical sciences; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Coprinus - genetics; Coprinus - growth & development; Coprinus - metabolism; Coprinus - physiology; Coprinus cinereus; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Gene Silencing; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Meiosis; Microbiology; Mycology; RNA Interference; RNA, Double-Stranded - genetics; RNA, Double-Stranded - metabolism; Spores, Fungal - growth & development
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.28209-0

1 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan 2 Department of Molecular Biology, Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan Correspondence Kengo Sakaguchi kengo{at}rs.noda.tus.ac.jp The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct ( LIM15 dsRNA) resulted in genomic insertion of LIM15 dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15 dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15 dsRNA. The LIM15/DMC1 -repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15 dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research. Abbreviations: DAPI, 4',6-diamidino-2-phenylindole; PAB, 4-aminobenzoic acid; RNAi, RNA interference; SC, synaptonemal complex The results of Southern analysis of pCc LIM15 dsRNA insertions in white-cap transformants of C. cinereus are shown in Supplementary Fig. S1 with the online version of this paper. Present address: Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02114, USA. Present address: Howard Hughes Medical Institute, Harvard-Partners Center for Genetics and Genomics, Harvard Medical School, Boston, MA 02115, USA.