Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing.
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 (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. 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 LIM15dsRNA. 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.
Namekawa
Satoshi H
SH
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan.
Iwabata
Kazuki
K
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan.
Sugawara
Hiroko
H
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan.
Hamada
Fumika N
FN
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan.
Koshiyama
Akiyo
A
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan.
Chiku
Hiroyuki
H
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan.
Kamada
Takashi
T
Department of Molecular Biology, Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
Sakaguchi
Kengo
K
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, Japan.
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D003302:Coprinus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D020868:Gene Silencing; D008540:Meiosis; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D013172:Spores, Fungal / Q000254:growth & development
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D012330:RNA, Double-Stranded; D000251:Adenosine Triphosphatases