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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 (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.
The results of Southern analysis of pCcLIM15dsRNA 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.
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