クワタ シゲル   Kuwata Shigeru
  桑田 茂
   所属   明治大学  農学部
   職種   専任教授
発表年月日 2013/03/16
発表テーマ DNA double-strand breaks generated by yeast endonuclease I-Sce I induce ectopic homologous recombination and targeted gene replacement in Magnaporthe oryzae
会議名 The 27 th Fungal Genetics Conference
主催者 Genetics Society of America
発表形式 ポスター
単独共同区分 共同
発表者・共同発表者 T. Arazoe, T. Younomaru, S. Ohsato, T. Arie, S. Kuwata
概要 The filamentous fungus Magnaporthe oryzae causes the rice blast disease that is one of the most destructive fungal diseases of cultivated rice plants. To control this fungal disease, many resistant genes have been introduced into cultivated rice germplasm, however, breakdowns of the resistance often occur within several years by rapid evolution of the fungus. Therefore, studies on the evolutionary mechanisms of the fungus are important for elucidation of the rapid evolution. We set out a novel detection/selection system of DNA double-strand breaks (DSBs)-mediated ectopic homologous recombination (HR) that is one of the evolutionary mechanisms. The system consists of two nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes as a donor and a recipient, and a yeast endonuclease I-Sce I gene as a DSB-inducer. In this system, ectopic HR can be detected and selected by restorations of YFP fluorescence and blasticidin S (BS)-resistance at a single cell level. These donor and recipient genes were simultaneously integrated into the M. oryzae genome and transformed lines were isolated. In the absence of the DSB-inducer, transformed lines showed relatively low frequencies of HR events (>2.1%). On the other hand, by integration of the DSB-inducer gene into transformed lines, we could observe the frequencies of DSB-mediated HR raising up to ~40%. This result clearly showed that DSB into a certain gene induce ectopic HR events between the gene and its homologs. Accordingly, we further applied I-Sce I mediated DSB for TGR in M. oryzae. To detect TGR, we constructed simple system using donor and recipient genes. The recipient gene was integrated into the M. oryzae genome and transformed lines were isolated. To recipient gene integrated lines, the donor gene was introduced and restorations of YFP fluorescence and BS-resistance were evaluated. As we expected, the TGR frequencies were increased at least 37-folds by I-Sce I co-transformation as compared with those obtained without I-Sce I. This result provides a new method using DSB for improving the TGR frequency in M. oryzae. Taken together, it is strongly suggested that DSBs can drive genomic rearrangement and accelerate pathogenic variability in M. oryzae through the ectopic HR between homologous sequences such as transposable elements and avirulence genes.