Functional genomics in the rice blastfungus, Magnapathe oryzae using an RNA siencing approach

• Project/Area Number: 17380030
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Plant pathology
• Research Institution: Kobe University
• Principal Investigator: NAKAYASHIKI Hitoshi Kobe University, Graduate School of Agricultural Science, Associate Professor (50252804)
• Co-Investigator(Kenkyū-buntansha): MAYAMA Shigeyuki Kobe University, Graduate School of Agricultural Science, Professor (00112251) ; TOSA Yukio Kobe University, Graduate Schcol of Agricultural Science, Professor (20172158)
• Project Period (FY): 2005 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥16,870,000 (Direct Cost: ¥15,700,000、Indirect Cost: ¥1,170,000); Fiscal Year 2007: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2006: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2005: ¥8,300,000 (Direct Cost: ¥8,300,000)
• Keywords: RNAi / RNA silencing / Maonaoorthe orvzae / ポストゲノム / 病原性遺伝子

Research Abstract

We developed an RNA silencing vector, pSilent-Duall (pSD1), with a convergent dual Pol II promoter system that provides a high-throughput platform for functional genomics research in filamentous fungi. In the pSD1 system, the target gene was designed to be transcribed as a chimeric RNA with enhanced green fluorescent protein (eGFP) RNA. This enabled us to efficiently screen the resulting transformants using GFP fluorescence as an indicator of gene silencing. A model study with the eGFP gene showed that pSD1-based vectors induced gene silencing via the RNAi pathway with slightly lower efficiency than did hairpin eGFP RNA-expressing vectors. To demonstrate the applicability of the pSD1 system for elucidating gene function in the rice blast fungus Magnaporthe oryzae, 37 calcium signaling-related genes that include almost all known calcium signaling proteins in the genome were targeted for gene silencing by the vector. Phenotypic analyses of the silenced transformants showed that at least 26, 35, and 15 of the 37 genes examined were involved in hyphal growth, sporulation and pathogenicity, respectively, in M oryzae. These included several novel findings such as that Pmcl-, Spfl- and Neo1-like Ca^<+>2 pumps, calreticulin, and calpactin heavy chain were essential for fungal pathogenicity.

Research Products

• [Journal Article] Systematic functional analysis of calcium signaling proteins in the genome of the rice blast fungus, Magnaporthe oryzae, using a high-throughput RNA silencing system. (2008)
  • Author(s): Nguyen, et. al.
  • Journal Title: Molecular Microbiology 68 Pages: 1348-1365
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] RNA interference: roles in fungal biology. (2008)
  • Author(s): Nakayashiki, et. al.
  • Journal Title: Current Opinion in Microbiology (in press)
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Speciation in Pyricularia inferred from multilocus phylogenetic analysis. (2008)
  • Author(s): Hirata, et. al.
  • Journal Title: Mycol Res. 111 Pages: 799-808
  • Peer Reviewed
• [Journal Article] Systematic functional analysis of calcium signaling proteins in the genome of the rice blast fungus, Magnaporthe oryzae, using a high-throughput RNA silencing system. (2008)
  • Author(s): Nguyen, et. al.
  • Journal Title: Molecular Microbiology (In press)
  • Peer Reviewed
• [Journal Article] siRNA-dependent and-independent posttranscriptional cosuppression of the LTR-retrotransposon MAGGY in the phytopathogenic fungus Magnaporthe oryaze. (2007)
  • Author(s): Murata, et. al.
  • Journal Title: Nucleic Acids Res. 35 Pages: 5987-5994
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] siRNA-dependent and -independent posttranscriptional cosuppression of the LTR-retrotransposon MAGGY in the phytopathogenic fungus Magnaporthe oryzae. (2007)
  • Author(s): Murata, et. al.
  • Journal Title: Nucleic Acids Res. 35 Pages: 5987-5994
  • Peer Reviewed
• [Journal Article] Evolution and diversification of RNA silencing proteins in fungi (2006)
  • Author(s): Nakayashiki H. et al.
  • Journal Title: J. Mol. Evol 63 Pages: 127-135
  • NAID: 120000949568
• [Journal Article] Specific cleavage of ribosomal RNA and mRNA during victorin-induced apoptotic cell death in oat (2006)
  • Author(s): Hoat, T. et al.
  • Journal Title: Plant J. 46 Pages: 922-933
• [Journal Article] The C-terminal chromodomain-like module in the integrase domain is crucial for high transposition efficiency of the retrotransposon MAGGY (2005)
  • Author(s): Nakayashiki, H. et al.
  • Journal Title: FEBS letters 579 Pages: 488-492
  • NAID: 120000942509
• [Journal Article] RNA silencing as a tool for exploring gene function in Ascomycete fungi (2005)
  • Author(s): Nakayashiki, H. et al.
  • Journal Title: Fungal Genet. & Biol. 42 Pages: 275-283
  • NAID: 120000942198
• [Journal Article] RNA silencing in fungi : Mechanisms and applications (2005)
  • Author(s): Nakayashiki H
  • Journal Title: FEBS letters 579 Pages: 5950-5957
  • NAID: 120000943890
• [Journal Article] (2007). Speciation in Pyricularia inferred from multilocus phylogenetic analysis
  • Author(s): Hirata, K, Kusaba, M, Chuma, I, Osue, J, Nakayashiki, H, Mayama, S, Tosa, Y
  • Journal Title: Mycol Res 111 Pages: 799-808
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] (2007). siRNAdependent and -independent posttranscriptional cosuppression of the LTR-retrotransposon MAGGY in the phytopathogenic fungus Magnaporthe oryzae
  • Author(s): Murata, N, Kadotani, N, Yamaguchi, M, Tosa, Y, Mayama, S, Nakayashiki, H
  • Journal Title: Nucleic Acids Res 35 Pages: 5987-5994
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] (2008)Systematic functional analysis of calcium signaling proteins in the genome of the rice blast fungus, Magnaporthe oryzae, using a high-throughput RNA silencing system
  • Author(s): Nguyen, B, Q, Kadotani, N, Kasahara, S, Tosa, Y, Mayama, S, Nakayashiki, H
  • Journal Title: Molecular Microbiology 68 Pages: 1348-1365
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] (2007)Analysis of calcium signaling proteins in the rice blast fungus, Magnaporthe oryzae, using an RNA silencing approach
  • Author(s): Quoc, N, B, Kadotani, N, Tosa, Y, Mayama, S, Nakayashiki, H
  • Journal Title: 4th International Rice blast conference. Changsha, China. Abstract 41
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Evolution and diversification of RNA silencing proteins in fungi
  • Author(s): Nakayashiki H. et al.
  • Journal Title: J.Mol.Evol (In press)
  • NAID: 120000949568
• [Journal Article] Specific cleavage of ribosomal RNA and mRNA during victorin-induced apoptotic cell death in oat
  • Author(s): Hoat, T. et al.
  • Journal Title: Plant J. (In press)
• [Presentation] Analysis of calcium signaling proteins in the rice blast fungus, Magnaporthe oryzae, using an RNA silencing approach. (2007)
  • Author(s): Nguyen, et. al.
  • Organizer: 4th International Rice blast conference
  • Place of Presentation: Changsha, China
  • Year and Date: 2007-10-11
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Analysis of calcium signaling proteins in the rice blast fungus, Magnaporthe oryzae, using an RNA silencing approach (2007)
  • Author(s): Quoc, N.B., Kadotani, N., Tosa, Y., Mayama, S., Nakayashiki, H
  • Organizer: 4th International Rice blast conference (Abstract P. 41)
  • Place of Presentation: Changsha, China
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] RNAi in fungi. In: RNAi (Advanced Methods) (2008)
  • Author(s): Nakayashiki, H.
  • Total Pages: 138
  • Publisher: Taylor and Francis. UK
  • Description: 「研究成果報告書概要(和文)」より
• [Book] RNAi in fungi. In: RNAi(Advanced Methods) (2008)
  • Author(s): Nakayashiki, H.
  • Total Pages: 138
  • Publisher: Taylor and Francis. UK
• [Book] RNA interference (RNAi) (eds. M. Latterich) (2007)
  • Author(s): Nakayashiki, H.
  • Publisher: Taylor and Francis. UK(in press)