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

使用 RNA 测序方法对稻瘟菌 Magnapathe oryzae 进行功能基因组学研究

• 批准号: 17380030
• 负责人: NAKAYASHIKI Hitoshi
• 金额: $ 10.8万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17380030/
• 关键词: RNAi; RNA silencing; Maonaoorthe orvzae; ポストゲノム; 病原性遺伝子

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.

我们开发了一个RNA沉默矢量Psilent-Duall（PSD1），并带有收敛的双Pol II启动子系统，该系统为丝状真菌的功能基因组学研究提供了高通量平台。在PSD1系统中，靶基因被设计为具有增强的绿色荧光蛋白（EGFP）RNA的嵌合RNA。这使我们能够使用GFP荧光作为基因沉默的指标有效地筛选了所得转化体。对EGFP基因的模型研究表明，基于PSD1的载体通过RNAi途径诱导基因沉默，其效率略低于发夹EGFP EGFP RNA的向量。为了证明PSD1系统在水稻爆炸真菌斑点孔中阐明基因功能的适用性，37个与基因组中几乎所有已知的钙信号蛋白的钙信号相关基因都靶向基因组中的所有已知钙信号蛋白。对沉默的转化体的表型分析表明，所检查的37个基因中的至少26、35和15个分别参与了Moryzae中的菌丝生长，孢子形成和致病性。其中包括几个新的发现，例如PMCL，SPFL和NEO1样的Ca^<+> 2泵，钙网蛋白和钙乳糖蛋白重链对于真菌致病性至关重要。

项目成果

Specific cleavage of ribosomal RNA and mRNA during victorin-induced apoptotic cell death in oat

• DOI: 10.1111/j.1365-313x.2006.02752.x
• 发表时间: 2006-06-01
• 期刊: PLANT JOURNAL
• 影响因子: 7.2
• 作者: Hoat, Trinh X.;Nakayashiki, Hitoshi;Mayama, Shigeyuki
• 通讯作者: Mayama, Shigeyuki

(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

(2008)使用高通量 RNA 沉默系统对稻瘟病菌 Magnaporthe oryzae 基因组中的钙信号蛋白进行系统功能分析

• 期刊: Molecular Microbiology 68
• 作者: Nguyen;B;Q;Kadotani;N;Kasahara;S;Tosa;Y;Mayama;S;Nakayashiki;H
• 通讯作者: H

The C-terminal chromodomain-like module in the integrase domain is crucial for high transposition efficiency of the retrotransposon MAGGY

整合酶结构域中的 C 端染色质结构域样模块对于反转录转座子 MAGGY 的高转座效率至关重要

• 发表时间: 2005
• 期刊: FEBS letters 579
• 作者: Nakayashiki;H.et al.
• 通讯作者: H.et al.

Speciation in Pyricularia inferred from multilocus phylogenetic analysis.

从多位点系统发育分析推断梨果属的物种形成。

• 发表时间: 2008
• 期刊: Mycol Res. 111
• 作者: Hirata;et. al.
• 通讯作者: et. al.

RNA silencing as a tool for exploring gene function in ascomycete fungi

• DOI: 10.1016/j.fgb.2005.01.002
• 发表时间: 2005-04-01
• 期刊: FUNGAL GENETICS AND BIOLOGY
• 影响因子: 3
• 作者: Nakayashiki, H;Hanada, S;Mayama, S
• 通讯作者: Mayama, S