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キク属植物におけるウイルス誘導性遺伝子サイレンシング系の確立に関する研究

村井 裕知 大阪府立大学 DOI:info:doi/10.24729/00017714

2022.07.05

概要

植物も我々動物と同じようにウイルスの感染宿主となり,病気を発症する.植物ウイルスによって引き起こされる病気によって,作物の収量低下・商品価値の低下が引き起こされ,年に300億ドルの経済的損失が生み出されている(Jones and Naidu, 2019).こうした植物ウイルスによる病害を抑制するために,形態的,生物学的,分子的などの様々な視点から植物ウイルス自体を理解するための研究がされてきた.なかでも,感染性cDNAクローンがbrome mosaic virus (BMV)で初めて作製されて以来(Ahlquist et al., 1984),様々な植物ウイルス種で感染性cDNAクローンが作製され(Boyer and Haenni, 1994),植物ウイルスの分子生物学的な実験が可能となった.それにより,植物ウイルスの遺伝子組み換えや変異体作製が容易に行えるようになり,ウイルスゲノムにコードされる遺伝子の機能解析が進んだ.例えば,キュウリモザイクウイルス(CMV)の移行タンパク質(MP)や外被タンパク質(CP)を欠失させた変異体は局部感染はするものの全身感染しなかった事から,ウイルスの移行に関与していることが示された(Suzuki et al., 1991).

感染性cDNAクローン技術の利用はこのようなウイルスの性質の解析に留まらず,外来遺伝子などの配列を植物ウイルスに導入して植物に接種することで,その遺伝子配列を植物に運ぶ技術へと発展していった.ウイルスは感染細胞で複製して隣接する細胞そして全身移行をするため,導入した遺伝子配列が増幅し植物体内に広がっていく.この性質を利用した技術を総じて植物ウイルスベクターと呼ぶ.導入する遺伝子配列の種類や,全長もしくは部分配列として導入するかによって,植物ウイルスベクターの用途は様々である(Fig.1-1).BMV感染性cDNAクローンが報告されて間もなく,植物ウイルスベクターに外来遺伝子を導入して植物細胞内で高発現を誘導し(Fig.1-1a),外来遺伝子高発現のプラットフォームとしての可能性が示唆された(French e tal., 1986).その後,数々の植物ウイルスおよび外来遺伝子の組み合わせで高発現ベクターが利用されてきた(Pogue et al., 2002).Icon Genetics社はタバコモザイクウイルス(TMV)を基にアグロインフィルトレーション接種のMagnifectionシステムを開発し(Marillonnet et al., 2004),現在ではMagnICONシステムと呼ばれる高発現系の植物ウイルスベクターの一つとして最も使用されている.また,これらのような外来遺伝子の高発現を目的とする利用方法だけでなく,生体内におけるウイルスの局在をマーキングする利用方法も開発された(Fig.1-1b).植物ウイルスベクターに緑色蛍光タンパク質(GFP)を導入することによって,植物ウイルス局在の継時的な観察が可能となり(Baulcombeetal.,1995),TMVの詳細な感染経路が明らかにされ,植物ウイルスの感染動態の解明に寄与した(Cheng et al., 2000).また,clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated (CRISPR-Casシステム)を利用したゲノム編集技術への応用も試みられている(Fig.1-1d).ガイドRNAを導入したタバコ茎えそウイルス(TRV)ベクターをCas9過剰発現Nicotiana benthamianaに接種することで,TRV感染細胞におけるゲノム編集が報告された(Alietal.,2015).さらに,Cas9とガイドRNAの両方を導入したbarley yellow striate mosaic virusベクターによるゲノム編集も報告され(Gaoetal.,2019),植物ウイルスベクターのゲノム編集への応用も日々改良が加えられている.上記のように,外来遺伝子の全長を発現させる用途とは異なり,宿主植物遺伝子の部分配列を導入する利用法も存在する(Fig.1-1c).当初,詳しいメカニズムは不明だったが,フィトエン不飽和化酵素遺伝子(PDS)部分配列を導入したTMVベクターがN.benthamianaに白化を誘導し(Kumagaietal.,1995),ウイルス誘導性遺伝子サイレンシング(VIGS)と呼ばれるようになった(vanKammen,1997).後に,VIGSは転写後遺伝子サイレンシング(PTGS)によって誘導されることが明らかになり,遺伝子機能解析への応用が提唱された(Baulcombe,1999).植物RNAウイルスが細胞で複製する際に形成される複製中間体2本鎖RNA(dsRNA)やゲノムRNAが高次構造を取り部分的に形成されたdsRNAがPTGSのトリガーとなる((Dalmay et al., 2000; Waterhouse et al., 1998).形成されたdsRNAは植物のDicerlikeenzymes(DCLs)によって20数塩基程のdsRNA,shortinterfering RNA(siRNA)へと切断される.siRNAはRNA-induced silencing complexes(RISC)に捕捉され,siRNAと相補的な配列を持つウイルスゲノムと結合し,RISCの主要な構成因子であるArgonauteタンパク質(AGO)がスライサー活性によってウイルスゲノムを分解する(Baulcombe, 2004; Bernstein et al., 2001; Hamilton and Baulcombe, 1999; Hammond et al., 2001).VIGSはこのPTGS機構を利用して遺伝子機能解析を行う.植物ウイルスベクターに宿主遺伝子の部分配列を導入し感染させることで,導入した部分配列に相補的な宿主遺伝子mRNAが分解され,表現型の変化が誘導される(Fig.1-2).VIGSは対象とする植物の遺伝子組み換えを必要としないため,遺伝子組み換えが困難な植物に有効な手法である.また,植物ウイルスベクターが植物体内で増殖する過程に依存しているため,早ければ数日,おおよそ数週間で表現型の変化を誘導することができ,アグロバクテリウム法や変異誘発などの従来法と比べて短期間で遺伝子機能解析が行える(Burch-Smithetal.,2004).遺伝子配列をウイルスベクターに導入するという性質上,標的とする遺伝子をあらかじめ設定することが可能であり,相同性が担保されれば,似た配列を持つ複数遺伝子を同時にサイレンシングすることもできる(Burch-Smithetal.,2004).

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