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Transcriptional regulation of genes involved in the production and secretion of phytoalexins in Nicotiana benthamiana

RIN, Soriya 名古屋大学

2021.03.31

概要

高等植物は、病原微生物の攻撃を受けた際に、病原菌の認識、細胞内情報伝達の活性化、抵抗性実行因子の遺伝子発現誘導とその活性化を経て病害抵抗性を発揮している。植物の抵抗性機構は、植物と病原菌の組み合わせによって多様であり、効果的な病害抵抗性応答は標的となる病原菌の持つ感染戦略によって大きく異なることが知られている。ジャガイモ疫病菌Phytophthora infestansは、世界4大作物であるジャガイモの最重要病害の1つである。ジャガイモを始めとするナス科植物の疫病菌抵抗性の分子機構については依然不明な点が多く、その解明は植物感染生理分野における重要研究課題である。

本学位論文では、ナス科のモデル植物であるベンサミアナタバコのジャガイモ疫病菌に対する抵抗性に必須な遺伝子群の発現制御機構について解析を行った。ベンサミアナタバコを含むNicotiana属植物は、ジャガイモ疫病菌に対して安定した抵抗性を示す。これまでの研究で、ベンサミアナタバコの生産するセスキテルペノイドの抗菌物資(ファイトアレキシン)が、ジャガイモ疫病菌に対する抵抗性に必須であることが明らかとなっている。そこで本研究では、Nicotiana属植物の主要なファイトアレキシンであるカプシジオールの生合成に関わる遺伝子群の病害抵抗性応答時における発現プロファイルを詳細に調べた。Nicotiana属植物において、カプシジオールはメバロン酸経路の7つの酵素およびファルネシル二リン酸合成酵素(FPPS)によって生産されるファルネシル二リン酸(FPP)を前駆体として、カプシジオール生産のための2つ酵素5-epi-aristolochene synthase(EAS)および5-epi-aristolochene dihydroxylase(EAH)による反応を経て生産される。ベンサミアナタバコのゲノムからはメバロン酸経路のそれぞれの酵素の遺伝子がいずれも複数(2-6コピー)見出され、それぞれの酵素遺伝子の一部あるいは全てが、ジャガイモ疫病菌の感染やジャガイモ疫病菌由来の分泌タンパク質であるINF1の処理によって発現誘導された。カプシジオール生産に特異的に機能するEASおよびEAHはそれぞれ10および6コピーが見出され、全ての遺伝子がジャガイモ疫病菌の感染に応答して発現誘導された。これらEASおよびEAHはゲノム中で相同遺伝子がタンデムに存在する例が見出されたことから、進化の過程で重複による遺伝子数の増加が起こり、ファイトアレキシンの生産性の向上に寄与したと推察された。

次に、ベンサミアナタバコの病害抵抗性誘導に中心的な役割を担うMAPキナーゼであるWIPK、SIPKおよびNTF4のファイトアレキシン生成における役割を調査した。ウイルス誘導型遺伝子サイレンシング(VIGS)法を用いて、ベンサミアナタバコのWIPK/SIPK/NTF4遺伝子の発現抑制を行なったところ、INF1の処理に応答したカプシジオール、デブネオール、カプシジオールアセテートなどのファイトアレキシン生成が顕著に抑制された。そこで、WIPK/SIPK/NTF4遺伝子の発現抑制を行なったベンサミアナタバコにおけるファイトアレキシン生成酵素遺伝子群の発現パターンを調査した。その結果、メバロン酸経路の酵素およびFPPSの発現はWIPK/SIPK/NTF4遺伝子の発現抑制への影響を殆ど受けなかったのに対して、カプシジオール生産特異的酵素をコードする6コピーのEAH遺伝子は全てMAPキナーゼの制御を受けていることが示された。この結果から、ファイトアレキシンの前駆体生成に関わる酵素遺伝子群とカプシジオール生成に特異的に機能す酵素遺伝子群が、病害抵抗性誘導時にいずれも発現誘導されるものの、その制御機構は異なる可能性が示された。

次に病害抵抗性誘導時に活性化されるカプシジオール分泌に関与するトランスポーター遺伝子NbABCG2aの発現制御機構を調べるため、そのプロモーター配列の解析を行った。NbABCG2aプロモーターに部分欠損や変異を導入し、その活性への影響をGFPの発現を指標として解析した。発現の程度が低下した変異プロモーターの配列解析から、病害抵抗性応答時に必要ないくつかのシス配列が特定された。特にエチレンへの応答に必須であるGCCbox、WRKY転写制御因子の結合配列であるW-boxがNbABCG2aプロモーターの病害抵抗性誘導時の活性化に必須であることが明らかとなった。また、エチレン応答に関与するEIN2遺伝子のサイレンシングによって、ベンサミアナの抵抗性が著しく低下し、特に病原菌の細胞への侵入頻度が高まることが示された。

本研究ではこれまで詳細が明らかにされていなかった、ナス科植物における抗菌物質生産に関する酵素遺伝子群の発現制御のメカニズムの一端を解明した。今後、これら遺伝子が独立あるいは一部共通した機構を介して発現制御されているその全容が明らかとなれば、モデル植物であるベンサミアナの病害抵抗性応答だけではなく、様々な重要作物を含むナス科植物の病害抵抗性機構の全容の解明につながる研究に発展できる可能性があり、植物科学の研究進展にも大きく貢献できると考えられる。

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