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生物間相互作用に基づくマツ材線虫病発病メカニズムの統合的理解

山口, 莉未 YAMAGUCHI, Rimi ヤマグチ, リミ 九州大学

2020.03.23

概要

マツ材線虫病は,北米原産のマツノザイセンチュウ[Bursaphelenchus xylophilus (Steiner & Buhrer)Nickle](pinewood nematode; 以下,PWN)によって引き起こされる劇症型森林病害である。クロマツ(Pinus thunbergii Parl.)やアカマツ(P. densiflora Sieb. et Zucc.)に代表される日本のマツ類は,本病に強い感受性を示す。そのため,1971 年に病原体が特定されて以来,これまで 40 年以上に亘り枯死メカニズムに関する知見が集積されてきた。これらの知見により,本病の病徴進展は感染初期と病徴進展期の 2 段階に分類されることが示され,病徴進展期へ移行する際の現象である PWNの顕著な増殖がマツを枯死に至らしめる要因であると考えられた。しかし,樹体内における PWNの増殖を可能とする宿主と病原体双方の要因についての情報は不足しており,結果としてなぜマツは発病に至るのかは明確でない。本研究では,PWN に特に強い感受性を示すクロマツを対象として,宿主と病原体間の相互作用,および相互作用の発現に関わる環境条件を視野に入れた,マツ材線虫病の発病メカニズム解明に向けての基礎研究を分子生物学的手法により行った。

はじめに,TaqMan qPCR 法を用いることで,マツ樹体内における PWN 頭数を樹体から分離することなく直接的に定量できる手法を確立した。本手法を用いて,PWN 感染後のクロマツ実生苗における PWN 頭数と植物の防御関連遺伝子である感染特異的(pathogenesis-related, PR)遺伝子群の発現を時空間的に評価した。感染初期では PWN は侵入箇所近傍を中心に分布していたが,離れた箇所でも少数検出された。一方で,PR 遺伝子群は全身で強く発現上昇していた。その後,PWN 頭数と PR 遺伝子群の発現量は苗全身で顕著に増加し,全ての個体が枯死に至った。これらのことから,PWN の速やかな分散に伴ってマツの防御反応は全身で誘導されるが,この防御反応は PWN の増殖抑制に効果的ではなく,さらなる防御反応の結果枯死に至ると考えられた。

続いて,クロマツ抵抗性 2 クローンおよび感受性 1 クローンを対象に,宿主と病原体のトランスクリプトームを同時に取得する dual RNA-sequencing(RNA-seq)解析を行った。宿主側の遺伝子発現に関しては,抵抗性クローン間でも遺伝子発現全体の動きは異なっており,抵抗性および感受性クローン間の発現変動遺伝子として宿主の病原体認識,細胞壁の形成と生合成,およびその他の防御関連遺伝子に関連する遺伝子群が特定された。病原体側のエフェクター候補遺伝子に関しては,植物の細胞壁分解など移動・増殖に関与する遺伝子群,および抗酸化や植物の防御反応の操作と関連する遺伝子群の発現が認められた。さらに,植物の細胞死を誘導させうる遺伝子の発現も認められた。宿主と病原体双方の情報から,クロマツの抵抗性因子は複数存在し,PWN による細胞の物理的な破壊や,エフェクターの浸透・拡散の抑制に効果のあると考えられる細胞壁の強化が抵抗性に関連する可能性が示された。

さらに,宿主と病原体間の相互作用の発現に作用する誘因であり,特に変温動物である PWN の増殖速度に影響を及ぼすと考えられる温度が異なる条件下で接種実験を行うことで,マツ樹体内における PWN の増殖過程と病徴進展との関連性について検討した。その結果,PWN の増殖に好適な温度条件下では,PWN の増殖と病徴進展は有効積算温度によってよく説明されることが明らかとなった。しかし,PWN の増殖に好適な温度条件下でない場合には,PWN の増殖頭数はマツ個体間でばらつきが認められ,部分枯死もしくは枯死に至った個体は認められなかった。以上のことから,温度は PWN の増殖速度を変化させることにより,マツ材線虫病の病徴進展に影響を及ぼす要因であることが示された。すなわち,PWN 頭数がある閾値に達するまでの時間がクロマツの発病率に関連する可能性があると推察された。

本研究では,樹体内の PWN 頭数を高精度に定量する TaqMan qPCR 法や,宿主と病原体のトランスクリプトームを同時に取得する dual RNA-seq 法を初めてマツ材線虫病の研究に適用することで,樹体内における PWN の動態とマツの防御反応,あるいは病徴進展との関連性について新たな知見を得ることができた。本研究と先行研究によって得られた知見から,クロマツは,PWN 侵入後,PWNの速やかな分散に伴う様々なエフェクター分泌に対し,全身で細胞死を伴う防御反応を行うが,この防御反応は PWN の移動能や耐性の高さのためその活動抑制に効果的ではなく,結果として PWNは顕著に増殖しマツは枯死に至ると考えられた。ただし,PWN が顕著に増殖する以前に,細胞壁の強化など PWN の活動抑制に有効な防御反応が誘導された場合には,マツは枯死を免れる可能性があることが示唆された。

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