NADPHオキシダーゼ活性の時空間的定量法の確立

• Project Area: Oxygen biology: a new criterion for integrated understanding of life
• Project/Area Number: 15H01398
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Nagoya University
• Principal Investigator: 吉岡 博文 名古屋大学, 生命農学研究科, 准教授 (30240245)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥13,000,000 (Direct Cost: ¥10,000,000、Indirect Cost: ¥3,000,000); Fiscal Year 2016: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2015: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
• Keywords: 活性酸素 / 植物免疫 / MAPキナーゼ / シグナル伝達 / リン酸化酵素

Outline of Annual Research Achievements

ベンサミアナタバコ (Nicotiana benthamiana) においては、NbRBOHBが病害応答性CDPKやRLCKによってリン酸化され、ROS生産を誘導する。本年度は、２種の蛍光タンパク質 (YFP、CFP)、NbRBOHB由来のリン酸化配列を用いて、CDPKまたはRLCKによるリン酸化によって生ずる構造の変化に伴って蛍光共鳴エネルギー移動 (Foster Resonance Energy Transfer: FRET) が起こる２つのセンサーの基本構造を構築し、FRET効率の最適化を図った。
FRETシステムは、センサーのリン酸化時と非リン酸化時で異なる波長の蛍光として観察されるため、CDPKまたはRLCKの活性化を生細胞で蛍光として観察できる。２遺伝子発現型バイナリーベクターをベースとして、作製したセンサーとエフェクターをそれぞれ35Sプロモーターの下流に挿入した。エフェクターには、病害応答性CDPKの恒常活性型変異体 (NbCDPK5VK) および非活性型変異体 (NbCDPK5VK-KM) を用いた。これらバイナリーベクターを、アグロインフィルタレーション法によってベンサミアナタバコに導入して発現させ、葉組織から粗抽出液を調製し、マルチプレートリーダーを用いてFRET蛍光 (YFP/CFP) の強度を評価した。しかし、現在までに良好なFRET蛍光は得られていない。そこで、NbRBOHB遺伝子の転写を制御するWRKY8転写因子を同定し、この転写因子を活性化するMAPKの活性化を可視化することを可能にしたMAPKバイオセンサーの開発に成功した。このセンサーをベンサミアナタバコに一過的に導入して発現させた結果、病原菌の感染シグナルに応答したFRET蛍光が観察された。

Research Progress Status

28年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

28年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] LIPM(フランス)
• [Journal Article] An NADPH oxidase RBOH functions in rice roots during lysigenous aerenchyma formation under oxygen-deficient conditions. (2017)
  • Author(s): Yamauchi, T., Yoshioka, M., Fukazawa, A., Mori, H., Nishizawa, N.K., Tsutsumi, N., Yoshioka, H. and Nakazono, M.
  • Journal Title: The Plant Cell Volume: 印刷中 Issue: 4 Pages: 775-790
  • DOI: 10.1105/tpc.16.00976
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] 植物免疫における活性酸素生成誘導のしくみ―植物はいかにして病原菌から身を護るのか？ (2017)
  • Author(s): 安達広明・吉岡博文
  • Journal Title: 化学と生物 Volume: 印刷中
  • NAID: 130007437458
• [Journal Article] SGT1 contributes to maintaining protein levels of MEK2DD to facilitate hypersensitive response-like cell death in Nicotiana benthamiana (2016)
  • Author(s): Kazuya Ichimura, Takehito Shinzato, Miki Edaki, Hirofumi Yoshioka, Ken Shirasu
  • Journal Title: Physiological and Molecular Plant Pathology Volume: 94 Pages: 47-52
  • DOI: 10.1016/j.pmpp.2016.04.001
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Hierarchical regulation of NADPH oxidase by protein kinases in plant immunity (2016)
  • Author(s): Yoshioka H., Adachi H., Nakano T., Miyagawa N., Asai S., Ishihama N., and Yoshioka M.
  • Journal Title: Physiological and Molecular Plant Pathology Volume: in press Pages: 20-26
  • DOI: 10.1016/j.pmpp.2016.03.004
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Nicotiana benthamiana MAPK-WRKY pathway confers resistance to a necrotrophic pathogen Botrytis cinerea. (2016)
  • Author(s): Adachi, H., Ishihama, N., Nakano, T., Yoshioka, M. and Yoshioka, H.
  • Journal Title: Plant Signaling & Behavior Volume: 11 Issue: 6 Pages: e1183085-e1183085
  • DOI: 10.1080/15592324.2016.1183085
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Nicotiana benthamiana MAPK-WRKY pathway confers resistance to a necrotrophic pathogen Botrytis cinerea. (2016)
  • Author(s): Adachi, H., Ishihama, N., Nakano, T., Yoshioka, M. and Yoshioka, H. (2016) Nicotiana benthamiana MAPK-WRKY pathway confers resistance to a necrotrophic pathogen Botrytis cinerea.
  • Journal Title: Plant Signal. Behav. Volume: in press
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A receptor pair with an integrated decoy converts pathogen disabling of transcription factors to immunity. (2015)
  • Author(s): Le Roux, C., Huet, G., Jauneau, A., Camborde, L., Tremousaygue, D., Kraut, A., Zhou, B., Levaillant, M., Adachi, H., Yoshioka, H., Raffaele, S., Berthome, R., Coute, Y., Parker, J.E. and Deslandes, L.
  • Journal Title: Cell Volume: 161 Issue: 5 Pages: 1074-1088
  • DOI: 10.1016/j.cell.2015.04.025
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Cell death-inducing stresses are required for defense activation in DS1-phosphatidic acid phosphatase-silenced Nicotiana benthamiana. (2015)
  • Author(s): Masahito Nakano, Hirofumi Yoshioka, Kouhei Ohnishi, Yasufumi Hikichi, Akinori Kiba
  • Journal Title: Journal of Plant Physiology Volume: 184 Pages: 15-19
  • DOI: 10.1016/j.jplph.2015.06.007
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Molecular characterization of LjABCG1, an ATP-binding cassette protein in Lotus japonicus. (2015)
  • Author(s): Sugiyama, A., Fukuda, S., Takanashi, K., Yoshioka, M., Yoshioka, H., Narusaka, Y., Narusaka, M., Kojima, M., Sakakibara, H., Shitan, N., Sato, S., Tabata, S., Kawaguchi, M. and Yazaki, K.
  • Journal Title: PLoS ONE Volume: 10 Issue: 9 Pages: e0139127-e0139127
  • DOI: 10.1371/journal.pone.0139127
  • NAID: 120005715333
  • Peer Reviewed / Open Access
• [Journal Article] WRKY transcription factors phosphorylated by MAPK regulate a plant immune NADPH oxidase in Nicotiana benthamiana. (2015)
  • Author(s): Adachi, H., Nakano, T., Miyagawa, N., Ishihama, N., Yoshioka, M., Katou, Y., Yaeno, T., Shirasu, K. and Yoshioka, H.
  • Journal Title: Plant Cell Volume: 27 Issue: 9 Pages: 2645-2663
  • DOI: 10.1105/tpc.15.00213
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] リン酸化反応が制御するROSバーストの分子機構． (2015)
  • Author(s): 吉岡博文・安達広明・石濱伸明・中野孝明・白石佑太郎・宮川典子・野村裕也・吉岡美樹・浅井秀太
  • Journal Title: 日本植物病理学会報 Volume: 81 Pages: 1-8
  • NAID: 130005129474
  • Peer Reviewed
• [Journal Article] Kinase-mediated orchestration of NADPH oxidase in plant immunity. (2015)
  • Author(s): Hiroaki Adachi and Hirofumi Yoshioka
  • Journal Title: Brief. Funct. Genomics Volume: 14 Issue: 4 Pages: 253-259
  • DOI: 10.1093/bfgp/elv004
  • Peer Reviewed
• [Presentation] Live-imaging of MAPK activity in plant immune responses (2017)
  • Author(s): 安達広明・石濱伸明・吉岡美樹・鳴坂真理・鳴坂義弘・吉岡博文
  • Organizer: 第58回日本植物生理学会年会
  • Place of Presentation: 鹿児島大学、鹿児島
  • Year and Date: 2017-03-16
• [Presentation] 植物免疫に関与するMAPキナーゼによる活性酸素生成機構 (2017)
  • Author(s): 吉岡博文
  • Organizer: 第４回新学術領域研究「酸素生物学」公開シンポジウム
  • Place of Presentation: 九州大学
  • Invited
• [Presentation] 免疫応答におけるSIPKの細胞内動態 (2016)
  • Author(s): 安達広明、波多江健太、吉岡美樹、鳴坂真理、鳴坂義弘、吉岡博文
  • Organizer: 平成28年度日本植物病理学会関西部会
  • Place of Presentation: 静岡県コンベンションアーツセンター，静岡
  • Year and Date: 2016-09-28
• [Presentation] Phosphorylation of rice RBOH by CERK1-associated RLCK mediates chitin-triggered ROS burst. (2016)
  • Author(s): Yoshioka, M., Yamaguchi, K., Fujiwara, M., Yoshimura, S., Kawasaki, T. and Yoshioka, H.
  • Organizer: XVII International Congress on Molecular Plant-Microbe Interactions.
  • Place of Presentation: Portland
  • Year and Date: 2016-07-17
  • Int'l Joint Research
• [Presentation] 葉緑体による活性酸素生成を介した植物免疫機構.「活性酸素が支える生体防御力」 (2016)
  • Author(s): 吉岡博文・安達広明
  • Organizer: 第27回日本生体防御学会学術総会
  • Place of Presentation: 九州大学
  • Year and Date: 2016-07-07
  • Invited
• [Presentation] Protein kinases hierarchically regulate a plant immune NADPH oxidase (2016)
  • Author(s): Yoshioka, H
  • Organizer: The 9th International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide
  • Place of Presentation: 仙台国際センター、仙台
  • Year and Date: 2016-05-20
  • Int'l Joint Research / Invited
• [Presentation] 植物免疫に関わるNADPH oxidaseの活性化機構 (2015)
  • Author(s): 吉岡博文
  • Organizer: 第38回日本分子生物学会年会、第88回日本生化学会大会合同大会
  • Place of Presentation: 神戸ポートピアホテル
  • Year and Date: 2015-12-01
  • Invited
• [Presentation] Protein kinase-mediated regulation of NADPH oxidase in plant immunity. (2015)
  • Author(s): Yoshioka, H., Adachi, H., Nakano, T., Miyagawa, N., Asai, S., Ishihama, N. and Yoshioka, M.
  • Organizer: 11th US-Japan Scientific Seminar
  • Place of Presentation: 香川
  • Year and Date: 2015-10-26
  • Int'l Joint Research / Invited
• [Presentation] MAPK-WRKY pathway up-regulates NbRBOHB gene and contributes to effector-triggered ROS burst in Nicotiana benthamiana. (2015)
  • Author(s): Adachi, H., Nakano, T., Miyagawa, N., Ishihama, N., Yoshioka, M., Katou, Y., Yaeno, T., Shirasu, K. and Yoshioka, H.
  • Organizer: The 4th International Conference on Biotic Plant Interactions.
  • Place of Presentation: 中国
  • Year and Date: 2015-08-26
  • Int'l Joint Research
• [Presentation] Transactivation of NbRBOHB gene via MAPK-WRKY pathway is essential for effector-triggered ROS burst in Nicotiana benthamiana. (2015)
  • Author(s): Adachi, H., Ishihama, N., Nakano, T., Miyagawa, N., Yoshioka, M., Yaeno, T., Shirasu, K. and Yoshioka, H.
  • Organizer: 12th International Conference on Reactive Oxygen and Nitrogen Species in Plants: from model systems to field.
  • Place of Presentation: イタリア
  • Year and Date: 2015-05-24
  • Int'l Joint Research
• [Presentation] RLCK-mediated phosphorylation of the N-terminus of RBOH regulates ROS burst in rice. (2015)
  • Author(s): Yoshioka, M., Yamaguchi, K., Fujiwara, M., Yoshimura, S., Kawasaki, T. and Yoshioka, H.
  • Organizer: 12th International Conference on Reactive Oxygen and Nitrogen Species in Plants: from model systems to field.
  • Place of Presentation: イタリア
  • Year and Date: 2015-05-24
  • Int'l Joint Research
• [Remarks]
  • URL: http://www.agr.nagoya-u.ac.jp/%7ebio4283/index.html
• [Remarks]
  • URL: http://www.agr.nagoya-u.ac.jp/~bio4283/index.html