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Novel MAP kinase substrates identified by solid-phase phosphorylation screening in Arabidopsis thaliana

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Abstract

Phosphorylation of substrate proteins by mitogen-activated protein kinases (MPKs) determines the specific cellular responses elicited by a particular extracellular stimulus. However, downstream targets of plant MPKs remain poorly characterized. In this study, 29 putative substrates of AtMPK3, AtMPK4 and AtMPK6 were identified by solid-phase phosphorylation screening of a λ phage expression library constructed from combined mRNAs from salt-treated, pathogen-treated and mechanically wounded Arabidopsis seedlings. To test the efficiency of this screening, we performed in vitro kinase assay with 10 recombinant fusion proteins. All proteins were phosphorylated by AtMPK3, AtMPK4 and AtMPK6, indicating the efficiency of this screening procedure. To confirm phosphorylation of isolated substrates by plant MPKs, we performed in-gel kinase assays. All test substrates were strongly phosphorylated by wounding or H2O2-activated AtMPK3 and AtMPK6. Three substrates, encoded by genes At2g41430, At2g41900, and At3g16770, were strongly phosphorylated, suggesting a function as AtMPK substrates. The type of screening provides a powerful way for identifying potential substrates of MAP kinases responsive to biotic and abiotic stresses.

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Abbreviations

MPK:

Mitogen-activated protein kinase

BWMK:

Blast- and wound-induced MAP kinase

GST:

Glutathione S-transferase

PAMP:

Pathogen-associated molecular pattern

PPPDB:

Plant protein phosphorylation database

MBP:

Myelin basic protein

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Acknowledgements

We thank Dr. Tony Hunter, Salk Institute, for the λGEX5 vector and the Arabidopsis Biological Resource Center (ABRC, Ohio State University) for providing atmpk6 mutant plants. This work was supported by grants from the Next-Generation BioGreen 21 Program (#PJ011091) funded by the Rural Development Administration, the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2016R1A2B4015859), and partly by Vietnam National Foundation (Grant No. 106-NN.02-2013.30) for Science and Technology Development (NAFOSTED).

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    Authors and Affiliations

    1. Division of Ecological Conservation, Bureau of Ecological Research, National Institute of Ecology, Geumgang-ro 1210, Seocheon, 33657, Republic of Korea

      Hyeong Cheol Park

    2. Faculty of Biotechnology, Vietnam National University of Agriculture, Hanoi, Vietnam

      Xuan Canh Nguyen

    3. Division of Applied Life Science (BK21 Plus PROGRAM), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Room No. 6-320, Jinju-daero 501, Jinju, 660-701, Republic of Korea

      Sunghwa Bahk, Byung Ouk Park, Min Chul Kim & Woo Sik Chung

    4. Plant Systems Engineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-806, Republic of Korea

      Ho Soo Kim

    5. Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

      Hans J. Bohnert

    6. College of Science, King Abdulaziz University, Jeddah, 21589, Kingdom of Saudi Arabia

      Hans J. Bohnert

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    1. Hyeong Cheol Park
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    Correspondence to Hyeong Cheol Park or Woo Sik Chung.

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    H. C. Park and X. C. Nguyen contributed equally to this work.

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    Park, H.C., Nguyen, X.C., Bahk, S. et al. Novel MAP kinase substrates identified by solid-phase phosphorylation screening in Arabidopsis thaliana . Plant Biotechnol Rep 10, 415–423 (2016). https://doi.org/10.1007/s11816-016-0412-9

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