A Fluorescence Anisotropy-Based Comprehensive Method for the In Vitro Screening of COI1–JAZs Agonists and Antagonists

  • Yousuke Takaoka
  • Kengo Hayashi
  • Kaho Suzuki
  • Ika Nurul Azizah
  • Minoru UedaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2085)


The phytohormone (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile) causes protein–protein interactions (PPI) between F-box Protein CORONATINE INSENSITIVE 1 (COI1) and JASMONATE ZIM DOMAIN (JAZ) transcriptional repressor. A total of 13 JAZ subtypes are encoded in the genome of Arabidopsis thaliana; however, their genetic redundancy obfuscates the individual function of each JAZ. One approach to decipher this redundant signaling network is chemical genetics, using small molecules specific to individual JAZ subtype, for which a reliable and high-throughput screening system of the ligands for all combinations of COI1–JAZs would be indispensable. In this chapter, we describe a fluorescence anisotropy-based quantitative screening system for the ligands of COI1–JAZ co-receptors. Our method is applicable to agonists and antagonists of the COI1–JAZs.

Key words

COI1-JAZ co-receptor Ligand screening Affinity Fluorescence anisotropy JAZ degron peptide Protein–protein interaction 



This work was supported by a Grant-in-Aid for Scientific Research (no. 26282207, 17H06407, 17H00885 to MU, and No. 25708026, 18H02101 to Y.T.), JSPS A3 Foresight Program (to MU), JSPS Core-to-core Program ‘Asian Chemical Biology Initiative’ (to MU), and JST PREST (JPMJPR16Q4 to YT).


  1. 1.
    Santner A, Estelle M (2009) Recent advances and emerging trends in plant hormone signalling. Nature 459(7250):1071–1078CrossRefGoogle Scholar
  2. 2.
    Chini A, Fonseca S, Fernandez G, Adie B, Chico JM, Lorenzo O, Garcia-Casado G, Lopez-Vidriero I, Lozano FM, Ponce MR, Micol JL, Solano R (2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448(7154):666–671CrossRefGoogle Scholar
  3. 3.
    Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K, He SY, Howe GA, Browse J (2007) JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling. Nature 448(7154):661–665CrossRefGoogle Scholar
  4. 4.
    Yan J, Zhang C, Gu M, Bai Z, Zhang W, Qi T, Cheng Z, Peng W, Luo H, Nan F, Wang Z, Xie D (2009) The Arabidopsis CORONATINE INSENSITIVE1 protein is a jasmonate receptor. Plant Cell 21(8):2220–2236CrossRefGoogle Scholar
  5. 5.
    Campos ML, Yoshida Y, Major IT, de Oliveira Ferreira D, Weraduwage SM, Froehlich JE, Johnson BF, Kramer DM, Jander G, Sharkey TD, Howe GA (2016) Rewiring of jasmonate and phytochrome B signalling uncouples plant growth-defense tradeoffs. Nat Commun 7:12570CrossRefGoogle Scholar
  6. 6.
    Dejonghe W, Russinova E (2017) Plant chemical genetics: from phenotype-based screens to synthetic biology. Plant Physiol 174(1):5–20CrossRefGoogle Scholar
  7. 7.
    Toth R, van der Hoorn RAL (2010) Emerging principles in plant chemical genetics. Trends Plant Sci 15(2):81–88CrossRefGoogle Scholar
  8. 8.
    Takaoka Y, Iwahashi M, Chini A, Saito H, Ishimaru Y, Egoshi S, Kato N, Tanaka M, Bashir K, Seki M, Solano R, Ueda M (2018) A rationally designed JAZ subtype-selective agonist of jasmonate perception. Nat Commun 9(1):3654CrossRefGoogle Scholar
  9. 9.
    Monte I, Hamberg M, Chini A, Gimenez-Ibanez S, Garcia-Casado G, Porzel A, Pazos F, Boter M, Solano R (2014) Rational design of a ligand-based antagonist of jasmonate perception. Nat Chem Biol 10(8):671–676CrossRefGoogle Scholar
  10. 10.
    Matiolli CC, Melotto M (2018) A comprehensive Arabidopsis yeast two-hybrid library for protein-protein interaction studies: a resource to the plant research community. Mol Plant-Microbe Interact 31(9):899–902CrossRefGoogle Scholar
  11. 11.
    Yan J, Yao R, Chen L, Li S, Gu M, Nan F, Xie D (2018) Dynamic perception of Jasmonates by the F-box protein COI1. Mol Plant 11(10):1237–1247CrossRefGoogle Scholar
  12. 12.
    Burger M, Mashiguchi K, Lee HJ, Nakano M, Takemoto K, Seto Y, Yamaguchi S, Chory J (2019) Structural basis of Karrikin and non-natural Strigolactone perception in Physcomitrella patens. Cell Rep 26(4):855–865CrossRefGoogle Scholar
  13. 13.
    Milroy LG, Grossmann TN, Hennig S, Brunsveld L, Ottmann C (2014) Modulators of protein-protein interactions. Chem Rev 114(9):4695–4748CrossRefGoogle Scholar
  14. 14.
    Takaoka Y, Nagumo K, Azizah IN, Oura S, Iwahashi M, Kato N, Ueda M (2019) A comprehensive in vitro fluorescence anisotropy assay system for screening ligands of the jasmonate COI1-JAZ co-receptor in plants. J Biol Chem 294(13):5074–5081CrossRefGoogle Scholar
  15. 15.
    Labbe CM, Laconde G, Kuenemann MA, Villoutreix BO, Sperandio O (2013) iPPI-DB: a manually curated and interactive database of small non-peptide inhibitors of protein-protein interactions. Drug Discov Today 18(19-20):958–968CrossRefGoogle Scholar
  16. 16.
    Sheard LB, Tan X, Mao H, Withers J, Ben-Nissan G, Hinds TR, Kobayashi Y, Hsu FF, Sharon M, Browse J, He SY, Rizo J, Howe GA, Zheng N (2010) Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468(7322):400–405CrossRefGoogle Scholar
  17. 17.
    Chini A, Gimenez-Ibanez S, Goossens A, Solano R (2016) Redundancy and specificity in jasmonate signalling. Curr Opin Plant Biol 33:147–156CrossRefGoogle Scholar
  18. 18.
    Thireault C, Shyu C, Yoshida Y, St Aubin B, Campos ML, Howe GA (2015) Repression of jasmonate signaling by a non-TIFY JAZ protein in Arabidopsis. Plant J 82(4):669–679CrossRefGoogle Scholar
  19. 19.
    Mizusawa K, Takaoka Y, Hamachi I (2012) Specific cell surface protein imaging by extended self-assembling fluorescent turn-on nanoprobes. J Am Chem Soc 134(32):13386–13395CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Yousuke Takaoka
    • 1
    • 2
  • Kengo Hayashi
    • 1
  • Kaho Suzuki
    • 1
  • Ika Nurul Azizah
    • 1
  • Minoru Ueda
    • 1
    • 3
    Email author
  1. 1.Department of Chemistry, Graduate School of ScienceTohoku UniversitySendaiJapan
  2. 2.Precursory Research for Embryonic Science and Technology (PREST)Japan Science and Technology AgencyTokyoJapan
  3. 3.Department of Molecular and Chemical Life Sciences, Graduate School of Life SciencesTohoku UniversitySendaiJapan

Personalised recommendations