In Vitro Ubiquitination Activity Assays in Plant Immune Responses

Part of the Methods in Molecular Biology book series (MIMB, volume 1578)


Ubiquitination is a central posttranslational modification that impinges on the fate of proteins. While attachment of K48-linked chains onto soluble proteins marks them for proteolysis via the 26S proteasome, mono-ubiquitination or K63-linked chains result in the endocytosis and sorting through the endomembrane system of integral membrane proteins, such as pattern recognition receptors. In vitro ubiquitination assays allow the biochemical analysis of all individual components of the ubiquitination machinery and its potential substrates. Here, we describe how to reconstitute the ubiquitination cascade in vitro and detail different variations of the assay, the required controls and how to interpret the obtained results.

Key words

Ubiquitination Posttranslational modification Degradation 



The author would like to acknowledge the funding from the Leibniz Association and the state of Saxony-Anhalt.


  1. 1.
    Furlan G, Klinkenberg J, Trujillo M (2012) Regulation of plant immune receptors by ubiquitination. Front Plant Sci. 3:238. doi: 10.3389/fpls.2012.00238 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Bates PW, Vierstra RD (1999) UPL1 and 2, two 405 kDa ubiquitin-protein ligases from Arabidopsis thaliana related to the HECT-domain protein family. Plant J 20:183–195. doi: 10.1046/j.1365-313x.1999.00590.x CrossRefPubMedGoogle Scholar
  3. 3.
    Miao Y, Zentgraf U (2010) A HECT E3 ubiquitin ligase negatively regulates Arabidopsis leaf senescence through degradation of the transcription factor WRKY53. Plant J 63:179–188. doi: 10.1111/j.1365-313X.2010.04233.x CrossRefPubMedGoogle Scholar
  4. 4.
    Stone SL, Hauksdóttir H, Troy A, Herschleb J, Kraft E, Callis J (2005) Functional analysis of the RING-type ubiquitin ligase of Arabidopsis. Plant Physiol 137:13–30. doi: 10.1104/pp.104.052423 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Mudgil Y, Shiu S, Stone SL, Salt JN, Goring DR (2004) A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-Box E3 ubiquitin ligase family. Plant Physiol 134:59–66. doi: 10.1104/pp.103.029553 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Ohi MD, Vander Kooi CW, Rosenberg JA, Chazin WJ, Gould KL (2003) Structural insights into the U-box, a domain associated with multi-ubiquitination. Nat Struct Biol 10:250–255. doi: 10.1038/nsb906 CrossRefPubMedGoogle Scholar
  7. 7.
    Wiborg J, O’Shea C, Skriver K (2008) Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases. Biochem J 413:447–457. doi: 10.1042/BJ20071568 CrossRefPubMedGoogle Scholar
  8. 8.
    Kraft E, Stone SL, Ma L, Su N, Gao Y, Lau O, Deng X, Callis J (2005) Genome analysis and functional characterization of the E2 and RING-type E3 ligase ubiquitination enzymes of Arabidopsis. Plant Physiol 139:1597–1611. doi: 10.1104/pp.105.067983 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Göhre V, Spallek T, Häweker H, Mersmann S, Mentzel T, Boller T, de Torres M, Mansfield JW, Robatzek S (2008) Plant pattern-recognition receptor FLS2 is directed for degradation by the bacterial ubiquitin ligase AvrPtoB. Curr Biol 18:1824–1832. doi: 10.1016/j.cub.2008.10.063 CrossRefPubMedGoogle Scholar
  10. 10.
    Lu D, Lin W, Gao X, Wu S, Cheng C, Avila J, Heese A, Devarenne TP, He P, Shan L (2011) Direct ubiquitination of pattern recognition receptor FLS2 attenuates plant innate immunity. Science 332:1439–1442. doi: 10.1126/science.1204903 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Gimenez-Ibanez S, Hann DR, Ntoukakis V, Petutschnig E, Lipka V, Rathjen JP (2009) AvrPtoB targets the LysM receptor kinase CERK1 to promote bacterial virulence on plants. Curr Biol 19:423–429. doi: 10.1016/j.cub.2009.01.054 CrossRefPubMedGoogle Scholar
  12. 12.
    Udeshi ND, Mertins P, Svinkina T, Carr SA (2013) Large-scale identification of ubiquitination sites by mass spectrometry. Nat Protoc 8:1950–1960. doi: 10.1038/nprot.2013.120 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Hatfield PM, Gosink MM, Carpenter TB, Vierstra RD (1997) The ubiquitin-activating enzyme (E1) gene family in Arabidopsis thaliana. Plant J 11:213–226. doi: 10.1046/j.1365-313X.1997.11020213.x CrossRefPubMedGoogle Scholar
  14. 14.
    Wen R, Torres-Acosta JA, Pastushok L, Lai X, Pelzer L, Wang H, Xiao W (2008) Arabidopsis UEV1D promotes lysine-63-linked polyubiquitination and is involved in DNA damage response. Plant Cell 20:213–227. doi: 10.1105/tpc.107.051862 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Callis J (2014) The ubiquitination machinery of the ubiquitin system. Arabidopsis Book. doi: 10.1199/tab.0174 PubMedPubMedCentralGoogle Scholar
  16. 16.
    Kim HT, Kim KP, Lledias F, Kisselev AF, Scaglione KM, Skowyra D, Gygi SP, Goldberg AL (2007) Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages. J Biol Chem 282:17375–17386. doi: 10.1074/jbc.M609659200 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Leibniz Institute of Plant BiochemistryHalle (Saale)Germany

Personalised recommendations