Analysis of PAMP-Triggered ROS Burst in Plant Immunity

  • Yuying Sang
  • Alberto P. MachoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)


The plant perception of pathogen-associated molecular patterns triggers a plethora of cellular immune responses. One of these responses is a rapid and transient burst of reactive oxygen species (ROS) mediated by plasma membrane-localized NADPH oxidases. The ROS burst requires a functional receptor complex and the contribution of several additional regulatory components. In laboratory conditions, the ROS burst can be detected a few minutes after the treatment with an immunogenic microbial elicitor. For these reasons, the elicitor-triggered ROS burst has been often exploited as readout to probe the contribution of plant components to early immune responses. Here, we describe a detailed protocol for the measurement of elicitor-triggered ROS burst in a simple, fast, and easy manner.

Key words

Reactive oxygen species ROS flg22 PAMP Immunity 



We thank Rosa Lozano-Durán for critical reading and suggestions on this manuscript. We also thank members of the Macho laboratory, past and present members of Cyril Zipfel’s group (The Sainsbury Laboratory, UK), and several other groups for their contribution to the optimization of this protocol over the years. Research in the Macho laboratory is supported by the Shanghai Center for Plant Stress Biology (Chinese Academy of Sciences), and the Chinese 1000 Talents Program.


  1. 1.
    Macho AP, Zipfel C (2014) Plant PRRs and the activation of innate immune signaling. Mol Cell 54:263–272CrossRefPubMedGoogle Scholar
  2. 2.
    Gilroy S, Suzuki N, Miller G, Choi WG, Toyota M, Devireddy AR, Mittler R (2014) A tidal wave of signals: calcium and ROS at the forefront of rapid systemic signaling. Trends Plant Sci 19(10):623–630CrossRefPubMedGoogle Scholar
  3. 3.
    Nathan C, Cunningham-Bussel A (2013) Beyond oxidative stress: an immunologist’s guide to reactive oxygen species. Nat Rev Immunol 13(5):349–361CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Suzuki N, Koussevitzky S, Mittler R, Miller G (2012) ROS and redox signalling in the response of plants to abiotic stress. Plant Cell Environ 35(2):259–270CrossRefPubMedGoogle Scholar
  5. 5.
    Marino D, Dunand C, Puppo A, Pauly N (2012) A burst of plant NADPH oxidases. Trends Plant Sci 17(1):9–15CrossRefPubMedGoogle Scholar
  6. 6.
    Kadota Y, Shirasu K, Zipfel C (2015) Regulation of the NADPH oxidase RBOHD during plant immunity. Plant Cell Physiol 56(8):1472–1480CrossRefPubMedGoogle Scholar
  7. 7.
    Nühse TS, Bottrill AR, Jones AME, Peck SC (2007) Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. Plant J 51(5):931–940CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhang J, Shao F, Li Y, Cui H, Chen L, Li H, Zou Y, Long C, Lan L, Chai J, Chen S, Tang X, Zhou J-M (2007) A Pseudomonas syringae effector inactivates MAPKs to suppress PAMP-induced immunity in plants. Cell Host Microbe 1(3):175–185CrossRefPubMedGoogle Scholar
  9. 9.
    Boutrot F, Segonzac C, Chang KN, Qiao H, Ecker JR, Zipfel C, Rathjen JP (2010) Direct transcriptional control of the Arabidopsis immune receptor FLS2 by the ethylene-dependent transcription factors EIN3 and EIL1. Proc Natl Acad Sci U S A 107(32):14502–14507CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hann DR, Dominguez-Ferreras A, Motyka V, Dobrev PI, Schornack S, Jehle A, Felix G, Chinchilla D, Rathjen JP, Boller T (2014) The Pseudomonas type III effector HopQ1 activates cytokinin signaling and interferes with plant innate immunity. New Phytol 201(2):585–598CrossRefPubMedGoogle Scholar
  11. 11.
    Macho AP, Boutrot F, Rathjen JP, Zipfel C (2012) ASPARTATE OXIDASE plays an important role in Arabidopsis stomatal immunity. Plant Physiol 159(4):1845–1856CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    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(5):423–429CrossRefPubMedGoogle Scholar
  13. 13.
    Monaghan J, Matschi S, Shorinola O, Rovenich H, Matei A, Segonzac C, Malinovsky FG, Rathjen JP, MacLean D, Romeis T, Zipfel C (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover. Cell Host Microbe 16(5):605–615CrossRefPubMedGoogle Scholar
  14. 14.
    Smith JM, Heese A (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae. Plant Methods 10(1):6CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18(3):265–276CrossRefPubMedGoogle Scholar
  16. 16.
    Mueller K, Chinchilla D, Albert M, Jehle AK, Kalbacher H, Boller T, Felix G (2012) Contamination risks in work with synthetic peptides: flg22 as an example of a pirate in commercial peptide preparations. Plant Cell 24(8):3193–3197CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lozano-Duran R, Bourdais G, He SY, Robatzek S (2014) The bacterial effector HopM1 suppresses PAMP-triggered oxidative burst and stomatal immunity. New Phytol 202(1):259–269CrossRefPubMedGoogle Scholar
  18. 18.
    Macho AP, Schwessinger B, Ntoukakis V, Brutus A, Segonzac C, Roy S, Kadota Y, Oh MH, Sklenar J, Derbyshire P, Lozano-Duran R, Malinovsky FG, Monaghan J, Menke FL, Huber SC, He SY, Zipfel C (2014) A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation. Science 343(6178):1509–1512CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.CAS Center for Excellence in Molecular Plant Sciences, Shanghai Center for Plant Stress BiologyShanghai Institutes of Biological Sciences, Chinese Academy of SciencesShanghaiChina

Personalised recommendations