Characterizing the Immune-Eliciting Activity of Putative Microbe-Associated Molecular Patterns in Tomato

  • Christopher R. ClarkeEmail author
  • Boris A. Vinatzer
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)


Detection of conserved microbe-associated molecular patterns (MAMPs), such as bacterial flagellin, is the first line of active defense in plants against pathogenic invaders. Successful pathogens must subvert this immune response to grow to high population density and cause disease. Flagellin from the bacterial pathogen Pseudomonas was the first identified bacterial MAMP and many species across the plant kingdom have sensitive perception systems for detecting the 22-amino acid epitope known as flg22. Tomato and several other solanaceous plants are also able to independently detect a second epitope of flagellin known as flgII-28. This chapter details four experimental protocols to identify and confirm the immune response-eliciting activity of flagellin and putative MAMPs with focus on the Pseudomonas–tomato pathosystem.

Key words

Microbe-associated molecular patterns (MAMPs) Flagellin flg22 flgII-28 Pseudomonas syringae Pattern-triggered immunity (PTI) 



Christopher Clarke is funded by a postdoctoral research fellowship from USDA-NIFA (2015-67012-22821) Work in the Vinatzer lab is funded by the NSF (IOS-1354215 and DEB-1241068). Funding to Vinatzer was also provided in part by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, US Department of Agriculture.


  1. 1.
    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
  2. 2.
    Kunze G et al (2004) The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16(12):3496–3507CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Felix G, Regenass M, Boller T (1993) Specific perception of subnanomolar concentrations of chitin fragments by tomato cells: induction of extracellular alkalinization, changes in protein phosphorylation, and establishment of a refractory state. Plant J 4(2):307–316CrossRefGoogle Scholar
  4. 4.
    Bittel P, Robatzek S (2007) Microbe-associated molecular patterns (MAMPs) probe plant immunity. Curr Opin Plant Biol 10(4):335–341CrossRefPubMedGoogle Scholar
  5. 5.
    Jones JDG, Dangl JL (2006) The plant immune system. Nature 444(7117):323–329CrossRefPubMedGoogle Scholar
  6. 6.
    Clarke CR et al (2013) Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility. New Phytol 200(3):847–860CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Sun W, Dunning FM, Pfund C, Weingarten R, Bent AF (2006) Within-species flagellin polymorphism in Xanthomonas campestris pv campestris and its impact on elicitation of Arabidopsis FLAGELLIN SENSING2-dependent defenses. Plant Cell 18(3):764–779CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Boller T, Felix G (2009) A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu Rev Plant Biol 60(1):379–406CrossRefPubMedGoogle Scholar
  9. 9.
    Zipfel C (2014) Plant pattern-recognition receptors. Trends Immunol 35(7):345–351CrossRefPubMedGoogle Scholar
  10. 10.
    Lacombe S et al (2010) Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial resistance. Nat Biotechnol 28(4):365–369CrossRefPubMedGoogle Scholar
  11. 11.
    Rosli HG et al (2013) Transcriptomics-based screen for genes induced by flagellin and repressed by pathogen effectors identifies a cell wall-associated kinase involved in plant immunity. Genome Biol 14(12):R139CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cai R et al (2011) The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity. PLoS Pathog 7(8):e1002130CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    McCann HC, Nahal H, Thakur S, Guttman DS (2012) Identification of innate immunity elicitors using molecular signatures of natural selection. Proc Natl Acad Sci 109(11):4215–4220CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Vinatzer BA, Monteil CL, Clarke CR (2014) Harnessing population genomics to understand how bacterial pathogens emerge, adapt to crop hosts, and disseminate. Annu Rev Phytopathol 52(1):19–43CrossRefPubMedGoogle Scholar
  15. 15.
    Adam L, Somerville SC (1996) Genetic characterization of five powdery mildew disease resistance loci in Arabidopsis thaliana. Plant J 9(3):341–356CrossRefPubMedGoogle Scholar
  16. 16.
    Chakravarthy S, Velásquez AC, Ekengren SK, Collmer A, Martin GB (2010) Identification of Nicotiana benthamiana genes involved in pathogen-associated molecular pattern–triggered immunity. Mol Plant Microbe Interact 23(6):715–726CrossRefPubMedGoogle Scholar
  17. 17.
    Melotto M, Underwood W, Koczan J, Nomura K, He SY (2006) Plant stomata function in innate immunity against bacterial invasion. Cell 126(5):969–980CrossRefPubMedGoogle Scholar
  18. 18.
    Zipfel C et al (2004) Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428(6984):764–767CrossRefPubMedGoogle Scholar
  19. 19.
    Mueller K et al (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
  20. 20.
    Segonzac C et al (2012) The shoot apical meristem regulatory peptide CLV3 does not activate innate immunity. Plant Cell 24(8):3186–3192CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Kvitko BH et al (2009) Deletions in the repertoire of Pseudomonas syringae pv. tomato DC3000 type III secretion effector genes reveal functional overlap among effectors. PLoS Pathog 5(4):e1000388CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Clarke CR, Hayes BW, Runde BJ, Wicker E, Vinatzer BA (2014) Eggplant and related species are promising genetic resources to dissect the plant immune response to Pseudomonas syringae and Xanthomonas euvesicatoria and to identify new resistance determinants. Mol Plant Pathol 15(8):814–822CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Plant Pathology, Physiology and Weed ScienceVirginia TechBlacksburgUSA

Personalised recommendations