Advertisement

Stomatal Bioassay to Characterize Bacterial-Stimulated PTI at the Pre-Invasion Phase of Infection

  • Jeanine Montano
  • Maeli MelottoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)

Abstract

Bacterium-triggered stomatal closure is a functional output of plant immunity, also known as stomatal defense. This is an early response mediated by the recognition of pathogen-associated molecular patterns (PAMPs) by the plant’s pathogen recognition receptors (PRRs). Several approaches to analyzing stomatal movement in response to bacteria have been described, but difficulties in fine-tuning the experimental procedures still exist. Here we provide a detailed method for assessing stomatal defense via high-quality microscopic imaging and explain trouble-shooting steps to obtaining robust data. Although this procedure requires minimal manipulation of the leaf sample, it is crucial to control all environmental conditions and extrinsic variables that could interfere with stomatal movement. The method described here is also suitable for in vivo characterization of stomatal response in new pathosystems and can be used in conjunction with other profiling assays to gain a detailed understanding of early PAMP-triggered immunity (PTI).

Key words

Stomatal movement regulation Guard cell Plant–microbe interaction Bacterial pathogens PTI Stomatal defense 

Notes

Acknowledgments

Research on stomatal defense in the Melotto laboratory is supported by NIH grant 5R01AI068718, USDA-NIFA grant CA-D-PLS-2291-CG, and the Center for Produce Safety.

References

  1. 1.
    Melotto M, Underwood W, He SY (2008) Role of stomata in plant innate immunity and foliar bacterial diseases. Annu Rev Phytopathol 46:101–122CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Dees M, Lysoe E, Nordskog B, Brurberg M (2015) Bacterial communities associated with surfaces of leafy greens: shift in composition and decrease in richness over time. Appl Environ Microbiol 81:1530–1539CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Lee J, Teitzel GM, Munkvold K, de Pozo O, Martin GB, Michelmore RW, Greenberg JT (2012) Type III secretion and effectors shape the survival and growth pattern of Pseudomonas syringae on leaf surfaces. Plant Physiol 158:1803–1818CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Underwood W, Melotto M, He SY (2007) Role of plant stomata in bacterial invasion. Cell Microbiol 9:1621–1629CrossRefPubMedGoogle Scholar
  5. 5.
    Roy D, Panchal S, Rosa B, Melotto M (2013) Escherichia coli O157:H7 induces stronger plant immunity than Salmonella enterica Typhimurium SL1344. Phytopathology 103:326–332CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Zhang W, He SY, Assmann S (2008) The plant innate immunity response in stomatal guard cells invokes G-protein-dependent ion channel regulation. Plant J 56:984–996CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Nicaise V, Roux M, Zipfel C (2009) Recent advances in PAMP-triggered immunity against bacteria: pathogen recognition receptors watch over and raise the alarm. Plant Physiol 150:1638–1647CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Melotto M, Underwood W, Koczan J, Nomura K, He SY (2006) Plant stomata function in innate immunity against bacterial invasion. Cell 126:969–980CrossRefPubMedGoogle Scholar
  9. 9.
    Katagiri F, Thilmony R, He SY (2002) The Arabidopsis thaliana-Pseudomonas syringae interaction. In: Somerville CR, Meyerowitz EM (eds) The Arabidopsis book. American Society of Plant Biologists, Rockville, MD. doi: 10.1199/tab.0039 http://www.aspb.org/publications/arabidopsis Google Scholar
  10. 10.
    Golberg D, Kroupitski Y, Belausov E, Pinto R, Sela S (2011) Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs. Int J Food Microbiol 145:250–257CrossRefPubMedGoogle Scholar
  11. 11.
    Chitrakar R, Melotto M (2010) Assessing stomatal response to live bacterial cells using whole leaf imaging. J Vis Exp (44). doi:10.3791/2185Google Scholar
  12. 12.
    Schellenberg B, Ramel C, Dudler R (2010) Pseudomonas syringae virulence factor syringolin A counteracts stomatal immunity by proteasome inhibition. Mol Plant Microbe Interact 23:1287–1293CrossRefPubMedGoogle Scholar
  13. 13.
    Mustilli AC, Merlot S, Vavasseur A, Fenzi F, Giraudat J (2002) Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production. Plant Cell 14:3089–3099CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Plant SciencesUniversity of CaliforniaDavisUSA

Personalised recommendations