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Automated Real-Time Monitoring of Extracellular pH to Assess Early Plant Defense Signaling

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Plant Peptide Hormones and Growth Factors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2731))

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Abstract

Extracellular alkalinization mediated by the inhibition of plasma membrane-located proton pumping ATPases hallmarks the initiation of defense signaling in plant cells. Early defense responses also include depolarization of the plasma membrane, increase in cytosolic Ca2+ concentration, and an oxidative burst. Together these early signaling events lead to the activation of plant immunity. The transient alkalinization response is triggered by well-studied pathogen-derived and plant endogenous elicitors, including, for example, bacterial flagellin, fungal chitin, and tomato systemin in both model and agronomic species. Employing cell suspension cultures, extracellular alkalinization can be easily assessed by measuring the elicitor-induced pH changes of the cultivating medium. Here, we provide a protocol for an improved alkalinization assay in a system which is able to simultaneously monitor multiple samples, and fully automatically transfer customizable real-time pH records. In this system flagellin, chitin and systemin elicit robust time- and dose-dependent responses, proving a powerful tool for assessing plant early defense signaling.

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References

  1. He Y, Zhou J, Shan L et al (2018) Plant cell surface receptor-mediated signaling – a common theme amid diversity. J Cell Sci 131. https://doi.org/10.1242/jcs.209353

  2. Saijo Y, Loo EP, Yasuda S (2018) Pattern recognition receptors and signaling in plant-microbe interactions. Plant J 93:592–613. https://doi.org/10.1111/tpj.13808

    Article  CAS  PubMed  Google Scholar 

  3. Bjornson M, Pimprikar P, Nürnberger T et al (2021) The transcriptional landscape of Arabidopsis thaliana pattern-triggered immunity. Nat Plants 7:579–586. https://doi.org/10.1038/s41477-021-00874-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 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 4:307–316. https://doi.org/10.1046/j.1365-313X.1993.04020307.x

    Article  CAS  Google Scholar 

  5. Felix G, Boller T (1995) Systemin induces rapid ion fluxes and ethylene biosynthesis in Lycopersicon peruvianum cells. Plant J 7:381–389. https://doi.org/10.1046/j.1365-313X.1995.7030381.x

    Article  CAS  Google Scholar 

  6. Felix G, Duran JD, Volko S et al (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18:165–276. https://doi.org/10.1046/j.1365-313x.1999.00265.x

    Article  Google Scholar 

  7. Schaller A, Oecking C (1999) Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants. Plant Cell 11:263–272. https://doi.org/10.1105/tpc.11.2.263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Jeworutzki E, Roelfsema MR, Anschutz U et al (2010) Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves ca-associated opening of plasma membrane anion channels. Plant J 62:367–378. https://doi.org/10.1111/j.1365-313X.2010.04155.x

    Article  CAS  PubMed  Google Scholar 

  9. Moroz N, Fritch KR, Marcec MJ et al (2017) Extracellular alkalinization as a defense response in potato cells. Front Plant Sci 8:32. https://doi.org/10.3389/fpls.2017.00032

    Article  PubMed  PubMed Central  Google Scholar 

  10. Haruta M, Sabat G, Stecker K et al (2014) A peptide hormone and its receptor protein kinase regulate plant cell expansion. Science 343:408–411. https://doi.org/10.1126/science.1244454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Haruta M, Gray WM, Sussman MR (2015) Regulation of the plasma membrane proton pump (H+-ATPase) by phosphorylation. Curr Opin Plant Biol 28:68–75. https://doi.org/10.1016/j.pbi.2015.09.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Haj Ahmad F, Wu X, Stintzi A et al (2019) The systemin signaling cascade as derived from time course analyses of the systemin-responsive phosphoproteome. Mol Cell Proteomics 18:1526–1542. https://doi.org/10.1074/mcp.RA119.001367

    Article  PubMed  PubMed Central  Google Scholar 

  13. Li X, Zhang J, Shi H et al (2022) Rapid responses: receptor-like kinases directly regulate the functions of membrane transport proteins in plants. J Int Plant Biol 64:1303–1309. https://doi.org/10.1111/jipb.13274

    Article  CAS  Google Scholar 

  14. Schaller A (1998) Action of proteolysis-resistant systemin analogues in wound signalling. Phytochemistry 47:605–612. https://doi.org/10.1016/S0031-9422(97)00523-2

    Article  CAS  PubMed  Google Scholar 

  15. Kunze G, Zipfel C, Robatzek S et al (2004) The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell 16:3496–3507. https://doi.org/10.1105/tpc.104.026765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Huffaker A, Pearce G, Ryan CA (2006) An endogenous peptide signal in Arabidopsis activates components of the innate immune response. Proc Natl Acad Sci U S A 103:10098–10103. https://doi.org/10.1073/pnas.0603727103

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zipfel C, Kunze G, Chinchilla D et al (2006) Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts agrobacterium-mediated transformation. Cell 125:749–760. https://doi.org/10.1016/j.cell.2006.03.037

    Article  CAS  PubMed  Google Scholar 

  18. Masachis S, Segorbe D, Turra D et al (2016) A fungal pathogen secretes plant alkalinizing peptides to increase infection. Nat Microbiol 1:16043. https://doi.org/10.1038/nmicrobiol.2016.43

    Article  CAS  PubMed  Google Scholar 

  19. Moroz N, Huffaker A, Tanaka K (2016) Extracellular alkalinization assay for the detection of early defense response. Curr Protoc Plant Biol 2(3):210–220. https://doi.org/10.1002/cppb.20057

    Article  CAS  Google Scholar 

  20. Stegmann M, Monaghan J, Smakowska-Luzan E et al (2017) The receptor kinase FER is a RALF-regulated scaffold controlling plant immune signaling. Science 355:287–289. https://doi.org/10.1126/science.aal2541

    Article  CAS  PubMed  Google Scholar 

  21. Chen Y-C, Siems WF, Pearce G et al (2008) Six peptide wound signals derived from a single precursor protein in Ipomoea batatas leaves activate the expression of the defense gene sporamin. J Biol Chem 283:11469–11476. https://doi.org/10.1074/jbc.M709002200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Chang X, Nick P (2012) Defence signalling triggered by Flg22 and Harpin is integrated into a different stilbene output in Vitis cells. PLoS One 7:e40446. https://doi.org/10.1371/journal.pone.0040446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Petre B, Hecker A, Germain H et al (2016) The poplar rust-induced secreted protein (RISP) inhibits the growth of the leaf rust pathogen Melampsora larici-Populina and triggers cell culture alkalinisation. Front Plant Sci 7:97. https://doi.org/10.3389/fpls.2016.00097

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

Our work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB1101 project D06) to Annick Stintzi and Andreas Schaller.

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Authors and Affiliations

  1. Department of Plant Physiology and Biochemistry, University of Hohenheim, Stuttgart, Germany

    Xu Wang, Rong Li, Annick Stintzi & Andreas Schaller

Authors
  1. Xu Wang
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  2. Rong Li
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  3. Annick Stintzi
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  4. Andreas Schaller
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Corresponding author

Correspondence to Andreas Schaller .

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Editors and Affiliations

  1. Department of Plant Physiology and Biochemistry, University of Hohenheim, Stuttgart, Baden-Württemberg, Germany

    Andreas Schaller

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Wang, X., Li, R., Stintzi, A., Schaller, A. (2024). Automated Real-Time Monitoring of Extracellular pH to Assess Early Plant Defense Signaling. In: Schaller, A. (eds) Plant Peptide Hormones and Growth Factors. Methods in Molecular Biology, vol 2731. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3511-7_13

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  • DOI: https://doi.org/10.1007/978-1-0716-3511-7_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3510-0

  • Online ISBN: 978-1-0716-3511-7

  • eBook Packages: Springer Protocols

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