Skip to main content
SpringerLink
Log in

Detection and Analyses of Endocytosis of Plant Receptor Kinases

  • Protocol
  • First Online:
Plant Receptor Kinases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1621))

  • 1529 Accesses

Abstract

Genetic dissection has led to a sophisticated understanding of receptor kinases in plant development and responses to abiotic and biotic stresses. Fluorescence confocal microscopy is essential to identify the (sub)cellular locations of resting and signaling receptor kinases that trigger molecular events in plant cells upon ligand perception. In this regard, the internalization of plasma membrane-localized FLAGELLIN SENSING 2 (FLS2) into endosomes induced by its ligand flg22, a peptide derived from bacterial flagellin, is a model system for studying activation status-dependent and endosomal receptor kinase trafficking routes and can be used in screens to identify pathogen effectors that target these trafficking routes for virulence promotion. In this chapter we describe approaches of visualizing fluorescently tagged FLS2, including protocols for flg22-induced endocytosis, instrument parameters, and image analysis. These approaches can be easily adapted for other receptor kinases, using the fast transient expression system in Nicotiana benthamiana for microscopic inspection.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 159.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. 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):379–406

    Article  CAS  PubMed  Google Scholar 

  2. Zipfel C, Robatzek S, Navarro L, Oakeley EJ, Jones JDG, Felix G, Boller T (2004) Bacterial disease resistance in Arabidopsis through flagellin perception. Nature 428(6984):764–767

    Article  CAS  PubMed  Google Scholar 

  3. Chinchilla D (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448:497–500

    Article  CAS  PubMed  Google Scholar 

  4. Beck M, Zhou J, Faulkner C, MacLean D, Robatzek S (2012) Spatio-temporal cellular dynamics of the arabidopsis flagellin receptor reveal activation status-dependent endosomal sorting. Plant Cell 24(10):4205–4219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Frescatada-Rosa M, Robatzek S, Kuhn H (2015) Should I stay or should I go? Traffic control for plant pattern recognition receptors. Curr Opin Plant Biol 28:23–29

    Article  CAS  PubMed  Google Scholar 

  6. Liebrand TWH, van den Berg GCM, Zhang Z, Smit P, Cordewener JHG, America AHP, Sklenar J, Jones AME, Tameling WIL, Robatzek S, Thomma BPHJ, Joosten MHAJ (2013) Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection. Proc Natl Acad Sci U S A 110(24):10010–10015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Peng K-C, Wang C-W, Wu C-H, Huang C-T, Liou R-F (2015) Tomato SOBIR1/EVR homologs are involved in elicitin perception and plant defense against the oomycete pathogen Phytophthora parasitica. Mol Plant-Microbe Interact 28(8):913–926

    Article  CAS  PubMed  Google Scholar 

  8. Mbengue M, Bourdais G (2016) Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases. Proc Natl Acad Sci U S A 113(39):11034–11039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. TO B, Belhaj K, Dagdas YF, Chaparro-Garcia A, Wu C-H, Cano LM, Kamoun S (2015) Rerouting of plant late endocytic trafficking toward a pathogen interface. Traffic 16(2):204–226

    Article  Google Scholar 

  10. Postma J, Liebrand TWH, Bi G, Evrard A, Bye RR, Mbengue M, Kuhn H, Joosten MHAJ, Robatzek S (2016) Avr4 promotes Cf-4 receptor-like protein association with the BAK1/SERK3 receptor-like kinase to initiate receptor endocytosis and plant immunity. New Phytol 210(2):627–642

    Article  CAS  PubMed  Google Scholar 

  11. TO B, Schornack S, Win J, Shindo T, Ilyas M, Oliva R, Cano LM, Jones AME, Huitema E, van der Hoorn RAL, Kamoun S (2011) Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface. Proc Natl Acad Sci U S A 108(51):20832–20837

    Article  Google Scholar 

  12. Chaparro-Garcia A, Schwizer S, Sklenar J, Yoshida K, Petre B, Bos JIB, Schornack S, Jones AME, TO B, Kamoun S (2015) Phytophthora infestans RXLR-WY effector AVR3a associates with dynamin-related protein 2 required for endocytosis of the plant pattern recognition receptor FLS2. PLoS One 10(9):e0137071

    Article  PubMed  PubMed Central  Google Scholar 

  13. Dagdas YF, Belhaj K, Maqbool A, Chaparro-Garcia A, Pandey P, Petre B, Tabassum N, Cruz-Mireles N, Hughes RK, Sklenar J, Win J, Menke F, Findlay K, Banfield MJ, Kamoun S, TO B (2016) An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor. eLife 5:e10856. doi:10.7554/eLife.10856

    Article  PubMed  PubMed Central  Google Scholar 

  14. Robatzek S, Chinchilla D, Boller T (2006) Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev 20(5):537–542

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Choi SW, Tamaki T, Ebine K, Uemura T, Ueda T, Nakano A (2013) RABA members act in distinct steps of subcellular trafficking of the FLAGELLIN SENSING2 receptor. Plant Cell 25(3):1174–1187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Spallek T, Beck M, Ben Khaled S, Salomon S, Bourdais G, Schellmann S, Robatzek S (2013) ESCRT-I mediates FLS2 Endosomal sorting and plant immunity. PLoS Genet 9(12):e1004035. doi:10.1371/journal.pgen.1004035

    Article  PubMed  PubMed Central  Google Scholar 

  17. Dettmer J, Hong-Hermesdorf A, Stierhof Y-D, Schumacher K (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18(3):715–730

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Geldner N (2009) Cell polarity in plants; a PARspective on PINs. Curr Opin Plant Biol 12(1):42–48

    Article  CAS  PubMed  Google Scholar 

  19. Smith JM, Leslie ME, Robinson SJ, Korasick DA, Zhang T, Backues SK, Cornish PV, Koo AJ, Bednarek SY, Heese A (2014) Loss of Arabidopsis thaliana Dynamin-related protein 2b reveals separation of innate immune signaling pathways. PLoS Pathog 10(12):e1004578. doi:10.1371/journal.ppat.1004578

    Article  PubMed  PubMed Central  Google Scholar 

  20. Ntoukakis V, Schwessinger B, Segonzac C, Zipfel C (2011) Cautionary notes on the use of C-terminal BAK1 fusion proteins for functional studies. Plant Cell 23(11):3871–3878

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ding S-W, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130(3):413–426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lu Y-J, Schornack S, Spallek T, Geldner N, Chory J, Schellmann S, Schumacher K, Kamoun S, Robatzek S (2012) Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking. Cell Microbiol 14(5):682–697

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, UK

    Jenna Loiseau & Silke Robatzek

Authors
  1. Jenna Loiseau
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Silke Robatzek
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Silke Robatzek .

Editor information

Editors and Affiliations

  1. Department of Biosciences, University of Oslo, Oslo, Norway

    Reidunn Birgitta Aalen

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Loiseau, J., Robatzek, S. (2017). Detection and Analyses of Endocytosis of Plant Receptor Kinases. In: Aalen, R. (eds) Plant Receptor Kinases. Methods in Molecular Biology, vol 1621. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7063-6_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7063-6_17

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7062-9

  • Online ISBN: 978-1-4939-7063-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation