Advertisement

BiFC Assay to Detect Calmodulin Binding to Plant Receptor Kinases

  • Cornelia Fischer
  • Margret Sauter
  • Petra Dietrich
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1621)

Abstract

Plant receptor-like kinases (RLKs) are regulated at various levels including posttranscriptional modification and interaction with regulatory proteins. Calmodulin (CaM) is a calcium-sensing protein that was shown to bind to some RLKs such as the PHYTOSULFOKINE RECEPTOR1 (PSKR1). The CaM-binding site is embedded in subdomain VIa of the kinase domain. It is possible that many more of RLKs interact with CaM than previously described. To unequivocally confirm CaM binding, several methods exist. Bimolecular fluorescence complementation (BiFC) and pull-down assays have been successfully used to study CaM binding to PSKR1 and are described in this chapter (BiFC) and in  Chapter 15 (pull down). The two methods are complementary. BiFC is useful to show localization and interaction of soluble as well as of membrane-bound proteins in planta.

Key words

Agrobacterium-mediated transformation of N. benthamiana BiFC Confocal laser scanning microscope Calmodulin Peptide receptor kinase Protein binding 

Notes

Acknowledgment

We would like to thank Heiner Busch for photography and the Deutsche Forschungsgemeinschaft for financial support.

References

  1. 1.
    Hartmann J, Fischer C, Dietrich P, Sauter M (2014) Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1. Plant J 78(2):192–202CrossRefPubMedGoogle Scholar
  2. 2.
    Hu C-D, Chinenov Y, Kerppola TK (2002) Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell 9(4):789–798CrossRefPubMedGoogle Scholar
  3. 3.
    Walter M, Chaban C, Schutze K, Batistic O, Weckermann K, Nake C, Blazevic D, Grefen C, Schumacher K, Oecking C, Harter K, Kudla J (2004) Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J 40(3):428–438CrossRefPubMedGoogle Scholar
  4. 4.
    Gehl C, Waadt R, Kudla J, Mendel RR, Hansch R (2009) New GATEWAY vectors for high throughput analyses of protein-protein interactions by bimolecular fluorescence complementation. Mol Plant 2(5):1051–1058CrossRefPubMedGoogle Scholar
  5. 5.
    Fischer C, Kugler A, Hoth S, Dietrich P (2013) An IQ domain mediates the interaction with calmodulin in a plant cyclic nucleotide-gated channel. Plant Cell Physiol 54(4):573–584CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33(5):949–956CrossRefPubMedGoogle Scholar
  7. 7.
    Ghosh I, Hamilton AD, Regan L (2000) Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc 122(23):5658–5659CrossRefGoogle Scholar
  8. 8.
    Kodama Y, Hu CD (2012) Bimolecular fluorescence complementation (BiFC): a 5-year update and future perspectives. Biotechniques 53(5):285–298CrossRefPubMedGoogle Scholar
  9. 9.
    Grefen C, Blatt MR (2012) A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC). Biotechniques 53(5):311–314CrossRefPubMedGoogle Scholar
  10. 10.
    Gookin TE, Assmann SM (2014) Significant reduction of BiFC non-specific assembly facilitates in planta assessment of heterotrimeric G-protein interactors. Plant J 80(3):553–567CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Horstman A, Nougalli Tonaco IA, Boutilier K, Immink RGH (2014) A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies. Int J Mol Sci 15(6):9628–9643CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Cornelia Fischer
    • 1
  • Margret Sauter
    • 2
  • Petra Dietrich
    • 1
  1. 1.Molecular Plant Physiology, Department of BiologyUniversity of Erlangen-NurembergErlangenGermany
  2. 2.Plant Developmental Biology and Plant PhysiologyUniversity of KielKielGermany

Personalised recommendations