Abstract
Protein–protein interactions (PPIs) play vital roles in all subcellular processes and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior to their application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI, and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced, and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2in1-cloning approach, and their application in plant cell biology: ratiometric Bimolecular Fluorescence Complementation (rBiFC), FRET Acceptor Photobleaching (FRET-AB), and Fluorescent Lifetime Imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Xing S, Wallmeroth N, Berendzen KW, Grefen C (2016) Techniques for the analysis of protein-protein interactions in vivo. Plant Physiol 171:727–758
Husbands AY, Aggarwal V, Ha T, Timmermans MC (2016) In planta single-molecule pull-down reveals tetrameric stoichiometry of HD-ZIPIII:LITTLE ZIPPER complexes. Plant Cell 28:1783–1794
Grefen C, Blatt MR (2012) A 2in1 cloning system enables ratiometric bimolecular fluorescence complementation (rBiFC). Biotechniques 53:311–314
Hecker A, Wallmeroth N, Peter S, Blatt MR, Harter K, Grefen C (2015) Binary 2in1 vectors improve in planta (co-) localisation and dynamic protein interaction studies. Plant Physiol 168:776–787
Ghosh I, Hamilton AD, Regan L (2000) Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc 122:5658–5659
Horstman A, Tonaco IA, Boutilier K, Immink RG (2014) A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies. Int J Mol Sci 15:9628–9643
Grefen C, Karnik R, Larson E, Lefoulon C, Wang YZ, Waghmare S, Zhang B, Hills A, Blatt MR (2015) A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion. Nat Plants 1:15108
Lipka E, Gadeyne A, Stockle D, Zimmermann S, De Jaeger G, Ehrhardt DW, Kirik V, Van Damme D, Muller S (2014) The phragmoplast-orienting kinesin-12 class proteins translate the positional information of the preprophase band to establish the cortical division zone in Arabidopsis thaliana. Plant Cell 26:2617–2632
Albert I, Bohm H, Albert M, Feiler CE, Imkampe J, Wallmeroth N, Brancato C, Raaymakers TM, Oome S, Zhang HQ, Krol E, Grefen C, Gust AA, Chai JJ, Hedrich R, Van den Ackerveken G, Nurnberger T (2015) An RLP23-SOBIR1-BAK1 complex mediates NLP-triggered immunity. Nat Plants 1:15140
Karnik R, Zhang B, Waghmare S, Aderhold C, Grefen C, Blatt MR (2015) Binding of SEC11 indicates its role in SNARE recycling after vesicle fusion and identifies two pathways for vesicular traffic to the plasma membrane. Plant Cell 27:675–694
Zhang B, Karnik R, Wang Y, Wallmeroth N, Blatt MR, Grefen C (2015) The arabidopsis R-SNARE VAMP721 interacts with KAT1 and KC1 K+ channels to moderate K+ current at the plasma membrane. Plant Cell 27:1697–1717
Kumar MN, Hsieh YF, Verslues PE (2015) At14a-Like1 participates in membrane-associated mechanisms promoting growth during drought in Arabidopsis thaliana. Proc Natl Acad Sci U S A 112:10545–10550
Le CT, Brumbarova T, Ivanov R, Stoof C, Weber E, Mohrbacher J, Fink-Straube C, Bauer P (2016) Zinc finger of Arabidopsis thaliana12 (ZAT12) interacts with FER-Like Iron Deficiency-Induced Transcription Factor (FIT) linking iron deficiency and oxidative stress responses. Plant Physiol 170:540–557
Forster T (1946) Energiewanderung und Fluoreszenz. Naturwissenschaften 33:166–175
Ishikawa-Ankerhold HC, Ankerhold R, Drummen GP (2012) Advanced fluorescence microscopy techniques—FRAP, FLIP, FLAP, FRET and FLIM. Molecules 17:4047–4132
Bucherl CA, Bader A, Westphal AH, Laptenok SP, Borst JW (2014) FRET-FLIM applications in plant systems. Protoplasma 251:383–394
Wallrabe H, Periasamy A (2005) Imaging protein molecules using FRET and FLIM microscopy. Curr Opin Biotechnol 16:19–27
De Los SC, Chang CW, Mycek MA, Cardullo RA (2015) FRAP, FLIM, and FRET: detection and analysis of cellular dynamics on a molecular scale using fluorescence microscopy. Mol Reprod Dev 82:587–604
Koncz C, Schell J (1986) The promoter of Tl-DNA Gene5 controls the tissue-specific expression of chimeric genes carried by a novel type of agrobacterium binary vector. Mol Gen Genet 204:383–396
Blatt MR, Grefen C (2014) Applications of fluorescent marker proteins in plant cell biology. Methods Mol Biol 1062:487–507
Mathur J, Koncz C (1997) Method for preparation of epidermal imprints using agarose. Biotechniques 22:280–282
Karnik A, Karnik R, Grefen C (2013) SDM-assist software to design site-directed mutagenesis primers introducing “silent” restriction sites. BMC Bioinformatics 14:105
Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025
Shuping Xing, Dietmar Gerald Mehlhorn, Niklas Wallmeroth, Lisa Yasmin Asseck, Ritwika Kar, Alessa Voss, Philipp Denninger, Vanessa Aphaia Fiona Schmidt, Markus Schwarzländer, York-Dieter Stierhof, Guido Grossmann, Christopher Grefen, (2017) Loss of GET pathway orthologs in causes root hair growth defects and affects SNARE abundance. Proceedings of the National Academy of Sciences 114(8):E1544–E1553
Acknowledgements
We are grateful to Eva Schwörzer and Laure Grefen for excellent technical support. Work in our lab is supported through seed funding of the SFB1101 and an Emmy Noether fellowship of the German Research Foundation (DFG) to C.G. (GR 4251/1-1).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Mehlhorn, D.G., Wallmeroth, N., Berendzen, K.W., Grefen, C. (2018). 2in1 Vectors Improve In Planta BiFC and FRET Analyses. In: Hawes, C., Kriechbaumer, V. (eds) The Plant Endoplasmic Reticulum . Methods in Molecular Biology, vol 1691. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7389-7_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7389-7_11
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7388-0
Online ISBN: 978-1-4939-7389-7
eBook Packages: Springer Protocols