Visualizing Protein-protein Interactions in Plants by Rapamycin-dependent Delocalization

Overview

Journal Plant Cell

Publisher Oxford University Press

Specialties Biology
Cell Biology

Date 2021 Apr 1

PMID 33793859

Citations 14

Authors

Joanna Winkler

Evelien Mylle

Andreas De Meyer

Benjamin Pavie

Julie Merchie

Peter Grones

Dani L Van Damme

Affiliations

Soon will be listed here.

Abstract

Identifying protein-protein interactions (PPIs) is crucial for understanding biological processes. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only a few tools allow complex multi-protein interactions to be visualized. Here, we present a conditional in vivo PPI tool for plant research that meets these criteria. Knocksideways in plants (KSP) is based on the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations of other PPI systems. This in vivo tool does not require spatial proximity of the bait and prey fluorophores and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore the visualization of the proteins in the absence of rapamycin acts as an internal control. We used KSP to confirm previously identified interactions in Nicotiana benthamiana leaf epidermal cells. Furthermore, the scripts that we generated allow the interactions to be quantified at high throughput. Finally, we demonstrate that KSP can easily be used to visualize complex multi-protein interactions. KSP is therefore a versatile tool with unique characteristics and applications that complements other plant PPI methods.

Citing Articles

A Hot-Swappable Genetic Switch: Building an inducible and trackable functional assay for the essential gene MEDIATOR 21.

Watson I, Maranas C, Nemhauser J, Leydon A bioRxiv. 2025; .

PMID: 39763940 PMC: 11702731. DOI: 10.1101/2024.12.16.628800.

ReLo is a simple and rapid colocalization assay to identify and characterize direct protein-protein interactions.

Salgania H, Metz J, Jeske M Nat Commun. 2024; 15(1):2875.

PMID: 38570497 PMC: 10991417. DOI: 10.1038/s41467-024-47233-4.

Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants.

Dragwidge J, Wang Y, Brocard L, De Meyer A, Hudecek R, Eeckhout D Nat Cell Biol. 2024; 26(3):438-449.

PMID: 38347182 PMC: 7615741. DOI: 10.1038/s41556-024-01354-6.

Phase separation-based visualization of protein-protein interactions and kinase activities in plants.

Safi A, Smagghe W, Goncalves A, Wang Q, Xu K, Fernandez A Plant Cell. 2023; 35(9):3280-3302.

PMID: 37378595 PMC: 10473206. DOI: 10.1093/plcell/koad188.

Protein-Protein Interactions in Abiotic Stress Signaling: An Overview of Biochemical and Biophysical Methods of Characterization.

Couee I, Gouesbet G Methods Mol Biol. 2023; 2642:319-330.

PMID: 36944886 DOI: 10.1007/978-1-0716-3044-0_17.

References

Kaplan-Levy R, Quon T, OBrien M, Sappl P, Smyth D . Functional domains of the PETAL LOSS protein, a trihelix transcription factor that represses regional growth in Arabidopsis thaliana. Plant J. 2014; 79(3):477-91. DOI: 10.1111/tpj.12574. View

Cabantous S, Nguyen H, Pedelacq J, Koraichi F, Chaudhary A, Ganguly K . A new protein-protein interaction sensor based on tripartite split-GFP association. Sci Rep. 2013; 3:2854. PMC: 3790201. DOI: 10.1038/srep02854. View

Hirst J, Schlacht A, Norcott J, Traynor D, Bloomfield G, Antrobus R . Characterization of TSET, an ancient and widespread membrane trafficking complex. Elife. 2014; 3:e02866. PMC: 4031984. DOI: 10.7554/eLife.02866. View

Myers C, Romanowsky S, Barron Y, Garg S, Azuse C, Curran A . Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes. Plant J. 2009; 59(4):528-39. DOI: 10.1111/j.1365-313X.2009.03894.x. View

Mehlmer N, Parvin N, Hurst C, Knight M, Teige M, Vothknecht U . A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana. J Exp Bot. 2012; 63(4):1751-61. PMC: 3971373. DOI: 10.1093/jxb/err406. View

Maruta N, Trusov Y, Botella J . Yeast Three-Hybrid System for the Detection of Protein-Protein Interactions. Methods Mol Biol. 2015; 1363:145-54. DOI: 10.1007/978-1-4939-3115-6_12. View

Noack L, Jaillais Y . Precision targeting by phosphoinositides: how PIs direct endomembrane trafficking in plants. Curr Opin Plant Biol. 2017; 40:22-33. DOI: 10.1016/j.pbi.2017.06.017. View

Houbaert A, Zhang C, Tiwari M, Wang K, de Marcos Serrano A, Savatin D . POLAR-guided signalling complex assembly and localization drive asymmetric cell division. Nature. 2018; 563(7732):574-578. DOI: 10.1038/s41586-018-0714-x. View

Nelson B, Cai X, Nebenfuhr A . A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007; 51(6):1126-36. DOI: 10.1111/j.1365-313X.2007.03212.x. View

10.

Ohashi-Ito K, Bergmann D . Regulation of the Arabidopsis root vascular initial population by LONESOME HIGHWAY. Development. 2007; 134(16):2959-68. PMC: 3145339. DOI: 10.1242/dev.006296. View

11.

Hughes K, Waters A . Rapid inducible protein displacement in and using knocksideways technology. Wellcome Open Res. 2017; 2:18. PMC: 5395084. DOI: 10.12688/wellcomeopenres.11005.1. View

12.

Rubio V, Shen Y, Saijo Y, Liu Y, Gusmaroli G, Dinesh-Kumar S . An alternative tandem affinity purification strategy applied to Arabidopsis protein complex isolation. Plant J. 2005; 41(5):767-78. DOI: 10.1111/j.1365-313X.2004.02328.x. View

13.

Tulin A, McClerklin S, Huang Y, Dixit R . Single-molecule analysis of the microtubule cross-linking protein MAP65-1 reveals a molecular mechanism for contact-angle-dependent microtubule bundling. Biophys J. 2012; 102(4):802-9. PMC: 3283812. DOI: 10.1016/j.bpj.2012.01.008. View

14.

Rosenfeldt G, Viana R, Mootz H, von Arnim A, Batschauer A . Chemically induced and light-independent cryptochrome photoreceptor activation. Mol Plant. 2009; 1(1):4-14. DOI: 10.1093/mp/ssm002. View

15.

Magliery T, Wilson C, Pan W, Mishler D, Ghosh I, Hamilton A . Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. J Am Chem Soc. 2005; 127(1):146-57. DOI: 10.1021/ja046699g. View

16.

Arora D, Abel N, Liu C, Van Damme P, Yperman K, Eeckhout D . Establishment of Proximity-Dependent Biotinylation Approaches in Different Plant Model Systems. Plant Cell. 2020; 32(11):3388-3407. PMC: 7610282. DOI: 10.1105/tpc.20.00235. View

17.

Van Damme D, Gadeyne A, Vanstraelen M, Inze D, Van Montagu M, De Jaeger G . Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways. Proc Natl Acad Sci U S A. 2010; 108(2):615-20. PMC: 3021081. DOI: 10.1073/pnas.1017890108. View

18.

Mair A, Xu S, Branon T, Ting A, Bergmann D . Proximity labeling of protein complexes and cell-type-specific organellar proteomes in enabled by TurboID. Elife. 2019; 8. PMC: 6791687. DOI: 10.7554/eLife.47864. View

19.

Simon M, Platre M, Marques-Bueno M, Armengot L, Stanislas T, Bayle V . A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants. Nat Plants. 2016; 2:16089. PMC: 4918763. DOI: 10.1038/nplants.2016.89. View

20.

Kirik A, Ehrhardt D, Kirik V . TONNEAU2/FASS regulates the geometry of microtubule nucleation and cortical array organization in interphase Arabidopsis cells. Plant Cell. 2012; 24(3):1158-70. PMC: 3336111. DOI: 10.1105/tpc.111.094367. View