Fluorescent Fusion Protein Expression in Plant Cells

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2023 Apr 24

PMID 37093472

Authors

Shuxian Huang

Zeng Yonglun

Affiliations

Soon will be listed here.

Abstract

Fluorescent proteins (FPs) revolutionized the cell biology research by visualizing the dynamics of cellular events. In fusion with the targeted proteins, the FPs can be utilized to monitor the protein dynamics and localization in cells. Recently, FPs have been used as reporters for live cell imaging to study the protein localization or organelles dynamics in plants, allowing cell biologists to explore the plant cell function by obtaining tremendous details of cell structures and functions in combination with confocal imaging. To facilitate the usage of fluorescent proteins for protein localization and dynamic analysis in plant cell biology research, here we describe the updated protocol of Agrobacterium-mediated transformation of Arabidopsis thaliana using fluorescent proteins to generate the stable expression transgenic plants for protein trafficking and localization study. We further use the GFP-tagged SDP1 (sugar-dependent protein) lipase, mCherry-tagged peroxisome marker, and BODYPY or Nile Red (lipid droplet staining dye) as examples to introduce the method for the protein localization analysis in plants.

Citing Articles

Imaging Plant Lipids with Fluorescent Reporters.

Li Y, Dai G, Zhang Y, Yao N Plants (Basel). 2025; 14(1.

PMID: 39795280 PMC: 11723198. DOI: 10.3390/plants14010015.

References

Snapp E . Fluorescent proteins: a cell biologist's user guide. Trends Cell Biol. 2009; 19(11):649-55. PMC: 2784028. DOI: 10.1016/j.tcb.2009.08.002. View

Kremers G, Gilbert S, Cranfill P, Davidson M, Piston D . Fluorescent proteins at a glance. J Cell Sci. 2010; 124(Pt 2):157-60. PMC: 3037093. DOI: 10.1242/jcs.072744. View

Katoh Y, Nozaki S, Hartanto D, Miyano R, Nakayama K . Architectures of multisubunit complexes revealed by a visible immunoprecipitation assay using fluorescent fusion proteins. J Cell Sci. 2015; 128(12):2351-62. DOI: 10.1242/jcs.168740. View

Chudakov D, Lukyanov S, Lukyanov K . Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 2005; 23(12):605-13. DOI: 10.1016/j.tibtech.2005.10.005. View

Zhang J, Campbell R, Ting A, Tsien R . Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol. 2002; 3(12):906-18. DOI: 10.1038/nrm976. View

Bayguinov P, Oakley D, Shih C, Geanon D, Joens M, Fitzpatrick J . Modern Laser Scanning Confocal Microscopy. Curr Protoc Cytom. 2018; 85(1):e39. DOI: 10.1002/cpcy.39. View

Valuchova S, Mikulkova P, Pecinkova J, Klimova J, Krumnikl M, Bainar P . Imaging plant germline differentiation within Arabidopsis flowers by light sheet microscopy. Elife. 2020; 9. PMC: 7012603. DOI: 10.7554/eLife.52546. View

Komis G, Mistrik M, Samajova O, Doskocilova A, Ovecka M, Illes P . Dynamics and organization of cortical microtubules as revealed by superresolution structured illumination microscopy. Plant Physiol. 2014; 165(1):129-48. PMC: 4012574. DOI: 10.1104/pp.114.238477. View

Betzig E, Patterson G, Sougrat R, Lindwasser O, Olenych S, Bonifacino J . Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006; 313(5793):1642-5. DOI: 10.1126/science.1127344. View

10.

Komis G, Mistrik M, Samajova O, Ovecka M, Bartek J, Samaj J . Superresolution live imaging of plant cells using structured illumination microscopy. Nat Protoc. 2015; 10(8):1248-63. DOI: 10.1038/nprot.2015.083. View

11.

Sparkes I, Runions J, Kearns A, Hawes C . Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc. 2007; 1(4):2019-25. DOI: 10.1038/nprot.2006.286. View

12.

Gao C, Luo M, Zhao Q, Yang R, Cui Y, Zeng Y . A unique plant ESCRT component, FREE1, regulates multivesicular body protein sorting and plant growth. Curr Biol. 2014; 24(21):2556-63. DOI: 10.1016/j.cub.2014.09.014. View

13.

Zeng Y, Li B, Ji C, Feng L, Niu F, Deng C . A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in . Proc Natl Acad Sci U S A. 2021; 118(17). PMC: 8092600. DOI: 10.1073/pnas.2021293118. View

14.

He F, Chen S, Ning Y, Wang G . Rice (Oryza sativa) Protoplast Isolation and Its Application for Transient Expression Analysis. Curr Protoc Plant Biol. 2019; 1(2):373-383. DOI: 10.1002/cppb.20026. View

15.

Miao Y, Jiang L . Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells. Nat Protoc. 2007; 2(10):2348-53. DOI: 10.1038/nprot.2007.360. View

16.

Clough S, Bent A . Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1999; 16(6):735-43. DOI: 10.1046/j.1365-313x.1998.00343.x. View

17.

Zhang X, Henriques R, Lin S, Niu Q, Chua N . Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc. 2007; 1(2):641-6. DOI: 10.1038/nprot.2006.97. View

18.

Boruc J, Van Damme D . Endomembrane trafficking overarching cell plate formation. Curr Opin Plant Biol. 2015; 28:92-8. DOI: 10.1016/j.pbi.2015.09.007. View

19.

Gadeyne A, Sanchez-Rodriguez C, Vanneste S, Rubbo S, Zauber H, Vanneste K . The TPLATE adaptor complex drives clathrin-mediated endocytosis in plants. Cell. 2014; 156(4):691-704. DOI: 10.1016/j.cell.2014.01.039. View

20.

Belda-Palazon B, Rodriguez L, Fernandez M, Castillo M, Anderson E, Gao C . FYVE1/FREE1 Interacts with the PYL4 ABA Receptor and Mediates Its Delivery to the Vacuolar Degradation Pathway. Plant Cell. 2016; 28(9):2291-2311. PMC: 5059795. DOI: 10.1105/tpc.16.00178. View