A bright green-colored bimolecular fluorescence complementation assay in living plant cells

Kodama, Yutaka

doi:10.5511/plantbiotechnology.10.1105a

Cited by 10 publications

(16 citation statements)

References 18 publications

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“…In this study, the fluorescence intensity of BiFC between psGN and psGC V163A was also compared with those of other GFPbased BiFCs in Arabidopsis protoplast cells ( Figure 3A). Consistent with the previous report in onion cells (Kodama 2011), complemented fluorescence between psGN and psGC was barely detectable, and the V163A mutation in psGC increased the fluorescence intensity in Arabidopsis cells (Figure 3B-D). Similarly, we also found that an F64L mutation increased the intensity of BiFC fluorescence, in a comparison between psGFPand eGFP-based BiFCs (Figure 3B-D).…”

Section: Bifc With Gfp Variants In Plant Cellssupporting

confidence: 81%

“…Although numerous laboratories employ GFP as a marker protein, the use of GFP-based BiFC has been limited due to its low fluorescent signal (Kodama 2011). In the present study, sfGFP-based BiFC was found to be the brightest system in plant cells; e.g.…”

Section: Discussionmentioning

confidence: 65%

“…Recently, we reported that the V163A mutation could improve fluorescence intensity of psGFP-based BiFC (psGFP V163A -based BiFC) (Kodama 2011). The V163A improves complementation efficiency of BiFC fragments rather than full-length psGFP folding efficiency, because full-length psGFP V163A protein emitted a comparable fluorescence with psGFP (Kodama 2011).…”

Section: Discussionmentioning

confidence: 99%

“…Each GFP variant was split between A155 and D156: psGN, psGC, eGN, eGC, frGN, frGC, sfGN and sfGC (Figures 1, 3A). In our previous study, a V163A mutation within the C-terminal fragment of psGFP (psGC V163A ) was reported to improve fluorescence intensity of the BiFC complex with psGN by 7-fold in onion epidermal cells (Kodama 2011). In this study, the fluorescence intensity of BiFC between psGN and psGC V163A was also compared with those of other GFPbased BiFCs in Arabidopsis protoplast cells ( Figure 3A).…”

Section: Bifc With Gfp Variants In Plant Cellsmentioning

confidence: 99%

“…Note that sGFP(S65T) is termed as "psGFP" in this study. To produce genes for eGFP and frGFP, the DpnI-mediated site-direct mutagenesis method (Fisher and Pei 1997;Kodama 2011) was used to introduce appropriate mutations (see Figure 1). A gene for sfGFP was generated as a synthetic gene (Operon Technologies Inc, Alabama, USA).…”

Section: Plasmid Constructionmentioning

confidence: 99%

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In planta comparative analysis of improved green fluorescent proteins with reference to fluorescence intensity and bimolecular fluorescence complementation ability

Fujii

Kodama

2015

Plant Biotechnology

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Green fluorescent protein (GFP) was discovered from the jellyfish Aequorea victoria, and several improvements have been carried out to change its physicochemical properties. The resulting improved GFP variants have been used as reporter proteins for bioimaging techniques in various research fields including plant science. Almost all GFP variants were developed using Escherichia coli to improve fluorescence properties in mammalian cells, but the impact in other organisms such as plant cells remains to be determined. In this study, we performed comparative analysis of four improved GFP variants, GFP-S65T, eGFP, frGFP and sfGFP, with reference to the fluorescence intensity in Arabidopsis protoplasts, and found that sfGFP is the brightest. Using non-fluorescent fragments from the GFP variants, we also conducted bimolecular fluorescence complementation (BiFC) assays to find appropriate fragment pairs of GFP-based BiFC for visualization of protein-protein interactions in living plant cells. Our observations revealed that the brightest is the sfGFP-based BiFC. Further, as an evaluation method for the sfGFP-based BiFC, a BiFC competition assay was successfully completed for the first time in planta. The present study provides useful information for selection and improvement of the GFP molecule and its application to BiFC technology in plants.Key words: Arabidopsis, BiFC, BiFC competition, GFP, protein-protein interaction.Since the first attempt to employ wild-type green fluorescent protein (wtGFP) as a reporter protein, it has been developed for bioimaging techniques in various research fields including plant science (Chalfie et al. 1994;Heim et al. 1995). wtGFP is a typical β-barrel structure harboring a chromophore spontaneously formed by the three residues (S65-T66-G67), and thereby exhibits green fluorescence without any cofactor (Tsien 1998). To date, wtGFP has been improved by targeted or random mutations to increase its fluorescence intensity, and almost all of the improvements were performed using Escherichia coli for suitable expression in mammalian cells (Shaner et al. 2005). The first improvement was a single mutation of serine to threonine residue at position 65 (S65T) for the chromophore (Figure 1); the mutation shifted the excitation peak to 490 nm from 395 nm and 470 nm of wtGFP (Heim et al. 1995). Compared with wtGFP, this peak-shifted GFP (psGFP) gives 6-fold brighter Abbreviations: BiFC, bimolecular fluorescence complementation; eGC, the C-terminal fragment of eGFP; eGFP, enhanced GFP; eGN, the N-terminal fragment of eGFP; EYFP, enhanced yellow fluorescent protein; frGC, the C-terminal fragment of frGFP; frGFP, folding reporter GFP; frGN, the N-terminal fragment of frGFP; GFP, green fluorescent protein; MXMT, 7-methylxanthine methyltransferase; psGC, the C-terminal fragment of psGFP; psGFP, peak-shifted GFP; psGN, the N-terminal fragment of psGFP; PEG, polyethylene glycol; RC, the C-terminal fragment of DsRED monomer; sfGC, the C-terminal fragment of sfGFP; sfGFP, superfolder GFP; sfGN, the N-...

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Section: Bifc With Gfp Variants In Plant Cellssupporting

confidence: 81%

Section: Discussionmentioning

confidence: 65%

Section: Discussionmentioning

confidence: 99%

Section: Bifc With Gfp Variants In Plant Cellsmentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

See 3 more Smart Citations

In planta comparative analysis of improved green fluorescent proteins with reference to fluorescence intensity and bimolecular fluorescence complementation ability

Fujii

Kodama

2015

Plant Biotechnology

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Subcellular Localization of Transiently Expressed Fluorescent Fusion Proteins

Collings

2013

Legume Genomics

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The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

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Novel Bimolecular Fluorescence Complementation (BiFC) Assay for Visualization of the Protein–Protein Interactions and Cellular Protein Complex Localizations

Shi,

Gao,

Zhang

et al. 2023

Mol Biotechnol

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A bright green-colored bimolecular fluorescence complementation assay in living plant cells

Cited by 10 publications

References 18 publications

In planta comparative analysis of improved green fluorescent proteins with reference to fluorescence intensity and bimolecular fluorescence complementation ability

In planta comparative analysis of improved green fluorescent proteins with reference to fluorescence intensity and bimolecular fluorescence complementation ability

Subcellular Localization of Transiently Expressed Fluorescent Fusion Proteins

Novel Bimolecular Fluorescence Complementation (BiFC) Assay for Visualization of the Protein–Protein Interactions and Cellular Protein Complex Localizations

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