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The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants.

Forner J, Binder S - BMC Plant Biol. (2007)

Bottom Line: The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed.In plants, eqFP611 is a suitable fluorescent reporter protein.Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molekulare Botanik, Universität Ulm, Ulm, Germany. joachim.forner@uni-ulm.de

ABSTRACT

Background: Intrinsically fluorescent proteins have revolutionized studies in molecular cell biology. The parallel application of these proteins in dual- or multilabeling experiments such as subcellular localization studies requires non-overlapping emission spectra for unambiguous detection of each label. In the red spectral range, almost exclusively DsRed and derivatives thereof are used today. To test the suitability of the red fluorescent protein eqFP611 as an alternative in higher plants, the behavior of this protein was analyzed in terms of expression, subcellular targeting and compatibility with GFP in tobacco.

Results: When expressed transiently in tobacco protoplasts, eqFP611 accumulated over night to levels easily detectable by fluorescence microscopy. The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed. When fused to N-terminal mitochondrial and peroxisomal targeting sequences, the red fluorescence was located exclusively in the corresponding organelles in transfected protoplasts. Upon co-expression with GFP in the same cells, fluorescence of both eqFP611 and GFP could be easily distinguished, demonstrating the potential of eqFP611 in dual-labeling experiments with GFP. A series of plasmids was constructed for expression of eqFP611 in plants and for simultaneous expression of this fluorescent protein together with GFP. Transgenic tobacco plants constitutively expressing mitochondrially targeted eqFP611 were generated. The red fluorescence was stably transmitted to the following generations, making these plants a convenient source for protoplasts containing an internal marker for mitochondria.

Conclusion: In plants, eqFP611 is a suitable fluorescent reporter protein. The unmodified protein can be expressed to levels easily detectable by epifluorescence microscopy without adverse affect on the viability of plant cells. Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

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Mitochondrially targeted eqFP611 and smGFP4 expressed from the same plasmid. Transfection of N. tabacum wild-type protoplasts with a construct encoding both eqFP611 and smGFP4 with mitochondrial presequences (pIVD144-eqFP611-IVD145-smGFP4). Pictures of the same cell, taken through MitoTracker (A) or FITC (B) filter sets, respectively. Scale bars: 10 μm. (C) Plasmid used for transfection. Black arrow, CaMV 35S: cauliflower mosaic virus 35S promoter; grey boxes, IVD(144)/(145): N-terminal 145 and 144 nucleotides of the IVD coding sequence, respectively; red box, eqFP611: eqFP611 coding sequence; green box, smGFP4: smGFP4 coding sequence; black box, NOS T: nopaline synthase terminator; white box, S: spacer sequence. H: HindIII, Sa: SacI, Sm: SmaI, Xh: XhoI, Xb: XbaI, P: PstI, B: BamHI, E: EcoRI restriction sites. Vector backbone: pUC19.
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Figure 6: Mitochondrially targeted eqFP611 and smGFP4 expressed from the same plasmid. Transfection of N. tabacum wild-type protoplasts with a construct encoding both eqFP611 and smGFP4 with mitochondrial presequences (pIVD144-eqFP611-IVD145-smGFP4). Pictures of the same cell, taken through MitoTracker (A) or FITC (B) filter sets, respectively. Scale bars: 10 μm. (C) Plasmid used for transfection. Black arrow, CaMV 35S: cauliflower mosaic virus 35S promoter; grey boxes, IVD(144)/(145): N-terminal 145 and 144 nucleotides of the IVD coding sequence, respectively; red box, eqFP611: eqFP611 coding sequence; green box, smGFP4: smGFP4 coding sequence; black box, NOS T: nopaline synthase terminator; white box, S: spacer sequence. H: HindIII, Sa: SacI, Sm: SmaI, Xh: XhoI, Xb: XbaI, P: PstI, B: BamHI, E: EcoRI restriction sites. Vector backbone: pUC19.

Mentions: Transformation of Nicotiana benthamiana leaves by injection of Agrobacterium tumefaciens [23] containing IFP fusion genes is another fast and simple method for the analysis of the subcellular localization of a protein. This procedure is presumably closer to the in vivo conditions than protoplast transfection, since the transformed cells remain in the original tissue context. In addition, this approach does not require the relatively laborious preparation of protoplasts. In this case, expression of the two fusion proteins from the same plasmid is advantageous, since a single transformation event is sufficient to ensure that every transformed cell contains both IFP genes. Apart from that, expressing both fluorescent proteins from the same plasmid under identical promoters should generate equal amounts of RFP and GFP within a cell. The entire procedure should be easier since only a single construct has to be handled. To investigate the feasibilty of this procedure, plasmid pIVD144-eqFP611-IVD145-smGFP4 containing both the eqFP611 and the smGFP4 genes with mitochondrial presequences each under control of a CaMV 35S promoter was constructed and first tested by transfection of tobacco protoplasts. Again, both red and green fluorescence could easily be detected in the same cell (Fig. 6). The fluorescence is found exclusively in mitochondria, the patterns of both red and green fluorescence being identical. This result is indistinguishable from the experiment with the same eqFP611 and smGFP4 expression cassettes encoded on two different plasmids (Fig. 3), but this time every transfected protoplast expressed both eqFP611 and smGFP4.


The red fluorescent protein eqFP611: application in subcellular localization studies in higher plants.

Forner J, Binder S - BMC Plant Biol. (2007)

Mitochondrially targeted eqFP611 and smGFP4 expressed from the same plasmid. Transfection of N. tabacum wild-type protoplasts with a construct encoding both eqFP611 and smGFP4 with mitochondrial presequences (pIVD144-eqFP611-IVD145-smGFP4). Pictures of the same cell, taken through MitoTracker (A) or FITC (B) filter sets, respectively. Scale bars: 10 μm. (C) Plasmid used for transfection. Black arrow, CaMV 35S: cauliflower mosaic virus 35S promoter; grey boxes, IVD(144)/(145): N-terminal 145 and 144 nucleotides of the IVD coding sequence, respectively; red box, eqFP611: eqFP611 coding sequence; green box, smGFP4: smGFP4 coding sequence; black box, NOS T: nopaline synthase terminator; white box, S: spacer sequence. H: HindIII, Sa: SacI, Sm: SmaI, Xh: XhoI, Xb: XbaI, P: PstI, B: BamHI, E: EcoRI restriction sites. Vector backbone: pUC19.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1904219&req=5

Figure 6: Mitochondrially targeted eqFP611 and smGFP4 expressed from the same plasmid. Transfection of N. tabacum wild-type protoplasts with a construct encoding both eqFP611 and smGFP4 with mitochondrial presequences (pIVD144-eqFP611-IVD145-smGFP4). Pictures of the same cell, taken through MitoTracker (A) or FITC (B) filter sets, respectively. Scale bars: 10 μm. (C) Plasmid used for transfection. Black arrow, CaMV 35S: cauliflower mosaic virus 35S promoter; grey boxes, IVD(144)/(145): N-terminal 145 and 144 nucleotides of the IVD coding sequence, respectively; red box, eqFP611: eqFP611 coding sequence; green box, smGFP4: smGFP4 coding sequence; black box, NOS T: nopaline synthase terminator; white box, S: spacer sequence. H: HindIII, Sa: SacI, Sm: SmaI, Xh: XhoI, Xb: XbaI, P: PstI, B: BamHI, E: EcoRI restriction sites. Vector backbone: pUC19.
Mentions: Transformation of Nicotiana benthamiana leaves by injection of Agrobacterium tumefaciens [23] containing IFP fusion genes is another fast and simple method for the analysis of the subcellular localization of a protein. This procedure is presumably closer to the in vivo conditions than protoplast transfection, since the transformed cells remain in the original tissue context. In addition, this approach does not require the relatively laborious preparation of protoplasts. In this case, expression of the two fusion proteins from the same plasmid is advantageous, since a single transformation event is sufficient to ensure that every transformed cell contains both IFP genes. Apart from that, expressing both fluorescent proteins from the same plasmid under identical promoters should generate equal amounts of RFP and GFP within a cell. The entire procedure should be easier since only a single construct has to be handled. To investigate the feasibilty of this procedure, plasmid pIVD144-eqFP611-IVD145-smGFP4 containing both the eqFP611 and the smGFP4 genes with mitochondrial presequences each under control of a CaMV 35S promoter was constructed and first tested by transfection of tobacco protoplasts. Again, both red and green fluorescence could easily be detected in the same cell (Fig. 6). The fluorescence is found exclusively in mitochondria, the patterns of both red and green fluorescence being identical. This result is indistinguishable from the experiment with the same eqFP611 and smGFP4 expression cassettes encoded on two different plasmids (Fig. 3), but this time every transfected protoplast expressed both eqFP611 and smGFP4.

Bottom Line: The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed.In plants, eqFP611 is a suitable fluorescent reporter protein.Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molekulare Botanik, Universität Ulm, Ulm, Germany. joachim.forner@uni-ulm.de

ABSTRACT

Background: Intrinsically fluorescent proteins have revolutionized studies in molecular cell biology. The parallel application of these proteins in dual- or multilabeling experiments such as subcellular localization studies requires non-overlapping emission spectra for unambiguous detection of each label. In the red spectral range, almost exclusively DsRed and derivatives thereof are used today. To test the suitability of the red fluorescent protein eqFP611 as an alternative in higher plants, the behavior of this protein was analyzed in terms of expression, subcellular targeting and compatibility with GFP in tobacco.

Results: When expressed transiently in tobacco protoplasts, eqFP611 accumulated over night to levels easily detectable by fluorescence microscopy. The native protein was found in the nucleus and in the cytosol and no detrimental effects on cell viability were observed. When fused to N-terminal mitochondrial and peroxisomal targeting sequences, the red fluorescence was located exclusively in the corresponding organelles in transfected protoplasts. Upon co-expression with GFP in the same cells, fluorescence of both eqFP611 and GFP could be easily distinguished, demonstrating the potential of eqFP611 in dual-labeling experiments with GFP. A series of plasmids was constructed for expression of eqFP611 in plants and for simultaneous expression of this fluorescent protein together with GFP. Transgenic tobacco plants constitutively expressing mitochondrially targeted eqFP611 were generated. The red fluorescence was stably transmitted to the following generations, making these plants a convenient source for protoplasts containing an internal marker for mitochondria.

Conclusion: In plants, eqFP611 is a suitable fluorescent reporter protein. The unmodified protein can be expressed to levels easily detectable by epifluorescence microscopy without adverse affect on the viability of plant cells. Its subcellular localization can be manipulated by N-terminal signal sequences. eqFP611 and GFP are fully compatible in dual-labeling experiments.

Show MeSH
Related in: MedlinePlus