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Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells.

Lee LY, Fang MJ, Kuang LY, Gelvin SB - Plant Methods (2008)

Bottom Line: Additional expression of mCherry indicates transfected cells and sub-cellular structures.Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-1 in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus.The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA. zofangmj@gate.sinica.edu.tw.

ABSTRACT

Background: The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed.

Results: We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP), and prey proteins are tagged with the N-terminal portions of either Venus (nVenus) or Cerulean (nCerulean). Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-1 in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus.

Conclusion: Multi-color BiFC is a useful technique to determine interactions simultaneously between a given" bait" protein and multiple "prey" proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

No MeSH data available.


Related in: MedlinePlus

Multi-color BiFC experiments using tobacco BY-2 suspension culture cells. In A and B, BY-2 protoplasts were transfected using electroporation and visualized using epifluorescence microscopy. In C, protoplasts were transfected by direct DNA uptake and visualized using laser scanning confocal microscopy. Labels above each set of panels indicate the various constructions introduced into the cells. Labels below each set of panels indicates the filter set/channel imaged. mCherry labels the entire cell, whereas mCherry-VirD2NLS labels only the nucleus. Note that, regardless of the tag, Impa-1 localizes to the cytoplasm and Impa-4 localizes predominantly to the nucleus, with some cytoplasmic staining. In Panel A, the blue signal outside the main imaged cell indicates autofluorescence from dead cells. DIC, differential interference contrast image.
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Figure 3: Multi-color BiFC experiments using tobacco BY-2 suspension culture cells. In A and B, BY-2 protoplasts were transfected using electroporation and visualized using epifluorescence microscopy. In C, protoplasts were transfected by direct DNA uptake and visualized using laser scanning confocal microscopy. Labels above each set of panels indicate the various constructions introduced into the cells. Labels below each set of panels indicates the filter set/channel imaged. mCherry labels the entire cell, whereas mCherry-VirD2NLS labels only the nucleus. Note that, regardless of the tag, Impa-1 localizes to the cytoplasm and Impa-4 localizes predominantly to the nucleus, with some cytoplasmic staining. In Panel A, the blue signal outside the main imaged cell indicates autofluorescence from dead cells. DIC, differential interference contrast image.

Mentions: Figure 3 shows results of the tobacco BY-2 transfection assays, as visualized both by epifluorescence and confocal microscopy. AtImpa-1-nVenus interacted with VirE2-cCFP in the cytoplasm, whereas AtImpa-4 simultaneously interacted with VirE2-nCerulean. This latter interaction occurred predominantly in the nucleus, but also weakly in the cytoplasm (see Figure 3A, fourth panel and Figures 3B and 3C, third panels). Nuclear localization of AtImpa-4 was confirmed by co-localization of the nuclear marker mCherry-VirD2NLS.


Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells.

Lee LY, Fang MJ, Kuang LY, Gelvin SB - Plant Methods (2008)

Multi-color BiFC experiments using tobacco BY-2 suspension culture cells. In A and B, BY-2 protoplasts were transfected using electroporation and visualized using epifluorescence microscopy. In C, protoplasts were transfected by direct DNA uptake and visualized using laser scanning confocal microscopy. Labels above each set of panels indicate the various constructions introduced into the cells. Labels below each set of panels indicates the filter set/channel imaged. mCherry labels the entire cell, whereas mCherry-VirD2NLS labels only the nucleus. Note that, regardless of the tag, Impa-1 localizes to the cytoplasm and Impa-4 localizes predominantly to the nucleus, with some cytoplasmic staining. In Panel A, the blue signal outside the main imaged cell indicates autofluorescence from dead cells. DIC, differential interference contrast image.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2572157&req=5

Figure 3: Multi-color BiFC experiments using tobacco BY-2 suspension culture cells. In A and B, BY-2 protoplasts were transfected using electroporation and visualized using epifluorescence microscopy. In C, protoplasts were transfected by direct DNA uptake and visualized using laser scanning confocal microscopy. Labels above each set of panels indicate the various constructions introduced into the cells. Labels below each set of panels indicates the filter set/channel imaged. mCherry labels the entire cell, whereas mCherry-VirD2NLS labels only the nucleus. Note that, regardless of the tag, Impa-1 localizes to the cytoplasm and Impa-4 localizes predominantly to the nucleus, with some cytoplasmic staining. In Panel A, the blue signal outside the main imaged cell indicates autofluorescence from dead cells. DIC, differential interference contrast image.
Mentions: Figure 3 shows results of the tobacco BY-2 transfection assays, as visualized both by epifluorescence and confocal microscopy. AtImpa-1-nVenus interacted with VirE2-cCFP in the cytoplasm, whereas AtImpa-4 simultaneously interacted with VirE2-nCerulean. This latter interaction occurred predominantly in the nucleus, but also weakly in the cytoplasm (see Figure 3A, fourth panel and Figures 3B and 3C, third panels). Nuclear localization of AtImpa-4 was confirmed by co-localization of the nuclear marker mCherry-VirD2NLS.

Bottom Line: Additional expression of mCherry indicates transfected cells and sub-cellular structures.Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-1 in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus.The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA. zofangmj@gate.sinica.edu.tw.

ABSTRACT

Background: The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed.

Results: We have constructed a series of gene expression vectors, based upon the pSAT series of vectors, to facilitate the practice of multi-color BiFC. The bait protein is tagged with the C-terminal portion of CFP (cCFP), and prey proteins are tagged with the N-terminal portions of either Venus (nVenus) or Cerulean (nCerulean). Interaction of cCFP-tagged proteins with nVenus-tagged proteins generates yellow fluorescence, whereas interaction of cCFP-tagged proteins with nCerulean-tagged proteins generates blue fluorescence. Additional expression of mCherry indicates transfected cells and sub-cellular structures. Using this system, we have determined in both tobacco BY-2 protoplasts and in onion epidermal cells that Agrobacterium VirE2 protein interacts with the Arabidopsis nuclear transport adapter protein importin alpha-1 in the cytoplasm, whereas interaction of VirE2 with a different importin alpha isoform, importin alpha-4, occurs predominantly in the nucleus.

Conclusion: Multi-color BiFC is a useful technique to determine interactions simultaneously between a given" bait" protein and multiple "prey" proteins in living plant cells. The vectors we have constructed and tested will facilitate the study of protein-protein interactions in many different plant systems.

No MeSH data available.


Related in: MedlinePlus