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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

Control experiments show specificity of multi-color BiFC experiments. Onion cells were transfected by particle bombardment 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-VirD2NLS labels the nucleus. In Panels A and B, respectively, VirE2-cCFP interacts with Impa-1-nVenus or Impa-4-nVenus expressed individually. Localization of yellow fluorescence is identical to that seen when both prey proteins are co-expressed with VirE2-cCFP. Note that in Panel C, yellow fluorescence is not reconstituted in the absence of interacting bait and prey proteins. DIC, differential interference contrast image. Size bars indicate 50 microns.
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Figure 5: Control experiments show specificity of multi-color BiFC experiments. Onion cells were transfected by particle bombardment 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-VirD2NLS labels the nucleus. In Panels A and B, respectively, VirE2-cCFP interacts with Impa-1-nVenus or Impa-4-nVenus expressed individually. Localization of yellow fluorescence is identical to that seen when both prey proteins are co-expressed with VirE2-cCFP. Note that in Panel C, yellow fluorescence is not reconstituted in the absence of interacting bait and prey proteins. DIC, differential interference contrast image. Size bars indicate 50 microns.

Mentions: To assure that, using these vectors, interaction of a bait protein in the presence of multiple prey proteins does not differ from interaction of a bait protein in the presence of a single prey protein, we co-expressed VirE2-cCFP with either AtImpa-1 or AtImpa4. Figures 5A and 5B show that, as described above, VirE2 interacts with AtImpa-1 in the cytoplasm and with AtImpa-4 predominantly in the nucleus of bombarded onion cells. In addition, we tested whether fluorescence complementation could occur in the absence of interacting bait and prey proteins. Figure 5C shows that when VirE2-cCFP was co-expressed with nVenus (empty-nVenus), no yellow fluorescence was observed. To assure that the onion cells had received the various BiFC constructs, we included in this latter control experiment a mCherry-VirD2NLS expression construct cloned into the same vector as the BiFC expression cassettes. Red fluorescence was detected and localized to the nucleus, indicating that the onion cell had received the various BiFC constructs. Thus, we did not detect reconstituted yellow fluorescence in the absence of appropriately tagged bait and prey proteins.


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)

Control experiments show specificity of multi-color BiFC experiments. Onion cells were transfected by particle bombardment 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-VirD2NLS labels the nucleus. In Panels A and B, respectively, VirE2-cCFP interacts with Impa-1-nVenus or Impa-4-nVenus expressed individually. Localization of yellow fluorescence is identical to that seen when both prey proteins are co-expressed with VirE2-cCFP. Note that in Panel C, yellow fluorescence is not reconstituted in the absence of interacting bait and prey proteins. DIC, differential interference contrast image. Size bars indicate 50 microns.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Control experiments show specificity of multi-color BiFC experiments. Onion cells were transfected by particle bombardment 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-VirD2NLS labels the nucleus. In Panels A and B, respectively, VirE2-cCFP interacts with Impa-1-nVenus or Impa-4-nVenus expressed individually. Localization of yellow fluorescence is identical to that seen when both prey proteins are co-expressed with VirE2-cCFP. Note that in Panel C, yellow fluorescence is not reconstituted in the absence of interacting bait and prey proteins. DIC, differential interference contrast image. Size bars indicate 50 microns.
Mentions: To assure that, using these vectors, interaction of a bait protein in the presence of multiple prey proteins does not differ from interaction of a bait protein in the presence of a single prey protein, we co-expressed VirE2-cCFP with either AtImpa-1 or AtImpa4. Figures 5A and 5B show that, as described above, VirE2 interacts with AtImpa-1 in the cytoplasm and with AtImpa-4 predominantly in the nucleus of bombarded onion cells. In addition, we tested whether fluorescence complementation could occur in the absence of interacting bait and prey proteins. Figure 5C shows that when VirE2-cCFP was co-expressed with nVenus (empty-nVenus), no yellow fluorescence was observed. To assure that the onion cells had received the various BiFC constructs, we included in this latter control experiment a mCherry-VirD2NLS expression construct cloned into the same vector as the BiFC expression cassettes. Red fluorescence was detected and localized to the nucleus, indicating that the onion cell had received the various BiFC constructs. Thus, we did not detect reconstituted yellow fluorescence in the absence of appropriately tagged bait and prey proteins.

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