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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

Schematic diagrams of the multi-cloning sites of the "recipient" plasmids. A, Plasmid based upon pUC119; B, T-DNA binary vector ocs-bar-RCS2-2 (pE3519). Pocs, octopine synthase promoter; bar, phosphinothricin/bialaphos/Basta resistance gene; Termocs, octopine synthase polyA addition signal; LB and RB, T-DNA left and right border repeat sequences, respectively.
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Figure 2: Schematic diagrams of the multi-cloning sites of the "recipient" plasmids. A, Plasmid based upon pUC119; B, T-DNA binary vector ocs-bar-RCS2-2 (pE3519). Pocs, octopine synthase promoter; bar, phosphinothricin/bialaphos/Basta resistance gene; Termocs, octopine synthase polyA addition signal; LB and RB, T-DNA left and right border repeat sequences, respectively.

Mentions: One of the versatile features of the pSAT series of vectors is that, by using expression cassettes flanked by different rare-cutting enzyme sites, multiple cassettes can be "loaded" into a common replicating plasmid or a T-DNA binary vector for simultaneous introduction into plant cells. Thus, we constructed each new expression cassette in the pSAT1 or pSAT1A vector series (flanked by AscI sites), pSAT4 or pSAT4A vector series (flanked by I-SceI sites), and pSAT6 (flanked by PI-PspI sites). As "recipient" vectors for these expression cassettes, we introduced a multiple rare-cutting site (RCS) sequence into pBluescript KS+ (pBS-RCS), pUC119 (pUC-RCS), and an altered version of the T-DNA binary vector pPZP-RCS2 [13]. pPZP-RCS2 was modified by placing a Pocs-bar-Termocs selection marker cassette into the EcoRI site of the binary vector, near the T-DNA left border, generating ocs-bar-RCS2-2 (pE3519). Figures 2A and 2B show maps of the pUC119 and T-DNA binary vectors, respectively.


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)

Schematic diagrams of the multi-cloning sites of the "recipient" plasmids. A, Plasmid based upon pUC119; B, T-DNA binary vector ocs-bar-RCS2-2 (pE3519). Pocs, octopine synthase promoter; bar, phosphinothricin/bialaphos/Basta resistance gene; Termocs, octopine synthase polyA addition signal; LB and RB, T-DNA left and right border repeat sequences, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Schematic diagrams of the multi-cloning sites of the "recipient" plasmids. A, Plasmid based upon pUC119; B, T-DNA binary vector ocs-bar-RCS2-2 (pE3519). Pocs, octopine synthase promoter; bar, phosphinothricin/bialaphos/Basta resistance gene; Termocs, octopine synthase polyA addition signal; LB and RB, T-DNA left and right border repeat sequences, respectively.
Mentions: One of the versatile features of the pSAT series of vectors is that, by using expression cassettes flanked by different rare-cutting enzyme sites, multiple cassettes can be "loaded" into a common replicating plasmid or a T-DNA binary vector for simultaneous introduction into plant cells. Thus, we constructed each new expression cassette in the pSAT1 or pSAT1A vector series (flanked by AscI sites), pSAT4 or pSAT4A vector series (flanked by I-SceI sites), and pSAT6 (flanked by PI-PspI sites). As "recipient" vectors for these expression cassettes, we introduced a multiple rare-cutting site (RCS) sequence into pBluescript KS+ (pBS-RCS), pUC119 (pUC-RCS), and an altered version of the T-DNA binary vector pPZP-RCS2 [13]. pPZP-RCS2 was modified by placing a Pocs-bar-Termocs selection marker cassette into the EcoRI site of the binary vector, near the T-DNA left border, generating ocs-bar-RCS2-2 (pE3519). Figures 2A and 2B show maps of the pUC119 and T-DNA binary vectors, respectively.

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