Limits...
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 onion epidermal cells. 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. 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 arrows indicate a gold particle in the nucleus. Imaging of gold particles results from 458 nm and 488 nm laser reflection. Because Cerulean images weakly using the Zeiss LSM510 Meta confocal microscope, the Cerulean images in Panels A and B were digitally enhanced by adjusting the brightness and contrast, in accordance with [22]. DIC, differential interference contrast image.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2572157&req=5

Figure 4: Multi-color BiFC experiments using onion epidermal cells. 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. 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 arrows indicate a gold particle in the nucleus. Imaging of gold particles results from 458 nm and 488 nm laser reflection. Because Cerulean images weakly using the Zeiss LSM510 Meta confocal microscope, the Cerulean images in Panels A and B were digitally enhanced by adjusting the brightness and contrast, in accordance with [22]. DIC, differential interference contrast image.

Mentions: Although A. tumefaciens can infect a wide variety of plants, including monocots [20], dicotyledonous plants such as tobacco are more "natural" hosts (i.e., plants which can develop Crown Gall disease). We therefore were interested in determining whether Agrobacterium VirE2 protein could interact with Arabidopsis importin α proteins in onion, which is not a natural Agrobacterium host. We introduced the various combinations of tagged protein-coding genes into onion epidermal cells by particle bombardment and visualized the sub-cellular sites of interaction using laser scanning confocal microscopy. Figure 4 shows that, as in tobacco BY-2 cells, VirE2 interacted with AtImpa-1 in the onion cytoplasm, and with AtImpa-4 predominantly in the onion nucleus. These experiments indicate that the sub-cellular sites of interaction of these proteins are not altered by the particular host, nor by the method of transgene delivery into these hosts.


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 onion epidermal cells. 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. 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 arrows indicate a gold particle in the nucleus. Imaging of gold particles results from 458 nm and 488 nm laser reflection. Because Cerulean images weakly using the Zeiss LSM510 Meta confocal microscope, the Cerulean images in Panels A and B were digitally enhanced by adjusting the brightness and contrast, in accordance with [22]. DIC, differential interference contrast image.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Multi-color BiFC experiments using onion epidermal cells. 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. 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 arrows indicate a gold particle in the nucleus. Imaging of gold particles results from 458 nm and 488 nm laser reflection. Because Cerulean images weakly using the Zeiss LSM510 Meta confocal microscope, the Cerulean images in Panels A and B were digitally enhanced by adjusting the brightness and contrast, in accordance with [22]. DIC, differential interference contrast image.
Mentions: Although A. tumefaciens can infect a wide variety of plants, including monocots [20], dicotyledonous plants such as tobacco are more "natural" hosts (i.e., plants which can develop Crown Gall disease). We therefore were interested in determining whether Agrobacterium VirE2 protein could interact with Arabidopsis importin α proteins in onion, which is not a natural Agrobacterium host. We introduced the various combinations of tagged protein-coding genes into onion epidermal cells by particle bombardment and visualized the sub-cellular sites of interaction using laser scanning confocal microscopy. Figure 4 shows that, as in tobacco BY-2 cells, VirE2 interacted with AtImpa-1 in the onion cytoplasm, and with AtImpa-4 predominantly in the onion nucleus. These experiments indicate that the sub-cellular sites of interaction of these proteins are not altered by the particular host, nor by the method of transgene delivery into these hosts.

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