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The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species.

Li JF, Park E, von Arnim AG, Nebenführ A - Plant Methods (2009)

Bottom Line: Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes.Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass.This method is potentially ideal for future automated high-throughput analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, USA. jli@molbio.mgh.harvard.edu.

ABSTRACT

Background: Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes.

Results: We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass.

Conclusion: The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis.

No MeSH data available.


Related in: MedlinePlus

FRET and BiFC evidence for HY5 homodimerization using the FAST assays. Arabidopsis seedlings were cocultivated with agrobacteria cells simultaneously carrying two FRET or BiFC constructs except for (B). A, The negative FRET control, soluble YFP and Cerulean alone, showed a low Nfret (normalized FRET) value in a representative region of the cotyledon. Note blue color. Scale bar = 60 μm. B, The positive FRET control, a YFP-Cerulean fusion, showed a high Nfret value in a representative region of the cotyledon. Note green to yellow color. The remaining Cerulean signal was barely visible due to the intensive energy transfer from Cerulean to YFP. Scale bar = 60 μm. C, YFP-HY5 and Cerulean-HY5 showed a medium Nfret value in a representative region of the cotyledon. Note light blue to green color. Scale bar = 60 μm. D, Quantification of the average Nfret value in a large number of cells for each FRET combination indicated. E, Reconstituted YFP fluorescence in a BiFC combination of YN-HY5 and YC-HY5 in a representative region of the cotyledon. Scale bar = 60 μm.
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Figure 5: FRET and BiFC evidence for HY5 homodimerization using the FAST assays. Arabidopsis seedlings were cocultivated with agrobacteria cells simultaneously carrying two FRET or BiFC constructs except for (B). A, The negative FRET control, soluble YFP and Cerulean alone, showed a low Nfret (normalized FRET) value in a representative region of the cotyledon. Note blue color. Scale bar = 60 μm. B, The positive FRET control, a YFP-Cerulean fusion, showed a high Nfret value in a representative region of the cotyledon. Note green to yellow color. The remaining Cerulean signal was barely visible due to the intensive energy transfer from Cerulean to YFP. Scale bar = 60 μm. C, YFP-HY5 and Cerulean-HY5 showed a medium Nfret value in a representative region of the cotyledon. Note light blue to green color. Scale bar = 60 μm. D, Quantification of the average Nfret value in a large number of cells for each FRET combination indicated. E, Reconstituted YFP fluorescence in a BiFC combination of YN-HY5 and YC-HY5 in a representative region of the cotyledon. Scale bar = 60 μm.

Mentions: In the FRET assay, we employed the normalized FRET (Nfret) calculation which removes spectral bleed-through and corrects for fluorophore expression level variation, and therefore is well-suited for widefield fluorescence microscopes [43]. For the negative FRET control, soluble YFP and Cerulean were found to be co-expressed in a large population of cotyledon cells after 40 hr cocultivation. As expected, these fluorescent proteins alone exhibited a low Nfret value (Figure 5A and 5D). By contrast, the positive FRET control of YFP-Cerulean fusion exhibited a high Nfret value (Figure 5B and 5D). In comparison, co-expressed YFP-HY5 and Cerulean-HY5 proteins showed a significant Nfret value (Figure 5C and 5D), which was markedly higher than that of the negative FRET control (Figure 5D), suggesting that HY5 proteins indeed homodimerize in vivo. The Nfret value of the HY5 homodimerization was lower than that of the positive FRET control (Figure 5D) as it resulted from a reversible protein-protein interaction rather than a permanent covalent attachment. Notably, although the expression levels of the fluorescent proteins in different cotyledon cells could be variable, we found that they did not affect the quantification of the Nfret value (see additional file 4; [43]), suggesting that the cell-to-cell variation of protein concentration does not prevent the FAST assay from producing reliable FRET data. The in vivo HY5 homodimerization detected by FRET could also be confirmed with the BiFC assay, where the reconstituted YFP fluorescence from YN-HY5 and YC-HY5 proteins was clearly visualized in the nuclei of many cotyledon cells after 40 hr cocultivation (Figure 5E).


The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species.

Li JF, Park E, von Arnim AG, Nebenführ A - Plant Methods (2009)

FRET and BiFC evidence for HY5 homodimerization using the FAST assays. Arabidopsis seedlings were cocultivated with agrobacteria cells simultaneously carrying two FRET or BiFC constructs except for (B). A, The negative FRET control, soluble YFP and Cerulean alone, showed a low Nfret (normalized FRET) value in a representative region of the cotyledon. Note blue color. Scale bar = 60 μm. B, The positive FRET control, a YFP-Cerulean fusion, showed a high Nfret value in a representative region of the cotyledon. Note green to yellow color. The remaining Cerulean signal was barely visible due to the intensive energy transfer from Cerulean to YFP. Scale bar = 60 μm. C, YFP-HY5 and Cerulean-HY5 showed a medium Nfret value in a representative region of the cotyledon. Note light blue to green color. Scale bar = 60 μm. D, Quantification of the average Nfret value in a large number of cells for each FRET combination indicated. E, Reconstituted YFP fluorescence in a BiFC combination of YN-HY5 and YC-HY5 in a representative region of the cotyledon. Scale bar = 60 μm.
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Related In: Results  -  Collection

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Figure 5: FRET and BiFC evidence for HY5 homodimerization using the FAST assays. Arabidopsis seedlings were cocultivated with agrobacteria cells simultaneously carrying two FRET or BiFC constructs except for (B). A, The negative FRET control, soluble YFP and Cerulean alone, showed a low Nfret (normalized FRET) value in a representative region of the cotyledon. Note blue color. Scale bar = 60 μm. B, The positive FRET control, a YFP-Cerulean fusion, showed a high Nfret value in a representative region of the cotyledon. Note green to yellow color. The remaining Cerulean signal was barely visible due to the intensive energy transfer from Cerulean to YFP. Scale bar = 60 μm. C, YFP-HY5 and Cerulean-HY5 showed a medium Nfret value in a representative region of the cotyledon. Note light blue to green color. Scale bar = 60 μm. D, Quantification of the average Nfret value in a large number of cells for each FRET combination indicated. E, Reconstituted YFP fluorescence in a BiFC combination of YN-HY5 and YC-HY5 in a representative region of the cotyledon. Scale bar = 60 μm.
Mentions: In the FRET assay, we employed the normalized FRET (Nfret) calculation which removes spectral bleed-through and corrects for fluorophore expression level variation, and therefore is well-suited for widefield fluorescence microscopes [43]. For the negative FRET control, soluble YFP and Cerulean were found to be co-expressed in a large population of cotyledon cells after 40 hr cocultivation. As expected, these fluorescent proteins alone exhibited a low Nfret value (Figure 5A and 5D). By contrast, the positive FRET control of YFP-Cerulean fusion exhibited a high Nfret value (Figure 5B and 5D). In comparison, co-expressed YFP-HY5 and Cerulean-HY5 proteins showed a significant Nfret value (Figure 5C and 5D), which was markedly higher than that of the negative FRET control (Figure 5D), suggesting that HY5 proteins indeed homodimerize in vivo. The Nfret value of the HY5 homodimerization was lower than that of the positive FRET control (Figure 5D) as it resulted from a reversible protein-protein interaction rather than a permanent covalent attachment. Notably, although the expression levels of the fluorescent proteins in different cotyledon cells could be variable, we found that they did not affect the quantification of the Nfret value (see additional file 4; [43]), suggesting that the cell-to-cell variation of protein concentration does not prevent the FAST assay from producing reliable FRET data. The in vivo HY5 homodimerization detected by FRET could also be confirmed with the BiFC assay, where the reconstituted YFP fluorescence from YN-HY5 and YC-HY5 proteins was clearly visualized in the nuclei of many cotyledon cells after 40 hr cocultivation (Figure 5E).

Bottom Line: Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes.Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass.This method is potentially ideal for future automated high-throughput analysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996-0840, USA. jli@molbio.mgh.harvard.edu.

ABSTRACT

Background: Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes.

Results: We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass.

Conclusion: The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis.

No MeSH data available.


Related in: MedlinePlus