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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

Transient expression of various constructs in Arabidopsis by the FAST assays. All cocultivations were carried out for 40 hr in the presence of 0.005% Silwet L-77 and bacteria of OD600 = 0.5. A, d35S::Peroxisome-CFP marker in wild-type seedling. Scale bar = 60 μm. B, d35S::YFP-FABD2 in wild-type seedling. Scale bar = 20 μm. C, d35S::YFP-MYA1CCGT in wild-type seedling. Scale bar = 60 μm. D, ubi-1::GUS in 33 wild-type seedling. Scale bar = 0.5 mm. E, MYA1pro::YFP-MYA1 in wild-type seedling. Scale bar = 60 μm. F, MYA1pro::YFP-MYA1 in mya1 mutant seedling. Scale bar = 60 μm. G, d35S::NLS-YFP-GUS in eif3h mutant seedling. Scale bar = 60 μm.
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Figure 2: Transient expression of various constructs in Arabidopsis by the FAST assays. All cocultivations were carried out for 40 hr in the presence of 0.005% Silwet L-77 and bacteria of OD600 = 0.5. A, d35S::Peroxisome-CFP marker in wild-type seedling. Scale bar = 60 μm. B, d35S::YFP-FABD2 in wild-type seedling. Scale bar = 20 μm. C, d35S::YFP-MYA1CCGT in wild-type seedling. Scale bar = 60 μm. D, ubi-1::GUS in 33 wild-type seedling. Scale bar = 0.5 mm. E, MYA1pro::YFP-MYA1 in wild-type seedling. Scale bar = 60 μm. F, MYA1pro::YFP-MYA1 in mya1 mutant seedling. Scale bar = 60 μm. G, d35S::NLS-YFP-GUS in eif3h mutant seedling. Scale bar = 60 μm.

Mentions: To ensure the reliability of the FAST system, the expression of a diverse array of constructs driven by various promoters in Arabidopsis seedlings with different genetic contexts was tested. We first employed this transient system to express d35S-driven organelle markers, namely Peroxisome-CFP and Mitochondria-YFP [25], in wild-type Arabidopsis seedlings. After 40 hr cocultivation, these two fluorescent proteins were visualized in numerous cotyledon cells (Figure 2A; data not shown) and were decorating different populations of motile punctate structures when co-expressed in the same cell (see additional file 5). By contrast, less than 5% of transformed cells demonstrated aberrant fluorescence distribution in the cytosol or nucleus in addition to proper organelle labeling. These abnormal cells all had exceptionally high levels of protein expression which could have been responsible for the partial mistargeting. Longer cocultivation (e.g. 72 hr) resulted in increased mistargeting of the organelle markers and cessation of the organelle movements (data not shown), suggesting that the extended cocultivation beyond the optimal time point (i.e., 36–40 hr) increased the stress to the cells and reduced the overall reliability of the assay. Besides compartment-targeted proteins, we also expressed a d35S-driven YFP-FABD2 construct, which clearly labeled the actin networks in the cytosol (Figure 2B) as described for its GFP version in a previous study [26]. Furthermore, we successfully expressed a d35S-driven YFP fusion of the truncated Arabidopsis myosin MYA1 protein including its C-terminal coiled-coil and globular tail domains, and found that this construct targeted to punctate structures in the cytosol (Figure 2C) as we had described earlier [27].


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)

Transient expression of various constructs in Arabidopsis by the FAST assays. All cocultivations were carried out for 40 hr in the presence of 0.005% Silwet L-77 and bacteria of OD600 = 0.5. A, d35S::Peroxisome-CFP marker in wild-type seedling. Scale bar = 60 μm. B, d35S::YFP-FABD2 in wild-type seedling. Scale bar = 20 μm. C, d35S::YFP-MYA1CCGT in wild-type seedling. Scale bar = 60 μm. D, ubi-1::GUS in 33 wild-type seedling. Scale bar = 0.5 mm. E, MYA1pro::YFP-MYA1 in wild-type seedling. Scale bar = 60 μm. F, MYA1pro::YFP-MYA1 in mya1 mutant seedling. Scale bar = 60 μm. G, d35S::NLS-YFP-GUS in eif3h mutant seedling. Scale bar = 60 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2693113&req=5

Figure 2: Transient expression of various constructs in Arabidopsis by the FAST assays. All cocultivations were carried out for 40 hr in the presence of 0.005% Silwet L-77 and bacteria of OD600 = 0.5. A, d35S::Peroxisome-CFP marker in wild-type seedling. Scale bar = 60 μm. B, d35S::YFP-FABD2 in wild-type seedling. Scale bar = 20 μm. C, d35S::YFP-MYA1CCGT in wild-type seedling. Scale bar = 60 μm. D, ubi-1::GUS in 33 wild-type seedling. Scale bar = 0.5 mm. E, MYA1pro::YFP-MYA1 in wild-type seedling. Scale bar = 60 μm. F, MYA1pro::YFP-MYA1 in mya1 mutant seedling. Scale bar = 60 μm. G, d35S::NLS-YFP-GUS in eif3h mutant seedling. Scale bar = 60 μm.
Mentions: To ensure the reliability of the FAST system, the expression of a diverse array of constructs driven by various promoters in Arabidopsis seedlings with different genetic contexts was tested. We first employed this transient system to express d35S-driven organelle markers, namely Peroxisome-CFP and Mitochondria-YFP [25], in wild-type Arabidopsis seedlings. After 40 hr cocultivation, these two fluorescent proteins were visualized in numerous cotyledon cells (Figure 2A; data not shown) and were decorating different populations of motile punctate structures when co-expressed in the same cell (see additional file 5). By contrast, less than 5% of transformed cells demonstrated aberrant fluorescence distribution in the cytosol or nucleus in addition to proper organelle labeling. These abnormal cells all had exceptionally high levels of protein expression which could have been responsible for the partial mistargeting. Longer cocultivation (e.g. 72 hr) resulted in increased mistargeting of the organelle markers and cessation of the organelle movements (data not shown), suggesting that the extended cocultivation beyond the optimal time point (i.e., 36–40 hr) increased the stress to the cells and reduced the overall reliability of the assay. Besides compartment-targeted proteins, we also expressed a d35S-driven YFP-FABD2 construct, which clearly labeled the actin networks in the cytosol (Figure 2B) as described for its GFP version in a previous study [26]. Furthermore, we successfully expressed a d35S-driven YFP fusion of the truncated Arabidopsis myosin MYA1 protein including its C-terminal coiled-coil and globular tail domains, and found that this construct targeted to punctate structures in the cytosol (Figure 2C) as we had described earlier [27].

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