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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

The FAST assays in other plant species. All cocultivations were carried out in the presence of 0.005% Silwet L-77 and bacteria of OD600 = 0.5. A, d35S::NLS-YFP-GUS expression in tobacco seedling after 40 hr of cocultivation. Scale bar = 0.5 mm. B, d35S::NLS-YFP-GUS expression in tomato seedling after 60 hr of cocultivation. Scale bar = 0.5 mm. C, Ubi-1::GUS expression in rice seedling after 6 days of cocultivation. Scale bar = 2 mm. D, Ubi-1::GUS expression in switchgrass seedling after 6 days of cocultivation. Scale bar = 2 mm.
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Figure 6: The FAST assays in other plant species. All cocultivations were carried out in the presence of 0.005% Silwet L-77 and bacteria of OD600 = 0.5. A, d35S::NLS-YFP-GUS expression in tobacco seedling after 40 hr of cocultivation. Scale bar = 0.5 mm. B, d35S::NLS-YFP-GUS expression in tomato seedling after 60 hr of cocultivation. Scale bar = 0.5 mm. C, Ubi-1::GUS expression in rice seedling after 6 days of cocultivation. Scale bar = 2 mm. D, Ubi-1::GUS expression in switchgrass seedling after 6 days of cocultivation. Scale bar = 2 mm.

Mentions: We further tested the applicability of the FAST assay in other representative dicot species such as tobacco and tomato using the NLS-YFP-GUS construct as a visible marker. After 40–60 hr cocultivation, bright nuclei labeled by YFP fluorescence were found throughout the cotyledons of tobacco and tomato seedlings (Figure 6A and 6B). The total area where a detectable expression of NLS-YFP-GUS protein occurred after 40 hr cocultivation was relatively small, though 60 hr cocultivation could typically generate a broader area with elevated protein expression levels (Figure 6B). We also tested the suitability of this transient assay in key monocot species like rice and switchgrass using a Ubi-1::GUS construct as a reporter. Transient expression was pronounced in these organisms if the cocultivation time was extended to 6 days. Interestingly, GUS expression could be observed in different tissues of rice seedlings including shoot and root (Figure 6C), while in swichgrass seedlings only the shoot had detectable GUS expression (Figure 6D). The detection of GUS expression in intact switchgrass tissues in our assay system was in contrast to previous observations where GUS expression was limited to cut surfaces or wound sites [20]. Thus, the FAST protocol appeared to improve transformation efficiency even without deliberate optimization of cocultivation conditions.


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)

The FAST assays in other plant species. All cocultivations were carried out in the presence of 0.005% Silwet L-77 and bacteria of OD600 = 0.5. A, d35S::NLS-YFP-GUS expression in tobacco seedling after 40 hr of cocultivation. Scale bar = 0.5 mm. B, d35S::NLS-YFP-GUS expression in tomato seedling after 60 hr of cocultivation. Scale bar = 0.5 mm. C, Ubi-1::GUS expression in rice seedling after 6 days of cocultivation. Scale bar = 2 mm. D, Ubi-1::GUS expression in switchgrass seedling after 6 days of cocultivation. Scale bar = 2 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: The FAST assays in other plant species. All cocultivations were carried out in the presence of 0.005% Silwet L-77 and bacteria of OD600 = 0.5. A, d35S::NLS-YFP-GUS expression in tobacco seedling after 40 hr of cocultivation. Scale bar = 0.5 mm. B, d35S::NLS-YFP-GUS expression in tomato seedling after 60 hr of cocultivation. Scale bar = 0.5 mm. C, Ubi-1::GUS expression in rice seedling after 6 days of cocultivation. Scale bar = 2 mm. D, Ubi-1::GUS expression in switchgrass seedling after 6 days of cocultivation. Scale bar = 2 mm.
Mentions: We further tested the applicability of the FAST assay in other representative dicot species such as tobacco and tomato using the NLS-YFP-GUS construct as a visible marker. After 40–60 hr cocultivation, bright nuclei labeled by YFP fluorescence were found throughout the cotyledons of tobacco and tomato seedlings (Figure 6A and 6B). The total area where a detectable expression of NLS-YFP-GUS protein occurred after 40 hr cocultivation was relatively small, though 60 hr cocultivation could typically generate a broader area with elevated protein expression levels (Figure 6B). We also tested the suitability of this transient assay in key monocot species like rice and switchgrass using a Ubi-1::GUS construct as a reporter. Transient expression was pronounced in these organisms if the cocultivation time was extended to 6 days. Interestingly, GUS expression could be observed in different tissues of rice seedlings including shoot and root (Figure 6C), while in swichgrass seedlings only the shoot had detectable GUS expression (Figure 6D). The detection of GUS expression in intact switchgrass tissues in our assay system was in contrast to previous observations where GUS expression was limited to cut surfaces or wound sites [20]. Thus, the FAST protocol appeared to improve transformation efficiency even without deliberate optimization of cocultivation conditions.

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