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

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.

Figure 1

Optimization of transient expression in the FAST assay. A, Effect of Silwet L-77 concentration on transient expression. Transient expression efficiency was quantified by the average luminescence intensity of each Arabidopsis seedling expressing a d35S::hRLuc construct when Silwet L-77 of indicated concentration and bacteria of OD600 = 0.3 were included in the cocultivation medium. The assay was carried out after 40 hr cocultivation. The negative control indicated by letter "C" was performed using Agrobacteria cells carrying no binary vector. B, Effect of bacteria density on transient expression. Transient expression efficiency was quantified by the average luminescence intensity of each Arabidopsis seedling expressing a d35S::hRLuc construct when 0.005% Silwet L-77 and bacteria of indicated density were included in the cocultivation medium. The assay was carried out after 40 hr cocultivation. C, Time-course for transient expression. Transient expression efficiency was quantified by the average luminescence intensity of each Arabidopsis seedling at different time points during the cocultivation when 0.005% Silwet L-77 and bacteria of OD600 = 0.5 were used in the cocultivation medium. D, Expression of a d35S::NLS-YFP-GUS construct in a representative Arabidopsis seedling after 36 hr cocultivation when 0.005% Silwet L-77 and bacteria of OD600 = 0.5 were used in the cocultivation medium. Scale bar = 0.3 mm. E, Western blot analysis of NLS-YFP-GUS expression in 8 transformed (NLS-YFP-GUS) or untransformed (C) Arabidopsis seedlings after 36 hr cocultivation when 0.005% Silwet L-77 and bacteria of OD600 = 0.5 were used in the cocultivation medium.

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.

Figure 3

Subcellular localization studies by the FAST assays. A, YFP-At3g51660 (green) targeted to peroxisomes labeled by Peroxisome-CFP (Magenta) as indicated by the merged image. Arabidopsis seedlings were cocultivated simultaneously with agrobacteria cells carrying d35S::YFP-At3g51660 construct and those carrying d35S::Peroxisome-CFP marker. B, At1g01170-YFP (green) targeted to mitochondria labeled by Mitochondria-CFP (Magenta) as indicated by the merged image. Transgenic Arabidopsis seedlings expressing Mitochondria-CFP marker were cocultivated with agrobacteria cells carrying d35S::At1g01170-YFP construct. C, At2g47840-YFP (green) targeted to the envelope of chloroplasts labeled by autofluorescence (Magenta) as indicated by the merged image. Arabidopsis seedlings were cocultivated with agrobacteria cells carrying d35S::At2g47840-YFP construct. D, At2g47840-YFP (green) targeted to the envelope of plastids labeled by Plastid-CFP (Magenta) as indicated by the merged image. Arabidopsis seedlings were cocultivated with agrobacteria cells simultaneously carrying d35S::At2g47840-YFP and d35S::Plastid-CFP constructs. Scale bar = 15 μm.

Figure 4

The FAST assay in 96-well plate. A, In a 4 × 4 grid of a 96-well plate, 2–3 Arabidopsis seedlings per well were soaked in 100 μL cocultivation medium containing 0.005% Silwet L-77 and bacteria of OD600 = 0.5. B-Q, Observation of protein expression after 40 hr cocultivation. B, ER-YFP; C, ER-CFP; D, Golgi-YFP; E, Golgi-CFP; F, Mitochondria-YFP; G, Mitochondria-CFP; H, Peroxisome-YFP; I, Peroxisome-CFP; J, Plastid-YFP; K, Plastid-CFP; L, YFP-HY5 labeling nucleus; M, Cerulean-HY5 labeling nucleus; N, YFP-At3g51660 labeling peroxisomes; O, At1g01170-YFP labeling mitochondria; P, At2g47840-YFP labeling plastids; Q, YFP-FABD2 labeling actin filaments. B-Q, Scale bar = 20 μm.

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.

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.