Abstract
BackgroundPlant 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.ResultsWe 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.ConclusionThe 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.
Highlights
Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes
Optimization of transient expression in the Fast Agro-mediated Seedling Transformation (FAST) assay A. tumefaciens cocultivation method has been successfully adopted for the transformation of a wide range of plant species [17,18,19,20,21], which encouraged us to test whether A. tumefaciens cocultivation could work for the transient transformation of Arabidopsis
When 4-day-old Arabidopsis Col-0 seedlings were cocultivated with A. tumefaciens GV3101 cells carrying the binary construct pVHK-NLS-YFP-GUS (Table 1), which allows the expression of a d35S promoter-driven nuclear targeted YFP-GUS fusion [22], only sporadic expression of this visible marker in cotyledon cells was detected under the fluorescence microscope
Summary
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. Sequencing of the complete genomes of the model plant Arabidopsis thaliana and several other plant species has stressed the need to understand the functions of large numbers of unknown genes encoded within these genomes. Experimental assays are required to confirm every in silico prediction or to resolve ambiguous or uncertain predictions. This means that a myriad of genes have to be expressed and analyzed in planta. Transgene expression in some cases could interfere with normal plant growth and development due to an overdose of the functional proteins or dominant negative effect of non-functional products [3]
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