Up to now, there was no available method that could achieve high transformation efficiency for transient gene expression in Arabidopsis with minimal manipulation. Pioneering efforts following the protocols similar to agro-infiltration of tobacco leaves led to rather limited success with great variability in Arabidopsis [10–13]. Very recently, an optimized A. tumefaciens vacuum infiltration protocol for young Arabidopsis seedlings had been described , which increased the overall success rate for obtaining transient expression in Arabidopsis. In this study, we explored a different strategy to transiently transform young Arabidopsis seedlings based on A. tumefaciens cocultivation, which we dubbed Fast Agro-mediated Seedling Transformation (FAST). Our data suggested that vacuum infiltration is not necessary as long as the surfactant Silwet L-77 is used in the FAST system (Figure 1; see additional file 2). Similarly, Silwet L-77 has previously been found to be critical in replacing laborious vacuum infiltration with the simple floral dip method in generating stably transformed Arabidopsis plants . In addition, we found here that Silwet L-77, even at suboptimal concentration, could partially substitute for the stressful wounding step in catalyzing the A. tumefaciens transformation of recalcitrant monocot species such as switchgrass (Figure 6D; ). The lack of a wounding-requirement for the Agrobacterium-mediated transformation of plants is also consistent with earlier studies [44, 45]. Thus, Silwet L-77 with a proper concentration is the key to achieve high transformation efficiency in this FAST system.
Following the FAST protocol, various constructs driven by different promoters were successfully expressed in young Arabidopsis seedlings with distinct genetic backgrounds including wild-type (Figure 1D; Figure 2A–E), mutant (Figure 2F and 2G) and transgenic seedlings (Figure 3B). The transient expression could occur in different organs of Arabidopsis seedlings including cotyledon, petiole and hypocotyl, and could occur in different cell types like epidermal and mesophyll cells (Figure 1D; see additional file 2). However, no transient expression could be detected in root (Figure 2D; data not shown). The tissue sensitivity of young Arabidopsis seedling to A. tumefaciens observed in this assay was in agreement with that described in a recent assay based on a vacuum infiltration method .
The FAST assay has advantages over other expression systems in planta
Compared with stable transgenic assay, the FAST assay is extremely simple and rapid. The entire assay from sowing seeds to protein detection could be readily completed within one week, in contrast to two to three months generally required for obtaining transgenic plants. Moreover, this transient assay allows the expression of deleterious proteins which would disrupt the Arabidopsis growth and development when expressed in transgenic lines.
In comparison with existing transient assays available for Arabidopsis, the FAST assay also provides several particular advantages: (i) only routine techniques and reagents are used in this assay, which breaks the constraint of specialized device such as a particle gun and reduces the overall cost for the experiments; (ii) unlike particle bombardment and protoplast transfection where high-quality plasmid DNA has to be prepared each time, A. tumefaciens cells used in this assay can be stored indefinitely and can be repeatedly generated from the glycerol stock before use; (iii) this transient assay could achieve higher co-transformation efficiency for two constructs when they are simultaneously carried in the same agrobacteria cell; (iv) a small-scale assay has already produced enough proteins for downstream analysis (e.g. western blot), and the protein production in this assay could be easily scaled up for other applications (e.g. pull-down assay); (v) the use of 4-day-old Arabidopsis seedlings instead of mature plants allows for rapid screening with minimal manipulations, which could even be adapted for a 96-well plate format (Figure 4). Unlike detached leaves, mesophyll protoplasts, or suspension cultured cells, the seedling as an intact plant should provide a more physiological environment for gene functional study. Compared with the recently described seedling vacuum infiltration approach , the FAST assay offers the advantage that neither the pressurizing device nor the supporting grid and fewer manual handling steps are required in the process.
The FAST assay is efficient in protein localization and protein-protein interaction studies
Although the variation of transformation efficiency from seedling to seedling is substantial in our transient system (see additional file 2) presumably due to an uneven distribution of agrobacteria during cocultivation, this has been significantly compensated by the advantage that the number of seedlings screenable in an assay could be very high. Thus, an overall constant output can be obtained. For a small-scale experiment using 30 seedlings, thousands of cells were readily transformed which could be screened under microscope within two hours and which should be more than enough to generate reliable protein localization or protein-protein interaction data.
The variation of gene expression from cell to cell is generally the major concern for a transient expression system. In the FAST assay, although we also detected various levels of protein expression in different cotyledon cells (Figure 2; see additional file 4), we have not noticed any alteration of protein targeting stemming from this variability except a partial mistargeting in a small percentage of cells (around 5%). This partial mistargeting likely was due to the extremely high concentration of introduced proteins in these cells, which may have led to the overflow of the compartment and a subsequent diffuse staining or aggregation of the proteins in the cytoplasm or nucleus. However, it is quite easy to gain thousands of transformed cells in a small-scale assay using 30 seedlings. Since the transient expression in each cell can be considered an independent event, the average distribution pattern of a given construct in a large population of cells should be considered typical. In protein-protein interaction studies, the variation of protein levels in different cells had negligible impact on FRET quantification when a proper algorithm such as Nfret  was used (see additional file 4).
Another possible concern is that high bacteria density and surfactant concentration may be stressful to the seedling cells and may generate secondary effects on protein localization. However, it has been suggested that the Agrobacterium infection generally would not change intracellular protein localization . Indeed, the overexpressed proteins in this study could faithfully target to cytoplasm, nucleus, organelles or specific cytoplasmic structures (e.g. actin filaments) in the vast majority of transformed cells at the appropriate observation time (i.e., after 36–40 hr cocultivation). Moreover, the vigorous movements of peroxisomes and mitochondria labeled by fluorescent organelle markers (see additional file 5) in the majority of transformed cells also is a reflection of their good physiological conditions, suggesting that this pathogen-associated transient assay was not very disturbing to normal cell physiology. Nevertheless, lower bacteria density and surfactant concentration in the cocultivation medium could create a milder environment which would be closer to the physiological conditions. In fact, we noticed that an A. tumefaciens density of OD600 = 0.1 (1.2 × 108 cfu/mL) and Silwet L-77 concentration as low as 0.002% could still generate large numbers of transformed cells and could on average reduce the overexpression level in the cell (data not shown).
To co-express two proteins in the same plant cell is the key for protein localization and protein-protein interaction studies. Here, we preferred to cocultivate the Arabidopsis seedlings with A. tumefaciens cells simultaneously carrying two compatible binary plasmids, a strategy modeled after the dual-binary T-DNA system . Since each binary plasmid contains the expression cassette for one protein, a theoretical 100% co-transformation efficiency could be guaranteed when a given plant cell is transformed by an A. tumefaciens cell. However, in practice, a co-transformation rate of approximately 70% was observed (data not shown). The reason behind this phenomenon is obscure but may be related to the nature of the different binary vectors and the encoded genes, as these two factors have previously been suggested to influence the transformation efficiency and the level of transient gene expression [3, 11, 15]. In protein-protein interaction studies, the high co-transformation efficiency enabled the co-expression of a protein pair (e.g. YFP-HY5 and Cerulean-HY5) in numerous cotyledon cells, which facilitated an easy acquisition of fluorescence data from a large number of cells for statistically significant FRET quantification and an easy detection of reconstituted YFP fluorescence in BiFC experiments.
Limitations of the FAST assay
While the FAST assay offers a number of advantages, it also comes with a few limitations that may reduce its usefulness in certain situations. For example, while we have observed high transformation rates in cotyledons, we did not detect any transgene expression in other tissues such as roots or true leaves, which could be relevant for interactions with endogenous proteins that are not expressed in cotyledons. Also, since cocultivation with A. tumefaciens could potentially induce host defenses as recently reported for A. tumefaciens infiltration , the FAST assay may not be appropriate for functional analysis of genes involved in plant defense response. However, this may not always be the case  and should be tested on a case by case basis.