The genus Populus, which includes poplar, aspen, and cottonwood species, can serve as a model organism for perennial woody plants. Populus trees have several potential biological advantages such as small genome size, over 30 species growing worldwide, rapid juvenile growth, ease of clonal propagation, simplicity of genetic transformation and regeneration, and extensive genetic maps. The first draft genome of black cottonwood (Populus trichocarpa) was published in 2006  and provided the first insights into the genomic organization of a tree species. The genome contains more than 45,000 protein-coding genes and ~12% of the genes show no similarity to genes in the model plant Arabidopsis thaliana. To unravel the biological function of Populus genes, transgenic trees are generated and characterized, providing insight in unique characteristics, life style, and biological organization of perennial woody plants. However, a limited number of Populus genes have been targeted in transgenic studies due to a lengthy transformation process and the need for characterization of many transgenic lines for each construct.
Transient transformation assays complement stable transformation and make gene function analysis more efficient. Transient gene expression techniques (e.g., biolistic bombardment, protoplast transfection, and Agrobacterium tumefaciens (Agrobacterium)-infiltration) are available for model plants such as Arabidopsis and rice as well as for crop plants such as corn, potato, soybean, tomato, and wheat [3–7]. In Populus spp., several studies have successfully demonstrated transient gene expression via biolistic bombardment, protoplast transfection, and Agrobacterium co-cultivation [8–13]. For example, leaf epidermal and guard cells were transiently transformed with a reporter construct in a hybrid poplar (Populus tremula × Populus alba) by particle bombardment . Transient transfection of Populus protoplasts isolated from leaf tissues and suspension culture cells was achieved either by means of electroporation or chemically with polyethylene glycol [8, 10, 12]. Agrobacterium co-cultivation was used to examine ectopic expression of a reporter gene in P. nigra P. tomentosa, and P. trichocarpa before the regeneration of stable transformants [9, 11, 13]. However, such transformation assays have certain disadvantages such as the requirement for expensive equipment and supplies associated with particle bombardment. In addition, the transformation efficiency is relatively low in the transfection of Populus protoplasts, and only a small piece of a tissue isolated from seedlings is used in Agrobacterium co-cultivation. To avoid these drawbacks, another transient assay such as Agrobacterium-mediated infiltration would be useful for Populus trees. This assay allows for a simple transformation process, easy operation, and high transformation efficiency in several plant species including Arabidopsis, grapevine, potato, switchgrass, tobacco, and tomato [6, 7, 14–17].
Transient transformation techniques are available for rapid in vivo analyses of gene function such as protein subcellular localization, protein-protein interaction, and promoter activity. For in vivo functional analyses, reporter genes such as green fluorescence protein (GFP), variants of GFP, and firefly luciferase (LUC) are common tools for molecular and cell biology studies. Protein subcellular localization, which is crucial for elucidating the cellular functions of proteins, is easily monitored by a transient expression of fluorescent fusion protein . In this assay, a reporter construct harboring the gene of interest is fused with GFP or its variants and is transiently transformed into plant cells where intracellular localization is visualized through fluorescence of the reporter gene. Fluorescent proteins are also used for in vivo protein-protein interactions. Interaction assays, such as bimolecular fluorescence complementation (BiFC) and fluorescence resonance energy transfer (FRET), allow visualization of protein-protein interaction and subcellular localization of target proteins . In these assays, transient co-transformation techniques with two different constructs are a convenient and practical alternative to generation of double-transformed transgenic plant and allow testing of several constructs and combinations. In addition to fluorescent proteins, LUC is used as a reporter gene mainly for measuring transcriptional activity. The LUC reporter is suitable for real-time monitoring of gene expression as it has a relatively short half-life compared to fluorescent proteins. For example, expression patterns of circadian clock-related genes, most of which show rhythmic expression in a day, are extensively examined by LUC reporter assays . Typically, these assays use bioluminescence to visualize diurnal or circadian rhythms that express the LUC gene driven by a clock promoter of transgenic plants. Although studies regarding the plant clock system have principally been conducted using stable transformants, published studies report successful transient LUC assays [21, 22].
In this study, we develop a transient transformation assay– Agrobacterium-mediated vacuum infiltration–for a model tree, hybrid aspen (P. tremula × P. tremuloides). The assay was optimized for aspen leaves and applicable to various cell types in leaf tissue. We investigated subcellular localization of Populus proteins and protein interaction by co-transformation of two different plasmids in aspen leaf cells. Furthermore, in vivo promoter activity of a hybrid aspen clock gene was measured using a LUC reporter assay. The transient transformation assay we developed is enhanced throughput and easily employed for rapid functional analyses of Populus gene and protein expression.