Systems biology aims to integrate large-scale data from the transcriptome, the proteome and the metabolome to provide a comprehensive view of the functional structure of a cell or organism. Such approaches require identification and integration of protein networks that rely on subcellular protein partitioning and interaction. To address the question of protein localization and interaction on a large scale, it is essential to express tagged proteins in cells in an efficient, rapid, versatile and non-destructive way. It is also important that the experiment is performed under physiological conditions, in particular by preserving tissue organization and cellular integrity and minimizing transformation and culture stresses as much as possible.
Several methods for transient transformation have been described in plants, particularly Arabidopsis. For example, Yoo et al. (2007) described an efficient and versatile transient assay involving transformation of mesophyll protoplasts. However, protoplasts are individual cells that have lost their identity and positional information, and which very quickly undergo profound de-differentiation processes accompanied by extensive chromatin remodeling (Tessadori et al., 2007). Alternatively, transient transformation of cultured Arabidopsis cells with Agrobacterium allows systematic subcellular protein localization or analysis of transcription factor–promoter interactions (Berger et al., 2007; Koroleva et al., 2005). However, like protoplasts, cultured Arabidopsis cells are physiologically distinct from native plant cells. They have lost their identity and cell-to-cell connections, and have often been sub-cultured for many years in synthetic medium which may cause extensive genetic and phenotypic changes. Moreover, cultured cells have limited genetic origins, and the considerable genetic diversity existing in Arabidopsis cannot be exploited using this method.
To avoid these drawbacks, transient plant transformation has been achieved through particle bombardment or Agrobacterium infiltration. Particle bombardment allows direct transformation of plant cells that are resistant to Agrobacterium infection, but shows relatively poor efficiency and induces significant mechanical stress. The most versatile and efficient method of transient plant transformation still relies on Agrobacterium-based infiltration. Agrobacterium can transfer foreign DNA into plant cells with intact cell walls and without excessive mechanical stress. Several reports have described protocols for transient transformation of Arabidopsis and tobacco leaves, stems and flowers by Agrobacterium, which mainly rely on the expression of GUS-based constructs (Kapila et al., 1997; Lee and Yang, 2006; Wroblewski et al., 2005; Yang et al., 2000). Assays for GUS activity are destructive and do not allow visualization of protein subcellular localization and interactions. Conversely, fluorescent protein (FP)-based constructs have been widely used to monitor protein subcellular localization and protein–protein interaction via fluorescence resonance energy transfer or bimolecular fluorescent complementation (BiFC). Indeed, tobacco epidermal cells have been used to efficiently express FP-based constructs with minimal stress responses. This allows in-depth analysis of endomembrane dynamics, mainly of the ER and Golgi apparatus (Batoko et al., 2000; Boevink et al., 1998; Nebenfuhr et al., 1999; Runions et al., 2006; daSilva et al., 2004), but also monitoring of protein–protein interactions (Bracha et al., 2002; Bracha-Drori et al., 2004; Citovsky et al., 2006; Walter et al., 2004; Zamyatnin et al., 2006). However, tobacco epidermal cells represent an heterologous system for expression of Arabidopsis proteins, and, like cell culture, cannot be used to test expression in an extensive collection of mutants and transgenic marker lines. Therefore, an efficient transient expression system in Arabidopsis seedlings for FP-based constructs is essential for functional analysis of protein networks in various genetic backgrounds. Manual leaf infiltration of FP constructs developed for tobacco has been scarcely used in Arabidopsis (Lavy et al., 2002), as the method induces mechanical and biotic stresses often associated with extensive non-specific autofluorescence that can impair FP detection. Direct transient transformation of seedlings with FP constructs has been documented but was not thoroughly tested (Lagrange et al., 2003). Recently, a new procedure for epidermal root cell transformation by Agrobacterium rhizogenes has been described, allowing dynamic analysis of endosomal compartments in root hair cells (Campanoni et al., 2007). Here, we describe the optimization of an infiltration protocol of Arabidopsis seedlings with Agrobacterium tumefaciens that allows rapid and efficient expression of fluorescent proteins (GFP, YFP, mRFP1, mCherry) in cotyledons and leaves. It is possible to label various cell types and all the subcellular compartments. Co-expression of GFP- and mRFP1-tagged proteins in the same cell allows direct co-localization studies, as illustrated by systematic analyses of the subcellular localization of enzymes involved in the sphingolipid metabolic pathway. Finally, we show that transient co-expression of proteins in Arabidopsis can be used for monitoring of protein–protein interactions by BiFC.