TARGET: a transient transformation system for genome-wide transcription factor target discovery.

Dear Editor, Determining the fundamental structure of gene regulatory networks (GRN) is a major challenge of systems biology. In particular, inferring GRN structure from comprehensive gene expression and transcription factor (TF)–promoter interaction data sets has become an increasingly sought-after aim in both fundamental and agronomical research in plant biology (Bonneau et al., 2007; Ruffel et al., 2010). A crucial step for the assessment of GRN is the identification of the direct TF-target genes. Transgenic plant lines expressing tagged versions of the TF-of-interest can be used together with transcriptomic and DNA-binding analyses to obtain high-confidence lists of direct targets (e.g. Mönke et al., 2012). However, the generation of such transgenics can be a limiting factor, especially in largescale studies or in non-model species. Transient transformation of protoplasts is therefore often employed for the study of TF–promoter interactions, using co-expression of effector constructs with a TF-of-interest and reporter constructs with a promoter-of-interest. We have developed a rapid technique to study the genome-wide effects of TF activation in protoplasts that uses transient expression of a glucocorticoid receptor (GR)-tagged TF. We demonstrate here that this system can be used to rapidly retrieve information on direct target genes in less than 2 weeks’ time. As a proof-of-principle candidate, we used the well-studied TF, ABSCICIC ACID INSENSITIVE 3 (ABI3; Koornneef et al., 1989; Mönke et al., 2012) and established the de novo identification of the abscisic acid response element (ABRE) and a majority of the previously classified direct targets. We have named this technique TARGET, for Transient Assay Reporting Genome-wide Effects of Transcription factors. Technically, plant protoplasts are transfected with a plasmid (pBeaconRFP_GR) that expresses the TF-of-interest fused to GR, which allows the controlled entry of the chimeric GR–TF into the nucleus by addition of the GR–ligand dexamethasone (DEX; Schena and Yamamoto, 1988). In addition, the vector contains a separate expression cassette with a positive fluorescent selection marker (red fluorescent protein; RFP) which enables fluorescence-activated cell sorting (FACS) of successfully transformed protoplasts (Figure 1A; Bargmann and Birnbaum, 2009). This purification step allows reliable qPCR or transcriptomic analysis of multiple independent transfections, which would otherwise be hampered by the presence of a population of untransformed cells that varies from experiment to experiment. Lastly, the effect of target gene induction by DEX treatment is measured in the presence or absence of the translation inhibitor cycloheximide (CHX), allowing for the distinction of direct and indirect target genes of the TF under study (Supplementary Data). pBeaconRFP_GR–ABI3 was used to transfect protoplasts prepared from the roots of Arabidopsis seedlings, where ABI3, known largely for its role in seed development, has also been shown to be involved in development (Brady et al., 2003). As a first test of the TARGET system, the expression of known direct ABI3 targets PER1 and CRU3 was assayed by qPCR. Compared to control gene expression, both PER1 and CRU3 showed significant induction of transcript levels upon DEX treatment in the ABI3–GR-transfected protoplasts in the presence of CHX (Figure 1B and 1C, and Supplementary Data). PER1 and CRU3 expression in protoplasts transformed with an empty vector control showed no significant induction by DEX treatment (Figure 1B and 1C). Significant induction of CRU3 expression could only be measured when CHX was present, indicating that the effects of CHX may in some cases facilitate ABI3 function. Enhancement of ABA signaling output by protein synthesis inhibitors that could explain this phenomenon has been noted before by independent studies (Reeves et al., 2011). For the transcriptomic analysis, using ATH1 Genome Array chips, a two-way analysis of variance (ANOVA) was performed, followed by a Tukey post hoc test to identify genes whose expression is differentially regulated in response to DEX treatment in the absence or presence of CHX (p < 0.05, fold change >1.5; Supplementary Data). Genes found to be significantly regulated by DEX treatment in the empty vector control were omitted from further analysis. This analysis yielded a total of 668 unique genes whose expression was affected by DEX-induced nuclear localization of ABI3, 227 regulated genes without CHX, and 458 regulated genes with CHX (microarray results were validated by qPCR; Supplementary Data). There was just a 17-gene overlap with and without CHX, reiterating that (as was seen for CRU3 in preliminary qPCR analysis) there are many genes whose response to GR–ABI3 was facilitated by the presence of the protein synthesis inhibitor CHX. The 210 genes regulated only in the absence of CHX were categorized