Rapid validation of transcriptional enhancers using agrobacterium-mediated transient assay

Abstract

Background: Enhancers are one of the most important classes of cis-regulatory elements (CREs) and play key roles in regulation of transcription in higher eukaryotes. Enhancers are difficult to identify because they lack positional constraints relative to their cognate genes. Excitingly, several recent studies showed that plant enhancers can be predicted based on their distinct features associated with open chromatin. However, experimental validation is necessary to confirm the predicted enhancer function.

Results: We developed a rapid enhancer validation system based on Nicotiana benthamiana. A set of 12 intergenic and intronic enhancers, identified in Arabidopsis thaliana, were cloned into a vector containing a minimal 35S promoter and a luciferase reporter gene, and were then infiltrated into N. benthamiana leaves mediated by agrobacterium. The enhancer activity of each candidate was quantitatively assayed based on bioluminescence measurement. The data from this luciferase-based validation was correlated with previous data derived from transgenic assays in A. thaliana. In addition, the relative strength of different enhancers for driving the reporter gene can be quantitatively compared. We demonstrate that this system can also be used to map the functional activity of a candidate enhancer under different environmental conditions.

Conclusions: In summary, we developed a rapid and efficient plant enhancer validation system based on a luciferase reporter and N. benthamiana-based leaf agroinfiltration. This system can be used for initial screening of leaf-specific enhancers and for validating candidate leaf enhancers from dicot species. It can potentially be used to examine the activity of candidate enhancers under different environmental conditions.

Keywords: Enhancer validation; Luciferase; Nicotiana benthamiana; Transient assay.

Fig. 1

Schematic representation of the enhancer validation pipeline using a luciferase-based transient assay. a Candidate enhancers were predicted based on DHSs and other chromatin datasets. b The predicted enhancer sequence was synthesized and cloned in the pCAMBIA-CRE-LUC vector containing a mini 35S promoter and the firefly luciferase reporter gene, and was transferred into Agrobacterium strain GV3101. c Each construct was agroinfiltrated into N. benthamiana leaves together with both positive and negative controls. d Bioluminescent data were collected using the NightSHADE LB 985 plant imaging system

Fig. 2

Measurement of enhancer activity based on bioluminescent imaging in vivo. a A representative N. benthamiana leaf infiltrated with constructs containing six different enhancers (sample 2, 3 and 5–8), the 35S promoter (positive control, sample1) and a mini35S promoter (negative control, sample 4). Data was collected at 30 h after agroinfiltration. Color scale of the luminescent signal intensity; purple, least intense signal; red, most intense signal; Inner gray for sample 1, over saturated intense signal. b Three-dimension bioluminescent signal of (A). c Three-dimension bioluminescent signal of (A) after excluding sample 1

Fig. 3

Relative signal intensity of candidate enhancers based on measurements using luciferase-based transient assays (left panel) and GUS-based transgenic assays in leaf (right panel). Left panel: Data from luciferase-based transient assays. Y-axis: 12 candidate enhancers along with two negative controls (N1 and mini35S); X-axis, relative bioluminescent signal strength normalized with enhancer C1R as standard 1. Data represent the mean ± SEM (n = 3). Right panel: Leaf data from GUS-based transgenic assays [12]. The strength of the enhancer in leaf is defined for three ordinal levels (+, ++, +++). No leaf GUS signal present as (−). DHS data associated with leaf tissue for each candidate enhancer is listed as yes (Y) or No (N). Color code for DHS: green, leaf-specific; blue, root-specific; purple, flower-specific; gray, not associated with DHS

Fig. 4

Enhancer and promoter activities associated with light/dark cycles. a Oscillation of the 35S promoter activity under 12 h light/12 h dark condition. b Oscillation of activities of seven enhancers under 12 h light/12 h dark condition. For data comparison between (a) and (b), the first peak value of the 35S promoter of (a) at 30 h was set as 1. c Luminescence activity of the 35S promoter under dark condition. d Luminescence activity of seven enhancers under dark condition. For data comparison between (c) and (d), the first peak value of the 35S promoter from (c) at 40 h was set as 1. Each data point was collected every 4 h after agroinfiltration. Data represent the mean ± SEM (n = 3)