Rapid validation of transcriptional enhancers using agrobacterium-mediated transient assay

Yuan Lin,Bo Zhu,Fanli Meng,Jiming Jiang,Chao Fang

doi:10.1186/s13007-019-0407-y

Abstract

BackgroundEnhancers 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.ResultsWe 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.ConclusionsIn 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.

Highlights

Cis-regulatory elements (CREs), which regulate gene expression, are fundamental contributors towards growth and development processes
The strength of each enhancer for driving the reporter gene can be quantitatively measured and compared. This system can be used for initial screening of leaf-specific enhancers and potentially for mapping functional activity of candidate enhancers under different environmental conditions
Development of a luciferase‐based system for enhancer validation We developed an agrobacterium-mediated transient assay for potential enhancer activity (Fig. 1)

Summary

Introduction

Cis-regulatory elements (CREs), which regulate gene expression, are fundamental contributors towards growth and development processes. Enhancers are one of the most common classes of CREs and are associated with the regulation of most genes in higher eukaryotes. Enhancers were found to be widely associated with transcriptional regulation in all higher eukaryotes. A few transcriptional enhancers have been discovered in plant species [5,6,7]. The “enhancer trapping” methodology was developed to capture functional enhancers genome-wide in several plant species [8,9,10,11]. Enhancers are one of the most important classes of cis-regulatory elements (CREs) and play key roles in regulation of transcription in higher eukaryotes. Experimental validation is necessary to confirm the predicted enhancer function

Methods

Results

Conclusion