Molecular screening tools to study Arabidopsis transcription factors

Abstract

In the model plant Arabidopsis thaliana, more than 2000 genes are estimated to encode transcription factors (TFs), which clearly emphasizes the importance of transcriptional control. Although genomic approaches have generated large TF open reading frame (ORF) collections, only a limited number of these genes is functionally characterized, yet. This review evaluates strategies and methods to identify TF functions. In particular, we focus on two recently developed TF screening platforms, which make use of publically available GATEWAY®-compatible ORF collections. (1) The Arabidopsis thaliana TF ORF over-Expression (AtTORF-Ex) library provides pooled collections of transgenic lines over-expressing HA-tagged TF genes, which are suited for screening approaches to define TF functions in stress defense and development. (2) A high-throughput microtiter plate based protoplast trans activation (PTA) system has been established to screen for TFs which are regulating a given promoter:Luciferase construct in planta.

A Genome-Scale Resource for the Functional Characterization of Arabidopsis Transcription Factors

“…This collection is compatible with recombination-based cloning, which allows bulk transfer of TF coding sequences into any destination plasmid such as the ones already created by the Arabidopsis community (Karimi et al, 2002; Rhee et al, 2003). The collection was conceived as a high-throughput resource to generate large genomic tools for the study of Arabidopsis TF function (Castrillo et al, 2011; Chang et al, 2013; Ou et al, 2011; Siggers et al, 2012; Wehner et al, 2011). One of the main goals of our project was to provide the highest clone quality.…”

“…Arabidopsis protoplasts were isolated from 4–5 weeks old Columbia ecotype (Col-0) seedlings using a procedure adapted from previously published protocols (Kim and Somers, 2010; Wu et al, 2009), and aliquoted in white 96-well plates (Evergreen Scientific). Protoplasts were co-transformed following a previously published procedure (Wehner et al, 2011), with a TF-overexpression (or control) vector (5 μg per 4 kb) and the reporter vector pOmegaCCA1-LUC_SK+ (Kim and Somers, 2010) (1 μg per 4 kb) that contains a CCA1::LUC+ reporter construct. After plasmid co-transformation, cells were resuspended in a solution containing 5% fetal bovine serum (Sigma) and 50 μM luciferin (Biosynth) as described before (Kim and Somers, 2010), and 96-well plates were covered with a transparent plastic lid.…”

NINJA-Associated ERF19 Negatively RegulatesArabidopsisPattern-Triggered Immunity

“…http://dx.doi.org/10.1101/180059 doi: bioRxiv preprint first posted online Aug. 23, 2017;7 the AtTORF-Ex collection (Weiste et al, 2007;Wehner et al, 2011) to B. cinerea. Notably, we found a transgenic line overexpressing ERF19/ERF019 (At1g22810, HA-ERF19) that developed increased disease lesions after drop-inoculation with B. cinerea spores (Supplemental Figures 1A-C).…”

Screen Identifying Arabidopsis Transcription Factors Involved in the Response to 9-Lipoxygenase-Derived Oxylipins

“…In order to get an insight into putative 9-LOX-oxylipin signaling pathways in plants, we set-up a screen aiming at TFs involved in the transcriptional response to 9-HOT. The At TORF-EX ( A rabidopsis t haliana T ranscription factor O pen R eading F rame Ex pression) collection holds seeds of transgenic TF overexpressing plants, which can be used for identifying regulators in development or stress response [ 22 , 23 ]. Using GATEWAY ® technology, a batch transformation procedure has been developed, which results in seed pools derived from transformants of 30–50 TF genes under control of the Cauliflower 35S promoter (35S).…”

“…Using GATEWAY ® technology, a batch transformation procedure has been developed, which results in seed pools derived from transformants of 30–50 TF genes under control of the Cauliflower 35S promoter (35S). Currently, this screening collection enables analysis of more than 700 TFs and has successfully been applied for several screening purposes [ 23 , 24 ]. Here, we identify and re-evaluate a set of TFs which when overexpressed result in tolerance to 9-HOT.…”