High‐throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function

Abstract

SUMMARYGenomic approaches have generated large Arabidopsis open reading frame (ORF) collections. However, molecular tools are required to characterize this ORFeome functionally. A high-throughput microtiter platebased protoplast transactivation (PTA) system has been established that can be used in a screening approach to define which transcription factor (TF) regulates a given promoter in planta. Using to this procedure, the transactivation properties of 96 TFs can be analyzed rapidly, making use of promoter:Luciferase (LUC)-reporters and luciferase imaging. Applying GATEWAY Ò technology, we have established a platform to assay more than 700 Arabidopsis TFs. As a proof-of-principle, the ethylene response factor (ERF) family has been studied to evaluate this system. Importantly, distinct subsets of related ERF factors were found to activate specifically the well described target promoters RD29A and PDF1.2 that are under control of DRE or GCC box cis-elements, respectively. Furthermore, several applications of the PTA system have been demonstrated, such as analysis of transcriptional repressors, salt-inducible gene expression or functional interaction of signaling molecules like kinases and TFs. This novel molecular tool will improve functional studies on transcriptional regulation in plants significantly.

Redundant ERF-VII Transcription Factors Bind to an Evolutionarily Conserved cis-Motif to Regulate Hypoxia-Responsive Gene Expression in Arabidopsis

“…To determine if these constructs were transactivated by RAP2.2, they were cotransfected into mesophyll protoplasts with N-terminally HA-tagged (stabilized) RAP2.2 (HA-RAP2.2) driven by the 35S promoter. Promoter activity was quantified by monitoring LUC activity relative to cotransfected Renilla Luciferase gene (RUC) (Wehner et al, 2011). HA-RAP2.2 strongly enhanced the basal activity of LBD41prom59-2 ( Figure 4C).…”

“…N-terminal HA-tagged effector constructs for the protoplast transfection, the effector control p35S: HA-GFP, and the LUC normalization vector p70SRUC have been described before (Stahl et al, 2004;Wehner et al, 2011;Klecker et al, 2014). For the construction of p35S:(MA)RAP2.2-hormone binding domain (HBD), the RAP2.2 coding sequence was amplified with a forward primer that replaced the second codon, the N-terminal Cys (C2), with Ala to remove susceptibility to N-end rule degradation.…”

A Shoot-Specific Hypoxic Response of Arabidopsis Sheds Light on the Role of the Phosphate-Responsive Transcription Factor PHOSPHATE STARVATION RESPONSE1

“…For the construction of the Firefly-Luciferase reporters pBT10-MDG3pro:LUC, pBT10-At5g20790pro:LUC, and pBT10-LBD41pro: LUC, the 59 upstream sequences (promoter) of MGD3, At5g20790, and LBD41 (-1,231, -719, and -1,649 bp from ATG, respectively) were amplified from genomic DNA of Arabidopsis Col-0, using specific PCR primer pairs (Supplemental Table S4). The MGD3pro PCR product was digested with EcoRI and NcoI, whereas At5g20790pro and LBD41pro were digested with NcoI and BamHI for insertion into the vector pBT10GAL4UAS (Wehner et al, 2011). For each construct, the same respective restriction enzymes were used to cut the vector and remove the GAL4UAS before ligation.…”

“…The fusion constructs with N-terminal hemagglutinin (HA)-tagged effectors p35S:PHR1-HA, p35S:PHL1-HA, p35S:HA-RAP2.2, and p35S:HA-RAP2.12 as well as all cloning vectors have been described elsewhere (Ehlert et al, 2006;Wehner et al, 2011). To create an HA-tagged effector control, the GFP coding sequence was recombined into the Gateway vector p35S:HA-GW (Ehlert et al, 2006).…”

“…For mesophyll protoplast isolation and transient transfection, all buffers and solutions were prepared as described previously (Wehner et al, 2011) and used at room temperature. Sixty rosette leaves from Arabidopsis Col-0 plants, grown for 3 to 4 weeks on soil under an 8-h-light/16-h-dark cycle (about 100 mmol m -2 s -1 ), were sliced into 1-mm strips and submerged in 10 mL of enzyme solution.…”