Unusual DNA-binding properties of the Arabidopsis thaliana WRKY50 transcription factor at target gene promoters
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ORIGINAL ARTICLE
Unusual DNA‑binding properties of the Arabidopsis thaliana WRKY50 transcription factor at target gene promoters Konstantin Kanofsky1 · Jendrik Rusche1 · Lea Eilert1 · Fabian Machens1,2 · Reinhard Hehl1 Received: 23 June 2020 / Accepted: 21 September 2020 © The Author(s) 2020
Abstract Key message WRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding. Abstract Based on the genome-wide prediction of WRKY50 target genes and the similarity of a WRKY50 binding site to WT-boxes in microbe-associated molecular pattern (MAMP)-responsive cis-regulatory modules (CRM), four WT-box containing CRMs from the promoter region of three WRKY50 target genes were investigated for their interaction with WRKY50. These target genes are DJ1E, WRKY30 and ATBBE4. Two of the four CRMs, one from DJ1E and one from WRKY30, were able to activate reporter gene expression in the presence of WRKY50. Activation requires the WT-boxes GGACTTTT, GGACTTTG from DJ1E and GGACTTTC from WRKY30. WRKY50 does not activate a second CRM from WRKY30 and the CRM from ATBBE4, both containing the WT-box TGACTTTT. In vitro gel-shift assays demonstrate WT-box-specific binding of the WRKY50 DNA-binding domain to all four CRMs. This work shows a high flexibility of WRKY50 binding site recognition beyond the classic W-box TTGACC/T. Keywords Electrophoretic mobility shift assay · Parsley protoplasts · Pep25 · Reporter gene · Transcriptional regulation · Transient gene expression
Introduction The transcriptional response of plants towards pathogens or MAMPs is mostly carried out by WRKY transcription factors (TFs). The classic WRKY binding site, the W-box, is enriched in promoters of pathogen-responsive genes (Maleck et al. 2000; Rinerson et al. 2015; Chen et al. 2019). In addition to WRKY TFs, other transcription factor families such as AP2/EREBP, bZIP, and MYB also contribute to pathogen-responsive gene expression (Rushton and Somssich 1998; Amorim et al. 2017). All TFs recognize a characteristic DNA binding site motif and the presence of such sequence motifs in a gene promoter allows the prediction Communicated by Neal Stewart. * Reinhard Hehl r.hehl@tu‑bs.de 1
Institut für Genetik, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany
Present Address: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam Science Park, Am Mühlenberg 1, Golm, 14476 Potsdam, Germany
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of how the gene is regulated (Hehl and Wingender 2001). To detect such sequences, promoters or gene identification numbers can be submitted to search databases of known regulatory sequences such as TRANSFAC, AGRIS, PlantCare, AthaMap, and PLACE (Higo et al. 1999; Lescot et al. 2002; Matys et al. 2003; Yilmaz et al. 2011; Hehl et al. 2016; Hehl and Bülow 2014). To identify gene groups that may be regulated by the same TF, promoters of co-regulated gene sets can be analysed simultaneously (Galuschka et al. 2007; Hehl and Bülow 2008, 2014). To date, binding sites for almost 1000 of the approx
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