Unusual DNA-binding properties of theArabidopsis thalianaWRKY50 transcription factor at target gene promoters

Key message WRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding. 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)-responsivecis-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 areDJ1E,WRKY30andATBBE4.Two of the four CRMs, one fromDJ1Eand one fromWRKY30, were able to activate reporter gene expression in the presence of WRKY50. Activation requires the WT-boxes GGACTTTT, GGACTTTG fromDJ1Eand GGACTTTC fromWRKY30. WRKY50 does not activate a second CRM fromWRKY30and the CRM fromATBBE4, 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.