Background The WT-boxes NGACTTTN are novel microbe-associated molecular pattern (MAMP)-responsive cis-regulatory sequences. Many of them are uncommon WRKY transcription factor (TF) binding sites. Results To understand their functional relevance, a genomic distribution analysis of the 16 possible WT-boxes and a functional analysis of a WT-box rich promoter was done. The genomic distribution analysis shows an enrichment of specific WT-boxes within 500 bp upstream of all Arabidopsis thaliana genes. Those that harbour a T 5′ to the core sequence GACTTT can also be part of the classic WRKY binding site the W-box TTGACT/C. The MAMP-responsive gene ATEP3, a class IV chitinase, harbours seven WT-boxes within its 1000 bp upstream region. In the context of synthetic promoters, the four proximal WT-boxes confer MAMP responsivity while the three WT-boxes further upstream have no effect. Rendering the nucleotides adjacent and in the vicinity of the WT-box core sequence reveals their functional importance for gene expression. A 158 bp long ATEP3 minimal promoter harbouring the two WT-boxes CGACTTTT, confers WT-box-dependent basal and MAMP-responsive reporter gene expression. The ATEP3 gene is a proposed target of WRKY50 and WRKY70. WRKY50 negatively regulates MAMP responsivity of the two WT-boxes CGACTTTT, while WRKY70 activates gene expression in a WT-box dependent manner. Both WRKY factors bind directly to the WT-box CGACTTTT. Conclusion In summary, WT-boxes are enriched in promoter regions and comprise novel and uncommon WRKY binding sites required for basal and MAMP-induced gene expression. WT-boxes not being part of a W-box may be a missing link for WRKY target gene prediction when these genes do not harbour a W-box.

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