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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., (© 2022. The Author(s).)

Key Message: Transcription factors from mammals and plants, which play a role in innate immunity, interact with the same microbe-associated molecular pattern (MAMP)-responsive sequences from Arabidopsis thaliana. The interaction of mouse NF-κB p65 with MAMP-responsive sequences containing the core motif GACTTT of the WT-box was investigated. This revealed one sequence, derived from the promoter of the A. thaliana gene At1g76960, a gene with unknown function, to activate NF-κB p65 dependent reporter gene expression in plant cells very strongly. A bioinformatic analysis predicts three putative NF-κB p65 binding sites in this sequence and all three sites are required for reporter gene activation and binding. The sequence is one of the weakest MAMP-responsive sequences previously isolated, but the introduction of a GCC-box increases its MAMP responsivity in combination with upstream WT-box sequences. Although a bioinformatic analysis of the unmutated cis-sequence only predicts NF-κB p65 binding, A. thaliana WRKY40 was selected in a yeast one-hybrid screen. WRKY40, which is a transcriptional repressor, requires the sequence TTTTCTA for direct binding. This sequence is similar to the WK-box TTTTCCAC, previously shown to interact with tobacco NtWRKY12. In summary, this work supports the similarity in binding site recognition between NF-κB and WRKY factors.