Genomic distribution and context dependent functionality of novel WRKY transcription factor binding sites

Abstract

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.

Multifaceted roles of WRKY transcription factors in abiotic stress and flavonoid biosynthesis

“…A platform for systematic investigations of WRKY family is provided by the expanding genome sequencing data from many plant species. The identification of genes crucial for specific processes has been made possible by genome sequences, which have enabled transcriptome profiling of some families (such as WRKY) during particular situations ( Arndt et al., 2022 ; Shende et al., 2022 ; Zhang et al., 2022e ). Furthermore, the genome-wide mapping of WRKY binding sites and target genes has been made possible by high-throughput sequencing after chromatin immunoprecipitation (ChIP-seq) ( Zhou et al., 2021 ; Xu et al., 2022 ).…”