Identification of a novel type of WRKY transcription factor binding site in elicitor-responsive cis-sequences from Arabidopsis thaliana
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Using a combination of bioinformatics and synthetic promoters, novel elicitor-responsive cis-sequences were discovered in promoters of pathogen-upregulated genes from Arabidopsis thaliana. One group of functional sequences contains the conserved core sequence GACTTTT. This core sequence and adjacent nucleotides are essential for elicitor-responsive gene expression in a parsley protoplast system. By yeast one-hybrid screening, WRKY70 was selected with a cis-sequence harbouring the core sequence GACTTTT but no known WRKY binding site (W-box). Transactivation experiments, mutation analyses, and electrophoretic mobility shift assays demonstrate that the sequence CGACTTTT is the binding site for WRKY70 in the investigated cis-sequence and is required for WRKY70-activated gene expression. Using several cis-sequences in transactivation experiments and binding studies, the CGACTTTT sequence can be extended to propose YGACTTTT as WRKY70 binding site. This binding site, designated WT-box, is enriched in promoters of genes upregulated in a WRKY70 overexpressing line. Interestingly, functional WRKY70 binding sites are present in the promoter of WRKY30, supporting recent evidence that both factors play a role in the same regulatory network.
KeywordsBioinformatics Gel shift experiments Plant-pathogen interaction Transient expression WT-box Yeast one-hybrid
We are grateful to Philipp Lissek and Lilith Christaler for plasmid constructs and for performing some of the transient reporter gene assays. David Havlik is gratefully acknowledged for advice in expressing WRKY70 in E. coli and Erik Hanko and Kai Zwara helped with greenhouse work. We also thank Norbert F. Käufer for critically reading the manuscript and Dietmar Stahl and Fridtjof Weltmeier for stimulating discussions. The TF-only yeast one-hybrid library of A. thaliana was obtained from the National Institute of Advanced Industrial Science and Technology (AIST), Japan. This work was supported by the Federal Ministry of Education and Research (BMBF) of Germany.
Conflict of interest
The authors declare that they have no conflict of interest.
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