Identification of a novel type of WRKY transcription factor binding site in elicitor-responsive cis-sequences from Arabidopsis thaliana

Plant Mol Biol. 2014 Mar;84(4-5):371-85. doi: 10.1007/s11103-013-0136-y. Epub 2013 Oct 9.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / cytology
  • Arabidopsis / genetics*
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / genetics*
  • Arabidopsis Proteins / metabolism
  • Base Sequence
  • Binding Sites / genetics
  • Electrophoretic Mobility Shift Assay
  • Fungal Proteins / genetics
  • Luciferases / genetics
  • Luciferases / metabolism
  • Membrane Glycoproteins / genetics
  • Mutation
  • Nucleotide Motifs / genetics*
  • Oligopeptides / genetics
  • Oligopeptides / metabolism
  • Phytophthora / genetics
  • Protein Binding
  • Protoplasts / metabolism
  • Response Elements / genetics
  • Sequence Homology, Nucleic Acid
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Transcriptional Activation
  • Two-Hybrid System Techniques

Substances

  • Arabidopsis Proteins
  • Fungal Proteins
  • Membrane Glycoproteins
  • Oligopeptides
  • Transcription Factors
  • WRKY70 protein, Arabidopsis
  • Luciferases