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

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.

A Brassica napus PHT1 phosphate transporter, BnPht1;4, promotes phosphate uptake and affects roots architecture of transgenic Arabidopsis

“…The Renilla luciferase gene was used as an internal control. Isolation and DNA transfection of Arabidopsis mesophyll protoplasts were performed as described previously (Machens et al 2014). The reporter construct, effector construct, and internal control were used for each co-transfection.…”

Analyses of Catharanthus roseus and Arabidopsis thaliana WRKY transcription factors reveal involvement in jasmonate signaling

“…The WRKY TF family is primarily a plant specific family with the exception of several examples in protozoa [29]. The WRKY domain is a 60 to 70 amino acid long DNA binding domain that recognizes the W-box (TTGACC/T); however, recent studies suggest this cis -element may be more degenerate and other components are involved for WRKY binding to DNA in response to a specific stimulus [30, 31]. The N-terminal portion of the WRKY domain is characterized by a highly conserved WRKYGQK motif whereas the C-terminal region of the domain contains either a Cys 2 -His 2 or Cys 2 -His-Cys zinc-finger [32].…”

'In silico expression analysis', a novel PathoPlant web tool to identify abiotic and biotic stress conditions associated with specific cis-regulatory sequences

“…This article describes the implementation of the web tool and a proof of concept analysis with known cis -sequences that confer stress-responsive gene expression. In addition, using the recently identified WRKY70 TF binding site CGACTTTT (15) and reporter gene technology in transgenic A. thaliana , the prediction made by an ‘ in silico expression analysis’ was confirmed.…”

“…Recently, a novel elicitor-responsive cis -regulatory sequence from A. thaliana , CGACTTTT, was predicted bioinformatically and was shown to be a binding site for the WRKY70 TF (5, 15). Although the cis -sequence, designated WT-box and bound by WRKY70, is enriched in promoters of genes upregulated in a WRKY70 overexpression line, the primary stimulus to which genes harbouring this sequence in their promoter respond to was unknown.…”

“…In the work presented here, the ‘ in silico expression analysis’ web tool was successfully used to determine the biotic stress response conditions for genes harbouring a novel cis -regulatory sequence designated WT-box (15). This sequence, CGACTTTT, was detected when pattern recognition programs were used on promoters of A. thaliana genes upregulated by pathogenic stimuli (5).…”