Abstract
Key messageWRKY50 from A. thaliana requires WT-boxes at target gene promoters for activation and binding.Based on the genome-wide prediction of WRKY50 target genes and the similarity of a WRKY50 binding site to WT-boxes in microbe-associated molecular pattern (MAMP)-responsive cis-regulatory modules (CRM), four WT-box containing CRMs from the promoter region of three WRKY50 target genes were investigated for their interaction with WRKY50. These target genes are DJ1E, WRKY30 and ATBBE4. Two of the four CRMs, one from DJ1E and one from WRKY30, were able to activate reporter gene expression in the presence of WRKY50. Activation requires the WT-boxes GGACTTTT, GGACTTTG from DJ1E and GGACTTTC from WRKY30. WRKY50 does not activate a second CRM from WRKY30 and the CRM from ATBBE4, both containing the WT-box TGACTTTT. In vitro gel-shift assays demonstrate WT-box-specific binding of the WRKY50 DNA-binding domain to all four CRMs. This work shows a high flexibility of WRKY50 binding site recognition beyond the classic W-box TTGACC/T.
Highlights
The transcriptional response of plants towards pathogens or microbe-associated molecular pattern (MAMP) is mostly carried out by WRKY transcription factors (TFs)
Since the identification of DJ1E, WRKY30, and ATBBE4 as target genes for WRKY50 by DNA Affinity Purification sequencing (DAP-seq) analysis does not necessarily mean that the WT-box containing cis-regulatory modules (CRM) are within the fragments bound by WRKY50, we used the genome browser at the Cistrome database to determine their position
The obtained results confirmed that the WT-box containing CRMs from the proposed target genes DJ1E, ATBBE4, and WRKY30 are all present within WRKY50bound DNA fragments (O’Malley et al 2016)
Summary
The transcriptional response of plants towards pathogens or MAMPs is mostly carried out by WRKY transcription factors (TFs). The classic WRKY binding site, the W-box, is enriched in promoters of pathogen-responsive genes (Maleck et al 2000; Rinerson et al 2015; Chen et al 2019). In addition to WRKY TFs, other transcription factor families such as AP2/EREBP, bZIP, and MYB contribute to pathogen-responsive gene expression (Rushton and Somssich 1998; Amorim et al 2017). In order to discover novel TF-binding sites, pattern recognition programs can be employed, that identify identical sequence patterns in the promoters of co-regulated genes. Examples of such programs include MEME, AlignACE, BioProspector, or CONSENSUS (Bailey and Elkan 1995; Hertz and Stormo 1999; Hughes et al 2000; Liu et al 2001).
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