Induced Genome-Wide Binding of Three Arabidopsis WRKY Transcription Factors during Early MAMP-Triggered Immunity

Abstract

During microbial-associated molecular pattern-triggered immunity (MTI), molecules derived from microbes are perceived by cell surface receptors and upon signaling to the nucleus initiate a massive transcriptional reprogramming critical to mount an appropriate host defense response. WRKY transcription factors play an important role in regulating these transcriptional processes. Here, we determined on a genome-wide scale the flg22-induced in vivo DNA binding dynamics of three of the most prominent WRKY factors, WRKY18, WRKY40, and WRKY33. The three WRKY factors each bound to more than 1000 gene loci predominantly at W-box elements, the known WRKY binding motif. Binding occurred mainly in the 500-bp promoter regions of these genes. Many of the targeted genes are involved in signal perception and transduction not only during MTI but also upon damage-associated molecular pattern-triggered immunity, providing a mechanistic link between these functionally interconnected basal defense pathways. Among the additional targets were genes involved in the production of indolic secondary metabolites and in modulating distinct plant hormone pathways. Importantly, among the targeted genes were numerous transcription factors, encoding predominantly ethylene response factors, active during early MTI, and WRKY factors, supporting the previously hypothesized existence of a WRKY subregulatory network. Transcriptional analysis revealed that WRKY18 and WRKY40 function redundantly as negative regulators of flg22-induced genes often to prevent exaggerated defense responses.

Figure 1.

Induction of WRKY18, WRKY40, and WRKY33 by flg22 Treatment. (A) RT-qPCR analysis of flg22-induced RNA levels. Total RNA was isolated from seedlings treated for 0, 1, 2, and 4 h with flg22 and analyzed by qPCR using gene-specific primers. Shown are the mean and sd (error bars) calculated from three biological replicates. (B) Immunoblot analysis of flg22-induced protein levels. Protein extracts from seedlings of the HA-tagged complementation lines were treated for indicated times with flg22 and subsequently analyzed by immunoblot using an anti-HA antibody. Ponceau S staining served as loading control.

Figure 2.

Distribution of flg22-Induced WRKY18, WRKY40, and WRKY33 Binding Regions in the Arabidopsis Genome. (A) Prevalence of WRKY binding regions in different genomic categories. Promoters are defined as the 1000-bp region upstream of the TSS. TTS refers to the 1000-bp region downstream of the 3′UTR, and genome regions located in between a TTS and the promoter of the next gene are regarded as intergenic. (B) Distance of WRKY binding region peaks to the transcription start site. The number of binding region peaks for each 50-bp region relative to the TSS is indicated.

Figure 3.

The W-Box Is the Predominant Motif within WRKY Binding Regions. (A) to (C) Motif position probability graphs for WRKY18 (A), WRKY40 (B), and WRKY33 (C) established by CentriMo motif search (Bailey and Machanick, 2012). Indicated are the most frequent motif, its rate of occurrence, and the probability of this motif occurring at a given position relative to the binding peak summit (0) in the 500-bp binding regions. The included P value describes the significance for central enrichment. (D) Distribution of W-box abundances in WRKY18, WRKY40, and WRKY33 binding regions.

Figure 4.

Overlap of WRKY18, WRKY40, and WRKY33 Target Gene Sets after 2 h flg22 Treatment. Indicated are the number of target genes in each section and the fraction of overlapping genes between each pair of WRKY target gene sets with respect to the smaller set.

Figure 5.

WRKY18, WRKY40, and WRKY33 Binding to the PEPR1, PROPEP2, and PROPEP3 Loci. IGV images of the PEPR1 (A) and PROPEP1-3 loci (B). Binding of WRKYs is visualized by read coverage histograms indicating sequencing read accumulation before (0 h) or after flg22 treatment (2 h). Wild-type samples served as negative control. The three lower tracks show the corresponding gene structures, position of W-boxes, and the direction of transcription (arrows).

Figure 6.

WRKY Factor Binding to Genes Related to Secondary Metabolism. WRKY18, WRKY40, and WRKY33 bind to genes involved in biosynthesis and transport of secondary metabolites camalexin and indole-glucosinolates (biosynthetic pathways adapted from Sønderby et al. [2010], Pfalz et al. [2011], and Møldrup et al. [2013]). Identified WRKY18, WRKY40, or WRKY33 target genes are indicated in yellow. TFs are underlined.

Figure 7.

DEGs in wrky18, wrky40, and wrky18 wrky40 Plants. (A) Number of significantly (FC ≥ 2, FDR < 0.05) up- or downregulated genes at 2 h flg22 treatment compared with 0 h in the indicated genotype. (B) Number of up- and downregulated DEGs (FDR < 0.05, FC ≥ 2) in the respective mutant lines compared with the wild type at 0, 1, or 2 h after flg22 treatment. (C) Overlap of the identified sets of DEGs in the respective mutant lines relative to the wild type at 2 h after flg22 treatment. The number of DEGs in each section is indicated.

Figure 8.

DRTs of WRKY18 and WRKY40. DRTs were identified by the overlaps of the WRKY18 and WRKY40 target gene sets with the sets of DEGs compared with the wild type in wrky18, wrky40, and wrky18 wrky40 mutants at 2 h after flg22 treatment. Indicated are the number of target genes, DEGs, and DRTs in the respective sections and the fraction DEGs identified as DRTs in each comparison.