CaWRKY40b in Pepper Acts as a Negative Regulator in Response to Ralstonia solanacearum by Directly Modulating Defense Genes Including CaWRKY40

Abstract

WRKY transcription factors (TFs) have been implicated in plant growth, development, and in response to environmental cues; however, the function of the majority of pepper WRKY TFs remains unclear. In the present study, we functionally characterized <i>CaWRKY40b</i>, a homolog of <i>AtWRKY40</i>, in pepper immunity. <i>Ralstonia solanacearum</i> inoculation (RSI) in pepper plants resulted in downregulation of <i>CaWRKY40b</i> transcript, and green fluorescent protein (GFP)-tagged CaWRKY40b was localized to the nuclei when transiently overexpressed in the leaves of <i>Nicotiana benthamiana</i>. Virus-induced gene silencing (VIGS) of <i>CaWRKY40b</i> significantly decreased pepper&rsquo; susceptibility to RSI. Consistently, the transient over-expression of <i>CaWRKY40b-SRDX</i> (chimeric repressor version of <i>CaWRKY40b</i>) triggered cell death, as indicated by darker trypan blue and DAB staining. CaWRKY40b targets a number of immunity-associated genes, including <i>CaWRKY40 JAR</i>, <i>RLK1</i>, <i>EIN3</i>, <i>FLS2</i>, <i>CNGIC8</i>, <i>CDPK13</i>, and <i>heat shock cognate protein 70</i> (<i>HSC70)</i>, which were identified by ChIP-seq and confirmed using ChIP-real time PCR. Among these target genes, the negative regulator HSC70 was upregulated by transient overexpression of <i>CaWRKY40b</i> and downregulated by silencing of <i>CaWRKY40b</i>, whereas other positive regulators as well as two non-target genes, <i>CaNPR1</i> and <i>CaDEF1</i>, were downregulated by the transient overexpression of <i>CaWRKY40b</i> and upregulated by <i>CaWRKY40b</i> silencing or transient overexpression of <i>CaWRKY40b-SRDX</i>. In addition, CaWRKY40b exhibited a positive feedback regulation at transcriptional level by directly targeting the promoter of itself. In conclusion, the findings of the present study suggest that <i>CaWRKY40b</i> acts as a negative regulator in pepper immunity against <i>R. solanacearum</i> by transcriptional modulation of a subset of immunity-associated genes; it also represses immunity in the absence of a pathogen, and derepresses immunity upon pathogen challenge.