ChiIV3 Acts as a Novel Target of WRKY40 to Mediate Pepper Immunity Against <i>Ralstonia solanacearum</i> Infection

Abstract

ChiIV3, a chitinase of pepper (<i>Capsicum annuum</i>), stimulates cell death in pepper plants. However, there are only scarce reports on its role in resistance against bacterial wilt disease such as that caused by <i>Ralstonia solanacearum</i> and their transcriptional regulation. In this study, the silencing of <i>ChiIV3</i> in pepper plants significantly reduced the resistance to <i>R. solanacearum</i>. The transcript of <i>ChiIV3</i> was induced by <i>R. solanacearum</i> inoculation (RSI) as well as exogenous application of methyl jasmonate and abscisic acid. The bioinformatics analysis revealed that the <i>ChiIV3</i> promoter consists of multiple stress-related <i>cis</i> elements, including six W-boxes and one MYB1AT. With the 5' deletion assay in the <i>ChiIV3</i> promoter, the W4-box located from -640 to -635 bp was identified as the <i>cis</i> element that is required for the response to RSI. In addition, the W4-box element was shown to be essential for the binding of the <i>ChiIV3</i> promoter by the WRKY40 transcription factor, which is known to positively regulate the defense response to <i>R. solanacearum</i>. Site-directed mutagenesis in the W4-box sequence impaired the binding of WRKY40 to the <i>ChiIV3</i> promoter. Subsequently, the transcription of <i>ChiIV3</i> decreased in <i>WRKY40</i>-silenced pepper plants. These results demonstrated that the expression of the defense gene <i>ChiIV3</i> is controlled through multiple modes of regulation, and WRKY40 directly binds to the W4-box element of the <i>ChiIV3</i> promoter region for its transcriptional regulation.