CaWRKY22 Acts as a Positive Regulator in Pepper Response to Ralstonia Solanacearum by Constituting Networks with CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58

Abstract

The WRKY web, which is comprised of a subset of WRKY transcription factors (TFs), plays a crucial role in the regulation of plant immunity, however, the mode of organization and operation of this network remains obscure, especially in non-model plants such as pepper (<i>Capsicum annuum</i>). Herein, <i>CaWRKY22</i>, a member of a subgroup of IIe WRKY proteins from pepper, was functionally characterized in pepper immunity against <i>Ralstonia Solanacearum</i>. CaWRKY22 was found to target the nuclei, and its transcript level was significantly upregulated by <i>Ralstonia Solanacearum</i> inoculation (RSI) and exogenously applied salicylic acid (SA), Methyl jasmonate (MeJA), or ethephon (ETH). Loss-of-function <i>CaWRKY22</i>, caused by virus-induced gene silencing (VIGS), enhanced pepper&rsquo;s susceptibility to RSI. In addition, the silencing of <i>CaWRKY22</i> perturbed the hypersensitive response (HR)-like cell death elicited by RSI and downregulated defense-related genes including <i>CaPO2</i>, <i>CaPR4</i>, <i>CaACC</i>, <i>CaBPR1</i>, <i>CaDEF1</i>, <i>CaHIR1</i>, and <i>CaWRKY40</i>. CaWRKY22 was found to directly bind to the promoters of <i>CaPR1</i>, <i>CaDEF1</i>, and <i>CaWRKY40</i> by chromatin immuno-precipitation (ChIP) analysis. Contrastingly, transient overexpression of <i>CaWRKY22</i> in pepper leaves triggered significant HR-like cell death and upregulated the tested immunity associated maker genes. Moreover, the transient overexpression of <i>CaWRKY22</i> upregulated the expression of <i>CaWRKY6</i> and <i>CaWRKY27</i> while it downregulated of the expression of <i>CaWRKY58</i>. Conversely, the transient overexpression of <i>CaWRKY6</i>, <i>CaWRKY27</i>, and <i>CaWRKY40</i> upregulated the expression of <i>CaWRKY22</i>, while transient overexpression of <i>CaWRKY58</i> downregulated the transcript levels of <i>CaWRKY22</i>. These data collectively recommend the role of CaWRKY22 as a positive regulator of pepper immunity against <i>R. Solanacearum</i>, which is regulated by signaling synergistically mediated by SA, jasmonic acid (JA), and ethylene (ET), integrating into WRKY networks with WRKY TFs including CaWRKY6, CaWRKY27, CaWRKY40, and CaWRKY58.