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

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 ( Capsicum annuum ). Herein, CaWRKY22 , a member of a subgroup of IIe WRKY proteins from pepper, was functionally characterized in pepper immunity against Ralstonia Solanacearum . CaWRKY22 was found to target the nuclei, and its transcript level was significantly upregulated by Ralstonia Solanacearum inoculation (RSI) and exogenously applied salicylic acid (SA), Methyl jasmonate (MeJA), or ethephon (ETH). Loss-of-function CaWRKY22 , caused by virus-induced gene silencing (VIGS), enhanced pepper’s susceptibility to RSI. In addition, the silencing of CaWRKY22 perturbed the hypersensitive response (HR)-like cell death elicited by RSI and downregulated defense-related genes including CaPO2 , CaPR4 , CaACC , CaBPR1 , CaDEF1 , CaHIR1 , and CaWRKY40 . CaWRKY22 was found to directly bind to the promoters of CaPR1 , CaDEF1 , and CaWRKY40 by chromatin immuno-precipitation (ChIP) analysis. Contrastingly, transient overexpression of CaWRKY22 in pepper leaves triggered significant HR-like cell death and upregulated the tested immunity associated maker genes. Moreover, the transient overexpression of CaWRKY22 upregulated the expression of CaWRKY6 and CaWRKY27 while it downregulated of the expression of CaWRKY58 . Conversely, the transient overexpression of CaWRKY6 , CaWRKY27 , and CaWRKY40 upregulated the expression of CaWRKY22 , while transient overexpression of CaWRKY58 downregulated the transcript levels of CaWRKY22 . These data collectively recommend the role of CaWRKY22 as a positive regulator of pepper immunity against R. Solanacearum , 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.