CaWRKY28 Cys249 is Required for Interaction with CaWRKY40 in the Regulation of Pepper Immunity to .

WRKY transcription factors have been implicated in plant response to pathogens but how WRKY-mediated networks are organized and operate to produce appropriate transcriptional outputs remains largely unclear. Here, we identify a member of the WRKY family from pepper (), CaWRKY28, that physically interacts with CaWRKY40, a positive regulator of pepper immunity and thermotolerance. We confirmed CaWRKY28-CaWRKY40 interaction by coimmunoprecipitation, bimolecular fluorescence complementation, and microscale thermophoresis. Our findings supported the idea that CaWRKY28 is a nuclear protein that acts as positive regulator in pepper responses to infection by the pathogenic bacterium . It performs its function not by directly modulating the W-box containing immunity-related genes but by promoting CaWRKY40 via physical interaction to bind and activate its immunity-related target genes, including , , , and , but not its thermotolerance-related target gene, . All of these data indicate that CaWRKY28 interacts with and potentiates CaWRKY40 in regulating immunity against infection but not thermotolerance. Importantly, we discovered that CaWRKY28 Cys249, shared by CaWRKY28 and its orthologs probably only in the family Solanaceae, is crucial for the CaWRKY28-CaWRKY40 interaction. These results highlight how CaWRKY28 associates with CaWRKY40 during the establishment of WRKY networks, and how CaWRKY40 achieves its functional specificity during pepper responses to infection.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.