CaWRKY28 Cys249 is required for interaction with CaWRKY40 in the regulation of pepper immunity to Ralstonia solanacearum.

Abstract

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 (Capsicum annuum), CaWRKY28, that physically interacts with CaWRKY40, a positive regulator of pepper immunity and thermotolerance. We confirmed CaWRKY28/CaWRKY40 interaction by co-immunoprecipitation, bimolecular fluorescence complementation and microscale thermophoresis. Our findings supported that CaWRKY28 is a nuclear protein that acts as positive regulator in pepper responses to the pathogenic bacterium Ralstonia solanacearum infection. 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 CaPR1, CaNPR1, CaDEF1 and CaABR1, but not its thermotolerance related target gene CaHSP24. All these data indicate that CaWRKY28 interacts with and potentiates CaWRKY40 in regulating immunity against R. solanacearum infection but not thermotolerance. Importantly, we discovered that CaWRKY28 Cys249, shared by CaWRKY28 and its orthologs probably only in the 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 R. solanacearum infection.