CaCBL1 Acts as a Positive Regulator in Pepper Response to Ralstonia solanacearum

Bacterial wilt caused by Ralstonia solanacearum is an important disease of pepper (Capsicum annuum), an economically important solanaceous vegetable worldwide, in particular, under high temperature (HT) conditions. However, the molecular mechanism underlying pepper immunity against bacterial wilt remains poorly understood. Herein, CaCBL1, a putative calcineurin B-like protein, was functionally characterized in the pepper response to R. solanacearum inoculation (RSI) under HT (RSI/HT). CaCBL1 was significantly upregulated by RSI at room temperature (RSI/RT), HT, or RSI/HT. CaCBL1-GFP fused protein targeted to whole epidermal cells of Nicotiana benthamiana when transiently overexpressed. CaCBL1 silencing by virus-induced gene silencing significantly enhanced pepper susceptibility to RSI under RT or HT, while its transient overexpression triggered hypersensitive response mimic cell death and upregulation of immunity-associated marker genes, including CabZIP63, CaWRKY40, and CaCDPK15, the positive regulators in the pepper response to RSI or HT found in our previous studies. In addition, by chromatin immunoprecipitation PCR and electrophoretic mobility shift assay, CaCBL1 was found to be directly targeted by CaWRKY40, although not by CaWRKY27 or CaWRKY58, via the W-box-2 within its promoter, and its transcription was found to be downregulated by silencing of CaWRKY40 while it was enhanced by its transient overexpression. These results suggest that CaCBL1 acts as a positive regulator in pepper immunity against R. solanacearum infection, constituting a positive feedback loop with CaWRKY40.