GSK3-like kinases have been mainly implicated in the brassinosteroids (BR) pathway and, therefore, in plant growth, development, and responses to abiotic stresses; however, their roles in plant immunity remain poorly understood. Herein, we present evidence that CaSK23, a putative GSK3/SHAGGY-like kinase in pepper, acts as a negative regulator in pepper’s response to Ralstonia solanacearum (R. solanacearum) inoculation (RSI). Data from quantitative RT-PCR (qRT-PCR) showed that the constitutively-expressed CaSK23 in pepper leaves was down-regulated by RSI, as well as by exogenously-applied salicylic acid (SA) or methyl jasomonate (MeJA). Silencing of CaSK23 by virus-induced gene silencing (VIGS) decreased the susceptibility of pepper plants to RSI, coupled with up-regulation of the tested genes encoding SA-, JA-, and ethylene (ET)-dependent pathogenesis-related (PR) proteins. In contrast, ectopic overexpression (OE) of CaSK23 conferred a compromised resistance of tobacco plants to RSI, accompanied by down-regulation of the tested immunity-associated SA-, JA-, and ET-dependent PR genes. In addition, transient overexpression of CaSK23 in pepper plants consistently led to down-regulation of the tested SA-, JA-, and ET-dependent PR genes. We speculate that CaSK23 acts as a negative regulator in pepper immunity and its constitutive expression represses pepper immunity in the absence of pathogens. On the other hand, its decreased expression derepresses immunity when pepper plants are attacked by pathogens.

Bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases in solanaceous plants, including peppers. It generally tends to be more serious under warm−temperature and moist (WM) conditions than at moist room−temperature (RM) conditions. Although immunity mechanisms at room temperature have been intensively studied, the mechanisms underlying WM conditions remain poorly understood. Herein, the pepper cysteine protease CaZingipain2 was expressed and functionally characterized in pepper immunity against R. solanacearum at WM conditions and at room temperature. The results showed that CaZingipain2 localized to the nucleus and was upregulated at the transcript level in pepper plants upon R. solanacearum infection under WM conditions (RSWM). Virus−induced gene silencing of CaZingipain2 significantly increased the susceptibility of pepper plants to RSWM, and was coupled with the downregulation of CaPRP1 and CaMgst3, which are specifically related to pepper immunity against RSWM, according to our previous studies, while its overexpression significantly reduced the susceptibility of N. benethamiana plants to RSWM compared to that of wild−type plants. In addition, our data showed that CaZingipain2 also acts positively in pepper immunity against R. solanacearum infection at room temperature by upregulating the SA− and JA−responsive PR genes, including CaNPR1 and CaDEF1. All these results indicate that CaZingipain2 improves pepper immunity against R. solanacearum under WM conditions and at room temperature by regulating different PR genes.

It has previously been found that CaWRKY40 is employed by pepper to activate immunity against Ralstonia solanacearum and to activate thermotolerance context-specifically, but the underlying mechanisms are not fully understood. Here, CaGβ, a subunit in the heterotrimeric G protein complex that was originally found to probably interact with CaWRKY40, was expressional and functionally characterized; the results showed that CaGβ was upregulated by R. solanacearum infection; its silencing by virus-induced silencing impaired pepper immunity against R. solanacearum infection, accompanied by downregulation of immunity-related marker genes, including CaPR1, CaDEF1, CaNPR1, and CaPR-STH2. In addition, CaGβ–CaWRKY40 interaction was confirmed by BiFC and pull-down assay using prokaryotically expressed proteins, and activations of immunity-related CaPR1, CaPR-STH2, and CaNPR1 by CaWRKY40 were all promoted, but the activation of thermotolerance-related CaHSP24 by CaWRKY40 blocked CaGβ through its interaction with CaWRKY40. All these data indicate that immunity against R. solanacearum and its antagonism to thermotolerance in pathogen-infected pepper plants are mediated by CaWRKY40 through physical interaction with CaGβ.