High temperature (HT), high humidity (HH), and pathogen infection often co-occur and negatively affect plant growth. However, these stress factors and plant responses are generally studied in isolation. The mechanisms of synergistic responses to combined stresses are poorly understood. We isolated the subgroup IIb WRKY family member CaWRKY6 from Capsicum annuum and performed quantitative real-time PCR analysis. CaWRKY6 expression was upregulated by individual or simultaneous treatment with HT, HH, combined HT and HH (HTHH), and Ralstonia solanacearum inoculation, and responded to exogenous application of jasmonic acid (JA), ethephon, and abscisic acid (ABA). Virus-induced gene silencing of CaWRKY6 enhanced pepper plant susceptibility to R. solanacearum and HTHH, and downregulated the hypersensitive response (HR), JA-, ethylene (ET)-, and ABA-induced marker gene expression, and thermotolerance-associated expression of CaHSP24, ER-small CaSHP, and Chl-small CaHSP. CaWRKY6 overexpression in pepper attenuated the HTHH-induced suppression of resistance to R. solanacearum infection. CaWRKY6 bound to and activated the CaWRKY40 promoter in planta, which is a pepper WRKY that regulates heat-stress tolerance and R. solanacearum resistance. CaWRKY40 silencing significantly blocked HR-induced cell death and reduced transcriptional expression of CaWRKY40. These data suggest that CaWRKY6 is a positive regulator of R. solanacearum resistance and heat-stress tolerance, which occurs in part by activating CaWRKY40.

The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper.

Plant responses to pathogen attacks and high-temperature stress (HTS) are distinct in nature but generally share several signaling components. How plants produce specific responses through these common signaling intermediates remains elusive. With the help of reverse-genetics approaches, we describe here the mechanism underlying trade-offs in pepper (Capsicum annuum) between growth, immunity, and thermotolerance. The NAC-type transcription factor CaNAC2c was induced by HTS and Ralstonia solanacearum infection (RSI). CaNAC2c-inhibited pepper growth, promoted immunity against RSI by activating jasmonate-mediated immunity and H2O2 accumulation, and promoted HTS responses by activating Heat shock factor A5 (CaHSFA5) transcription and blocking H2O2 accumulation. We show that CaNAC2c physically interacts with CaHSP70 and CaNAC029 in a context-specific manner. Upon HTS, CaNAC2c–CaHSP70 interaction in the nucleus protected CaNAC2c from degradation and resulted in the activation of thermotolerance by increasing CaNAC2c binding and transcriptional activation of its target promoters. CaNAC2c did not induce immunity-related genes under HTS, likely due to the degradation of CaNAC029 by the 26S proteasome. Upon RSI, CaNAC2c interacted with CaNAC029 in the nucleus and activated jasmonate-mediated immunity but was prevented from activating thermotolerance-related genes. In non-stressed plants, CaNAC2c was tethered outside the nucleus by interaction with CaHSP70, and thus was unable to activate either immunity or thermotolerance. Our results indicate that pepper growth, immunity, and thermotolerance are coordinately and tightly regulated by CaNAC2c via its inducible expression and differential interaction with CaHSP70 and CaNAC029.

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.

WRKY transcription factors (TFs) have been implicated in plant growth, development, and in response to environmental cues; however, the function of the majority of pepper WRKY TFs remains unclear. In the present study, we functionally characterized CaWRKY40b, a homolog of AtWRKY40, in pepper immunity. Ralstonia solanacearum inoculation (RSI) in pepper plants resulted in downregulation of CaWRKY40b transcript, and green fluorescent protein (GFP)-tagged CaWRKY40b was localized to the nuclei when transiently overexpressed in the leaves of Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of CaWRKY40b significantly decreased pepper’ susceptibility to RSI. Consistently, the transient over-expression of CaWRKY40b-SRDX (chimeric repressor version of CaWRKY40b) triggered cell death, as indicated by darker trypan blue and DAB staining. CaWRKY40b targets a number of immunity-associated genes, including CaWRKY40 JAR, RLK1, EIN3, FLS2, CNGIC8, CDPK13, and heat shock cognate protein 70 (HSC70), which were identified by ChIP-seq and confirmed using ChIP-real time PCR. Among these target genes, the negative regulator HSC70 was upregulated by transient overexpression of CaWRKY40b and downregulated by silencing of CaWRKY40b, whereas other positive regulators as well as two non-target genes, CaNPR1 and CaDEF1, were downregulated by the transient overexpression of CaWRKY40b and upregulated by CaWRKY40b silencing or transient overexpression of CaWRKY40b-SRDX. In addition, CaWRKY40b exhibited a positive feedback regulation at transcriptional level by directly targeting the promoter of itself. In conclusion, the findings of the present study suggest that CaWRKY40b acts as a negative regulator in pepper immunity against R. solanacearum by transcriptional modulation of a subset of immunity-associated genes; it also represses immunity in the absence of a pathogen, and derepresses immunity upon pathogen challenge.