CaSTH2 disables CaWRKY40 from activating pepper thermotolerance and immunity against Ralstonia solanacearum via physical interaction.

CaWRKY40 coordinately activates pepper immunity against Ralstonia solanacearum infection (RSI) and high temperature stress (HTS), forms positive feedback loops with other positive regulators and is promoted by CaWRKY27b/CaWRKY28 through physical interactions; however, whether and how it is regulated by negative regulators to function appropriately remain unclear. Herein, we provide evidence that CaWRKY40 is repressed by a SALT TOLERANCE HOMOLOG2 in pepper (CaSTH2). Our data from gene silencing and transient overexpression in pepper and epoptic overexpression in Nicotiana benthamiana plants showed that CaSTH2 acted as negative regulator in immunity against RSI and thermotolerance. Our data from BiFC, CoIP, pull down, and MST indicate that CaSTH2 interacted with CaWRKY40, by which CaWRKY40 was prevented from activating immunity or thermotolerance-related genes. It was also found that CaSTH2 repressed CaWRKY40 at least partially through blocking interaction of CaWRKY40 with CaWRKY27b/CaWRKY28, but not through directly repressing binding of CaWRKY40 to its target genes. The results of study provide new insight into the mechanisms underlying the coordination of pepper immunity and thermotolerance.

Identification of WRKY Family Members and Characterization of the Low-Temperature-Stress-Responsive WRKY Genes in Luffa (Luffa cylindrica L.).

The plant-specific WRKY transcription factor family members have diverse regulatory effects on the genes associated with many plant processes. Although the WRKY proteins in Arabidopsis thaliana and other species have been thoroughly investigated, there has been relatively little research on the WRKY family in Luffa cylindrica, which is one of the most widely grown vegetables in China. In this study, we performed a genome-wide analysis to identify L. cylindrica WRKY genes, which were subsequently classified and examined in terms of their gene structures, chromosomal locations, promoter cis-acting elements, and responses to abiotic stress. A total of 62 LcWRKY genes (471-2238 bp) were identified and divided into three phylogenetic groups (I, II, and III), with group II further divided into five subgroups (IIa, IIb, IIc, IId, and IIe) in accordance with the classification in other plants. The LcWRKY genes were unevenly distributed across 13 chromosomes. The gene structure analysis indicated that the LcWRKY genes contained 0-11 introns (average of 4.4). Moreover, 20 motifs were detected in the LcWRKY proteins with conserved motifs among the different phylogenetic groups. Two subgroup IIc members (LcWRKY16 and LcWRKY31) contained the WRKY sequence variant WRKYGKK. Additionally, nine cis-acting elements related to diverse responses to environmental stimuli were identified in the LcWRKY promoters. The subcellular localization analysis indicated that three LcWRKY proteins (LcWRKY43, LcWRKY7, and LcWRKY23) are localized in the nucleus. The tissue-specific LcWRKY expression profiles reflected the diversity in LcWRKY expression. The RNA-seq data revealed the effects of low-temperature stress on LcWRKY expression. The cold-induced changes in expression were verified via a qRT-PCR analysis of 24 differentially expressed WRKY genes. Both LcWRKY7 and LcWRKY12 were highly responsive to the low-temperature treatment (approximately 110-fold increase in expression). Furthermore, the LcWRKY8, LcWRKY12, and LcWRKY59 expression levels increased by more than 25-fold under cold conditions. Our findings will help clarify the evolution of the luffa WRKY family while also providing valuable insights for future studies on WRKY functions.

CaIAA2-CaARF9 module mediates the trade-off between pepper growth and immunity.

To challenge the invasion of various pathogens, plants re-direct their resources from plant growth to an innate immune defence system. However, the underlying mechanism that coordinates the induction of the host immune response and the suppression of plant growth remains unclear. Here we demonstrate that an auxin response factor, CaARF9, has dual roles in enhancing the immune resistance to Ralstonia solanacearum infection and in retarding plant growth by repressing the expression of its target genes as exemplified by Casmc4, CaLBD37, CaAPK1b and CaRROP1. The expression of these target genes not only stimulates plant growth but also negatively impacts pepper resistance to R. solanacearum. Under normal conditions, the expression of Casmc4, CaLBD37, CaAPK1b and CaRROP1 is active when promoter-bound CaARF9 is complexed with CaIAA2. Under R. solanacearum infection, however, degradation of CaIAA2 is triggered by SA and JA-mediated signalling defence by the ubiquitin-proteasome system, which enables CaARF9 in the absence of CaIAA2 to repress the expression of Casmc4, CaLBD37, CaAPK1b and CaRROP1 and, in turn, impeding plant growth while facilitating plant defence to R. solanacearum infection. Our findings uncover an exquisite mechanism underlying the trade-off between plant growth and immunity mediated by the transcriptional repressor CaARF9 and its deactivation when complexed with CaIAA2.

Transcription factor CaHDZ15 promotes pepper basal thermotolerance by activating HEAT SHOCK FACTORA6a.

High temperature stress (HTS) is a serious threat to plant growth and development and to crop production in the context of global warming, and plant response to HTS is largely regulated at the transcriptional level by the actions of various transcription factors (TFs). However, whether and how homeodomain-leucine zipper (HD-Zip) TFs are involved in thermotolerance are unclear. Herein, we functionally characterized a pepper (Capsicum annuum) HD-Zip I TF CaHDZ15. CaHDZ15 expression was upregulated by HTS and abscisic acid in basal thermotolerance via loss- and gain-of-function assays by virus-induced gene silencing in pepper and overexpression in Nicotiana benthamiana plants. CaHDZ15 acted positively in pepper basal thermotolerance by directly targeting and activating HEAT SHOCK FACTORA6a (HSFA6a), which further activated CaHSFA2. In addition, CaHDZ15 interacted with HEAT SHOCK PROTEIN 70-2 (CaHsp70-2) and glyceraldehyde-3-phosphate dehydrogenase1 (CaGAPC1), both of which positively affected pepper thermotolerance. CaHsp70-2 and CaGAPC1 promoted CaHDZ15 binding to the promoter of CaHSFA6a, thus enhancing its transcription. Furthermore, CaHDZ15 and CaGAPC1 were protected from 26S proteasome-mediated degradation by CaHsp70-2 via physical interaction. These results collectively indicate that CaHDZ15, modulated by the interacting partners CaGAPC1 and CaHsp70-2, promotes basal thermotolerance by directly activating the transcript of CaHSFA6a. Thus, a molecular linkage is established among CaHsp70-2, CaGAPC1, and CaHDZ15 to transcriptionally modulate CaHSFA6a in pepper thermotolerance.

Ralstonia solanacearum effector RipAK suppresses homodimerization of the host transcription factor ERF098 to enhance susceptibility and the sensitivity of pepper plants to dehydration.

Plants have evolved a sophisticated immune system to defend against invasion by pathogens. In response, pathogens deploy copious effectors to evade the immune responses. However, the molecular mechanisms used by pathogen effectors to suppress plant immunity remain unclear. Herein, we report that an effector secreted by Ralstonia solanacearum, RipAK, modulates the transcriptional activity of the ethylene-responsive factor ERF098 to suppress immunity and dehydration tolerance, which causes bacterial wilt in pepper (Capsicum annuum L.) plants. Silencing ERF098 enhances the resistance of pepper plants to R. solanacearum infection not only by inhibiting the host colonization of R. solanacearum but also by increasing the immunity and tolerance of pepper plants to dehydration and including the closure of stomata to reduce the loss of water in an abscisic acid signal-dependent manner. In contrast, the ectopic expression of ERF098 in Nicotiana benthamiana enhances wilt disease. We also show that RipAK targets and inhibits the ERF098 homodimerization to repress the expression of salicylic acid-dependent PR1 and dehydration tolerance-related OSR1 and OSM1 by cis-elements in their promoters. Taken together, our study reveals a regulatory mechanism used by the R. solanacearum effector RipAK to increase virulence by specifically inhibiting the homodimerization of ERF098 and reprogramming the transcription of PR1, OSR1, and OSM1 to boost susceptibility and dehydration sensitivity. Thus, our study sheds light on a previously unidentified strategy by which a pathogen simultaneously suppresses plant immunity and tolerance to dehydration by secreting an effector to interfere with the activity of a transcription factor and manipulate plant transcriptional programs.

Temperature-dependent action of pepper mildew resistance locus O 1 in inducing pathogen immunity and thermotolerance.

Plant diseases tend to be more serious under conditions of high-temperature/high-humidity (HTHH) than under moderate conditions, and hence disease resistance under HTHH is an important determinant for plant survival. However, how plants cope with diseases under HTHH remains poorly understood. In this study, we used the pathosystem consisting of pepper (Capsicum annuum) and Ralstonia solanacearum (bacterial wilt) as a model to examine the functions of the protein mildew resistance locus O 1 (CaMLO1) and U-box domain-containing protein 21 (CaPUB21) under conditions of 80% humidity and either 28 °C or 37 °C. Expression profiling, loss- and gain-of-function assays involving virus-induced gene-silencing and overexpression in pepper plants, and protein-protein interaction assays were conducted, and the results showed that CaMLO1 acted negatively in pepper immunity against R. solanacearum at 28 °C but positively at 37 °C. In contrast, CaPUB21 acted positively in immunity at 28 °C but negatively at 37 °C. Importantly, CaPUB21 interacted with CaMLO1 under all of the tested conditions, but only the interaction in response to R. solanacearum at 37 °C or to exposure to 37 °C alone led to CaMLO1 degradation, thereby turning off defence responses against R. solanacearum at 37 °C and under high-temperature stress to conserve resources. Thus, we show that CaMLO1 and CaPUB21 interact with each other and function distinctly in pepper immunity against R. solanacearum in an environment-dependent manner.

Differential CaKAN3-CaHSF8 associations underlie distinct immune and heat responses under high temperature and high humidity conditions.

High temperature and high humidity (HTHH) conditions increase plant susceptibility to a variety of diseases, including bacterial wilt in solanaceous plants. Some solanaceous plant cultivars have evolved mechanisms to activate HTHH-specific immunity to cope with bacterial wilt disease. However, the underlying mechanisms remain poorly understood. Here we find that CaKAN3 and CaHSF8 upregulate and physically interact with each other in nuclei under HTHH conditions without inoculation or early after inoculation with R. solanacearum in pepper. Consequently, CaKAN3 and CaHSF8 synergistically confer immunity against R. solanacearum via activating a subset of NLRs which initiates immune signaling upon perception of unidentified pathogen effectors. Intriguingly, when HTHH conditions are prolonged without pathogen attack or the temperature goes higher, CaHSF8 no longer interacts with CaKAN3. Instead, it directly upregulates a subset of HSP genes thus activating thermotolerance. Our findings highlight mechanisms controlling context-specific activation of high-temperature-specific pepper immunity and thermotolerance mediated by differential CaKAN3-CaHSF8 associations.

SlWRKY30 and SlWRKY81 synergistically modulate tomato immunity to Ralstonia solanacearum by directly regulating SlPR-STH2.

Bacterial wilt is a devastating disease of tomato (Solanum lycopersicum) caused by Ralstonia solanacearum that severely threatens tomato production. Group III WRKY transcription factors (TFs) are implicated in the plant response to pathogen infection; however, their roles in the response of tomato to R. solanacearum infection (RSI) remain largely unexplored. Here, we report the crucial role of SlWRKY30, a group III SlWRKY TF, in the regulation of tomato response to RSI. SlWRKY30 was strongly induced by RSI. SlWRKY30 overexpression reduced tomato susceptibility to RSI, and also increased H2O2 accumulation and cell necrosis, suggesting that SlWRKY30 positively regulates tomato resistance to RSI. RNA sequencing and reverse transcription-quantitative PCR revealed that SlWRKY30 overexpression significantly upregulated pathogenesis-related protein (SlPR-STH2) genes SlPR-STH2a, SlPR-STH2b, SlPR-STH2c, and SlPR-STH2d (hereafter SlPR-STH2a/b/c/d) in tomato, and these SlPR-STH2 genes were directly targeted by SlWRKY30. Moreover, four group III WRKY proteins (SlWRKY52, SlWRKY59, SlWRKY80, and SlWRKY81) interacted with SlWRKY30, and SlWRKY81 silencing increased tomato susceptibility to RSI. Both SlWRKY30 and SlWRKY81 activated SlPR-STH2a/b/c/d expression by directly binding to their promoters. Taking these results together, SlWRKY30 and SlWRKY81 synergistically regulate resistance to RSI by activating SlPR-STH2a/b/c/d expression in tomato. Our results also highlight the potential of SlWRKY30 to improve tomato resistance to RSI via genetic manipulations.

MADS-box protein AGL8 interacts with chromatin-remodelling component SWC4 to activate thermotolerance and environment-dependent immunity in pepper.

Pepper (Capsicum annuum) employs distinct defence responses against Ralstonia solanacearum infection (RSI); however, the mechanisms by which pepper activates these defence responses in a context-dependent manner is unclear. Here we study pepper plants defence response to RSI under room temperature-high humidity (RSRT, 28 °C / 90%) and high temperature-high humidity (RSHT, 37 °C / 90%) conditions, and non-infected plants under high temperature-high humidity (HTHH, 42 °C / 90%) stress. Herein, we found that the MADS-box transcription factor CaAGL8 was up-regulated by HTHH stress and RSRT or RSHT, and its silencing significantly reduced pepper thermotolerance and susceptibility to infection under both room and high temperature-high humidity (RSRT and RSHT). This was coupled with down-regulation of CaSTH2 and CaDEF1 upon RSRT, down-regulation of CaMgst3 and CaPRP1 upon RSHT, and down-regulation of CaHSP24 upon HTHH. In contrast, the ectopic overexpression of CaAGL8 significantly increased the resistance of Nicotiana benthamiana plants to RSRT, RSHT, and HTHH. In addition, CaAGL8 was found to interact with CaSWC4, which acted as a positive regulator of the pepper response to RSRT, RSHT, and HTHH. Silencing of either CaAGL8 or CaSWC4 blocked the hypersensitive response (HR) cell death and context-dependent up-regulation of defence-related genes triggered by the other. Importantly, enrichment of H4K5Ac, H3K9Ac, H3K4me3, and H3K9me2 on the tested defence-related genes was context- and gene-specifically regulated through synergistic interaction between CaSWC4 and CaAGL8. Our results indicate that pepper employs CaAGL8 to modulate chromatin remodelling by interacting with CaSWC4, thereby activating defence responses to RSRT, RSHT, and HTHH.

PMT6 Is Required for SWC4 in Positively Modulating Pepper Thermotolerance.

High temperature stress (HTS), with growth and development impairment, is one of the most important abiotic stresses frequently encountered by plants, in particular solanacaes such as pepper, that mainly distribute in tropical and subtropical regions. Plants activate thermotolerance to cope with this stress; however, the underlying mechanism is currently not fully understood. SWC4, a shared component of SWR1- and NuA4 complexes implicated in chromatin remodeling, was previously found to be involved in the regulation of pepper thermotolerance, but the underlying mechanism remains poorly understood. Herein, PMT6, a putative methyltranferase was originally found to interact with SWC4 by co-immunoprecipitation (Co-IP)-combined LC/MS assay. This interaction was further confirmed by bimolecular fluorescent complimentary (BiFC) and Co-IP assay, and PMT6 was further found to confer SWC4 methylation. By virus-induced gene silencing, it was found that PMT6 silencing significantly reduced pepper basal thermotolerance and transcription of CaHSP24 and significantly reduced the enrichment of chromatin-activation-related H3K9ac, H4K5ac, and H3K4me3 in TSS of CaHSP24, which was previously found to be positively regulated by CaSWC4. By contrast, the overexpression of PMT6 significantly enhanced basal thermotolerance of pepper plants. All these data indicate that PMT6 acts as a positive regulator in pepper thermotolerance, likely by methylating SWC4.

N-Methyltransferase CaASHH3 Acts as a Positive Regulator of Immunity against Bacterial Pathogens in Pepper.

Proteins with conserved SET domain play a critical role in plant immunity. However, the means of organization and functions of these proteins are unclear, particularly in non-model plants such as pepper (Capsicum annum L.). Herein, we functionally characterized CaASHH3, a member of class II (the ASH1 homologs H3K36) proteins in pepper immunity against Ralstonia solanacearum and Pseudomonas syringae pv tomato DC3000 (Pst DC3000). The CaASHH3 was localized in the nucleus, and its transcript levels were significantly enhanced by R. solanacearum inoculation (RSI) and exogenous application of salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), and abscisic acid (ABA). Knockdown of CaASHH3 by virus-induced gene silencing (VIGS) compromised peppers' resistance to RSI. Furthermore, silencing of CaASHH3 impaired hypersensitive-response (HR)-like cell death response due to RSI and downregulated defense-associated marker genes, including CaPR1, CaNPR1, and CaABR1. The CaASHH3 protein was revealed to affect the promoters of CaNPR1, CaPR1, and CaHSP24. Transiently over-expression of CaASHH3 in pepper leaves elicited HR-like cell death and upregulated immunity-related marker genes. To further study the role of CaASHH3 in plant defense in vivo, CaASHH3 transgenic plants were generated in Arabidopsis. Overexpression of CaASHH3 in transgenic Arabidopsis thaliana enhanced innate immunity against Pst DC3000. Furthermore, CaASHH3 over-expressing transgenic A. thaliana plants exhibited upregulated transcriptional levels of immunity-associated marker genes, such as AtNPR1, AtPR1, and AtPR2. These results collectively confirm the role of CaASHH3 as a positive regulator of plant cell death and pepper immunity against bacterial pathogens, which is regulated by signaling synergistically mediated by SA, JA, ET, and ABA.

The CaPti1-CaERF3 module positively regulates resistance of Capsicum annuum to bacterial wilt disease by coupling enhanced immunity and dehydration tolerance.

Bacterial wilt, a severe disease involving vascular system blockade, is caused by Ralstonia solanacearum. Although both plant immunity and dehydration tolerance might contribute to disease resistance, whether and how they are related remains unclear. Herein, we showed that immunity against R. solanacearum and dehydration tolerance are coupled and regulated by the CaPti1-CaERF3 module. CaPti1 and CaERF3 are members of the serine/threonine protein kinase and ethylene-responsive factor families, respectively. Expression profiling revealed that CaPti1 and CaERF3 were upregulated by R. solanacearum inoculation, dehydration stress, and exogenously applied abscisic acid (ABA). They in turn phenocopied each other in promoting resistance of pepper (Capsicum annuum) to bacterial wilt not only by activating salicylic acid-dependent CaPR1, but also by activating dehydration tolerance-related CaOSM1 and CaOSR1 and inducing stomatal closure to reduce water loss in an ABA signaling-dependent manner. Our yeast two hybrid assay showed that CaERF3 interacted with CaPti1, which was confirmed using co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that upon R. solanacearum inoculation, CaPR1, CaOSM1, and CaOSR1 were directly targeted and positively regulated by CaERF3 and potentiated by CaPti1. Additionally, our data indicated that the CaPti1-CaERF3 complex might act downstream of ABA signaling, as exogenously applied ABA did not alter regulation of stomatal aperture by the CaPti1-CaERF3 module. Importantly, the CaPti1-CaERF3 module positively affected pepper growth and the response to dehydration stress. Collectively, the results suggested that immunity and dehydration tolerance are coupled and positively regulated by CaPti1-CaERF3 in pepper plants to enhance resistance against R. solanacearum.

CabZIP23 Integrates in CabZIP63-CaWRKY40 Cascade and Turns CabZIP63 on Mounting Pepper Immunity against Ralstonia solanacearum via Physical Interaction.

CabZIP63 and CaWRKY40 were previously found to be shared in the pepper defense response to high temperature stress (HTS) and to Ralstonia solanacearum inoculation (RSI), forming a transcriptional cascade. However, how they activate the two distinct defense responses is not fully understood. Herein, using a revised genetic approach, we functionally characterized CabZIP23 in the CabZIP63-CaWRKY40 cascade and its context specific pepper immunity activation against RSI by interaction with CabZIP63. CabZIP23 was originally found by immunoprecipitation-mass spectrometry to be an interacting protein of CabZIP63-GFP; it was upregulated by RSI and acted positively in pepper immunity against RSI by virus induced gene silencing in pepper plants, and transient overexpression in Nicotiana benthamiana plants. By chromatin immunoprecipitation (ChIP)-qPCR and electrophoresis mobility shift assay (EMSA), CabZIP23 was found to be directly regulated by CaWRKY40, and CabZIP63 was directly regulated by CabZIP23, forming a positive feedback loop. CabZIP23-CabZIP63 interaction was confirmed by co-immunoprecipitation (CoIP) and bimolecular fluorescent complimentary (BiFC) assays, which promoted CabZIP63 binding immunity related target genes, including CaPR1, CaNPR1 and CaWRKY40, thereby enhancing pepper immunity against RSI, but not affecting the expression of thermotolerance related CaHSP24. All these data appear to show that CabZIP23 integrates in the CabZIP63-CaWRKY40 cascade and the context specifically turns it on mounting pepper immunity against RSI.

CaSWC4 regulates the immunity-thermotolerance tradeoff by recruiting CabZIP63/CaWRKY40 to target genes and activating chromatin in pepper.

Pepper (Capsicum annuum) responds differently to high temperature stress (HTS) and Ralstonia solanacearum infection (RSI) but employs some shared transcription factors (TFs), such as CabZIP63 and CaWRKY40, in both cases. How the plant activates and balances these distinct responses, however, was unclear. Here, we show that the protein CaSWC4 interacts with CaRUVBL2 and CaTAF14b and they all act positively in pepper response to RSI and thermotolerance. CaSWC4 activates chromatin of immunity or thermotolerance related target genes of CaWRKY40 or CabZIP63 by promoting deposition of H2A.Z, H3K9ac and H4K5ac, simultaneously recruits CabZIP63 and CaWRKY40 through physical interaction and brings them to their targets (immunity- or thermotolerance-related genes) via binding AT-rich DNA element. The above process relies on the recruitment of CaRUVBL2 and TAF14 by CaSWC4 via physical interaction, which occurs at loci of immunity related target genes only when the plants are challenged with RSI, and at loci of thermotolerance related target genes only upon HTS. Collectively, our data suggest that CaSWC4 regulates rapid, accurate responses to both RSI and HTS by modulating chromatin of specific target genes opening and recruiting the TFs, CaRUVBL2 and CaTAF14b to the specific target genes, thereby helping achieve the balance between immunity and thermotolerance.

RNAi-Based Gene Silencing of RXLR Effectors Protects Plants Against the Oomycete Pathogen Phytophthora capsici.

Phytophthora capsici is a broad-host range oomycete pathogen that can cause severe phytophthora blight disease of pepper and hundreds of other plant species worldwide. Natural resistance against P. capsici is inadequate, and it is very difficult to control by most of existing chemical fungicides. Therefore, it is urgent to develop alternative strategies to control this pathogen. Recently, host-induced or spray-induced gene silencing of essential or virulent pathogen genes provided an effective strategy for disease controls. Here, we demonstrate that P. capsici can effectively take up small interfering RNAs (siRNAs) from the environment. According to RNA-seq and quantitative reverse transcription PCR analysis, we identified four P. capsici RXLR effector genes that are significantly up-regulated during the infection stage. Transient overexpression and promote-infection assays indicated that RXLR1 and RXLR4 could promote pathogen infection. Using a virus-induced gene silencing system in pepper plants, we found that in planta-expressing RNA interference (RNAi) constructs that target RXLR1 or RXLR4 could significantly reduce pathogen infection, while co-interfering RXLR1 and RXLR4 could confer a more enhanced resistance to P. capsici. We also found that exogenously applying siRNAs that target RXLR1 or RXLR4 could restrict growth of P. capsici on the pepper and Nicotiana benthamiana leaves; when targeting RXLR1 and RXLR4 simultaneously, the control effect was more remarkable. These data suggested that RNAi-based gene silencing of RXLR effectors has great potential for application in crop improvement against P. capsici and also provides an important basis for the development of RNA-based antioomycete agents.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A CaCDPK29-CaWRKY27b module promotes CaWRKY40-mediated thermotolerance and immunity to Ralstonia solanacearum in pepper.

CaWRKY40 in pepper (Capsicum annuum) promotes immune responses to Ralstonia solanacearum infection (RSI) and to high-temperature, high-humidity (HTHH) stress, but how it interacts with upstream signalling components remains poorly understood. Here, using approaches of reverse genetics, biochemical and molecular biology we functionally characterised the relationships among the WRKYGMK-containing WRKY protein CaWRKY27b, the calcium-dependent protein kinase CaCDPK29, and CaWRKY40 during pepper response to RSI or HTHH. Our data indicate that CaWRKY27b is upregulated and translocated from the cytoplasm to the nucleus upon phosphorylation of Ser137 in the nuclear localisation signal by CaCDPK29. Using electrophoretic mobility shift assays and microscale thermophoresis, we observed that, due to the replacement of Q by M in the conserved WRKYGQK, CaWRKY27b in the nucleus failed to bind to W-boxes in the promoters of immunity- and thermotolerance-related marker genes. Instead, CaWRKY27b interacted with CaWRKY40 and promoted its binding and positive regulation of the tested marker genes including CaNPR1, CaDEF1 and CaHSP24. Notably, mutation of the WRKYGMK motif in CaWRKY27b to WRKYGQK restored the W-box binding ability. Our data therefore suggest that CaWRKY27b is phosphorylated by CaCDPK29 and acts as a transcriptional activator of CaWRKY40 during the pepper response to RSI and HTHH.

Solanaceous plants switch to cytokinin-mediated immunity against Ralstonia solanacearum under high temperature and high humidity.

Plant diseases generally tend to be more serious under conditions of high temperature and high humidity (HTHH) than under ambient temperature, but plant immunity against pathogen attacks under HTHH remains elusive. Herein, we used pepper as an example to study how Solanaceae cope with Ralstonia solanacearum infection (RSI) under HTHH by performing RNA-seq combined with the reverse genetic method. The result showed that immunities mediated by salicylic acid (SA) and jasmonic acid (JA) in pepper roots were activated by RSI under ambient temperature. However, upon RSI under HTHH, JA signalling was blocked and SA signalling was activated early but its duration was greatly shortened in pepper roots, instead, expression of CaIPT5 and Glutathione S-transferase encoding genes, as well as endogenous content of trans-Zeatin, were enhanced. In addition, by silencing in pepper plants and overexpression in Nicotiana benthamiana, CaIPT5 was found to act positively in the immune response to RSI under HTHH in a way related to CaPRP1 and CaMgst3. Furthermore, the susceptibility of pepper, tomato and tobacco to RSI under HTHH was significantly reduced by exogenously applied tZ, but not by either SA or MeJA. All these data collectively suggest that pepper employs cytokinin-mediated immunity to cope with RSI under HTHH.

A cysteine-rich receptor-like protein kinase CaCKR5 modulates immune response against Ralstonia solanacearum infection in pepper.

BACKGROUND: Cysteine-rich receptor-like kinases (CRKs) represent a large subfamily of receptor-like kinases and play vital roles in diverse physiological processes in regulating plant growth and development. RESULTS: CaCRK5 transcripts were induced in pepper upon the infection of Ralstonia solanacearum and treatment with salicylic acid. The fusions between CaCRK5 and green fluorescence protein were targeted to the plasma membrane. Suppression of CaCRK5 via virus-induced gene silencing (VIGS) made pepper plants significantly susceptible to R. solanacearum infection, which was accompanied with decreased expression of defense related genes CaPR1, CaSAR8.2, CaDEF1 and CaACO1. Overexpression of CaCRK5 increased resistance against R. solanacearum in Nicotiana benthamiana. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation coupled with quantitative real-time PCR analysis revealed that a homeodomain zipper I protein CaHDZ27 can active the expression of CaCRK5 through directly binding to its promoter. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses suggested that CaCRK5 heterodimerized with the homologous member CaCRK6 on the plasma membrane. CONCLUSIONS: Our data revealed that CaCRK5 played a positive role in regulating immune responses against R. solanacearum infection in pepper.

CaAIL1 Acts Positively in Pepper Immunity against Ralstonia solanacearum by Repressing Negative Regulators.

APETALA2 (AP2) subfamily transcription factors participate in plant growth and development, but their roles in plant immunity remain unclear. Here, we discovered that the AP2 transcription factor CaAIL1 functions in immunity against Ralstonia solanacearum infection (RSI) in pepper (Capsicum annuum). CaAIL1 expression was upregulated by RSI, and loss- and gain-of-function assays using virus-induced gene silencing and transient overexpression, respectively, revealed that CaAIL1 plays a positive role in immunity to RSI in pepper. Chromatin immunoprecipitation sequencing (ChIP-seq) uncovered a subset of transcription-factor-encoding genes, including CaRAP2-7, CaGATA17, CaGtf3a and CaTCF25, that were directly targeted by CaAIL1 via their cis-elements, such as GT or AGGCA motifs. ChIP-qPCR and electrophoretic mobility shift assays confirmed these findings. These genes, encoding transcription factors with negative roles in immunity, were repressed by CaAIL1 during pepper response to RSI, whereas genes encoding positive immune regulators such as CaEAS were derepressed by CaAIL1. Importantly, we showed that the atypical EAR motif (LXXLXXLXX) in CaAIL1 is indispensable for its function in immunity. These findings indicate that CaAIL1 enhances the immunity of pepper against RSI by repressing a subset of negative immune regulators during the RSI response through its binding to several cis-elements in their promoters.