CaWRKY40b in Pepper Acts as a Negative Regulator in Response to Ralstonia solanacearum by Directly Modulating Defense Genes Including CaWRKY40

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.


Introduction
During the course of evolution, plants have developed a sophisticated defense mechanism for counteracting diverse pathogens. In addition to the physical barriers for damage prevention, plants possess two layers of inducible immunities termed as pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) and effector triggered immunity (ETI). The perception of conserved microbe-associated molecular patterns (MAMPs) in plant triggers PTI via pattern recognition receptors (PRRs) on the plant cell surface. However, ETI in plants is triggered by the perception of strain-specific pathogen effectors that are delivered into host cells via intracellular R proteins, which are generally coupled with hypersensitive response (HR) cell death. Interconnected in a zig-zag manner [1-6], PTI and ETI share a highly overlapping signaling network [7][8][9]. A key step in both PTI and ETI is massive reprogramming that is mediated by various transcription factors (TFs) [10][11][12], which play vital roles in plant responses to the pathogen attack [13,14]. However, the roles of the majority of TFs in plant immunity and how these translate upstream stress signals into appropriate transcriptional outputs remain to be elucidated.
WRKYs constitute one of the largest plant TF families and have been implicated in plant growth and development as well as responses to various environmental stresses. These are named after their one or two WRKY domains, which comprise a highly conserved amino acid sequence WRKYGQK, together with a zinc-finger-like motif [15]. WRKY TFs have the ability to activate or repress transcription by directly binding to the W-box (TTGACC/T) within the promoters of their target genes [16], thereby playing important roles in the regulation of plant responses to pathogen and herbivore attacks as well as abiotic stresses, including heat stress, drought, and salinity. A subset of WRKYs have been found to be involved in plant immunity [17][18][19][20][21]. For example, among the 72 WRKY TFs in Arabidopsis, 49 AtWRKY genes are differentially expressed during the infection with Pseudomonas syringae or application of exogenous salicylic acid (SA) [22]. Genetic evidence demonstrates that WRKY8 [23], WRKY22 [24], WRKY25 [25,26], WRKY28 [23], WRKY33 [25,27], WRKY38 [28], WRKY48 [23], AtWRKY54 [29], WRKY62 [28], AtWRKY72 [30], and WRKY75 [31] act as positive regulators, whereas WRKY11, WRKY17 [32], WRKY40 [33], and WRKY60 [34] act as negative regulators in basal defense or immunity. WRKY TFs are part of a WRKY network and play important roles in the regulation of plant immunity [35,36]. However, the exact functions of other pathogen-responsive WRKY TFs and the modes of coordination among multiple WRKY TFs participating in plant immunity in non-model plant families such as the Solanaceae remain unclear.
Pepper (Capsicum annuum) is an important vegetable and a typical member of the Solanaceae. It is distributed in uplands during warm seasons and is constantly challenged with various soil-borne pathogens. The frequent occurrence of high temperature and high humidity (HTHH) conditions usually causes severe diseases in pepper plants. Breeding and the application of pepper cultivars that are highly resistant to disease is one of the most efficient approaches to solving the problem caused by diseases in pepper production, and a better understanding of the mechanism underlying pepper immunity against various pathogens may facilitate in the genetic improvement of pepper disease resistance. Although WRKY TFs play important roles in plant immunity, the number of WRKY TFs in pepper that have been characterized in terms of function and expression in relation to plant immunity is limited. They include: CaWRKY1 [37], CaWRKY-a [38,39], CaWRKY6 [3], CaWRKYb [40], CaWRKYd [41], CaWRKY2 [42], CaWRKY27 [43], CaWRKY40 [4], and CaWRKY58 [44]. In addition, some pepper WRKY TFs such as CaWRKY6 [3] and CaWRKY40 [4] also act as positive regulators of responses to high temperature stress, probably reflecting the evolution of special immunity under the combined pressure of HTHH and soil-borne pathogens. However, the exact function of the majority of the WRKY TFs in pepper remains elusive. In the present study, we focused on evaluating the function of CaWRKY40b in R. solanacearum-infected C. annuum plants. CaWRKY40b was found to act as a negative regulator in pepper immunity by directly targeting immunity-associated genes, including CaWRKY40.

The Expression of CaWRKY40b Is Modulated Transcriptionally by Ralstonia solanacearum Inoculation (RSI)
The presence of the subset of pathogen-responsive CGTGA motif [45] and W-box bound by WRKY TFs [16,17] in the CaWRKY40b promoter region implies its possible transcriptional response to pathogens. To test this possibility, the expression of CaWRKY40b in pepper plants against R. solanacearum was assessed by qRT-PCR. The results revealed a downregulation of CaWRKY40b by RSI in the pepper leaves from 6 hours post inoculation (hpi) to 48 hpi (Figure 1), indicating that CaWRKY40b might be involved in the response of pepper to RSI.

The Expression of CaWRKY40b Is Modulated Transcriptionally by Ralstonia solanacearum Inoculation (RSI)
The presence of the subset of pathogen-responsive CGTGA motif [45] and W-box bound by WRKY TFs [16,17] in the CaWRKY40b promoter region implies its possible transcriptional response to pathogens. To test this possibility, the expression of CaWRKY40b in pepper plants against R. solanacearum was assessed by qRT-PCR. The results revealed a downregulation of CaWRKY40b by RSI in the pepper leaves from 6 hours post inoculation (hpi) to 48 hpi (Figure 1), indicating that CaWRKY40b might be involved in the response of pepper to RSI. solanacearum-inoculated leaves after inoculation with virulent R. solanacearum strain FJC100301 (OD600nm = 0.8) were compared to that in control plants at relative expression level of "1′. Experiments were performed thrice in triplicate biological repeats. Data are expressed as the mean ± SD of three samples, each containing three plants. Different letters indicate significant differences as determined by Fisher's protected LSD test: uppercase letters, p < 0.01; lower case letters, p < 0.05. (B) CaWRKY40b transcript levels measured at different time points in R. solanacearum-inoculated leaves after inoculation with virulent R. solanacearum strain FJC100301 (OD600nm = 0.8) were compared to that in control plants at relative expression level of "1 . Experiments were performed thrice in triplicate biological repeats. Data are expressed as the mean ± SD of three samples, each containing three plants. Different letters indicate significant differences as determined by Fisher's protected LSD test: uppercase letters, p < 0.01; lower case letters, p < 0.05.

CaWRKY40b Localizes Exclusively to the Nuclei
To localize CaWRKY40b at subcellular level, a fused CaWRKY40b-GFP protein was expressed in N. benthamiana leaves by infiltrating GV3101 cells containing 35S::CaWRKY40b-GFP and using 35S::GFP construct as a control. The GFP signal was observed under a laser scanning confocal microscope (LSCM). In CaWRKY40b-GFP transiently overexpressed epidermal cells of N. benthamiana leaves, the GFP signal was exclusively observed in the nuclei, whereas the GFP signals in the epidermal cells of 35S::GFP-infiltrated N. benthamiana leaves were found across the entire cell, including plasma membrane, cytosol, and nucleus ( Figure 2).

CaWRKY40b Localizes Exclusively to the Nuclei
To localize CaWRKY40b at subcellular level, a fused CaWRKY40b-GFP protein was expressed in N. benthamiana leaves by infiltrating GV3101 cells containing 35S::CaWRKY40b-GFP and using 35S::GFP construct as a control. The GFP signal was observed under a laser scanning confocal microscope (LSCM). In CaWRKY40b-GFP transiently overexpressed epidermal cells of N. benthamiana leaves, the GFP signal was exclusively observed in the nuclei, whereas the GFP signals in the epidermal cells of 35S::GFP-infiltrated N. benthamiana leaves were found across the entire cell, including plasma membrane, cytosol, and nucleus ( Figure 2).

Silencing of CaWRKY40b Enhances the Resistance of Pepper to RSI
To investigate the possible role of CaWRKY40b in the response of pepper to RSI, loss of function analysis was conducted by VIGS in pepper plants. To silence CaWRKY40b in pepper plants, two fragments in the 3′ UTR or ORF of CaWRKY40b were employed. The specificity of these two fragments was further confirmed by whole-genome search (http://passport.pepper.snu.ac.kr). GV3101 cells containing TRV::CaWRKY40b were infiltrated into the leaves of 20-day-old pepper seedlings. The silencing process was monitored by TRV::PDS pepper plants. Using real-time RT-PCR, the transcription of CaWRKY40b was assayed and after 20 days post inoculation (dpi), the transcript level of CaWRKY40b in TRV::CaWRKY40b-1 and TRV::CaWRKKY40b-2 pepper plants was <10% of that in the TRV::00 plants ( Figure 3A).
The TRV::CaWRKY40b-1, CaWRKKY40b-2 and TRV::00 pepper plants were inoculated with R. solanacearum cells FJC100301 [4] at 3 dpi. A clear wilting phenotype was observed in the majority of FJC100301-inoculated TRV::00 pepper plants, whereas no obvious or only slight wilting was observed in both TRV::CaWRKY40b-1 and TRV::CaWRKY40b-2 pepper plants ( Figure 3B). To accurately quantify the extent of disease in R. solanacearum-inoculated plants, we determined the relative disease index from 1 dpi to 8 dpi in both the TRV::CaWRKY40b-1 TRV::CaWRKY40b-2 and TRV:00 pepper plants. To quantify the growth of R. solanacearum in the inoculated pepper rootstocks, the cfu of the rootstocks of R. solanacearum inoculated pepper plants was measured. TRV2::CaWRKY40b-1 and TRV2::CaWRKY40b-2 consistently showed a significant decrease in disease symptoms as well as pathogen growth, respectively, compared to the wild-type plants ( Figure  3C,D).

Silencing of CaWRKY40b Enhances the Resistance of Pepper to RSI
To investigate the possible role of CaWRKY40b in the response of pepper to RSI, loss of function analysis was conducted by VIGS in pepper plants. To silence CaWRKY40b in pepper plants, two fragments in the 3 UTR or ORF of CaWRKY40b were employed. The specificity of these two fragments was further confirmed by whole-genome search (http://passport.pepper.snu.ac.kr). GV3101 cells containing TRV::CaWRKY40b were infiltrated into the leaves of 20-day-old pepper seedlings. The silencing process was monitored by TRV::PDS pepper plants. Using real-time RT-PCR, the transcription of CaWRKY40b was assayed and after 20 days post inoculation (dpi), the transcript level of CaWRKY40b in TRV::CaWRKY40b-1 and TRV::CaWRKKY40b-2 pepper plants was <10% of that in the TRV::00 plants ( Figure 3A).
The TRV::CaWRKY40b-1, CaWRKKY40b-2 and TRV::00 pepper plants were inoculated with R. solanacearum cells FJC100301 [4] at 3 dpi. A clear wilting phenotype was observed in the majority of FJC100301-inoculated TRV::00 pepper plants, whereas no obvious or only slight wilting was observed in both TRV::CaWRKY40b-1 and TRV::CaWRKY40b-2 pepper plants ( Figure 3B). To accurately quantify the extent of disease in R. solanacearum-inoculated plants, we determined the relative disease index from 1 dpi to 8 dpi in both the TRV::CaWRKY40b-1 TRV::CaWRKY40b-2 and TRV:00 pepper plants. To quantify the growth of R. solanacearum in the inoculated pepper rootstocks, the cfu of the rootstocks of R. solanacearum inoculated pepper plants was measured. TRV2::CaWRKY40b-1 and TRV2::CaWRKY40b-2 consistently showed a significant decrease in disease symptoms as well as pathogen growth, respectively, compared to the wild-type plants ( Figure 3C,D). , the mean ± SD was calculated from four independent duplicates, with each duplicate consisting of 10 plants. Asterisks in C indicate statistically significant differences compared to the mock treatment by the LSD test (* p < 0.05, ** p < 0.01). Different letters in (A-D) indicate significant differences among means as determined by Fisher's protected LSD test: uppercase letters, p < 0.01; lower case letters, p < 0.05.

Transient Overexpression of CaWRKY40b-SRDX Triggered Intensive Cell Death in Pepper Plants
The effect of transient overexpression of CaWRKKY40b and its chimeric repressor version CaWRKY40b-SRDX [46,47] on the hypersensitive response (HR) cell death in leaves of pepper plants was investigated. GV3101 cells containing construct 35S::CaWRKY40b, 35S::CaWRKY40b-SRDX and 35S::00 were infiltrated into the pepper leaves. Transient overexpression of CaWRKY40b-HA in pepper leaves was detected by immunoblotting (IB) against the antibody of HA at 48 hpi ( Figure  4A). A clear cell death and darker staining of trypan blue and DAB were consistently noticed around infiltrated sites of Agrobacterium cells containing 35S::CaWRKY40b-SRDX; however, no obvious cell death or darker staining of trypan blue or DAB was observed in the CaWRKY40b-transiently overexpressing and in the mock-treated pepper leaves ( Figure 4B). Furthermore, a higher ion leakage was triggered by 35S::CaWRKY40b-SRDX than that by 35S::CaWRKY40b ( Figure 4C). In (A,C,D), the mean ± SD was calculated from four independent duplicates, with each duplicate consisting of 10 plants. Asterisks in C indicate statistically significant differences compared to the mock treatment by the LSD test (* p < 0.05, ** p < 0.01). Different letters in (A-D) indicate significant differences among means as determined by Fisher's protected LSD test: uppercase letters, p < 0.01; lower case letters, p < 0.05.

Transient Overexpression of CaWRKY40b-SRDX Triggered Intensive Cell Death in Pepper Plants
The effect of transient overexpression of CaWRKKY40b and its chimeric repressor version CaWRKY40b-SRDX [46,47]   CaWRKY40b-SRDX, but not by that of CaWRKY40b and displayed by phenotype, DAB, and trypan blue staining at 4 dpi, respectively. Bar = 100 µm. (C) Quantification of electrolyte leakage as ion conductivity to assess the cell death response in leaf disks, the means ± SD were calculated from four samples, each containing six disks. Capital and lowercase letters above the bars indicate significant difference at p < 0.01 and p < 0.05, respectively, as analyzed by Fisher's protected LSD test.

Transcriptional Modulation of Marker Genes by Transient Overexpression and Virus-Induced Silencing of CaWRKY40b
Chromatin Immunoprecipitation sequencing (ChIP-seq) was performed to identify the potential target genes of CaWRKY40b ( Figure S2). To do this, GV3101 cells harboring 35S::CaWRKY40b-HA was infiltrated into leaves of six-week-old pepper plants and maintained in the greenhouse. Forty-eight hours post infiltration, the infiltrated leaves were sampled for chromatin isolation, the chromatins were sheared into fragments of 300-500 bps in length and were immunoprecipitated with antibodies of HA, the sequencing of immunoprecipitated DNA was performed on Illumina HiSeq2500 resulted in about one million reads, in which more than 8 hundred binding sites were identified. Gene ontology (GO) was analyzed to determine the biological and functional processes of CaWRKY40b target genes ( Figure S2 and Table S1). As the role of CaWRKY40b in pepper immunity was the focus of the present study, immunity associated genes were selected from the target genes of CaWRKY40b to confirm the role of CaWRKY40b and to test the possible mode of action, including CA00g87690 (CaWRKY40), CA05g11520 (jasmonic acid-amido synthetase JAR1-like isoform X2, JAR1), CA06g24540 (heat shock cognate 70 kDa protein 1, HSC70), CA01g13570 (ETHYLENE INSENSITIVE 3-like, EIN3), CA02g12020 (LRR receptor-like serine/threonine-protein kinase FLS2), CA10g01730 (LRR receptor-like serine/threonine-protein kinase At3g47570 isoform X1, RLK1), CA05g11620 (putative cyclic nucleotide-gated ion channel 8, CNGIC8), and CA09g10430 (calcium-dependent protein kinase 13, CDPK13). The result from ChIP-PCR showed that DNA within the promoter regions of these genes enriched in the CaWRKY40b bound DNA, attesting the validity of the ChIP-seq results (Table S1 and Figure S3).
To test whether CaWRKY40b can control the transcriptional expression of all the genes directly targeted by CaWRKY40b, their transcription was comparatively tested in CaWRKY40b transiently overexpressed and CaWRKY40b-silenced pepper plants by real-time RT-PCR. The results demonstrated that the transcription of CaWRKY40, JAR1, BPR1, EIN3, FLS2, RLK1, CNGIC8, and CDPK13 was significantly enhanced by RSI, and the transcription of these genes was significantly higher in CaWRKY40b-silenced plants than in the CaWRKY40b un-silenced control plants with or without RSI. In contrast, the transcription of CaHSC70, which was downregulated by RSI, was lower in CaWRKY40b-silenced pepper plants than that in the control plants ( Figure 5). Consistent to the result in CaWRKY40b-silenced pepper plants, transient overexpression of CaWRKY40b-SRDX significantly downregulated CaHSC70, while elevated the transcript levels of the other tested genes compared to the control ( Figure 6). (B) Intensive cell death was triggered by transient overexpression of CaWRKY40b-SRDX, but not by that of CaWRKY40b and displayed by phenotype, DAB, and trypan blue staining at 4 dpi, respectively. Bar = 100 µm. (C) Quantification of electrolyte leakage as ion conductivity to assess the cell death response in leaf disks, the means ± SD were calculated from four samples, each containing six disks. Capital and lowercase letters above the bars indicate significant difference at p < 0.01 and p < 0.05, respectively, as analyzed by Fisher's protected LSD test.

Transcriptional Modulation of Marker Genes by Transient Overexpression and Virus-Induced Silencing of CaWRKY40b
Chromatin Immunoprecipitation sequencing (ChIP-seq) was performed to identify the potential target genes of CaWRKY40b ( Figure S2). To do this, GV3101 cells harboring 35S::CaWRKY40b-HA was infiltrated into leaves of six-week-old pepper plants and maintained in the greenhouse. Forty-eight hours post infiltration, the infiltrated leaves were sampled for chromatin isolation, the chromatins were sheared into fragments of 300-500 bps in length and were immunoprecipitated with antibodies of HA, the sequencing of immunoprecipitated DNA was performed on Illumina HiSeq2500 resulted in about one million reads, in which more than 8 hundred binding sites were identified. Gene ontology (GO) was analyzed to determine the biological and functional processes of CaWRKY40b target genes ( Figure S2 and Table S1). As the role of CaWRKY40b in pepper immunity was the focus of the present study, immunity associated genes were selected from the target genes of CaWRKY40b to confirm the role of CaWRKY40b and to test the possible mode of action, including CA00g87690 (CaWRKY40), CA05g11520 (jasmonic acid-amido synthetase JAR1-like isoform X2, JAR1), CA06g24540 (heat shock cognate 70 kDa protein 1, HSC70), CA01g13570 (ETHYLENE INSENSITIVE 3-like, EIN3), CA02g12020 (LRR receptor-like serine/threonine-protein kinase FLS2), CA10g01730 (LRR receptor-like serine/threonine-protein kinase At3g47570 isoform X1, RLK1), CA05g11620 (putative cyclic nucleotide-gated ion channel 8, CNGIC8), and CA09g10430 (calcium-dependent protein kinase 13, CDPK13). The result from ChIP-PCR showed that DNA within the promoter regions of these genes enriched in the CaWRKY40b bound DNA, attesting the validity of the ChIP-seq results (Table S1 and Figure S3).
To test whether CaWRKY40b can control the transcriptional expression of all the genes directly targeted by CaWRKY40b, their transcription was comparatively tested in CaWRKY40b transiently overexpressed and CaWRKY40b-silenced pepper plants by real-time RT-PCR. The results demonstrated that the transcription of CaWRKY40, JAR1, BPR1, EIN3, FLS2, RLK1, CNGIC8, and CDPK13 was significantly enhanced by RSI, and the transcription of these genes was significantly higher in CaWRKY40b-silenced plants than in the CaWRKY40b un-silenced control plants with or without RSI. In contrast, the transcription of CaHSC70, which was downregulated by RSI, was lower in

The Expression of CaWRKY40b Was Directly and Transcriptionally Regulated by CaWRKY40b Itself
As CaWRKY40b itself was identified by ChIP-seq as the target gene of CaWRKY40b, the possible binding of CaWRKY40b to its own promoter was also confirmed by ChIP-seq ( Figure 7A). The possible self-regulation of CaWRKY40b was also tested comparatively by assaying the effect of transient overexpression of CaWRKY40b on the expression of GUS-driven pCaWRKY40b and on the transcription of CaWRKY40b. The results presented that the transient overexpression of CaWRKY40b significantly increased GUS expression ( Figure 7B) as well as the transcriptional level of CaWRKY40b by real-time RT-PCR using a pair of specific primers based on the sequence of 3′-UTR of CaWRKY40b ( Figure 7C). qRT-PCR analysis of the expression of immunity-associated marker genes in 35S::00, 35S::CaWRKY40b and 35S::CaWRKY40b-SRDX constructs. Data display the means ± SD of four independent biological replicates, each containing three leaves. Capital and lowercase letters above the bars indicate significantly different means (p < 0.01) and significantly different means (p < 0.01), respectively, as analyzed by Fisher's protected LSD test.

The Expression of CaWRKY40b Was Directly and Transcriptionally Regulated by CaWRKY40b Itself
As CaWRKY40b itself was identified by ChIP-seq as the target gene of CaWRKY40b, the possible binding of CaWRKY40b to its own promoter was also confirmed by ChIP-seq ( Figure 7A). The possible self-regulation of CaWRKY40b was also tested comparatively by assaying the effect of transient overexpression of CaWRKY40b on the expression of GUS-driven pCaWRKY40b and on the transcription of CaWRKY40b. The results presented that the transient overexpression of CaWRKY40b significantly increased GUS expression ( Figure 7B) as well as the transcriptional level of CaWRKY40b by real-time RT-PCR using a pair of specific primers based on the sequence of 3 -UTR of CaWRKY40b ( Figure 7C). Data are the means ± SD of four independent biological replicates. Capital and lowercase letters above the bars indicate significantly different means at p < 0.01 and p < 0.05, respectively, as analyzed by Fisher's protected LSD test.

Discussion
Data mainly from model plants including Arabidopsis and rice suggest that WRKY TFs play important roles in plant immunity, but their role and the underlying mechanism remain poorly understood. Despite functional characterization of several members of this family in pepper [3,4,37,38,40,42,43,48,49], the function of majority of members in this family in pepper remains unknown. The findings of the present study suggest that CaWRKY40b acts as a negative regulator in pepper immunity against RSI by directly modulating the transcription of a subset of immunity-associated genes.
WRKY proteins are characterized by their one or two highly conserved WRKY domains and bind to the target genes promoters mainly by means of the typical W-box [16,17]. The presence of highly conserved WRKY domains in CaWRKY40b and their binding to W-box-containing promoters of their potential target genes as indicated by ChIP assay and real-time PCR suggest that overexpressing leaves, the primer pair based on the 3 UTR was used. Data are the means ± SD of four independent biological replicates. Capital and lowercase letters above the bars indicate significantly different means at p < 0.01 and p < 0.05, respectively, as analyzed by Fisher's protected LSD test.

Discussion
Data mainly from model plants including Arabidopsis and rice suggest that WRKY TFs play important roles in plant immunity, but their role and the underlying mechanism remain poorly understood. Despite functional characterization of several members of this family in pepper [3,4,37,38,40,42,43,48,49], the function of majority of members in this family in pepper remains unknown. The findings of the present study suggest that CaWRKY40b acts as a negative regulator in pepper immunity against RSI by directly modulating the transcription of a subset of immunity-associated genes.
WRKY proteins are characterized by their one or two highly conserved WRKY domains and bind to the target genes promoters mainly by means of the typical W-box [16,17]. The presence of highly conserved WRKY domains in CaWRKY40b and their binding to W-box-containing promoters of their potential target genes as indicated by ChIP assay and real-time PCR suggest that CaWRKY40b is a member of the WRKY family of pepper. In addition, our results indicate that CaWRKY40b acts as a negative regulator in the response of pepper to R. solanacearum, and silencing of CaWRKY40b with two independent VIGS vectors significantly and consistently decreased the growth of R. solanacearum and the susceptibility of pepper plants to RSI. Similarly, transient overexpression of CaWRKY40 chimeric repressor version (e.g., CaWRKY40-SRDX) [46,47] triggered intensive HR cell death in pepper leaves whereas that of CaWRKY40b did not. The role of CaWRKY40b as a negative regulator in pepper immunity is similar to that of its homolog, AtWRKY40, in Arabidopsis [34], but different from its ortholog CaWRKY40 in pepper [4]. Further investigation into how this functional difference between CaWRKY40 and CaWRKY40b is structurally determined would provide new insights into pepper immunity. Because CaWRKY40b was downregulated by RSI, we hypothesize that the constitutive expression of CaWRKY40b blocks defense responses to minimize the unnecessary resource cost for defense responses in the absence of pathogens, whereas its downregulation might derepress immunity with RSI challenge. Besides CaWRKY40b, CaWRKY1 [37] and CaWRKY58 [44] have been characterized in pepper as negative regulators in the immunity [44,50]. Gene silencing of CaWRKY40b in this study or CaWRKY1 and CaWRKY58 in previous studies [37,44] significantly decreased the susceptibility of pepper to pathogen attack, thereby suggesting their functional specificities. The existence of multiple negative regulators might favor plants to avert inappropriate activation of different defense responses or activate defense responses against pathogens with different lifestyles.
The role of CaWRKY40b as a negative regulator in pepper immunity was further supported by its direct modulation of the transcription of a subset of immunity-associated genes. These genes originally identified by ChIP-seq, including CaWRKY40, JAR, HSC70, RLK1, EIN3, FLS2, CNGIC8, and CDPK13, which act as positive regulators in plant immunity, were downregulated by transient overexpression of CaWRKY40b, but upregulated by silencing of CaWRKY40b. Although the exact roles of the tested target genes in pepper immunity remain to be identified, JAR [51][52][53] and EIN3 [54,55] have been implicated in JA and ET signaling pathways, which ubiquitously exist in the immune system of different plant species. RLK1 [56,57] and FLS2 [58][59][60][61], which are PRRs crucial for perception of conserved MAMPs to activate PTI, are conserved among different plant species [5,6]. Most importantly, our previous study provided concrete evidence that CaWRKY40 acts as positive regulator in the response of pepper to RSI [3,4]. Therefore, we hypothesize that CaWRKY40b functions as a negative regulator in pepper immunity at least partially by repressing these positive regulators. In contrast, HSC70, a negative regulator in plant immunity [62] was downregulated by CaWRKY40b silencing but enhanced by overexpression of CaWRKY40b, indicating that CaWRKY40b also acts as a negative regulator by activating negative regulators, and CaWRKY40b possesses dual functionality, acting either as a repressor or as an activator in a promoter-context dependent manner, similar to AtWRKY33 [63]. In addition to these target genes, CaNPR1 [3,4,[64][65][66] and CaDEF1 [67][68][69], which were found to be non-target genes of CaWRKY40b, were also consistently upregulated by CaWRKY40b silencing and downregulated by transient overexpression of CaWRKY40b. All these findings support the role of CaWRKY40b as a negative regulator that directly regulates immunity at multiple levels, including signaling regulatory proteins, TFs, and PR proteins.
WRKY genes have been suggested to be functionally connected by forming transcriptional networks [35,36]. Our data in the present study illustrate that CaWRKY40, which was previously found to be transcriptionally modulated directly by CaWRKY6 [3], is directly targeted and transcriptionally regulated by CaWRKY40b. Additionally, typical W-boxes were also found to be enriched in the promoters of CaWRKY6 and CaWRKY40, as well as the promoter of CaWRKY40b ( Figure 1A), indicating the participation of these WRKYs in a WRKY web during the regulation of pepper responses to R. solanacearum. The present study also revealed that CaWRKY40b positively modulates the transcription of CaWRKY40b by directly binding to the promoter of CaWRKY40b. A similar positive feedback regulation has been frequently reported in the WRKY network [35,70].
In sum, CaWRKY40b acts as a negative regulator of pepper resistance to RSI by transcriptionally regulating a set of immunity-associated genes, including CaWRKY40. Its downregulation upon RSI derepresses immunity, but its upregulation in healthy plants impairs pepper immunity.

Plant Materials and Growth Conditions
Seeds of pepper (C. annuum) inbred line 68-2, which has an intermediate level of resistance to R. solanacearum, and Nicotiana benthamiana were obtained from the pepper breeding group at the Fujian Agriculture and Forestry University (Fuzhou, China). Seeds were sown in in plastic pots containing soil with a peat moss:perlite ratio of (2:1, v/v) at 25 ± 2 • C, 70% relative humidity, 60-70 µmol photons·m −2 ·s −1 , and 16 h light/8 h dark [71,72].

R. solanacearum Inoculation
A virulent R. solanacearum strain FJC100301 was isolated from wilted pepper samples at our laboratory in Fujian Province (China) and amplified as described elsewhere [4]. The R. solanacearum strain was cultured in PSA medium (200 g/L of potato, 20 g/L of sucrose, 3 g/L of beef extract, and 5 g/L of tryptone) at 28 ± 2 • C and 200 rpm and then re-suspended in a 10 mM MgCl 2 solution. The bacterial cell solution used for inoculation was diluted to 10 8 cfu·mL −1 (OD600 = 0.8). To inoculate pepper plants with R. solanacearum by root irrigation, pepper plants grown in pots were placed in a tray containing Hoagland's nutrient solution supplemented with the FJC100301 suspension. To inoculate pepper leaves with R. solanacearum, 10 µL of the R. solanacearum suspension (OD600 = 0.8) was infiltrated into the top third leaves of pepper plants at the eight-leaf stage using a needleless syringe, whereas pepper plants inoculated with 10 mM MgCl 2 was used as mock control.

Vector Construction
To construct vectors for overexpression, subcellular localization, and ChIP assay, the full-length ORF of CaWRKY40b (with or without a termination codon) was cloned into the entry vector pDONR207 by BP reaction. This entry vector was then cloned into destination vectors pMDC83 and 3687-HA by LR reaction using the Gateway ® cloning technique (Invitrogen, Carlsbad, CA, USA). In addition, the EAR repression domain (SRDX) was fused to the 3 terminus of the ORF of CaWRKY40b; the resulting CaWRKY40b-SRDX was cloned into pEarleyGate201 as previously described. To construct the vector for VIGS, a 229-bp fragment in 3 -untranslated region (UTR) of CaWRKY40b and a fragment in its ORF were selected for vector construction, and their specificities were confirmed by BLAST against the genome sequence in the databases of CM334 (http://peppergenome.snu.ac.kr/) and Zunla-1 (http://peppersequence.genomics.cn/page/species/blast.jsp). The specific fragment was cloned into the entry vector pDONR207, and then cloned into the PYL279 vector.

Subcellular Localization
Agrobacterium tumefaciens strain GV3101 containing the constructs 35S::CaWRKY40b-GFP or 35S::GFP (used as a control) were grown overnight, respectively, and then resuspended in the induction medium. Bacterial suspensions (OD600 = 0.8) were injected into N. benthamiana leaves using a syringe without a needle. At 48 hpi, GFP fluorescence was imaged using a Laser Scanning Confocal Microscope (TCS SP8, Leica, Solms, Germany) at an excitation wavelength of 488 nm and a 505-530 nm band-pass emission filter.

VIGS of CaWRKY40b in Pepper Plants
For VIGS of CaWRKY40b in 68-2 line pepper plants, A. tumefaciens strain GV3101 harboring PYL192, PYL 279-CaWRKY40b1 and PYL279-CaWRKY40b2 or PYL279 (resuspended in the induction medium at a 1:1 ratio, OD600 = 0.6) were co-infiltrated into the cotyledons of two-week-old pepper plants. The detailed process was conducted according to our previous studies [3,4].

Transient Overexpression of CaWRKY40b in Pepper Leaves
For transient overexpression analysis, A. tumefaciens strain GV3101 harboring the 35S::CaWRKY40b or 35S::CaWRKY40b-HA, 35S::CaWRKY40b-SRDX or 35S::00 vector (the empty vector was used as control) was both shaking-grown overnight. The agrobacterium pellets were collected by centrifugation and resuspended in the induction medium (10 mM MES, 10 mM MgCl 2 , 200 µM acetosyringone, pH 5.6). The adjusted bacterial suspension (OD600 = 0.8) was vacuum-infiltrated into the leaves of 6-week-old pepper plants using a needleless syringe and the injected leaves were maintained in the greenhouse. The samples were collected at the indicated time points for further analysis.

Histochemical Staining
Staining of the leaves with trypan blue and diaminobenzidine (DAB) was used as described elsewhere [73] and in our previous studies [3,74].

Quantitative Real-Time RT-PCR
To determine the relative transcript accumulations of target genes, real-time PCR was performed using specific primers (Table S2) according to manuals of BIO-RAD real-time PCR system (Foster City, CA, USA) and SYBR Premix Ex Taq II system (TaKaRa). Total RNA extraction and real-time RT-PCR were performed as earlier described [3,74]. Four independent biological replicates of each treatment were performed. The Livak method [75] was used to analyze the real-time PCR data. The data were expressed as a normalized relative expression level (2 −∆∆Ct ) of the respective genes. The transcript accumulation of CaActin (GQ339766) and 18S ribosomal RNA (EF564281) were, respectively, used to normalize the relative transcriptional level of each sample.

ChIP Analysis
ChIP assays were performed by following a previously described protocol with slight modifications [76]. Three to four fully expanded leaves of plants at the eight-leaf stage were inoculated with GV3101 cells containing 35S:CaWKRY40b-HA or 35S:00 (used as a control). The inoculated leaves were collected at 24 hpi; about 4 g of leaves were treated with 1.0% formaldehyde for 8 min, to which 3 M glycine was added to a final concentration of 0.125 M. The sample was then vacuumed for 5 min to stop cross linking. Nuclear extracts were isolated and were resuspended with the extraction buffer I (0.4 M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 5 mM β-mercaptoethanol, 1 U protease Inhibitors), II (0.25 M sucrose, 10 mM Tris-Cl, pH 8.0, 10 mM MgCl 2 , 1% Triton X-100, 5 mM β-mercaptoethanol, 1 µL protease inhibitors), and III (1.7 M sucrose, 10 mM Tris-HCl, pH 8.0, 2 mM MgCl 2 , 0.15% Triton X-100, 5 mM β-mercaptoethanol, 1 µL protease Inhibitors) sequentially, and then digested with micrococcal nuclease (Takara, Dalian, China), according to the manufacturer's instructions. Magnetic beads (Invitrogen, Carlsbad, CA, USA) linked to the antibody of HA (anti-HA tag rabbit polyclonal antibody, Sigma, St. Louis, MO, USA) were added to the digested samples, and then eluted. Later, the protein-DNA complex was digested with 2 mL of 10 mg·mL −1 proteinase K and incubated at dry bath with 45 • C for 1 h and same volume of Tris-saturated phenol:chloroform:isoamyl alcohol (25:24:1 v/v) was used to extract the DNA solution twice. Then, DNA was precipitated by adding 3 mL of 100% ethanol, 1/10 volume of 3 M NaOAc, and 1 mL of 2 M glycogen along with overnight incubation at −20 • C. DNA was pelleted by spinning for 20 min at 16,700× g. The DNA pellets were washed with 80% ethanol, dried at room temperature, resuspended in 50 µL TE buffer, and stored at −20 • C until use for ChIP-seq and ChIP-PCR. For ChIP-seq, the immunoprecipited DNA samples were used to generate sequencing libraries bearing barcodes using a NEBNext ChIP-seq Library PreReagent Set for Illumina kit (New England Biolabs, Ipswich, MA, USA). Sequencing was performed on Illumina HiSeq2500 at Nevogene (Beijing, China).
ChIP-seq data analysis was performed following the method used by Liu et al. [63]. Quantitative real-time PCR was used to analyze the immune-precipitated DNA for enrichment of CaWRKY40b at the promoter region of the target genes. Fold increases of immune-precipitated DNA were calculated relative to the input DNA and the internal control CaACTIN or 18S rRNA. ChIP-PCR was performed at least in triplicate.

Fluorometric GUS Enzymatic Assay
A fluorometric GUS enzymatic assay for measuring GUS activity in pepper plant extracts was performed by adopting a previously described protocol [4].

Immunoblotting
Total protein extracts were incubated with anti-HA agarose (Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4 • C. Beads were collected and washed with Tris-buffered saline and Tween-20 (0.05%). Eluted proteins were analyzed by immune-blotting using an anti-HA-peroxidase antibody (Abcam, Cambridge, UK).

Quantification of R. solanacearum Growth in Pepper Plants
The growth of R. solanacearum was quantified by measuring the colony-forming units (cfu), and the rootstocks of R. solanacearum-inoculated pepper plants were harvested at indicated time points, which were ground into powder in liquid nitrogen. For every 1.0 g of powder, 1.0 mL of 10 mM MgCl 2 solution was added and spun down, the supernatant was collected and diluted into 10,000 times, and 1 µL of the supernatant was added to the PSA plate, which was kept at 28 • C. Approximately 48 h later, the cfus were calculated.