A Cysteine-Rich Receptor-Like Protein Kinase CaCKR5 Contributes to Immune Defense Against Ralstonia Solanacearum in Pepper

Background: Cysteine-rich receptor-like kinases (CRKs) represent a large subfamily of receptor-like kinases and have important roles in numerous different physiological processes in plants. Results: CaCRK5 transcripts were observed to accumulate after the inoculation of R. solanacearum and treatment with salicylic acid. The fusion between CaCRK5 and the green uorescence protein was targeted to the plasma membrane. Suppression of CaCRK5 via virus-induced gene silencing made pepper plants signicantly susceptible to R. solanacearum infection, which was accompanied by decreased expression of defense related genes CaPR1, CaSAR8.2, CaDEF1 and CaACO1. Overexpression of CaCRK5 in tobacco conferred increased resistance against R. solanacearum. Furthermore, electrophoretic mobility shift assay and chromatin immunoprecipitation with quantitative real-time PCR analysis veried that a homeodomain zipper I protein CaHDZ27 can bind to the CaCRK5 promoter, and directly active its expression. Yeast two-hybrid and bimolecular uorescence complementation analyses suggested that CaCRK5 could heterodimerize with the homologous member CaCRK6 in the plasma membrane. Conclusions: Our data indicated that CaCRK5 played a positive role in pepper resistance against R. The experiments were repeated three times with similar results. b Phenotypes of control and VIGS pepper plants. After the VIGS was established, 4 week old plants were inoculated with R. solanacearum. Photographs were taken at 10 d after R. solanacearum infection. c Rate of disease index on a scale of 0 to 4 was calculated in CaCRK5 silenced and control plants. d Growth of R. solanacearum in leaves of TRV:CaCRK5 and plants at 3 d post leaf inoculation of R. solanacearum. The data are shown as means ± SD from three independent repeats (n=3, *, P<0.05, Student’s t-test). e Visualization of diaminobenzidine (DAB) and trypan blue staining 48 h post leaf inoculation with R. solanacearum. f The expression levels of defense related genes in control (TRV:00) and CaCRK5-silenced (TRV:CaCRK5) plants. statistically

Some CRKs has been functionally characterized in the response to pathogen infection. For example, CRK45 acted as a positive regulator in Arabidopsis disease resistance. CRK45 overexpression enhanced resistance to Pseudomonas syringae, whereas the crk45 mutant was more sensitive to P. syringae [13] . Overexpression of CRK4, CRK6 and CRK36 in Arabidopsis increased disease resistance to the bacterial pathogen Pst DC3000 through a positive role in the PTI response by association with the FLS2 [14]. Intriguingly, recent studies revealed that some CRKs form a receptor complex with RLKs and play roles in transducing signals in the plant immune. For example, increased expression of CRK28 in Arabidopsis enhanced ROS burst and disease resistance toward P. syringae. CRK28 associated with FLS2/BAK1 immune complex as well as associated with the closely related CRK29 [15]. However, whether members of this large CRK subfamily in pepper regulate defense responses are unknown.
Here, we report the identi cation of a CRK coding genes CaCRK5 that function as a positive regulator in the pepper response to R. solanacearum infection. Suppression of CaCRK5 in pepper plants via virusinduced gene silencing (VIGS) increased susceptibility to pathogen. Overexpression of CaCRK5 in tobacco enhanced resistance to pathogen. In addition, CaCRK5 was targeted and up-regulated by CaHDZ27. Lastly, we demonstrate that CaCRK5 could interact with its homologous member CaCRK6 in the plasma membrane.

Plant materials and Pathogen inoculation
Pepper (Capsicum annuum cultivar L. cv. Fj8) was obtained from the pepper breeding group at Fujian Agriculture and Forestry University (FAFU), it exhibted medium-resistance to R. solanacearum that was identi ed by Prof. Shuilin He at FAFU in China. Pepper and tobacco (Nicotiana benthamiana) plants were grown in plastic pots containing steam-sterilized soil at 26°C with a long-day photoperiod (16 h light/8 h dark) with 60% relative humidity.
Virulent strains FJC100301 of R. solanacearum was used in this study. The R. solanacearum strain was grown in PSA medium. Bacteria cell solution was harvested with sterile 10 mM MgCl 2 and re-suspended to 1.0×10 8 CFU/mL. For pepper inoculation, seedlings were irrigated by cutting the roots and pouring 30 mL bacterial inoculum into each pot. For leaf inoculation, the leaf was in ltrated with 10 μL of the R. solanacearum suspension using a syringe without a needle. For each plant, a disease index on a scale of 0 to 4 was calculated as described previously.

Subcellular Localization
For the subcellular localization assay, CaCRK5 were cloned into pMDC83 vector, resulting in 35S:CaCRK5-GFP construct. The plasmids were transformed into A. tumefaciens GV1301. The transient expression assays were conducted as described previously [44]. Fluorescence of GFP in the transformed leaves was imaged using a confocal laser scanning microscope (TCSSP8; Leica, Solms, Germany).

Virus-Induced Gene Silencing
The tobacco rattle virus (TRV)-based virus-induced gene silencing system was used for gene silencing in pepper. To achieve CaCRK5 speci c silencing, about 200 bp DNA fragment in their 3'-untranslated region were PCR ampli ed and cloned into TRV2 to generate TRV2:CaCRK5. A. tumefaciens GV3101 carrying TRV1 with TRV2:CaCRK5 was co-in ltrated into the fully expanded cotyledons of pepper plants. Then, the plants were incubated at 16°C for 56 h, and grown at 26°C. The experiments were performed with at least 50 plants per treatment and repeated three times. The rate of the disease index was calculated as previously described [44].
RNA extraction and quantitative real-time RT-PCR Total RNA was extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). First-strand cDNAs were synthesized using One Step PrimeScript TM cDNA Synthesis Kit (TaKaRa, Shigo, Japan).The real-time RT-PCR analysis was performed using a Bio-Rad real-time PCR system (Foster City, CA, USA.) and the SYBR Premix ExTaq II system (Takara). The relative expression of the CaCRK5 and defense-related genes were calculated with the 2 −ΔΔCt method. CaActin or NtActin served as the reference gene to normalize the total amount of cDNA in each reaction. The primers were listed in Additional le 3.
Yeast two-hybrid analysis The pGADT7 vector was used for GAL4 AD, and the pGBKT7 vector was used for GAL4 BD. ORFs of CaCRK5 and CaCRK6 were ampli ed and cloned into pGADT7 to generate constructs designated AD-CaCRK5 and AD-CaCRK6. The cDNAs of CaCRK5 and CaCRK6 were ampli ed and cloned into pGBKT7 to generate constructs designated BD-CaCRK5 and BD-CaCRK6. The corresponding AD and BD plasmids were co-transformed into Y2H Gold yeast strain. Their interaction was identi ed by spot assay. The transformants were tested on SD (Synthetic Dextrose) screening medium. The plates were incubated at 30°C till the formation of colonies.

Bimolecular uorescence complementation (BiFC) assay
To generate BiFC constructs, CaCRK5 and CaCRK6 full length cDNAs were cloned into puc-SPYNE GW and puc-SPYCE GW vectors, which contain the N-terminal or C-terminal fragment of YFP (nYFP or cYFP, respectively). For transient expression, A. tumefaciens strain GV3101 carrying each construct was used to in ltrate leaves of 5-week-old N. benthamiana plants. For microscopic analysis, lower epidermis cells were observed using a confocal laser scanning microscope

Tobacco transformation
Transgenic tobacco plants were generated by using A. tumefaciens mediated tobacco leaf disc transformation [45]. The CaCRK5 coding region was cloned into pK7WG2 vector to get 35S:CRK5 constructs. Two constructs were introduced into A. tumefaciens GV3101 and used to transform N.
benthamiana. Transformed seed stock was selected for kanamycin resistance by planting seeds on Murashige and Skoog agar plates containing 50 µg of kanamycin per milliliter.

Histochemical staining
Histochemical staining was performed as previously described [44]. The in ltrated leaves were stained with trypan blue to visualize the cell death response and DAB solution for H 2 O 2 detection.
Electrophoretic mobility shift assay (EMSA) Expression of the CaHDZ27-GST fusion protein was induced in transformed E. coli BL21 cells, by adding IPTG (isopropyl β-D-1-thiogalactopyranoside, 0.05 mM) at 37°C for 4 h. The recombinant proteins were extracted from the cells and puri ed using BeaverBeads™ GSH protein puri ed kit according to manufacturer's instructions.
The EMSA was performed as described previously [46]. Cy5-labelled DNA fragments were synthesized and used as probes, while unlabeled DNA of the same sequence was used as a competitor.

Chromatin immunoprecipitation (ChIP)-qPCR analysis
ChIP assay was performed using the method as described previously [44]. Chromatin was extracted from pepper leaves in ltrated with 35S:HA-CaHDZ27 and cross-linked with 1% formaldehyde (v/v). The chromatin was broken into fragments by probe sonicator to an average length of 300 to 500 bps. The fragmented DNA was immunoprecipitated with anti-HA antibody. Enrichment of DNA samples was analyzed by quantitative real-time PCR.

Results
CaCRK5 encodes a pathogen induced receptor-like kinase of the arginine aspartate (RD) family In cDNA-AFLP experiments designed to isolate genes involved in the pepper resistance to the infection of R. solanacearum, a partial cDNA fragment of CaCRK5 was obtained. As its expression was signi cantly upregulated after inoculation of R. solanacearum, we decided to study the function of this gene further. CaCRK5 cDNA clones were isolated from a cDNA library made from R. solanacearum inoculated leaves of pepper inbred line CM334. The proteins deduced from the cDNA clones of CaCRK5 contained 669 residues. SMART (http://smart.embl-heidelberg.de/) analysis of the domain architecture showed that their primary structures were composed of two cysteine-rich DUF26 domains, a transmembrane region and a serine/threonine kinase domain , and therefore CaCRK5 belongs to the family of cysteine-rich kinases [10]. In addition, CaCRK5 contains a conserved arginine-aspartic (RD) motif in the protein kinase domain (Fig.1). For most RD kinases, the phosphorylation in the activation loop is crucial for triggering kinase activity which usually displays phosphorylation/autophosphorylation ability. Non-RD kinases usually exhibit lower kinase activities because of the lack of activation loop autophosphorylation. RD and non-RD kinases often cooperate to control innate immune signaling in plants [16][17][18][19].
Genome-wide identi cation of CRK family in pepper In the start of this work, we evaluate the CRK gene family in the pepper genome, Hidden Markov Model (HMM) pro le of the Stress-antifung domain (PF01657) was used to search the pepper genome database PGP (http://peppergenome.snu.ac.kr/). The Arabidopsis CRK gene family sequences [10] were also used as query sequences to search against the PGP and the NCBI database. All of them were further subjected to domain analysis using NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART, to ensure the presence of three important domains for typical CRK proteins including stressantifung domains, transmembrane domain, and kinase domain. A total of 27 CRK genes (CaCRKs) were identi ed in the pepper cultivar CM334 genome, which were numbered from CaCRK1 to CaCRK27 according to their localization on chromosomes. Except for CaCRK13/24/25 with only one DUF26 domain, most CRKs contained two DUF26 domains. The length of CaCRK proteins varied from 332 to 1120aa, and molecular weights from 36.76 kDa to 128.25 kDa. The predicted isoelectric points of CaCRK ranged from 5.53 to 9.37(Additional le 1).
Chromosome physical localization analysis revealed that 27 CaCRKs were distributed on 7 of the 12 chromosomes in pepper. Chromosomes 2 contained the largest number of CRKs with 9 genes (34.6%), chromosomes 12 had only 1 gene. Gene clustering was the most common feature of the CRK genes distribution (Fig. 2a). Phylogenetic analysis was performed using the full length protein sequences of CRK from pepper, and an unrooted phylogenetic tree constructed by the neighbor-joining (NJ) method.
Expression of CaCRK5 response to infection of R. solanacearum and treatment with exogenous SA, MeJA and ETH To characterize the expression pattern of CaCRK5 in detail, quantitative real-time PCR (qRT-PCR) analyses were performed. 6-week-old pepper plants were leaf-inoculated with R. solanacearum. CaCRK5 was rapidly up-regulated over time after inoculation of R. solanacearum, and the highest level of expression was observed at 12 h post inoculation (hpi) with about 12.4-fold of that in control plant (Fig. 3a). Next, we investigated the expression of CaCRK5 treated with the defense-related signaling molecules salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). The analysis showed the expression of CaCRK5 was increased after SA treatment and reached a peak at 48h, and no signi cant change was observed after methy jasmonate (MeJA) or ethephon (ETH) treatment (Fig. 3b). These results supported that CaCRK5 was involved in pepper defense against R. solanacearum invasion.

Subcellular localization of CRK5
Since CaCRK5 encoded a potential transmembrane domain, it was predicted to localize to plasma membrane. To test the hypothesis, CaCRK5 was fused with green uorescent protein (GFP) under the control of the CaMV 35S promoter. CBL1n protein, which is known to be localized to the plasma membrane [21], was fused with red uorescent protein (RFP). 35S:GFP, 35S:CaCRK5-GFP were transiently co-expressed with 35S:CBL1n-RFP in leaf epidermal cells of N. benthamiana. As shown in Fig.4, 35S:GFP construct served as a negative control, and green uorescence was ubiquitously distributed throughout the cell. CaCRK5-GFP green uorescence overlapped very closely with the red uorescence in the plasma membrane suggesting that they are localized on the plasma membrane in the plant cell.

Silencing of CaCRK5 in pepper plants increases susceptibility to R. solanacearum infection
To assess the role of CaCRK5 in the interaction between pepper and R. solanacearum, loss-of-function experiments in pepper seedlings by virus-induced gene silencing (VIGS) were performed [22]. As shown in Additional le 2, photobleaching was observed in newly emerged true leaves of plants in ltrated with Agrobacterium carrying CaPDS, indicating that the VIGS system worked e ciently. qRT-PCR analysis showed that expression of CaCRK5 in pepper leaves was signi cantly down-regulated during R. solanacearum infection in VIGS plants (Fig. 5a), indicating that CaCRK5 were e ciently silenced.
CaCRK5 silenced and control (TRV:00) pepper plants were subjected to R. solanacearum challenge. Phenotypic analysis indicated that CaCRK5 silenced pepper plants showed more severe disease symptoms than control plants after R. solanacearum infection (Fig. 5b). From 6 days post inoculation with R. solanacearum, CRK5 silenced pepper plants exhibited signi cant delay of wilt symptom. The disease index of CaCRK5 silenced plants was signi cantly increased, compared to the control plants (Fig. 5c). To address whether silencing of CaCRK5 affect growth of R. solanacearum, the bacterial population was determined. As shown in Fig. 5d, growth of R. solanacearum was signi cantly enhanced in CaCRK5 silenced plants 3 day post leaf-inocualtion, compared with control plants. Based on these observations, we believed that CaCRK5 participate defense responses in pepper.
Next, we investigated the cell death and oxidative burst in CaCRK5 silenced and the control leaves. Trypan blue and DAB staining con rmed that hypersensitive cell death and H 2 O 2 accumulation were signi cantly reduced in CaCRK5 silenced leaves 48 h after inoculation with R. solanacearum (Fig. 5e), indicating that CaCRK5 play pivotal roles in early defense response associated with cell death during R. solanacearum infection. We further determined the effects of CaCRK5 silencing on the expression of defense related genes in pepper during R. solanacearum infection. qRT-PCR analyses showed that CaCRK5 silencing in pepper leaves signi cantly attenuated expression of defense related genes, including CaNPR1 [23], CaSAR8.2 [24], CaDEF1 [25] and CaACO1 [26], during R. solanacearum infection (Fig. 5f).
Overexpression of CaCRK5 in tobacco reduces susceptibility to R. solanacearum infection A gain-of-function approach was also employed to study the function of CaCRK5 in the defense response. Due to technical di culties, we used the tobacco plant (Nicotiana benthamiana), which is also a host for R. solanacearum. At least 10 transgenic tobacco lines were obtained and con rmed by kanamycin resistance analysis. Two T 3 CaCRK5 overexpressed lines exhibited constitutive expression (Fig. 6a), and were used in subsequent experiments.
Wild-type and transgenic plants (L3 and L7) at 4 weeks old were root-inoculated with R. solanacearum. The effect of CaCRK5 overexpression on development of bacterial wilt disease was observed. As shown in Fig. 6b, the CaCRK5 overexpressed plants showed much weaker symptoms at 12 d post-inoculation, compared with the wild-type. The wild-type plants showed wilt symptom from 6 day after inoculation and completely died 14 d after inoculation with R. solanacearum. The CaCRK5 overexpressing plants were observed signi cant delay of wilt symptom compared to wild-type plants (Fig. 6c), showing that overexpression of CaCRK5 conferred increased disease tolerance to R. solanacearum in tobacco. In addition, we also checked the expression of defense related genes, including NtPR2, NtPR3, NtHSR201 and NtHSR505. qRT-PCR analysis indicated that expressions of these tested defense related genes were increased in CaCRK5 overexpressed plants compared with wild-type plants, during the inoculation of R. solanacearum (Fig. 6d). Collectively, these data suggested that CaCRK5 overexpression enhanced defense responses against R. solanacearum infection in tobacco.

CaCRK5 is directly regulated by transcription factor CaHDZ27
To better understand the regulatory mechanism of CaCRK5, a 2000-bp promoter region upstream of the CaCRK5 coding sequence was identi ed. Sequence analysis using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) suggested that the cis-elements in the promoter of CaCRK5 included two TCA-element involved in salicylic acid responsiveness, ve binding sites for MYB transcription factor, and one binding sites for MYC. In addition, a known binding site (CAATTATTG) for the HD-Zip subfamily I member CaHDZ27 was located between positions -625 and -617. As CaHDZ27 also regulate the defense in pepper against R. solanacearum infecction [27], we speculated that CaCRK5 might be targeted by CaHDZ27.
An electrophoretic mobility shift assay (EMSA) was conducted to access the interaction of CaHDZ27 with the CaCKR5 promoter, and CaHDZ27 was found to bind to the Cy5-labeled CaCKR5 promoter probe. Moreover, binding was gradually attenuated by increasing concentrations of unlabeled probe, indicating that CaHDZ27 binds speci cally to the CAATTATTG in the CaCKR5 promoter in vivo (Fig. 7a). Chromatin immunoprecipitation (ChIP)-qPCR was conducted to con rm interaction of CaHDZ27 and the CaCKR5 promoter in vitro. The pepper leaves in ltrated with Agrobacterium strain GV3101 containing 35S:HA-CaHDZ27 or 35S:HA were harvested for ChIP assay. Chromatin from these pepper leaves was immunoprecipitated using anti-HA antibodies and enrichment of DNA sample was determined by qRT-PCR. The result showed that CaHDZ27 was signi cantly enriched in the CaCKR5 promoter, and the enrichment was signi cantly enhanced by the inoculation of R. solanacearum (Fig. 7b and c), suggesting that CaHDZ27 could bind to the CAATTATTG in the CaCKR5 promoter in vitro, and binding was increased by the R. solanacearum infection.
To further investigate the CaCKR5 regulated by CaHDZ27 in the transcriptional level, we transiently expressed 35S:CaHDZ27-GFP in pepper leaves, and 35S:GFP in ltrated leaves were used as a negative control. qRT-PCR analysis demonstrated that the transcript level of CaCKR5 was increased in 35S:CaHDZ27-GFP in ltrated leaves compared with the control (Fig. 8a). Using anti-GFP immune-blotting, CaHDZ27 was con rmed to be expressed in pepper leaves (Fig. 8b). We used a VIGS approach to silence CaHDZ27. The transcript level of CaCKR5 is reduced signi cantly in CaHDZ27-silenced plants compared with TRV:00 in ltrated plants, 48 h post the inoculation of R. solanacearum (Fig. 8c and d). These data suggested that the transcriptional expression of CaCKR5 was positively regulated by CaHDZ27.

CaCRK5 interacts with CaCRK6
Previous studies indicated that plants employ immune receptor complex for sensing microbe-derived molecular patterns and effectors to trigger inducible immune defenses [28][29][30][31], and a few CRKs have been known to function in association with each other, such as AtCRK28/AtCRK29 [15] and AtCRK39/AtCRK40 [32]. Thus, we investigated the ability of CaCRK5 to heterodimerize with the closely related CaCRK6, which has 77.4% amino acid sequence identity to CaCRK5.
The cDNAs of CaCRK5 and CaCRK6 were cloned into the pGBKT7 and pGADT7 vectors separately, and generated DNA-binding domain (BD) and activation domain (AD) fusions. BD-CaCRK5/AD-CaCRK6 and BD-CaCRK6/AD-CaCRK5 transformant yeast cells can grow on SD/-Trp-Leu-His-Ade medium, as did positve control (pGBKT7-53/pGADT7-T). In contrast, no growth was observed in the negative controls. The result indicated that CaCRK5 interacts with CaCRK6 each other in yeast cells (Fig. 9a). To provide more evidence for the interaction between CaCRK5 and CaCRK6, we performed bimolecular uorescence complementation (BiFC) assays. CaCRK5 and CaCRK6 were fused to the N-and C-terminal ends of yellow uorescent protein (YFP) to generate CaCRK5-nYFP/CaCRK6-cYFP and CaCRK5-cYFP/CaCRK6-nYFP, respectively. Then, these constructs were transiently co-expressed in Nicotiana benthamiana leaves. At 48h post in ltration, the YFP uorescence signals were checked by by confocal microscopy. As showed in Fig. 9b, YFP uorescence was oboserved at the plasma membrane in N. benthamiana leaves. These results indicate that CaCRK5 heterodimerizes with CaCRK6 at the plasma membrane of plant cells.

Discussion
The tandem repeats contributed to the expansion of the CaCRK family in pepper CRKs are a subfamily of plant RLKs. In this study, 27 CaCRKs were identi ed in pepper genome; many CRKs were present in tandem repeat which made physical clusters at speci c positions in the chromosome. Previous studies showed that a large proportion of the RLK gene family is found in tandem repeats [33][34][35]. Tandem repeats are enriched in stress-responsive genes [36][37][38]. The expansion and degree of tandem repeats are signi cantly correlated with the stress responsiveness of RLK genes [36]. The tandem repeats of CaCRKs in pepper may facilitate the stress adaptive evolution.
CaCRK5 played a positve role in the pepper resistance against R. solanacearum Previous studies have shown that some CRK members including AtCRK29 in Arabidopsis were induced in response to treatment with pathogens, SA and g22 [15,39,40], suggesting a role for these CRKs in plant immunity. The CaCRK5 closest homologue in Arabidopsis is AtCRK29 protein with 41.6% identity to CaCRK5. In the study, expressions of CaCRK5 were increased over time after inoculation of R. solanacearum, with high levels of gene expression at the early stage of infection. CaCRK5 was strongly up-regulated after treatment with SA, and no signi cant change of CRK5 was observed treated with MeJA and ETH.
Based on the subcellular localizations of GFP fusions, CaCRK5 are probably targeted to the plasma membrane. Several CRK proteins also have been reported to be localized in the plasma membrane, including CRK4, CRK6, and CRK36 in Arabidopsis [14] and GbCRK18 in cotton [41]. The TRV-based VIGS system was used to investigate the function of CaCRK5 in the response to R. solanacearum infection. CaCRK5 was transcriptionally regulated directly by CaHDZ27 HD-Zip transcription factors can be divided into four subfamilies (HD-Zip I-IV) based on their sequence similarities [42], and HD-Zip I proteins were reported to play roles in biotic and abiotic stress responses [43]. CaHDZ27, a HD-Zip I member, acts a positive regulator in pepper response to R. solanacearum infection [27]. Here we showed that CaHDZ27 acts as a positive regulator of CaCRK5, CaHDZ27 binds to a 9-bp CAATTATTG motif of the CaCRK5 promoter revealed by EMSA and ChIP-qPCR. Expression of CaCRK5 is increased in CaHDZ27 transit expressed pepper leaves, and decreased in CaHDZ27 silenced pepper plants with the inoculation of R. solanacearum. Moreover, CaCRK5 play a positive role in pepper resistance agianst R. solanacearum infection, consistent with the role of CaHDZ27. These results suggest that CaCRK5 and CaHDZ27 coordinate to regulate the pepper defense against R. solanacearum infection.

CaCRK5 heterodimerizes with CaCRK6
Yeast two-hybrid and bimolecular uorescence complementation analyses indicated that CaCRK5 heterodimerizes with CaCRK6 in vivo in both yeast and plant cells. Recent evidence suggest that pattern recognition receptors (PRRs) enable the rapid recruitment of other RLKs, including CRKs, to enhance PRR signaling intensity [15,39]. CaCRK5 might act in concert with CaCRK6, and other RLKs in the pepper defense against R. solanacearum infection.

Conclusions
In summary, our data suggest that CaCRK5 may signi cantly contribute to immune defense against R. solanacearum in pepper and tobacco. VIGS in pepper plants and gain-of-function analyses of CaCRK5 in tobacco, revealed that CaCRK5 positively regulates plant immune responses. In addition, CaHDZ27 promotes CaCRK5 expression by directly binding to its promoter. We propose that the pepper defense response mediated by CaCRK5 could be enhanced by CaHDZ27.    Fluorescence was examined using a confocal microscope. GFP, green uorescent protein; RFP, red uorescent protein. Bars, 50μm.

Figure 6
Page 20/23 Effect of CaCRK5 overexpression in tobacco on bacterial wilt disease by inoculation with R. solanacearum. a Expression levels of CaCRK5 in WT (wild-type) and transgenic tobacco lines (L3 and L7 and wild-type. Four-week-old wild-type and transgenic tobacco seedlings were leaf-inoculated with R. solanacearum. After 48 h, total RNAs were extracted for qRT-PCR assays. Data represent means ± SD (n = 3). Asterisks indicate signi cant differences according to Student's t test, *P < 0.05.

Figure 7
CaHDZ27 binds to the CAATTATTG motif in the promoter of CaCKR5. a EMSA was used to analyze the interaction of CaHDZ27-GST and a Cy5-labeled probe in vitro. The probe sequences containing the CAATTATTG motif in the CaCKR5 promoter are shown. CaHDZ27-GST puri ed protein was incubated with Cy5 uorochrome labeled wild-type probe. Non labeled probes at 10-and 100-fold concentrations were added for the competition test. b Schematic representation of the CaCKR5 promoter used for ChIP-qPCR analysis. the blue lines indicate the sequence regions that were used for ChIP-qPCR. c, ChIP-qPCR assays showed the binding of CaHDZ27 to CaCKR5 promoter in vitro. DNA samples were coimmunoprecipitated with anti-HA antibody. The ChIP value was normalized to its respective input DNA value. Different letters indicate signi cant differences, as determined by LSD test (P<0.01).

Figure 8
The