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

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. 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. Our data revealed that CaCRK5 played a positive role in regulating immune responses against R. solanacearum infection in pepper.


Background
When plants are attacked by pathogens, plant immune receptors can detect pathogen infection and elicit a battery of defense responses. As the first line of plant defense, pattern recognition receptors (PRRs) recognize microbe-associated molecular patterns (MAMPs) that are released by the pathogen during infection and trigger plant immunity [1]. An increasing body of evidence suggests that the surface-localized PRRs are either receptorlike kinases (RLKs) or receptor-like proteins (RLPs) in plants [2,3]. Well-characterized PRRs include the Arabidopsis leucine-rich repeat (LRR) receptor kinases FLA-GELLIN SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), which can recognize bacterial flagellin [4] and EF-Tu [5], respectively. RLKs comprise a superfamily of transmembrane proteins, which play a critical role in various signal transduction pathway. In higher plants, there are about 610 RLKs in model plants Arabidopsis [6] and near 1000 in rice [7]. A typical RLK contains an extracellular receptor, a transmembrane domain and an intracellular Ser/Thr kinase domain [6]. Although roles for most RLKs are unknown, many RLKs were reported to regulate plant physiological processes, including development, hormone perception and defense responses [8].
Cysteine-rich RLKs (CRKs) are characterized by the presence of one to four copies of Domain of Unknown Function 26 (DUF26) and a C-X8-C-X2-C motif in the extracellular receptor region at the N-terminus [9]. The conserved Cys residues might be required to form the three-dimensional structure of the protein through disulfide bonds [10], can mediate protein-protein interactions [11]. It has been reported that CRKs are involved in the regulation of immune responses [12][13][14]. Most of the CRKs in Arabidopsis are induced by pathogen attack and SA application at the transcriptional level [15,16]. Some of the CRKs have been functionally characterized in the response to the pathogen infection. For example, CRK45 served as a positive regulator in Arabidopsis disease resistance. CRK45 overexpression enhanced resistance to Pseudomonas syringae, whereas the crk45 mutant was more susceptible to P. syringae [16]. LecRK-VI.2, a positive regulator of pattern-triggered immunity (PTI), induces the expression of 7 CRK genes (CRK4/6/ 7/13/23/36/37) in Arabidopsis. Among them, overexpression of CRK4, CRK6 and CRK36 increased disease resistance to the bacterial pathogen Pst DC3000 through a positive regulation of PTI response [17]. Moreover, recent studies revealed that some CRKs could form a receptor complex with RLKs and play roles in transducing PTI signals in the plant. It has been reported that increased expression of CRK28 in Arabidopsis enhanced ROS burst and disease resistance to P. syringae. CRK28 associated with FLS2/brassinosteroid insensitive 1associated kinase 1 (FLS2/BAK1) immune complex as well as associated with the closely related CRK29 in Arabidopsis [18]. However, it remains unknown how CRKs regulate defense responses in pepper.
Wilt disease is one of the most devastating bacterial diseases in the world that is mainly caused by the soilborne bacterium R. solanacearum [19,20]. The pathogen infects a wide range of crops of economic importance, but is particularly devastating to Solanaceae plants, such as tomato, tobacco and pepper [21]. Here, we report the identification of a CRK protein, CaCRK5, which functions as a positive regulator in the pepper response to R. solanacearum infection. Suppression of CaCRK5 in pepper plants via virus-induced gene silencing (VIGS) increased susceptibility to the pathogen. In contrast, overexpression of CaCRK5 in N. benthamiana enhanced resistance to pathogen. In addition, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-qPCR analysis revealed that transcription factor CaHDZ27 up-regulates the expression of CaCRK5 by binding to its promoter. Furthermore, the data from yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses indicate that CaCRK5 interacted with its homolog CaCRK6 on the plasma membrane. These results uncover that CaCRK5 plays a vital role in the regulatory network regulating pepper response to R.solanacearum infection, suggesting that CaCRK5 is an effective target for improvement of pepper resistance to wilt disease.

Results
CaCRK5 encodes a pathogen induced receptor-like kinase of the arginine aspartate (RD) family In cDNA-AFLP (cDNA amplified fragment length polymorphism) experiments designed to isolate genes involved in the pepper resistance to the infection of R. solanacearum, a partial cDNA fragment of CaCRK5 was obtained [22]. As its expression was significantly 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 predicted that CaCKR5 was composed of two cysteine-rich DUF26 domains (PFAM01657, Stressantifung domain), a transmembrane region and a serine/ threonine kinase domain (PFAM07714), and therefore CaCRK5 belongs to the family of cysteine-rich kinases [10]. In addition, CaCRK5 contains a conserved arginineaspartic (RD) motif in the protein kinase domain (Additional file 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 [23][24][25][26].

Genome-wide identification of CRK family in pepper
To gain more information about CRK in pepper, we evaluate the CRK gene family in the pepper genome, Hidden Markov Model (HMM) profile of the DUF26 domain (PFAM01657) was used to search the pepper cultivar CM334 proteins database (http://cab.pepper.snu. ac.kr/). The Arabidopsis CRK gene family sequences [10] were also used as query sequences to search against the PGP (Pepper Genome Platform) and the NCBI database. The identified candidates were further subjected to domain analysis using NCBI-CDD and SMART, to ensure the presence of three essential domains for specific CRK proteins including stress-antifung domains, a transmembrane domain, and a kinase domain. A total of 27 CRK genes (CaCRKs) were identified in the pepper cultivar CM334 genome, 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 amino acid number composing of CaCRK proteins varied from 332 to 1120, 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 file 2).
Chromosome physical localization analysis revealed that 27 CaCRKs were distributed on 7 of the 12 chromosomes in pepper genome. Chromosome 2 contained the largest number of CRKs with 9 genes (34.6 %), whereas chromosomes 12 contained only 1 gene. Gene clustering was the most common feature of the CRK genes distribution. 22 CRKs were identified in tandem repeats on the same chromosome, with no or only one intervening annotated gene. (e.g. CaCRK1-7, CaCRK10/11, CaCRK14/15, CaCRK16-19, CaCRK20/21, CaCRK22/23 and CaCRK24-26). 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. The result showed that all CRKs in pepper were classified into four subfamilies designated I to IV according to the phylogenetic relationship. IV is the largest subfamily that contains 13 CRKs, III is the smallest subfamily and comprised of only two CRKs (CaCRK10/11). Subfamily II contains 5 CRKs (CRK13-15 and CRK20/21), and I contains 7 CRKs (CaCRK1-7) which all locate on chromosome 2. CRKs that located in tandem repeats clustered in the same clade in the phylogenetic tree, except CaCRK22/23 (Additional file 3). These results suggested that the expansion of CRKs in pepper might be due to tandem duplication. This distribution and physical clustering pattern are consistent with CRKs in Arabidopsis [18] and soybean [27].
Expression of CaCRK5 in response to the 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 leafinoculated 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. 1a). Next, we determined the response of CaCRK5 to the defense-related signaling molecules salicylic acid (SA), methy jasmonate (MeJA) and ethephon (ETH). The results showed that the expression of CaCRK5 was significantly increased after SA treatment and reached a peak at 48 h. In contrast, indistinguishable change was observed when treated with MeJA or ETH (Fig. 1b). These results suggested that CaCRK5 was involved in pepper defense against R. solanacearum invasion, likely through SA-mediated defense signaling.

Subcellular localization of CaCRK5
Since CaCRK5 encoded a potential transmembrane domain, it was predicted to localize on the plasma membrane. To test the hypothesis, CaCRK5 was fused with green fluorescent protein (GFP) under the control of the CaMV 35 S promoter. CBL1n protein, which is known localized on the plasma membrane [28], was fused with red fluorescent protein (RFP). 35 S:GFP, 35 S:CaCRK5-GFP were transiently co-expressed with 35 S:CBL1n-RFP in leaf epidermal cells of N. benthamiana. As shown in Fig. 2, 35S:GFP construct served as a negative control, and green fluorescence was ubiquitously distributed throughout the cell. CaCRK5-GFP green fluorescence overlapped very closely with the red fluorescence on the plasma membrane suggesting the plasma membrane localization 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 [29]. Specific fragment for CaCKR5 gene was used to generate VIGS construct, and standard VIGS procedure was performed with pepper phytoene desaturase (CaPDS) construct as the positive control. As shown in Additional file 4, photobleaching was observed in newly emerged true leaves of plants infiltrated with Agrobacterium carrying CaPDS, indicating that the VIGS system worked efficiently.
CaCRK5 silenced (TRV:CaCRK5) and empty vector control (TRV:00) pepper plants were subjected to R. solanacearum challenge. qRT-PCR analysis showed that expression of CaCRK5 in pepper leaves was significantly down-regulated during R. solanacearum infection in VIGS plants (Fig. 3a), indicating that CaCRK5 were efficiently silenced. Phenotypic analysis indicated that CaCRK5 silenced pepper plants showed more severe disease symptoms than control plants after R. solanacearum infection (Fig. 3b). From 6 days post-inoculation with R. solanacearum, CRK5 silenced pepper plants exhibited a more rapid disease development. The disease index of CaCRK5 silenced plants was significantly increased, compared to the control plants (Fig. 3c). To address whether silencing of CaCRK5 affects the growth of R. solanacearum, the bacterial population was determined. As shown in Fig. 3d, the growth of R. solanacearum was  significantly 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 confirmed that hypersensitive cell death and H 2 O 2 accumulation were significantly reduced in CaCRK5 silenced leaves 48 h after inoculation with R. solanacearum (Fig. 3e), indicating that CaCRK5 play pivotal roles in early defense response associated with hypersensitive response 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 significantly attenuated expression of defenserelated genes, including CaNPR1 [30], CaSAR8.2 [31], CaDEF1 [32] and CaACO1 [33], during R. solanacearum infection (Fig. 3f).
Overexpression of CaCRK5 in N. benthamiana 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 the difficulty in getting regenerated plants for pepper genetic transformation, we choosed N. benthamiana plant, which is also a host for R. solanacearum. At least 10 transgenic N. benthamiana lines were obtained and confirmed by kanamycin resistance analysis. Two T 3 CaCRK5 overexpressed lines exhibited constitutive expression ( Fig. 4a and Additional file 5), 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 determined. As shown in Fig. 4b, 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 Four-week-old WT and transgenic N. benthamiana 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 significant differences according to Mann-Whitney U test, *P < 0.05 died 14 d after inoculation with R. solanacearum. The CaCRK5 overexpressed plants exhibited a significant delay of wilt symptom compared to wild-type plants (Fig. 4c), showing that overexpression of CaCRK5 conferred increased disease tolerance to R. solanacearum in N. benthamiana. In addition, the expression of defenserelated genes, including NtPR2, NtPR3, NtHSR201 and NtHSR505, were determined by qRT-PCR. The analysis indicated that expressions of these tested defense-related genes were increased in CaCRK5 overexpressed plants compared to wild-type plants, during the inoculation of R. solanacearum (Fig. 4d). Collectively, these data suggested that CaCRK5 overexpression enhanced defense responses against R. solanacearum infection in N. benthamiana.

The expression of 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 identified. 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, five 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 infection [34], we speculated that CaCRK5 might be targeted by CaHDZ27.
An electrophoretic mobility shift assay (EMSA) was conducted to access the interaction between CaHDZ27 and the CaCKR5 promoter, which found CaHDZ27 binds to the Cy5-labeled CaCKR5 promoter probe. Moreover, binding was gradually attenuated by increasing unlabeled probe concentrations, indicating that CaHDZ27 binds specifically to the CAATTATTG in the CaCKR5 promoter in vitro ( Fig. 5a and Additional file Fig. 5 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 purified protein was incubated with Cy5-fluorochrome 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 black line indicates the sequence region that was used for ChIP-qPCR. c, ChIP-qPCR assay was performed to verify the binding between CaHDZ27 and CaCKR5 promoter in vivo. DNA samples were co-immunoprecipitated with anti-HA antibody. The ChIP value was normalized to its respective input DNA value. Different letters indicate significant differences, as determined by the LSD test (P < 0.01) 5). Chromatin immunoprecipitation (ChIP)-qPCR was conducted to confirm interaction of CaHDZ27 and the CaCKR5 promoter in vivo. 2 days post infiltration with Agrobacterium strain GV3101 containing 35 S:HA-CaHDZ27 or 35 S:HA, the pepper leaves were collected 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 significantly enriched in the CaCKR5 promoter, and the enrichment was significantly enhanced by the inoculation of R. solanacearum (Fig. 5b and c), suggesting that CaHDZ27 could bind to the CAATTATTG in the CaCKR5 promoter in vitro, and the binding was enhanced by the R. solanacearum infection.
To further investigate the CaCKR5 regulation by CaHDZ27 at the transcriptional level, we transiently expressed 35 S:CaHDZ27-GFP in pepper leaves, and 35 S:GFP infiltrated leaves were used as a negative control. qRT-PCR analysis demonstrated that the transcript level of CaCKR5 was increased in 35 S:CaHDZ27-GFP infiltrated leaves compared with the control (Fig. 6a). The presence of CaHDZ27 in pepper leaves was confirmed by immune blot with an anti-GFP antibody ( Fig. 6b and Additional file 5). When CaHDZ27 was silenced by VIGS, the transcript level of R. solanacearuminduced CaCKR5 is reduced significantly in CaHDZ27silenced plants compared with TRV:00 infiltrated plants ( Fig. 6c 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 MAMPs and effectors to trigger inducible immune defenses [35][36][37][38], and a few CRKs have been reported to function in association with each other, such as AtCRK28/AtCRK29 [18] and AtCRK39/AtCRK40 [39]. Based on these reports, we assume that CaCRK5 possibly formed a complex with other homologs including CaCRK6. Thus, we investigated whether CaCRK5 can 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, BD-CaCRK6/AD-CaCRK5 and pGBKT7-53/pGADT7-T (positive control) transformant yeast cells can grow on SD/-Trp-Leu-His-Ade medium. In contrast, no growth was observed in the negative controls. The result indicated that CaCRK5 interacts with CaCRK6 each other in yeast cells (Fig. 7a). To further confirm the interaction between CaCRK5 and CaCRK6, we performed bimolecular fluorescence complementation (BiFC) assays. CaCRK5 and CaCRK6 were fused to the N-and C-terminal ends of yellow fluorescent protein (YFP) to generate CaCRK5-nYFP/CaCRK6-cYFP and CaCRK5-cYFP/CaCRK6-nYFP, respectively. Then, these constructs were transiently co-expressed in N. benthamiana leaves. At 48 h post infiltration, the YFP fluorescence signals were determined by confocal microscopy. As showed in Fig. 7b, YFP fluorescence was observed on the plasma membrane in N. benthamiana leaves. These results indicate that CaCRK5 heterodimerizes with CaCRK6 on 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 identified in pepper genome; 22 CRKs were present in tandem repeat which made physical clusters at specific positions in the chromosome. Previous studies showed that a large proportion of the RLK genes are found in tandem repeats [40][41][42]. Tandem repeats tend to be involved in stress response in plants [43][44][45]. The expansion and degree of tandem repeats are significantly correlated with the stress responsiveness of RLK genes [43]. The tandem repeats of CaCRKs in pepper may facilitate the stress adaptive evolution.
CaCRK5 played a positive 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 flg22 [18,46,47], suggesting a role for these CRKs in plant immunity. The CaCRK5 closest homolog 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 significant change of CRK5 was observed treated with MeJA and ETH. Signaling mediated by SA, JA or ET is involved in plant defense response to different pathogens [48][49][50]. SA generally acts in positively in plant response to biotrophic pathogen [51], while JA and ET are usually regarded as the primary regulators during plant response to necrotrophic pathogens [52]. As CaCRK5 was induced by the infection of R. solanacearum and SA, and not by MeJA and ETH, we speculate that CaCRK5 acts as positive regulator during the early period of pepper response to R. solanacearum, when R. solanacearum is on its biotrophic stage.
Based on the subcellular localizations of GFP fusions, CaCRK5 is very likely localized on the plasma membrane. Several other CRKs also reported to localize on the plasma membrane, including CRK4, CRK6, and CRK36 in Arabidopsis [17] and GbCRK18 in cotton [53]. The TRV-based VIGS system was used to investigate the function of CaCRK5 in the response to R. solanacearum infection. Expression of CaCRK5 was significantly decreased in CaCRK5-silenced pepper plants during R. solanacearum infection. CaCRK5-silenced pepper plants resulted in an increased susceptibility to R. solanacearum, and pepper leaves significantly compromised

CaCRK5 was transcriptionally regulated directly by CaHDZ27
The HD-Zip transcription factors consist of four subfamilies (HD-Zip I-IV) based on their sequence similarities [54], and HD-Zip I was reported to play roles in biotic and abiotic stress responses [55]. CaHDZ27, a Fig. 7 CaCRK5 interacts with CaCRK6. a Yeast two-hybrid (Y2H) assay. CaCRK5-BD and CaCRK6-AD were co-transformed into 'Y2H Gold' yeast cells, which were grown on SD/-Trp-Leu and SD/-Trp-Leu-His-Ade for 3 d. EV indicates the empty vector for pGBKT7. b Bimolecular fluorescence complementation (BiFC) assay. YFP fusion constructs were transiently co-expressed in N. benthamiana leaves, and BiFC signals were observed under a fluorescence microscope. Bars = 50 μm. The experiment was repeated independently three times with a similar result HD-Zip I member, acts a positive regulator in pepper response to R. solanacearum infection [34]. 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 transiently 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 against 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
CRK can form heterodimerization with closely related homologs [18,39]. Yeast two-hybrid and bimolecular fluorescence complementation analyses indicated that CaCRK5 heterodimerizes with CaCRK6 in vivo in both yeast and plant cells. Recent evidence suggests that pattern recognition receptors (PRRs) enable the rapid recruitment of other RLKs, including CRKs, to enhance PRR signaling intensity [18,46]. 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 significantly contributes to immune defense against R. solanacearum in pepper and N. benthamiana. VIGS in pepper plants and gain-of-function analyses of CaCRK5 in N. benthamiana revealed that CaCRK5 positively regulates plant immune responses. In addition, CaHDZ27 promotes the expression of CaCRK5 by directly binding to its promoter. We propose that the pepper defense response mediated by CaCRK5 might be enhanced by CaHDZ27. CaCRK5 is involved in the pepper innate immune response and may be promising target for genetic engineering to enhance pepper resistance against wilt disease.

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), which exhibited medium-resistance to R. solanacearum infection identified by Prof. Shuilin He at FAFU in China. Pepper and N. 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. R. solanacearum strain was grown on casamino peptone agar (CPG) plates containing triphenyltetrazolium chloride (TZC), and a single colony was cultured in PSA (potato saccharose agar) medium at 28°C for 2 days. Bacteria cell solution was harvested with sterile 10 mM MgCl 2 , and the suspension was adjusted to 1.0 × 10 8 CFU/mL. Inoculation of bacteria was carried out by root irrigation. After cutting the plant roots, 30 mL bacterial solution was introduced into the soil in each pot. To inoculate plant leaves, 10 µL of the suspension of R. solanacearum was infiltrated into the leaf of 6-week-old pepper plant using a syringe without a needle, and sterilized MgCl 2 solution was infiltrated in mock-treated plants. For each plant, a disease index on a scale of 0 to 4 was calculated as described previously [22].

Bacterial strain growth
The growth of R. solanacearum was measured by quantifying bacterial multiplication in pepper leaves. To do this, four leaf discs of 0.5 cm diameter were collected per samples at 3 days post inoculation, and were ground in 1 mL of sterile water. Diluent was plated on TZC solid medium and was incubated at 28°C for 48 h. Colonies were counted to determine as CFUs per square centimeter of leaf tissue. Pathogen growth experiment was repeated three times, and contained at least three plants per genotype.

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 or CaHDZ27 specific silencing, the DNA fragments in 3'-untranslated region of CaCRK5 (5-ACTATTCTCACAGCGAC-3 and 5-GAATTCGA ACAAATAACA-3) and CaHDZ27 (5-TTCCACAA GAGAATAGTG-3 and 5-GGAACAAAGCTAATAAA-3) were PCR amplified, then cloned into pTRV2 vector containing part of the tobacco rattle virus (TRV) genome to generate TRV2:CaCRK5 or TRV2:CaHDZ27. These plasmids were introduced into A. tumefaciens strain GV3101 by freeze-thaw method. The Phytoene Desaturase (PDS) is used as an indicator gene, its expression is reduced sufficiently to yield a photobleached phenotype. A. tumefaciens GV3101 carrying TRV1 with pTRV2:00 (empty vector) or TRV2:CaCRK5 or TRV2: CaHDZ27 was co-infiltrated 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.

RNA extraction and quantitative real-time RT-PCR
Leaves from three different pepper or N. benthamiana plants were harvested, and total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was determined by Nanodrop 2000 (Thermo Fisher Scientific), and the integrity was examined using agarose gel electrophoresis. Reverse transcription was performed using HiScript® Q RT SurperMix for qPCR reagent Kit with gDNA wiper according to the manufacturer's instructions (Vazyme), and a nontemplate negative control was used to detect the primer dimerization. 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. To characterize the expression of the defenserelated genes (CaNPR1, CaSAR8.2, CaDEF1 and CaACO1) in CaCRK5 silenced pepper, the relative transcript levels were normalized to the control (TRV:00) using the CaActin and Ca18S rRNA genes as internal references. To assess the expression of the defense defense-related genes (NtPR2, NtPR3, NtHSR201 and NtHSR505) in CaCRK5 transgenic N. benthamiana plants, the relative transcript levels were normalized to the wild type using the NtActin and NtEF1α genes as internal references. The primers were listed in Additional file 6, and melting curves were listed in Additional file 7.

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 amplified and cloned into pGADT7 to generate constructs designated AD-CaCRK5 and AD-CaCRK6. The cDNAs of CaCRK5 and CaCRK6 were amplified 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. Transformants containing the indicated plasmids were spotted onto the SD (Synthetic Dextrose) screening medium, and incubated at 30°C till the formation of colonies. After selection on SD/-Trp-Leu medium, yeast cells were transferred to SD/-Trp-Leu-His-Ade medium for evaluation of interaction.

Bimolecular fluorescence complementation (BiFC) assay
To generate BiFC constructs, CaCRK5 and CaCRK6 full length cDNAs were cloned into puc-SPYNE GW (CaCRK5-YFP N and CaCRK6-YFP N ) and puc-SPYCE GW (CaCRK5-YFP C and CaCRK6-YFP C ) vectors, which contain the N-terminal or C-terminal fragment of YFP (nYFP or cYFP, respectively). Since CabZIP63 acts as a transcription factor, CabZIP63-YFP N and CabZIP63-YFP C were used as the negative control of CaCRK6-YFP N and CaCRK6-YFP C , respectively. These constructs were introduced into A. tumefaciens strain GV3101 by freeze-thaw method. For transient expression, cells mixture of A. tumefaciens strain GV3101 carrying each construct was used to infiltrate leaves of 4-week-old N. benthamiana plants. For microscopic analysis, lower epidermis cells were observed using a confocal laser scanning microscope.

N. benthamiana transformation
Transgenic N. benthamiana plants were generated by using A. tumefaciens mediated N. benthamiana leaf disc transformation [56]. The CaCRK5 coding region was cloned into pK7WG2 vector to get 35 S:CaCRK5 construct, then it was introduced into A. tumefaciens GV3101 and used to transform N. benthamiana. Fresh leaf discs were cut from N.benthamiana, and immersed in the GV3101 cell suspension (OD = 0.6) for 7-10 min. After drying on sterile paper, leaf discs were transferred to basal Murashige and Skoog (MS) agar medium and incubated at 28°C for 2-3 days in the dark. After cleaning with sterile water, leaf discs were placed on the selection medium containing kanamycin. At least ten independent N. benthamiana transgenic lines were obtained, and checked the expression of CaCRK5 by RT-PCR. The selected transgenic lines were selfpollinated, T 3 seeds were obtained and planted on MS agar plates containing 50 µg of kanamycin per milliliter for use in the study.

Histochemical staining
Histochemical staining was performed as previously described [22]. The infiltrated 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 purified using BeaverBeads™ GSH protein purified kit according to manufacturer's instructions.
The EMSA was performed as described previously [57]. 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 [22]. Chromatin was extracted from pepper leaves 2 days post infiltration with 35 S: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. The values are presented as the relative enrichment ratio, and the value for the negative control (35 S:HA, mock treatment) was set to "1". Primers used for ChIP-qPCR are listed in Additional file 6.
Essential information of all CKRs in pepper including molecular weight and the theoretical pI were evaluated using the ProtParam tool (http://web.expasy.org/ protparam/). An unrooted phylogenetic tree was built by MEGA version 7.0 software [62], using neighbor-joining method with 1000 bootstrap trials. The chromosomal localization of CRK gene family members in pepper were verified from the CM334 (v1.55) proteins database, chromosomal images were drawn using MapInspect 1.0 software (https://mapinspect.software.informer.com/).