Identification of CBL and CIPK gene families and functional characterization of CaCIPK1 under Phytophthora capsici in pepper (Capsicum annuum L.)

Background Calcineurin B-like proteins (CBLs) are major Ca2+ sensors that interact with CBL-interacting protein kinases (CIPKs) to regulate growth and development in plants. The CBL-CIPK network is involved in stress response, yet little is understood on how CBL-CIPK function in pepper (Capsicum annuum L.), a staple vegetable crop that is threatened by biotic and abiotic stressors. Results In the present study, nine CaCBL and 26 CaCIPK genes were identified in pepper and the genes were named based on their chromosomal order. Phylogenetic and structural analysis revealed that CaCBL and CaCIPK genes clustered in four and five groups, respectively. Quantitative real-time PCR (qRT-PCR) assays showed that CaCBL and CaCIPK genes were constitutively expressed in different tissues, and their expression patterns were altered when the plant was exposed to Phytophthora capsici, salt and osmotic stress. CaCIPK1 expression changed in response to stress, including exposure to P. capsici, NaCl, mannitol, salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), ethylene (ETH), cold and heat stress. Knocking down CaCIPK1 expression increased the susceptibility of pepper to P. capsici, reduced root activity, and altered the expression of defense related genes. Transient overexpression of CaCIPK1 enhanced H2O2 accumulation, cell death, and expression of genes involved in defense. Conclusions Nine CaCBL and 26 CaCIPK genes were identified in the pepper genome, and the expression of most CaCBL and CaCIPK genes were altered when the plant was exposed to stress. In particular, we found that CaCIPK1 is mediates the pepper plant’s defense against P. capsici. These results provide the groundwork for further functional characterization of CaCBL and CaCIPK genes in pepper.

helix motif containing 12 residues of +X• + Y• + Z•-Y•-X••-Z, where the letters represent the ligands involved in metal coordination, and the dots represent the intervening residues [5]. CBL-interacting protein kinases (CIPKs) belong to the SnRK3 protein family that contain a Ser/Thr protein kinase domain [6]. CIPKs phosphorylate CBLs at a conserved Ser residue in CBL's PFPF motif [7]. CIPKs frequently have a conserved N-terminal kinase domain, serine-threonine kinases domain, NAF/FISL motif and C-terminal regulatory domain [8,9]. In addition to these domains, the C-terminus of CIPKs contain a protein-phosphatase interaction (PPI) domain, which interacts with phosphatase 2C (PP2C) proteins [10]. The interaction between the CBL and CIPK is involved in a Ca 2+ -decoding system called the CBL-CIPK network [11].
Recent studies have found that CIPKs regulate cell signaling across plants species. For instance, the apple CIPK protein kinase MdSOS2L1 interacts with MdCBL1, MdCBL4 and MdCBL10 to increase the levels of antioxidant metabolites and enhance salt tolerance in apple and tomato [30].
Overexpression of MdCIPK22 increases ABA sensitivity in an MdAREB2 (ABA responsive element binding factors) dependent manner, and MdCIPK13 phosphorylates MdSUT2.2 (sucrose transporter) to regulate salt tolerance [31,32]. Overexpressing SlSOS2 (SlCIPK24) increases tolerance against salinity in tomato [33]. In wheat, TaCIPK23 is involved in ABA and drought stress responses, as well as the crosstalk between ABA signaling and drought [34].
Pepper (Capsicum annuum L.) is an important horticultural crop that belongs to Solanaceae, and is a rich source of vitamins, minerals and nutrients that are of great importance for human health [35,36]. Nevertheless, the cultivation of pepper is threatened by biotic and abiotic stressors, such as pathogens, drought, salinity, and low temperature. Phytophthora capsici (P. capsici) is a devastating soil-borne pathogen that is causing significant damage to pepper crops worldwide by causing damping-off, seedling blight, and plant death [37,38]. Infection of plants by pathogens leads to the activation of pattern-triggered immunity (PTI) and effectortriggered immunity (ETI), and infection by microbes leads to micro-associated molecular patterns (MAMPs) of PTI [39]. OsCIPK14 and OsCIPK15 regulate MAMPs defense signaling pathways in rice, and TaCIPK5 positively regulates resistance against stripe rust fungus in wheat with TaCBL4 [40]. However, whether CBL and CIPK genes are involved in mediating stress response in pepper remains unclear, and studying the CBL-CIPK network is important for furthering our understanding of how pepper plants respond to biotic and abiotic stressors. Here, we identified two gene families (CaCBL and CaCIPK) in the pepper genome. Phylogenetic analyses were performed to investigate the evolutionary relationships of the nine CaCBL and 26 CaCIPK members. A comprehensive analysis of gene structure, protein motif conservation, chromosomal location, gene duplication, stress-related cis-elements, and prediction of protein-protein interaction networks were conducted to further understand the structure and relationship of CaCBL and CaCIPK genes. Furthermore, we examined the expression profiles of CaCBL and CaCIPK genes in pepper plants of various developmental stages and those exposed to biotic and abiotic stressors. We found that CaCIPK1 expression was induced by exposure to biotic and abiotic stressors, and we utilized the virus-induced gene silencing (VIGS) system to investigate the function of CaCIPK1 in pepper plants infected with P. capsici.

Identification of CaCBL and CaCIPK genes in pepper
To identify CBLs and CIPKs in pepper, a HMM (Hidden Markov Model) analysis was performed against the CM334 (Capsicum annuum Cultivars in Mexico) and Zunla-1 (Capsicum annuum Cultivars in China) genomic databases. All putative genes were surveyed to verify the presence of conserved domains. Nine CBL and 26 CIPK genes were identified and named by chromosomal order (Additional file 1, Additional file 2). CaCBLs had four EF-hands, and CaCIPKs had a protein kinase catalytic domain (PKC), the 24-amino acid NAF/FISL motif and a PPI motif, similar to other genes in these families.

Phylogenetic and sequence analysis of CaCBL and CaCIPK genes
To understand the evolutionary relationship of CBL and CIPK proteins among pepper and other plants, a neighbor-joining (NJ) tree was constructed using amino acid sequences from Capsicum annuum, Arabidopsis thaliana, Manihot esculenta, Populus trichocarpa, Oryza sativa, Triticum aestivum, and Brassica napus ( Fig. 1; Additional file 4). There were multiple CBL and CIPK sequences from wheat, potentially due to its hexaploidy, therefore only one sequence from each set of homologous gene sequences was used for the analysis. Sixty CBLs were clustered into four different groups (I to VI) with high bootstrap values (Fig. 1a). Three CBL genes (CaCBL3, CaCBL4 and CaCBL9) were categorized into group I, CaCBL5 was a part of group II, CaCBL1, − 2, and − 6 belonged to group III, and CaCBL7 and CaCBL8 clustered into group IV. CaCBL7 was orthologous to AtCBL7, and CaCBL8 was orthologous to wheat and rice CBLs. There were no sequences available for some CIPK genes, such as TaCIPK6, TaCIPK12, and TaCIPK13 [41].
A total 188 of CIPK genes from seven species were clustered into five subfamilies and assigned names (A to E) (Fig. 1b). Group A contained CaCIPK1, − 6, − 9, − 10, Multiple sequence alignment (MSA) revealed that CaCBLs have four EF-hand domains and a conserved PFPF motif (Additional file 5A). In addition, five CaCBLs harbored a conserved myristoylation motif (MGXXXS/T) on the N-terminus, including CaCBL1, − 2, − 3, − 4, Fig. 1 The phylogenetic analysis of CBL (a) and CIPK (b) gene families from pepper, Arabidopsis cassava, canola, rice, poplar and wheat. Full-length protein sequences were used to construct Neighbor-Joining (NJ) trees by MEGA-X program with pairwise deletion, Poisson correction and bootstrap value 1000. Different species are denoted using different symbols. Subfamilies are distinguished with different colors. CBLs were classified into four groups, and CIPK were classified into five groups and − 6. CaCBL5 had an extended N-terminus, which contained a transmembrane helix. CaCBL8 possessed the consensus motif for the tonoplast targeting sequences (TTS, MSQCXDGXKHXCXSXXXCF). MEME analysis showed that CaCBLs have six different conserved motifs, and these were named Motif-1 to Motif-6 ( Fig. 2a; Additional file 6).

Interaction network of CaCBL and CaCIPK members
To investigate the relationship of CaCBLs and CaCIPKs, a protein-protein interaction network was built using homologs of the Arabidopsis interaction network (Additional file 9). Nine CaCBLs were homologous to five AtCBLs, and 26 CaCIPKs were homologous to 13 AtCIPKs. The co-expression network of CBLs and CIPK was calculated using the Pearson correlation coefficient Cis-acting elements of CaCBL and CaCIPK genes in pepper To better understand how CaCBL and CaCIPK genes are regulated, the 1500 bp upstream sequences of the coding region were detected by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [42] to identify cis-acting elements (upstream sequences are shown in Additional file 10). Thirteen cis-elements, including ABRE (abscisic acid responsiveness), ARE (anaerobic induction), CE3 (ABA and VP1 responsiveness), CGTCA-motif (MeJA-responsiveness), GC-motif (enhancer-like element involved in anoxic specific inducibility), HSE (heat stress responsiveness), LTR (low-temperature responsiveness), MBS (MYB binding site involved in drought-inducibility), SARE and TCA-element (salicylic acid responsiveness), TC-rich (defense and stress), TGA-box (auxin-responsiveness) and WUN-motif (wound-responsiveness) were mapped onto the promoter regions (Fig. 4). ABRE elements were found in the promoter regions of five CaCBL genes (55.6%) including CaCBL1, − 3, − 7, − 8, and − 9, Interestingly, the promoter region of some CIPKs harbored at least one HSE elements, and the promoter of CaCIPK15 had the highest number of HSE elements (6). In addition, the WUN-motif was the least common element in these genes, and only in two CaCBL (CaCBL4, − 6) and five CaCIPK genes (CaCIPK3, − 9, − 12, − 18, − 22) contained WUN-motifs upstream of their promoter. All cis-elements that were identified in this analysis are involved in stress response and hormone signaling.

Subcellular localization of CaCIPK proteins
To explore the potential function of CaCIPK, we selected one member from every subfamily to study its subcellular localization. The subcellular localizations were detected in epidermal cells of Nicotiana benthamiana by Agrobacterium-mediated transient expression. CaCIPK1 was localized to the nucleus, plasma membrane and cytoplasm, CaCIPK5 and CaCIPK20 were localized to the plasma membrane, and CaCIPK7 was localized to the nucleus and plasma membrane. In particular, the CaCIPK15-GFP fusion protein was localized to organelles or other structures in the cytoplasm (Additional file 11).

Gene expression patterns in response to various stresses and developmental stages of pepper
The CBL-CIPK network regulates stress response against biotic and abiotic stressors. In order to explore whether the expression of CaCBL and CaCIPK genes change when exposed to various stressors, including NaCl, mannitol, incompatible PC strain and compatible HX-9 strain of P. capsici, we conducted qRT-PCR analyses to study the expression patterns of CaCBLs and CaCIPKs.
We chose samples at 6 h post treatment (hpt) for abiotic stress and 12 h post-inoculation (hpi) for biotic stress, and compared expression to plants that were collected at the same time that were not exposed to biotic or abiotic stress (Fig. 5a).
In response to abiotic stresses, five CaCBL genes were up-regulated when exposed to NaCl and seven CaCBL genes were up-regulated when exposed to mannitol.
CaCBL3 was up-regulated by 4.25-fold when plants were exposed to NaCl and by 8.77-fold when exposed to mannitol. CaCBL2 and CaCBL9 were down-regulated when plants were treated with high levels of NaCl and mannitol. In the CaCIPK family, CaCIPK1, − 2, − 3, − 4, , but were not affected by NaCl stress. The same pattern was found in CaCBL8, indicating that they are co-regulated in response to osmotic stress.
To further investigate the spatial expression patterns of CaCBLs and CaCIPKs in pepper, qRT-PCR analysis was conducted on root, stem, leaf, flower, green fruit and red fruit. Expression levels of all genes were calculated relative to the expression levels of CaCIPK1 in the stem (Fig. 5b). CaCBL and CaCIPK genes were constitutively expressed in different tissues. Most CaCBL and CaCIPK genes showed higher expression in the root and Expression pattern of CaCIPK1 during stress and hormone treatment Our results above showed that CaCIPK1 expression increased under different treatments, especially when plants were infected with P. capsici and exposed to high levels of mannitol (Fig. 5). To obtain better insight into CaCIPK1 is a member of the CIPK family, which interacts with Ca 2+ sensors. Therefore, leaves were sprayed with different concentrations of CaCl 2 to explore whether CaCIPK1 was involved in the plant's response to Ca 2+ stress (Fig. 6).
CaCIPK1 expression was induced when leaves were sprayed with SA, MeJA, ABA and ETH. When sprayed with SA, CaCIPK1 was up-regulated and reached to a Expression patterns of CaCBLs and CaCIPKs in plants exposed to P. capsici, NaCl, and mannitol (a), and in different tissues (b). The grey means N/A. The samples in (a) were collected at different time points (6 hpt and 12 hpi for abiotic and biotic stresses respectively). "F" in (b) represents fruit. Actin (AY572427.1) is used as the internal control. Relative transcript levels were calculated using the comparative threshold (2 −ΔΔCT ) method, and normalized using log2. The heat map was created by MeV peak (6.36-fold) at 6 hpt, and was down-regulated at subsequent time points. When leaves were sprayed with ABA and ETH, the highest CaCIPK1 expression levels (3.46-fold and 3.77-fold respectively) were recorded at 6 hpt. Plants treated with MeJA showed highest CaCIPK1 expression level at 3 hpt (7.98-fold). In response to cold stress, CaCIPK1 expression steadily increased and reached highest expression level (3.28-fold) at 24 hpt. After heat stress, CaCIPK1 expression changed dynamically, and was significantly up-regulated (14.34-fold) at 24 hpt. In addition, expression of CaCIPK1 varied under different concentration of CaCl 2 at 6 hpt. CaCIPK1 expression increased with exposure to higher concentrations of CaCl 2 , and reached a 5.55-fold up-regulation at 10 mM. CaCIPK1 expression was down-regulated when exposed to CaCl 2 concentrations above 50 mM.

VIGS of CaCIPK1 increased pepper sensitivity to P. capsici
To further explore the function of CaCIPK1 in the pepper's response to P. capsici infection, CaCIPK1 loss-of-function peppers were generated by VIGS using the AA3 cultivar. At 5 weeks post-inoculation, the positive control with pTRV2:CaPDS (phytoene desaturase gene) showed photobleaching phenotypes. qRT-PCR analysis confirmed that CaCIPK1 was silenced in the leaves, and CaCIPK1 expression in the silenced plants (pTRV2: CaCIPK1) was 50 to 80% lower than the negative control (pTRV2:00) (Fig. 7a). To ensure the construct specifically targeted CaCIPK1, the CaCIPK1 sequence was aligned with its homologous genes (CaCIPK17 and − 19) (Additional file 12A), and we found that the expression of CaCIPK17 and CaCIPK19 was not suppressed in pTRV2:CaCIPK1 plants (Additional file 12B). The sensitivity of CaCIPK1-silenced plants to the avirulent P. capsici (PC strain) was explored using detached leaves assays. Leaves from negative control and CaCIPK1-silenced plants were sampled to match the same region that showed photobleaching in the positive control sample. Leaves were injected with zoospore suspension of the PC strain. Slight disease symptoms were detected at 2-day post inoculation (dpi) in the CaCIPK1-silenced plants, and these symptoms gradually became apparent at 4 dpi. In contrast, very few disease lesions were observed in the control (Fig. 7b). Quantitative analysis of the lesion area revealed that CaCIPK1-silenced plants had significantly larger lesion areas (34.28%) than the control (1.42%) (Fig. 7c).
To understand the molecular mechanisms underlying the increased sensitivity to the PC strain in the CaCIPK1 knocked down plants, expression levels of defense related genes were measured. Defense genes that were studied included CaPR1 (pathogenesis-related gene 1) [43], CaDEF1 (defensin gene) [44], CaSAR8.2 (systemic acquired resistance gene) [45], CaPO1 (peroxidase) [46], and CaCAT (catalase, highly homologous with AtCAT), and their expression patterns were examined at 2, 4, and 7 dpi (Fig. 7a). Knocking down CaCIPK1 resulted in a significant decline in the expression of CaPR1, CaSAR8.2 and CaDEF1. Control plants had 4.83-fold higher expression of CaPR1 than the CaCIPK1-silenced plants at 2 dpi. CaSAR8.2 and CaDEF1 showed 80% decrease in expression in CaCIPK1-silenced plants at both 2 dpi and 7 dpi. The expression of CaPO1 and CaCAT were significantly up-regulated in CaCIPK1-silenced plants, and the fold change was as high as 5-fold and 4-fold compared to the control at 4 dpi, respectively.
Furthermore, the vigor of the metabolism in the root system was measured by assessing root activity using triphenyltetrazolium chloride (TTC) [47]. TCC was reduced in the control and CaCIPK1-silenced plants that were treated with P. capsici (Fig. 7d). Root activity was reduced in both plants; however, CaCIPK1-silenced plants that were infected with P. capsici had significant lower root activity than control plants. The lowest activity was recorded in CaCIPK1-silenced plants at 4 dpi, where the activity was reduced by 57.3% compared to the control.

Transient expression of CaCIPK1 in pepper leaves
Knocking down CaCIPK1 increased the expression of CaPO1 and CaCAT, two genes that regulate ROS levels, therefore we explored whether CaCIPK1 is involved in ROS accumulation. The vectors 35S:00 and 35S:CIPK1 were ectopically overexpressed in pepper leaves using Agrobacterium. We measured cell death, H 2 O 2 production, and expression of defense-related genes at 24 h post agroinfiltration. Transient expression of CaCIPK1 induced cell death and enhanced H 2 O 2 accumulation compared with samples infected with the empty vector control (Fig. 8a, b). CaCIPK1 expression increased by 126.76-fold in the infected samples compared to the control, and CaPR1, CaDEF1, CaSAR8.2 expression increased by 16.11-fold, 3.96-fold and 136.54-fold, respectively (Fig. 8c).

Discussion
Calcium is a core regulator of plant development and responses to the environment [3]. The Ca 2+ sensor CBLs and their target protein kinases CIPKs, comprise a complicated signaling network that allows plants to adapt to developmental and environmental stress. CBL-CIPK modules are involve in ion channel, phytohormones signaling network, growth and development [1,[12][13][14][15]. Past studies showed that multiple CBL and CIPK genes are found in many plant species, such as Arabidopsis thaliana (10 CBLs and 26 CIPKs), Manihot esculenta (8 CBLs and 26 CIPKs), Oryza sativa (10 CBLs and 34 CIPKs), Brassica napus (7 CBLs and 23 CIPKs), Populus trichocarpa (10 CBLs and 27 CIPKs), Triticum aestivum (24 CBLs and 79 CIPKs loci in sub-genomes A, B, and D respectively), and Vitis vinifera (8 CBLs and 20 CIPKs) [14,40,41,[48][49][50][51][52][53][54]. In this study, nine CBL and 26 CIPK genes were identified using the CM334 [55] and Zunla-1 [56] pepper genomes. MSA of CaCBLs found that all members contained four EF-hands, which consisted of 12 relatively conserved amino acids. Interestingly, the amino acids numbers between adjacent EF-hand were same. EF1 and EF2 were 23 amino acids apart, EF2 and EF3 were 25 amino acids apart, and 32 amino acids separated EF3 from EF4. The number of amino acids separating adjacent EF-hands were not consistent with findings from Arabidopsis thaliana, Oryza sativa and Vitis vinifera, where there were 22 amino acids between EF1 and EF2 [49,52]. To further explore the numbers of amino acids between EF-hands in other species, CBLs in Arabidopsis thaliana, Capsicum annuum, Manihot esculenta, Populus trichocarpa, and Triticum aestivum were tested again for protein structure. We found there were consistently 23 amino acids between EF-1 and EF-2, unlike previous studies that identified 22 amino acids, and this is potentially due to differences in methods for counting amino acids between the domains (Additional file 13).
To uncover the potential function of CaCBLs and CaCIPKs, we performed phylogenetic analysis and MSA ( Fig. 1; Additional file 5). Of the genes that belonged to group II (Fig. 1a), CaCBL5 had a transmembrane helix that may target the protein to the plasma membrane, similar to AtCBL10, OsCBL9, OsCBL10, and TaCBL9 [40]. Group IV contained the tonoplast targeting sequences (TTS). However, one member, CaCBL7, did not have the tonoplast targeting motif, similar to AtCBL7 [57,58]. Compared with Arabidopsis, CaCBL8 and AtCBL2/3 contained TTS, suggesting that it is localized to the tonoplast [40,59]. Additionally, five CaCBLs (CaCBL1, − 2, − 3, − 4 and − 6) had a myristoylation motif in the Nterminus, and this motif is required for binding to the membrane and interacting with SOS3 under salt stress [7,60]. In addition, there was a PFPF motif at the C-terminus, and the motif contained a conserved serine residue known to interact with CIPKs. Interestingly, CIPKs in group B, such as CaCIPK2, − 4, − 11, − 13, − 20, − 25, and − 26, contained more than 10 introns, making this an intron-rich clade ( Fig. 1b; Additional file 3). CaCIPKs in other groups contained zero or one intron, suggesting that they are an intron-poor clade. CIPK genes were also classified into intron-rich and intron-poor clades, such as in Arabidopsis thaliana, Triticum aestivum, and Oryza sativa [41,52]. However, the number of introns in CBLs did not show a specific pattern. Hence the classification method using intron numbers is unique to the CIPK family. CaCIPKs had the conserved N-terminal kinase domain, C-terminal regulatory domain and NAF/FISL motif (Additional file 5B). The NAF domain consists of the conserved amino acids asparagine (N), alanine (A), and phenylalanine (F), and mediates the interaction with CBL. Next to the NAF motif, there was the PPI domain, which interacts with PP2C [8][9][10]. The N-terminal catalytic kinase domain has an ATP binding site and an activation loop [61].
In the gene duplication analysis, we found that the CaCBL family underwent two segmental duplication events (CaCBL1, − 2, − 6, and CaCBL7, − 8) in group III and group IV. The CaCIPK family harbored two segmental duplication events (CaCIPK6, − 9, − 10, and CaCIPK18, − 21) in group A, and three (CaCIPK2, − 4, − 25, CaCIPK3,-11,-22, and CaCIPK13, − 26) in group B. Tandem duplication events were not detected in CaCBL and CaCIPK genes. These results suggested that segmental duplications may have contributed to the complexity and diversity of both gene families. Additionally, the Ka/Ks ratio of CaCBL and CaCIPK genes (less than 1) inferred that the duplicated genes were maintained by purifying selection. The earliest duplication was for CaCIPK18 and CaCIPK21, and it was estimated to have occurred around 106.34 million years ago (Additional file 8).
The role of CBL-CIPK complexes in regulating plant development and response to environmental stress has been studied in different plant species, such as Arabidopsis and wheat [1,3,61]. To better understand the function of CaCBLs and CaCIPKs, we compared genes belonging to these families from pepper and Arabidopsis using a phylogenetic analysis (Additional file 14). CaCBL3 and CaCIPK20 were orthologous to AtCBL5 and AtCIPK24, respectively. AtCBL5-AtCIPK24 proteins are localized to the plasma membrane. Overexpression of AtCBL5 enhances salt and drought stress tolerance, and AtCIPK24 modulates cellular responses to salt stress and activate the Na + /H + antiport activity of SOS1 [19,21,61,62]. In pepper, CaCBL3 was up-regulated when exposed to salt (4.25-fold) and mannitol (8.77-fold) stress, and CaCIPK20 was also up-regulated when plants were exposed to mannitol (7.90-fold). MBS elements regulate gene expression under drought conditions, and these elements were found in the promoter region of CaCBL3 and CaCIPK20, suggesting that CaCBL3 may interact with CaCIPK20 to regulate drought stress tolerance (Figs. 4, 5a). PAT10 (Protein S-Acyl Transferase10)-CBL2/3-CIPK9/17 complexes regulate ABA signaling during stomatal movement in Arabidopsis [15,17]. CaCBL7 and CaCBL8 were orthologous to AtCBL2 and AtCBL3, respectively. They contained ABRE elements in the promoter regions, suggesting that they may be regulated by PAT in ABA signaling. In wheat, TaCBL4 interacts with TaCIPK5 to positively modulate wheat resistance against fungus [40]. The genes orthologous to TaCBL4 and TaCIPK5, CaCBL9 and CaCIPK9/10, were up-regulated when plants were exposed to the PC strain, and down-regulated when exposed to the HX-9 strain. We speculate that CaCBL9-CaCIPK9/10 may regulate the pepper plant's resistance against P. capsici.
The expression levels of CaCBLs and CaCIPKs were detected by qRT-PCR in plants exposed to pathogen, salt, and mannitol (Fig. 5a). However, expression patterns of CaCBL and CaCIPK genes were variable within the different subfamilies. For instance, CaCBL3 and CaCBL4 were up-regulated when exposed to stress, but CaCBL9 was up-regulated in plants treated with mannitol and incompatible P. capsici. CaCIPK13 was upregulated by exposure to salt (10.87-fold), mannitol (89.38-fold) and incompatible P. capsici (35.20-fold), while CaCIPK26 was slightly up-regulated by exposure to mannitol (3.17-fold) and incompatible P. capsici (2.94-fold). CaCIPK9 was derived from a segmental duplication of CaCIPK10, and these two genes showed similar expression patterns. Nevertheless, other paralogs did not show similar patterns. In contrast, many CaCBLs and CaCIPKs genes were expressed at higher levels during different developmental stages (Fig. 5b). For instance, CaCIPK18 was expressed at the highest level in all stages, specifically in the red fruit (227.27-fold), inferring that it may have vital functions during different developmental stages. The expression level of CaCIPK4 was lower than other genes in all tissues, and its expression levels did not change when plants were exposed to P. capsici. Therefore, CBL-CIPK may play an important role in the response to biotic and abiotic stimuli, rather than development in pepper.
Five genes from each subfamily in CaCIPKs were chosen for subcellular localization assay. CaCIPK1 was localized to the nucleus, plasma membrane and cytoplasm, similar to TaCIPK14 and TaCIPK23 [34,63]. Overexpression of TaCIPK14 enhances cold and salt stress tolerance in tobaccos. CaCIPK1 and TaCIPK14 belong to the same clade in Group I, therefore CaCIPK1 may also be involved in regulating tolerance against abiotic stress. CaCIPK5 and CaCIPK20 were localized to the plasma membrane. CaCIPK7 was localized to the nucleus and plasma membrane, similar to AtCIPK21, MeCIPK23 and BnaCIPK24 [64][65][66]. CaCIPK15 was targeted to organelles in the cytoplasm, deviating from the patterns observed in other CaCIPKs in this study (Additional file 11). In most plants, CIPKs are recruited by CBLs to the plasma membrane or tonoplatst to form a complex [21,67], and it is likely that the interaction between CBLs and CIPKs influenced the plasma membrane localization of the five CIPKs were analyzed in this study.
While the role of the CBL-CIPK network in regulating response against abiotic stress is well documented, it remains unclear whether this network also regulates responses against biotic stress. OsCIPK14 and OsCIPK15 are rapidly induced by MAMPs in rice, and RNAi against these genes reduces sensitivity to Trichoderma viride/ethylene-inducing xylanase [68]. The TaCBL4-TaCIPK5 complex positively contributes to the interaction of wheat and Puccinia triiformis f. sp. tritici through ROS signaling [40]. CaCIPK1 was strongly upregulation in the root of plants that were infected with incompatible (51.68-fold) and compatible (16.34-fold) P. capsici, indicating that CaCIPK1 may be involved in resistance against P. capsici in pepper. Moreover, the subcellular localization of CaCIPK1 was similar to TaCIPK14 and TaCIPK23. Overexpression of TaCIPK14 enhances cold and salt stress tolerance and TaCIPK23 positively regulates drought stress and ABA responses [34,63], and CaCIPK1 may have similar functions. Meanwhile, the CaCIPK1 promoter region contained ABRE, HSE, MBS, LTR, and TCA cis-acting elements, which are involved in ABA signaling, heat stress, drought, low temperature and SA (Fig. 4). The presences of these elements suggest that CaCIPK1 is involved in abiotic and phytohormone stress response, and we tested the expression pattern of CaCIPK1 to verify this hypothesis (Fig. 6). SA, MeJA, ABA, and ETH are central regulators of defensive signaling and plant innate immunity [69,70]. The results implied that CaCIPK1 may play a crucial role in defensive and innate immunity in pepper.
To confirm that CaCIPK1 is involved in mediating the interaction between pepper and incompatible P. capsici, TRV-VIGS was used to successfully knock down CaCIPK1. The silenced leaves were detached [71] and inoculated with the PC strain. There were larger lesions in the CaCIPK1-silenced plants compared to the control (24.06-fold), indicating that knocking down of CaCIPK1 increased sensitivity to incompatible P. capsici (Fig. 7c). Similar results were observed in wheat, where knockdown of TaCBL4 and TaCIPK5 reduced the defense response of wheat against stripe rust fungus [40]. To further study the role of CaCIPK1 in plants that were infected with the PC strain, root activity assays were conducted to detect the effects of the fungus on CaCIPK1-silenced plants. When the inoculation time was prolonged, root activity in CaCIPK1-silenced plants was lower than control plants, and there was a significant decrease in root activity at 4 and 7 dpi (Fig. 7b). These results showed that CaCIPK1-silenced plants had reduced resistance against incompatible P. capsici compared with control plants. In addition, CaPR1, CaSAR8.2, and CaDEF1 expression levels were lower in CaCIPK1-silenced plants that were exposed to PC (Fig. 7a). CaPR1 and CaSAR8.2 are genes involved in pathogenesis and systemic acquired resistance, and are induced by SA signaling when plants are exposed to biotic stress [40,43]. Interestingly, CaCIPK1 expression was induced by SA (Fig. 6). Taken together, CaCIPK1 may be involved in the SA pathway to defend the pepper plant against incompatible P.capsici. CaCIPK1-silenced plants that were exposed to PC had higher expression levels of CaPO1 and CaCAT, which may lead to lower H 2 O 2 accumulation. Previous studies have postulated that the accumulation of ROS (e.g., H 2 O 2 ) and the release of Ca 2+ positively regulate each other [72]. To verify whether CaCIPK1 is involved in H 2 O 2 signaling, CaCIPK1 was transiently over-expressed in pepper. Plants over-expressing CaCIPK1 had higher cell death and H 2 O 2 accumulation, and up-regulation of defense-related genes CaPR1 (16.11-fold) and CaSAR8.2 (136.54-fold) (Fig.  8). Thus, we hypothesize that CaCIPK1 is involved in H 2 O 2 and SA signaling to modulate P. capsici tolerance by interacting with CBLs. Nevertheless, more studies are necessary to clarify the molecular mechanisms by which CaCIPK1 regulates the resistance of pepper against P. capsici.

Conclusions
The CBL-CIPK signaling pathway is a Ca 2+ -related pathway that regulates the plant's response to environmental stimuli and ion stress [1]. Here, we identified nine CaCBL and 26 CaCIPK genes in pepper and most genes were highly expressed in different developmental stages. These genes also showed varying responses to biotic and abiotic stressors, suggesting that they may be crossing nodes of different signaling networks. Furthermore, CaCIPK1 expression levels changed in response to various stresses, including exposure to P. capsici, abiotic stress, and phytohormones. Knockdown of CaCIPK1 decreased the resistance of pepper against P. capsici, and changed the expression of defense related genes and root activity. Transient expression of CaCIPK1 in pepper leaves enhanced H 2 O 2 accumulation and cell death. In brief, our study establishes a basic foundation for further research on the function of CaCBL and CaCIPK genes, and report a preliminarily exploration of the role of CaCIPK1 in pepper's resistance to P. capsici. Further investigations are required to reveal the mechanism by which CaCIPK1 regulates resistance against P. capsici in pepper.

Genome-wide identification of CBL and CIPK genes in pepper
The proteome of pepper was downloaded from the Pepper Genome Database (CM334, http://peppergenome. snu.ac.kr/) and Zunla-1 (http://peppersequence.genomics.cn/). CBLs contain four unique EF-hands, and the CIPKs have a highly conserved protein kinase domain and NAF domain. The HMM profile of EF-hand (PF00036) and NAF (PF03822) were obtained from the Pfam (http://pfam.sanger.ac.uk/) protein family database. These domains were used as queries to search the pepper genome database with the BLASTP program (Evalue ≤1.0E-3) [73]. All candidate CaCBLs were submitted to InterProScan (http://www.ebi.ac.uk/Tools/Inter-ProScan) and SMART (http://smart.embl-heidelberg.de) to ensure the presence of the four EF-hand domains, and the same method was used to ensure CaCIPKs contained the Pkinase domain (IPR000719) and the NAF domain (IPR004041). Sequences were aligned with ClustalX (version 2.1) (http://www.clustal.org) to ensure that the CBL and CIPK genes that were identified in the analysis aligned to the CM334 and Zunla-1 genome sequences. When the sequences were different in CM334 and Zunla-1 databases, we designed the two sets of primers that were specific to the gene of interest in the different strains, and the PCR products were used to clone the gene. The sequences were aligned with ClustalX to identify the sequence of the gene of interest in cultivar AA3. The deduced amino acid and CDS sequences, theoretical isoelectric point (pI), instability index (with a value < 40 regarded as stable) [74] and protein molecular weight (MW) were analyzed by ExPASY (https://web.expasy.org/translate/; https://web. expasy.org/ protparam/). The WoLF PSORT program (http://wolfpsort.org/) was used to predict the subcellular localizations. Nomenclature of the CaCBL and CaCIPK genes were based on their chromosomal order.

Phylogenetic analysis
The full-length amino acid sequences of CBL and CIPK protein from Capsicum annuum, Arabidopsis thaliana, Manihot esculenta, Oryza sativa, Brassica napus, Populus trichocarpa, and Triticum aestivum, were aligned as an unrooted neighbor-joining phylogenetic tree using MEGA-X [75] with the bootstrap test replicated 1000 times. The classification of CaCBLs and CaCIPKs were based on previous research in Arabidopsis thaliana and Populus trichocarpa [50] and had high bootstrap values (> 50). The full-length amino acid sequences of genes from the species listed above were acquired from NCBI databases (http://www.ncbi.nlm.nih.gov/), Plant Genome Resource (https://phytozome.jgi.doe.gov/pz/portal.html), and TAIR database (https://www.arabidopsis.org/index. jsp). The sequences are displayed in Additional file 4.

Sequence analysis
The Multiple Sequence Alignment was executed using ClustalX to detect conserved domains. The position and number of introns in CaCBL and CaCIPK were visualized using Gene Structure Display Server (GSDS, http://gsds. cbi.pku.edu.cn/index.php) [76]. The conserved motifs of CaCBLs and CaCIPKs were identified and analyzed using protein sequences in the MEME online tool (http:// meme-suite.org/tools/meme). The site distribution was selected as any number of repetitions, the optimum width of motifs ranged from 10 to 50 (10 to 200 for CIPK), and the maximum numbers of motifs were identified as 6 for CaCBLs and 10 for CaCIPKs. Motifs with position pvalues less than 0.0001 are shown.

Chromosomal location and gene duplication
The chromosomal location of CaCBL and CaCIPK genes were identified using MapDraw [77]. Tandem duplications were defined as adjacent homologous genes on the same chromosome with a distance of less than 50-kb [78]. If they were paralogs located on duplicated chromosomal blocks, they were defined as a segmental duplication event [79]. The non-synonymous substitutions (Ka) and synonymous substitutions (Ks) were calculated by MEGA-X and DnaSP v6 [75,80]. The divergence time (Mya, million years ago) was calculated as T = Ks / (2 × 6.1 × 10 − 9 ) × 10 − 6 [81].

Prediction of protein-protein interaction network
The protein-protein interaction relationships were tested to establish the genome-wide regulation network. Since there were no references about the interaction of CBL and CIPK proteins in pepper, homologous genes from Arabidopsis was used to predict the protein-protein interaction network for CBLs and CIPKs from pepper. First, CBL and CIPK proteins from Arabidopsis were analyzed using Arabidopsis Interactions Viewer (http://bar. utoronto.ca/interactions/cgi-bin/arabidopsis_interac tions_viewer.cgi). The homologs were identified in pepper, and the corresponding interaction network was created by Cytoscape 3.6.0 (National Institute of General Medical Sciences, MD, USA) [82].

Subcellular localization
The full-length cDNA sequences of CaCIPK1, CaCIPK5, CaCIPK7, CaCIPK15, and CaCIPK20 were cloned from the root tissue of AA3 and inserted into the pVBG2307 vector [84], which was modified with the green fluorescent protein (GFP) under the control of the 35S promoter. Constructs were introduced into the Agrobacterium strain GV3101. Agrobacterium cultures were grown overnight, and resuspended in 10 mM MES (pH 5.7) with 400 mM acetosyringone (3,5-dimethoxy-4′-hydroxy-acetophenone). Subcellular localization was observed following methods described by Wydro et al. [85]. The infiltrated Nicotiana benthamiana leaves were imaged 2 days after agroinfiltration using the OLYMPUS BX63 automated fluorescence microscope (OLYMPUS Corporation, Tokyo, Japan).

Plant materials, RNA extraction and quantitative RT-PCR
The pepper cultivar AA3 was obtained from the College of Horticulture, Northwest A&F University. AA3 is compatible with the HX-9 strain of P. capsici (virulent) and incompatible with the PC strain (avirulent). The plants were grown on soil and vermiculite (1:1), and cultivated in growth chambers (24/20°C day/night temperature and 16/8 h day/night photoperiod). When plants reached the 6-8 true leaves stage, zoospore suspension of P. capsici was inoculated using the root drenching method as described previously [86]. Root samples were collected from treated (P. capsici) and control (treated with sterile water) plants, and collected at 0 and 12 hpi and immediately stored at − 80°C. To induce NaCl and mannitol stress, seedlings at 6-8 true leaves stage were prehydroponically cultivated in sterile water for 2 days, and then treated with NaCl (200 mM) and mannitol (300 mM) hydroponically. Root samples were collected at 0 and 6 hpt [84]. Root, stem, leaf, flower, green fruits and red fruits were collected from normal AA3 plants grown in soil, frozen in liquid nitrogen and stored at − 80°C for tissue-specific experiments.
Total RNA was extracted using the Trizol Reagent (Invitrogen, Carlsbad, CA), and reverse-transcribed using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China). Primer Premier 5.0 was used to design primer pairs against CaCBL and CaCIPK genes for Quantitative Real-Time PCR analysis. The primer's specificity was tested by NCBI Primer BLAST (https://www.ncbi.nlm.nih. gov/tools/primer-blast/index.cgi) and checked by electrophoresis in 1.5% (w/v) agarose gel. All the primers used in this study are listed in Additional file 15. Actin was used as the reference gene [87]. Quantitative real-time PCR (qRT-PCR) was used to detect expression levels by SYBR Green Supermix (Takara, Dalian, China) on IQ5.0 Bio-Rad iCycler thermocycler (Bio-Rad, Hercules, CA, USA). All experiments included three independent biological replicates. The relative expression levels of pepper CBL and CIPK genes were calculated using the comparative 2 −ΔΔCT method [88]. After normalizing dates by log2, a heatmap was drawn by Multi experiment viewer (MeV, http://www.tm4.org/mev.html).

Expression profile of CaCIPK1
The pepper line AA3 was grown to the 6-8 true leaf stage and sprayed with 5 mM SA, 50 μM MeJA, 0.57 μM ABA and 10 mM ETH solutions to detect changes in the expression level of CaCIPK1. Control plants were treated with sterile water. Plants that were at the same growth stage were placed at 4°C and 42°C for cold and heat stress, and leaves were collected at 0, 3, 6, 12, and 24 hpt. For CaCl 2 stress, plants were sprayed with 0, 1, 10, 50, 100 mM CaCl 2 and leaves were collected at 6 hpt. The samples were frozen in liquid nitrogen and stored at − 80°C for RNA extraction.

Virus-induced gene silencing (VIGS) of CaCIPK1 in pepper
To construct the tobacco rattle virus (TRV) vector, primers for CaCIPK1 were designed by Sol Genomics Network (http://vigs.solgenomics.net/). A 245 bp fragment of the CaCIPK1 ORF was amplified using the specific primer pair vigs-F and vigs-R from cDNA isolated from the AA3 root grown under normal conditions (Additional file 12). The fragment was inserted into the original TRV vector for gene silencing. Agrobacterium strain GV3101 harboring pTRV1 was mixed at a 1:1 ratio with pTRV2 (negative control), pTRV2:CaPDS (silencing the phytoene desaturase gene, which induces bleaching) and pTRV2:CaCIPK1. The mixture was injected into fully extended cotyledons leaves [89]. Plants were cultivated in growth chambers (22/18°C day/ night temperature and 16/8 h day/night photoperiod). After 4 weeks, when the positive control (pTRV2:CaPDS) showed the photobleaching phenotype, leaf samples were collected from pTRV2:CaCIPK1 and pTRV2 to test the silencing efficiency by qRT-PCR. pTRV2:CaCIPK1 and control plants were treated with incompatible P. capsici (PC strain), and the third to fifth leaf from top of the control and CaCIPK1 silenced plants were picked. Leaves were washed with sterile water and injected with a 20 μL zoospore suspension (1 × 10 5 zoospores mL − 1 ) of incompatible P. capsici. Leaves were then moved into petri dishes and sealed with parafilm [86].