**3. Discussion**

Chitin-binding protein (CBP) is an important biotic and abiotic resistance responsive multigene family in plant [20,32,33,43], which play an important role to enhance resistance against stresses in different crops [15]. Chitin-binding proteins are well characterized class of PR proteins [44] which are speculated to be involved in the production of proline due to proline and glycine-rich region [20]. The number of chitin-binding protein genes varies in different plant species. Formerly, 24 chitins in *Arabidopsis thaliana*, 37 in *Oryza sativa* and 17 chitin genes in *Saccharum officinarum* were reported [20,32].

In the past, no comprehensive study has been conducted on genome-wide identification and characterization of chitin-binding proteins in pepper. Therefore, in the current study, we retrieved 16 CaChi genes from "CM334" and "Zunla-1" databases of pepper genome. Previously chitins were also found in different plant species [15,20,32,45]. The structural analysis revealed that out of 16 CaChi, eight (50%) CaChi contained introns in which seven genes (43.75%) had only one intron while *CaChiIV1* contain two introns (Figure 3c). Consequently, previously studies on the chitin genes in brassica and banana showed contrasting results [33,46]. The reason may be due to expansion in CaChi family in pepper plant and it may be concluded that the CaChi have undergone diverse gene structure changes during evolution process. The ORFs analysis revealed that the amino acid (aa) sequences ranged from 85 aa (*CaChiVI1*) to 331 aa (*CaChiI1*). The subcellular location of all the CaChi in pepper were predicted, and found that they exist in chloroplast, extracellular region, nucleus, cytoplasmic and vacuolar locations (Table 1). Nishizawa et al. (1999) and Collingel et al. (1993) [47,48] also identified chitins genes in *Oryza sativa*, *Pisum sativum* and *Hordeum vulgare* and described their subcellular localization in vacuole and extracellular.

Previous studies revealed that the nomenclature of the chitins gene family had seven (I–VII) distinct classes [4,15,33]. Therefore, in our study, we also classified CaChi genes with previous criteria into four classes (classes I, III, IV and VI). Parallel, results were also obtained by Backiyarani et al. (2015) [46]. Thus, there is strong possibility that the classification may be due to the homology of the protein sequences and the presence of core conserved domain and probably also in function. The phylogenetic analysis showed that CaChi genes can be divided into four distinct classes: *CaChiI*, *CaChiIII*, *CaChiIV* and *CaChiVI*. It was noticed that sequences contain the glyco\_hydro\_19 super family, chitinase\_glyco\_hydro\_19 and barwin domains, and they were classified into classes I, IV and VI, respectively (Figure 2). Typical CaChi exhibited higher similarity in the sequence of their conserved domains but an obvious diversity in gene structure and protein size (Figure 1 and Table S2), implying an evolutionary relationship between CaChi genes. It shows that CaChi share a common ancestor and some similar biological functions [32,49].

The chromosomal locations exposed that CaChi were detected on seven diverse chromosomes of *Capsicum annuum*, the highest number of CaChi genes (5) were found on chromosome 3 followed by 7 which had four CaChi. In the duplication and transposition analysis of CaChi, we obtained five clusters of segmental duplication and two genes tandemly duplicated on chromosome 8 (Figure 4). Similarly, our findings are also supported by Cannon et al. (2004) and Backiyarani et al. (2015) [46,50], who also found that tandem and segmental duplication events in *Musa* spp. Therefore, the expansion of a gene family might be segmental and tandem duplication or transposition is the core evolutionary tools [50]. Thus, comparing with tandem duplication and transposition, segmental duplication happens more frequently because of polyploidy in utmost plants, which conserve numerous duplicated chromosomal blocks in their genomes [50]. These conclusions suggest that the pepper CaChi genes endured a complicated evolutionary history during the gene expansion and functional divergence.

Tissue specific expression is a common characteristic of the genes of a certain protein family in plants, which often reflects the functional collaborative and/or differences of the family members [51]. The preceding studies also shed light on developmentally regulated plant chitinases, clarifying their role in the specific physiological processes [52]. To further clarify the possible functions of the CaChi in the growth and development of *Capsicum annuum*, the transcription profiles of CaChi were studied through qRT-qPCR in different tissues. The results showed that mostly CaChi genes exhibited the highest expression during seed formation, followed by the stem, flower, leaf, root and green-fruit (Figure 10B), while the lowest expression was detected in red-fruit. This is similar to previous studies on *Brassica*, where comparatively higher expression levels were observed in flower followed by stem, leaves and roots [33]. Furthermore, Su et al. (2015) [53] also detected chitin gene expression in sugarcane and found high expression in stem pit compared with leaf, and stem epidermis. The transcriptomic

analysis as shown in Figure 10A also showed the same expressions in most of the tested tissues but in some case, the transcriptional level is changes may be because of different cultivar was used and some other environmental factors may be involved. The pepper CaChi were expressed in an organ-specific way, suggesting the probable functions in distinct biological processes. CaChi genes suggesting their specific roles in heading stage implying its vital roles in growth regulation and stress response in pepper plant.

Plant diseases activated by fungal pathogens are one of the main concerns and promote defense to a plant pathogen is a difficult mechanism, which includes the triggering of several immune responses [1]. Several resistance genes, including pathogenesis related proteins, have been isolated and were used to improve the defense to different disease in plants. The pathogenesis related proteins are present during hypersensitive response to pathogens of bacteria, virus, fungi and are responsible for the induced resistance in plants. Although it is found that PR proteins not only develop resistance in plants but also play role against pathogen as well as abiotic stresses in susceptible condition [54,55]. In nature, the PR proteins are widely found in the form of chitins genes and play crucial role in plant defense system against the pathogen attack. Multiple antifungal chitinases (CH1, CH2 and CH3) have been reported in various crops such as, from *Sorghum bicolor* [15,56]. Recently, wheat class VII chitinases showed broad-spectrum antifungal activity against *Alternaria* sp., *Sarocladium oryzae*, *Fusarium sp.*, *C. falcatum*, *Pestalotia theae* and *Rhizoctonia solani*. The partial mRNA sequences of the chitins *ScChiB1* were amplified from both cultivars (red rot-compatible and incompatible) of sugarcane [57]. In addition, chitin family genes were found to be involved in proline synthesis and primarily associated with defense and resistance against pathogens [32,49], and Western blotting study revealed greater and faster accumulation of chitin genes in a red rot-resistant cultivar [58].

The cellular predicted function of CaChi proteins indicated that they had mainly involved in defense response to fungi, bacterium infection, cell wall macromolecule catabolic process, chitin catabolic process and also in other stresses. The molecular functions of chitinase activities and chitin binding were predicted during GO analysis (Figure 6). Our results correlate with the results of Kovacs et al. (2013) and Rahul et al. (2013) [21,57], who find the functions of chitins genes in *Musa* spp. and *Saccharum officinarum*, respectively. In the current research work, during *Phytophthora capsici* inoculation (0–7 hpi), the transcription level of at least 11 CaChi (*CaChiI2*, *CaChiI3*, *CaChiIII1*, *CaChiIII2*, *CaChiIII4*, *CaChiIII6*, *CaChiIII7*, *CaChiIV1*, *CaChiVI1*, *CaChiVI2* and *CaChiVI3*) were highly induced by both strains (Figure 7). Moreover, the transcript levels of *CaChiI2*, *CaChiIII4* and *CaChiIII7* were higher in response to the PC-strain, whereas *CaChiI3*, *CaChiIII2*, *CaChiIV1*, *CaChiIV2*, *CaChiVI2* and *CaChiVI3* showed higher expression after inoculation with the HX-9 strain versus the PC-strain. These results suggest that CaChi genes are pathogen-inducible and perhaps also participate in immune system of pepper plant, thus helping in disease resistance. It has been found that CaChi are responsive to several biotic stresses and their transcripts were greatly increased (Figure 7). An earlier study also showed that chitins genes were induced by a fungus via *Phytophthora capsici* [49], whereas a class III sugarcane chitinase gene (*ScChi*) was exposed to be induced by *S. scitamineum* [59]. The expression of the *CABPR1* gene in pepper was higher in the virulent strain interaction versus the avirulent interaction [60]. However, in contrast to our previous studies [61], it was found that the expression of most *CaSBPs* was comparatively greater in the avirulent interaction than in the compatible interaction. Some other studies have revealed that the expression of oxysterol-binding protein gene (*CanOBP*) and a novel peroxidase gene (*CanPOD*) were higher in the incompatible interaction [62,63] and the defense-related genes such as b-1, 3-glucanase gene (*CABGLU*), disease-associated protein gene (*CABPR1*), and peroxidase gene (*CAPO1*) were expressed in a similar pattern, after the inoculation of virulent and avirulent strains of *Phytophthora capsici* on the roots of pepper as reported by Wang et al. (2013) [62]. The differences in the expression patterns of CaChi and other defense-related genes might be because of dissimilarities in the inoculation of *Phytophthora capsici* strains, duration of infection, cultivar or the variation in their compatibility systems.

Earlier studies have shown that chitins genes were expressed in different patterns in response to various abiotic stresses [16,59,64]. Based on GO analysis, CaChi genes imply a role in different stresses, in support of our study. Kumar et al. (2017) [65] also investigated the *OsWRKY71* gene enhanced cold tolerance and mainly involved in metabolic as well as in regulatory pathways in rice, while Eroglu and Aksoy (2017) [66] studied COP9 response under Fe deficiency in Arabidopsis. Hence, we extended our study to investigate the expression analysis of eight representative CaChi after salt (NaCl), cold (6 ◦C) and drought (mannitol) stresses (Figure 8). The results revealed that *CaChiI3*, *CaChiIII2*, and *CaChiIVI* genes showed significant response to mannitol, while *CaChiIII4*, *CaChiIII7*, *CaChiIVI CaChiIV2* and *CaChiV12* exhibited greater response to cold stress. Therefore, we assumed that chilling induced intracellular Ca2+ overload may enhance the ROS production which is key component response to chilling stress. The *CaChiIII6*, *CaChiIII7* and *CaChiIVI* genes expression was maximum at 6 hpt salt stress treatment. These results are supported by Yin et al. (2014) [67] who studied *CaAQP* gene in pepper under salt stress and conform the highest expression at 4 h salt stress but in latter hours the expression was dramatically reduced. The reason for downregulation of permeability of membrane is it results in limitation of water loss from vacuole. These effects propose that the dissimilar pepper chitin-binding protein have separate functions in response to numerous environmental stresses. However, as discussed above, different CaChi exhibit strong spatiotemporal and tissue-specific expressions, demonstrating an obvious collaborative and/or divergence in both biological roles and evolutionary relationship of the chitin-binding protein family genes in pepper.

Chitin-binding protein in plants are responsive for the certain level of abiotic (low temperature, drought, heavy metals and salt) stresses and plant hormones [1,58,59]. Previous reports show that MeJA. SA and ethylene are involved in signal compounds inducing two kinds of defense such as for induced systemic resistance (ISR) and systemic acquired resistance (SAR) [68]. The basic defense to biotrophic pathogens is mediated by SA [3]. In plant responses to environmental cues, MeJA plays the fastest role in resistance reaction via signal molecule reaction center and the genes which are related to MeJA showed upregulation, causing hyper accumulation of MeJA under biotic and abiotic stresses [69]. Several hormonal responsive elements were located in the promoter regions of CaChi, e.g. for MeJA (CGTCA-motif) [70] and SA (TCA-element) [71]. In light of this evidence, pepper plants were exposed to SA and MeJA stresses and their effects on the expression levels were investigated. The expression pattern of CaChi could be differentially regulated by MeJA, ABA and SA (Figure 9). External application of SA lead in an increased accumulation of *CaChiI1*, *CaChiIII2*, *CaChiIII6*, *CaChiIV1*, *CaChiIV2* and *CaChiIV2* expression, *CaChiIII7*, *CaChiIV1* and *CaChiVI2* showed maximum expression level against MeJA and *CaChiIII2*, *CaChiIII6*, *CaChiIII7* and *CaChiV12* exhibit increased transcription by ABA application. It should be noticed that the expression profiles of the members of CaChi have distinct characteristics approach in response to these hormone treatments. For example, Guo et al. (2013) [72] reported that the ABA responsive genes in pepper reduce cold stress injuries and help plants to combat unfavorable environment. Similarly, the evaluated level of these hormones may enhance the antioxidant activity also reduce the accumulation of reactive oxygen species (ROS) and thus play a crucial role in signaling pathways and ultimately in plant defense system.

For functional characterization of *CaChiIV1* gene initially, we searched the dataset of *cis-*regulatory elements in the promoter region and then we successfully knocked them down in pepper plant. Further, we performed an expression analysis under salt stress condition (Figure 11A). The results displayed significant response to salt stress as well as the defense related gene when compared to control plants. Similar to our results, rice *OsDIRs* exhibit greater response to salt stress as compared with mock-treated control seedlings [73]. Wu et al. (2009) [74] also reported similar finding during their studies on dirigent protein gene from the resurrection plant *Boea hygrometrica*. In current study, we also performed the detached leaf assay of the *CaChiIV1*-silenced gene with control plants treated with salt stress. The silenced plants showed the reduction in chlorophyll content, suggesting that due to knock down of *CaChiIV1* gene the pepper leaves are more susceptible to the NaCl stress (Figure 12). In pepper, the dehydrin *CaDHN1* silenced plants showed decrease in chlorophyll contents after three

days of salt treatments [75]. Thus, the degree of leaf senescence in *CaChiIV1*-silenced plants are greater than control. Furthermore, the TTC reductase activity in the roots of pepper plant was measured after NaCl stress in both silenced and control plant with a duration of 0–24 h. the significant reduction in root activity was seen in silenced than control plants (Figure 11D). The root activity was significantly reduced of the silenced plants in avirulent strain of *P. capsica* than virulent strain [62]. Moreover, the *CaPTI1* gene in pepper also showed the significant differences in root activity [76]. The reason plants are susceptible to salt stress is that it causes severe injury in root tips and may lead to reduction in root activity. In conclusion, the *CaChiIV1* gene demonstrated a crucial role in biological processes and functionally involved in NaCl stress and defense response in pepper plant. three days of salt treatments [75]. Thus, the degree of leaf senescence in *CaChiIV1*-silenced plants are greater than control. Furthermore, the TTC reductase activity in the roots of pepper plant was measured after NaCl stress in both silenced and control plant with a duration of 0–24 h. the significant reduction in root activity was seen in silenced than control plants (Figure 11D). The root activity was significantly reduced of the silenced plants in avirulent strain of *P. capsica* than virulent strain [62]. Moreover, the *CaPTI1* gene in pepper also showed the significant differences in root activity [76]. The reason plants are susceptible to salt stress is that it causes severe injury in root tips and may lead to reduction in root activity. In conclusion, the *CaChiIV1* gene demonstrated a crucial role in biological processes and functionally involved in NaCl stress and defense response in pepper plant.

*Int. J. Mol. Sci.* **2018**, *19*, 2216 17 of 26

to knock down of *CaChiIV1* gene the pepper leaves are more susceptible to the NaCl stress (Figure 12). In pepper, the dehydrin *CaDHN1* silenced plants showed decrease in chlorophyll contents after

**Figure 12.** The *CaChiIV1*-silenced pepper plants reduced resistance to NaCl stress. Leaf discs phenotypes (0.5 cm in diameter) of the *TRV2:CaChiIV1* and TRV:00 plants in response to 0 mM, 100 mM and 300 mM NaCl stress after 48 h. **Figure 12.** The *CaChiIV1*-silenced pepper plants reduced resistance to NaCl stress. Leaf discs phenotypes (0.5 cm in diameter) of the *TRV2:CaChiIV1* and TRV:00 plants in response to 0 mM, 100 mM and 300 mM NaCl stress after 48 h.

#### **4. Materials and Methods 4. Materials and Methods**

#### *4.1. Identification and Sequence Analysis of CaChi Genes Family in Pepper 4.1. Identification and Sequence Analysis of CaChi Genes Family in Pepper*

To identify the CaChi family members based on the conserved domain, accession No. "PF00187.17" was collected from Pfam database (http://pfam.xfam.org/) as described in our previous study [61,77]. To further authenticate the CaChi family members, the CaChi were aligned with DNAMAN to cross check in both CM334 (Available online: http://peppergenome.snu.ac.kr/download.php) [40] and Zunla-1 (Available online: http://peppersequence.genomics.cn/) [78] databases of pepper, while the obtained sequences were from the latest versions, i.e., v1.55 and v2.0, respectively. Furthermore, gene-specific primer pairs (Table S1) were designed by using Primer Premier 6.0 (Premier Biosoft International, Redwood City, To identify the CaChi family members based on the conserved domain, accession No. "PF00187.17" was collected from Pfam database (http://pfam.xfam.org/) as described in our previous study [61,77]. To further authenticate the CaChi family members, the CaChi were aligned with DNAMAN to cross check in both CM334 (Available online: http://peppergenome.snu.ac.kr/download.php) [40] and Zunla-1 (Available online: http://peppersequence.genomics.cn/) [78] databases of pepper, while the obtained sequences were from the latest versions, i.e., v1.55 and v2.0, respectively. Furthermore, gene-specific primer pairs (Table S1) were designed by using Primer Premier 6.0 (Premier Biosoft International, Redwood City, CA, USA) to amplify the different target regions.
