The CaChiVI2 Gene of Capsicum annuum L. Confers Resistance Against Heat Stress and Infection of Phytophthora capsici

Extreme environmental conditions seriously affect crop growth and development, resulting in substantial reduction in yield and quality. However, chitin-binding proteins (CBP) family member CaChiVI2 plays a crucial role in eliminating the impact of adverse environmental conditions, such as cold and salt stress. Here, for the first time it was discovered that CaChiVI2 (Capana08g001237) gene of pepper (Capsicum annuum L.) had a role in resistance to heat stress and physiological processes. The full-length open-reading frame (ORF) of CaChiVI2 (606-bp, encoding 201-amino acids), was cloned into TRV2:CaChiVI2 vector for silencing. The CaChiVI2 gene carries heat shock elements (HSE, AAAAAATTTC) in the upstream region, and thereby shows sensitivity to heat stress at the transcriptional level. The silencing effect of CaChiVI2 in pepper resulted in increased susceptibility to heat and Phytophthora capsici infection. This was evident from the severe symptoms on leaves, the increase in superoxide (O2–) and hydrogen peroxide (H2O2) accumulation, higher malondialdehyde (MDA), relative electrolyte leakage (REL) and lower proline contents compared with control plants. Furthermore, the transcript level of other resistance responsive genes was also altered. In addition, the CaChiIV2-overexpression in Arabidopsis thaliana showed mild heat and drought stress symptoms and increased transcript level of a defense-related gene (AtHSA32), indicating its role in the co-regulation network of the plant. The CaChiVI2-overexpressed plants also showed a decrease in MDA contents and an increase in antioxidant enzyme activity and proline accumulation. In conclusion, the results suggest that CaChiVI2 gene plays a decisive role in heat and drought stress tolerance, as well as, provides resistance against P. capsici by reducing the accumulation of reactive oxygen species (ROS) and modulating the expression of defense-related genes. The outcomes obtained here suggest that further studies should be conducted on plants adaptation mechanisms in variable environments.


INTRODUCTION
In vivo cultivated crops inevitably suffer adverse effects of biotic (pathogens, diseases, etc.) and abiotic (temperature, heavy metals, salinity, and drought) stresses Kilasi et al., 2018). Consequently, there are heavy losses in both quality and quantity with estimated yield decreases up to 50% (Maxmen, 2013;Wang et al., 2013b). Being sessile in nature, plants face a number of unfavorable environmental conditions. Though they have evolved an array of sophisticated mechanisms to combat these stresses, the combination of stresses may adversely affect plant physiology and productivity (Obata et al., 2015). Over the period of time, plants have evolved various pathways to adapt to the changing environmental conditions in order to survive and reproduce. These have been studied extensively with considerable emphasis on individual stresses at the molecular levels (Ramegowda and Senthil-kumar, 2015). Besides, in plants undergoing stress, many proteins are denatured resulting in loss of natural functions causing serious losses in yield and quality (Li et al., 2010;Khurana et al., 2013;Zhai et al., 2017). When plants are in severe stress, they reprogramme a number of biochemical reactions. For example, reduction of proteins that can bind and disassemble the aggregations for refolding to form native proteins and as a result avoid damages due to aggregations (Shan et al., 2007;Lambert et al., 2011;Ruibal et al., 2013;Personat et al., 2014;Li Z. et al., 2016). Additionally, the mechanism of plant adaptation is initiated by the combined effect of reactive oxygen species (ROS) and stress-induced signaling pathways, which are activated together and provide an immediate response to various external stimuli (Sewelam et al., 2016). Another elegant response to different environmental stimuli, such as high temperature, drought, and pathogen infection, is that accompanied by secondary messenger signaling pathways. These pathways are activated by the imbalance of intracellular concentrations of various molecular compounds (Ranty et al., 2016). Abscisic acid (ABA) is linked with the plants responses to biotic and abiotic stresses through regulation of stomatal apertures and up-regulation of defense-related genes (Lim et al., 2015;Sah et al., 2016). Sets of defense-related genes against heat shock and drought stress, i.e., HSP20, PR-2, and CBP gene families, are reported to have a role in effector-triggered immunity (ETI), relying on intensity and time of the interacting signaling components (Tsuda and Katagiri, 2010;Thomma et al., 2011). Among the defenserelated genes, chitin-binding protein genes family contributes significantly in plants adaptability and tolerance (Hejgaard et al., 1992;Ali et al., 2018). There are some CBP-encoded enzymes, which show high response during environmental stresses such as cold and high salt concentration. Furthermore, they play their contributing role in physiological processes of plants, such as ethylene production and embryogenesis (Fukamizo et al., 2003;Hamid et al., 2013). Previously, we identified 16 putative genes of CBP family in pepper plant. The transcriptomic analysis revealed that CaChiVI2 gene had the most remarkable motif "AAAAAATTTC." It was heat stress responsive protein highly induced by ABA hormone .
Pepper (Capsicum annuum L.) is a profitable crop extensively used as a green vegetable, as a spice in food, and as an innate source of natural coloring Baoling, 2018). Pepper is thermophilic in nature, i.e., its growth and development is susceptible to extreme temperature and water deficiency (Guo et al., 2014). Pepper crop faces a number of challenges during cultivation period, especially in the summer, when water is deficient and temperature is too high. Such conditions favor Phytophthora blight disease, caused by the oomycete Phytophthora capsici. The infection significantly inhibits pollination, fertilization, and seed setting (Erickson and Markhart, 2002;Pagamas and Nawata, 2008). The severity of the P. capsici infection can have a devastating effect on plant morphology and growth. These include symptoms such as damping-off, leaf browning, senescence wilting, and dwarfing that cause plant death ultimately (Hausbeck and Lamour, 2004;Sy et al., 2005;Lamour et al., 2012;Zhang H.-X. et al., 2016).
Thus, it was essential to investigate this issue. Due to lack of conclusive evidence, it was difficult to prove that CaChiVI2 gene was potentially involved in heat tolerance, drought stress, and P. capsici infection. Therefore, we extended our work to characterize CaChiVI2 in pepper plant for the mentioned purpose. The gene is known for its role in regulating stress responses. The purpose of this study was to generate a meaningful background for further research through silencing of the target gene in pepper and over-expressing in Arabidopsis via transgenic approaches. Moreover, we were also interested in the physiological responses and changes induced by heat stress and disease susceptibility or tolerance through inoculation with P. capsici.

Plant Growth Conditions
Pepper (Capsicum annuum L.) cultivar AA3 maintained in Vegetable Plant Biotechnology and Germplasm Innovation lab, Northwest A&F University-China was studied in the present work. The growth conditions for pepper seedlings were 22/18 • C (day/night) temperature with a 16 h photoperiod (i.e., 16 h light and 8 h dark cycle) and 65% relative humidity. Arabidopsis ecotype Columbia-0 (Col-0) was also cultivated at 25/20 • C (day/night) temperature and the above-mentioned photoperiod and humidity conditions for overexpression of CaChiVI2-gene.

Sequence Alignment and in silico Analysis
The protein sequences of the chitin-binding protein family (CBP) were aligned by ClustalW 1 . The phylogenetic tree was built using iTOL 2 (Letunic and Bork, 2016). Cis-regulatory elements (1500bp upstream region) were searched by PlantCARE online server 3 (Lescot et al., 2002). Publicly available transcriptomic data of root and leaf for pepper cultivar "Zunla" were obtained from the online server 4 , and the genomic database was generated by following Liu Z. et al. (2017) and Yu et al. (2017) method and the data were presented in line-graphs (CaChiVI2 sequence shown in Supplementary Table S1).

Quantitative Real-Time PCR Analysis
The total-RNA was extracted as explained in our recently published papers Khan et al., 2018). The first chain was synthesized by the Primer Script TM Kit (TaKaRa, Dalian, China). The iQ5.0 Bio-Rad iCycler thermocycler (Bio-Rad, Hercules, CA, United States) was used for qRT-PCR, and SYBRR Premix Ex TaqTM II (TaKaRa) was used for the reaction. Pepper ubiquitin-binding gene CaUBI3 (Wan et al., 2011) and Arabidopsis Atactin2 was used as a reference. Relative gene expression levels were calculated according to the comparative threshold (2 − CT ) technique (Schmittgen and Livak, 2008;Muhammad et al., 2018). All primer pairs (Supplementary Table S2) used for qRT-PCR were designed by NCBI Primer-BLAST. The expression levels were normalized and presented the mean and standard deviation (+SD) of data were obtained from three independent biological experiments with three replicates.

Protein Localization of CaChiVI2
The transient transformation technique was performed using Nicotiana benthamiana epidermal cells to detect the protein localization of target gene (Jin et al., 2019;Ma et al., 2019). The ORF fragment (1244-bp) of CaChiVI2 (primers in Supplementary Table S3) was inserted into the pVBG2307 vector regulated by CaMV 35S promoter and then transformed into GV3101 (Agrobacterium tumefaciens). The pVBG2307:GFP vector without the CaChiVI2 gene was used as a control. The Agrobacterium cells were infiltrated into 4-weeks-old tobacco plant leaves . In a growth chamber, agroinfiltrated plants were transferred for 2-3 days. OLYMPUS BX63 automated fluorescence microscope was used for the determination of epidermal cells (Olympus, Tokyo, Japan).

Virus Induce Gene Silencing Assay of CaChiVI2
The virus induce gene silencing (VIGS) technique was performed for the knock-down of pepper CaChiVI2 gene, following the same approach explained by Liu et al. (2016). While for the construction of TRV2:CaChiVI2 vector, 230-bp CDS fragment using the specific primer pair (Supplementary Table S4) of CaChiVI2 gene with restriction enzymes sites EcoRI and XhoI was amplified through PCR. CaChiVI2 was cloned into the TRV2 vector, while TRV2:00 vector was used as a negative control, whereas the TRV2:CaPDS (phytoene desaturase gene) was used as a positive control. Subsequently, the vector was used to transform into an Agrobacterium tumefaciens strain (GV3101) using the freeze-thaw method (Wang et al., 2013c). Consequently, the TRV2:00, TRV2:CaPDS and TRV2:CaChiVI2 were activated with OD 600 = 1.0. Afterward, the suspensions were infiltrated into the fully extended cotyledons leaves of pepper plants by using a 1.0 mL sterilized needleless syringe (Li et al., 2014;Feng et al., 2019). The infiltrated pepper plants were kept at 18-22 • C in the growth chamber, maintaining the 16h/8h light/dark photoperiod as mentioned by Wang et al. (2013a). The leaf samples of the control and CaChiVI2-silenced plants were collected after 45 days when the TRV2:CaPDS injected leaves exhibited photo-bleaching phenotype and the silencing efficiency was measured by qRT-PCR. For the precision of results, the experiment was performed with three independent biological replicates.

Generation of CaChiVI2 Overexpressed Arabidopsis Lines
The full-length of CaChiVI2 ORF fragment was cloned by cutting with restriction enzymes XbaI and KpnI. The amplified product from cDNA was recovered through a gene-specific primer pair (Supplementary Table S5) and further transferred into a pVBG2307 expression vector. For overexpression analysis, recombinant fusion vector was used to transform into Arabidopsis plants (ecotype Columbia-0, Col-0) via Agrobacterium tumefaciens strain GV3101 (Clough and Bent, 1998;Guo et al., 2014). Positive transgenic lines were selected on MS-medium containing 50 mmol/L kanamycin. The transgenic lines were further grown for homozygosity till T 3 generation. The T 3 seeds were used for further experimental treatment.

P. capsici Preparation and Inoculation
Phytophthora capsici (PC strain) was obtained from our laboratory and the technique used for its inoculation was similar as described in our previous studies Ali et al., 2019). The roots were sampled at different days of interval (0, 1, 2, 4, and 8 dpi), immediately frozen in liquid nitrogen and stored at −80 • C for further study. The authentication of data was checked by a repeat of three times for each treatment.

Stress Treatments and Samples Collection
To examine the transcript pattern of CBP family genes, pepper seedlings (8-weeks-old) at the stage of six to eight true leaves were used for basic thermo-tolerance treatment. Pepper seedlings were incubated at 45 • C and samples were collected at 0, 1, 3, 6, 12, and 24 h post-treatment. CaChiVI2-silenced and TRV2:00 (control) plants were used to analyze the functions of CaChiVI2 under heat and P. capsici inoculation. The seedlings were exposed to 45 • C for heat stress, and samples were collected at different intervals of 0, 0.5, 1, 3, 6, 12, and 24 h. During stress treatment, seedlings were watered to avoid drought stress. Heat treated pepper plant leaves were collected for the validation of MDA content, total chlorophyll content and relative electrolyte leakage (REL) analysis at 0, 12 and 24 h.
The CaChiVI2-overexpressed Arabidopsis lines (OE2, OE3, OE5, OE6, and OE7) were selected. T 3 homozygous lines (8days old seedlings) grown on MS-media were incubated at 45 • C to give heat stress for a period of 2 h. The seedlings were then shifted to growth chamber (22 • C) for 7 days to recover and the survival rate was recorded . For drought stress, seeds were grown on MS-medium containing 0, 100-and 200-mM mannitol and the root length was measured 8 days post sowing. Furthermore, 3-weeks-old CaChiVI2-overexpressed and wild-type Arabidopsis lines grown in soil-media were used for the analysis of heat and drought stress tolerance.
The Arabidopsis plants were incubated for a period of 16 h at 40 • C for heat stress treatment. Plants were irrigated frequently during treatment to avoid drought stress. Furthermore, for drought stress, water was withheld from the seedlings for 5days, while in control treatment, seedlings were provided with standard watering conditions. Leaf samples were collected for MDA, proline, and extraction of RNA. Randomly six separate seedlings were used for sample collection and then instantly frozen in liquid nitrogen and stored at −80 • C. The experiment was conducted with three independent biological replicates for the accuracy of data.

Physiological Attributes
To measure the contributing parameters of CaChiVI2-silenced pepper plants and CaChiVI2-overexpressed Arabidopsis plants, samples (0.5 g) were collected and finely grinded in liquid nitrogen. The malondialdehyde (MDA) content was measured through thiobarbituric acid (TBA) reaction method (De Vos et al., 1991). For this, 1.5 mL of the extract supernatant was mixed with 2 mL 0.6% (w/v) TBA solution dissolved in 5% (v/v) trichloroacetic acid (TCA) and heated in boiling water for 10 min. The supernatant was used for determination of MDA at 450 and 532 nm wavelength and subtracted from the absorbance at 600 nm. The activity of superoxide dismutase (SOD) was estimated by the inhibition of nitro-blue tetrazolium (NBT). The supernatant volume was illuminated at 4000 Lux for 20 min, then SOD activity was quantified spectrophotometrically at 560 nm. Control was determined in dark (Stewart and Bewley, 1980). Peroxidase (POD) activity was determined through guaiacol method (Bestwick et al., 1998). The reaction mixture used consisted of 50 mL 0.05 M phosphate buffer (pH7.8), 19 µL 30% H 2 O 2 (v/v) and 28 µL guaiacol. The enzyme extract (0.5 mL) was added and a total of 3.0 mL of the reaction mixture was placed into a cuvette. The increase in absorbance was recorded at 470 nm at 30 s intervals for 3 min. Proline estimation was done according to Bates et al. (1973). A 2 ml of aqueous extract was mixed with 2 ml of glacial acetic acid and 2 ml of acid ninhydrin reagent (1.25 g of ninhydrin, 30 ml of glacial acetic acid, and 20 ml of 6 M orthophosphoric acid) and heated at 100 • C for 30 min. After cooling, the reaction mixture was partitioned against toluene (4 ml) and the absorbance of the organic phase was firm at 520 nm. The resulting values were compared with a standard curve constructed using known amounts of proline (Sigma, St Louis, MO, United States).
Histochemical staining's were performed to detect superoxide radical (O 2 − ) and hydrogen peroxide (H 2 O 2 ) in leaves. The leaves were inserted in 0.1% nitro-blue tetrazolium (NBT) with 50 mM potassium phosphate buffer (pH 7.8) for O 2 − as described by Liu et al. (2012). For H 2 O 2 detection, 3,3 -diaminobenzidine (DAB) solution was used in agroinfiltrated leaves. The leaves were incubated in 1.0 mg/mL DAB-HCl solution at room temperature, covered in dark for 12 h. Then de-stained by boiling in 95% ethanol for 5 min until the brown H 2 O 2 spots on the leaves appeared (Thordal-Christensen et al., 1997;Kim et al., 2012). Relative electrolyte leakage (REL) was measured as per Feng et al. (2019), and the electrical conductivity percentage was calculated as REL (%) = C1/C2 × 100. For chlorophyll content, the readings were recorded on spectrophotometer after extracting into 80% (v/v) acetone (Arkus et al., 2005). Spectrophotometer (UV-1201 Shimadzu spectrophotometer, Japan) was used for all measurements.

Statistical Analyses
IBM SPSS Statistics 25, United States were used for the statistical analysis. Significance differences between individual treatments were further analyzed at P ≤ 0.05 through Duncan Multiple Range (DMR) test. The analyzed data were expressed as mean and standard deviation (±SD). Three individual experiments were performed, and the data set for each biological replicate was used separately for analysis. The data were plotted by GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, United States).

Cloning and in silico Analysis of Pepper CaChiVI2
As previously reported, the evolutionary relationship between CaChiVI2 and pepper CBP homologs in other species was based on phylogenetic analysis . The analysis revealed that pepper CBP could be classified into four main classes with potentially similar functions. Among all the chitinbinding protein (CBP) family genes, we identified a putative gene named CaChiVI2 (Capana08g001237) (Figure 1A), which is re-derived from 'Zunla' database. The CaChiVI2 was cloned using cDNA extracted from pepper leaves of AA3 pureline. The full-length CDS of CaChiVI2 cDNA consists of 606-bp and encodes 201 amino acids, while the genomic sequence contains 1203 nucleotides (Supplementary Table S6), including two exons and one intron . To investigate the possible cis-acting elements involved in the heat stimulation of defense-related genes, the 1.5 kb upstream region from the start codon (ATG) of all the CBP genes was analyzed with PlantCARE online server. The in silico analysis exhibited that heat stress elements (HSE) were available in the promoter region of 13 out of 16 members ( Figure 1B) while the highest number (4) of HSE was found in CaChiIV1 and CaChiVI2.
For further insight into the transcriptomic characteristics of CaChiVI2 in pepper roots and leaves under heat stress, we initially practiced in silico analysis from a publicly available transcriptomic database of pepper (Zunla cultivar) (Liu F. et al., 2017). The diagrams display the index of a transcript ranging from yellow to red (Figure 2A). The transcript level of CaChiVI2 in pepper revealed higher variance in distinct parts and at time intervals under heat stress, as demonstrated in Figures 2B,C. Moreover, the highest expression level (52.5) was noted in leaf tissue at 12 h interval followed by 35.5 at 6 h interval. In roots, the highest level (30.9) was recorded at 6 h interval. Overall, the in silico analysis revealed that expression in the leaf tissue was more elevated than the root.

Protein Localization of CaChiVI2
The ORF fragment of CaChiVI2 was recombined with the expression vector pVBG2307 that contained 35S promoter and reporter genes for green fluorescence protein (GFP). The pVBG2307:GFP and pVBG2307:CaChiVI2:GFP fused plasmids were infiltrated into Nicotiana benthamiana plants for CaChiVI2 expression in epidermal tissue (Jin et al., 2019;Ma et al., 2019). The confocal laser micrographs exhibit that 35S:CaChiVI2:GFP fused protein were localized only in the cytoplasm of the cell (Figure 3). On the other side, the pVBG2307:GFP (mock) vector, used as a control, was give signal in three main parts of the cell including cell membrane, cytoplasm and nucleus.

Expression Pattern of CBP Genes Under Heat Stress
The seedlings of pepper plant were incubated at 45 • C for heat stress to examine the transcript levels of CBP genes using qRT-PCR. The investigation revealed that different expression levels were found in different genes (Figure 4). Among all 16 genes, 14 were upregulated, while two genes (CaChiIII3 and CaChiIII5) were downregulated at each time point. The CaChiI1 initially expressed abruptly (14.5) at 1-h post-treatment then downregulated at all time points, whereas CaChiI2, CaChiI3, CaChiIII2, CaChiIII6, CaVhiVI1, CaChiVI2, and CaChiVI4 progressively showed increased expression at each time point, till slight downregulation at 24 hpt. The CaChiVI2 transcript level was the highest (39.76) at 12 hpt. Compared to other genes, its expression was significantly higher at every time point. Some members of CBP (CaChiIII1, CaChiIII4, CaChiIV1, and CaChiVI3) showed higher expression at some time points, though irregular changes were noticed in their expression level (Figure 4). Additionally, two genes (CaChiIII7 and CaChiIV2) exhibited no remarkable changes in transcript levels against heat stress and their expression was not predominantly upregulated at various interval of time.

CaChiVI2-Silencing Effect on Heat Stress Response and P. capsici Tolerance
After 6-weeks of inoculation, the leaves of the positively controlled (pTRV2:CaPDS) plants exhibited photo-bleaching phenotype, which demonstrate the success of VIGS (Figure 5A). The silencing efficiency of pTRV2:CaChiVI2 (CaChiVI2silenced) and pTRV2:00 (control) plants were detected by qRT-PCR. As shown in Figure 5A, there was no visual difference between pTRV2:CaChiVI2 and pTRV2:00 plants which grown in normal conditions and the silencing efficiency was around 71% (Figure 5B). Thus, CaChiVI2-silenced and controlled plants were used for further research.
After heat stress treatment, the expression of CaChiVI2 was checked in both pTRV2:CaChiVI2 and pTRV2:00 plants. A remarkable difference was recorded in pTRV2:00 and pTRV2:CaChiVI2 samples at all the time points, which revealed that the expression level of CaChiVI2 was lower in pTRV2:CaChiVI2 compared to pTRV2:00. A huge difference of >50% was found between pTRV2:CaChiVI2 and pTRV2:00 at 6 h post-treatment (hpt) with values of 3.65 and 1.96 respectively ( Figure 6A).
The regulation of other defense-related genes after the silencing of CaChiVI2 showed that the transcript levels of resistance responsive genes, such as CaHSP70-2 and CaDEF1 (Do et al., 2004) increased in both CaChiVI2-silenced and control plants. Their transcript was significant lower in CaChiVI2silenced plants comparatively control (TRV2:00) plants, for all the tested time points (Figures 6B,C). For examination of functional specificity of CaChiVI2 under biotic stress, the TRV2:CaChiVI2 (silenced) and TRV2:00 (controlled) pepper plants were infected with P. capsici. A higher transcript level of CaChiVI2 was detected in control plants compared with CaChiVI2-silenced plants, with CaChiVI2 expressing 66% more in pTRV2:00 plants. As shown in Figure 6D, the silencing of CaChiVI2-gene had a significant tolerance role against P. capsici.
We further explored the expression levels of other stress responsive genes to find the interaction/role of CaChiVI2 with other resistance responsive genes by altering their expression. It was noted that after P. capsici inoculation, CaDEF1 (Do et al., 2004) and CaPR1 gave a positive response. Their transcript level in the pTRV2:00 plants was better than that of the TRV2:CaChiVI2 plants at all tested time points (Figures 6E,F).
After heat stress treatment, the MDA contents and REL in CaChiVI2-silenced plants were significantly higher than control plants (Figures 6G,H). More severe symptoms of wilting and yellowing were observed in CaChiVI2-silenced plants, which indicated that chlorophyll contents degraded more in pTRV2:CaChiVI2 plants ( Figure 6I). Moreover, the recovery efficiency was also checked which revealed that pTRV2:00 plants recovered their growth quicker than TRV2:CaChiVI2 plants, when incubated at normal temperature (22 • C) (Figure 7).
Production of ROS is a typical consequence once plants are in stress condition. We noticed a higher accumulation of superoxide (O 2 − ) and hydrogen peroxide (H 2 O 2 ) using NBT and DAB staining respectively. After heat stress, a higher production of O 2 − was detected in CaChiVI2-silenced plants, leaves (darkblue color) compared with control plants (light-blue color) FIGURE 4 | The transcript level of CBP genes in response to heat stress. The plant tissues were sampled at different time points (0, 1, 3, 6, 12, and 24 h post heat treatment) and were examined by qRT-PCR. The mean values and ± SDs for three replicates are presented. Different letters (a-f) on the bars in each histogram represent significant differences at P ≤ 0.05 using Duncan Multiple Range (DMR) test.  ( Figure 8A). After 3 days of inculcation, the Phytophthora capsici lesions were detected on the isolated leaves of both TRV2:CaChiVI2 and TRV2:00 pepper plants. However, the infected areas and H 2 O 2 accumulation were more pronounced in silenced plant versus control ( Figure 8B). Quantitative analysis exhibited that disease infected area of TRV2:CaChiVI2 plants (>74%) was significantly more expanded than the TRV2:00 plants (11.6%) ( Figure 8C).

Overexpression of CaChiVI2 Improve the Tolerance Against Heat Stress Damage in Arabidopsis
In order to select the best thermo-tolerant homozygous T 3 lines for further analysis, wild-type (WT) and five CaChiVI2overexpressed (OE2, OE3, OE5, OE6, and OE7) Arabidopsis lines were cultured on MS-media and were kept at 45 • C (high temperature) for 2h. The CaChiVI2-overexpressed plants were then kept at 22 • C for 7-days to recover and their survival rate was measured. The results exhibited that the survival rate of OE3, OE6, and OE7 lines was higher than WT, OE2, and OE5-lines (Figures 9A,B). Besides, some WT and transgenic Arabidopsis lines were grown in normal environmental conditions to detect the transcript levels of CaChiVI2 gene. As presented in Figure 9C, the transcript levels of CaChiVI2 in OE3, OE6, and OE7 lines were significantly higher than the WT seedlings. Thus, the OE3, OE6, and OE7 were selected for further studies.
The seedlings were then grown in composite soil and treated with 40 • C temperature for 16 h. As a result, the leaves of WT seedlings wilted severely, while mildly in case of CaChiVI2-OE seedlings. Ten days after heat treatment, the WT did not show progressive growth as compared to transgenic lines, as shown in Figure 9D. However, the defense-related gene (AtHSA32) was highly expressed in both WT and transgenic Arabidopsis. Additionally, the expression level of AtHSA32 in transgenic-lines (OE3, OE6, and OE7) was substantially higher than WT plants (see Figure 9E). Moreover, the accumulation of MDA contents in CaChiVI2-overexpressed lines was lower (Figure 9F), while SOD, POD activity and proline contents of CaChiVI2-OE seedlings were higher as compared to WT (Figures 9G-I).

Overexpression of CaChiVI2 Enhances Endurance to Drought Stress in Arabidopsis
The CaChiVI2 overexpressed transgenic lines were investigated for drought stress tolerance on MS-medium containing 0, 100, and 200 mM mannitol. There were no differences in the germination rates of WT plants and transgenic lines after 5 days. However, the root length of CaChiVI2-OE lines was substantially longer than the WT seedlings (Figures 10A,B). Besides, the WT and transgenic seedlings were grown in soil-media where water was withheld for 5 days. As a result, the WT seedlings dehydrated and displayed severe wilting. In comparison, slight damage was observed in transgenic seedlings and the rate of recovery was also faster than WT plants ( Figure 10C). Nevertheless, the drought stress-related gene AtHSA32 was elicited in both transgenic and WT Arabidopsis, however its transcript level was greater in transgenic seedlings compared to wild-type plants ( Figure 10D). Besides, the MDA content was substantially lower and proline significantly higher in CaChiVI2-OE seedlings as compared to WT (Figures 10E,F). Additionally, the accumulation of O 2 − was lower in transgenic seedlings (Figure 10G).

DISCUSSION
The CBP is a stress-responsive multigene family, which regulates resistance against biotic and abiotic stresses (Xu et al., 2007;Ahmed et al., 2012;Su et al., 2015). The specificity of CBP-family regarding various stresses is confirmed in wheat crop (Singh et al., 2007). However, study on the characterization of pepper CBP genes, especially in relation to heat and drought stress is still not conclusive. Therefore, to fill the gap and disclose more CBP functions, CaChiVI2 gene from the Capsicum annuum L. genome database was chosen for further analysis. Likewise other CBP members, CaChiVI2 also had four heat shock elements (HSE's) in their promoter region (Pastuglia et al., 1997;Ali et al., 2018). To confirm its function, the CaChiVI2 in pepper was successfully knockdown by VIGS, which correspondingly reduced resistance against heat stress and P. capsici infection (Figures 6A,D). Additionally, CaChiVI2 knockdown affect the expression of other stress responsive genes, i.e., CaDEF1 and CaHSP70-2 under heat stress and CaPR1 and CaDEF1 against P. capsici infection, which showed its role in defense mechanism of pepper plant. This statement is supported by Jin et al. (2016) through studying the function of CaPTI1 in pepper plant. The findings indicate that CaChiVI2 has a crucial role in enhancing pepper plants endurance against high temperature and P. capsici infection.
Reactive oxygen species signal components such as H 2 O 2 and O 2 − play a significant role in plants responses to unfavorable environmental conditions. They perform multiple roles by acting directly in the initial defense. However, the over-accumulation of H 2 O 2 and O 2 − under stress results in an active damage to the cell structures, photosynthesis and natural intracellular environment (Uzilday et al., 2012;Tuteja et al., 2014;Srivastava et al., 2016;  Haq et al., 2019). Therefore, the contents of H 2 O 2 and O 2 − are often used to find the damage levels of plant cells . The detached leaves assay of CaChiVI2-silenced and control (TRV2:00) plants after the heat and P. capsici treatments were established to assess the ROS activities. As a result, more infection and ROS accumulation were found in the leaves of CaChiVI2-silenced versus control, which exhibited that knockdown of CaChiVI2 increased sensitivity and plants became more prone to stresses (Figures 8A,B). The outcome followed the similar pattern as explained in previous study by Zhang et al. (2018), and our conclusion are also consistent with Su et al. (2015). Contrarily, when CaChiVI2 overexpressed in Arabidopsis, low O 2 − accumulation was recorded in overexpressed plants as compared to WT under drought stress ( Figure 10G). This indicates the role of CaChiVI2 in the defense system of a plant and also suggests that CaChiVI2 confers the plants ability to reduce the H 2 O 2 and O 2 − concentrations thus evading the injuries caused by ROS under heat, drought and P. capsici infection.
Previously, overexpressed tobacco plants confirmed resistance to high temperature and oxidative stress exhibiting higher physiological indicators than WT plants Zang et al., 2018) and higher survival rate and root length was observed under high temperature and NaCl stress . CaChiVI2 overexpression showed moderate heat and drought stress symptoms and increased the resistance of Arabidopsis plants (Figures 9D, 10C). The transcriptional activation/up-regulation of CaChiVI2 is necessary for immediate resistance against heat in CaChiVI2-OE lines (Figure 9C), signifying that CaChiVI2 not only responds to heat stress but also participates in drought stress. These facts confirm the earlier conclusions that CBP genes play an important role in environmental stresses (Hamid et al., 2013). Furthermore, resistance to high temperature and drought stress is also regulated by other genes in Arabidopsis, such as acquired thermotolerance related gene AtHSA32 is strongly activated by heat stress (Charng et al., 2006;Burke and Chen, 2015;Iurlaro et al., 2016). In our study, the transcript level of AtHSA32 gene was highly provoked by high temperature and drought stress in CaChiVI2-OE Arabidopsis as compared to WT plants ( Figure 9E). It suggests that CaChiVI2 may possibly be involved in heat stress endurance by altering the transcript level of stress Phenotypes, the transcript level of AtHSA32, MDA contents, SOD, POD activity and Proline accumulation measured in transgenic lines (OE3, OE6, and OE7) and WT plants after 40 • C heat stress treatment for 16 h. Arabidopsis seedlings grown at 22 • C were used as control. The bars indicated the mean values ± SDs for three replicates. The small letters (a-m) and asterisk (*) in corresponding graphs denote significant differences at P ≤ 0.05. responsive genes. However, further study is needed to understand the exact mechanism.
Malondialdehyde (MDA) is an important product of membrane lipid peroxidation (LPO), which is considered as a reliable biochemical oxidative stress marker (Del Rio et al., 1996;Koźmińska et al., 2019). Based on our findings, it can be determined that the CaChiVI2-silenced plants exhibited an increase in MDA level, though the CaChiVI2-overexpressed Arabidopsis plants showed a decrease in MDA level (Figures 6G,  9F, 10E). The oxidative stress has shown correlation with MDA level (Gechev et al., 2002;Madhusudhan et al., 2009). It suggests that this might be due to the silencing effect of CaChiVI2 leading to damage in the plasma membrane. Whereas, the CaChiVI2 overexpressed Arabidopsis plants tolerate heat and the plasma membrane damages due to drought stress.
To mitigate the damages of ROS in unfavorable environmental conditions, plants develop special mechanisms to scavenge glut ROS through antioxidant enzymes such as SOD, POD, CAT, and APX and non-enzymatic antioxidants (Vitamin-C and proline content). These antioxidant enzymes and proline reduce oxidative damage caused by stress (Schöffl et al., 1999;Feng et al., 2019). During the scavenging of ROS, the SOD first decomposes O 2 − to H 2 O 2 and then the H 2 O 2 is scavenged by peroxidase (POD) in the cytosol and the outer cellular space (Gill and Tuteja, 2010;Uzilday et al., 2012). It is also investigated that the CBD having cysteine and the hinge region, which is saturated by proline and glycine. The biosynthesis of proline decreases the injury caused by ROS (Schöffl et al., 1999), while in many plant species proline has been considered one of the most common compatible osmolyte for cellular osmotic adjustment which are conferred by the high salinity, water deficit and other stresses (Szabados and Savoure, 2010;Al Hassan et al., 2017;Kumar et al., 2017). Likewise, in our study, the CaChiVI2-overexpressed Arabidopsis revealed significantly higher SOD, POD activities and proline contents than WT plants under heat stress (Figures 9G-I, 10F). Therefore, the previous findings of Sun et al. (2019), where transgenic Arabidopsis revealed better resistance to high temperature by accumulating more CAT, SOD and POD activity compared to wild-type plants, support our results. Interestingly in our study, the observation of CaChiVI2-OE lines showed higher proline biosynthesis relative to WT plants under heat and drought stress conditions (Figures 9I, 10F). Therefore, it can be concluded that the regulation of CaChiVI2 under CaMV35S promoter is clearly overexpressed and significantly influences the antioxidant enzymes machinery and also proline biosynthesis in response to heat and drought stress. In plants the electron transport chain regulated by mitochondrial second terminal oxidase (alternative oxidase-AOX) is also vital for the defensive machinery of a plant (Dong et al., 2009). Keeping in view this theory, we may conclude that the overexpression of CaChiVI2 upregulates the expression of AOX gene and thereby participates in the scavenging of ROS pathway and improves the overall defense system. Our findings clearly demonstrate that CaChiVI2 may tolerate high temperature and drought condition through the ROS-scavenging pathway. However, before declaring the potential role of CaChiVI2 as a novel stress-responsive gene, it is required to evaluate the exact mechanisms and regulation under heat signaling pathways.
The subcellular localization of CBP members may show their associated functions. Some CBP genes in other plants such as ScChiI1 have been localized in the cytoplasm and the plasma membrane (Su et al., 2015) and ChiIV3 in the plasma membrane (Liu Z. et al., 2017). In Oryza sativa, CBP proteins are located in cytoplasm and mitochondria and are thought to be associated with the thylakoid membrane under heat stress (Xu et al., 2007). The transient expression of CaChiVI2 in N. benthamiana leaves confirmed the localization of CaChiVI2 proteins only in the cytoplasm, indicating a vital role in the cytoplasm of a cell (Figure 3). Our key findings from testing the cloned CaChiVI2 gene are that heat stress and subsequently silencing by VIGS, caused severe damages to plant morphology and physiology. The ectopic expression of CaChiVI2 in Arabidopsis thaliana showed lethal damage due to heat and drought condition by increasing the transcript of resistance responsive genes. In the light of previous studies, we can conclude that CaChiVI2 may be acting as a transcriptional activator and provide an innate response to high temperature and drought condition.

CONCLUSION
Our results validated that CaChiVI2 knockdown decreased the stress tolerance ability of pepper plants against heat and P. capsici infection; additionally, the physiological and morphological attributes were also altered at certain level. The co-regulatory response of other defensive genes such as CaDEF1, CaPR1, and CaHSA70-2 not only confirmed the functional diversity of CaChiVI2 gene, but also introduce the important role in stress tolerance mechanism. In contrast, CaChiVI2overexpressed plants showed mild symptoms, decreased heat and drought stress with the elevated transcript levels of the defense-related gene, including AtHSA32. The identified tasks of CaChiVI2 gene may provide some solid proofs for advance study of pepper plant adaptation mechanisms in response to sever environmental conditions.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
The study presented in the manuscript did not involve any experimentation on human or animal subjects.

AUTHOR CONTRIBUTIONS
MHA and Z-HG conceived and designed the research. MKA conducted the experiments and wrote the manuscript. KA and IM analyzed the data. SH and AK performed the in silico analysis. AK, MHA, and HU critically revised the manuscript. Z-HG and GL contributed reagents and funded the project. All the authors read and approved the manuscript.