To investigate the role of the peroxidase gene in pepper plant response to pathogen infection, the CanPOD gene was identified and characterized. The full sequence of CanPOD cDNA comprised of 1353-bp, containing the start code ATG, stop code TAG, a 5′-untranslated region (UTR) of 67-bp, an open reading frame (ORF) of 975-bp and a 3′-UTR of 311-bp with a poly (A⁺) tail of 28-bp. The cDNA sequence has been submitted to GenBank as accession number FJ596178. The ORF encodes a predicted protein of 324 amino acids. The calculated molecular mass of the mature protein is 34.93 kDa, with an estimated pI of 9.220. TMpred showed that CanPOD protein contained three inside to outside helices and one outside to inside helice, which score more than 500. Therefore, CanPOD protein contained transmembrane stuctures. The CanPOD protein would be a secretory protein with signal peptide and transmembrane structures, the resorts of NCBI Blast also sustain this viewpoint. CanPOD protein contained four conserved domains (Figure 1). The rectangle indicates the heme binding site with 29 residues; ellipse indicates the active site with 31 residues; # indicates the substrate binding site with 10 residues; Δ indicates the calcium binding sites with 9 residues. Eight conserved cysteine residues (C38, C71, C76, C118, C124, C203, C229 and C320) yielded four disulfide bridges.

Multiple amino acid sequence alignment revealed that the deduced amino acid sequence of CanPOD displayed substantial homology with other proteins (Figure 2), including Nicotiana tabacum peroxidase (BAA82306.1, 88%), Vitis vinifera peroxidase 4 (XP_002278996.1, 78%), Rubia cordifolia peroxidase 6 (ADN96693.1, 78%), Glycine max peroxidase 52-like (XP_003523269.1, 81%), Ipomoea batatas anionic peroxidase swpb3 (AAP42508.1, 82%), Gossypium hirsutum class III peroxidase (AAP76387.1, 82%), Catharanthus roseus putative secretory peroxidase (AAX44001.2, 78%) and Medicago truncatula Peroxidase (XP_003616748.1, 77%), respectively.

To evaluate the molecular evolutionary relationships of CanPOD against other peroxidases, a phylogenetic tree was carried out using Mega5.1. Two clusters were formed using the full-length cDNA sequences of 18 peroxidase genes from Catharanthus roseus, Arachis, Catharanthus roseus, Glycine max, Gossypium hirsutum, Ipomoea batatas, Medicago truncatula, Nicotiana tabacum, Quercus, Rubia cordifolia, Solanum tuberosum and Vitis vinifera (Figure 3). CanPOD gene (FJ596178) was grouped together with POD a2 from Solanum tuberosum, which is a secretory peroxidase, belongs to the plant peroxidase super family. All of the bioinformatics analysis results suggested that CanPOD should be a plant peroxidase.

In order to investigate the expression levels of CanPOD gene in different tissues of pepper cultivar A3 plants, total RNA was extracted from roots, stems, leaves, flowers and immature fruits. The results were shown in Figure 4. Differences were observed among the different tissues of pepper plants. In general, the highest expression levels of CanPOD gene were detected in leaves, followed by roots, stems and flowers, respectively. However, CanPOD gene was slightly expressed in immature fruits. The results suggest that CanPOD gene is differentially expressed in the tissues of pepper cultivar A3 plant. This finding is in agreement with previous studies showing that the expression of many peroxidase genes is tissue specific [6,7,9,14,16,20]. The relatively high expression of CanPOD gene in leaves suggests that CanPOD gene may be associated with defense responses in leaves.

Plants possess an elaborate and finely regulated defense system to defend against various biotic and abiotic stresses [27]. To study the relationship between the CanPOD gene and stress response of pepper, the expression patterns of CanPOD gene in response to diverse stresses were measured, including biotic stress (pathogen infection) and abiotic stresses (cold, salt, drought, SA and MeJA), using Real-Time RT-PCR.

To examine the effect of loss-of-function of the CanPOD gene in pepper plants, VIGS was performed in pepper cultivar A3 using the tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) technique [14,43–46]. A fragment from the 3′ end of the CanPOD open reading frame was cloned into the pTRV2 vector and generated pTRV2-CanPOD vector (Figure 6). Empty vector (pTRV-00) was used as a negative control.

The pTRV2-CaPDS, which silences phytoene desaturase gene (PDS) and induces a photo-bleaching phenotype, was used as a positive control to determine the success of gene silencing. As shown in Figure 7a, symptoms of photo-bleaching were occurred on the leaves of CaPDS silenced plants. This provided the evidence that VIGS is successful in our study. There was no morphological difference between the control plant (pTRV-00) and the CanPOD silenced plant (pTRV2-CanPOD) 35 d after inoculation (Figure 7a). Meanwhile, the efficiency of the VIGS was investigated by Real-Time RT-PCR (Figure 7b). The results revealed that the levels of CanPOD expression in CanPOD silenced plant (pTRV2-CanPOD) were reduced drastically comparing to the control pepper plant (pTRV-00). These RT-PCR data indicate that CanPOD was silenced effectively in pepper plants. In order to further confirm the efficiency of VIGS on CanPOD, the peroxidase (POD) enzymatic activity in pepper leaves was tested in the control plant (pTRV-00) and CanPOD silenced plant (pTRV2-CanPOD). As shown in Figure 7c, the activities of POD enzyme were decreased in the CanPOD silenced plants comparing to the control plant, which were essentially identical to the results from the Real-Time RT-PCR, proving the validity of RT-PCR analysis. These findings further suggest that VIGS was successful and effective for CanPOD silencing on pepper plants.

It is well known that peroxidase be induced by various pathogens infection [12,14,20,27]. The CanPOD expression in pepper leaves was significantly induced in the incompatible interaction with avirulent strain of P. capsici. To determine the role played by CanPOD in the basal defense response of pepper, we analyzed the levels of resistance to P. capsici infection in leaves of CanPOD silenced plants. Five weeks after VIGS, the fourth leaves from the top of control plants (pTRV2-00) and silenced pepper plants (pTRV2-CanPOD) were detached and inoculated by addition of 20ul zoospores suspension (10⁴ cfu mL⁻¹) of P. capsici by using a syringe without needle. Disease symptoms were observed in the CanPOD silenced plant leaves 3 d after inoculation. However, the control (pTRV-00) plant leaves did not exhibit any cell death or disease symptoms (Figure 8). Therefore, we demonstrate that CanPOD-silenced plants are more susceptible to P. capsici infection than the control plants. One paper had reported similar results to our study [14]. As a whole, the CanPOD gene is involved in the defense responses to pathogen infection of pepper plants.

Pepper cultivar A3 was used in this study, which provided by pepper breeding group in Northwest A&F University, China. Seeds were soaked in warm water (55 °C) for approximately 20 min to promote germination. The seeds were rinsed twice every day and then placed on moist gauze in an incubator (28 °C, 60% relative humidity in darkness). When the seeds were at least 80% germinated, they were sown in a soil mix [grass charcoal/perlite (3/1, v/v)] in plastic pots. Pepper plants were grown in a growth chamber with a 16 h light and 8 h dark photoperiod at 25 °C.

Preparation of P. capsici inocula was described previously [47]. The avirulent strain PC of P. capsici was grown on potato dextrose agar (PDA) medium in darkness at 28 °C for 7 days. The mycelia were scraped and incubated under fluorescent light for 2 days to promote sporangium formation. Zoospores were prepared by sporulating cultures with sterile distilled water and incubating at 4 °C for 1 h followed by 30 min at 28 °C to initiate zoospore release. The zoospores were collected by filtering through four layers of cheesecloth and estimated using a hemacytometer. The concentration of the zoospore suspension was adjusted to 1 × 10⁴ zoospores per millimeter with sterile water.

For pathogen stress treatment, pepper plants at six-true leaf stage were inoculated by addition of 2.5 mL zoospores suspension (10⁴ cfu. mL⁻¹) of P. capsici each pot.

For virus-induced gene silencing (VIGS) of CanPOD gene in pepper plants, the fourth leaves from the top of control plants (pTRV2-00) and silenced pepper plants (pTRV2-CanPOD) after inoculation 5 weeks were collected and sterilized with 75% ethanol for 1 min, washed three times with sterile water, then injected by addition of 20ul zoospores suspension (10⁴ cfu. mL⁻¹) of P. capsici by using a syringe without needle. After inoculation, the leaves were grown in a growth chamber at 28 °C under a 16 h light/8 h dark photoperiod cycle with 60% relative humidity.

Total RNA was extracted from young leaves of pepper cultivar A3 using the Trizol (Invitrogen) method. To isolate the complete 5′ and 3′ regions of the putative CanPOD gene, the rapid amplification of cDNA ends (RACE) method was used. First-strand cDNA synthesis was performed using Smart RACE cDNA amplification kit (Clontech). Gene-specific primer GSP1: (5′-GCTTCGTCAATGGATGTGATGGA-3′) was used for 3′-RACE and the gene-specific primer GSP2: (5′-TGGAAGAAAAGGCGAAGCAGAGA-3′) was used for 5′-RACE, respectively. The universal primers for 5′ and 3′ RACE are given in the kit. The full-length cDNA sequence of CanPOD gene was obtained by PCR amplification using forward (5′-GGTGCTCAACACACACTTTA-3′) and reverse (5′-TTGTTTAAGTTATTCATGAT-3′) primers. The PCR products were cloned into pMD19-T vector (Takara) and sequenced by Shanghai GeneCore Biotechnologies Co. (GeneCore).

NCBI bioinformatics tools [48] were used to compare and analyze the nucleotide and protein sequences data. Multiple sequence alignments of CanPOD gene with peroxidase from other species were performed by the Clustal X1.83 analysis program of DNAstar software (LaserGene, Madison, WI, USA). The phylogenetic tree was constructed using Mega5.1 by the neighbor-joining method. Conserved domains in protein were identified in Conserved Domain database [49].

Total RNA was extracted from pepper leaves of different time points after diverse stress treatments using Trizol (Invitrogen) method. The concentration of total RNA was measured spectrophotometrically using a NanoDrop instrument (Thermo Scientific NanoDrop 2000C Technologies, Wilmington, DE, USA), and the purity was assessed using the A260/280 and A260/230 ratios provided by NanoDrop. For quantitative Real-Time RT-PCR analysis, first strand cDNA was synthesized from 500 ng total RNA using a PrimeScript™ Kit (TaKaRa, Bio Inc, China) following the manufacturer’s protocols. Real-Time RT-PCR was carried out using SYBR® Premix Ex Taq™ II (TaKaRa, Bio Inc., China). Real-Time RT-PCR analysis was performed on a 20 μL mixture containing 10.0 μL SYBR^® Premix Ex Taq™ II, 2.0 μL diluted cDNA, 0.8 μL of forward and reverse primers. The amplification was completed with the following cycling parameters: 95 °C for 1 min, followed by 45 cycles at 95 °C for 10 s, 52 °C for 30 s, and 72 °C for 30 s. The gene of Ubi3 (AY486137.1) was used as internal control (reference gene) in this study [50]. The primer sequences used for Real-Time RT-PCR were shown in Table 1. The relative expression levels of gene were determined by using comparative threshold method of 2^−ΔΔCt[51]. All samples were performed in triplicate and each had at least three independent biological replicates.

The tobacco rattle virus (TRV)-based VIGS system was used for gene silencing in pepper plants as described by [14,52]. Phytoene desaturase (PDS) encodes an enzyme involved in the carotenoid biosynthesis pathway [53]. The CaPDS (phytoene desaturase from C. annuum, GenBank: X68058.1) has been used as a visual marker for the effectiveness of VIGS in pepper plants. A fragment of coding region of CaPDS gene was amplified using gene-specific primers (Forward: 5′-GGGGAATTCTGTTGTCAAAACTCCAAGGTCTGTA-3′ with an EcoRI restriction site and Reverse: 5′-GGGGGATCCTTTCTCCCACTTGGTTCACTCTTGT-3′ with a BamHI restriction site). The resulting product was inserted into pTRV2 vector to generate pTRV2-CaPDS. A fragment of coding region of CanPOD gene was amplified using gene-specific primers (Forward: 5′-GGGTCTAGAGTGCTCAACACACACTTTATTCTTCTC-3′ with an XbaI restriction site and Reverse: 5′-GGGGGATCCCCAAGAATGACAACAGAGTCCCTA-3′ with a Bam HI restriction site). The resulting product was inserted into pTRV2 vector to generate pTRV2-CanPOD. The pTRV1, pTRV2, pTRV2-CaPDS and pTRV2-CanPOD vectors were transformed into the Agrobacterium tumefaciens strain GV3101, respectively. Agrobacterium tumefaciens strain GV3101 carrying pTRV1 were respectively mixed with pTRV2, pTRV2-CaPDS and pTRV2-CanPOD at a 1:1 ratio. The suspensions of agrobacterium inocula containing pTR1 and pTRV2-00, pTRV2-CaPDS or pTRV2-CanPOD (OD₆₀₀ = 1.0) was infiltrated into the fully expanded cotyledons of pepper plants using a 1.0 mL sterilized syringe without a needle. The agrobacterium-inoculated pepper plants were grown in a growth chamber at 18 °C in darkness for 2 days with 45% relative humidity, and then transferred into a growth chamber at 22 °C under a 16 h light/8 h dark photoperiod cycle with 60% relative humidity. Leaves of control plants (pTRV2-00) and silenced pepper plants (pTRV2-CanPOD) after inoculation five weeks were used for CanPOD gene analysis.

Activities of Peroxidase (POD) enzyme were quantified using the technique of Beffa [54]. The samples were collected from leaves of control plants (pTRV2-00) and silenced plants (pTRV2-CanPOD) 35 d after inoculation and stored at −80 °C. The lyophilised leaf powder (approximately 1.0 g) was ground in a mortar and homogenised with 5.0 mL pre-chilled extraction buffer (0.1 M potassium phosphate buffer (pH 7.5), 1mM EDTA, and 4% polyvinylpyrrolidone). The homogenate was centrifuged at 12,000 rpm for 25 min. The supernatant fraction was used as a crude extract for the enzyme activity assays. All procedures were carried out at 4 °C. The reaction mixtures contained 25 μL 50 mM H₂O₂, 5 μL 250 mM guaiacol, 195 μL 12.5 mM 3,3-dimethylglutaric acid (3,3-DGA)-NaOH (pH 6.0) and 25 μL enzyme extract. The optical density was recorded at 470 nm. The amount of enzyme required for the formation of 1 μmol tetraguaiacol min⁻¹ at room temperature was defined as 1 unit (U) of PX activity. Each treatment was conducted in three independent experiments and each measurement was repeated three times.

All data were expressed as the mean ± SD of three independent replicates (n = 3). Data from replicates of the same experiment were pulled together for one-way analysis of Variance (ANOVA), and differences among means of treatments were determined using the least significant difference (LSD). Statistical procedures were performed using Statistical Analysis System software (SAS Institute, version 8.2). Values of p ≤ 0.05 were considered statistically significant.