Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora

This is the first report to present a full-length cDNA (designated HbPR-1) encoding a putative basic HbPR-1 protein from rubber tree (Hevea brasiliensis) treated with salicylic acid. It was characterized and also expressed in Nicotiana benthamiana using Agrobacterium-mediated transient gene expression system in order to investigate the role of HbPR-1 gene in rubber tree against its oomycete pathogen Phytopthora palmivora and to produce recombinant HbPR-1 protein for microbial inhibition test. The HbPR-1 cDNA was 647 bp long and contained an open reading frame of 492 nucleotides encoding 163 amino acid residues with a predicted molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56, demonstrating that HbPR-1 protein belongs to the basic PR-1 type. The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop. Expression and purification of recombinant HbPR-1 protein were successful using Agrobacterium-mediated transient expression and one-step of affinity chromatography. Heterologous expression of HbPR-1 in N. benthamiana reduced necrosis areas which were inoculated with P. palmivora zoospores, indicating that the expressed HbPR-1 protein played an important role in plant resistance to pathogens. The purified recombinant HbPR-1 protein was found to inhibit 64% of P. palmivora zoospore germination on a water agar plate compared with control, suggesting that it was an antimicrobial protein against P. palmivora.


Introduction
Growth and productivity of plants depend on their ability in responding and adapting to external stresses. Plants defend themselves against external stresses such as pathogen invasion by light and 12 h dark for 1 day. The 22-day-old rubber seedlings were sprayed with 4 mM salicylic acid (SA) solution to induce production of PR-1 proteins. Rubber leaves were collected 24 h after SA treatment for total RNA extraction.
N. benthamiana seedlings were grown in pots under growth chamber conditions at 25°C, 60% humidity, 16 h light/8 h dark cycle for 6 weeks.
Preparation of Phytophthora palmivora zoospores P. palmivora PP3-3 isolated from a diseased papaya plant was cultured on 10% unclarified V8 agar medium at room temperature for 5 days. The culture was covered with sterile distilled water and incubated at 4°C for 30 min then shaken at 50 rpm at room temperature for 30 min to release the motile zoospores.
Amplification and cloning of HbPR-1 Molecular cloning of HbPR-1 consisted of three steps: Amplification of HbPR-1 partial fragment by PCR, 3 0 and 5 0 rapid amplification of cDNA ends (RACE) PCR and HbPR-1 fulllength cDNA amplification. Total RNA was extracted from leaves of rubber plants which were treated with 4 mM SA for 24 h using RNeasy 1 Plant Mini Kit (Qiagen, https://www.qiagen. com) following the manufacturer's instructions. The total RNAs were treated with RNase-free DNaseI (Qiagen) at 37°C for 30 min to eliminate genomic DNA. The reaction was inactivated by heating at 65°C for 10 min. The purity of total RNAs was confirmed based on A260 nm/ A280 nm ratio >1.8. The first-stranded cDNAs were synthesized using the SuperScript III reverse transcriptase RT-PCR system (Invitrogen, https://www.thermofisher.com) according to the manufacturer's instructions. PCR was performed with the degenerate primers, dFPR-1: 5 0 -TTCACAACCAGGCACGAGS(G/C)A GCGGT-3 0 and dRPR-1: 5 0 -ATGGTTCCACCATTGT TACACCTCAC-3 0 using Taq DNA polymerase (Invitrogen). The degenerate primers were designed based on the conserved sequences of two PR-1 genes (Arabidopsis thaliana: NM_127025.2 and Eutrema wasabi: AB271488.1). The reaction was performed with PCR condition of initial denaturation step at 95°C for 5 min, followed by 40 cycles of denaturation step at 95°C for 1 min, annealing step at 55°C for 1 min, extension step 72°C for 1 min and final extension step at 72°C for 10 min. The PCR products were visualized on 1.2% agarose gels. The PCR product of expected size was excised and purified using the QIAquick gel extraction kit (Qiagen) before ligating into pGEM 1 -T Easy plasmid (Promega, https://worldwide.promega. com). The ligation mixtures were transformed into E. coli JM109 competent cells (Promega) and plated on McConkey Agar/amp plates. Colonies were selected for culture and plasmid extraction using E.Z.N.A 1 Plasmid DNA Mini Kit I (OMEGA bio-tek, http://omegabiotek. com) then subjected to DNA sequencing. The sequence of full-length HbPR-1cDNA was determined using the 5 0 /3 0 RACE kit, 2 nd Generation (Roche, https://lifescience.roche.com) according to the manufacturer's instructions. 3 0 -RACE HbPR-1 primer (5 0 -TCTTGTGCATTCCAGCA ATC-3 0 ) was designed based on the sequence of the partial PCR fragment mentioned above and the result from 3 0 -RACE was used to design the 5 0 -RACE HbPR-1 primer (5 0 -ACGTGT TTTCTGTTCATTAATAAGTGAAGAAGC-3 0 ). To confirm the assembled HbPR-1 full-length sequence, PCR amplification was applied using specific primers designed according to the sequences of 5 0 -and 3 0 -RACE product as follows: FHbPR-1full (5 0 -CATCCATTGCCT AAGATCTTAAACAAACTC-3 0 ) and RHbPR-1full (5 0 -ACGTGTTTTCTGTTCATTAA TAAGT GAAGAAGC-3 0 ). The PCR reaction was performed using the EmeraldAmp 1 PCR Master Mix (Takara, http://www.clontech.com) and carried out at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 62°C for 1 min, 72°C for 1 min and a final extension 72°C for 10 min. The amplicon was cloned into pGEM 1 -T Easy plasmid (Promega) and transformed into E. coli JM109 competent cells (Promega). The positive clones were selected for plasmid extraction and DNA sequencing.

Plasmid construction and transformation into Agrobacterium tumefaciens
The pGD_HbPR-1 plasmid construction was performed by cloning PCR-amplified DNA fragment corresponding to HbPR-1 protein-encoding sequence into the binary pGD vectors [48]. The pGEM 1 -T Easy_HbPR-1 plasmid containing HbPR-1 coding sequence was used as template for PCR. The oligonucleotides FPR-1_SalI (5 0 -gcggtcgacATGGTGTTC TGCAA GAATTC-3 0 ) and RPR-1_BamHI (5 0 -gcgggatccTTAgtggtgatggtgatggtgATAAGG TTTCTGCCCAAC-3 0 ) were used for fragment amplification. The introduced SalI and BamHI restriction sites were underlined. The italic letters represent the hexahistidine-tag sequences. The PCR was performed using the Phusion 1 HF DNA polymerase (New England Biolabs Inc., https://www.neb.com) and was carried out at 98°C for 30 s, followed by 35 cycles of 98°C for 15 s, 60°C for 15 s, 72°C for 1 min and a final extension 72°C for 10 min. The amplified fragment was cloned into SalI and BamHI sites of pGD [48]. The pGD_HbPR-1 plasmid containing HbPR-1 gene insert with the correct nucleotide sequences was used to electroporate into A. tumefaciens C58-C1.

Transient expression of HbPR-1 in N. benthamiana
Transient expression of HbPR-1 in N. benthamiana was conducted according to the agroinfiltration method described by Kruger et al. [49]. A. tumefaciens strain C58-C1 carrying the pGD_HbPR-1 plasmid and A. tumefaciens strain GV3101 carrying the pJL3-p19 plasmid [50] were grown in Luria-Bertani media supplemented with appropriate antibiotics at 28°C. Overnight agrobacterial cultures were collected from plates and re-suspended in infiltration/ induction media (10 mM MgCl 2 , 10 mM MES, pH 5.6, and 150 μM acetosyringone). The A. tumefaciens cultures of pGD_HbPR-1 or pJL3-p19 were prepared with an optical density (OD600) of 0.4 for infiltration of single strain. For co-infiltration, two cultures were prepared with OD600 of 0.8 and then mixed with equal volumes of each one. The mixtures were kept for 3 h at room temperature. The infiltration/induction media buffer (mock, negative control) or the mixtures were infiltrated into 6-week-old leaves of N. benthamiana plants using 1 mL needleless syringe to the abaxial surface of fully expanded leaves. Analysis of PR-1 protein expression and isolation of intercellular fluids were conducted at 48 h after infiltration.
Analysis of PR-1 protein expression in N. benthamiana PR-1 protein expression in N. benthamiana was evaluated by Western blot analysis. Total proteins were obtained as described: leaf discs were collected using No.7 cork borer and transferred into 2-mL screw cap tube. The sample was disrupted by bead homogenizer method using grinding balls and a FastPrep 1 -24 homogenizer (MP Biomedicals). The homogenates were extracted with 2x Laemmli buffer [51], boiled for 5 min and centrifuged at 13,000 rpm for 5 min. The supernatant was loaded onto 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot.

Purification of the PR-1 protein
The intercellular fluid containing the hexahistidine-tagged HbPR-1 was used for purification of HbPR-1 protein expressed in N. benthamiana plants. NaCl and imidazole stock solutions were slowly added to 20-mL of intercellular fluid to a final concentration of 500 mM NaCl and 20 mM imidazole. The mixture was loaded into 2 mL of the Complete his-tag purification resin (Roche) that was pre-equilibrated with wash buffer (25 mM HEPES, pH 7.5, 500 mM NaCl, 20 mM imidazole, 10% glycerol). After washing with wash buffer, the bound proteins were eluted with multiple 1 mL of elution buffer (wash buffer with different concentrations of 50-250 mM imidazole). 1 mL fractions were collected and analyzed by 15% SDS-PAGE to determine the presence of the hexahistidine-tagged HbPR-1. The fractions containing the purified proteins were pooled and then desalted and concentrated using Amicon 1 ultra-15 Centrifugal Filters (Merck, http://www.merckmillipore.com) according to the manufacturer's protocol. The protein concentration was determined using the Bradford protein assay kit (Bio-Rad, http://www. bio-rad.com).

SDS-PAGE and Western blot analysis
The obtained proteins were analyzed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue as previously described by Sambrook et al. [53] or the proteins were transferred to a polyvinylidenedifluoride (PVDF) membrane (Thermoscientific, https://www.thermofisher.com) for Western blot. HRP conjugated anti-His monoclonal antibody His-probe (H-3) (sc-8036 HRP, Santa Cruz Biotechnology, INC., http://www.scbt.com) was used for the detection of hexahistidine-tagged HbPR-1 proteins, which were visualized with 1-Step Ultra TMB-Blotting Solution (Thermoscientific). The size of proteins was estimated with Precision plus protein™ all blue standards (10-250 kDa, BIO-RAD).

The role of PR-1 in the protection of plant against the pathogen
The role of PR-1 protein in plant defense response against the pathogen was conducted using N. benthamiana leaves expressing HbPR-1 by Agrobacterium-mediated transient expression.
The leaves were infiltrated with Agrobacterium carrying HbPR-1 gene and then inoculated with zoospores of the oomycete pathogen P. palmivora. The leaf was divided into two halves based on the midvein. The first half was co-infiltrated with A. tumefaciens strain C58-C1 carrying the pGD_HbPR-1 plasmids and A. tumefaciens strain GV3101 carrying the pJL3-p19 plasmid. The other half which served as control was infiltrated only with A. tumefaciens strain GV3101 carrying the pJL3-p19 plasmid. After 24 h of infiltration, 10-uL of 5×10 2 zoospore/mL or 1×10 3 zoospore/mL was inoculated on 90 leaves from 30 different plants (for each zoospore suspension concentration). Disease incidence and lesion diameter on each leaf were recorded on 3, 4 and 5 d after inoculation.
Effect of recombinant PR-1 protein on P. palmivola zoospore germination The effect of the PR-1 protein on inhibition of P. palmivora zoospore germination was evaluated using sterile distilled water as negative control and antibiotic G418 as positive control. Zoospore suspension (2×10 5 zoospores/mL) was prepared in sterile distilled water from 5-dayold cultures of P. palmivora. Fifty microlitters of this zoospore suspension were added to equal volume of purified HbPR-1 (200 μg/mL), G418 antibiotic (200 μg/mL) or sterile distilled water. After 30 min, 10 μL of each mixture were dropped on 1.5% water agar plates which were then incubated at room temperature for 2 h. Germinated zoospores were counted under an optical microscope and the germination rates were calculated. The experiment was performed with three replicates.

Statistical analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) with Duncan's multiple range test using SPSS Statistics 17.0 software. Statistical significance was taken at P<0.05. All results were presented as the means±SE.

Isolation and molecular characterization of HbPR-1
The HbPR-1 (PR-1 of Hevea brasiliensis or rubber tree) cDNA was successfully isolated from 24 h SA-treated rubber tree leaves using RT-PCR and RACE techniques. The full-length cDNA of HbPR-1 had a total length of 647 nucleotides containing a 34 bp of 5 0 -untranslated region (UTR) located upstream of a start codon (ATG) and 121 bp of 3 0 -UTR that ended with a poly (A) tail. The putative polyadenylation signal sequences, ATAAA, were located downstream of a stop codon (TAA) at nucleotide position 606. The ORF was 492 bp which corresponds to a predicted translated product of 163 amino acids (S1 Fig). The complete cDNA sequence of HbPR-1 gene has been submitted to the GenBank with an accession number KM514666.
The deduced amino acids of HbPR-1 were predicted to have a molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56. HbPR-1 possessed a hydrophobic signal peptide as predicted using SignalP4.1 program. The predicted cleavage site of signal peptide and mature protein was between amino acid A (Alanine) and amino acid Q (Glutamine) (Fig 1). Moreover, by using the primary amino acid sequence of HbPR-1 protein, a high score from the TMHMM result indicated that 163 amino acids of HbPR-1 protein were located on the outside of the cell membrane and the amino acid residues Met1-Ala25 matched the signal peptide with probability score of 0.70. The Protter results also suggested that HbPR-1 protein is located at the extracellular side of the cell membrane. Moreover, Protter tool showed that the twenty-five amino acid residues on the N-terminus end of PR-1 protein have the potential as a signal peptide (Fig  2A). All above analyses suggest that the HbPR-1 protein is secreted into the extracellular space in leaves of rubber tree.
The mature HbPR-1 protein contained six conserved cysteine residues (Fig 1). BLASTP search revealed that HbPR-1 amino acid sequences had the conserved domain of SCP (sperm coating protein)-PR-1_like: SCP-like extracellular protein domain. SCP-like extracellular protein domain may act as an antifungal agent or be involved in cell wall loosening. This family also includes CRISPs (cysteine rich secretory proteins) which combine SCP with a Cterminal cysteine rich domain [54]. Corresponding to the result from ScanProsite tool analysis, HbPR-1 protein contained two domains; CRISP family signature 1 (CRISP_1) at position 120-130 (GHYTQVVWRNS) and CRISP family signature 2 (CRISP_2) at position 146-157 (FIGCNYDPPGNF) (Fig 2).
The deduced amino acid sequences of HbPR-1 were compared to the PR-1 genes of different plant species (Citrus sinensis, Vitis vinifera, Prunus mum, Glycine max, Malus domstica, Ficus pumila var. awkeotsang) using the CLUSTALX program (Fig 1). Similarity searches of the HbPR-1 protein against the non-redundant database of GenBank™ were conducted using the BLASTP program. The amino acid sequences of HbPR-1 have close sequence identity to plant PR-1 proteins in both monocots and dicots (Fig 1).

Homology modeling and analysis of HbPR-1 protein
The predicted 3D model of HbPR-1 protein was simulated using SWISS-MODEL and I-TAS-SER server (Fig 2B and 2C). The homologous template structures of HbPR-1 protein were collected from the RSCB databank, including the NMR solution structure of the chain A of Solanum lycopersicum pathogenesis-related protein P14a, the x-ray diffraction structure of Vespula vulgaris pathogenesis-related protein superfamily, human Golgi-associated plant pathogenesis-related protein and human Glioma pathogenesis-related protein 1 (PDB ID: 1CFE, 1QNX, 1SMB and 3Q2U, respectively). The predicted 3D structure of HbPR-1 was composed of four α-helices, three β-sheets, seven strands, and one junction loop (Fig 2B and 2C). To assess the quality of the predicted model of HbPR-1, the Ramachandran plot analysis was used to visualize backbone dihedral angles ψ against φ of amino acid residues that reflects the stability of the HbPR-1 structure prediction. The amino acid analysis result for HbPR-1 structure prediction showed 98.60% residues plotted in the allowed regions, suggesting that the HbPR-1 structure prediction has good stability (S2 Fig). According to the predicted 3D structure of the HbPR-1 protein (Fig 2), the pairs of cysteine residues that form the disulfide bridges were identified as Cys69/Cys140, Cys113/Cys119 and Cys135/Cys146, respectively. Moreover, the CRISP family signature 1 (amino acid residues at position 120-130) and CRISP family signature 2 (amino acid residues at position 146-157) were also identified ( Fig 2C).
Expression of recombinant HbPR-1 in N. benthamiana. In order to investigate the function of HbPR-1 gene, agroinfiltration was used for transient expression of HbPR-1 fused with the hexahistidine-tag at the amino terminus in N. benthamiana leaves. Total proteins and intercellular fluids were extracted from leaves infiltrated with single strain or a combination of A. tumefaciens cultures expressing pJL3-p19 or pGD_HbPR-1. Recombinant HbPR-1 protein was detected by Western blot with HRP conjugated anti-His monoclonal antibody. A single band which corresponds to molecular weight of the expected HbPR-1 protein (17 kDa) was observed in samples from leaves co-infiltrated with A. tumefaciens strains expressing pJL3-p19 and pGD_HbPR-1 (Fig 4). However, the band was not detected in the samples from leaves infiltrated only with A. tumefaciens expressing pJL3-p19 and the mock control, suggesting that this band represents HbPR-1 protein (Fig 4). The HbPR-1 protein from single infiltration was barely detected and therefore much less than that from co-expression of HbPR-1 and p19, indicating that Agrobacterium-mediated transient expression was enhanced by p19 gene silencing suppressor protein. In addition, the band was observed from intercellular fluids indicating that the recombinant HbPR-1 protein was an extracellular protein, which is consistent with the predicted results using Protter and TMHMM program that the HbPR-1 protein was located at the extracellular side of the cell membrane. The above results demonstrated that expression of HbPR-1 by agroinfiltration was successful, which promised successful purification of HbPR-1 from isolated intercellular fluid.
Purification of recombinant HbPR-1 protein expressed in N. benthamiana. To investigate whether the recombinant HbPR-1 protein expressed in the extracellular space of N. benthamiana can be purified by affinity chromatography, the intercellular fluids isolated from N. benthamiana leaves co-expressing p19 and HbPR-1 proteins were applied onto a column packed with Complete his-tag resin. The bound HbPR-1 was eluted from the column by elution buffer containing different concentrations of 50-250 mM imidazole. The fractions containing HbPR-1 were pooled, desalted and concentrated.
The intercellular fluids before purification ( Fig 5A) and the purified protein ( Fig 5B) were run on SDS-PAGE and stained with Coomassie Brilliant Blue. The purified HbPR-1 protein appeared as a single band with an apparent molecular mass of approximately 17 kDa (Fig 5B), the same size as the distinct band which was only present in intercellular fluids collected from leaves co-expressing p19 and HbPR-1 but not in the control (Fig 5A). HbPR-1 was purified to a high purity after one-step purification process with affinity column, indicating that the system established for HbPR-1 purification was very efficient.

Role of HbPR-1 in plant defense
To evaluate the effect of overexpression of HbPR-1 genes on generation of N. benthamiana resistance to the oomycete pathogen P. palmivora, N. benthamiana leaves were infiltrated with A. tumefaciens cultures of pJL3-p19 (negative control) or co-infiltrated with A. tumefaciens cultures of pJL3-p19 and pGD_HbPR-1. The infiltrated leaves were then inoculated with 10-μL of 5×10 2 zoospore/mL or 1×10 3 zoospore/mL of P. palmivora. The localized necrosis area was observed after inoculation. The expression of HbPR-1 significantly reduced the localized necrosis area (Fig 6). When inoculated with 5×10 2 zoospores/mL, the expression of HbPR-1 reduced necrosis areas by 94.4, 92.0 and 87.6% on 3, 4 and 5 dpi respectively compared to control. Similar reduction of necrosis areas were also observed when inoculated with 1×10 3 zoospores/mL, with reductions of 83.0, 85.6 and 84.5%, respectively (Fig 6A). These results indicated that the expressed HbPR-1 protein played an important role in N. benthamiana resistance to P. palmivora.
Inhibition of HbPR-1 protein on P. palmivora zoospore germination To determine the function of recombinant HbPR-1 protein on inhibiting P. palmivora zoospore germination, zoospores of P. palmivora were incubated with purified HbPR-1 protein for Expression of HbPR-1 from Rubber Tree in Nicotiana benthamiana 30 min and then grown on a water agar plate for 2 h. The zoospore germination was observed and compared with the one in sterile distilled water and antibiotic G418. HbPR-1 exhibited strong inhibition of P. palmivora zoospore germination compared with the negative control (sterile distilled water) whereas G418 showed complete inhibition (Fig 7). The recombinant HbPR-1 inhibited approximately 64% of zoospore germination compared with control. This result indicated that recombinant HbPR-1 protein was an efficient antimicrobial protein against P. palmivora.

Discussion
The full-length cDNA of HbPR-1 had a total length of 647 nucleotides which were predicted to be translated to a product of 163 amino acids with a molecular mass of 17,681 Da and an isoelectric point (pI) of 8.56. Based on the isoelectric point (pI), the PR-1 proteins were classified Expression of HbPR-1 from Rubber Tree in Nicotiana benthamiana into two groups as acidic and basic forms [7]. Previously, two rice PR-1 genes were identified to encode an acidic PR1a protein (pI 4.4) and a basic PR1b protein (pI 8.0) [11]. In addition, an acidic PR-1 protein (pI 5.14) was identified from Oryza grandiglumis leaves [15]. Here we characterized HbPR-1 protein as belonging to the basic PR-1 type.
The mature HbPR-1 protein contained six conserved cysteine residues and two CRISPs (cysteine rich secretory proteins) domains. Generally, mature PR-1 proteins contain six conserved cysteine residues which form disulfide bridges, and exhibit high sequence conservation  level in all plant families [7]. CRISPs are thought to be involved in the plant defense against pathogens and plant stress resistance [55][56]. Since the amino acid sequence of domains in PR-1 proteins showed high conservation in plants, Van Loon and Van Strien [7] suggested that these domains play an important role in the PR-1 protein function.
The 3D structure of HbPR-1 predicted in this study was composed of four α-helices, three β-sheets, seven strands and one junction loop. This was in accordance with the PR-1-type proteins which generally have a specific structure with four α-helices and several β-sheets arranged in antiparallel between helices [6][7]57]. Moreover, all PR-1 proteins also contain six cysteines, ones of a number of conserved residues found in these proteins. These findings indicate that a protein module having been preserved during evolution is defined by the unique molecular structure of PR-1 which must serve important functions [10]. According to the potential binding sites of the predicted structure, the HbPR-1 had the important putative sites that could bind three molecules: Glycerol, Zn 2+ and EAH (5S,7E,9E,11Z,14Z)-5-hydroxyicosa-7,9,11,14-tetraenoic acid). Amino acid residues showed a possible interaction with fatty acid (EAH) and glycerol which is a component of fat molecules, suggesting that amino acid residues of HbPR-1 protein could bind lipid molecules which are components of microbial cell wall. Park et al. [58] found that purified PR-1 protein from pumpkin rind inhibited growth of various fungal pathogens and had membrane permeabilization activity. The damage of the fungal cellular membrane is directly induced by PR-1, resulting in a leakage of cytoplasmic components to the exterior of cell. Therefore, the prediction results in this work could lead to a further study on the PR-1-lipid interaction in order to understand the role of PR-1 in the antimicrobial activity and plant defense. Moreover, the His73 residue on CRISP_1 domain and His121 of HbPR-1 could bind Zn 2+ . CRISPs which have been identified as a toxin family in most animal venoms can bind Zn 2+ at their N-terminal PR-1 domain, but their function remains unknown [59]. Zinc coordination environments in proteins have been defined as catalysis, cocatalysis, structure and interface [60]. Thus, it is probable that PR-1 in plants binds Zn 2+ in order to obtain correct folding of the polypeptide chain. PR-1 may also bind other molecules containing Zn 2+ , which involves in plant defense.
To date, much of the information about the roles and biochemical functions of PR-1 is still unknown. However, it has been reported that PR-1 enhances plant resistance to various pathogens.
Ectopic expression of a pepper basic pathogenesis-related protein 1 gene (CABPR1) in tobacco plants (N. tabacum cv. xanthi) enhanced resistance to the pathogens Ralstonia solanacearum, Pseudomonas syringae pv. tabaci and P. nicotianae [1]. Transgenic tobacco plants overexpressing a basic-type PR-1 gene showed significantly greater antibacterial resistance to P. syringae pv. tabaci than when overexpressing an acidic-type PR-1 gene or control [61]. The overexpression of a pepper basic PR protein 1 gene in tobacco plants may activate a signal transduction pathway that may be related to the resistance against pathogen disease. It may involve reactive oxygen species (ROS) or ion flux generation that causes the activation of defense mechanisms [1]. Although the key mechanisms are still unknown, a role for PR-1 in programmed cell-death (PCD)-related pathways was supported by the fact that PR-1 proteins are pathogen-inducible and frequently isolated from HR-associated plant tissues [9]. Moreover, HbPR-1 protein is an extracellular protein that can effectively prevent pathogen invasions. Although the extracellular defense-related proteins are in a location that they can contact invading pathogens before the tissue has been penetrated, it takes time before the proteins start accumulation. Therefore, effective pathogens' passing into further tissues is likely to occur before the induced proteins become sufficiently active. As a result, the function of these proteins may be effective against following invaders, or involve the development of SAR which provides biochemical barrier against subsequent infections [10].
The recombinant HbPR-1 protein was found to be an antimicrobial protein against P. palmivora, which is in agreement with previous reports showing that PR-1 proteins exhibited a broad-spectrum antimicrobial activity. Zhu et al. [6] expressed and purified N. benthamiana PR-1 (NbPR-1) possessing antimicrobial activity against multiple phytopathogenic fungi including Bipolaris maydis, Fusarium graminearum, Aspergillus oryzae, A. niger and Sclerotinia sclerotiorum. The recombinant Nepenthes mirabilis PR-1 (NmPR-1) protein was found to confer antibacterial activity against both gram-negative P. syringae pv. glycenia and Escherichia coli and gram-positive bacteria Bacillus subtilis [57]. Moreover, purified PR-1 protein from pumpkin rind has antifungal activity against F. moniliforme, F. oxysporum, F. solani, Collectrichum coccodes, Botryris cinerea, Rhizoctonia solani, Candida albicans and A. flavus. PR-1 is thought to damage the fungal cellular membrane and inhibit fungal growth through osmosis or efflux of intracellular components [58]. It has been reported that PR-1 of tobacco and tomato exhibits anti-oomycete activity [1]. Similarly, the obtained results from this study showed that HbPR-1 had an activity against oomycete P. palmivora. The inhibitory effects of plant PR-1 on many pathogens imply that HbPR-1 may also inhibit other pathogens in addition to P. palmivora. The graphic shows that 72.1% (101 residues) of HbPR-1 amino acids was plotted in the most favored regions, 23.6% (33 residues) in additional allowed regions, 2.9% (4 residues) in generously allowed regions and 1.4% (2 residues) in disallowed regions, respectively. The 85.9% (140 residues) was plotted in non-glycine and non-proline residues, 1.2% (2 residues) represented in the end-residues (excl. Gly and Pro), 8.6% (14 residues) was the glycine residues and 4.3% (7 residues) was the proline residues. (TIF)