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

Khunjan U, Ekchaweng K, Panrat T, Tian M, Churngchow N - PLoS ONE (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand.

ABSTRACT
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.

No MeSH data available.


The putative binding sites of HbPR-1 protein.(A) The putative binding sites of HbPR-1 protein were predicted by the I-TASSER. The result suggested the possible locations of HbPR-1 protein interacting with glycerol (red-yellow color), Zn2+ (navy blue color) and EAH (pink-red color). (B-D) The putative residues of HbPR-1 protein that might be bound to the glycerol molecule (B), Zn2+ ion (C) and EAH molecule (D), respectively.
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pone.0157591.g003: The putative binding sites of HbPR-1 protein.(A) The putative binding sites of HbPR-1 protein were predicted by the I-TASSER. The result suggested the possible locations of HbPR-1 protein interacting with glycerol (red-yellow color), Zn2+ (navy blue color) and EAH (pink-red color). (B-D) The putative residues of HbPR-1 protein that might be bound to the glycerol molecule (B), Zn2+ ion (C) and EAH molecule (D), respectively.

Mentions: For further investigation of HbPR-1 amino acids within the 3D predicted structure, the potential binding sites were predicted using I-TASSER server. The I-TASSER prediction results suggested that the HbPR-1 structure had the important putative sites that could bind three molecules: Glycerol, Zn2+ and EAH (5S,7E,9E,11Z,14Z)-5-hydroxyicosa-7,9,11,14-tetraenoic acid) (Fig 3). With the prediction sites of the HbPR-1 model, the amino acid residue Leu23, Asn24, Ser28, Gln59 and Lys136-137 showed a possible interaction with Glycerol (Fig 3B). The His73 residue on CRISP_1 domain and His121 of HbPR-1 could bind Zn2+ ion (Fig 3C). Amino acid residue Ala35, Ala39, Ala42, Asn82, Ala95, Val96, Trp99, Val100, Lys103, His121 and Cys142 within α-helix H1 and H3 were possibly bound to EAH molecule (Fig 3D). To further analyze HbPR-1 structure in details, we have predicted the protein-protein interaction sites by using the eFindSite server. The eFindSite prediction result suggested that HbPR-1 had a high confidential score of protein-protein binding with the thirteen interfacial residues: Asn70, Val72-Asn76, Gln118, Gly120, Asn156-Phe157, and Gly159-Lys161, respectively. Furthermore, the eFindSite prediction hinted that the Asn156 residue was the hydrogen bond binding and the His73 residue was aromatic interaction site.


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

Khunjan U, Ekchaweng K, Panrat T, Tian M, Churngchow N - PLoS ONE (2016)

The putative binding sites of HbPR-1 protein.(A) The putative binding sites of HbPR-1 protein were predicted by the I-TASSER. The result suggested the possible locations of HbPR-1 protein interacting with glycerol (red-yellow color), Zn2+ (navy blue color) and EAH (pink-red color). (B-D) The putative residues of HbPR-1 protein that might be bound to the glycerol molecule (B), Zn2+ ion (C) and EAH molecule (D), respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940168&req=5

pone.0157591.g003: The putative binding sites of HbPR-1 protein.(A) The putative binding sites of HbPR-1 protein were predicted by the I-TASSER. The result suggested the possible locations of HbPR-1 protein interacting with glycerol (red-yellow color), Zn2+ (navy blue color) and EAH (pink-red color). (B-D) The putative residues of HbPR-1 protein that might be bound to the glycerol molecule (B), Zn2+ ion (C) and EAH molecule (D), respectively.
Mentions: For further investigation of HbPR-1 amino acids within the 3D predicted structure, the potential binding sites were predicted using I-TASSER server. The I-TASSER prediction results suggested that the HbPR-1 structure had the important putative sites that could bind three molecules: Glycerol, Zn2+ and EAH (5S,7E,9E,11Z,14Z)-5-hydroxyicosa-7,9,11,14-tetraenoic acid) (Fig 3). With the prediction sites of the HbPR-1 model, the amino acid residue Leu23, Asn24, Ser28, Gln59 and Lys136-137 showed a possible interaction with Glycerol (Fig 3B). The His73 residue on CRISP_1 domain and His121 of HbPR-1 could bind Zn2+ ion (Fig 3C). Amino acid residue Ala35, Ala39, Ala42, Asn82, Ala95, Val96, Trp99, Val100, Lys103, His121 and Cys142 within α-helix H1 and H3 were possibly bound to EAH molecule (Fig 3D). To further analyze HbPR-1 structure in details, we have predicted the protein-protein interaction sites by using the eFindSite server. The eFindSite prediction result suggested that HbPR-1 had a high confidential score of protein-protein binding with the thirteen interfacial residues: Asn70, Val72-Asn76, Gln118, Gly120, Asn156-Phe157, and Gly159-Lys161, respectively. Furthermore, the eFindSite prediction hinted that the Asn156 residue was the hydrogen bond binding and the His73 residue was aromatic interaction site.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand.

ABSTRACT
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.

No MeSH data available.