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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 cellular localization and predicted structure of HbPR-1.(A) HbPR-1 protein localization predicted by the Protter server. The result suggested that the HbPR-1 protein was located at the extracellular side of the cell membrane. The twenty-five amino acids (red color) at the N-terminus represent the predicted signaling peptide. (B-C) The cartoon representation of the model of HbPR-1 protein predicted using the SWISS-MODEL and I-TASSER server. (B) The graphical display of the 2D topology of the predicted HbPR-1 model. (C) The cartoon structure representation of HbPR-1 3D model with the four α-helices, three β-sheets, seven strands and one junction loop.
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pone.0157591.g002: The putative cellular localization and predicted structure of HbPR-1.(A) HbPR-1 protein localization predicted by the Protter server. The result suggested that the HbPR-1 protein was located at the extracellular side of the cell membrane. The twenty-five amino acids (red color) at the N-terminus represent the predicted signaling peptide. (B-C) The cartoon representation of the model of HbPR-1 protein predicted using the SWISS-MODEL and I-TASSER server. (B) The graphical display of the 2D topology of the predicted HbPR-1 model. (C) The cartoon structure representation of HbPR-1 3D model with the four α-helices, three β-sheets, seven strands and one junction loop.

Mentions: 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.


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 cellular localization and predicted structure of HbPR-1.(A) HbPR-1 protein localization predicted by the Protter server. The result suggested that the HbPR-1 protein was located at the extracellular side of the cell membrane. The twenty-five amino acids (red color) at the N-terminus represent the predicted signaling peptide. (B-C) The cartoon representation of the model of HbPR-1 protein predicted using the SWISS-MODEL and I-TASSER server. (B) The graphical display of the 2D topology of the predicted HbPR-1 model. (C) The cartoon structure representation of HbPR-1 3D model with the four α-helices, three β-sheets, seven strands and one junction loop.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0157591.g002: The putative cellular localization and predicted structure of HbPR-1.(A) HbPR-1 protein localization predicted by the Protter server. The result suggested that the HbPR-1 protein was located at the extracellular side of the cell membrane. The twenty-five amino acids (red color) at the N-terminus represent the predicted signaling peptide. (B-C) The cartoon representation of the model of HbPR-1 protein predicted using the SWISS-MODEL and I-TASSER server. (B) The graphical display of the 2D topology of the predicted HbPR-1 model. (C) The cartoon structure representation of HbPR-1 3D model with the four α-helices, three β-sheets, seven strands and one junction loop.
Mentions: 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.

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.