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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.


Amino acid sequence alignment of the HbPR-1 from H. brasiliensis with its homologous proteins of different plant species.The sequences of the plant PR-1 proteins were obtained from the GenBank database: Citrus sinensis (XP_006486822.1), Vitis vinifera (XP_ 002273416.1), Prunus mum (XP_008236225.1), Glycine max (XP_003545771.1), Malus domstica (XP_008370577.1), Ficus pumila var. awkeotsang (AFK93500.1). The sequences were aligned by Clustal-X (Thompson et al., 2001). The conserved amino acid sequences were highlighted in red which indicates 100% conserved sequences. The arrowhead indicated the cleavage site between the signal peptide and the mature protein. The positions of the cysteine residues forming disulfide linkages were shown as C. CRISP family signature 1 (CRISP_1) were highlighted in green and CRISP family signature 2 (CRISP_2) were highlighted in blue.
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pone.0157591.g001: Amino acid sequence alignment of the HbPR-1 from H. brasiliensis with its homologous proteins of different plant species.The sequences of the plant PR-1 proteins were obtained from the GenBank database: Citrus sinensis (XP_006486822.1), Vitis vinifera (XP_ 002273416.1), Prunus mum (XP_008236225.1), Glycine max (XP_003545771.1), Malus domstica (XP_008370577.1), Ficus pumila var. awkeotsang (AFK93500.1). The sequences were aligned by Clustal-X (Thompson et al., 2001). The conserved amino acid sequences were highlighted in red which indicates 100% conserved sequences. The arrowhead indicated the cleavage site between the signal peptide and the mature protein. The positions of the cysteine residues forming disulfide linkages were shown as C. CRISP family signature 1 (CRISP_1) were highlighted in green and CRISP family signature 2 (CRISP_2) were highlighted in blue.

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)

Amino acid sequence alignment of the HbPR-1 from H. brasiliensis with its homologous proteins of different plant species.The sequences of the plant PR-1 proteins were obtained from the GenBank database: Citrus sinensis (XP_006486822.1), Vitis vinifera (XP_ 002273416.1), Prunus mum (XP_008236225.1), Glycine max (XP_003545771.1), Malus domstica (XP_008370577.1), Ficus pumila var. awkeotsang (AFK93500.1). The sequences were aligned by Clustal-X (Thompson et al., 2001). The conserved amino acid sequences were highlighted in red which indicates 100% conserved sequences. The arrowhead indicated the cleavage site between the signal peptide and the mature protein. The positions of the cysteine residues forming disulfide linkages were shown as C. CRISP family signature 1 (CRISP_1) were highlighted in green and CRISP family signature 2 (CRISP_2) were highlighted in blue.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0157591.g001: Amino acid sequence alignment of the HbPR-1 from H. brasiliensis with its homologous proteins of different plant species.The sequences of the plant PR-1 proteins were obtained from the GenBank database: Citrus sinensis (XP_006486822.1), Vitis vinifera (XP_ 002273416.1), Prunus mum (XP_008236225.1), Glycine max (XP_003545771.1), Malus domstica (XP_008370577.1), Ficus pumila var. awkeotsang (AFK93500.1). The sequences were aligned by Clustal-X (Thompson et al., 2001). The conserved amino acid sequences were highlighted in red which indicates 100% conserved sequences. The arrowhead indicated the cleavage site between the signal peptide and the mature protein. The positions of the cysteine residues forming disulfide linkages were shown as C. CRISP family signature 1 (CRISP_1) were highlighted in green and CRISP family signature 2 (CRISP_2) were highlighted in blue.
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.