Limits...
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.


Transient expression of HbPR-1 protein in N. benthamiana.Total proteins and intercellular fluids were isolated from agroinfiltrated N. benthamiana plants and were visualized by Western blot analysis. Lane M represents protein standard and lanes 1–4 represent total proteins isolated from infiltrated N. benthamiana leaves with infiltration buffer (Mock, lane 1), A. tumefaciens GV3101 expressing pJL3-p19 (lane 2), A. tumefaciens C58C1 expressing pGD_HbPR-1 (lane 3) and a mixture of A. tumefaciens strains expressing pJL3-p19 and pGD_HbPR-1 (lane 4). Lane 5 and 6 represent intercellular fluids isolated from infiltrated N. benthamiana leaves with A. tumefaciens GV3101 expressing pJL3-p19 (lane 5) and a mixture of A. tumefaciens strains expressing pJL3-p19 and pGD_HbPR-1 (lane 6), respectively. The numbers on the left represent the size of molecular weight markers.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4940168&req=5

pone.0157591.g004: Transient expression of HbPR-1 protein in N. benthamiana.Total proteins and intercellular fluids were isolated from agroinfiltrated N. benthamiana plants and were visualized by Western blot analysis. Lane M represents protein standard and lanes 1–4 represent total proteins isolated from infiltrated N. benthamiana leaves with infiltration buffer (Mock, lane 1), A. tumefaciens GV3101 expressing pJL3-p19 (lane 2), A. tumefaciens C58C1 expressing pGD_HbPR-1 (lane 3) and a mixture of A. tumefaciens strains expressing pJL3-p19 and pGD_HbPR-1 (lane 4). Lane 5 and 6 represent intercellular fluids isolated from infiltrated N. benthamiana leaves with A. tumefaciens GV3101 expressing pJL3-p19 (lane 5) and a mixture of A. tumefaciens strains expressing pJL3-p19 and pGD_HbPR-1 (lane 6), respectively. The numbers on the left represent the size of molecular weight markers.

Mentions: 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.


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)

Transient expression of HbPR-1 protein in N. benthamiana.Total proteins and intercellular fluids were isolated from agroinfiltrated N. benthamiana plants and were visualized by Western blot analysis. Lane M represents protein standard and lanes 1–4 represent total proteins isolated from infiltrated N. benthamiana leaves with infiltration buffer (Mock, lane 1), A. tumefaciens GV3101 expressing pJL3-p19 (lane 2), A. tumefaciens C58C1 expressing pGD_HbPR-1 (lane 3) and a mixture of A. tumefaciens strains expressing pJL3-p19 and pGD_HbPR-1 (lane 4). Lane 5 and 6 represent intercellular fluids isolated from infiltrated N. benthamiana leaves with A. tumefaciens GV3101 expressing pJL3-p19 (lane 5) and a mixture of A. tumefaciens strains expressing pJL3-p19 and pGD_HbPR-1 (lane 6), respectively. The numbers on the left represent the size of molecular weight markers.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0157591.g004: Transient expression of HbPR-1 protein in N. benthamiana.Total proteins and intercellular fluids were isolated from agroinfiltrated N. benthamiana plants and were visualized by Western blot analysis. Lane M represents protein standard and lanes 1–4 represent total proteins isolated from infiltrated N. benthamiana leaves with infiltration buffer (Mock, lane 1), A. tumefaciens GV3101 expressing pJL3-p19 (lane 2), A. tumefaciens C58C1 expressing pGD_HbPR-1 (lane 3) and a mixture of A. tumefaciens strains expressing pJL3-p19 and pGD_HbPR-1 (lane 4). Lane 5 and 6 represent intercellular fluids isolated from infiltrated N. benthamiana leaves with A. tumefaciens GV3101 expressing pJL3-p19 (lane 5) and a mixture of A. tumefaciens strains expressing pJL3-p19 and pGD_HbPR-1 (lane 6), respectively. The numbers on the left represent the size of molecular weight markers.
Mentions: 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.

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.