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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.


Related in: MedlinePlus

Purification of HbPR-1 protein expressed in the extracellular space of N. benthamiana leaves.(A) SDS-PAGE of the intercellular fluids isolated from N. benthamiana leaves. Lane 1 indicates intercellular fluid isolated from N. benthamiana leaves infiltrated with A. tumefaciens strain GV3101 carrying the pJL3-p19 and lane 2 indicates intercellular fluid isolated from N. benthamiana leaves co-infiltrated with A. tumefaciens C58-C1 carrying the pGD_HbPR-1 and A. tumefaciens GV3101 carrying the pJL3-P19. (B) SDS-PAGE of the purified HbPR-1 protein. The protein was purified from intercellular fluid shown in A (lane 2) by affinity chromatography with complete his-tag resin. Lane M indicates protein standard and the numbers on the left represent the size of molecular weight markers.
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pone.0157591.g005: Purification of HbPR-1 protein expressed in the extracellular space of N. benthamiana leaves.(A) SDS-PAGE of the intercellular fluids isolated from N. benthamiana leaves. Lane 1 indicates intercellular fluid isolated from N. benthamiana leaves infiltrated with A. tumefaciens strain GV3101 carrying the pJL3-p19 and lane 2 indicates intercellular fluid isolated from N. benthamiana leaves co-infiltrated with A. tumefaciens C58-C1 carrying the pGD_HbPR-1 and A. tumefaciens GV3101 carrying the pJL3-P19. (B) SDS-PAGE of the purified HbPR-1 protein. The protein was purified from intercellular fluid shown in A (lane 2) by affinity chromatography with complete his-tag resin. Lane M indicates protein standard and the numbers on the left represent the size of molecular weight markers.

Mentions: 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.


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)

Purification of HbPR-1 protein expressed in the extracellular space of N. benthamiana leaves.(A) SDS-PAGE of the intercellular fluids isolated from N. benthamiana leaves. Lane 1 indicates intercellular fluid isolated from N. benthamiana leaves infiltrated with A. tumefaciens strain GV3101 carrying the pJL3-p19 and lane 2 indicates intercellular fluid isolated from N. benthamiana leaves co-infiltrated with A. tumefaciens C58-C1 carrying the pGD_HbPR-1 and A. tumefaciens GV3101 carrying the pJL3-P19. (B) SDS-PAGE of the purified HbPR-1 protein. The protein was purified from intercellular fluid shown in A (lane 2) by affinity chromatography with complete his-tag resin. Lane M indicates protein standard and 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.g005: Purification of HbPR-1 protein expressed in the extracellular space of N. benthamiana leaves.(A) SDS-PAGE of the intercellular fluids isolated from N. benthamiana leaves. Lane 1 indicates intercellular fluid isolated from N. benthamiana leaves infiltrated with A. tumefaciens strain GV3101 carrying the pJL3-p19 and lane 2 indicates intercellular fluid isolated from N. benthamiana leaves co-infiltrated with A. tumefaciens C58-C1 carrying the pGD_HbPR-1 and A. tumefaciens GV3101 carrying the pJL3-P19. (B) SDS-PAGE of the purified HbPR-1 protein. The protein was purified from intercellular fluid shown in A (lane 2) by affinity chromatography with complete his-tag resin. Lane M indicates protein standard and the numbers on the left represent the size of molecular weight markers.
Mentions: 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.

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.


Related in: MedlinePlus