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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

Effect of purified HbPR-1 on germination of P. palmivora zoospores.(A) The percentage of zoospore germination after zoospores were treated with purified PR-1 or sterile distilled water (control) or G418 for 30 min and then grown on 1.5% water agar for 2 h. Data represent the average of percentage of zoospore germination with standard error of three replicates. Bars with different letters indicated statistically significant differences at P<0.05. (B) Photographs of zoospores of P. palmivora grown on 1.5% water agar after treatments.
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pone.0157591.g007: Effect of purified HbPR-1 on germination of P. palmivora zoospores.(A) The percentage of zoospore germination after zoospores were treated with purified PR-1 or sterile distilled water (control) or G418 for 30 min and then grown on 1.5% water agar for 2 h. Data represent the average of percentage of zoospore germination with standard error of three replicates. Bars with different letters indicated statistically significant differences at P<0.05. (B) Photographs of zoospores of P. palmivora grown on 1.5% water agar after treatments.

Mentions: To determine the function of recombinant HbPR-1 protein on inhibiting P. palmivora zoospore germination, zoospores of P. palmivora were incubated with purified HbPR-1 protein for 30 min and then grown on a water agar plate for 2 h. The zoospore germination was observed and compared with the one in sterile distilled water and antibiotic G418. HbPR-1 exhibited strong inhibition of P. palmivora zoospore germination compared with the negative control (sterile distilled water) whereas G418 showed complete inhibition (Fig 7). The recombinant HbPR-1 inhibited approximately 64% of zoospore germination compared with control. This result indicated that recombinant HbPR-1 protein was an efficient antimicrobial protein against P. palmivora.


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)

Effect of purified HbPR-1 on germination of P. palmivora zoospores.(A) The percentage of zoospore germination after zoospores were treated with purified PR-1 or sterile distilled water (control) or G418 for 30 min and then grown on 1.5% water agar for 2 h. Data represent the average of percentage of zoospore germination with standard error of three replicates. Bars with different letters indicated statistically significant differences at P<0.05. (B) Photographs of zoospores of P. palmivora grown on 1.5% water agar after treatments.
© Copyright Policy
Related In: Results  -  Collection

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pone.0157591.g007: Effect of purified HbPR-1 on germination of P. palmivora zoospores.(A) The percentage of zoospore germination after zoospores were treated with purified PR-1 or sterile distilled water (control) or G418 for 30 min and then grown on 1.5% water agar for 2 h. Data represent the average of percentage of zoospore germination with standard error of three replicates. Bars with different letters indicated statistically significant differences at P<0.05. (B) Photographs of zoospores of P. palmivora grown on 1.5% water agar after treatments.
Mentions: To determine the function of recombinant HbPR-1 protein on inhibiting P. palmivora zoospore germination, zoospores of P. palmivora were incubated with purified HbPR-1 protein for 30 min and then grown on a water agar plate for 2 h. The zoospore germination was observed and compared with the one in sterile distilled water and antibiotic G418. HbPR-1 exhibited strong inhibition of P. palmivora zoospore germination compared with the negative control (sterile distilled water) whereas G418 showed complete inhibition (Fig 7). The recombinant HbPR-1 inhibited approximately 64% of zoospore germination compared with control. This result indicated that recombinant HbPR-1 protein was an efficient antimicrobial protein against P. palmivora.

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