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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.


Effect of HbPR-1 on the protection of N. benthamiana against P. palmivora.Half leaf was infiltrated with A. tumefaciens GV3101 carrying the pJL3-p19 (p19) and the other half was infiltrated with A. tumefaciens C58-C1 carrying the pGD_HbPR-1 and A. tumefaciens GV3101 carrying the pJL3-p19 (p19+HbPR-1). After 24 h, the leaves were inoculated with 10-μL of 1×103 zoospores/mL. Data represented the average diameter of lesion area with standard error from 90 different leaves of 30 plants. Bars with different letters within day indicated statistically significant differences at P<0.05 according to the Duncan’s multiple range test. (A) Average lesion area of N. benthamiana after inoculation with 1×103 zoospores/mL P. palmivora on 3, 4 and 5 days post inoculation (dpi). (B) Photographs of representative N. benthamiana leaves expressing p19 or p19 together with HbPR-1 at 0 and 5 dpi.
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pone.0157591.g006: Effect of HbPR-1 on the protection of N. benthamiana against P. palmivora.Half leaf was infiltrated with A. tumefaciens GV3101 carrying the pJL3-p19 (p19) and the other half was infiltrated with A. tumefaciens C58-C1 carrying the pGD_HbPR-1 and A. tumefaciens GV3101 carrying the pJL3-p19 (p19+HbPR-1). After 24 h, the leaves were inoculated with 10-μL of 1×103 zoospores/mL. Data represented the average diameter of lesion area with standard error from 90 different leaves of 30 plants. Bars with different letters within day indicated statistically significant differences at P<0.05 according to the Duncan’s multiple range test. (A) Average lesion area of N. benthamiana after inoculation with 1×103 zoospores/mL P. palmivora on 3, 4 and 5 days post inoculation (dpi). (B) Photographs of representative N. benthamiana leaves expressing p19 or p19 together with HbPR-1 at 0 and 5 dpi.

Mentions: To evaluate the effect of overexpression of HbPR-1 genes on generation of N. benthamiana resistance to the oomycete pathogen P. palmivora, N. benthamiana leaves were infiltrated with A. tumefaciens cultures of pJL3-p19 (negative control) or co-infiltrated with A. tumefaciens cultures of pJL3-p19 and pGD_HbPR-1. The infiltrated leaves were then inoculated with 10-μL of 5×102 zoospore/mL or 1×103 zoospore/mL of P. palmivora. The localized necrosis area was observed after inoculation. The expression of HbPR-1 significantly reduced the localized necrosis area (Fig 6). When inoculated with 5×102 zoospores/mL, the expression of HbPR-1 reduced necrosis areas by 94.4, 92.0 and 87.6% on 3, 4 and 5 dpi respectively compared to control. Similar reduction of necrosis areas were also observed when inoculated with 1×103 zoospores/mL, with reductions of 83.0, 85.6 and 84.5%, respectively (Fig 6A). These results indicated that the expressed HbPR-1 protein played an important role in N. benthamiana resistance to 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 HbPR-1 on the protection of N. benthamiana against P. palmivora.Half leaf was infiltrated with A. tumefaciens GV3101 carrying the pJL3-p19 (p19) and the other half was infiltrated with A. tumefaciens C58-C1 carrying the pGD_HbPR-1 and A. tumefaciens GV3101 carrying the pJL3-p19 (p19+HbPR-1). After 24 h, the leaves were inoculated with 10-μL of 1×103 zoospores/mL. Data represented the average diameter of lesion area with standard error from 90 different leaves of 30 plants. Bars with different letters within day indicated statistically significant differences at P<0.05 according to the Duncan’s multiple range test. (A) Average lesion area of N. benthamiana after inoculation with 1×103 zoospores/mL P. palmivora on 3, 4 and 5 days post inoculation (dpi). (B) Photographs of representative N. benthamiana leaves expressing p19 or p19 together with HbPR-1 at 0 and 5 dpi.
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pone.0157591.g006: Effect of HbPR-1 on the protection of N. benthamiana against P. palmivora.Half leaf was infiltrated with A. tumefaciens GV3101 carrying the pJL3-p19 (p19) and the other half was infiltrated with A. tumefaciens C58-C1 carrying the pGD_HbPR-1 and A. tumefaciens GV3101 carrying the pJL3-p19 (p19+HbPR-1). After 24 h, the leaves were inoculated with 10-μL of 1×103 zoospores/mL. Data represented the average diameter of lesion area with standard error from 90 different leaves of 30 plants. Bars with different letters within day indicated statistically significant differences at P<0.05 according to the Duncan’s multiple range test. (A) Average lesion area of N. benthamiana after inoculation with 1×103 zoospores/mL P. palmivora on 3, 4 and 5 days post inoculation (dpi). (B) Photographs of representative N. benthamiana leaves expressing p19 or p19 together with HbPR-1 at 0 and 5 dpi.
Mentions: To evaluate the effect of overexpression of HbPR-1 genes on generation of N. benthamiana resistance to the oomycete pathogen P. palmivora, N. benthamiana leaves were infiltrated with A. tumefaciens cultures of pJL3-p19 (negative control) or co-infiltrated with A. tumefaciens cultures of pJL3-p19 and pGD_HbPR-1. The infiltrated leaves were then inoculated with 10-μL of 5×102 zoospore/mL or 1×103 zoospore/mL of P. palmivora. The localized necrosis area was observed after inoculation. The expression of HbPR-1 significantly reduced the localized necrosis area (Fig 6). When inoculated with 5×102 zoospores/mL, the expression of HbPR-1 reduced necrosis areas by 94.4, 92.0 and 87.6% on 3, 4 and 5 dpi respectively compared to control. Similar reduction of necrosis areas were also observed when inoculated with 1×103 zoospores/mL, with reductions of 83.0, 85.6 and 84.5%, respectively (Fig 6A). These results indicated that the expressed HbPR-1 protein played an important role in N. benthamiana resistance to 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.