Molecular Cloning of HbPR-1 Gene from Rubber Tree, Expression of HbPR-1 Gene in Nicotiana benthamiana and Its Inhibition of Phytophthora palmivora

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.

Fig 1. 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.

Fig 2. The putative cellular localization and predicted structure of HbPR-1.

(A) HbPR-1 protein localization predicted by the Protter server. The result suggested that the HbPR-1 protein was located at the extracellular side of the cell membrane. The twenty-five amino acids (red color) at the N-terminus represent the predicted signaling peptide. (B-C) The cartoon representation of the model of HbPR-1 protein predicted using the SWISS-MODEL and I-TASSER server. (B) The graphical display of the 2D topology of the predicted HbPR-1 model. (C) The cartoon structure representation of HbPR-1 3D model with the four α-helices, three β-sheets, seven strands and one junction loop.

Fig 3. The putative binding sites of HbPR-1 protein.

(A) The putative binding sites of HbPR-1 protein were predicted by the I-TASSER. The result suggested the possible locations of HbPR-1 protein interacting with glycerol (red-yellow color), Zn²⁺ (navy blue color) and EAH (pink-red color). (B-D) The putative residues of HbPR-1 protein that might be bound to the glycerol molecule (B), Zn²⁺ ion (C) and EAH molecule (D), respectively.

Fig 4. 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.

Fig 5. 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.

Fig 6. 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.

Fig 7. 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.