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.

The plant defense and pathogen counterdefense mediated by Hevea brasiliensis serine protease HbSPA and Phytophthora palmivora extracellular protease inhibitor PpEPI10

“…tumefaciens stain C58-C1 carrying pGD- HbSPA , pGD- HbSPB , or pGD- HbSPC , with or without A . tumefaciens stain GV3101 carrying pJL3- P19 , was achieved by agroinfiltration following the methods previously described [ 36 ]. The expression of His-tagged HbSPA, HbSPB, and HbSPC proteins was analyzed by Western blot using HRP conjugated anti-His monoclonal antibody His-probe (H-3) (sc-8036HRP, Santa Cruz Biotechnology, INC., http://www.scbt.com ) as described previously [ 36 ].…”

“…Leaves of 6-week-old N . benthamiana plants were infiltrated with a mixture of Agrobacterium stain C58-C1 carrying pGD- HbSPA and Agrobacterium stain GV3101 carrying pJL3- P19 on one half leaf (delineated by the midvein), and on another half Agrobacterium stain GV3101 carrying pJL3- P19 was infiltrated to serve as a control, following the methods described previously [ 36 ]. After that, plants were maintained in the laboratory under continuous fluorescent lighting for protein expression.…”

A secreted protein of 15 kDa plays an important role in Phytophthora palmivora development and pathogenicity

“…The pJL-TRBO-Ppal15kDaA (2020) 10:2319 | https://doi.org/10.1038/s41598-020-59007-1 www.nature.com/scientificreports www.nature.com/scientificreports/ and pJL-TRBO-Ppal15kDaB plasmids containing the correct Ppal15kDaA and Ppal15kDaB nucleotide sequences were used to transform A. tumefaciens stain GV3101 by electroporation. Transient protein expression in N. benthamiana was performed using A. tumefaciens GV3101 carrying the above plasmids or pJL-TRBO-G expressing GFP 35 (as a control) as described previously by Khunjan et al 56 .…”

Sulphated Polysaccharide from Acanthophora spicifera Induced Hevea brasiliensis Defense Responses Against Phytophthora palmivora Infection

“…In contrast, the sulphated polysaccharide (SPS) from H. musciformis enhances expression of genes involving SA and subsequently activated the JA/ET signaling pathway [12]. P. palmivora infection is inhibited in N. benthamina that expresses the PR-1 protein; furthermore, the PR-1 protein acts as an antifungal protein by inhibiting zoospore germination [31]. The cell wall of the oomycete mainly consists of β-1,3-glucan [32] which is a substrate of β-1,3-glucanase.…”

“…The GenBank accession no. KM514666 [31], AY325498 [41], and KM979450 [42] were used in the design of the specific primers of HbPR-1 , HbGLU, and HbASI genes, respectively. A housekeeping gene, the mitosis protein YLS8 gene ( HbMito ) of H. brasiliensis (GenBank accession no.…”