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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

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          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.

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          Evaluation of time profile reconstruction from complex two-color microarray designs

          Ana Fierro, Raphaël Thuret, Kristof Engelen … (2008)
          Background As an alternative to the frequently used "reference design" for two-channel microarrays, other designs have been proposed. These designs have been shown to be more profitable from a theoretical point of view (more replicates of the conditions of interest for the same number of arrays). However, the interpretation of the measurements is less straightforward and a reconstruction method is needed to convert the observed ratios into the genuine profile of interest (e.g. a time profile). The potential advantages of using these alternative designs thus largely depend on the success of the profile reconstruction. Therefore, we compared to what extent different linear models agree with each other in reconstructing expression ratios and corresponding time profiles from a complex design. Results On average the correlation between the estimated ratios was high, and all methods agreed with each other in predicting the same profile, especially for genes of which the expression profile showed a large variance across the different time points. Assessing the similarity in profile shape, it appears that, the more similar the underlying principles of the methods (model and input data), the more similar their results. Methods with a dye effect seemed more robust against array failure. The influence of a different normalization was not drastic and independent of the method used. Conclusion Including a dye effect such as in the methods lmbr_dye, anovaFix and anovaMix compensates for residual dye related inconsistencies in the data and renders the results more robust against array failure. Including random effects requires more parameters to be estimated and is only advised when a design is used with a sufficient number of replicates. Because of this, we believe lmbr_dye, anovaFix and anovaMix are most appropriate for practical use.
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            An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.

            Olivier Voinnet, Susana Rivas, Pere Mestre … (2003)
            Transient gene expression is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is vacuum-infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic protein expression ceases after 2-3 days. Here, we show that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. We describe a system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced 50-folds or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. The effect of p19 was not saturated in cells that had received up to four individual T-DNAs and persisted until leaf senescence. Because of its simplicity and rapidity, we anticipate that the p19-enhanced expression system will have value in industrial production as well as a research tool for isolation and biochemical characterisation of a broad range of proteins without the need for the time-consuming regeneration of stably transformed plants.
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              Protein structure homology modeling using SWISS-MODEL workspace.

              Lorenza Bordoli, Florian Kiefer, Konstantin Arnold … (2009)
              Homology modeling aims to build three-dimensional protein structure models using experimentally determined structures of related family members as templates. SWISS-MODEL workspace is an integrated Web-based modeling expert system. For a given target protein, a library of experimental protein structures is searched to identify suitable templates. On the basis of a sequence alignment between the target protein and the template structure, a three-dimensional model for the target protein is generated. Model quality assessment tools are used to estimate the reliability of the resulting models. Homology modeling is currently the most accurate computational method to generate reliable structural models and is routinely used in many biological applications. Typically, the computational effort for a modeling project is less than 2 h. However, this does not include the time required for visualization and interpretation of the model, which may vary depending on personal experience working with protein structures.
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                Author and article information

                Contributors
                Chunxian Chen: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, CA USA )
                ISSN (Electronic): 1932-6203
                Publication date (Electronic): 23 June 2016
                Publication date Collection: 2016
                Volume: 11
                Issue: 6
                Electronic Location Identifier: e0157591
                Affiliations
                [1 ]Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand
                [2 ]Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, Manoa, HI, United States of America
                [3 ]Digital Media Program, Prince of Songkla University International College, Prince of Songkla University, Hat Yai, Songkhla, Thailand
                [4 ]Center for Genomics and Bioinformatics Research, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, Thailand
                USDA/ARS, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: NC MT UK. Performed the experiments: UK TP KE. Analyzed the data: NC MT UK. Contributed reagents/materials/analysis tools: NC MT. Wrote the paper: UK. MT NC: Revised and proofread the paper.

                Article
                Publisher ID: PONE-D-16-09457
                DOI: 10.1371/journal.pone.0157591
                PMC ID: 4940168
                PubMed ID: 27337148
                SO-VID: 8a623dcd-fcb9-4f9c-8762-24c6094206ac
                License:

                This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

                History
                Date received : 20 March 2016
                Date accepted : 1 June 2016
                Page count
                Figures: 7, Tables: 0, Pages: 20
                Funding
                Funded by: The Office of the Higher Education Commission Strategic Scholarships Fellowships Frontier Research Networks (Specific for Southern region)
                Award Recipient :
                Funded by: The government budget of Prince of Songkla University
                Funded by: The grant from the Biochemistry Excellence Strengthening Program, Faculty of Science, Prince of Songkla University
                Funded by: The Research Funding of the Graduate School, Prince of Songkla University
                This work was funded by the Office of the Higher Education Commission Strategic Scholarships Fellowships Frontier Research Networks (Specific for Southern region), the government budget of Prince of Songkla University, a grant from the Biochemistry Excellence Strengthening Program, Faculty of Science, Prince of Songkla University 4, and research funding of the Graduate School, Prince of Songkla University.
                Categories
                Subject: Research Article
                Subject: Biology and Life Sciences
                Subject: Biochemistry
                Subject: Proteins
                Subject: Recombinant Proteins
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Bacteria
                Subject: Agrobacteria
                Subject: Agrobacterium Tumefaciens
                Subject: Biology and Life Sciences
                Subject: Microbiology
                Subject: Plant Microbiology
                Subject: Agrobacteria
                Subject: Agrobacterium Tumefaciens
                Subject: Biology and Life Sciences
                Subject: Plant Science
                Subject: Plant Microbiology
                Subject: Agrobacteria
                Subject: Agrobacterium Tumefaciens
                Subject: Biology and Life Sciences
                Subject: Molecular Biology
                Subject: Macromolecular Structure Analysis
                Subject: Protein Structure
                Subject: Protein Structure Prediction
                Subject: Biology and Life Sciences
                Subject: Biochemistry
                Subject: Proteins
                Subject: Protein Structure
                Subject: Protein Structure Prediction
                Subject: Biology and Life Sciences
                Subject: Plant Science
                Subject: Plant Anatomy
                Subject: Leaves
                Subject: Biology and Life Sciences
                Subject: Biochemistry
                Subject: Plant Biochemistry
                Subject: Biology and Life Sciences
                Subject: Plant Science
                Subject: Plant Biochemistry
                Subject: Physical Sciences
                Subject: Chemistry
                Subject: Polymer Chemistry
                Subject: Macromolecules
                Subject: Polymers
                Subject: Elastomers
                Subject: Rubber
                Subject: Physical Sciences
                Subject: Materials Science
                Subject: Materials by Structure
                Subject: Polymers
                Subject: Elastomers
                Subject: Rubber
                Subject: Biology and Life Sciences
                Subject: Plant Science
                Subject: Plant Pathology
                Subject: Plant Pathogens
                Subject: Biology and Life Sciences
                Subject: Agriculture
                Subject: Crop Science
                Subject: Crops
                Subject: Fruits
                Subject: Figs
                Subject: Biology and Life Sciences
                Subject: Organisms
                Subject: Plants
                Subject: Fruits
                Subject: Figs
                Custom metadata
                Data Availability All relevant data are within the paper and its Supporting Information files.

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