1Wageningen UR Plant Breeding, Wageningen University, 2Key Laboratory of Horticultural Plant Biology, Huazhong Agricultural University, Ministry of Education, National Centre for Vegetable Improvement (Central China), Potato Engineering and Technology Research Centre of Hubei Province, Huazhong Agricultural University
Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.
Published January 3, 2014. Keywords: Plant Biology, Genetics, Bioengineering, Plants, Genetically Modified, DNA, Plant Immunity, Plant Diseases, Genes, Genome, Plant Pathology, Effectoromics, Agroinfiltration, PVX agroinfection, potato, Nicotiana benthamiana, high-throughput, functional genomics
JoVE Immunology and Infection
1Plant Sciences Institute, Agricultural Research Service, United States Department of Agriculture, 2Molecular Plant Pathology Laboratory, Agricultural Research Service, United States Department of Agriculture
A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.
Published February 14, 2013. Keywords: Virology, Plant Biology, Infection, Molecular Biology, Biochemistry, Proteins, Chemicals and Drugs, Analytical, Diagnostic and Therapeutic Techniques and Equipment, Technology, Industry, Agriculture, Chemistry and materials, Virus-like particles (VLPs), VLP, sulfhydryl-reactive chemistries, labeling, cross-linking, multivalent display, Maize rayado fino virus, mosaic virus, virus, nanoparticle, drug delivery, peptides, Nicotiana benthamiana, plant model
1Plant Pathology and Plant-Microbe Biology, Cornell University, 2Boyce Thompson Institute for Plant Research
Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.
Published June 10, 2009. Keywords: Plant Biology, Virus-induced gene silencing (VIGS), RNA interference (RNAi), Tobacco Rattle Virus (TRV) vectors, Nicotiana benthamiana, tomato
1Fraunhofer USA Center for Molecular Biotechnology
Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.
Published April 19, 2014. Keywords: Plant Biology, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
1The College of Technology and Innovation, Center for Infectious Disease and Vaccinology, The Biodesign Institute, Arizona State University
Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.
Published July 23, 2013. Keywords: Plant Biology, Genetics, Molecular Biology, Cellular Biology, Virology, Microbiology, Bioengineering, Plant Viruses, Antibodies, Monoclonal, Green Fluorescent Proteins, Plant Proteins, Recombinant Proteins, Vaccines, Synthetic, Virus-Like Particle, Gene Transfer Techniques, Gene Expression, Agroinfiltration, plant infiltration, plant-made pharmaceuticals, syringe agroinfiltration, vacuum agroinfiltration, monoclonal antibody, Agrobacterium tumefaciens, Nicotiana benthamiana, GFP, DsRed, geminiviral vectors, imaging, plant model
1Department of Biotechnology, University of Verona, Verona, Italy, 2Department of Internal Medicine, University of Perugia, Perugia, Italy
In this study the expression of a target human recombinant protein in different production platforms was compared. We focused on traditional fermenter-based cultures and on plants, describing the set-up of each system and highlighting, on the basis of the reported results, the inherent limits and advantages for each platform.
Published March 23, 2015. Keywords: Plant Biology, Plant biotechnology, transient expression, stable expression, transgenic plant, Nicotiana tabacum, Nicotiana benthamiana, Baculovirus/insect cells, recombinant protein
JoVE Immunology and Infection
1Plant Biology Division, The Samuel Roberts Noble Foundation
Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.
Published August 23, 2013. Keywords: Virology, Plant Biology, Infection, Genetics, Molecular Biology, Cellular Biology, Physiology, Genomics, Pathology, plants, Nonhost Resistance, Virus-induced gene silencing, VIGS, disease resistance, gene silencing, Pseudomonas, GFPuv, sequencing, virus, Nicotiana benthamiana, plant model
1Synthetic Biology Platform, Wyss Institute for Biologically Inspired Engineering, Harvard University, 2Department of Systems Biology, Harvard Medical School, 3Department of Biotechnology, Delft University of Technology
This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.
Published April 18, 2014. Keywords: Environmental Sciences, Plant Leaves, Synthetic Biology, Plants, Genetically Modified, DNA, Plant, RNA, Gene Targeting, Plant Physiological Processes, Genes, Gene gun, Gibson assembly, Nicotiana benthamiana, Alternative splicing, confocal microscopy, chloroplast, peroxisome
1School of Life Sciences, University of Warwick, 2The Sainsbury Laboratory, Norwich Research Park, 3Research School of Biology, The Australian National University
We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.
Published February 22, 2014. Keywords: Plant Biology, plant-microbe interactions, protein complex purification, mass spectrometry, protein phosphorylation, Prf, Pto, AvrPto, AvrPtoB
1Department of Biochemical, Cellular and Molecular Biology, University of Tennessee, Knoxville, 2Advanced Microscopy and Imaging Facility, University of Tennessee, Knoxville
Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.
Published October 13, 2014. Keywords: Plant Biology, High-pressure freezing, freeze substitution, transmission electron microscopy, ultrastructure, Nicotiana benthamiana, Arabidopsis thaliana, imaging, cryofixation, dehydration
1Institute for Molecular Biotechnology, RWTH Aachen University, 2Institute for Molecular Biology and Applied Ecology, Fraunhofer Gesellschaft
We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.
Published January 31, 2014. Keywords: Bioengineering, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
JoVE Immunology and Infection
1Department of Plant Pathology and Microbiology, University of California, Riverside, 2Department of Bioengineering, University of California, Riverside
A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.
Published March 1, 2012. Keywords: Immunology, Agrobacterium, Brome mosaic virus, Nicotiana benthamiana, encapsidation, dissociation, in vitro assembly, Nano technology
1Department of Biomedical Engineering, Case Western Reserve University, 2Department of Biomedical Engineering, Radiology, and Materials Science and Engineering, Case Western Reserve University
Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.
Published November 16, 2012. Keywords: Cancer Biology, Bioengineering, Biomedical Engineering, Molecular Biology, Virology, Oncology, Viral nanoparticles, bioconjugate chemistry, tumor xenograft mouse model, fluorescence imaging
1Department of Plant Pathology and Microbiology, Institute for Plant Genomics and Biotechnology, Texas A&M University
Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.
Published May 6, 2011. Keywords: Plant Biology, Cell death, GUS, Transient expression, Nicotiana benthamiana.
1Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 2Bio-Medical Engineering Department, NED University of Engineering and Technology
Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.
Published August 27, 2010. Keywords: Cellular Biology, Symplastic transport, transient expression, microbombardment, fluorescent protein, plant, confocal microscopy
1Department of Food and Environmental Sciences, University of Helsinki, 2Institute of Biotechnology, University of Helsinki
A method is described for efficient purification of twin-Strep-tagged fusion proteins and their specific complexes on modified streptavidin (Strep-Tactin) resin covalently cross-linked with Bis(sulfosuccinimidyl) suberate (BS3). The method has the advantages of fast speed, good target protein recovery and high purity, and is compatible with subsequent analysis by mass spectrometry.
Published April 20, 2014. Keywords: Biochemistry, Strep-tag, fusion protein, Strep-Tactin, protein complex purification, bis(sulfosuccinimidyl) suberate, BS3, protein cross-linking, protein structure stabilization, proteomics, mass spectrometry
JoVE Immunology and Infection
1Boyce Thompson Institute for Plant Research, 2Plant Pathology and Plant-Microbe Biology, Cornell University
A cell death-based assay for PTI in Nicotiana benthamiana plants is described.
Published September 9, 2009. Keywords: Jove Infectious Diseases, Plant Biology, plant immunity, pathogen-associated molecular pattern (PAMP), PAMP-triggered immunity (PTI), effector-triggered immunity (ETI), Nicotiana benthamiana
1Department of Biology, University of Western Ontario, 2Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada
We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.
Published September 16, 2011. Keywords: Plant Biology, protein interaction, Gateway, Bimolecular fluorescence complementation, Confocal microscope, Agrobacterium, Nicotiana benthamiana, Arabidopsis
1Department Biologie I, Botanik, Ludwig-Maximilians-Universität, München
Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.
Published March 9, 2014. Keywords: Plant Biology, Tetratricopeptide repeat domain, chaperone, chloroplasts, endoplasmic reticulum, HSP90, Toc complex, Sec translocon, BiFC
1Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York
Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular “tags” for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.
Published March 26, 2014. Keywords: Biochemistry, Ubiquitin/proteasome system, 26S proteasome, protein degradation, proteasome inhibitor, Western blotting, plant genetic transformation