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
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