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  JoVE Developmental Biology


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October, 2006
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 JoVE Biology

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

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

JoVE 50971

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.

 JoVE Immunology and Infection

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

1Plant Sciences Institute, Agricultural Research Service, United States Department of Agriculture, 2Molecular Plant Pathology Laboratory, Agricultural Research Service, United States Department of Agriculture

JoVE 50084

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.

 JoVE Biology

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

1Plant Pathology and Plant-Microbe Biology, Cornell University, 2Boyce Thompson Institute for Plant Research

JoVE 1292

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

 JoVE Biology

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

1Fraunhofer USA Center for Molecular Biotechnology

JoVE 51204

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.

 JoVE Biology

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

1The College of Technology and Innovation, Center for Infectious Disease and Vaccinology, The Biodesign Institute, Arizona State University

JoVE 50521

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.

 JoVE Biology

A Comparative Analysis of Recombinant Protein Expression in Different Biofactories: Bacteria, Insect Cells and Plant Systems

1Department of Biotechnology, University of Verona, Verona, Italy, 2Department of Internal Medicine, University of Perugia, Perugia, Italy

JoVE 52459

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.

 JoVE Immunology and Infection

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

1Plant Biology Division, The Samuel Roberts Noble Foundation

JoVE 51033

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.

 JoVE Environment

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

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

JoVE 51234

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

 JoVE Biology

Identification of Post-translational Modifications of Plant Protein Complexes

1School of Life Sciences, University of Warwick, 2The Sainsbury Laboratory, Norwich Research Park, 3Research School of Biology, The Australian National University

JoVE 51095

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.

 JoVE Biology

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

1Department of Biochemical, Cellular and Molecular Biology, University of Tennessee, Knoxville, 2Advanced Microscopy and Imaging Facility, University of Tennessee, Knoxville

JoVE 51844

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.

 JoVE Bioengineering

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

1Institute for Molecular Biotechnology, RWTH Aachen University, 2Institute for Molecular Biology and Applied Ecology, Fraunhofer Gesellschaft

JoVE 51216

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.

 JoVE Immunology and Infection

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

1Department of Plant Pathology and Microbiology, University of California, Riverside, 2Department of Bioengineering, University of California, Riverside

JoVE 3645

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.

 JoVE Bioengineering

Viral Nanoparticles for In vivo Tumor Imaging

1Department of Biomedical Engineering, Case Western Reserve University, 2Department of Biomedical Engineering, Radiology, and Materials Science and Engineering, Case Western Reserve University

JoVE 4352

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.

 JoVE Biology

A β-glucuronidase (GUS) Based Cell Death Assay

1Department of Plant Pathology and Microbiology, Institute for Plant Genomics and Biotechnology, Texas A&M University

JoVE 2680

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.

 JoVE Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

1Department of Biochemistry and Cell Biology, State University of New York at Stony Brook, 2Bio-Medical Engineering Department, NED University of Engineering and Technology

JoVE 2208

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.

 JoVE Biology

One-step Purification of Twin-Strep-tagged Proteins and Their Complexes on Strep-Tactin Resin Cross-linked With Bis(sulfosuccinimidyl) Suberate (BS3)

1Department of Food and Environmental Sciences, University of Helsinki, 2Institute of Biotechnology, University of Helsinki

JoVE 51536

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.

 JoVE Immunology and Infection

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

1Boyce Thompson Institute for Plant Research, 2Plant Pathology and Plant-Microbe Biology, Cornell University

JoVE 1442

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

 JoVE Biology

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

1Department of Biology, University of Western Ontario, 2Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada

JoVE 3473

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.

 JoVE Biology

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

1Department Biologie I, Botanik, Ludwig-Maximilians-Universität, München

JoVE 51327

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.

 JoVE Biology

Assaying Proteasomal Degradation in a Cell-free System in Plants

1Department of Biochemistry and Cell Biology, Stony Brook University, State University of New York

JoVE 51293

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.

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