Split green fluorescent protein as a tool to study infection with a plant pathogen, Cauliflower mosaic virus
| Autor: | Carine Alcon, Catherine Curie, Daniel Gargani, Beatriz Dáder, Jaclyn S Zhou, Jean-Luc Macia, Véronique Brault, James C. K. Ng, Myriam Burckbuchler, Martin Drucker |
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| Přispěvatelé: | Biologie et Génétique des Interactions Plante-Parasite (UMR BGPI), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro), Santé de la vigne et qualité du vin (SVQV), Université de Strasbourg (UNISTRA)-Institut National de la Recherche Agronomique (INRA), Biochimie et Physiologie Moléculaire des Plantes (BPMP), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Department of Microbiology and Plant Pathology, INRA SPE department, Agence Nationale de la Recherche : 12-BSV7-005-01, Human Frontier Science Program : RGP0013/2015, European Project: 609398,EC:FP7:PEOPLE,FP7-PEOPLE-2013-COFUND,AGREENSKILLSPLUS(2014), Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Université de Strasbourg (UNISTRA), Université de Montpellier (UM)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro)-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Leaves Physiology [SDV]Life Sciences [q-bio] Arabidopsis Plant Science Plant Genetics medicine.disease_cause Biochemistry Genetically Modified Plants 01 natural sciences Green fluorescent protein Fluorescence Microscopy Caulimovirus Viral factory Plant Genomics Medicine and Health Sciences Arabidopsis thaliana pathologie végétale ComputingMilieux_MISCELLANEOUS Microscopy Viral Genomics 0303 health sciences Microscopy Confocal Multidisciplinary biology Plant Anatomy Genetically Modified Organisms Microbiology and Parasitology Eukaryota Light Microscopy food and beverages Genomics Plants Plants Genetically Modified Microbiologie et Parasitologie Body Fluids Blood Experimental Organism Systems Engineering and Technology Medicine Anatomy Genetic Engineering Research Article Biotechnology Viral protein Recombinant Fusion Proteins Arabidopsis Thaliana Science Green Fluorescent Proteins Mutagenesis (molecular biology technique) Bioengineering Context (language use) Brassica Microbial Genomics Research and Analysis Methods Green Fluorescent Protein Microbiology Virus Viral Proteins 03 medical and health sciences Model Organisms Plant and Algal Models Virology Genetics medicine [SDV.BV]Life Sciences [q-bio]/Vegetal Biology Plant Diseases 030304 developmental biology fungi Organisms Biology and Life Sciences Proteins Blood Serum biology.organism_classification Plant Leaves Luminescent Proteins caulimovirus mosaïque du chou fleur Mutagenesis Site-Directed Animal Studies Plant Biotechnology Cauliflower mosaic virus Immune Serum [SDV.AEN]Life Sciences [q-bio]/Food and Nutrition 010606 plant biology & botany |
| Zdroj: | PLoS ONE PLoS ONE, Public Library of Science, 2019, 14 (3), pp.e0213087. ⟨10.1371/journal.pone.0213087⟩ PLoS ONE, Vol 14, Iss 3, p e0213087 (2019) PloS One Plos One 3 (14), . (2019) PLoS ONE, 2019, 14 (3), pp.e0213087. ⟨10.1371/journal.pone.0213087⟩ |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0213087⟩ |
| Popis: | The split GFP technique is based on the auto-assembly of GFP when two polypeptides– GFP1-10 (residues 1–214; the detector) and GFP11 (residues 215–230; the tag)–both non- fluorescing on their own, associate spontaneously to form a fluorescent molecule. We evaluated this technique for its efficacy in contributing to the characterization of Cauliflower mosaic virus (CaMV) infection. A recombinant CaMV with GFP11 fused to the viral protein P6 (a key player in CaMV infection and major constituent of viral factory inclusions that arise during infection) was constructed and used to inoculate transgenic Arabidopsis thaliana expressing GFP1-10. The mutant virus (CaMV11P6) was infectious, aphid-transmissible and the insertion was stable over many passages. Symptoms on infected plants were delayed and milder. Viral protein accumulation, especially of recombinant 11P6, was greatly decreased, impeding its detection early in infection. Nonetheless, spread of infection from the inoculated leaf to other leaves was followed by whole plant imaging. Infected cells dis- played in real time confocal laser scanning microscopy fluorescence in wild type-looking virus factories. Thus, it allowed for the first time to track a CaMV protein in vivo in the context of an authentic infection. 11P6 was immunoprecipitated with anti-GFP nanobodies, present- ing a new application for the split GFP system in protein-p The split GFP technique is based on the auto-assembly of GFP when two polypeptides– GFP1-10 (residues 1–214; the detector) and GFP11 (residues 215–230; the tag)–both non- fluorescing on their own, associate spontaneously to form a fluorescent molecule. We evaluated this technique for its efficacy in contributing to the characterization of Cauliflower mosaic virus (CaMV) infection. A recombinant CaMV with GFP11 fused to the viral protein P6 (a key player in CaMV infection and major constituent of viral factory inclusions that arise during infection) was constructed and used to inoculate transgenic Arabidopsis thaliana expressing GFP1-10. The mutant virus (CaMV11P6) was infectious, aphid-transmissible and the insertion was stable over many passages. Symptoms on infected plants were delayed and milder. Viral protein accumulation, especially of recombinant 11P6, was greatly decreased, impeding its detection early in infection. Nonetheless, spread of infection from the inoculated leaf to other leaves was followed by whole plant imaging. Infected cells dis- played in real time confocal laser scanning microscopy fluorescence in wild type-looking virus factories. Thus, it allowed for the first time to track a CaMV protein in vivo in the context of an authentic infection. 11P6 was immunoprecipitated with anti-GFP nanobodies, presenting a new application for the split GFP system in protein-protein interaction assays and proteomics. Taken together, split GFP can be an attractive alternative to using the entire GFP for protein tagging. |
| Databáze: | OpenAIRE |
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