Split green fluorescent protein as a tool to study infection with a plant pathogen, Cauliflower mosaic virus

Dáder Beatriz, Burckbuchler Myriam, Macia Jean Luc, Alcon Carine, Curie Catherine, Gargani Daniel, Zhou Jaclyn S., Ng James, Brault Véronique, Drucker Martin. 2019. Split green fluorescent protein as a tool to study infection with a plant pathogen, Cauliflower mosaic virus. PloS One, 14 (3):e0213087, 18 p.

https://doi.org/10.1371/journal.pone.0213087

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Quartile : Q2, Sujet : MULTIDISCIPLINARY SCIENCES

Liste HCERES des revues (en SHS) : oui

Thème(s) HCERES des revues (en SHS) : Psychologie-éthologie-ergonomie; Staps

Résumé : 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.

Mots-clés Agrovoc : Arabidopsis thaliana, virus des végétaux, caulimovirus mosaïque du chou fleur, Myzus persicae, transmission des maladies, plante transgénique, protéine végétale

Mots-clés géographiques Agrovoc : France

Agences de financement européennes : European Commission

Agences de financement hors UE : Institut National de la Recherche Agronomique, Agence Nationale de la Recherche, Human Frontier Science Program

Programme de financement européen : FP7

Projets sur financement : (FRA) Interactions virales avec la plante hôte contrôlent la transmission ultérieure par vecteur, (EU) AgreenSkills+

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