title: Potatovirus X and Tobacco mosaic virus-based vectors compatible with the Gateway cloning system.
abstract: Virus-based expression vectors are important tools for high-level production of foreign proteins and for gene function analysis through virus induced gene silencing. To exploit further their advantages as fast, high yield replicons, a set of vectors was produced by converting and adapting Potato virus X (PVX) and Tobacco mosaic virus (TMV)-based vectors to allow easy cloning of foreign sequences by the Gateway cloning system. Target genes were cloned efficiently by recombination and successfully expressed in Nicotiana benthamiana following inoculation by Agrobacterium (agroinfection). Using green fluorescent protein (GFP) as marker, high-level expression with both PVX-GW and TMV-GW vectors was confirmed. A Gateway inserted phytoene desaturase gene (pds) fragment in PVX-GW and TMV-GW vectors (PVX-GW-PDS and TMC-GW-PDS), induced gene silencing of the endogenous pds gene in N. benthamiana as evidenced by chlorotic leaves. The PVX-GW vector was adapted further by cloning the GFP gene upstream of the Gateway sequences, allowing the easy production of GFP fusions after recombination of a target gene. Subcellular localization of resulting GFP fusion was validated by recombining and expressing the coat protein gene from Tomato chlorotic mottle virus, revealing its nuclear localization. A PVX-GW transient expression assay of a nucleocapsid protein gene fragment of Tomato spotted wilt virus and of a single chain antibody against this protein was shown to confer effective resistance to TSWV infection.
authors.affiliation: Laboratory of Virology, Wageningen University, Wageningen, The Netherlands. lacorte@cenargen.embrapa.br
authors.forename: Cristiano
authors.initials: C
authors.lastname: Lacorte
authors.forename: Simone G
authors.initials: SG
authors.lastname: Ribeiro
authors.forename: Dick
authors.initials: D
authors.lastname: Lohuis
authors.forename: Rob
authors.initials: R
authors.lastname: Goldbach
authors.forename: Marcel
authors.initials: M
authors.lastname: Prins
mesh_terms: D053839:Begomovirus / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D020868:Gene Silencing; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics; D017863:Potexvirus / Q000235:genetics*; D012231:Rhizobium / Q000235:genetics; D013194:Staining and Labeling / Q000379:methods; D014026:Nicotiana; D014027:Tobacco Mosaic Virus / Q000235:genetics*; D017865:Tospovirus / Q000235:genetics
chemical_list: D036022:Capsid Proteins; D049452:Green Fluorescent Proteins; D010088:Oxidoreductases; C022398:phytoene dehydrogenase