title: Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L.
abstract: Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm - with or without anchoring them in the plasma membrane -, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised either by adding a C-terminal stabilisation signal or by anchoring the protein on the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.
authors.affiliation: Institute of Plant Biology, Biological Research Centre, Szeged, Hungary.
authors.forename: B
authors.initials: B
authors.lastname: Morgun
authors.forename: A
authors.initials: A
authors.lastname: Richter
authors.forename: S D
authors.initials: SD
authors.lastname: Deshmukh
authors.forename: V
authors.initials: V
authors.lastname: Stepanyuk
authors.forename: Katalin
authors.initials: K
authors.lastname: Kálai
authors.forename: G
authors.initials: G
authors.lastname: Nagy
authors.forename: L
authors.initials: L
authors.lastname: Hufnagel
authors.forename: Noémi
authors.initials: N
authors.lastname: Lukács
mesh_terms: D000906:Antibodies / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005838:Genotype; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D010935:Plant Diseases / Q000821:virology; D030821:Plants, Genetically Modified; D017800:Potyvirus / Q000235:genetics; D012330:RNA, Double-Stranded / Q000276:immunology*; D014026:Nicotiana / Q000033:anatomy & histology / Q000235:genetics / Q000378:metabolism* / Q000821:virology
chemical_list: D000906:Antibodies; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D012330:RNA, Double-Stranded