INTRACELLULAR EXPRESSION OF TMV-SPECIFIC SINGLE-CHAIN FV FRAGMENTS LEADS TO IMPROVED VIRUS-RESISTANCE IN NICOTIANA-TABACUM

We evaluated the concept for protection of plants against virus infect ion based on the expression of single-chain Fv (scFv) fragments in the apoplasm or cytosol of transgenic plants. Cloned cDNA of a tobacco mo saic virus (TMV)-specific scFv antibody, which binds to intact virions , was integrated into the plant expression vector pSS and used for Agr obacterium-mediated transformation of Nicotiana tabacum cv. Xanthi-nc. Regenerated transgenic tobacco plants were analysed by northern blot, western blot and ELISA to assess expression and functionality of reco mbinant antibody (rAb) fragments. A significant increase of scFv level s in T-1 progeny was obtained for plants secreting apoplastic scFv ant ibodies but not for scFvs expressed in the cytosol. Bioassays revealed that T-1 progeny producing scFvs in different plant cell compartments showed different levels of resistance upon inoculation with TMV. The most dramatic reduction of necrotic local lesion numbers upon virus in fection was observed in T-1 plants expressing scFv fragments in the cy tosol. Infectivity could be reduced by more than 90%, despite the obse rvation that protein expression levels for functional scFv antibodies were very low. Furthermore, upon inactivation of the N-resistance gene at elevated temperature, a significant portion of the T-1 progenies i nhibited systemic virus spread, indicating that expression of TMV-spec ific cytosolic scFvs confers virus resistance in these transgenic plan ts. Moreover, inoculation of protoplasts isolated from transgenic and non-transgenic tobacco plants with TMV-RNA demonstrated that accumulat ion of virus particles is affected by cytosolic scFv expression.