Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in shape Nicotiana tabacum

We evaluated the concept for protection of plants against virus infection based on the expression of single-chain Fv (scFv) fragments in the apoplasm or cytosol of transgenic plants. Cloned cDNA of a tobacco mosaic virus (TMV)-specific scFv antibody, which binds to intact virions, was integrated into the plant expression vector pSS and used for Agrobacterium-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 recombinant antibody (rAb) fragments. A significant increase of scFv levels in T₁ progeny was obtained for plants secreting apoplastic scFv antibodies but not for scFvs expressed in the cytosol. Bioassays revealed that T₁ 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 infection was observed in T₁ plants expressing scFv fragments in the cytosol. Infectivity could be reduced by more than 90%, despite the observation 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 T₁ progenies inhibited systemic virus spread, indicating that expression of TMV-specific cytosolic scFvs confers virus resistance in these transgenic plants. Moreover, inoculation of protoplasts isolated from transgenic and non-transgenic tobacco plants with TMV-RNA demonstrated that accumulation of virus particles is affected by cytosolic scFv expression.