No abstract

Hybridoma cell lines secreting monoclonal antibodies to TSWV were characterized, and one cell line was selected for cloning of the antibody genes. Universal degenerate oligonucleotides were designed to prime PCR amplification of the variable regions of the heavy and light chains of a monoclonal antibody. The PCR products were ligated to produce one single-chain variable fragment (scFv) antibody gene construct that was used to transform Nicotiana tabacum 'Turk'. PCR analyses showed 48% of the 120 regenerated R0 plants contained TSWV scFv genes. The integration and transcription of the scFv gene in the transgenic tobacco plants were confirmed by Southern and northern blot hybridizations, respectively. The expression of TSWV scFv genes in transformed plants was examined by ELISA. TSWV scFv antibodies accumulated to only low levels in transformed plants. Antigen-binding analysis showed that plant-produced TSWV scFv antibodies had the same antigen-binding specificity as the murine TSWV monoclonal antibody. Two TSWV scFv-transformed plants were resistant to TSWV systemic infection as determined by the absence of systemic infection symptoms following TSWV inoculation. The level of virus resistance was correlated with the level of the transgene RNA transcript and scFv antibody production. This resistance was inherited into the R1 generation, and was specific to a lettuce isolate of TSWV (TSWV-L). No resistance was found to Tobacco mosaic virus (TMV) or Cucumber mosaic virus (CMV).