Showing papers by "Tony A. Kavanagh published in 2004"

T7 RNA Polymerase-Directed Expression of an Antibody Fragment Transgene in Plastids Causes a Semi-Lethal Pale-Green Seedling Phenotype

Abstract: A T7 promoter-controlled transgene, AbL, encoding a camel single-domain antibody fragment that binds to the model antigen chicken egg-white lysozyme was introduced into the plastid genome of tobacco. AbL expression was activated in the transplastomic line by introducing a nuclear transgene, ST7, encoding a light-regulated plastid-targeted T7RNAP by cross-pollination. The resulting AbL × ST7 progeny seedlings developed a pale-green phenotype and ceased growth soon after germination. High levels of AbL transcripts accumulated in AbL × ST7 seedlings and expression of functional AbL antibody was detected by ELISA. Transplastomic AbL plants were also crossed with nuclear-transformed tobacco plants containing a salicylic acid-inducible transgene encoding a plastid-targeted T7RNAP (PR-T7 transgene). The resulting AbL × PR-T7 progeny were wild-type in appearance but were slow growing and prone to wilting even when provided with adequate water. Although AbL transcription was inducible by treating AbL × PR-T7 leaves with salicylic acid, high levels of T7RNAP-dependent AbL transcripts also accumulated in the absence of induction. However, AbL antibody did not accumulate at levels detectable by immunoblotting or ELISA in AbL × PR-T7 plants despite the fact that total leaf RNA containing AbL transcripts was capable of directing AbL antibody synthesis in an E. coli-derived in vitro translation system.