T7 RNA polymerase-directed expression of an antibody fragment transgene in plastids causes a semi-lethal pale-green seedling phenotype.
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 x ST7 progeny seedlings developed a pale-green phenotype and ceased growth soon after germination. High levels of AbL transcripts accumulated in AbL x 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 x 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 x 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 x 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.
Magee
Alan M
AM
Plant Molecular Biology Laboratory, Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland.
Coyne
Seamus
S
Murphy
David
D
Horvath
Eva M
EM
Medgyesy
Peter
P
Kavanagh
Tony A
TA
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003433:Crosses, Genetic; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D005804:Genes, Lethal; D005822:Genetic Vectors; D007128:Immunoglobulin Fragments / Q000235:genetics*; D009113:Muramidase / Q000276:immunology; D010641:Phenotype; D030821:Plants, Genetically Modified; D018087:Plastids / Q000235:genetics*; D011058:Pollen; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012231:Rhizobium / Q000235:genetics; D036226:Seedlings / Q000235:genetics / Q000254:growth & development; D014026:Nicotiana / Q000235:genetics / Q000254:growth & development; D014158:Transcription, Genetic; D014764:Viral Proteins
D017931:DNA Primers; D007128:Immunoglobulin Fragments; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D009113:Muramidase