Disruption of essential plastid gene expression caused by T7 RNA polymerase-mediated transcription of plastid transgenes during early seedling development.
Transcription of plastid transgenes by plastid-targeted T7 RNA polymerase (ptT7RNAP) during early seedling development in tobacco was associated with a pale-green leaf phenotype, depletion of plastid rRNAs and arrest of shoot development. Extensive analysis of mutant seedlings at the transcript level using DNA microarrays and RNA gel blotting revealed severe disruption of plastid rRNA accumulation at 4-days post-germination and reduced transcript accumulation for the essential gene clpP. Several nuclear genes encoding plastid proteins were differentially regulated in mutant seedlings over time. Ef-Tu was upregulated at 4-days post-germination and then subsequently downregulated, while RbcS was already downregulated at this early time point. The downregulation of nuclear genes encoding plastid proteins suggests disruption of plastid-to-nucleus signalling. In contrast, transcripts of three plastid genes showed increased accumulation in mutant seedlings. Transcripts of ndhC and ndhK accumulated at high levels possibly due to T7RNAP-mediated enhancement of transcription, while ptT7RNAP-mediated transcription through the phage T7 Tphi terminator into the adjacent plastome increased the level of accD transcripts. The leakiness of the Tphi terminator has implications for the use of T7RNAP-based expression systems in plastid biotechnology.
Magee
Alan M
AM
Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland.
MacLean
Daniel
D
Gray
John C
JC
Kavanagh
Tony A
TA
D017123:Bacteriophage T7 / Q000201:enzymology; D002467:Cell Nucleus / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000494:pharmacology*; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D020411:Oligonucleotide Array Sequence Analysis; D030821:Plants, Genetically Modified / Q000235:genetics; D018087:Plastids / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000254:growth & development* / Q000378:metabolism; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics; D014158:Transcription, Genetic; D019076:Transgenes / Q000502:physiology*; D014764:Viral Proteins / Q000494:pharmacology*
D012333:RNA, Messenger; D018749:RNA, Plant; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases