Assessment of the effectiveness of a nuclear-launched TMV-based replicon as a tool for foreign gene expression in plants in comparison to direct gene expression from a nuclear promoter.
An environmentally safe Tobacco Mosaic Virus (TMV)-based expression replicon was constructed that lacks movement protein (MP) and coat protein (CP), and which expresses the green fluorescent protein (GFP) gene from a full CP subgenomic promoter. The TMV replicon, whose cDNA was positioned between an enhanced Cauliflower Mosaic Virus 35S promoter (CaMV) and a self-cleaving hammerhead ribozyme with a downstream nopaline synthase gene polyadenylation signal [nos-poly(A)], was assessed for its effectiveness to accumulate GFP upon agroinfiltration into plant leaves compared to a control construct in which GFP was directly expressed from the enhanced CaMV 35S promoter. It was determined that individually expressing cells produced ca. 9-fold more GFP from the TMV-based replicon than from the enhanced 35S promoter. In contrast, GFP measurements from total leaf extracts determined that leaves infiltrated with the TMV-based replicon produced ca. 7-fold less GFP than the control construct. These apparently contradictory results can be explained by the low infectivity of the TMV-based replicon as it was found that the number of foci expressing GFP produced in leaves agroinfiltrated with the TMV-based replicon was ca. 66-fold lower than produced by the control.
Man
Michal
M
Department of Plant Sciences, the George S. Wise Faculty of Life Sciences, Tel Aviv University, 69778, Tel Aviv, Israel.
Epel
Bernard L
BL
D002467:Cell Nucleus / Q000235:genetics* / Q000821:virology; D003593:Cytoplasm / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000821:virology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012093:Replicon / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics / Q000821:virology; D014027:Tobacco Mosaic Virus / Q000235:genetics*
D012333:RNA, Messenger; D049452:Green Fluorescent Proteins