An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus.
Transient gene expression is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is vacuum-infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells. However, the utility of the system is limited because the ectopic protein expression ceases after 2-3 days. Here, we show that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. We describe a system based on co-expression of a viral-encoded suppressor of gene silencing, the p19 protein of tomato bushy stunt virus (TBSV), that prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Expression of a range of proteins was enhanced 50-folds or more in the presence of p19 so that protein purification could be achieved from as little as 100 mg of infiltrated leaf material. The effect of p19 was not saturated in cells that had received up to four individual T-DNAs and persisted until leaf senescence. Because of its simplicity and rapidity, we anticipate that the p19-enhanced expression system will have value in industrial production as well as a research tool for isolation and biochemical characterisation of a broad range of proteins without the need for the time-consuming regeneration of stably transformed plants.
Voinnet
Olivier
O
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
Rivas
Susana
S
Mestre
Pere
P
Baulcombe
David
D
D015870:Gene Expression; D020868:Gene Silencing; D018551:Solanum lycopersicum / Q000821:virology; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000235:genetics*; D017864:Tombusvirus / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D014764:Viral Proteins