We have developed an efficient, versatile, and user-friendly viral engineering and expression system that is based on
in planta
assembly of functional viral vectors from separate pro-vector modules. With this new system, instead of supplying a plant cell with a complete viral vector as a mature viral particle, an RNA or a linear DNA molecule, we use agrobacteria to deliver various modules that are assembled inside the cell with the help of a site-specific recombinase. The resulting DNA is transcribed, and undesired elements such as recombination sites are spliced out, generating a fully functional RNA replicon. The proposed protocol allows us, by simply treating a plant with a mixture of two or more agrobacteria carrying specific prefabricated modules, to rapidly and inexpensively assemble and test multiple vector/gene combinations, without the need to perform the various engineering steps normally required with alternative protocols. The process described here is very fast (expression requires 3–4 days); it provides very high protein yield (up to 80% of total soluble protein); more than before, it is carried out using
in vivo
manipulations; it is based on prefabricated genetic modules that can be developed/upgraded independently; and it is inherently scalable.
Pronounced variability of transgene expression and transgene silencing are commonly observed among independent plant lines transformed with the same construct. Single-copy T-DNA lines harboring reporter genes of various kind and number under the control of a strong promoter were established in Arabidopsis thaliana for a comprehensive analysis of transgene expression. Characterization of 132 independent transgenic lines revealed no case of silencing as a result of site of T-DNA integration. Below a certain number of identical transgenes in the genome, gene copy number and expression were positively correlated. Expression was high, stable over all generations analyzed, and of a comparable level among independent lines harboring the same copy number of a particular transgene. Conversely, RNA silencing was triggered if the transcript level of a transgene surpassed a gene-specific threshold. Transcript level–mediated silencing effectively accounts for the pronounced transgene expression variability seen among transformants. It is proposed that the RNA sensing mechanism described is a genome surveillance system that eliminates RNA corresponding to excessively transcribed genes, including transgenes, and so plays an important role in genome defense
Summary
Transgene expression was analysed in Arabidopsis T‐DNA transformants carrying defined numbers and arrangement of different reporter genes. All transgenes were placed under the control of the strong constitutive CaMV 35S promoter. High, stable transgene expression was observed in plants containing two copies of the β‐glucuronidase (GUS) gene, two or four copies of the green fluorescent protein (GFP) gene and two, four or six copies of the streptomycin phosphotransferase (SPT) gene. Thus, the mere presence of multiple promoter and/or transgene sequences did not result in gene silencing. In none of the cases analysed were tandem repeat arrangements of transgenes and/or inverted repeat (IR) T‐DNA structures sufficient to trigger silencing of the different reporter genes. Instead, post‐transcriptional gene silencing (PTGS) correlated with the copy number of the highly expressed transgenes. Twelve copies of the SPT and four copies of the GUS gene triggered silencing. Silencing is frequently associated with repetitive T‐DNA structures. We favour the idea that in many cases this may be attributed to the high transgene doses rather than the repeat arrangements themselves.
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