In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium.
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.
Marillonnet
Sylvestre
S
Icon Genetics, Biozentrum Halle, Weinbergweg 22, D-06120 Halle/Saale, Germany.
Giritch
Anatoli
A
Gils
Mario
M
Kandzia
Romy
R
Klimyuk
Victor
V
Gleba
Yuri
Y
D004269:DNA, Bacterial / Q000378:metabolism*; D005822:Genetic Vectors; D030821:Plants, Genetically Modified; D012328:RNA Viruses / Q000378:metabolism*; D012231:Rhizobium
D004269:DNA, Bacterial; C036483:T-DNA