Plant biotechnology relies on two approaches for delivery and expression of heterologous genes in plants: stable genetic transformation and transient expression using viral vectors. Although much faster, the transient route is limited by low infectivity of viral vectors carrying average-sized or large genes. We have developed constructs for the efficient delivery of RNA viral vectors as DNA precursors and show here that Agrobacterium-mediated delivery of these constructs results in gene amplification in all mature leaves of a plant simultaneously (systemic transfection). This process, called "magnifection", can be performed on a large scale and with different plant species. This technology combines advantages of three biological systems (the transfection efficiency of A. tumefaciens, the high expression yield obtained with viral vectors, and the post-translational capabilities of a plant), does not require genetic modification of plants and is faster than other existing methods.

Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that were found to be noncompeting. Unlike vectors derived from the same virus, noncompeting vectors effectively coexpress the heavy and light chains in the same cell throughout the plant body, resulting in yields of up to 0.5 g of assembled mAbs per kg of fresh-leaf biomass. This technology allows production of gram quantities of mAbs for research purposes in just several days, and the same protocol can be used on an industrial scale in situations requiring rapid response, such as pandemic or terrorism events.monoclonal antibody ͉ potato virus X ͉ tobacco mosaic virus A lthough the ability of plants to express full-size human antibodies was discovered 17 years ago (1-3), the idea of industrial-scale antibody production in plants has been abandoned by most companies, mostly because of limitations of existing expression protocols. Stably transformed (transgenic) plants are able to express correctly folded and functional antibodies of both the IgG and IgA classes, but yields are generally very low (usually in the range of 1-40 g͞g of fresh biomass); in addition, the time necessary to generate the first grams of research antibody material is very long, requiring Ͼ2 years (4-8).Transient expression systems, on the other hand, allow production of research quantities of antibody material much faster. However, the early versions of transfection systems, such as Agrobacterium-mediated transient expression or viral vectormediated expression, cannot provide for high-level coexpression of two or several polypeptides necessary for the assembly of heterooligomeric proteins, in particular IgG antibodies (9-12).Recently, we have developed a scalable transient expression technology (magnifection) that is based on replication of viral vectors delivered to multiple parts of a plant body (systemic delivery) by Agrobacterium (13). Such a technology is in essence an en masse infiltration of whole, mature plants with a diluted agrobacteria suspension carrying T-DNAs encoding viral replicons. The magnifection process allows expression of various proteins, but, until now, it has been used to express only single-polypeptide proteins or homooligomers (14). Attempts to express two or more different polypeptides from one viral replicon failed because of drastically reduced expression levels obtained with bicistronic constructs (unpublished results).Therefore, we decided to explore expression protocols that involve two or more viral replicons. We report here a general solution for coexpression of high amounts of two heterologous polypeptides by using two d...

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.

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