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.

A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator ( En/Spm ) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (d Spm ) element with a phosphinothricin herbicide resistance ( BAR ) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable d Spm transpositions. Treatments for both positive ( BAR ) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which ‫ف‬ 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.

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The mechanisms involved in the posttranslational targeting of membrane proteins are not well understood. The light-harvesting chlorophyll proteins (LHCP) of the thylakoid membrane are a large family of hydrophobic proteins that are targeted in this manner. They are synthesized in the cytoplasm, translocated across the chloroplast envelope membranes into the stroma, bound by a stromal factor to form a soluble intermediate, ''transit complex'', and then integrated into the thylakoid membrane by a GTP dependent reaction. Signal recognition particle (SRP), a cytoplasmic ribonucleoprotein, is known to mediate the GTP dependent cotranslational targeting of proteins to the endoplasmic reticulum. We show that chloroplasts contain an SRP consisting of, cpSRP54, a homologue of SRP54 and a previously undescribed 43-kDa polypeptide (cpSRP43) instead of an RNA. We demonstrate that both subunits of cpSRP are required for the formation of the transit complex with LHCP. Furthermore, cpSRP54, cpSRP43, and LHCP are sufficient to form a complex that appears to be identical to authentic transit complex. We also show that the complex formed between LHCP and cpSRP, together with an additional soluble factor(s) are required for the proper integration of LHCP into the thylakoid membrane. It appears that the expanded role of cpSRP in posttranslational targeting of LHCP has arisen through the evolution of the 43-kDa protein.The insertion of proteins into membranes is a fundamental process essential for the vitality of all organisms. The paradigm for this process is the targeting mediated by signal recognition particle (SRP), a cytoplasmic ribonucleoprotein. In prokaryotes, the SRP-RNA binds a single 54-kDa-polypeptide subunit, while in eukaryotes, five additional subunits are bound. One of the distinctive features of this targeting mechanism is cotranslational protein insertion. The synthesis of hydrophobic protein domains at the membrane circumvents potential protein folding problems that might otherwise occur in an aqueous environment. However not all hydrophobic proteins are targeted cotranslationally. For example, the major proteins of the thylakoid membrane, the light harvesting chlorophyll proteins (LHCP), are targeted by a posttranslational mechanism. LHCP form a large family of related proteins that have three to four transmembrane domains. They are synthesized in the cytoplasm, and are targeted to the thylakoid membrane through three aqueous compartments: the cytoplasm, the inter-envelope space, and the stroma (1, 2). The factors that mediate the posttranslational targeting of members of the LHCP family have not been definitively identified.The LHCP are inserted into the thylakoid membrane in a reaction requiring GTP and stroma (3-5). It was found previously that a stromal factor binds LHCP to form a soluble intermediate, designated transit complex, that maintains the solubility of these proteins as they are transported through the stroma (6, 7). The transit complex is the only soluble form of LHCP that accumulates when ...

SummaryBased on homologies between the yeast DMCl and the lily LIM15 meiosis-specific genes, degenerate PCR primers were designed that amplified the Arabidopsis DMCl gene