John A. Lindbo

TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector.

Transient expression is a rapid, useful approach for producing proteins of interest in plants. Tobacco mosaic virus (TMV)-based transient expression vectors can express very high levels of foreign proteins in plants. However, TMV vectors are, in general, not efficiently delivered to plant cells by agroinfection. It was determined that agroinfection was very efficient with a 35S promoter-driven TMV replicon that lacked the TMV coat protein gene sequence. This coat protein deletion vector had several useful features as a transient expression system, including improved ease of use, higher protein expression rates, and improved biocontainment. Using this TMV expression vector, some foreign proteins were expressed at levels of 3 to 5 mg/g fresh weight of plant tissue. It is proposed that this new transient expression vector will be a useful tool for expressing recombinant proteins in plants for either research or production purposes.

High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors

BackgroundPlants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.ResultsWe have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.ConclusionThese modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.

Functional Modulation of the Geminivirus AL2 Transcription Factor and Silencing Suppressor by Self-Interaction

ABSTRACT The DNA genomes of geminiviruses have a limited coding capacity that is compensated for by the production of small multifunctional proteins. The AL2 protein encoded by members of the genus Begomovirus (e.g., Tomato golden mosaic virus) is a transcriptional activator, a silencing suppressor, and a suppressor of a basal defense. The related L2 protein of Beet curly top virus (genus Curtovirus) shares the pathogenicity functions of AL2 but lacks transcriptional activation activity. It is known that AL2 and L2 can suppress local silencing by interacting with adenosine kinase (ADK) and can suppress basal defense by interacting with SNF1 kinase. However, how the activities of these viral proteins are regulated remains an unanswered question. Here, we provide some answers by demonstrating that AL2, but not L2, interacts with itself. The zinc finger-like motif (CCHC) is required but is not sufficient for AL2 self-interaction. Alanine substitutions for the invariant cysteine residues that comprise the motif abolish self-interaction or cause aberrant subnuclear localization but do not abolish interaction with ADK and SNF1. Using bimolecular fluorescence complementation, we show that AL2:AL2 complexes accumulate primarily in the nucleus, whereas AL2:ADK and L2:ADK complexes accumulate mainly in the cytoplasm. Further, the cysteine residue mutations impair the ability of AL2 to activate the coat protein promoter but do not affect local silencing suppression. Thus, AL2 self-interaction correlates with nuclear localization and efficient activation of transcription, whereas AL2 and L2 monomers can suppress local silencing by interacting with ADK in the cytoplasm.

Tobamovirus Transient Expression Vectors: Tools for Plant Biology and High-Level Expression of Foreign Proteins in Plants

The majority of viruses infecting higher plants have single-stranded, positive (or message-) sense RNA genomes. Infectious transcripts can be synthesized in vitro from full-length cDNA clones to study RNA virus biology, develop methods of disease control, and construct plant expression vectors (10,22). Tobamoviruses have a long history of experimentation and represent efficient genetic systems (2,3,4). Vectors based on the tobacco mosaic virus (TMV) genome were among the first to be developed (8) and have particular advantages for novel application(s) in the expression of foreign sequences in plants. These advantages include: 1. Speed: TMV vectors carrying foreign genes move rapidly and systemically in plants (generally 7–10 days post inoculation). 2. High expression levels: TMV produces more virus-encoded protein per infected cell than any other known plant virus [up to 10% of dry weight in TMV-infected tobacco plants; (1)]. Foreign proteins (18,21) and peptides (26) produced systemically in plants by TMV vectors can accumulate to 1–5% of total soluble protein. The levels of foreign proteins expressed from viral vectors are generally much higher than that obtainable from stably transformed cell lines, transgenic organisms, or plasmid DNA-based transient expression systems. 3. Protein targeting: It is possible to direct the foreign protein to various subcellular locations including the endomembrane system, the cytosol, organelles, or even virion particles. Careful study and control of subcellular targeting of foreign proteins or peptides can maximize their specific activity, stability, and greatly facilitate their purification (21,27). 4. Wide range of potential applications: TMV expression vectors have been used as research tools to study plant biosynthetic pathways, screen gene libraries and to express proteins toxic to plant and non-plant systems. Examples include: 1) The carotenoid biosynthetic pathway in plants has been altered by up or down-regulating enzymes responsible for the synthesis of key isoprenoid intermediates (19). The levels of intermediate products were altered up to 50 fold by expressing sequences in the plant cytoplasm in either sense or anti-sense orientation. These dramatic alterations in secondary metabolite accumulation can only be achieved by regulated induction in vegetative tissues, and are predicted to be lethal if constitutively expressed using transgenic technologies. 2) A plant gene library has been constructed in a TMV vector in order to identify expressed sequences that induce cell death in plants (C. Holt, pers. commun.). 3) Peptides or proteins such as antimicrobial peptides, animal hormones, and growth regulators that would be predicted to be toxic or accumulate poorly in microbial or transgenic expression systems have been expressed in plants from TMV vectors (27). 5. Low frequency of sequence drift: Contrary to initial predictions, foreign sequences propagated in TMV-based vectors accumulate very few point mutations during multiple passages in whole plants (15). 6. Broad host range: cDNA clones are available for many tobamoviruses. Functional hybrid vectors are compatible with an array of plant species. 7. Plants as ideal hosts: The greatest advantages in using plant viruses as expression vectors are the characteristics of their hosts. Transfected leaves are one of the most economical sources of biomass for commercial product development and can be inexpensively scaled to meet production requirements. 8. Proven utility in laboratory and field-scale production: Recombinant TMV vectors have been tested in five outdoor field trials to date. Expression characteristics, host range, persistence in the environment, and large-scale plant extraction procedures have all been evaluated (12). Multi-ton extraction of tobacco tissue grown in the field has resulted in the purification of kilogram (kg) quantities of recombinant viruses for development of vaccines and anti-microbial peptides (27). 9. No biological vector: Because TMV is mechanically spread in nature (28), recombinant vectors are contained to inoculated fields (12).

Epigenetic Modification of Plants with Systemic RNA Viruses

Knowledge of gene function is critical to the development of new plant traits for improved agricultural and industrial applications. Viral expression vectors offer a rapid and proven method to provide epigenetic expression of foreign sequences throughout infected plants. Expression of these sequences from viral vectors can lead to gain- or loss-of-function phenotypes, allowing gene function to be determined by phenotypic or biochemical effects in the infected plant. Tobacco mosaic virus and barley stripe mosaic virus expression vectors have been developed to express foreign gene sequences in dicotyledonous and monocotyledonous hosts, respectively. Large-scale application of both viral vector systems for gene function discovery in Nicotiana and barley hosts resulted in high infection rates and produced distinctive visual phenotypes in approximately 5% of transfected plants. Novel genes expressing potential herbicide target proteins in addition to genes promoting stem elongation, leaf development and apical dominance were identified in the large-scale screening. This report illustrates the adaptability of viral vectors for gene function discovery in higher plants.

Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression

The effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and analysis system developed for continual monitoring of GFP expression. Suppressors were introduced into lima bean cotyledonary tissues either as 3′-GFP translational fusions or as co-introductions with the GFP gene on a separate plasmid. The resultant transient expression profiles for each suppressor depended on whether the suppressor was introduced as a fusion or co-introduced on separate plasmids. As co-introductions, the silencing suppressors HCPro (from Tobacco etch virus), p19 (from Tomato bushy stunt virus), γb (from Barley stripe mosaic virus) and p21 (from Beet yellows virus) led to an almost twofold increase in initial GFP expression levels, followed by a rapid decline. In contrast, fusions of HCPro, p19, and γb to the 3′-end of GFP resulted in slightly lower but more prolonged GFP expression. Compared with the co-introductions, all GFP::Suppressor translational fusions gave reduced GFP fluorescence levels, suggesting interference of the fusion partner with GFP fluorescence. Regardless of the configuration, introductions of the silencing suppressors AL2 (from Tomato golden mosaic virus) and 126-kDa protein (from Tobacco mosaic virus) resulted in very low GFP fluorescence. This is the first report that directly compares the effects of a large number of viral suppressors of silencing on transient transgene expression using both translational fusions and co-introductions.

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants.

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing.

Viral Vector Expression of Foreign Proteins in Plants

It is now fairly routine to engage plant viruses to express foreign proteins in plants. Plant viruses have several features that make them quite useful for vectoring foreign genes into whole plants. The majority of viruses infecting higher plants have single-stranded, positive-sense RNA genomes. Infectious transcripts can be synthesized in vitro from full-length cDNA clones to study RNA virus biology, develop methods of disease control and construct plant expression vectors (Goldbach and Hohn, 1997; Scholthof et al., 1996). Tobamoviruses are among the most studied and well-understood viruses and represent superbly efficient genetic systems (Dawson et al., 1986; Dawson et al.,1990; Dawson, 1992). Vectors based on the tobacco mosaic virus (TMV) were among the first to be developed (Donson et al., 1991) and have several advantages for novel applications in the expression of foreign sequences in plants. These advantages include speed, high expression levels, broad host-range, and controlled containment, as transmission occurs only by mechanical means.