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First published online August 24, 2007; 10.1104/pp.107.106377 Plant Physiology 145:1232-1240 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
TRBO: A High-Efficiency Tobacco Mosaic Virus RNA-Based Overexpression Vector1,[C],[OA]Department of Plant Pathology, The Ohio State University/Ohio Agricultural Research and Development Center, Wooster, Ohio 44691
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.
As interest in proteomics, biochemistry, and protein structure increases, there is an increasing need for efficient, easy-to-use recombinant protein expression systems. For researchers interested in expressing recombinant proteins in plants, there are multiple transient expression systems available. However, every protein expression system has inherent advantages and disadvantages. Improving transient expression vectors so they are easier to use, more cost effective, and produce higher levels of recombinant proteins will be of use to the wide variety of researchers who use recombinant proteins in research or development. A recent study has shown that producing recombinant plant proteins in plant cells, instead of yeast (Saccharomyces cerevisiae) or Escherichia coli cells, is more likely to result in the production of properly folded, active plant proteins (Popescu et al., 2007  ).
A variety of viral- and nonviral-based plant transient protein expression systems have been described in the literature (Voinnet et al., 2003
Other transient expression systems are based on plant viruses, such as Tobacco mosaic virus (TMV), for example. Detailed descriptions of TMV and TMV-based transient expression vectors have been described elsewhere (Pogue et al., 1998
Plants can be inoculated with TMV vectors through a process called agroinfection. In agroinfection, A. tumefaciens was used to deliver T-DNA composed of 35S promoter-driven TMV cDNA to plant cells. Transcription of T-DNA in the plant nucleus generated RNA that was capable of initiating self-replication in the cytoplasm. Multiple reports have documented the low agroinfection efficiency of the typical 35S-driven TMV vector (Turpen et al., 1993 Here, we report on the construction of an improved agroinfection-compatible TMV vector that lacks the TMV CP gene coding sequence. This modification resulted in a vector with several significant improvements, such as (1) much higher agroinfection efficiency; (2) higher recombinant protein expression levels; and (3) inability to form virus particles during its infection/replication cycle. This new expression vector is called the TMV RNA-based overexpression (TRBO) vector. Here, we demonstrate that the TRBO vector can produce up to 100 times more recombinant protein than the P19-enhanced agroinfiltration transient expression system described above. It is proposed that, because of its efficacy and ease of use, the TRBO vector will be a useful transient expression vector for production of recombinant proteins in plants for either research or production purposes.
Plasmids Constructed for This Experiment
We previously constructed an agroinfection-compatible full-length TMV expression vector called pJL24 (Fig. 1
) that expressed all of the TMV genes in addition to a foreign, inserted gene (Lindbo, 2007
Agroinfection with the TRBO Replicon Is Very Efficient
Previous work with the TMV vector contained in pJL24 determined that ectopic coexpression of an RNA-silencing suppressor gene (such as the P19 gene from Tomato bushy stunt virus) was needed to obtain the highest agroinfection rates for this vector (Lindbo, 2007
Because the pJL-TRBO-G vector had a higher rate of agroinfection than the vector pJL24, we determined whether dilute suspensions of A.t./pJL-TRBO-G alone could be used to efficiently inoculate leaves. Half-leaves of Nicotiana benthamiana plants were infiltrated with various dilutions of A.t./pJL-TRBO-G cells. Leaves were observed daily with a hand-held UV lamp to monitor the progress and extent of agroinfection, as demonstrated by GFP expression from the TRBO-G replicon. Results are shown in Figure 3 . Even in leaves infiltrated with A. tumefaciens cells diluted 1:300 from a starting optical density (OD600) of 1.0, nearly all cells of the infiltrated zone expressed GFP by 4 dpi, as determined by visual inspection of infiltrated leaves under a hand-held UV lamp. Thus, plants can be efficiently agroinoculated with the TRBO-G replicon over a wide range of A.t./pJL-TRBO-G cell densities.
To further demonstrate that the agroinfection rate of A.t./pJL-TRBO-G is higher than that of A.t./pJL24, a 1:100 dilution (from a starting OD of 1.0) of A.t./pJL24 cells was infiltrated into N. benthamiana leaves with or without A.t./pJL3:P19 cells. The image in Figure 3B is a photograph (3 dpi) of such an infiltrated leaf under UV illumination. Again, coinfiltration of a mixture of A.t./pJL24 and A.t./pJL3:P19 cell suspensions dramatically increased the agroinfection rate of the pJL24 vector. The amount of GFP-expressing tissue in this treatment appeared similar visually to the amount of GFP-expressing tissue (at 3 dpi) in leaves infiltrated with 1:150 or 1:300 dilutions of A.t./pJL-TRBO-G cells (Fig. 3A). This further demonstrates that the agroinfection rate of pJL-TRBO-G is significantly higher than that of pJL24 and that even diluted A.t./pJL-TRBO-G cell suspensions can be used to efficiently inoculate leaves in the absence of an ectopically expressed RNA-silencing suppressor, such as P19.
Because the TMV CP is required for systemic movement (Donson et al., 1991
TRBO Vector Expressed Very High Levels of Recombinant Protein To compare the amounts of GFP produced from the TMV vectors JL24 and TRBO-G, or from the transient coexpression of 35S:GFP and the RNA-silencing suppressor protein P19, a plate-based GFP fluorescence assay was used. Purified His-6-tagged GFP, purified from TRBO-G-infected plants by metal affinity chromatography, was used as a standard. Leaves of N. benthamiana were infiltrated with one of the following A. tumefaciens cell suspensions: A.t./p35S:GFP + A.t./pJL3:P19 (each at an OD600 of 0.5); A.t./pJL24 + A.t./pJL3:P19 (each at an OD600 of 0.5); or A.t./pJL-TRBO-G (OD600 0.02). Protein samples from infiltrated tissues were prepared at 5 or 6 dpi. Dilutions of protein extracts and purified His-6-tagged GFP were transferred into wells of a 96-well plate (in triplicate). GFP fluorescence levels were recorded on a Perkin-Elmer HTS 7000 BioAssay plate reader. The results are shown in Figure 5 . The TRBO-G replicon expressed up to 100 times more GFP than was obtained from cointroducing T-DNAs for 35S:GFP and 35S:P19 into plants, and 2 to 3 times more GFP than the TMV vector JL24. Similar results were also obtained from an ELISA assay, using anti-GFP-specific antibodies (data not shown). In multiple repetitions of this experiment, the relative expression levels from the different expression systems were always consistent. The TRBO-G replicon always expressed significantly more GFP than the other transient expression systems examined.
Temporal Analysis of Protein Expression from TRBO After pJL-TRBO-G T-DNA is transcribed, RNA initiates self-replication and gene expression in the cytoplasm. Because the TRBO-G replicon expressed the TMV movement protein, it moved cell to cell in the inoculated (infiltrated) leaf. The result of this movement is that individual GFP-expressing foci on a leaf enlarge as the virus moves cell to cell over time. This can be observed by comparing the sizes of individual GFP-expressing cell foci in 3- and 4-dpi images (Fig. 3A) of leaves infiltrated with 1:500 dilutions of A.t./pJL-TRBO-G. When leaves were infiltrated with higher concentrations of A.t./pJL-TRBO-G cells, the large number of GFP-expressing cells in the infiltrated zone made it difficult to identify an individual focus. Regardless, as replication and cell-to-cell movement of the replicon progressed, the amount of GFP expressed in the infiltrated leaf increased. After a certain point, the steady-state level of GFP in infiltrated tissue appeared to reach a plateau. To demonstrate the temporal nature of protein expression from the TRBO-G replicon, extracts were prepared from inoculated tissue at various days postinfiltration. Total soluble protein extracts were analyzed by SDS-PAGE and Coomassie Blue staining (Fig. 6 ). TRBO-G-expressed GFP accumulation appeared to plateau by 4 to 6 dpi, consistent with the increase in GFP activity that is observed by viewing infiltrated leaves under UV illumination. Samples in lanes 3 to 6 went through a freeze-thaw cycle that results in the precipitation of a significant portion of the Rubisco large subunit protein, with little or no effect on the solubility of the GFP (J. Lindbo, unpublished data).
Expression of Various Proteins from the TRBO Replicon To further demonstrate the utility of the TRBO expression replicon, genes of various sizes were cloned into the pJL-TRBO plasmid. N. benthamiana plants were infiltrated with suspensions of A. tumefaciens cells transformed with the various plasmids. Several (4–6) days postinfiltration, total soluble protein extracts were prepared from agroinfiltrated tissue. Extracts were separated on SDS-PAGE gels and stained with Coomassie Blue (Fig. 7A ). Because some of the recombinant proteins expressed from TRBO had C-terminal amino acid tags of His-6(HA)2 (where HA is the influenza hemagglutinin peptide YPYDVPDYA), some extracts (Fig. 7B) were also subjected to immunoblot analysis using anti-HA primary antibodies (Invitrogen). The results of this analysis demonstrated that different recombinant proteins accumulated to different levels in plants. Some (e.g. adenosine kinase) accumulated to greater levels than GFP. Other proteins accumulated at lower levels. The results also demonstrated that TRBO can be used to express His-6- and epitope-tagged recombinant proteins. Although several of the lesser accumulating proteins had His-6(HA)2 C-terminal amino acid tags, it is not proposed that this peptide tag was solely responsible for the lower accumulation levels. However, this tag may have an effect on the final level of accumulation on some proteins. For example, His-6(HA)2-tagged GFP (Fig. 7, lane 3) did accumulate to slightly lower levels than that of nontagged GFP.
We recently reported on the construction of a full-length TMV vector that could be efficiently delivered to cells by agroinfection provided a 35S-driven RNA-silencing suppressor gene was cointroduced at the same time (Lindbo, 2007 ). In this study, we describe a CP deletion mutant of that 35S-driven TMV vector. This vector was efficiently delivered to cells by agroinfection regardless of the coexpression of an RNA-silencing suppressor protein. These data demonstrated that the sequences in or around the TMV CP gene had a significant, negative impact on agroinfection efficiency. The negative effect of the TMV CP sequence could be at least partially neutralized by ectopic expression of a suppressor of RNA silencing. Therefore, it is hypothesized that the RNA sequence of the TMV CP subgenomic promoter and CP ORF may be a potent inducer of RNA silencing. Since their inception, the majority of the published work on TMV vector development has focused on generating TMV expression vectors that expressed the TMV CP and therefore moved systemically in plants. This was in part because early TMV vectors were very inefficiently delivered to plants by hand rubbing of naked TMV vector RNAs (transcribed in vitro from TMV cDNA clones) onto plant leaves. As a result, the only way to obtain significant quantities of TMV vector-infected plant tissue was to allow the vector to move systemically in the plant. By dramatically improving the agroinfection rate of a TMV vector, we have made it possible to reliably infect large amounts of plant tissue with a TMV vector in the absence of systemic movement of the vector. This makes it possible to use TMV vectors that do not express the CP. This approach allows for several advantages, including higher protein expression levels from the TMV vector in a shorter time frame and the absence of large amounts of TMV CP in infected tissue. All of these advantages make it easier to produce and purify recombinant proteins of interest from plants with a TMV vector.
It is interesting that the agroinfectivity and expression capacity of the CP deletion vector described here are comparable to a TMV CP deletion vector described by Marillonnet et al. (2005)
The CP deletion vector TRBO described in this article had several advantages over a full-length TMV vector. The TRBO vector had a dramatically higher agroinfection rate. Because of this, plants could be efficiently inoculated even with very dilute suspensions of A. tumefaciens. This is important for two reasons. First, there are some plant species that demonstrate a hypersensitive response when infiltrated with high-density A. tumefaciens suspensions (Wroblewski et al., 2005
One of the challenges of working with plant virus expression vectors is the tendency of vector deletion mutants to appear in systemically infected portions of inoculated plants (Dawson et al., 1989 Another advantage of TRBO is that it does not produce TMV CP. Because TMV CP is required for systemic movement, TRBO is not capable of systemic movement in plants. It also will not produce virions in plants. This has definitive biocontainment and protein purification advantages. First, this feature reduces the chances for inadvertent plant-to-plant movement of the vector. Second, when extracting proteins from TRBO-infected tissue, the recombinant protein of interest does not need to be purified away from virion particles. If one is using a viral vector that does generate virus particles (such as JL24), efforts must be taken to both separate virion particles from the recombinant protein of interest and also to inactivate virus particles in any extracts of infected plant materials. These issues are not a concern with TRBO because it does not generate virus particles. It is important to realize that not every protein expressed from a viral vector will necessarily accumulate to very high levels. The results in Figure 7 demonstrate that some proteins accumulate to levels greater than GFP. Other proteins accumulate to levels significantly lower than GFP. For example, in Figure 7, A and B, lane 7, the recombinant protein expressed could only be detected by immunoblotting (western-blotting) procedures and not by Coomassie Blue staining. There are no doubt effects of protein stability involved in the final accumulation level of any protein. Regardless, the high expression capacity of the TRBO expression vector provides an excellent opportunity to produce detectable levels of recombinant protein in plants in a very short time frame.
The TMV overexpression vector in pJL-TRBO, which lacks the TMV CP gene, has several useful advantages over a TMV vector that does express the CP gene. These advantages include higher agroinfection rates, easier scale up, higher protein expression levels, and biocontainment/protein purification advantages. The TRBO expression vector can express proteins at up to 100 times the level of the routinely used enhanced Agrobacterium transient expression method of cointroduction of a gene of interest and the P19 RNA-silencing suppressor gene into plants. It is proposed that this vector will be very useful for researchers interested in rapidly expressing recombinant proteins in plants. The ease of use of this expression vector system will make it accessible to a wide range of researchers in plant biology and biotechnology.
Plasmid Construction
The construction of pJL24, pCB:GFP, and pJL3:P19 have been described previously (Lindbo, 2007
PCR products of the following genes were cloned into PacI- and AvrII-digested pJL-TRBO: (1) the cycle 3 mutant version of the gfp gene (gfpc3; Crameri et al., 1996
Binary plasmids purified from E. coli cultures were transformed into A. tumefaciens GV3101 using the freeze-thaw method (Chen et al., 1994
N. benthamiana plants were grown in a growth chamber with an 18-h photoperiod, 25°C to 27°C. For GFP photography, plants were photographed with a Cannon G6 digital camera equipped with a Tiffen Deep Yellow 15 filter. Plants were illuminated with a hand-held long-wave UV lamp (UVP Blak-Ray model UVL-56).
Total soluble protein extracts of agroinfiltrated plant tissue were prepared by freezing tissue samples in liquid nitrogen and then grinding in the presence of 4 volumes/g fresh weight, 50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20, 0.1% β-mercaptoethanol. Extracts were clarified by centrifuging for 15 min at 12,000g to 15,000g at 4°C. Clarified supernatant was stored at –20°C. Equal volumes of clarified extract of each treatment were combined with SDS-PAGE loading dye (Laemmli, 1970
Samples of clarified plant protein extracts, prepared as described above (or standards of purified GFP), were diluted in 50 mM carbonate buffer (pH 9.6). Protein samples in wells of a 96-well plate (Costar; white polystyrene) were read on a Perkin-Elmer HTS 7000 BioAssay reader with 405-nm excitation/535-nm emission filters.
His-6 C-terminally tagged GFP was expressed in plants from a TRBO replicon by agroinfection. Plant tissue was collected at approximately 5 dpi and ground in 4 volumes of extraction buffer (50 mM phosphate, pH 8.0, 10 mM Tris, pH 8.0, 500 mM NaCl, 0.1% Tween 20, 0.1% Nonidet P-40, 0.1% β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride). Extract was filtered through cheesecloth, then centrifuged at 12,000g, 4°C for 20 min. Clarified supernatant was then passed through a –20°C freeze-thaw cycle. After thawing, samples were centrifuged (as before). Imidazole was added to supernatant for a final concentration of 10 mM. One-half milliliter of washed nickel nitilotriacetic acid agarose beads (Qiagen) was added to 8 to 10 mL of extract and incubated at 4°C on a rocker for 1 to 2 h. The column was washed in 20 to 25 column volumes of wash buffer (50 mM phosphate, pH 8.0, 500 mM NaCl, 0.1% Tween 20, 20 mM imidazole). Bound His-tagged GFP was eluted with 250 mM imidazole in 1x phosphate-buffered saline. The eluted fraction was dialyzed twice (6 h to overnight) into 1,000 volumes of 1x phosphate-buffered saline (11.9 mM phosphate, pH 7.4, 137 mM NaCl, 2.7 mM KCl) at 4°C. Protein concentration was estimated using bicinchoninic acid assay (Pierce) and bovine serum albumin as a standard.
We thank Shannon Woody for excellent technical assistance. Received July 28, 2007; accepted August 16, 2007; published August 24, 2007.
1 This work was supported by a grant from The Ohio State University/OARDC Research Enhancement Competitive Grants Program.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: John A. Lindbo (john_lindbo2003{at}yahoo.com).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.106377 * E-mail john_lindbo{at}yahoo.com.
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