A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression

Agrobacterium-mediated gene transfer, or agroinfiltration, can be a highly efficient method for transforming and inducing transient transgene expression in plant tissue. The technique uses the innate DNA secretion pathway of Agrobacterium tumefaciens to vector a particular plasmid-encoded segment of DNA from the bacteria to plant cells. Vacuum is often applied to plant tissue submerged in a suspension of A. tumefaciens to improve agroinfiltration. However, the effects of vacuum application on agroinfiltration and in planta transient transgene expression have not been well quantified. Here we show that vacuum application and release act to drive A. tumefaciens suspension into the interior of leaf tissue. Moreover, the amount of suspension that enters leaves can be predicted based on the vacuum intensity and duration. Furthermore, we show that transient expression levels of an agroinfiltrated reporter gene vary in response to the amount of A. tumefaciens vacuum infiltrated into leaf tissue, suggesting that vacuum infiltration conditions can be tailored to achieve optimal transient transgene expression levels after agroinfiltration.     

Depletion of the NbCORE receptor drastically improves agroinfiltration productivity in older Nicotiana benthamiana plants

Nicotiana benthamiana is increasingly used for transient gene expression to produce antibodies, vaccines, and other pharmaceutical proteins but transient gene expression is low in fully developed, 6–8-week old plants. This low gene expression is thought to be caused by the perception of the cold shock protein (CSP) of Agrobacterium tumefaciens. The CSP receptor is contested because both NbCSPR and NbCORE have been claimed to perceive CSP. Here, we demonstrate that CSP perception is abolished in 6-week-old plants silenced for NbCORE but not NbCSPR. Importantly, older NbCORE-silenced plants support a highly increased level of GFP fluorescence and protein upon agroinfiltration. The drastic increase in transient protein production in NbCORE-depleted plants offers new opportunities for molecular farming, where older plants with larger biomass can now be used for efficient protein expression.     

Optimization of the bioprocessing conditions for scale‐up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system

Use of transient expression for the rapid, large‐scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale‐up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha‐1‐antitrypsin (rAAT) by co‐infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD₆₀₀ of 0.5 and a 0.25‐min vacuum infiltration, and multiple 1‐min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post‐induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves

We have constructed a matched set of binary vectors designated pGD, pGDG and pGDR for the expression and co-localization of native proteins and GFP or DsRed fusions in large numbers of plant cells. The utility of these vectors following agroinfiltration into leaves has been demonstrated with four genes from Sonchus yellow net virus, a plant nucleorhabdovirus, and with a nucleolar marker protein. Of the three SYNV proteins tested, sc4 gave identical localization patterns at the cell wall and nucleus when fused to GFP or DsRed. However, some differences in expression patterns were observed depending on whether DsRed or GFP was the fusion partner. In this regard, the DsRed:P fusion showed a similar pattern of localization to GFP:P, but localized foci appeared in the nucleus and near the periphery of the nucleus. Nevertheless, the viral nucleocapsid protein, expressed as a GFP:N fusion, co-localized with DsRed:P in a subnuclear locale in agreement with our previous observations (Goodin et al., 2001). This locale appears to be distinct from the nucleolus as indicated by co-expression of the N protein, DsRed:P and a nucleolar marker AtFib1 fused to GFP. The SYNV M protein, which is believed to be particularly prone to oligomerization, was detectable only as a GFP fusion. Our results indicate that agroinfiltration with bacteria containing the pGD vectors is extremely useful for transient expression of several proteins in a high proportion of the cells of Nicotiana benthamiana leaves. The GFP and DsRed elements incorporated into the pGD system should greatly increase the ease of visualizing co-localization and interactions of proteins in a variety of experimental dicotyledonous hosts.     

Attachment of Agrobacterium tumefaciens to leaf tissue in response to infiltration conditions

Transient expression of recombinant proteins in plant tissues following Agrobacterium‐mediated gene transfer is a promising technique for rapid protein production. However, transformation rates and transient expression levels can be sub‐optimal depending on process conditions. Attachment of Agrobacterium tumefaciens to plant cells is an early, critical step in the gene transfer pathway. Bacterial attachment levels and patterns may influence transformation and, by extension, transient expression. In this study, attachment of A. tumefaciens to lettuce leaf tissue was investigated in response to varying infiltration conditions, including bacterial density, surfactant concentration, and applied vacuum level. Bacterial density was found to most influence attachment levels for the levels tested (10⁸, 10⁹, and 10¹⁰ CFU/mL), with the relationship between bacterial density and attachment levels following a saturation trend. Surfactant levels tested (Break‐Thru S240: 1, 10, 100, and 1,000 µL/L) also had a significant positive effect on bacterial attachment while vacuum level (5, 25, and 45 kPa) did not significantly affect attachment in areas exposed to bacteria. In planta transgene transient expression levels were measured following infiltration with 10⁸, 10⁹, and 10¹⁰ CFU/mL bacterial suspension. Notably, the highest attachment level tested led to a decrease in transient expression, suggesting a potential link between bacterial attachment levels and downstream phenomena that may induce gene silencing. These results illustrate that attachment can be controlled by adjusting infiltration conditions and that attachment levels can impact transgene transient expression in leaf tissue. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1137–1144, 2014     

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).     

A procedure for the transient expression of genes by agroinfiltration above the permissive threshold to study temperature‐sensitive processes in plant–pathogen interactions

Localized expression of genes in plants from T‐DNAs delivered into plant cells by Agrobacterium tumefaciens is an important tool in plant research. The technique, known as agroinfiltration, provides fast, efficient ways to transiently express or silence a desired gene without resorting to the time‐consuming, challenging stable transformation of the host, the use of less efficient means of delivery, such as bombardment, or the use of viral vectors, which multiply and spread within the host causing physiological alterations themselves. A drawback of the agroinfiltration technique is its temperature dependence: early studies have shown that temperatures above 29 °C are nonpermissive to tumour induction by the bacterium as a result of failure in pilus formation. However, research in plant sciences is interested in studying processes at these temperatures, above the 25 °C experimental standard, common to many host–environment and host–pathogen interactions in nature, and agroinfiltration is an excellent tool for this purpose. Here, we measured the efficiency of agroinfiltration for the expression of reporter genes in plants from T‐DNAs at the nonpermissive temperature of 30 °C, either transiently or as part of viral amplicons, and envisaged procedures that allow and optimize its use for gene expression at this temperature. We applied this technical advance to assess the performance at 30 °C of two viral suppressors of silencing in agropatch assays [Potato virus Y helper component proteinase (HCPro) and Cucumber mosaic virus 2b protein] and, within the context of infection by a Potato virus X (PVX) vector, also assessed indirectly their effect on the overall response of the host Nicotiana benthamiana to the virus.     

Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce

Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for the generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties owing to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that are effective, safe, low cost, and amenable to large-scale manufacturing.     

High-level transient expression of recombinant protein in lettuce

Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations < or = 100 ppm in the vacuum infiltration did not affect GUS expression, while infiltration with an A. tumefaciens density of 10(7) and 10(8) colony forming units/mL, incubation at 29 degrees C, and a surfactant concentration of 1,000 ppm significantly reduced expression. Incubation in continuous light caused lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta.     

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other''s results.     

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Background

Plants 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.

Results

We 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.

Conclusion

These 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.     

A novel and fully scalable Agrobacterium spray‐based process for manufacturing cellulases and other cost‐sensitive proteins in plants

Transient transfection of plants by vacuum infiltration of Agrobacterium vectors represents the state of the art in plant‐based protein manufacturing; however, the complexity and cost of this approach restrict it to pharmaceutical proteins. We demonstrated that simple spraying of Nicotiana plants with Agrobacterium vectors in the presence of a surfactant can substitute for vacuum inoculation. When the T‐DNA of Agrobacterium encodes viral replicons capable of cell‐to‐cell movement, up to 90% of the leaf cells can be transfected and express a recombinant protein at levels up to 50% of total soluble protein. This simple, fast and indefinitely scalable process was successfully applied to produce cellulases, one of the most volume‐ and cost‐sensitive biotechnology products. We demonstrate here for the first time that representatives of all hydrolase classes necessary for cellulosic biomass decomposition can be expressed at high levels, stored as silage without significant loss of activity and then used directly as enzyme additives. This process enables production of cellulases, and other potential high‐volume products such as noncaloric sweetener thaumatin and antiviral protein griffithsin, at commodity agricultural prices and could find broad applicability in the large‐scale production of many other cost‐sensitive proteins.     

Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large‐scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal‐derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal‐derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design‐of‐experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two‐fold increase in OD₆₀₀ compared to YEB medium during a 4‐L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal‐derived components, thus facilitating the GMP‐compliant large‐scale transient expression of recombinant proteins in plants.     

Large-scale production of foreign proteins via the novel plant transient expression system in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Transient expression of foreign genes by Agrobacterium infiltration is a versatile technique that can be used as a rapid tool for functional protein production in plants. A reproducible protocol of large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L. was established in our study. Non-detached plants from soil-independent culture were used as the target organ, and vacuum infiltrating mediated by Agrobacterium tumefaciens harboring green fluorescent protein (GFP) gene was performed. Step-by-step optimization was performed and showed that the quality of plant material as well as agro-infiltration conditions were the major factors influencing the gene expression. Monitoring the transient GFP expression daily, the highest expression level was achieved on the 8th day post-infiltration. Evidence of anti-acidic fibroblast growth factor-single chain variable fragment (anti-aFGF-scFv) gene expression in pea seedling was also achieved using agro-mediated vacuum infiltration system. Our work proves that the system is suitable for the largescale production of pharmaceutical proteins. The in planta infiltration system described here provides a powerful tool to explore easily gene expression in Pisum sativum L. avoiding tissue culture steps and the labor-intensive generation of transgenic plants.     

Comparison of Expression Vectors for Transient Expression of Recombinant Proteins in Plants

Production of recombinant proteins in plants is of increasing importance for practical applications. However, the production of stable transformed transgenic plants is a lengthy procedure. Transient expression, on the other hand, can deliver recombinant proteins within a week, and many viral vectors have been constructed for that purpose. Each of them is reported to be highly efficient, robust and cost-effective. Here, a variety of expression vectors which were designed for transient and stable plant transformation, including pPZP3425, pPZP5025, pPZPTRBO, pJLTRBO, pEAQ-HT and pBY030-2R, was compared for the expression of green fluorescent protein and β-glucuronidase in Nicotiana benthamiana by Agrobacterium-mediated transient expression. Our results show that pPZPTRBO, pJLTRBO and pEAQ-HT had comparable expression levels without co-infiltration of a RNA-silencing inhibitor. The other vectors, including the non-viral vectors pPZP5025 and pPZP3425, needed co-infiltration of the RNA-silencing inhibitor P19 to give good expression levels.     

AgroLux: bioluminescent Agrobacterium to improve molecular pharming and study plant immunity

Agroinfiltration in Nicotiana benthamiana is widely used to transiently express heterologous proteins in plants. However, the state of Agrobacterium itself is not well studied in agroinfiltrated tissues, despite frequent studies of immunity genes conducted through agroinfiltration. Here, we generated a bioluminescent strain of Agrobacterium tumefaciens GV3101 to monitor the luminescence of Agrobacterium during agroinfiltration. By integrating a single copy of the lux operon into the genome, we generated a stable ‘AgroLux’ strain, which is bioluminescent without affecting Agrobacterium growth in vitro and in planta. To illustrate its versatility, we used AgroLux to demonstrate that high light intensity post infiltration suppresses both Agrobacterium luminescence and protein expression. We also discovered that AgroLux can detect Avr/Cf-induced immune responses before tissue collapse, establishing a robust and rapid quantitative assay for the hypersensitive response (HR). Thus, AgroLux provides a non-destructive, versatile and easy-to-use imaging tool to monitor both Agrobacterium and plant responses.     

High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system

Recombinant hepatitis B surface antigen (HBsAg) constitutes currently used vaccines against hepatitis B virus, and has been successfully employed as a carrier for foreign epitopes. With the aim of developing an inexpensive, easily administered vaccine source for global immunization, several groups have expressed HBsAg in plant systems. Transgenic plant-derived HBsAg assembles into virus-like particles (VLPs) and is immunogenic in both mice and humans. However, HBsAg expression is relatively low in transgenic plant systems. The time-consuming and labour-intensive process of generating transgenic plants also significantly limits high-throughput analyses of various HBsAg fusion antigens. In this paper, the high-yield rapid production of HBsAg in plant leaf using a novel viral transient expression system is described. Nicotiana benthamiana leaves infiltrated with the MagnICON viral vectors produced HBsAg at high levels, averaging 295 µg/g leaf fresh weight at 10 days post-infection, as measured by a polyclonal enzyme-linked immunosorbent assay. Transiently expressed HBsAg accumulated as the full-length product, formed disulphide-linked dimers, displayed the conformational 'a' antigenic determinant and assembled into VLPs. Immunization of mice with partially purified HBsAg elicited HBsAg-specific antibodies. Furthermore, it was found that transient production of HBsAg using vacuum infiltration of whole plants, rather than syringe infiltration of leaves, was readily scalable, and greatly improved the accumulation of correctly folded HBsAg that displays the protective 'a' determinant.     

Agrobacterium-Mediated Transient Gene Expression and Silencing: A Rapid Tool for Functional Gene Assay in Potato

Potato is the third most important food crop worldwide. However, genetic and genomic research of potato has lagged behind other major crops due to the autopolyploidy and highly heterozygous nature associated with the potato genome. Reliable and technically undemanding techniques are not available for functional gene assays in potato. Here we report the development of a transient gene expression and silencing system in potato. Gene expression or RNAi-based gene silencing constructs were delivered into potato leaf cells using Agrobacterium-mediated infiltration. Agroinfiltration of various gene constructs consistently resulted in potato cell transformation and spread of the transgenic cells around infiltration zones. The efficiency of agroinfiltration was affected by potato genotypes, concentration of Agrobacterium, and plant growth conditions. We demonstrated that the agroinfiltration-based transient gene expression can be used to detect potato proteins in sub-cellular compartments in living cells. We established a double agroinfiltration procedure that allows to test whether a specific gene is associated with potato late blight resistance pathway mediated by the resistance gene RB. This procedure provides a powerful approach for high throughput functional assay for a large number of candidate genes in potato late blight resistance.     

Rapid,in situ detection of Agrobacterium tumefaciens attachment to leaf tissue

Attachment of the plant pathogen Agrobacterium tumefaciens to host plant cells is an early and necessary step in plant transformation and agroinfiltration processes. However, bacterial attachment behavior is not well understood in complex plant tissues. Here we developed an imaging‐based method to observe and quantify A. tumefaciens attached to leaf tissue in situ. Fluorescent labeling of bacteria with nucleic acid, protein, and vital dyes was investigated as a rapid alternative to generating recombinant strains expressing fluorescent proteins. Syto 16 green fluorescent nucleic acid stain was found to yield the greatest signal intensity in stained bacteria without affecting viability or infectivity. Stained bacteria retained the stain and were detectable over 72 h. To demonstrate in situ detection of attached bacteria, confocal fluorescent microscopy was used to image A. tumefaciens in sections of lettuce leaf tissue following vacuum‐infiltration with labeled bacteria. Bacterial signals were associated with plant cell surfaces, suggesting detection of bacteria attached to plant cells. Bacterial attachment to specific leaf tissues was in agreement with known leaf tissue competencies for transformation with Agrobacterium. Levels of bacteria attached to leaf cells were quantified over time post‐infiltration. Signals from stained bacteria were stable over the first 24 h following infiltration but decreased in intensity as bacteria multiplied in planta. Nucleic acid staining of A. tumefaciens followed by confocal microscopy of infected leaf tissue offers a rapid, in situ method for evaluating attachment of A. tumefaciens' to plant expression hosts and a tool to facilitate management of transient expression processes via agroinfiltration. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Development of a new tobamovirus-based viral vector for protein expression in plants

Plants are becoming an interesting alternative system for the heterologous production of pharmaceutical proteins, providing a more scalable, cost-effective, and biologically safer option than the current expression systems. The development of plant virus expression vectors has allowed rapid and high-level transient expression of recombinant genes, and, in turn, provided an attractive plant-based production platform. Here we report the development of vectors based on the tobamovirus Pepper mild mottle virus (PMMoV) to be used in transient expression of foreign genes. In this PMMoV vector, a middle part of the viral coat protein gene was replaced by the green fluorescent protein (GFP) gene, and this recombinant genome was assembled in a binary vector suitable for plant agroinoculation. The accumulation of GFP was evaluated by observation of green fluorescent signals under UV light and by western blotting. Furthermore, by using this vector, the multiepitope gene for chikungunya virus was successfully expressed and confirmed by western blotting. This PMMoV-based vector represents an alternative system for a high-level production of heterologous protein in plants.