Transgenic Tobacco Cells Research Articles

An efficient protocol for the extraction of pigment-free active polyphenol oxidase and soluble proteins from plant cells.

The elimination of brownish pigments from plant protein extracts has been a challenge in plant biochemistry studies. Although numerous approaches have been developed to reduce pigments for enzyme assays, none has been able to completely remove pigments from plant protein extracts for biochemical studies. A simple and effective protocol was developed to completely remove pigments from plant protein extracts. Proteins were extracted from red anthocyanin-rich transgenic and greenish wild-type tobacco cells cultured on agar-solidified Murashige and Skoog medium. Protein extracts from these cells were brownish or dark due to the pigments. Four approaches were comparatively tested to show that the diethylaminoethyl (DEAE)-Sephadex anion exchange gel column was effective in completely removing pigments to obtain transparent pigment-free protein extracts. A Millipore Amicon® Ultra 10K cut-off filter unit was used to effectively desalt proteins. Moreover, the removal of pigments significantly improved the measurement accuracy of total soluble proteins. Furthermore, enzymatic assays using catechol as a substrate coupled with high-performance liquid chromatography analysis demonstrated that the pigment-free proteins not only showed polyphenol oxidase (PPO) activity but also enhanced the catalytic activity of PPO. Taken together, this protocol is effective for extracting pigment-free plant proteins for plant biochemistry studies. A simple and effective protocol was successfully developed to not only completely and effectively remove anthocyanin and polyphenolics-derived quinone pigments from plant protein extracts but also to decrease the effects of pigments on the measurement accuracy of total soluble proteins. This robust protocol will enhance plant biochemical studies using pigment-free native proteins, which in turn increase their reliability and sensitivity.

Promoter Variation of the Key Apple Fruit Texture Related Gene MdPG1 and the Upstream Regulation Analysis

MdPG1 encoding polygalacturonase in apple (Malus × domestica) is a key gene associated with fruit firmness and texture variations among apple cultivars. However, the causative variants of MdPG1 are still not known. In this study, we identified a SNPA/C variant within an ERF-binding element located in the promoter region of MdPG1. The promoter containing the ERF-binding element with SNPA, rather than the SNPC, could be strongly bound and activated by MdCBF2, a member of the AP2/ERF transcription factor family, as determined by yeast-one-hybrid and dual-luciferase reporter assays. We also demonstrated that the presence of a novel long non-coding RNA, lncRNAPG1, in the promoter of MdPG1 was a causative variant. lncRNAPG1 was specifically expressed in fruit tissues postharvest. lncRNAPG1 could reduce promoter activity when it was fused to the promoter of MdPG1 and a tobacco gene encoding Mg-chelatase H subunit (NtCHLH) in transgenic tobacco cells but could not reduce promoter activity when it was supplied in a separate gene construct, indicating a cis-regulatory effect. Our results provide new insights into genetic regulation of MdPG1 allele expression and are also useful for the development of elite apple cultivars.

Optimizing the particle bombardment conditions in cell suspension cultures of Nicotiana tabacum and expression of the recombinant antihypertensive amarantin

The first successful attempt to produce transgenic tobacco cell suspensions expressing a modified version of amarantin, through direct gene transfer using particle gun is described. The physical parameters for transformation were optimized using the gene encoding β-glucuronidase (gusA) as the reporter gene. Statistical analysis revealed that helium pressure, target distance, and osmotic pretreatment had significant influence on transformation efficiency. Of the different variables evaluated, a combination of sorbitol 0.5 M pretreatment, shooting distance of 7 cm and firing pressure of 900 psi were found ideal. Selection of putative transgenic calli was done on Hygromicine-selection medium. Presence of the transgene was confirmed using histological glucuronidase assay and PCR analysis. Accumulation and stable expression of the recombinant amarantin was detected using Western blot analysis. Transformation efficiency in terms of biochemical GUS assay was of 60%. The present results demonstrate the possibility of incorporating and expressing transgene encoding recombinant proteins of interest, in tobacco cell suspension through biolistic delivery system.

Salt-inducible expression of OsJAZ8 improves resilience against salt-stress

BackgroundProductivity of important crop rice is greatly affected by salinity. The plant hormone jasmonate plays a vital role in salt stress adaptation, but also evokes detrimental side effects if not timely shut down again. As novel strategy to avoid such side effects, OsJAZ8, a negative regulator of jasmonate signalling, is expressed under control of the salt-inducible promoter of the transcription factor ZOS3–11, to obtain a transient jasmonate signature in response to salt stress. To modulate the time course of jasmonate signalling, either a full-length or a dominant negative C-terminally truncated version of OsJAZ8 driven by the ZOS3–11 promoter were expressed in a stable manner either in tobacco BY-2 cells, or in japonica rice.ResultsThe transgenic tobacco cells showed reduced mortality and efficient cycling under salt stress adaptation. This was accompanied by reduced sensitivity to Methyl jasmonate and increased responsiveness to auxin. In the case of transgenic rice, the steady-state levels of OsJAZ8 transcripts were more efficiently induced under salt stress compared to the wild type, this induction was more pronounced in the dominant-negative OsJAZ8 variant.ConclusionsThe result concluded that, more efficient activation of OsJAZ8 was accompanied by improved salt tolerance of the transgenic seedlings and demonstrates the impact of temporal signatures of jasmonate signalling for stress tolerance.

Metabolomics-guided investigations of unintended effects of the expression of the hydroxycinnamoyl quinate hydroxycinnamoyltransferase (hqt1) gene from Cynara cardunculus var. scolymus in Nicotiana tabacum cell cultures

Chlorogenic acids (CGAs) are phenolic compounds biosynthesized in the phenylpropanoid pathway, with hydroxycinnamoyl quinate hydroxycinnamoyltransferase (HQT) as the key enzyme. Variation of CGAs has been noted in different plants, with globe artichoke (Cynara cardunculus var. scolymus L.) producing high amounts and a diverse spectrum of CGAs in its leaves. In the current study, the effect of overexpression of the hqt1 transgene from globe artichoke in tobacco was evaluated at the metabolome level. Here, metabolomic approaches based on ultra-high performance liquid chromatography coupled to mass spectrometry, together with chemometric models such as principal component analysis and orthogonal partial least square discriminant analysis, were employed to evaluate altered metabolic changes due to hqt1 overexpression. CGA profiles (caffeoylquinic acids: 3-CQA, 4-CQA and 5-CQA; p-coumaroylquinic acids: 4-pCoQA and 5-pCoQA; and 4,5-di-caffeoylquinic acid) of transgenic tobacco cell cultures were detected at lower concentrations than in the wild type. Interestingly, the cells were found to rather accumulate, as an unintended effect, abscisic acid - and benzoic acid derivatives. The results suggest that insertion of hqt1 in tobacco, and overexpression in undifferentiated cells, led to rechannelling of the phenylpropanoid pathway to accumulate benzoic acids. These findings proved to be contrary to the results shown elsewhere in leaf tissues, thus indicating differential metabolic control and regulation in the undifferentiated cell culture system.

Iron deficiency stress can induce MxNAS1 protein expression to facilitate iron redistribution in Malus xiaojinensis.

Iron (Fe) is a vital trace element in plants, and deficiency of this element in apple trees can reduce fruit quality. Nicotianamine (NA) is known to play an important role in Fe transport and endogenous hormone balance. In the present study, we investigated the role of a nicotianamine synthase 1 gene (MxNas1) in an apple species, Malus xiaojinensis, that has a more Fe-efficient genotype than other apple species and ecotypes. To characterise the response of M.xiaojinensis to Fe deficiency, we used quantitative Q-PCR to determine the level of expression of MxNas1 and Western blot to measure protein levels. Immunohistochemical staining and GFP fluorescence localisation of the MxNAS1 protein were also carried out. HPLC and polarised absorption spectrophotometry were performed to investigate the effects of overexpression of MxNas1 in order to elucidate the role of MxNAS1 in the cellular uptake of active Fe in tobacco suspension cells. We found that MxNas1 expression and protein levels were higher under Fe deficiency stress than under Fe sufficiency. Immunohistochemical staining showed that MxNAS1 was localised mainly in the epidermal and vascular tissues of the roots, vascular tissues of the stem and palisade cells of mature leaves, and in parenchyma cells of young leaves. MxNAS1 was mainly localised in the plasma membranes and vesicles of protoplasts. In addition, overexpression of MxNas1 in stable transgenic tobacco cells increased NA and active Fe content under Fe sufficiency. The results suggest that MxNas1 expression in M.xiaojinensis is induced in response to Fe deficiency stress, resulting in higher levels of the protein. MxNAS1 may be involved in the redistribution of Fe in M.xiaojinensis under Fe deficiency.

Structured plant metabolomics for the simultaneous exploration of multiple factors.

Multiple factors act simultaneously on plants to establish complex interaction networks involving nutrients, elicitors and metabolites. Metabolomics offers a better understanding of complex biological systems, but evaluating the simultaneous impact of different parameters on metabolic pathways that have many components is a challenging task. We therefore developed a novel approach that combines experimental design, untargeted metabolic profiling based on multiple chromatography systems and ionization modes, and multiblock data analysis, facilitating the systematic analysis of metabolic changes in plants caused by different factors acting at the same time. Using this method, target geraniol compounds produced in transgenic tobacco cell cultures were grouped into clusters based on their response to different factors. We hypothesized that our novel approach may provide more robust data for process optimization in plant cell cultures producing any target secondary metabolite, based on the simultaneous exploration of multiple factors rather than varying one factor each time. The suitability of our approach was verified by confirming several previously reported examples of elicitor–metabolite crosstalk. However, unravelling all factor–metabolite networks remains challenging because it requires the identification of all biochemically significant metabolites in the metabolomics dataset.

Scale-up of transgenic tobacco cells that express intimin of enterohemorrhagic Escherichia coli O157:H7 for use as a transitional platform for an oral cattle vaccine

Intimin is the primary adhesin protein of enterohemorrhagic Escherichia coli O157:H7, a pathogen carried by cattle and transmitted to humans via contaminated food and water. This work describes the rapid production of 1.3 kg dry weight of transgenic tobacco cells expressing the C-terminal 261 amino acids of intimin (Int261) for subsequent oral vaccine testing to reduce cattle colonization by E. coli O157:H7. Both a stirred-tank bioreactor (60 L) and a less capital-intensive oxygenated carboy culture were shown feasible for generating this scale of transgenic plant tissue culture biomass. Antibiotic selection pressure was not required during scale-up to maintain Int261 expression over the 13-wk culture period. Extended medium autoclave times of up to 90 min used for bioreactor sterilization had only minimal impact on nutrient uptake, culture growth, and intimin expression. Plant tissue was transformed, produced in vitro, and available for feeding studies in a fraction of the time required to develop and grow transgenic plants.

Expression of a taste-modifying protein, miraculin, in by-2 tobacco cell line (Nicotiana tabacum L. Cv Bright Yellow 2)

Miraculin is a taste-modifying and low calorie protein isolated from the red berries of Richadella dulcifica, a shrub native to West Africa. In this study, a synthetic miraculin gene was designed to optimize its codon usage for BY-2 tobacco cell lines. This gene was inserted into two constructs including pBI121/HSP-pro:Mir:HSP-ter that contains HSP promoter and HSP terminator isolated from Arabidopsis thaliana for enhancing production of miraculin and pBI121/35S-pro:Mir:NOS-ter for transformation into BY-2 tobacco cell lines. The presence and expression of miraculin gene in BY-2 tobacco cell lines were verified by PCR and Western blot, respectively. It was demonstrated that transgenic BY-2 tobacco cell lines grew stably in liquid media after 4-5 times of transplant. The expression of miraculin gene in transgenic BY-2 tobacco cell lines resulted in the accumulation of miraculin protein with molecular mass of approximately 43-45 kDa. Besides, it was observed that the expression of miraculin in HSP-pro:Mir:HSP-ter transformed BY-2 tobacco cell lines is better than the expression of miraculin in 35S-pro:Mir:HSP-ter transformed ones. For the first time, miraculin recombinant protein has been expressed in BY-2 tobacco cells providing a great potential for research and production of miraculin protein at industrial scale.

Expression of Multiple Taenia Solium Immunogens in Plant Cells Through a Ribosomal Skip Mechanism.

Taenia solium cysticercosis is a major parasitic disease that affects the human health and the economy in underdeveloped countries. Porcine cysticercosis, an obligatory stage in the parasite life cycle, is a suitable target for vaccination. While several recombinant and synthetic antigens proved to be effective as vaccines, the cost and logistic difficulties have prevented their massive use. Taking this into account, a novel strategy for developing a multi-epitope low-cost vaccine is herein explored. The S3Pvac vaccine components (KETc1, KETc12, KETc7, and GK1 [KETc7]) and the protective HP6/TSOL18 antigen were expressed in a Helios2A polyprotein system, based on the 'ribosomal skip' mechanism mediated by the 2A sequence (LLNFDLLKLAGDVESNPG-P) derived from the Foot-and-mouth disease virus, which induces self-cleavage events at a translational level. This protein arrangement was expressed in transgenic tobacco cells. The inserted sequence and its transcript were detected in several Helios2A lines, with some lines showing recombinant protein accumulation levels up to 1.3 µg/g of fresh weight in leaf tissues. The plant-derived Helios2A vaccine was recognized by antibodies in the cerebral spinal fluid from neurocysticercosis patients and elicited specific antibodies in BALB/c immunized mice. These evidences point to the Helios2A polyprotein as a promising system for expressing multiple antigens of interest for vaccination and diagnosis in one single construction.

Grape expansins, VvEXPA14 and VvEXPA18 promote cell expansion in transgenic Arabidopsis plant

We herein report the molecular characterization of grape expansins, VvEXPA14 and VvEXPA18. VvEXPA14 and VvEXPA18 transcription was detected in grape berries at 2 weeks post flowering and their expression was upregulated during growth phase I, the period of rapid berry expansion. Experiments using VvEXPA14:GFP or VvEXPA18:GFP fusion proteins demonstrated that both expansins were localized at cell wall, suggesting that both expansins may function on cell wall. Transgenic tobacco cells overexpressing VvEXPA14 or VvEXPA18 exhibited cell expansion. In addition, VvEXPA14- or VvEXPA18-overexpressing Arabidopsis plants showed primary root elongation associated with cell expansion. VvEXPA14 overexpression enlarged rosette leaves in Arabidopsis transgenic plants, although the height of the plants remained unchanged. Since plant expansin play role in cell wall loosing and softening, these results underscore the possibility that VvEXPA14 and VvEXPA18 are involved in cell expansion and plant growth.

Overexpression of the CaTIP1-1 pepper gene in tobacco enhances resistance to osmotic stresses.

Both the gene expression and activity of water channel protein can control transmembrane water movement. We have reported the overexpression of CaTIP1-1, which caused a decrease in chilling tolerance in transgenic plants by increasing the size of the stomatal pore. CaTIP1-1 expression was strongly induced by salt and mannitol stresses in pepper (Capsicum annuum). However, its biochemical and physiological functions are still unknown in transgenic tobacco. In this study, transient expression of CaTIP1-1-GFP in tobacco suspension cells revealed that the protein was localized in the tonoplast. CaTIP1-1 overexpressed in radicle exhibited vigorous growth under high salt and mannitol treatments more than wild-type plants. The overexpression of CaTIP1-1 pepper gene in tobacco enhanced the antioxidant enzyme activities and increased transcription levels of reactive oxygen species-related gene expression under osmotic stresses. Moreover, the viability of transgenic tobacco cells was higher than the wild-type after exposure to stress. The pepper plants with silenced CaTIP1-1 in P70 decreased tolerance to salt and osmotic stresses using the detached leaf method. We concluded that the CaTIP1-1 gene plays an important role in response to osmotic stresses in tobacco.

Molecular Farming – Production of pharmaceuticals in plants and plant cells

Many different plant-based systems have been used to produce valuable small molecules or recombinant proteins but only a small number have made the leap from an experimental platform to a viable commercial process. This reflects a combination of factors, principally the technical issues that must be addressed to achieve competitive performance, the economic principles that need to be satisfied to ensure manufacturing processes are financially viable and sustainable, and the regulatory demands that must be met to ensure that products manufactured in plants are safe, efficacious and meet the quality standards demanded by the regulators [1,2]. In general, plants and plant cells are ideal production platforms, which can be easily scaled up to provide valuable products for pharmaceutical, cosmetic and industrial applications. Typically plant systems are grown in containment under defined conditions, either in the greenhouse or in bioreactors, allowing process controls to regulate growth and product formation, thus ensuring regulatory compliance. In this presentation, the advantages and disadvantages of commonly used plant-based systems are presented as well as strategies for recovering the final product. In many cases production and downstream processes have to be optimized to establish a commercially viable platform and therefore novel strategies for strain and process optimization, helping to increase yields and scalability are presented. These approaches include the optimization of expression vectors and recombinant protein targeting [3], the testing of different plant platforms [4], the use of statistical experimental techniques to optimise medium and cultivation conditions [5], establishment of elite lines by flow cytometry [6], as well as strategies for scale up and GMP-compliant production [7].

Assessment of cultivation factors that affect biomass and geraniol production in transgenic tobacco cell suspension cultures.

A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.

Molecular pharming in plants and plant cell cultures: a great future ahead?

Plant biotechnology may not be a familiar concept to the general public, but it is a rapidly developing field of research that involves the use of plants, plant tissues and plant cell cultures to make or modify products and processes. The versatility of plants and plant cells can be harnessed to produce diverse products, including valuable proteins. This is often described as ‘molecular farming’ and it requires the introduction of foreign DNA into plants or plant cells, turning them into factories for the production of specific recombinant protein products. The term ‘molecular pharming’ is often used instead to highlight the production of protein-based biopharmaceuticals, which contributes to the sustainable production of drugs that promote human and animal wellbeing. Both terms also apply to the production of valuable secondary metabolites such as the anticancer drugs paclitaxel, vincristine and vinblastine, but we will focus on recombinant proteins and their use as biopharmaceuticals in this article. The biopharmaceutical markets have expanded rapidly over the last 20 years, and are projected to more than double in volume over the next decade from US$200 billion in 2013 to at least US$500 billion in 2020. The two major biopharmaceutical production systems are microbes (mainly Escherichia coli and yeast) and mammalian cells such as the Chinese hamster ovary platform. In both cases, productivity has increased substantially over the last decade due to process optimization, platform standardization and genetic improvements. Both the US FDA and European Medicines Agency are familiar with these systems, and standard protocols can be followed to ensure the approval of new products. However, equivalent protocols are only just emerging for plant-based production systems, and only one plant-derived biopharmaceutical protein is currently on the market. With their established production infrastructure and regulatory framework, microbial and mammalian production systems have raced far ahead of their plant-based counterparts. No company will change their production host without a clear economic benefit, nor will they consider plants and plant cells for new products if there is no advantage over their incumbent technology. Furthermore, new companies will not base their manufacturing on a second-best option. Therefore, plant-based systems must begin to compete head-to-head with the established systems and, on a technological basis, we can already identify the areas where plantbased systems have the advantage, namely in terms of speed, improved product quality and scalability. The international success story of molecular pharming began in 2006 with the US Department of Agriculture approval of a poultry vaccine against Newcastle disease developed by Dow AgroSciences (IN, USA) [1,2]. The vaccine was manufactured in transgenic tobacco cell suspension cultures and was a benchmark for the regulatory acceptance of plants as a manufacturing platform, Molecular pharming in plants and plant cell cultures: a great future ahead?

Alteration of the proteostasis network of plant cells promotes the post-endoplasmic reticulum trafficking of recombinant mutant (L444P) human β-glucocerebrosidase

Gaucher disease is a prevalent lysosomal storage disease characterized by a deficiency in the activity of lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a L444P missense mutation in the GCase protein, which results in its disrupted folding in the endoplasmic reticulum (ER) and impaired post-ER trafficking. To determine whether the post-ER trafficking of this severely malfolded protein can be restored, we expressed the mutant L444P GCase as a recombinant protein in transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cells, in which the GCase variant was equipped with a plant signal peptide to allow for secretion upon rescued trafficking out of the ER. The recombinant L444P mutant GCase was retained in the plant endoplasmic reticulum (ER). Kifunensine and Eeyarestatin I, both inhibitors of ER-associated degradation (ERAD), and the proteostasis regulators, celastrol and MG-132, increased the steady-state levels of the mutant protein inside the plant cells and further promoted the post-ER trafficking of L444P GCase, as indicated by endoglycosidase-H sensitivity- and secretion- analyses. Transcript profiling of genes encoding ER-molecular chaperones, ER stress responsive proteins, and cytoplasmic heat shock response proteins, revealed insignificant or only very modest changes in response to the ERAD inhibitors and proteostasis regulators. An exception was the marked response to celastrol which reduced the steady-state levels of cytoplasmic HSP90 transcripts and protein. As HSP90 participates in the targeting of misfolded proteins to the proteasome pathway, its down-modulation in response to celastrol may partly account for the mechanism of improved homeostasis of L444P GCase mediated by this triterpene.

Mechanism Of Oral Tolerance Induced By Bioencapsulated Coagulation Factor IX In Hemophilia B Mice

Currently, the most serious complication in hemophilia therapy is the development of neutralizing antibodies (inhibitors) against the therapeutic coagulation factor. Inhibitor formation in treatment of hemophilia B (deficiency in factor IX, FIX) is less frequent than in hemophilia A. However, FIX inhibitors tend to be high-titer, and 25-50% of such patients develop anaphylactic reactions against FIX. Clinical immune tolerance induction (ITI) protocols (to eliminate the inhibitors) are lengthy, expensive, and are often terminated in hemophilia B due to anaphylaxis or nephrotic syndrome. In order to establish a protocol for prevention of FIX inhibitors in high-risk patients (i.e. those with gene deletions or other severe mutations) while avoiding immune suppressive drugs or genetic manipulation, we have developed a source of bioencapsulated human FIX. Oral delivery of plant leaf material, expressing CTB-FIX in chloroplasts, effectively suppresses inhibitor formation and prevents anaphylaxis in FIX replacement therapy in hemophilia B mice of the C3H/HeJ F9-/- strain (PNAS 107:7101, 2010). This is accomplished by delivery of the fusion molecule to the epithelium of Peyer's Patches (PP), which expresses high levels of GM1, the receptor for the CTB transmucosal carrier. Subsequently, FIX antigen is transferred to dendritic cells (DCs) in the PP and also into the circulation. Mice fed twice per week for 2 months fail to form inhibitors during a 2-month treatment phase (1 IU rFIX, IV, once/week), lack IgE formation and fatal anaphylactic reactions (which occur in control mice), but still form non-inhibitory IgG1 at reduced titers. An oral dose of FIX antigen equivalent to ∼1.5 IU rFIX/kg is sufficient to achieve tolerance. To define the underlying mechanism, we analyzed DC and lymphocyte populations in different compartments (n=4-6 for all experimental groups and data sets). Compared to control mice, which received FIX only or were fed with WT plant material and treated with FIX, tolerized mice had a marked increase in the frequencies of CD103+ DC and pDC in mesenteric lymph nodes (MLN) and PP. Both DC subsets are known to play crucial roles in oral tolerance and Treg induction. A minor, non-significant increase pDCs was also seen in the spleen. Interestingly, tolerized mice also showed a significant increase in the frequencies of CD4+ T cells expressing gut homing receptors (CCR9+ and α4β7), including in the spleen and thus indicating interaction between the gut and systemic immune systems. The spleen is a major site for systemic immune surveillance and critical for responses to intravenous FIX. Splenocytes re-stimulated in vitro with hF.IX were anayzed by RT-PCR array. We found that Th1 and Th2 cytokines (IL2, IL-4, IL-13, IFN-γ) were downregulated by oral FIX delivery, whereas immune suppressive cytokines (IL-10, TGF-β), and the Treg marker Foxp3 were upregulated, suggesting a switch from an effector to a suppressive response. Moreover, the frequency of LAP+CD4+CD25-FoxP3- T cells (which suppress responses via TGF-β) was dramatically increased in FIX-fed mice in PP (from <5% to 10-20%), and to lesser extent in spleen and MLN. Although the overall frequency of CD4+CD25+FoxP3+ Treg was not altered, tolerized mice had a significant increase in LAP+ cells in that population, indicating Treg activation. Interestingly, we failed to prevent anaphylaxis or inhibitor formation in C3H/HeJ [F9-/- x IL-10-/-] mice (n=7 per group, 42% mortality after 4th FIX injection, >5 BU in surviving animals). Therefore, both IL-10 and TGF-βare integral components of the tolerance mechanism. In summary, oral delivery of CTB-FIX antigen bioencapsulated in plant cells prevents anaphylaxis and inhibitor formation against FIX via induction of a complex immune regulatory network, involving increases in Treg-promoting subsets of DC, induction/activation of LAP+ Treg, and active cytokine-mediated suppression. Preliminary data suggest that the regimen can also be used to desensitize mice from FIX. While animal experiments performed thus far have utilized transgenic tobacco cells, we have now generated and characterized FIX-transplastomic lettuce expressing CTB-FIX, which will be tested towards translation of this approach. Disclosures:Daniell:Bayer: Research Funding. Herzog:Genzyme: AAV technology, AAV technology Patents & Royalties.

Safety and immunogenicity of bacterial and tobacco plant cell line derived recombinant native and mutant Escherichia coli heat-labile toxin in chickens

The safety and immunogenicity of the mammalian mucosal adjuvants, Escherichia coli wild-type heat-labile holotoxin (LT) and E. coli mutant LT (LTA-K63/LTB), were examined in 1-day-old chicks and 10-day-old to 21-day-old broilers. Biologically active, E. coli recombinant wild-type LT and recombinant LTA-K63/LTB produced in a transgenic Nicotiana tabacum (NT-1) tobacco cell line (SLT102) were tested for safety and antigenicity following various routes of administration. Safety was assessed by clinical signs, body weight gain, gross organ pathology and wet organ weight, and histopathology. Antigenicity was assessed using LT-B-specific serum IgG enzyme-linked immunosorbent assay. Parenteral administration of E. coli recombinant wild-type LT did not have any discernible effect on bird health and was well tolerated at levels up to 400 µg per dose. Recombinant, SLT102-derived mutant LT derived from SLT102 cells retained in vitro ganglioside binding and was safe and antigenic following repeated mucosal administration to birds. The highest systemic LT-B-specific IgG titres were detected in birds that received three on-feed doses of SLT102-derived mutant LT. Among the various SLT102-derived mutant LT preparations tested, whole, wet cells or whole cell lysates were the most antigenic. These results demonstrate for the first time that E. coli-derived recombinant, wild-type LT holotoxin is well tolerated following multiple administrations to young birds at body weight doses previously reported to be enteropathogenic and toxic in mammalian species. Moreover, these data also demonstrate the feasibility of using recombinant wild-type and mutant LT produced in transgenic NT-1 tobacco cells as safe and potent vaccine adjuvants in poultry.

Functional ectodomain of the hemagglutinin-neuraminidase protein is expressed in transgenic tobacco cells as a candidate vaccine against Newcastle disease virus

Recently, the use of plants for the production of recombinant proteins has been well demonstrated with promising outcomes. In this study, an efficient Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells system expressing the ectodomain of hemagglutinin-neuraminidase (eHN) protein from Newcastle disease virus (NDV) strain AF2240 was established. Transgenic tobacco BY-2 cell cultures expressing the immunogenic eHN protein were generated and the translation efficiency of eHN protein was enhanced using the 5′-untranslated region of Nicotiana tabacum alcohol dehydrogenase gene (NtADH 5′-UTR) under the control of strong cauliflower mosaic virus (CaMV 35S) promoter. Transgenic lines verified by real-time PCR showed high level of eHN mRNA transcripts and immunoblotting confirmed the presence of 66 kD eHN protein. The eHN protein was stably produced in an average of 0.2–0.4 % total soluble protein. Green fluorescent protein-tagged eHN protein was expressed and localized at the cytosol of BY-2 cell. All mice receiving purified eHN protein from transgenic tobacco BY-2 cells produced specific anti-NDV antibodies. We concluded that plant made eHN elicit immune response and can serve as candidate vaccine against NDV.

Chloroplast‐targeted bacterial RecA proteins confer tolerance to chloroplast DNA damage by methyl viologen or UV‐C radiation in tobacco (Nicotiana tabacum) plants

The nature and importance of the DNA repair system in the chloroplasts of higher plants under oxidative stress or UV radiation-induced genotoxicity was investigated via gain-of-functional approaches exploiting bacterial RecAs. For this purpose, transgenic tobacco (Nicotiana tabacum) plants and cell suspensions overexpressing Escherichia coli or Pseudomonas aeruginosa RecA fused to a chloroplast-targeting transit peptide were first produced. The transgenic tobacco plants maintained higher amounts of chloroplast DNA compared with wild-type (WT) upon treatments with methyl viologen (MV), a herbicide that generates reactive oxygen species (ROS) in chloroplasts. Consistent with these results, the transgenic tobacco leaves showed less bleaching than WT following MV exposure. Similarly, the MV-treated transgenic Arabidopsis plants overexpressing the chloroplast RecA homologue RECA1 showed weak bleaching, while the recA1 mutant showed opposite results upon MV treatment. In addition, when exposed to UV-C radiation, the dark-grown E. coli RecA-overexpressing transgenic tobacco cell suspensions, but not their WT counterparts, resumed growth and greening after the recovery period under light conditions. Measurements of UV radiation-induced chloroplast DNA damage using DraI assays (Harlow et al. 1994) with the chloroplast rbcL DNA probe and quantitative PCR analyses showed that the transgenic cell suspensions better repaired their UV-C radiation-induced chloroplast DNA lesions compared with WT. Taken all together, it was concluded that RecA-overexpressing transgenic plants are endowed with an increased chloroplast DNA maintenance capacity and enhanced repair activities, and consequently have a higher survival tolerance to genotoxic stresses. These observations are made possible by the functional compatibility of the bacterial RecAs in chloroplasts.