Transplastomic Tobacco Research Articles

Stable expression of pentameric capsomeres, HPV-16 L1 with glutathione-S-transferase in transplastomic tobacco: a step towards affordable capsomere-based HPV vaccine

Human Papillomavirus (HPV) is a single major source in causing cervical cancer worldwide which is the most common cause of death in females. Around 85% of the cervical cancer cases occur in developing countries where existing vaccines are not available or unaffordable. Hence, alternative platforms are considered necessary for the development of cost-effective vaccines against HPV for their availability in developing countries. Plants offer unique advantages for vaccine production over fermenter-based systems. Pentameric capsomeres ofHPVhave been reported to generate elevated immune responses against HPV. In present study a modified HPV-16 L1 (L1 2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) into tobacco plastids. Seven transplastomic lines of Nicotiana benthamiana were generated using biolistic transformation technique. Site specific integration of the GST-L1 2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homoplasmy of all transplastomic lines.AntigencaptureELISAwith theconformationspecific antibody Ritti01, showed protein expression as well as the retention of immunogenic epitopes of L1 protein. GST-L1 expressing tobacco plants were completely normal in their morphology. Taken together, this data contribute another step forward towards thedevelopmentof cost-effectiveplant-madevaccinesagainstHPV for resource poor countries.

Optimization of transplastomic production of hemicellulases in tobacco: effects of expression cassette configuration and tobacco cultivar used as production platform on recombinant protein yields

BackgroundChloroplast transformation in tobacco has been used extensively to produce recombinant proteins and enzymes. Chloroplast expression cassettes can be designed with different configurations of the cis-acting elements that govern foreign gene expression. With the aim to optimize production of recombinant hemicellulases in transplastomic tobacco, we developed a set of cassettes that incorporate elements known to facilitate protein expression in chloroplasts and examined expression and accumulation of a bacterial xylanase XynA. Biomass production is another important factor in achieving sustainable and high-volume production of cellulolytic enzymes. Therefore, we compared productivity of two tobacco cultivars – a low-alkaloid and a high-biomass - as transplastomic expression platforms.ResultsFour different cassettes expressing XynA produced various mutant phenotypes of the transplastomic plants, affected their growth rate and resulted in different accumulation levels of the XynA enzyme. The most productive cassette was identified and used further to express XynA and two additional fungal xylanases, Xyn10A and Xyn11B, in a high-biomass tobacco cultivar. The high biomass cultivar allowed for a 60% increase in XynA production per plant. Accumulation of the fungal enzymes reached more than 10-fold higher levels than the bacterial enzyme, constituting up to 6% of the total soluble protein in the leaf tissue. Use of a well-characterized translational enhancer with the selected expression cassette revealed inconsistent effects on accumulation of the recombinant xylanases. Additionally, differences in the enzymatic activity of crude plant extracts measured in leaves of different age suggest presence of a specific xylanase inhibitor in the green leaf tissue.ConclusionOur results demonstrate the pivotal importance of the expression cassette design and appropriate tobacco cultivar for high-level transplastomic production of recombinant proteins.

Towards Generation of Transplastomic Tobacco, Expressing a 35 kdaProtein as an Antigen: A Step towards Affordable Plant made Vaccine against Mycobacterium

Plants offer the unique opportunity to be engineered as bio-factories for the production of different antigens, enzymes and vaccines. Recently chloroplast transformation has gained strong interest in the field of Plant Made Vaccines (PMVs). An efficient cross-protective antigen against Mycobacterium is a 35 kDa protein encoded by mmpI gene in Mycobacterium leprae. Although vaccines against Mycobacterium infections are commercially available, they are neither affordable nor available for patients in resource poor countries. Thus, alternative cost-effective vaccine production approaches need to be developed. In the current study we have reported the possibility to generate transplastomic tobacco carrying 35-kDa protein conferring cross-protective resistance against two different pathogens Mycobacterium leprae and Mycobacterium avium. The mmpI gene along with an adjuvant Lymphotoxin-beta (LTB) was successfully transformed into tobacco chloroplast by the Polyethylene glycol (PEG) mediated transformation method. The PEG transformation method provides an effective and cost efficient transformation procedure and can easily be adopted in resource-poor countries in comparison to biolistic transformation. Integration of LTB and mmpI genes into transplastomic plant genome was confirmed by PCR analysis. In total four transplastomic lines were generated which were healthy and normal. All plants had regular growth pattern, they were able to reach maturity and produce viable seeds. Taken together, the data presented in the study is a valuable step forward to pave the way in the development of cost effective and easily administrable PMVs for resource-poor countries.

Characterisation of synthetically developed cry1Ab gene in transgenic tobacco chloroplasts

Synthetic cry1Ab gene was developed with optimised codons and expressed in the tobacco plastome. Characterisation of the synthetic cry1Ab gene was carried out on homoplasmic transgenic shoots. To purify the homoplasmic shoots, the proliferating heteroplasmic shoots were subjected to various rounds of selection and regeneration on spectinomycin-containing regeneration medium of plant medium. The PCR-screened homoplasmic shoots were placed on MS medium for root induction and proliferation. The functionally expressed CRY protein was detected using ImmunoStrips, and the amount of toxin protein in transplastomic tobacco plants was measured by DAS-ELISA, specific for cry1Ab gene product. The accumulation of Bacillus thuringiensis (Bt) toxin protein in transgenic chloroplasts confirmed that the synthetic gene could be expressed in other crop plants using constitutive and tissue specific regulatory sequences.

Overexpression of plastidial thioredoxins f and m differentially alters photosynthetic activity and response to oxidative stress in tobacco plants

Plants display a remarkable diversity of thioredoxins (Trxs), reductases controlling the thiol redox status of proteins. The physiological function of many of them remains elusive, particularly for plastidial Trxs f and m, which are presumed based on biochemical data to regulate photosynthetic reactions and carbon metabolism. Recent reports revealed that Trxs f and m participate in vivo in the control of starch metabolism and cyclic photosynthetic electron transfer around photosystem I, respectively. To further delineate their in planta function, we compared the photosynthetic characteristics, the level and/or activity of various Trx targets and the responses to oxidative stress in transplastomic tobacco plants overexpressing either Trx f or Trx m. We found that plants overexpressing Trx m specifically exhibit altered growth, reduced chlorophyll content, impaired photosynthetic linear electron transfer and decreased pools of glutathione and ascorbate. In both transplastomic lines, activities of two enzymes involved in carbon metabolism, NADP-malate dehydrogenase and NADP-glyceraldehyde-3-phosphate dehydrogenase are markedly and similarly altered. In contrast, plants overexpressing Trx m specifically display increased capacity for methionine sulfoxide reductases, enzymes repairing damaged proteins by regenerating methionine from oxidized methionine. Finally, we also observed that transplastomic plants exhibit distinct responses when exposed to oxidative stress conditions generated by methyl viologen or exposure to high light combined with low temperature, the plants overexpressing Trx m being notably more tolerant than Wt and those overexpressing Trx f. Altogether, these data indicate that Trxs f and m fulfill distinct physiological functions. They prompt us to propose that the m type is involved in key processes linking photosynthetic activity, redox homeostasis and antioxidant mechanisms in the chloroplast.

Suppression of Inhibitor Formation Against Factor VIII in Hemophilia A Mice by Oral Delivery of Bioencapsulated Antigen

Abstract 14Approximately 25% of hemophilia A patients develop inhibitors against factor VIII during replacement therapy by infusion of factor VIII concentrates, rendering this treatment ineffective. Elimination of this antibody response is currently achieved by highly expensive immune tolerance induction (ITI) protocols involving prolonged administration of FVIII. No prophylactic immune tolerance protocols are available. To overcome these limitations, this study seeks to develop a cost-effective approach for tolerance induction by oral delivery of human factor VIII (hF.VIII) immunogenic domains expressed in chloroplasts and bioencapsulated in plant cells. Previously, we have shown that this approach effectively suppresses inhibitor formation and anaphylaxis against factor IX in hemophilia B mice (PNAS 107:7101, 2010). Bioencapsulation protects protein antigens from gastric enzymes and acidic environment of the stomach, resulting in antigen release to the immune system via digestion of plant cell walls by microbes that colonizing the gut. The transplastomic tobacco plants created expressed the heavy chain (HC, A1-A2), A3 and C2 domains fused to the transmucosal carrier cholera toxin B subunit (CTB) to facilitate GM1 receptor mediated delivery. Besides a GPGP hinge, a furin cleavage site was introduced to link CTB with the different domains of hF.VIII coding sequence for proper folding and release of hF.VIII domains into the circulatory or immune system. PCR and/or Southern blot analysis was carried out to confirm site-specific transgene integration. Western blot analysis showed expected size fusion protein band in all four transplastomic lines expressing CTB-HC, CTB-A2, CTB-A3 and CTB-C2 fusion protein. The GM1-ganglioside receptor binding ELISA assay with chloroplast synthesized CTB-C2 and CTB-A2 fusion protein showed equivalent absorbance when compared to the purified CTB, confirming the correct folding and disulfide-bond formation of CTB pentamers within transformed chloroplasts. Transplastomic leaves expressed CTB-HC, CTB-A2, CTB-A3 and CTB-C2 in the range of 0.4–1.4%, 0.1–0.2%, 0.3–0.7% and 3.0–9.1% in the total leaf protein. Leaf materials were ground in liquid nitrogen and orally delivered to male hemophilia A mice (C57BL6/129 F8e16 −/−) for tolerance induction. In a first set of experiments, 125 mg plant material was used per oral dose, representing a mix of an approximately equal amount of HC-CTB and C2-CTB fusion proteins. Gavages were performed twice per week for 8 weeks. Control mice were fed with wild-type plant material. During the last 4 weeks, all mice (n=6 per group) were additionally treated with recombinant B domain deleted human F.VIII (intravenous injection of 1 IU once per week). By the end of the experiment, control mice had formed IgG2a (up to 0.9 μg/ml) and IgG2b (up to 1.7 μg/ml) titers against hFVIII, which were undetectable in hF.VIII-fed mice. Moreover, the control mice formed very high-titer IgG1 against hF.VIII (ranging from 7–24 μg/ml), resulting in an inhibitor titer of up to 400 BU (with an average of 211±126 BU). In contrast, hFVIII-fed mice only developed 1.9±0.6 μg/ml IgG1 and 30±12 BU, representing a highly significant (P=0.006 and P=0.001, respectively) 7–10 fold reduction in antibody formation upon factor replacement therapy. These data demonstrate that hF.VIII antigen can be produced by transplastomic technology and provide first proof-of-principle that oral delivery of bioencapsulated hF.VIII antigen is effective in controlling inhibitor development. Current work focuses on further optimization, and generation of an edible crop plant (lettuce) expressing hFVIII domains in the chloroplast for future translational studies is well on its way. Disclosures:Daniell:Bayer: Research Funding.

Dual targeting of a mature plastoglobulin/fibrillin fusion protein to chloroplast plastoglobules and thylakoids in transplastomic tobacco plants

Plastoglobules (PG) are lipid droplets in chloroplasts and other plastid types having important functions in lipid metabolism. Plastoglobulins (PGL) also known as fibrillins (FBN) are evolutionary conserved proteins present at the PG surface but also to various extents at the thylakoid membrane. PGLs are thought to have structural functions in PG formation and maintenance. The targeting of an Arabidopsis PGL (PGL34) to PG required the full protein sequence with the exception of a short C-terminal stretch. This indicated that PGL targeting relies on correct folding rather than a discrete sequence. PGLs lack strongly hydrophic regions and may therefore extrinsically associate with PG and thylakoid membranes via interaction with hydrophilic headgroups of surface lipids. Here, we report on the expression of the Arabidopsis plastoglobulin of 35kD (PGL35 or FBN1a) expressed as a mature protein fused to HIVp24 (human immunodeficiency virus capsid particle p24) or HCV (hepatitis C virus core protein) in transplastomic tobacco. A PGL35-HIVp24 fusion targeted in part to plastoglobules but a larger proportion was recovered in the thylakoid fraction. The findings indicate that transplastomic PGL35-HIVp24 folded correctly after its synthesis inside the chloroplast and then dually targeted to plastoglobules as well as thylakoid membranes.

Improvement of vitamin E quality and quantity in tobacco and lettuce by chloroplast genetic engineering.

Vitamin E (tocopherol: Toc) is an important lipid-soluble antioxidant synthesized in chloroplasts. Among the 8 isoforms of vitamin E, α-Toc has the highest activity in humans. To generate transgenic plants with enhanced vitamin E activity, we applied a chloroplast transformation technique. Three types of the transplastomic tobacco plants (pTTC, pTTMT and pTTC-TMT) carrying the Toc cyclase (TC) or γ-Toc methyltransferase (γ-TMT) gene and the TC plus γ-TMT genes as an operon in the plastid genome, respectively, were generated. There was a significant increase in total levels of Toc due to an increase in γ-Toc in the pTTC plants. Compared to the wild-type plants, Toc composition was altered in the pTTMT plants. In the pTTC-TMT plants, total Toc levels increased and α-Toc was a major Toc isoform. Furthermore, to use chloroplast transformation to produce α-Toc-rich vegetable, TC-overexpressing transplastomic lettuce plants (pLTC) were generated. Total Toc levels and vitamin E activity increased in the pLTC plants compared with the wild-type lettuce plants. These findings indicated that chloroplast genetic engineering is useful to improve vitamin E quality and quantity in plants.

Production of a Subunit Vaccine Candidate against Porcine Post-Weaning Diarrhea in High-Biomass Transplastomic Tobacco

Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeGntd/dsc, engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc). The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeGntd/dsc per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue) and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeGntd/dsc protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeGntd/dsc in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeGntd/dsc protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD.

Alteration of Photosynthate Partitioning by High-Level Expression of Phosphoglucomutase in Tobacco Chloroplasts

Plastidial phosphoglucomutase (PGM) plays an important role in starch synthesis and degradation. Nonetheless, the impact of enhanced plastidial PGM activity on metabolism in photosynthetic tissue is yet to be elucidated. In this study, we generated transplastomic tobacco plants overproducing Arabidopsis thaliana plastidial PGM (AtptPGM) in chloroplasts and analyzed the consequent metabolic and physiological parameters in the transplastomic plants. AtptPGM accumulated in the chloroplasts to up to 16% of total soluble protein in the leaves. PGM activity in leaves increased 100-fold relative to that of wild-type plants. The transplastomic plants were phenotypically indistinguishable in their growth rates, photosynthetic activities, and starch synthesis from wild-type plants, but hexose partitioning in the light period was dramatically different. Furthermore, alteration of extracellular invertase activity was observed in the lower leaves of the transplastomic plants. These observations suggest that high-level expression of plastidial PGM alters hexose partitioning in light periods via modification of extracellular invertase activity.

Stable expression of a fungal laccase protein using transplastomic tobacco

Laccase catalyzes the oxidation of various phenolic compounds that can be used in a wide range of industrial applications such as waste detoxification and the textile industry. In the present study, we generated transplastomic tobacco plants to develop a reliable commercial source of laccase production. The stability of the laccase protein in the transgenic plants was increased by using the enhancer sequence from green fluorescent protein, resulting in three independent lines with high levels of laccase accumulation (up to 2 % of total protein); significant laccase activity, however, was not detected. Interestingly, the transplastomic lines showed slightly retarded vegetative growth, with a light green leaf color in comparison with the control, which may be attributable to copper deficiency induced by ligand chelation by abundantly produced laccase. These results suggest that the tobacco chloroplast is an efficient system for the mass production of laccase protein, but further studies are needed to obtain active enzyme.

Nuclear genome diversity in somatic cells is accelerated by environmental stress

DNA transfer to the nucleus from prokaryotic ancestors of the cytoplasmic organelles (mitochondria and plastids) has occurred during endosymbiotic evolution in eukaryotes. In most eukaryotes, organelle DNA transfer to nucleus is a continuing process. The frequency of DNA transposition from plastid (chloroplast) to nucleus has been measured in tobacco plants (Nicotiana tabacum) experimentally. We have monitored the effects of environmental stress on the rate of DNA transfer from plastid to nucleus by exploiting nucleus-specific reporter genes in two transplastomic tobacco lines. DNA migration from plastids to the nucleus is markedly increased by mild heat stress. In addition, insertions of mitochondrial DNA into induced double-strand breaks are observed after heat treatment. These results show that movement of organelle DNA to the nucleus is remarkably increased by heat stress.

Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco

Fragments from camelid single-chain antibodies known as VHHs or nanobodies represent a valuable tool in diagnostics, investigation and passive immunity therapy. Here, we explored different strategies to improve the accumulation of a neutralizing VHH antibody against rotavirus in tobacco transplastomic plants. First, we attempted to express the VHH in the chloroplast stroma and then two alternative strategies were carried out to improve the expression levels: expression as a translational fusion to the β-glucuronidase enzyme (GUS-E-VHH), and redirection of the VHH into the thylakoid lumen (pep-VHH). Every attempt to produce transplastomic plants expressing the VHH in the stroma was futile. The transgene turned out to be unstable and the presence of the VHH protein was almost undetectable. Although pep-VHH plants also presented some of the aforementioned problems, higher accumulation of the nanobody was observed (2-3% of the total soluble proteins). The use of β-glucuronidase as a partner protein turned out to be a successful strategy and expression levels reached 3% of the total soluble proteins. The functionality of the VHHs produced by pep-VHH and GUS-E-VHH plants was studied and compared with that of the antibody produced in Escherichia coli. This work contributes to optimizing the expression of VHH in transplastomic plants. Recombinant proteins could be obtained either by accumulation in the thylakoid lumen or as a fusion protein with β-glucuronidase, and both strategies allow for further optimization.

Environmental stress increases the entry of cytoplasmic organellar DNA into the nucleus in plants

Mitochondria and chloroplasts (photosynthetic members of the plastid family of cytoplasmic organelles) in eukaryotic cells originated more than a billion years ago when an ancestor of the nucleated cell engulfed two different prokaryotes in separate sequential events. Extant cytoplasmic organellar genomes contain very few genes compared with their candidate free-living ancestors, as most have functionally relocated to the nucleus. The first step in functional relocation involves the integration of inactive DNA fragments into nuclear chromosomes, and this process continues at high frequency with attendant genetic, genomic, and evolutionary consequences. Using two different transplastomic tobacco lines, we show that DNA migration from chloroplasts to the nucleus is markedly increased by mild heat stress. In addition, we show that insertion of mitochondrial DNA fragments during the repair of induced double-strand breaks is increased by heat stress. The experiments demonstrate that the nuclear influx of organellar DNA is a potentially a source of mutation for nuclear genomes that is highly susceptible to temperature fluctuations that are well within the range experienced naturally.

Increased accumulation and stability of rotavirus VP6 protein in tobacco chloroplasts following changes to the 5′ untranslated region and the 5′ end of the coding region

Rotavirus is the main cause of gastroenteritis in children worldwide, and the World Health Organisation has recommended that a rotavirus vaccine should be included in all infant immunization programmes. VP6 is the most immunogenic rotavirus subunit and is a potential target for an oral subunit vaccine. VP6 accumulated at up to 3% of total soluble protein in the young leaves of transplastomic tobacco plants, but the protein was unstable and was lost as the leaves aged. The aim of this study was to alter the 5'-untranslated region (5'-UTR) and the 5' end of the coding region of VP6 cDNA in an attempt to increase the expression and stability of VP6 protein in tobacco chloroplasts. The inclusion of the 5'-UTR from gene 10 of bacteriophage T7 (T7g10) and the addition of 15 nucleotides, encoding five additional amino acid residues, at the 5' end of the coding region increased the expression to >15% of total leaf protein and stabilized the protein in ageing leaves. Plants containing VP6 expression constructs with the rbcL 5'-UTR and with the native VP6 5' end of the coding region produced VP6 protein at only 1.9% of total leaf protein. Both the T7g10 5'-UTR and the additional 15 nucleotides increased transcript accumulation and translational efficiency compared with VP6 constructs containing the rbcL 5'-UTR. The VP6 protein produced from all gene constructs appeared to be susceptible to proteolytic processing at its N-terminal region. However, in all transplastomic lines, VP6 proteins assembled into the trimeric form found in the rotavirus capsid.

Effects of <i>GFP</i> Transplastomic Tobacco on Soil Enzyme Activities

In this study, the GFP transplastomic tobacco (TG) and non-transgenetic (WT) were compared to study the effects of GFP transplastomic tobacco on soil enzyme activities. The activities of some soil enzymes (urease, cellulase, protease, and invertase) were determined at seeding stage, vegetative stage, flowering stage, and senescing stage. The results showed that there was no significant difference on the activities of urease, cellulase, protease, and invertase between GFP transplastomic tobacco and non-transgenetic at same growing stage. No matter transplastpmic tobacco or controls, the soil enzyme activities of the vegetative stage and flowering stage were higher than seeding stage and senescing stage. The results suggest that the effects of GFP transplastomic tobacco to major soil enzyme activities were not significant.

Isoleucine 309 acts as a C 4 catalytic switch that increases ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) carboxylation rate in Flaveria

Improving global yields of important agricultural crops is a complex challenge. Enhancing yield and resource use by engineering improvements to photosynthetic carbon assimilation is one potential solution. During the last 40 million years C(4) photosynthesis has evolved multiple times, enabling plants to evade the catalytic inadequacies of the CO(2)-fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco). Compared with their C(3) ancestors, C(4) plants combine a faster rubisco with a biochemical CO(2)-concentrating mechanism, enabling more efficient use of water and nitrogen and enhanced yield. Here we show the versatility of plastome manipulation in tobacco for identifying sequences in C(4)-rubisco that can be transplanted into C(3)-rubisco to improve carboxylation rate (V(C)). Using transplastomic tobacco lines expressing native and mutated rubisco large subunits (L-subunits) from Flaveria pringlei (C(3)), Flaveria floridana (C(3)-C(4)), and Flaveria bidentis (C(4)), we reveal that Met-309-Ile substitutions in the L-subunit act as a catalytic switch between C(4) ((309)Ile; faster V(C), lower CO(2) affinity) and C(3) ((309)Met; slower V(C), higher CO(2) affinity) catalysis. Application of this transplastomic system permits further identification of other structural solutions selected by nature that can increase rubisco V(C) in C(3) crops. Coengineering a catalytically faster C(3) rubisco and a CO(2)-concentrating mechanism within C(3) crop species could enhance their efficiency in resource use and yield.

Expression of human soluble tumor necrosis factor (TNF)-related apoptosis-inducing ligand in transplastomic tobacco

The soluble extracellular domain of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (sTRAIL) can, as the whole length TRAIL protein, bind with its receptors and specifically induce the apoptosis of cancer cells; therefore, it has been developed as a potential therapeutic agent for various cancer treatments. As it has become an attractive technology for foreign protein production, especially for production of biopharmaceuticals, chloroplast engineering was applied in this study to express human sTRAIL protein in tobacco. Two transplastomic lines were obtained. Southern blot showed that sTRAIL gene was inserted into the right site of the tobacco chloroplast genome. RT-PCR results also confirmed that the foreign gene is transcribed in both lines. However, western blot showed that only one line accumulated sTRAIL protein stably, while the other line lost the ability to accumulate this protein after several rounds of subcultures. The possible reason for this unexpected phenomenon is discussed. Key words: Chloroplast transformation, sTRAIL, pharmaceutical protein, expression, tobacco chloroplast.

Exploration of horizontal gene transfer between transplastomic tobacco and plant-associated bacteria

The likelihood of gene transfer from transgenic plants to bacteria is dependent on the transgene copy number and on the presence of homologous sequences for recombination. The large number of chloroplast genomes in a plant cell as well as the prokaryotic origin of the transgene may thus significantly increase the likelihood of gene transfer from transplastomic plants to bacteria. In order to assess the probability of such a transfer, bacterial isolates, screened for their ability to colonize decaying tobacco plant tissue and possessing DNA sequence similarity to the chloroplastic genes accD and rbcL flanking the transgene (aadA), were tested for their ability to take up extracellular DNA (broad host-range pBBR1MCS-3-derived plasmid, transplastomic plant DNA and PCR products containing the genes accD-aadA-rbcL) by natural or electrotransformation. The results showed that among the 16 bacterial isolates tested, six were able to accept foreign DNA and acquire the spectinomycin resistance conferred by the aadA gene on plasmid, but none of them managed to integrate transgenic DNA in their chromosome. Our results provide no indication that the theoretical gene transfer-enhancing properties of transplastomic plants cause horizontal gene transfer at rates above those found in other studies with nuclear transgenes.

Immunogenicity of chloroplast‐derived HIV‐1 p24 and a p24‐Nef fusion protein following subcutaneous and oral administration in mice

High-level expression of foreign proteins in chloroplasts of transplastomic plants provides excellent opportunities for the development of oral vaccines against a range of debilitating or fatal diseases. The HIV-1 capsid protein p24 and a fusion of p24 with the negative regulatory protein Nef (p24-Nef) accumulate to ∼4% and ∼40% of the total soluble protein of leaves of transplastomic tobacco (Nicotiana tabacum L.) plants. This study has investigated the immunogenicity in mice of these two HIV-1 proteins, using cholera toxin B subunit as an adjuvant. Subcutaneous immunization with purified chloroplast-derived p24 elicited a strong antigen-specific serum IgG response, comparable to that produced by Escherichia coli-derived p24. Oral administration of a partially purified preparation of chloroplast-derived p24-Nef fusion protein, used as a booster after subcutaneous injection with either p24 or Nef, also elicited strong antigen-specific serum IgG responses. Both IgG1 and IgG2a subtypes, associated with cell-mediated Th1 and humoral Th2 responses, respectively, were found in sera after subcutaneous and oral administration. These results indicate that chloroplast-derived HIV-1 p24-Nef is a promising candidate as a component of a subunit vaccine delivered by oral boosting, after subcutaneous priming by injection of p24 and/or Nef.