Immunogenicity of EIT chimeric protein expressed in transplastomic tobacco plants towards development of an oral vaccine against Escherichia coli O157:H7

Chloroplast genetic engineering offers an opportunity for high level expression and cost-effective recombinant protein production. Escherichia coli O157:H7 is one of the most important zoonotic pathogens causing hemorrhagic colitis (HC) and the life-threatening hemolytic-uremic syndrome in humans worldwide. The occurrence of zoonotic E. coli O157:H7 outbreaks in recent years has led to increased efforts in the development of safe and cost-effective immunogenic antigens against E. coli O157:H7. EspA and Tir/Intimin proteins are the important virulence factors which are encoded by the LEE locus of enterohemorrhagic E. coli. In this study, we hypothesized that the high level expression of the chimeric form of these effectors in chloroplasts and using tobacco transplastomic plants as an oral delivery system for the development of an edible-base vaccine would induce an immune response for the prevention of E. coli 0157:H7 attachment and colonization in animal model mice. The prokaryotic codon-optimized EIT protein was expressed in plastid genome via chloroplast transformation. Putative transplastomic plants were analyzed by PCR, and Southern blot analysis confirming chloroplast integration and homoplasmy in the T1 progeny. Immunoblotting and ELISA assays demonstrated that the EIT protein was expressed in chloroplasts and accumulated up to 1.4 % of total soluble protein in leaf tissue. In mice orally immunized with transplastomic tobacco plant leaves, high immunological responses (IgG and IgA specific antibodies) were detected in serum and feces. Finally, the challenging assay with E. coli O157:H7 in immunized mice showed reduced bacterial shedding.     

Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer

Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues. R. E. Soria-Guerra and A. G. Alpuche-Solís contributed equally to this work.     

Transient expression of recombinant tissue plasminogen activator (rt-PA) gene in cucurbit plants using viral vector

Objective

To use a transient expression system to express a truncated human tissue plasminogen activator (K2S) gene in cucurbit plants.

Results

The recombinant tissue plasminogen activator protein (K2S form) was expressed in active form in cucurbit plants. Its molecular weight was 43 kDa. The plant-derived rt-PA was determined using goat anti-rabbit antibody by western blotting. Among the infected lines, the highest expression of rt-PA was 62 ng/100 mg per leaf tissue as measured by ELISA. The enzymatic activity of the plant-derived rt-PA was 0.8 IU/ml.

Conclusions

The K25 form of rt-PA was expressed for the first time using the viral expression system. Plant-derived rt-PA showed similar potency to commercially-available PA.

     

Multiple elements of the S 2 -RNase promoter from potato (Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco

A functional analysis of the promoter of the S ₂ -RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S ₂ -RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S ₂ promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterised by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S ₁ -RNase and S ₂ -RNase promoters, termed motif I and motif III, are located in a fragment of the S ₂ promoter extending from position ?200 to bp ?100, and motif II, located between bp ?498 and ?480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified within motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S ₂ -RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other tissues.     

BSD2 is a Rubisco‐specific assembly chaperone,forms intermediary hetero‐oligomeric complexes,and is nonlimiting to growth in tobacco

The folding and assembly of Rubisco large and small subunits into L₈S₈ holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco‐BSD2 assembly complexes in the model C₃ plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.m²) stably integrated in putative intermediary Rubisco complexes that account for <0.2% of the L₈S₈ pool. RNAi‐silencing BSD2 production in transplastomic tobacco producing bacterial L₂ Rubisco had no effect on leaf photosynthesis, cell ultrastructure, or plant growth. Genetic crossing the same RNAi‐bsd2 alleles into wild‐type tobacco however impaired L₈S₈ Rubisco production and plant growth, indicating the only critical function of BSD2 is in Rubisco biogenesis. Agrobacterium mediated transient expression of tobacco, Arabidopsis, or maize BSD2 reinstated Rubisco biogenesis in BSD2‐silenced tobacco. Overexpressing BSD2 in tobacco chloroplasts however did not alter Rubisco content, activation status, leaf photosynthesis rate, or plant growth in the field or in the glasshouse at 20°C or 35°C. Our findings indicate BSD2 functions exclusively in Rubisco biogenesis, can efficiently facilitate heterologous plant Rubisco assembly, and is produced in amounts nonlimiting to tobacco growth.     

Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.     

Characterization of the GLP13 gene promoter in Arabidopsis thaliana

In transgenic plants, for many applications it is important that the inserted genes are expressed in a tissue-specific manner. This in turn could help better understanding their roles in plant development. Germin-like proteins (GLPs) play diverse roles in plant development and defense responses. In order to understand the functions and regulation of the GLP13 gene, its promoter (762 bp) was cloned and fused with a β-glucuronidase (GUS) reporter gene for transient expression in Arabidopsis thaliana and tobacco (Nicotiana tabacum cv. K326). Histochemical analysis of the transgenic plants showed that GUS was specifically expressed in vascular bundles predominantly in phloem tissue of all organs in Arabidopsis. Further analyses in transgenic tobacco also identified similar GUS expression in the vascular bundles.     

Tobacco plastid ribosomal protein S18 is essential for cell survival

Plastid genomes contain a conserved set of genes most of which are involved in either photosynthesis or gene expression. Among the ribosomal protein genes present in higher plant plastid genomes, rps18 is special in that it is absent from the plastid genomes of several non-green unicellular organisms, including Euglena longa and Toxoplasma gondii. Here we have tested whether the ribosomal protein S18 is required for translation by deleting the rps18 gene from the tobacco plastid genome. We report that, while deletion of the rps18 gene was readily obtained, no homoplasmic Δrps18 plants or leaf sectors could be isolated. Instead, segregation into homoplasmy led to severe defects in leaf development suggesting that the knockout of rps18 is lethal and the S18 protein is required for cell survival. Our data demonstrate that S18 is indispensable for plastid ribosome function in tobacco and support an essential role for plastid translation in plant development. Moreover, we demonstrate the occurrence of flip-flop recombination on short inverted repeat sequences which generates different isoforms of the transformed plastid genome that differ in the orientation a 70 kb segment in the large single-copy region. However, infrequent occurrence of flip-flop recombination and random segregation of plastid genomes result in the predominant presence of only one of the isoforms in many tissue samples. Implications for the interpretation of chloroplast transformation experiments and vector design are discussed.     

Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase

Schistosoma mansoni is a blood fluke parasite that causes schistosomiasis, a debilitating disease of global public health importance. These relatively large parasites are able to survive prolonged periods in the human vasculature without inducing stable blood clots around them. We show here that the intravascular life stages (schistosomula and adult males and females) can all promote significant plasminogen (PLMG) activation in the presence of tissue plasminogen activator (tPA). This results in the generation of the potent fibrinolytic agent plasmin which could degrade blood clots forming around the worms in vivo. We demonstrate that S. mansoni enolase (SmEno) is a host-interactive tegumental enzyme that, in recombinant form, can bind PLMG and promote its activation. Like classical members of the enolase protein family, SmEno can catalyze the interconversion of 2-phospho-D-glycerate (2-PGA) and phosphoenolpyruvate (PEP). The enzyme has maximal activity at pH 7.5, requires Mg²⁺ for optimal activity and can be inhibited by NaF but not mefloquin. Suppressing expression of the SmEno gene significantly diminishes enolase mRNA levels, protein levels and surface enzyme activity but, surprisingly, does not affect the ability of the worms to promote PLMG activation. Thus, while SmEno can enhance PLMG activation, our analysis suggests that it is not the only contributor to the parasite’s ability to perform this function. We show that the worms possess several other PLMG-binding proteins in addition to SmEno and these may have a greater importance in schistosome-driven PLMG activation.     

Expression of Brassica oleracea FtsZ1-1 and MinD alters chloroplast division in Nicotiana tabacum generating macro- and mini-chloroplasts

FtsZ1-1 and MinD plastid division-related genes were identified and cloned from Brassica oleracea var. botrytis. Transgenic tobacco plants expressing BoFtsZ1-1 or BoMinD exhibited cells with either fewer but abnormally large chloroplasts or more but smaller chloroplasts relative to wild-type tobacco plants. An abnormal chloroplast phenotype in guard cells was found in BoMinD transgenic tobacco plants but not in BoFtsZ1-1 transgenic tobacco plants. Transgenic tobacco plants bearing the macro-chloroplast phenotype had 10 to 20-fold increased levels of total FtsZ1-1 or MinD, whilst the transgenic tobacco plants bearing the mini-chloroplast phenotype had lower increased FtsZ1-1 or absence of detectable MinD. We also described for the first time, plastid transformation of macro-chloroplast bearing tobacco shoots with a gene cassette allowing for expression of green fluorescent protein (GFP). Homoplasmic plastid transformants from normal chloroplast and macro-chloroplast tobacco plants expressing GFP were obtained. Both types of transformants accumulated GFP at ~6% of total soluble protein, thus indicating that cells containing macro-chloroplasts can regenerate shoots in tissue culture and can stably integrate and express a foreign gene to similar levels as plant cells containing a normal chloroplast size and number.     

Flower Formation in Excised Tobacco Stem Segments: III. Deoxyribonucleic Acid Content in Stem Tissue of Vegetative and Flowering Tobacco Plants

A method has been developed that extracts DNA from stem tissue of flowering tobacco plants, Nicotiana tabacum cv. Wis. 38. The DNA content of stem tissue from a flowering tobacco plant is correlated with its capacity to flower in vitro. Stem segments known to form 100% floral buds contain 10 times more DNA per gram fresh weight than segments that form 5% floral buds and 95% vegetative buds, and in the uppermost 28 centimeters of flowering tobacco plant stems the DNA content decreases roughly in parallel with the floral gradient.     

The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.     

Some Physiological Parameters and Seed Lipid Composition of Transgenic Tobacco Plant

To see the effects of foreign gene introduction on the physiological performance and the quality and quantity of seed lipids, we studied transgenic tobacco plant as a model system, as tobacco seeds are oil seeds. Using Agrobacterium Ti plasmid based vectors, tobacco plants cv Petit Havana were transformed by NPT II gene as selectable marker. Transformed T₀ generation plants raised in tissue culture were transferred to pots and selfed. From the seeds, T₁ generation plants were grown in pots and their physiological performance was assessed. The transgenic plants showed slightly slower rates of germination and growth. Total chlorophyll content, chlorophyll a/b ratio and specific leaf weight, however, remained unchanged. The transgenic plants also had delayed flowering. However, total protein, lipid content and fatty acid composition of lipids of seeds in transgenic plants did not show appreciable difference from the seeds from control plants. Thus the physiological cost of transgenic plant for the extra genetic load was only marginal, if any.     

Targeted overexpression of the Escherichia coli MinC protein in higher plants results in abnormal chloroplasts

Higher plant chloroplast division involves some of the same types of proteins that are required in prokaryotic cell division. These include two of the three Min proteins, MinD and MinE, encoded by the min operon in bacteria. Noticeably absent from annotated sequences from higher plants is a MinC homologue. A higher plant functional MinC homologue that would interfere with FtsZ polymerization, has yet to be identified. We sought to determine whether expression of the bacterial MinC in higher plants could affect chloroplast division. The Escherichia coli minC (EcMinC) gene was isolated and inserted behind the Arabidopsis thaliana RbcS transit peptide sequence for chloroplast targeting. This TP-EcMinC gene driven by the CaMV 35S² constitutive promoter was then transformed into tobacco (Nicotiana tabacum L.). Abnormally large chloroplasts were observed in the transgenic plants suggesting that overexpression of the E. coli MinC perturbed higher plant chloroplast division.     

Transgenic tobacco plants expressing PicW gene from Picea wilsonii exhibit enhanced freezing tolerance

A cold-induced gene of 669 bp in length without introns, PicW, was cloned from Picea. wilsonii, a cold tolerant conifer species. Sequence analysis showed that it was a member of the dehydrin family because of its conserved amino acid constitution and protein secondary structure. The protein was rich in hydrophilic amino acids such as alanine, lysine, glutamic acid, glutamine and threonine, but devoid of two hydrophobic amino acids, cysteine and tryptophan. The PicW gene contained five repeated motifs homologous to the core K-segment in dehydrins. Protein secondary structure prediction showed that PicW comprised 29 % α-helix, mostly in the K-homologous segment, and random coils. The PicW gene was cloned into the expression vector PEZR(K)-LC under the 35S promoter and transformed into tobacco plants. After treatment at ?5 °C for 3 h, all of the tobacco plants were wilting. However, the transgenic plants showed better growth performance than wild-type plants. Further tests of physiological indexes including relative electrolyte leakage and malondialdehyde content, proline and soluble sugar content also revealed significant differences between the wild-type and transgenic tobacco plants. It was concluded that the PicW gene could be an important gene resource for freezing-tolerant plant breeding.     

Monocotyledonous C4 NADP+ -malate dehydrogenase is efficiently synthesized,targeted to chloroplasts and processed to an active form in transgenic plants of the C3 dicotyledon tobacco

Chloroplastic NADP⁺-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbonfixation pathway of some C₄ plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C₃ plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C₃-C₄ photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic proteinimport machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of l-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.     

High expression of foot-and-mouth disease virus structural protein VP1 in tobacco chloroplasts

A tobacco chloroplast expression vector, pTRVP1, containing the foot-and-mouth disease virus (FMDV) VP1 gene and the selective marker aadA gene, was constructed and transferred to tobacco by biolistic method. Three resistant lines were obtained through spectinomycin selection, and each transgenic line was subjected to a second round of spectinomycin selection. PCR and PCR southern blot analysis revealed that the VP1 gene had integrated into the chloroplast genome. Western blot and quantification ELISA assays indicated that the VP1 gene was expressed in tobacco chloroplasts and accounted for 2–3% of total soluble protein. This suggested that plant chloroplasts were an efficient expression system for the potential production of recombinant antigens in plants.     

Expression of photosynthesis gene-promoter fusions in leaf epidermal cells of transgenic tobacco plants

Fusions of the promoter regions of the pea plasto-cyanin, pea ferredoxin: NADP⁺ reductase and tobacco rbcS genes to the β-glucuronidase (GUS) reporter gene have been introduced into tobacco via Agro-bacterium-mediated transformation, and epidermal peels of the lower leaf surface of tissue-cultured and greenhouse-grown plants examined histochemically for GUS activity. For each of the constructs, GUS was detected in epidermal cells as well as in stomatal guard cells. Epidermal peels from plants in tissue culture stained more readily than those from greenhouse-grown plants. Light and electron microscopy clearly demonstrated the presence of chloroplasts in epidermal cells of tobacco leaves. These results provide further evidence for the correlation between the presence of chloroplasts and the expression of nuclear genes for photosynthesis components.