Mária Oszvald

Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm.

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a trangenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.

Expression of cholera toxin B subunit in transgenic rice endosperm.

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for GM1-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.

Overproduction of an Arabidopsis aldo–keto reductase increases barley tolerance to oxidative and cadmium stress by an in vivo reactive aldehyde detoxification

In this work, we expressed an Arabidopsis thaliana-coded protein (AKR4C9) in transgenic barley to study its enzymatic activity and to enhance the reactive aldehyde neutralizing capacity (part of the oxidative stress tolerance) of transgenic plants. Total leaf protein was extracted from transgenic plants expressing either C or N-terminally His-tagged aldo–keto reductase (AKR) enzyme and purified by affinity chromatography. The Arabidopsis-coded enzyme showed moderate activity against the synthetic reactive aldehyde, glutaraldehyde, and low but detectable enzyme activity against fructose with a low Michaelis–Menten constant (Km value). Activity of the C and the N-terminally His-tagged AKRs were found to be in the same range. Glutaraldehyde was also tested in vivo by spraying onto the leaves of the plants. The reactive aldehyde tolerance of both wild type and transgenic plants, as well as the general physiological effects of this reactive aldehyde treatment were evaluated. The growth rate was found to decrease in all (both wild type and transgenic) plants. The high AKR-expressing transgenic plants showed a lower respiratory rate, and they also showed higher fresh weight, higher chlorophyll content and photosynthetic activity, indicating a higher reactive aldehyde tolerance. Cadmium (Cd) treatment was also performed to validate this result. Cd caused strong lipid peroxidation; however, the Arabidopsis enzyme lowered the reactive aldehyde content as expected. This is the first report in which kinetic parameters of the fructose reduction by the stress inducible plant AKR enzyme are presented. Furthermore, data on the effects of a reactive aldehyde treatment on intact plants are also provided.

Comparative Study of the Effect of Incorporated Individual Wheat Storage Proteins on Mixing Properties of Rice and Wheat Doughs

The aim of this work was to compare the effects of incorporated wheat storage proteins on the functional properties of rice and wheat flours. The advantage of rice as a base flour compared to wheat is that it does not contain any wheat flour components and, therefore, has no interactive effect between wheat glutenin proteins. The incorporation of individual HMW glutenin subunit proteins (Bx6, Bx7, and By8) in different ratios had significant positive effects on the mixing requirements of both rice and wheat doughs. Reconstitution experiments using two x+y type HMW-GS pairs together with a bacterially expressed LMW-GS have been also carried out in this study. The largest effects of polymer formation and mixing properties of rice flour dough were observed when Bx and By subunits were used in a 1:1 ratio and HMW and LMW glutenin subunits in a 1:3 ratio. However, using the same subunit ratios in wheat as the base flour, these synergistic effects were not observed.

Wheat Storage Proteins in Transgenic Rice Endosperm

Transgenic rice seed expressing wheat HMW glutenin subunit was characterized to study the effects of the wheat prolamin on the protein expression pattern and protein size distribution in the endosperm and the functional and rheological properties of the rice flour and dough. Significant differences were found in the protein expression pattern between the transgenic and wild type samples. Comparing the protein expression profiles of transgenic and nontransgenic plants, combined with proteomic-based studies, indicated increased protein disulfide isomerase (PDI) levels in the transgenic rice lines. The accurate molecular size of HMW-GS in rice endosperm was identified by MALDI-TOF-MS analysis. The expressed wheat HMW (subunit 1Dx5) GS showed a positive effect on the functional properties of rice dough by significantly increasing the size distribution of the polymeric protein fraction and modifying the dough mixing parameters.

Synthesis and assembly ofEscherichia coli heat-labile enterotoxin B subunit in transgenic rice (Oryza sativa L.)

Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as a potent mucosal immunogen and immunoadjuvant for co-administered antigens. The synthetic LTB (sLTB) was modified based on plant optimized codon usage, and fused to a translation signal (the Kozak sequence) in the front of start codon and the ER retention signal, SEKDEL, in the c-teminus of sLTB gene. The sLTB and the wild-type LTB gene (wLTB) were located into plant expression vectors under the control of the wheat Bx17 HMW (High Molecular Weight) glutenin endosperm-specific promoter containing the first intron of the rice actin 1 gene. Both genes were introduced into rice cells (Oryza sativa L.) via particle bombardment mediated transformation. The integration of LTB gene into the chromosome of transgenic plants was confirmed by genomic DNA PCR amplification methods. The transcription and translation of the LTB genes were demonstrated by reverse-transcription PCR (RT-PCR) and Western blot analyses, respectively. The LTB proteins produced in the seed tissues of transgenic rice showed binding affinity for GM1 ganglioside, a receptor for biologically active LTB, suggesting the plant-produced LTB are capable of forming active pentamers. The expression level of sLTB was higher than wLTB in transgenic rice plants and was up to 2.7% of the total soluble proteins of the seed tissues.

Improved reactive aldehyde, salt and cadmium tolerance of transgenic barley due to the expression of aldo–keto reductase genes

Under various stress conditions, plant cells are exposed to oxidative damage which triggers lipid peroxidation. Lipid peroxide breakdown products include protein crosslinking reactive aldehydes. These are highly damaging to living cells. Stress-protective aldo–keto reductase (AKR) enzymes are able to recognise and modify these molecules, reducing their toxicity. AKRs not only modify reactive aldehydes but may synthesize osmoprotective sugar alcohols as well. The role of these mixed function enzymes was investigated under direct reactive aldehyde, heavy metal and salt stress conditions. Transgenic barley (Hordeum vulgare L.) plants constitutively expressing AKR enzymes derived from either thale cress (Arabidopsis thaliana) (AKR4C9) or alfalfa (Medicago sativa) (MsALR) were studied. Not only AKR4C9 but MsALR expressing plants were also found to produce more sorbitol than the non-transgenic (WT) barley. Salinity tolerance of genetically modified (GM) plants improved, presumably as a consequence of the enhanced sorbitol content. The MsALR enzyme expressing line (called 51) exhibited almost no symptoms of salt stress. Furthermore, both transgenes were shown to increase reactive aldehyde (glutaraldehyde) tolerance. Transgenic plants also exhibited better cadmium tolerance compared to WT, which was considered to be an effect of the reduction of reactive aldehyde molecules. Transgenic barley expressing either thale cress or alfalfa derived enzyme showed improved heavy metal and salt tolerance. Both can be explained by higher detoxifying and sugar alcohol producing activity. Based on the presented data, we consider AKRs as very effective stress-protective enzymes and their genes provide promising tools in the improvement of crops through gene technology.

Effects of Wheat Storage Proteins on the Functional Properties of Rice Dough

The objective of this work was to develop a rice flour based procedure for in vitro structure-function studies of wheat proteins. Rice flour has an advantage over wheat flour, because the signal/noise ratio should be higher after the incorporation of the wheat prolamins into the protein matrix of the dough. A reduction/oxidation procedure has been developed to incorporate glutenin subunits into the polymeric structure of rice dough protein. The results indicated that incorporation of bulk fractions of HMW and LMW glutenin subunits increased the mixing requirements of the dough, whereas simple addition resulted in weaker dough. The incorporation studies of individual HMW subunits (Bx6, Bx7, and By8) demonstrated that rice flour can be used to study and compare the functional properties of different glutenin subunits.

The trafficking pathway of a wheat storage protein in transgenic rice endosperm.

Background and Aims The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these. Methods The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies. Key results The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells. Conclusions The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm.

Plastid Molecular Pharming I. Production of Oral Vaccines via Plastid Transformation.

BACKGROUND Vaccines produced in plants have opened up new opportunities in vaccination. OBJECTIVE Among the various categories of vaccines, the recombinant vaccine is generally regarded as the most economical and safest type because it cannot cause disease and does not require large-scale cultivation of pathogens. Due to the low cost of their cultivation, plants may represent viable alternative platforms for producing subunit vaccines. Genetic engineering of plastids is the innovation of the last three decades and has numerous benefits when compared to nuclear transformation. Due to the high level of expression, oral vaccines produced in transplastomic plants do not have to be purified as they can be consumed raw, which, therefore, reduces the cost of preparation, transportation and handling of the vaccines. Oral vaccination also excludes the risk of other infections or contaminations, while compartmentation of the plant cell provides an excellent encapsulation to the antigen within the plastid. RESULTS & CONCLUSION Herein we review the main biotechnological and immunological aspects of the progress achieved in the field of plastid derived edible vaccines during the last decade. As there is a public debate against genetically modified crops, the advantages and limitations of oral vaccines are also discussed.