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Chronic Hepatitis B Virus (HBV) infection leads to severe liver pathogenesis associated with significant morbidity and mortality. As no curable medication is yet available, vaccination remains the most costeffective approach to limit HBV spreading and control the infection. Although safe and efficient, the standard vaccine based on production of the small (S) envelope protein in yeast fails to elicit an effective immune response in about 10% of vaccinated individuals, which are at risk of infection. One strategy to address this issue is the development of more immunogenic antigens. Here we describe a novel HBV antigen obtained by combining relevant immunogenic determinants of S and large (L) envelope proteins. Our approach was based on the insertion of residues 21-47 of the preS1 domain of the L protein (nomenclature according to genotype D), involved in virus attachment to hepatocytes, within the external antigenic loop of S. The resulting S/preS121-47 chimera was successfully produced in HEK293T and Nicotiana benthamiana plants, as a more economical recombinant protein production platform. Comparative biochemical, functional and electron microscopy analysis indicated assembly of the novel antigen into subviral particles in mammalian and plant cells. Importantly, these particles preserve both S- and preS1-specific epitopes and elicit significantly stronger humoral and cellular immune responses than the S protein, in both expression systems used. Our data promote this antigen as a promising vaccine candidate to overcome poor responsiveness to the conventional, S protein-based, HBV vaccine.

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The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases ( e.g . cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca s ativa ) using Agrobacterium - mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2 Δ N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2 Δ N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.

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Chrysanthemum stunt viroid (CSVd) was first reported in US in the 1940s and is widespread in the world wherever chrysanthemum is grown. Cryotherapy of shoot tips, a new biotechnology developed in the recent years, is a novel application of plant cryopreservation techniques that allows pathogen eradication at a high frequency. Existing studies have proven that this technique can efficiently eradicate pathogens such as virus, phytoplasma and bacterium. However, up to now, there has been no report on viroid eradication. In the present study, we attempted to establish a droplet vitrification cryotherapy method for Argyranthemum and to apply it to eradicate CSVd. Results obtained so far demonstrated that cryotherapy of shoot tips alone failed to eradicate CSVd from the infected shoot tips of Argyranthemum maderense ‘Yellow Empire’. Using in situ hybridization of CSVd and histological analysis, we found that CSVd can invade meristematic cells and at the same time, these cells were able to survive following cryotherapy. These findings explained why cryotherapy of shoot tips alone could not be efficient enough to eradicate CSVd from the diseased materials. Further studies combining cold treatment with cryotherapy are under investigation for CSVd eradication.

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Euphorbia pulcherrima, poinsettia, is a non-food and non-feed vegetatively propagated ornamental plant. Appropriate plant height is one of the most important traits in poinsettia production and is commonly achieved by application of chemical growth retardants. To produce compact poinsettia plants with desirable height and reduce the utilization of growth retardants, the Arabidopsis SHORT INTERNODE (AtSHI) gene controlled by the cauliflower mosaic virus 35S promoter was introduced into poinsettia by Agrobacterium-mediated transformation. Three independent transgenic lines were produced and stable integration of transgene was verified by PCR and Southern blot analysis. Reduced plant height (21–52%) and internode lengths (31–49%) were obtained in the transgenic lines compared to control plants. This correlates positively with the AtSHI transcript levels, with the highest levels in the most dwarfed transgenic line (TL1). The indole-3-acetic acid (IAA) content appeared lower (11–31% reduction) in the transgenic lines compared to the wild type (WT) controls, with the lowest level (31% reduction) in TL1. Total internode numbers, bract numbers and bract area were significantly reduced in all transgenic lines in comparison with the WT controls. Only TL1 showed significantly lower plant diameter, total leaf area and total dry weight, whereas none of the AtSHI expressing lines showed altered timing of flower initiation, cyathia abscission or bract necrosis. This study demonstrated that introduction of the AtSHI gene into poinsettia by genetic engineering can be an effective approach in controlling plant height without negatively affecting flowering time. This can help to reduce or avoid the use of toxic growth retardants of environmental and human health concern. This is the first report that AtSHI gene was overexpressed in poinsettia and transgenic poinsettia plants with compact growth were produced.

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In 1998 Plum pox virus (PPV) wasdetected for the first time in Norway. Virus-like symptoms were observed on several trees in a collection ofplum cultivars at Njøs Research Station in the Sogn og Fjordane County in WestNorway. The Norwegian Food SafetyAuthority and the Norwegian Crop Research Institute immediately startedsurveying other variety collections around the country, nuclear stock materialand orchards in all important plum-growing areas. Since 1998 we have surveyed themain part of the commercial plum orchards in Norway. About 75 000 individualtrees have been tested. About 1 % of the trees have been found infected by PPV.Only the PPV-D strain has been found. It is suspected that the main infectionsource was infected plums or apricots imported to Njøs around 1970 or earlier.In most plum orchards in Norway,the spread of PPV by aphids is relatively slow. Therefore, we expect to be ableto eradicate PPV from commercial plum orchards in the near future.The eradication work iscontinuing.

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Virus og fytoplasma i julestjerne har vært et forskningsfelt ved Bioforsk Plantehelse siden 1990-tallet. Vi har kombinert dette med ny kunnskap om gen-transformering. I 2009, etter flere års FoU-arbeid, er vi kommet så langt at vi har dyrket virusresistent julestjerne i et mindre forsøk i et gartneri. Nå er det naturlig å summere opp resultater vi har oppnådd og muligheter som åpner seg.

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Abstract Poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch), is a contemporary symbol of Christmas in most parts of the world. Today, Europe and North America represent the largest volume of production and sales, but demand is growing quickly in the other regions as poinsettia becomes more popular each year. In Norway, poinsettia is one of the most important pot plants, with a yearly production close to 6 million plants. Its ornamental value and innovation potential have laid the foundation for extensive research in Norway and elsewhere. Poinsettia mosaic virus (PnMV) can cause diseases in modern poinsettia cultivars. PnMV is a single-stranded, positive-sense RNA virus that belongs to the family Tymoviridae. Infection of poinsettia plants with PnMV results in mosaic symptoms during parts of the growing season, which in turn decreases the commercial value of this ornamental plant. Thus, growers are interested in the potential benefits of growing PnMV-free poinsettias. PnMV-free poinsettia plants can be obtained by heat treatment or in vitro culture of apical meristems, which are time-consuming and cost-ineffective methods. There is a need for a new and effective alternative approach, like Agrobacterium-mediated transformation, which can overcome these difficulties. Therefore, we have developed an Agrobacterium-mediated transformation approach for poinsettia for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring three hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75 % of the explants produced somatic embryos. In five experiments utilizing 868 explants, 18 independent transgenic lines were generated. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were detected among transformants. Northern blot analysis confirmed the production of transgene-derived small interfering RNAs (siRNAs). Transgenic lines showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA). The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.

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Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.

Sammendrag

Poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch), is a contemporary symbol of Christmas in most parts of the world. Today, Europe and North America represent the largest volume of production and sales, but demand is growing quickly in the other regions as poinsettia becomes more popular each year. In Norway, poinsettia is one of the most important pot plants, with a yearly production close to 6 million plants. Its ornamental value and innovation potential have laid the foundation for extensive research in Norway and elsewhere. Poinsettia mosaic virus (PnMV) is a RNA virus that belongs to the family Tymoviridae. Infection of poinsettia plants with PnMV results in mosaic symptoms during parts of the growing season and decrease the commercial value of this ornamental plant. Thus, growers are interested in the potential benefits of growing PnMV-free poinsettias. PnMV-free poinsettia plants can be obtained by heat treatment or in vitro culture of apical meristems, which are time-consuming and cost-ineffective methods. There is thus an urgent need for a new approach, like Agrobacterium-mediated transformation, which can overcome these difficulties. We have therefore developed an Agrobacterium-mediated transformation approach for poinsettia. Transgenic poinsettia plants with improved resistance against PnMV by expressing hairpin RNA constructs which targeted various regions of the virus genome were produced. Mechanical inoculation of PnMV and subsequent enzyme-linked immunosorbent assay (ELISA) confirmed the PnMV resistance. The siRNA analysis has demonstrated gene silencing mediated resistance. The PnMV resistant transgenic poinsettia lines produced are in the process of being commercialized. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.

Sammendrag

Poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch), is a contemporary symbol of Christmas in most parts of the world. Today, Europe and North America represent the largest volume of production and sales, but demand is growing quickly in the other regions as poinsettia becomes more popular each year. In Norway, poinsettia is one of the most important pot plants, with a yearly production close to 6 million plants. Its ornamental value and innovation potential have laid the foundation for extensive research in Norway and elsewhere. Two viruses i.e. poinsettia mosaic virus (PnMV) and poinsettia cryptic virus (PnCV) can cause diseases in modern poinsettia cultivars. PnMV gives visible symptoms in poinsettia during parts of the growing season. Growers show great interest in the potential benefits of growing PnMV-free poinsettias. Traditionally, PnMV-free poinsettia plants were obtained by in vitro culture of apical meristems. However, this is a time-consuming method and the regenerated new PnMV-free poinsettia has sometimes lost the branching characteristic which is important for poinsettia. We have therefore developed an Agrobacterium-mediated transformation approach for poinsettia. Using this method, we have produced transgenic poinsettia with improved resistance against PnMV by expressing three hairpin (hp) RNA gene constructs which targeted various regions of the virus genome. Molecular analyses have confirmed the stable integration of transgenes into the poinsettia genome. This is the first report describing Agrobacterium-mediated transformation of poinsettia. The PnMV resistant transgenic poinsettia lines produced are of commercial potential. The methodology developed could also facilitate the further improvement of this ornamental plant with the aims of enhancing its disease resistance, quality traits, desirable colour and ornamental value. We have also transformed N. benthemiana to reveal the relationship of different vector constructs and the RNA silencing mediated PnMV resistance. This result will imrpove our understanding of RNA silencing mediated resistance through genetic engineering.

Sammendrag

Poinsettia, Euphorbia pulcherrima Willd & Klotsch, is an ornamental flower which grows in many countries. Two viruses i.e. poinsettia mosaic virus (PnMV) and poinsettia cryptic virus (PnCV) can cause diseases on modern poinsettia cultivars. To produce virus-resistant poinsettia plants, genetic transformation has been considerated. Since there is no established method available for the transformation of poinsettia, we have chosen an electrophoresis-based transformation method to generate poinsettia transformants. The main advantage of this method is that we can avoid the time-consuming tissue culture and regeneration procedure. To develop a reliable transformation method, the green fluorescent protein (GFP) gene has been used as a reporter gene. Here, we report our preliminary results.

Sammendrag

Poinsettia (Euphorbia pulcherrima) is a major ornamental pot plant in many countries. Two viruses, poinsettia mosaic virus (PnMV) and poinsettia cryptic virus, often infect modern poinsettia cultivars. To produce virus-resistant poinsettia plants, genetic engineering has been considered. Since there is no established method available for the transformation of poinsettia, we have chosen an electrophoresisbased transformation method to generate poinsettia transformants. The main advantage of this method is that we can avoid the time-consuming tissue culture and regeneration procedure. To develop a reliable transformation method, the green fluorescent protein (GFP) gene has been used as a reporter gene. Here we report our preliminary results.

Sammendrag

Blomster er både dekorative og viktig i hverdagslivet vårt. Euphorbia pulcherrima Willd. Ex Klotzsch, julestjerne (poinsettia på engelsk) er en av de blomstene som har stor symbolverdi og som dyrkes i mange land i verden (Ecke III et al. 2004). Julestjernen har blitt vår mest populære juleblomst. Det produseres faktisk mellom 5 og 6 millioner julestjerner årlig i Norge (Hagen 2004). Julestjernen framviser store variasjoner i forskjellige deler av verden, fra å være høye og busklignende til kompakte potteblomster med mange grener. Mange patogener og skadegjørere kan angripe julestjerne, blant annet virus. Poinsettia mosaic virus (PnMV) og poinsettia latent virus (PnLV) er to virus som ofte infiserer julestjerne. PnMV forårsaker symptomer på bladene slik at kvaliteten blir redusert i følsomme sorter. Vi ønsker å lage PnMV-resistent julestjerne for å kunne få til en julestjerne av ennå høyere kvalitet. I tillegg ønsker vi å bruke samspillet mellom PnMV og julestjerneplanta som et modellsystem for å bygge opp kompetansen på genmodifisering av planter.