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Abstract

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.

Abstract

Phosphorus (P) is one of the main limiting factors for crop productivity while rhizosphere organic anions have been hypothesized to play an important role in P acquisition. Sampling in a long-term field experiment was carried out in order to understand the impact of long-term differences in P fertilization on secretion of organic anions under field conditions. Rhizosphere organic anions were extracted and analyzed every week from three leaves stage to completed flowering stage of wheat (Triticum aestivum) grown on plots that have received 0 (P0) or 48 (P48) kg P ha−1 year−1 since 1966. The study showed that it is possible to extract and quantify rhizosphere organic anions from field plots. In P48 plots, root P concentrations decreased around 40 % at the early stages (before heading), compared with the first sampling, and then increased slightly, while plants grown in P0 plots showed the opposite trend. Malate was the main organic anion secreted throughout all the wheat growth stages. Rhizosphere citrate and malate showed negative and positive correlations (P < 0.05) respectively with root P concentrations at 29 and 42 days after emergence (DAE). Rhizosphere organic anion concentrations were quite low until 29 DAE and then increased up to 4–10-fold until 42 DAE; these concentrations declined at later stages, indicating that root-released organic anions may have been affected by developmental stage and root P concentration. The present study provides valuable information about the relationship between rhizosphere organic anions and various P concentrations of wheat grown in the field.

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Abstract

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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Abstract

Cereal cyst nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands and are secreted into plant tissues through the stylet. To understand the function of nematode effectors in parasitic plants, we cloned predicted effectors genes from Heterodera avenae and transiently expressed them in Nicotiana benthamiana. Infiltration assays showed that HaEXPB2, a predicted expansin-like protein, caused cell death in N. benthamiana. In situ hybridization showed that HaEXPB2 transcripts were localised within the subventral gland cells of the pre-parasitic second-stage nematode. HaEXPB2 had the highest expression levels in parasitic second-stage juveniles. Subcellular localization assays revealed that HaEXPB2 could be localized in the plant cell wall after H. avenae infection.This The cell wall localization was likely affected by its N-terminal and C-terminal regions. In addition, we found that HaEXPB2 bound to cellulose and its carbohydrate-binding domain was required for this binding. The infectivity of H. avenae was significantly reduced when HaEXPB2 was knocked down by RNA interference in vitro. This study indicates that HaEXPB2 may play an important role in the parasitism of H. avenae through targeting the host cell wall.

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Abstract

Abstract Dengue fever is a disease in many parts of the tropics and subtropics and about half the world’s popula- tion is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Cur- rently there is only one vaccine (DengvaxiaÒ) available (limited to a few countries) on the market since 2015 after half a century’s intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibod- ies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype- specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion con- struct combining EDIII polypeptides from all four ser- otypes was also attempted. Transplastomic EDIII- expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a GatewayÒ plastid transformation vector for inducible transgene expression.

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Abstract

Many arable lands have accumulated large reserves of residual phosphorus (P) and a relatively large proportion of soil P is less available for uptake by plants. Root released organic anions are widely documented as a key physiological strategy to enhance P availability, while limited information has been generated on the contribution of rhizosphere organic anions to P utilization by crops grown in agricultural soils that are low in available P and high in extractable Ca, Al, and Fe. We studied the role of rhizosphere organic anions in P uptake from residual P in four common crops Triticum aestivum, Avena sativa, Solanum tuberosum, and Brassica napus in low- and high-P availability agricultural soils from long-term fertilization field trials in a mini-rhizotron experiment with four replications. Malate was generally the dominant organic anion. More rhizosphere citrate was detected in low P soils than in high P soil. B. napus showed 74–103% increase of malate in low P loam, compared with clay loam. A. sativa had the greatest rhizosphere citrate concentration in all soils (5.3–15.2 μmol g−1 root DW). A. sativa also showed the highest level of root colonization by arbuscular mycorrhizal fungi (AMF; 36 and 40%), the greatest root mass ratio (0.51 and 0.66) in the low-P clay loam and loam respectively, and the greatest total P uptake (5.92 mg P/mini-rhizotron) in the low-P loam. B. napus had 15–44% more rhizosphere acid phosphatase (APase) activity, ~0.1–0.4 units lower rhizosphere pH than other species, the greatest increase in rhizosphere water-soluble P in the low-P soils, and the greatest total P uptake in the low-P clay loam. Shoot P content was mainly explained by rhizosphere APase activity, water-soluble P and pH within low P soils across species. Within species, P uptake was mainly linked to rhizosphere water soluble P, APase, and pH in low P soils. The effects of rhizosphere organic anions varied among species and they appeared to play minor roles in improving P availability and uptake.

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Abstract

Phosphorus (P) is an important element for crop productivity and is widely applied in fertilizers. Most P fertilizers applied to land are sorbed onto soil particles, so research on improving plant uptake of less easily available P is important. In the current study, we investigated the responses in root morphology and root-exuded organic acids (OAs) to low available P (1 mM P) and sufficient P (50 mM P) in barley, canola and micropropagated seedlings of potato— three important food crops with divergent root traits, using a hydroponic plant growth system.We hypothesized that the dicots canola and tuber-producing potato and the monocot barley would respond differently under various P availabilities. WinRHIZO and liquid chromatography triple quadrupole mass spectrometry results suggested that under low P availability, canola developed longer roots and exhibited the fastest root exudation rate for citric acid. Barley showed a reduction in root length and root surface area and an increase in root-exudedmalic acid under low-P conditions. Potato exuded relativelysmall amounts of OAs under low P, while therewas a marked increase in root tips. Based on the results, we conclude that different crops show divergent morphological and physiological responses to low P availability, having evolved specific traits of root morphology and root exudation that enhance their P-uptake capacity under low-P conditions. These results could underpin future efforts to improve P uptake of the three crops that are of importance for future sustainable crop production.

Abstract

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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Abstract

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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Abstract

Abstract Strict control of morphogenesis is essential in production of potted poinsettia. Commonly, this is obtained by the use of plant growth retardants (PGRs), often in combination with early morning temperature drops. Due to negative effects on human health and the environment, the use of PGRs is becoming restricted. Also, energy-saving growth regimes and periods of high temperatures limit effective use of temperature drops. In the present study the use of a high proportion of blue (B) light provided by light emitting diodes [LEDs, 20% blue (B), 80% red (R)] was compared with traditional high pressure sodium (HPS) lamps (5% B) providing similar phytochrome photostationary state to produce compact poinsettia plants. Both in the greenhouse and growth chamber, all cultivars were 20–34% shorter for LED compared to HPS grown plants. Also, leaf and bract area as well as chlorophyll content and total dry matter accumulation were lower under LED. The LED did not delay bract color formation, visible cyathia and flowering compared to HPS, and no difference in post production performance (cyathia/bract abscission or necrosis) between the two light treatments was found. The effect of end of day-red (EOD-R) lighting combination with LED and HPS supplemental lamps during the photoperiod in the greenhouse was also investigated. Reduced stem extension (13%) was observed under HPS only and for one of the two cultivars tested, whereas under the LED regime, there was no effect of EOD-R lighting.

Abstract

The global spread of dengue fever threatens a large percentage of the world’s population. The disease causes great human suffering, a high mortality from dengue haemorrhagic fever and its complications, and major costs. There is currently no vaccine to prevent dengue virus infection. Our project aims to express a tetravalent vaccine candidate in tobacco chloroplasts, a cost effective system, and hence to contribute to innovation and bio-economy as a long term goal.

Abstract

The effects of genetically modified (GM) maize (Zea mays L.) expressing the Bacillus thuringiensis Berliner Cry1Fa2 protein (Bt) and phosphinothricin or glyphosate herbicide tolerance on soil chemistry (organic matter, N, P, K and pH), compared with non-GM controls, were assessed in field and pot experiments. In the field experiment, NH4+ was significantly higher in soil under the crop modified for herbicide tolerance compared to the control (mean values of 11 and 9.6 mg N/kg respectively) while P was significantly higher in soil under the control compared to under the GM crop (mean values of 6.9 and 6.4 dg P/kg, respectively). No significant differences were found as a result of growing Bt/herbicide tolerant maize. In the pot experiment, using soils from three sites (Gongzhuling, Dehui and Huadian), significant effects of using Bt maize instead of conventional maize were found for all three soils. In the Gongzhuling soil, P was significantly higher in soil under the control compared to under the GM crop (mean values of 4.8 and 4.0 dg P/kg, respectively). For the Dehui soil, the pH was significantly higher in soil under the control compared to under the GM crop (mean values for {H+} of 1.1 and 2.4 μM for the control and the GM crop respectively). In the Huadian soil, organic matter and total N were both higher in soil under the GM crop than under the control. For organic matter, the mean values were 3.0 and 2.9% for the GM crop and the control, respectively, while for total nitrogen the mean values were 2.02 and 1.96‰ for the GM crop and the control respectively. Our results indicate that growing GM crops instead of conventional crops may alter soil chemistry, but not greatly, and that effects will vary with both the specific genetic modification and the soil.

Abstract

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.