To document

Abstract

Hepatitis B and C viruses chronically affect approximately 3.5% of the global population, causing more than 800,000 deaths yearly due to severe liver pathogenesis. Current HBV vaccines have significantly contributed to the reduction of chronic HBV infections, supporting the notion that virus eradication is a feasible public health objective in the near future. In contrast to HBV, a prophylactic vaccine against HCV infection is not available yet; however, intense research efforts within the last decade have significantly advanced the field and several vaccine candidates are shortlisted for clinical trials. A successful vaccine against an infectious disease of global importance must not only be efficient and safe, but also easy to produce, distribute, administer, and economically affordable to ensure appropriate coverage. Some of these requirements could be fulfilled by oral vaccines that could complement traditional immunization strategies. In this review, we discuss the potential of edible plant-based oral vaccines in assisting the worldwide fight against hepatitis B and C infections. We highlight the latest research efforts to reveal the potential of oral vaccines, discuss novel antigen designs and delivery strategies, as well as the limitations and controversies of oral administration that remain to be addressed to make this approach successful.

To document

Abstract

Difenoconazole is a widely used triazole fungicide that has been frequently detected in the environment, but comprehensive study about its environmental fate and toxicity of potential transformation products (TPs) is still lacking. Here, laboratory experiments were conducted to investigate the degradation kinetics, pathways, and toxicity of transformation products of difenoconazole. 12, 4 and 4 TPs generated by photolysis, hydrolysis and soil degradation were identified via UHPLC-QTOF/MS and the UNIFI software. Four intermediates TP295, TP295A, TP354A and TP387A reported for the first time were confirmed by purchase or synthesis of their standards, and they were further quantified using UHPLC-MS/MS in all tested samples. The main transformation reactions observed for difenoconazole were oxidation, dechlorination and hydroxylation in the environment. ECOSAR prediction and laboratory tests showed that the acute toxicities of four novel TPs on Brachydanio rerio, Daphnia magna and Selenastrum capricornutum are substantially lower than that of difenoconazole, while all the TPs except for TP277C were predicted chronically very toxic to fish, which may pose a potential threat to aquatic ecosystems. The results are important for elucidating the environmental fate of difenoconazole and assessing the environmental risks, and further provide guidance for scientific and reasonable use.

To document

Abstract

Cardiomyopathy syndrome (CMS) is a severe cardiac disease occurring in the grow-out sea phase of farmed Atlantic salmon with approximately 100 outbreaks annually in Norway. Piscine myocarditis virus (PMCV) is believed to be the causative agent of CMS. There is no vaccine available to control CMS, partially because PMCV withstands propagation in known cell cultures. In the present study, we selected the putative capsid protein of PMCV as the candidate antigen for immunization experiments and produced it in the plant Nicotiana benthamiana by transient expression. The recombinant PMCV antigen formed virus-like particles (VLPs). To evaluate the efficacy of the plant made VLP vaccine, a PMCV infection model was established. In an experimental salmon vaccination trial, the VLP vaccine triggered innate immunity, and indicative but not significant inhibition of viral replication in heart, spleen and kidney tissues was observed. Similarly, a reduction of inflammatory lesions in cardiomyocytes and subendocardial infiltration by mononuclear leukocytes were observed. Therefore, there was no difference in efficacy or immune response observed post the plant made PMCV VLP antigen vaccination. Taken together, this study has demonstrated that plant made VLP antigens should be investigated further as a possible platform for the development of PMCV antigens for a CMS vaccine.

To document

Abstract

Chronic infection with hepatitis C virus (HCV) remains a leading cause of liver-related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus-neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N-glycosylation pattern. Here we report the first evidence of successful production of the full-length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium-mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N-glycan analysis of HCV-E2 produced in N. benthamiana and mammalian cells indicated host-specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant-derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV-neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost-effective production platforms for the development of HCV vaccines.

Abstract

Aquaculture has undergone rapid development in the past decades. It provides a large part of high-quality protein food for humans, and thus, a sustainable aquaculture industry is of great importance for the worldwide food supply and economy. Along with the quick expansion of aquaculture, the high fish densities employed in fish farming increase the risks of outbreaks of a variety of aquatic diseases. Such diseases not only cause huge economic losses, but also lead to ecological hazards in terms of pathogen spread to marine ecosystems causing infection of wild fish and polluting the environment. Thus, fish health is essential for the aquaculture industry to be environmentally sustainable and a prerequisite for intensive aquaculture production globally. The wide use of antibiotics and drug residues has caused intensive pollution along with risks for food safety and increasing antimicrobial resistance. Vaccination is the most effective and environmentally friendly approach to battle infectious diseases in aquaculture with minimal ecological impact and is applicable to most species of farmed fish. However, there are only 34 fish vaccines commercially available globally to date, showing the urgent need for further development of fish vaccines to manage fish health and ensure food safety. Plant genetic engineering has been utilized to produce genetically modified crops with desirable characteristics and has also been used for vaccine production, with several advantages including cost-effectiveness, safety when compared with live virus vaccines, and plants being capable of carrying out posttranslational modifications that are similar to naturally occurring systems. So far, plant-derived vaccines, antibodies, and therapeutic proteins have been produced for human and animal health. However, the development of plant-made vaccines for animals, especially fish, is still lagging behind the development of human vaccines. The present review summarizes the development of fish vaccines currently utilized and the suitability of the plant-production platform for fish vaccine and then addresses considerations regarding fish vaccine production in plants. Developing fish vaccines by way of plant biotechnology are significant for the aquaculture industry, fish health management, food safety, and human health.

To document

Abstract

The demand for animal protein has increased considerably worldwide, especially in China, where large numbers of livestock and poultry are produced. Antibiotics have been widely applied to promote growth and prevent diseases. However, the overuse of antibiotics in animal feed has caused serious environmental and health risks, especially the wide spread of antimicrobial resistance (AMR), which seriously affects animal and human health, food safety, ecosystems, and the sustainable future development of animal protein production. Unfortunately, AMR has already become a worldwide challenge, so international cooperation is becoming more important for combatting it. China’s efforts and determination to restrict antibiotic usage through law enforcement and effective management are of significance. In this review, we address the pollution problems of antibiotics; in particular, the AMR in water, soil, and plants caused by livestock and poultry manure in China. The negative impact of widespread and intensive use of antibiotics in livestock production is discussed. To reduce and mitigate AMR problems, we emphasize in this review the development of antibiotic substitutes for the era of antibiotic prohibition.

To document

Abstract

Propionate and propionyl-CoA accumulation have been associated with the development of mitochondrial dysfunction. In this study, we show that propionate induces intestinal damage in zebrafish when fed a high-fat diet (HFD). The intestinal damage was associated with oxidative stress owing to compromised superoxide dismutase 2 (Sod2) activity. Global lysine propionylation analysis of the intestinal samples showed that Sod2 was propionylated at lysine 132 (K132), and further biochemical assays demonstrated that K132 propionylation suppressed Sod2 activity. In addition, sirtuin 3 (Sirt3) played an important role in regulating Sod2 activity via modulating de-propionylation. Finally, we revealed that intestinal oxidative stress resulting from Sod2 propionylation contributed to compositional change of gut microbiota. Collectively, our results in this study show that there is a link between Sod2 propionylation and oxidative stress in zebrafish intestines and highlight the potential mechanism of intestinal problems associated with high propionate levels.

To document

Abstract

Probiotics confer a health benefit on the host and could be used as a good alternative to antibiotics. Probiotics are strain‐specific when exerting their function, so it is necessary to identify them to strain level. In recent years, intra‐species molecular typing and identification methods have developed rapidly, which commonly are used for typing the main pathogenic bacteria and rare for studies on probiotic typing, whilst it is imperative. This article describes molecular typing methods including AFLP, RAPD, PFGE, ribotyping, MLST, rep‐PCR and whole‐genome sequencing to identity some aquatic probiotics approved by the Ministry of Agriculture of China, which are Bifidobacterium, Enterococcus, Lactobacillus, Pediococcus, Aspergillus, Bacillus, Rhodopseudomonas palustris and Streptococcus thermophilus. In addition, the principles, applications, advantages and disadvantages of these typing methods are also discussed.

To document

Abstract

Hyperspectral imaging has many applications. However, the high device costs and low hyperspectral image resolution are major obstacles limiting its wider application in agriculture and other fields. Hyperspectral image reconstruction from a single RGB image fully addresses these two problems. The robust HSCNN-R model with mean relative absolute error loss function and evaluated by the Mean Relative Absolute Error metric was selected through permutation tests from models with combinations of loss functions and evaluation metrics, using tomato as a case study. Hyperspectral images were subsequently reconstructed from single tomato RGB images taken by a smartphone camera. The reconstructed images were used to predict tomato quality properties such as the ratio of soluble solid content to total titratable acidity and normalized anthocyanin index. Both predicted parameters showed very good agreement with corresponding “ground truth” values and high significance in an F test. This study showed the suitability of hyperspectral image reconstruction from single RGB images for fruit quality control purposes, underpinning the potential of the technology—recovering hyperspectral properties in high resolution—for real-world, real time monitoring applications in agriculture any beyond.

To document

Abstract

Diseases caused by viruses threaten the production industry and food safety of aquaculture which is a great animal protein source. Grass carp reovirus (GCRV) has caused tremendous loss, and the molecular function of viral proteins during infection needs further research, as for most aquatic viruses. In this study, interaction between GCRV major outer capsid protein VP4 and RIG-I, a critical viral RNA sensor, was screened out by GST pull-down, endogenous immunoprecipitation and subsequent LC-MS/MS, and then verified by co-IP and an advanced farred fluorescence complementation system. VP4 was proved to bind to the CARD and RD domains of RIG-I and promoted K48-linked ubiquitination of RIG-I to degrade RIG-I. VP4 reduced mRNA and promoter activities of key genes of RLR pathway and sequential IFN production. As a consequence, antiviral effectors were suppressed and GCRV replication increased, resulting in intensified cytopathic effect. Furthermore, results of transcriptome sequencing of VP4 stably expressed CIK (C. idella kidney) cells indicated that VP4 activated the MyD88-dependent TLR pathway. Knockdown of VP4 obtained opposite effects. These results collectively revealed that VP4 interacts with RIG-I to restrain interferon response and assist GCRV invasion. This study lays the foundation for anti-dsRNA virus molecular function research in teleost and provides a novel insight into the strategy of immune evasion for aquatic virus.

To document

Abstract

Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway. To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis 1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrN and ywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrN and BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 1416.47 U/mL/OD600, which was about 121% higher than that of the wild-type strain. Furthermore, LC–MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6 was classified as a member of the α/β hydrolase family with a conserved “nucleophile-acid-histidine” catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

To document

Abstract

Aquaculture is a fast‐growing and rapidly expanding industry in the world. Probiotics are widely used in aquaculture and have provided benefits to aquatic animal health, and it is also a promising alternative to antibiotics for control of fish diseases. With the development of biotechnology, new expression systems and novel techniques for the surface display of heterologous proteins in surface of probiotics cells have been developed. This review provides an overview of the systems and strategies for displaying functional proteins on the surface of probiotics commonly used in aquaculture, which are Bacillus subtilis, Bacillus thuringiensis, lactic acid bacteria represented by Lactococcus lactis and yeast. Their applications in aquaculture especially for oral vaccine development afforded by this technology and prospects and challenges associated with this technology are also highlighted.

Abstract

Endogenous antimicrobial peptides (AMPs) are evolutionarily ancient factors of innate immunity, which are produced by all multicellular organisms and play a key role in their protection against infection. Red king crab (Paralithodes camtschaticus), also called Kamchatka crab, is widely distributed and the best known species of all king crabs belonging to the family Lithodidae. Despite their economic importance, the genetic resources of king crabs are scarcely known and no fullgenome sequences are available to date. Therefore, analysis of the red king crab transcriptome and identifcation and characterization of its AMPs could potentially contribute to the development of novel antimicrobial drug candidates when antibiotic resistance has become a global health threat. In this study, we sequenced the P. camtschaticus transcriptomes from carapace, tail fap and leg tissues using an Illumina NGS platform. Libraries were systematically analyzed for gene expression profles along with AMP prediction. By an in silico approach using public databases we defned 49 cDNAs encoding for AMP candidates belonging to diverse families and functional classes, including buforins, crustins, paralithocins, and ALFs (anti-lipopolysaccharide factors). We analyzed expression patterns of 27 AMP genes. The highest expression was found for Paralithocin 1 and Crustin 3, with more than 8,000 reads. Other paralithocins, ALFs, crustins and ubiquicidins were among medium expressed genes. This transcriptome data set and AMPs provide a solid baseline for further functional analysis in P. camtschaticus. Results from the current study contribute also to the future application of red king crab as a bio-resource in addition to its being a known seafood delicacy.

To document

Abstract

To meet increasing demand for animal protein, swine have been raised in large Chinese farms widely, using antibiotics as growth promoter. However, improper use of antibiotics has caused serious environmental and health risks, in particular Antimicrobial resistance (AMR). This paper reviews the consumption of antibiotics in swine production as well as AMR and the development of novel antibiotics or alternatives in China. The estimated application of antibiotics in animal production in China accounted for about 84240 tons in 2013. Overuse and abuse of antibiotics pose a great health risk to people through food-borne antibiotic residues and selection for antibiotic resistance. China unveiled a national plan to tackle antibiotic resistance in August 2016, but more support is needed for the development of new antibiotics or alternatives like plant extracts. Antibiotic resistance has been a major global challenge, so international collaboration between China and Europe is needed.

To document

Abstract

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7Arec) was compared with the native fungal enzyme (TrCel7Anat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7Arec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.

To document

Abstract

Soybean cyst nematode (SCN, Heterodera glycines (I.)) is one of the most important soil-borne pathogens for soybeans. In plant parasitic nematodes, including SCN, lysozyme plays important roles in the innate defense system. In this study, two new lysozyme genes (Hg-lys1 and Hg-lys2) from SCN were cloned and characterized. The in situ hybridization analyses indicated that the transcripts of both Hg-lys1 and Hg-lys2 accumulated in the intestine of SCN. The qRT-PCR analyses showed that both Hg-lys1 and Hg-lys2 were upregulated after SCN second stage juveniles (J2s) were exposed to the Gram-positive bacteria Bacillus thuringiensis, Bacillus subtilis or Staphylococcus aureus. Knockdown of the identified lysozyme genes by in vitro RNA interference caused a significant decrease in the survival rate of SCN. All of the obtained results indicate that lysozyme is very important in the defense system and survival of SCN.

To document

Abstract

Cereal cyst nematode (CCN, Heterodera avenae) presents severe challenges to wheat (Triticum aestivum L.) production worldwide. An investigation of the interaction between wheat and CCN can greatly improve our understanding of how nematodes alter wheat root metabolic pathways for their development and could contribute to new control strategies against CCN. In this study, we conducted transcriptome analyses of wheat cv. Wen 19 (Wen19) by using RNA-Seq during the compatible interaction with CCN at 1, 3 and 8 days past inoculation (dpi). In total, 71,569 transcripts were identified, and 10,929 of them were examined as differentially expressed genes (DEGs) in response to CCN infection. Based on the functional annotation and orthologous findings, the protein phosphorylation, oxidation-reduction process, regulation of transcription, metabolic process, transport, and response process as well as many other pathways previously reported were enriched at the transcriptional level. Plant cell wall hydrolysis and modifying proteins, auxin biosynthesis, signalling and transporter genes were up-regulated by CCN infection to facilitate penetration, migration and syncytium establishment. Genes responding to wounding and jasmonic acid stimuli were enriched at 1 dpi. We found 16 NBS-LRR genes, 12 of which were down-regulated, indicating the repression of resistance. The expression of genes encoding antioxidant enzymes, glutathione S-transferases and UDP-glucosyltransferase was significantly up-regulated during CCN infection, indicating that they may play key roles in the compatible interaction of wheat with CCN. Taken together, the results obtained from the transcriptome analyses indicate that the genes involved in oxidation-reduction processes, induction and suppression of resistance, metabolism, transport and syncytium establishment may be involved in the compatible interaction of Wen 19 with CCN. This study provides new insights into the responses of wheat to CCN infection. These insights could facilitate the elucidation of the potential mechanisms of wheat responses to CCN.

To document

Abstract

Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S)envelope protein are currently used in universal vaccination programs and achieve protective immuneresponse in more than 90% of recipients. However, new vaccination strategies are necessary for successfulimmunization of the remaining non- or low-responders. We have previously characterized a novel HBVchimeric antigen, which combines neutralization epitopes of the S and the preS1 domain of the large (L)envelope protein (genotype D). The S/preS121–47chimera produced in mammalian cells and Nicotianabenthamiana plants, induced a significantly stronger immune response in parenterally vaccinated micethan the S protein. Here we describe the transient expression of the S/preS121–47antigen in an edibleplant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral admin-istration of adjuvant-free Lactuca sativa expressing the S/preS121–47antigen, three times, at 1lg/dose,was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies wereable to neutralize HBV infection in an NTCP-expressing infection system (HepG2-NTCP cell line) moreefficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results sup-port the S/preS121–47antigen as a promising candidate for future development as an edible HBV vaccine.

To document

Abstract

Dengue fever is a mosquito (Aedes aegypti) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD‐TDV), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoral and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.

To document

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.

To document

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.

To document

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.

To document

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.

To document

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.

To document

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.

To document

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.

To document

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.

Abstract

We describe the cloning and characterization of a single copy gene from Trichoderma atroviride P1 encoding a novel 30 kDa chitinase, Ech30. Ech30 is a family 18 chitinase showing low sequence similarity to other Trichoderma chitinases. Real-time quantitative RT-PCR studies revealed that expression of the ech30 gene was induced by the presence of Botrytis cinerea in plate confrontation assays, but hardly by chitin in liquid cultures. Studies of Ech30 purified from an Escherichia coli strain overexpressing the ech30 gene devoid of the leader sequence and a predicted intron, showed that the gene encodes an active chitinase, which, as expected for family 18 chitinases, is inhibited by allosamidin.

20140613 Kålproduksjon i Heia

Division of Biotechnology and Plant Health

LowImpact - ChiNor solutions for Low Impact climate smart vegetable production with reduced pesticide residues in food, soil and water resources


Biochar technologies show promise as tools for climate smart and environmentally friendly agricultural production, both as tools to improve soil quality and impact greenhouse gas emission from soils and to reduce pesticide pollution to the environment and pesticide residues in food. However, there is a lack of studies integrating these concerns and designing joint solutions.

Active Updated: 23.07.2021
End: jun 2022
Start: jan 2019
HCV talk Biotechnology Havana 2017 Dec 6, 2017-bilde
SmartVac - Next Generation Viral Hepatitis B and C vaccine development in plantsand algae using advanced biotechnological tools


Hepatitis B (HBV) and C viruses (HCV) infect the human liver, triggering persistent inflammation and eventually cirrhosis and hepatocellular carcinoma (HCC), the second leading cause of cancer-related mortality worldwide. Currently, more than 500 million people are chronically infected with HBV or HCV and at high risk of developing end stage liver disease and HCC.

Active Updated: 27.08.2021
End: may 2023
Start: jun 2019