Plant Molecular Farming
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Plant Molecular Farming
A news site for the activities of members of the International Society for Plant Molecular Farming, as well as for papers of interest in the field
Curated by Ed Rybicki
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Plants produce ‘green vaccine’ against norovirus 

In a new study, Andrew G. Diamos and Hugh S. Mason of the Biodesign Center for Immunology, Vaccines and Virotherapy describe a trial vaccine against norovirus. The innovative therapeutic is produced using a plant-based system, which offers many advantages over traditional routes of pharmaceutical...
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Developing a flexible, high‐efficiency Agrobacterium‐mediated sorghum transformation system with broad application

Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi‐arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium‐mediated transformation of sorghum. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free ‘quality events’ ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84‐7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9‐mediated gene knockouts in Tx430.
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Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.
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Plant‐made E2 glycoprotein single‐dose vaccine protects pigs against classical swine fever 

Classical Swine Fever Virus (CSFV) causes classical swine fever, a highly contagious hemorrhagic fever affecting both feral and domesticated pigs. Outbreaks of CSF in Europe, Asia, Africa, and South America had significant adverse impacts on animal health, food security and the pig industry. The disease is generally contained by prevention of exposure through import restrictions (e.g. banning import of live pigs and pork products), localized vaccination programs, and culling of infected or at‐risk animals, often at very high cost. Current CSFV modified live virus vaccines are protective, but do not allow differentiation of infected from vaccinated animals (DIVA), a critical aspect of disease surveillance programs. Alternatively, first generation subunit vaccines using the viral protein E2 allow for use of DIVA diagnostic tests, but are slow to induce a protective response, provide limited prevention of vertical transmission, and may fail to block viral shedding. CSFV E2 subunit vaccines from a baculovirus/insect cell system have been developed for several vaccination campaigns in Europe and Asia. However, this expression system is considered expensive for a veterinary vaccine and is not ideal for wide‐spread deployment. To address the issues of scalability, cost of production, and immunogenicity, we have employed an Agrobacterium‐mediated transient expression platform in Nicotiana benthamiana and formulated the purified antigen in novel oil‐in‐water emulsion adjuvants. We report the manufacturing of adjuvanted, plant‐made CSFV E2 subunit vaccine. The vaccine provided complete protection in challenged pigs, even after single‐dose vaccination, which was accompanied with strong virus neutralization antibody responses.
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This Company Is Testing A Flu Vaccine Made In Tobacco -- And Philip Morris Is On Board

This Company Is Testing A Flu Vaccine Made In Tobacco -- And Philip Morris Is On Board | Plant Molecular Farming | Scoop.it
As the FDA meets to plan next year's flu vaccine, companies like Medicago are testing innovative technologies designed to minimize the risk of another devastating outbreak.
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A plant-produced vaccine protects mice against lethal West Nile virus infection without enhancing Zika or dengue virus infectivity

West Nile virus (WNV) has caused multiple global outbreaks with increased frequency of neuroinvasive disease in recent years. Despite many years of research, there are no licensed therapeutics or vaccines available for human use. One of the major impediments of vaccine development against WNV is the potential enhancement of infection by related flaviviruses in vaccinated subjects through the mechanism of antibody-dependent enhancement of infection (ADE). For instance, the recent finding of enhancement of Zika virus (ZIKV) infection by pre-exposure to WNV further complicates the development of WNV vaccines. Epidemics of WNV and the potential risk of ADE by current vaccine candidates demand the development of effective and safe vaccines. We have previously reported that the domain III (DIII) of the WNV envelope protein can be readily expressed in Nicotiana benthamiana leaves, purified to homogeneity, and promote antigen-specific antibody response in mice. Herein, we further investigated the in vivo potency of a plant-made DIII (plant-DIII) in providing protective immunity against WNV infection. Furthermore, we examined if vaccination with plant-DIII would enhance the risk of a subsequent infection by ZIKV and Dengue virus (DENV). Plant-DIII vaccination evoked antigen-specific cellular immune responses as well as humoral responses. DIII-specific antibodies were neutralizing and the neutralization titers met the threshold correlated with protective immunity by vaccines against multiple flaviviruses. Furthermore, passive administration of anti-plant DIII mouse serum provided full protection against a lethal challenge of WNV infection in mice. Notably, plant DIII-induced antibodies did not enhance ZIKV and DENV infection in Fc gamma receptor-expressing cells, addressing the concern of WNV vaccines in inducing cross-reactive antibodies and sensitizing subjects to subsequent infection by heterologous flavivirus. This study provides the first report of a WNV subunit vaccine that induces protective immunity, while circumventing induction of antibodies with enhancing activity for ZIKV and DENV infection.
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Tobacco plants as life-savers

Tobacco plants as life-savers | Plant Molecular Farming | Scoop.it
Queensland University of Technology (QUT), Australia, is the sole international cooperation partner in an AUD$10.5 million European project to develop new tobacco varieties that can be used as biofactories for pharmaceutical
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Enhanced transport of plant‐produced rabies single‐chain antibody‐RVG peptide fusion protein across an in cellulo blood–brain barrier device

Enhanced transport of plant‐produced rabies single‐chain antibody‐RVG peptide fusion protein across an in cellulo blood–brain barrier device | Plant Molecular Farming | Scoop.it
The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in plant
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Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination

Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination | Plant Molecular Farming | Scoop.it
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 infection
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How research into glowing fungi could lead to trees lighting our streets

How research into glowing fungi could lead to trees lighting our streets | Plant Molecular Farming | Scoop.it
How research into bioluminescent fungi could lead to trees replacing street lighting
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Plant-based “meat” is so tasty that Europe’s meat industry has to bite back

Plant-based “meat” is so tasty that Europe’s meat industry has to bite back | Plant Molecular Farming | Scoop.it
THE “kapsalon” is a healthy mix of chips, melted Gouda cheese, shawarma, lettuce and garlic sauce and is a tried and tested hangover cure in the Netherlands. So naturally, a butcher’s shop on the Spui, in The Hague, put it on its takeaway menu, alongside burgers and sausage rolls.
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Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector

Rapid Construction of Complex Plant RNA Virus Infectious cDNA Clones for Agroinfection Using a Yeast-E. coli-Agrobacterium Shuttle Vector | Plant Molecular Farming | Scoop.it
The availability of infectious full-length clone is indispensable for reverse genetics studies of virus biology, pathology and construction of viral vectors. However, for RNA viruses with large genome sizes or those exhibiting inherent cloning difficulties, procedure to generate biologically active complementary DNA (cDNA) clones can be time-consuming or technically challenging. Here we have constructed a yeast-Escherichia coli-Agrobacterium shuttle vector that enables highly efficient homologous recombination in yeast for assembly of Agrobacterium compatible plant virus clones. Using this vector, we show that infectious cDNA clones of a plant negative-stranded RNA virus, sonchus yellow net rhabdovirus, can be rapidly assembled. In addition, one-step assembly of infectious clones of potato virus Y in yeast, either with or without intron, was readily achieved from as many as eight overlapping DNA fragments. More importantly, the recovered yeast plasmids can be transformed directly into Agrobacterium for inoculation, thereby obviating the E. coli cloning steps and associated toxicity issues. This method is rapid, highly efficient and cost-effective and should be readily applicable to a broad range of plant viruses.
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iBio/BRU Molecular Farming Workshop

iBio/BRU Molecular Farming Workshop Press Release - South Africa   iBio Inc and the University of Cape Town’s Biopharming Research Unit (BRU) announce a Molecular Farming Workshop to be held in Franschhoek in the Western Cape of South Africa, on November 3rd and 4th, 2017. The workshop will bring together public and private entities driving…
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Plants are way more energy efficient than we thought

Plants are way more energy efficient than we thought | Plant Molecular Farming | Scoop.it
Scientists thought plants wasted almost a third of the energy they made with photosynthesis. New research finds otherwise.
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Plant‐expressed Fc‐fusion protein tetravalent dengue vaccine with inherent adjuvant properties

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly‐immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor‐targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell‐mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen‐specific CD4+ and CD8+ T‐cell proliferation, IFN‐γ and antibody production. The purified polymeric fraction of dengue PIGS (D‐PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low‐affinity Fcγ receptors on antigen‐presenting cells. These results show that the plant‐expressed D‐PIGS have the potential for translation towards a safe and easily scalable single antigen‐based tetravalent dengue vaccine.
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Simultaneous CRISPR/Cas9‐mediated editing of cassava eIF4E isoforms nCBP‐1 and nCBP‐2 reduces cassava brown streak disease symptom severity and incidence

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome‐linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E‐1, eIF(iso)4E‐2, novel cap‐binding protein‐1 (nCBP‐1), and nCBP‐2. Protein‐protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9‐mediated genome editing was employed to generate ncbp‐1, ncbp‐2, and ncbp‐1/ncbp‐2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp‐1/ncbp‐2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titer in storage roots relative to wild‐type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.
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Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to Rice tungro spherical virus

Rice tungro disease (RTD) is a serious constraint in rice production across tropical Asia. RTD is caused by the interaction between Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. RTSV resistance found in traditional cultivars has contributed to a reduction in the incidence of RTD in the field. Natural RTSV resistance is a recessive trait controlled by the translation initiation factor 4 gamma gene (eIF4G). The Y1059 V1060 V1061 residues of eIF4G are known to be associated with the reactions to RTSV. To develop new sources of resistance to RTD, mutations in eIF4G were generated using the CRISPR/Cas9 system in the RTSV-susceptible variety IR64, widely grown across tropical Asia. The mutation rates ranged from 36.0% to 86.6%, depending on the target site, and the mutations were successfully transmitted to the next generations. Among various mutated eIF4G alleles examined, only those resulting in in-frame mutations in SVLFPNLAGKS residues (mainly NL), adjacent to the YVV residues, conferred resistance. Furthermore, our data suggest that eIF4G is essential for normal development, as alleles resulting in truncated eIF4G could not be maintained in homozygous state. The final products with RTSV resistance and enhanced yield under glasshouse conditions were found to no longer contain the Cas9 sequence. Hence, the RTSV-resistant plants with the novel eIF4G alleles represent a valuable material to develop more diverse RTSV-resistant varieties.

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Virus-like particles that display Zika virus envelope protein domain III induce potent neutralizing immune responses in mice

Virus-like particles that display Zika virus envelope protein domain III induce potent neutralizing immune responses in mice | Plant Molecular Farming | Scoop.it
Several Zika virus (ZIKV) vaccine candidates have recently been described which use inactivated whole virus, DNA or RNA that express the virus’ Envelope (E) glycoprotein as the antigen. These were successful in stimulating production of virus-targete
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Canadian Cannabis Companies Correct After Capital Raising Crescendo

Canadian Cannabis Companies Correct After Capital Raising Crescendo | Plant Molecular Farming | Scoop.it
Stocks of publicly-traded Canadian cannabis producers have declined sharply after hitting all-time highs in January. The introduction of an ETF right before the end of 2017 and momentum trading helped push the stocks up to levels that weren't sustainable, and the companies raised $1.2 billion.
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Scientists’ approach to saving the “Chocolate Trees” from extinction

Scientists’ approach to saving the “Chocolate Trees” from extinction | Plant Molecular Farming | Scoop.it
Scientists predicted that cacao trees may be extinct by 2050 due to factors including climate change, pests and fungal infections. To save chocolate, "the food of the gods", researchers within the chocolate industry is trying to use the innovative CRISPR genome editing technology to save these delicate plants. Genome editing for cacao is an effective way to build tougher cacao plants and help them survive the climate change. In fact, genetically modified chocolate is already on the market. Despite so, a chocolate deficit is still in our near future due to the increasing demand for it. Recently, the candy company, Mars, invested $1 billion into their "Sustainability Generation" R&D project and is teaming up with scientists to use the most effective genome editing tool, CRISPR/Cas9 technology, for editing and breeding stronger cacao plants. As part of their "Cocoa Sustainability Approach", Mars has already sequenced the cocoa genome and share their results to the public in 2013. With their new plan for making super chocolate, hopefully, we could avoid the predicted chocolate deficit from occurring.
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Characterization of scientific studies usually cited as evidence of adverse effects of GM food/feed

Characterization of scientific studies usually cited as evidence of adverse effects of GM food/feed | Plant Molecular Farming | Scoop.it
GM crops are the most studied crops in history. Approximately 5% of the safety studies on them show adverse effects that are a cause for concern and tend to be featured in media reports. Althoug
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Homer's Addictive ToMacco

The Simpson's tomatoes turned out to mix with the tobacco seeds. Chief Wiggum and Ralph buy it as it's very addictive.
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Friday Think: The curious process of turning plants into vaccine “factories”

Friday Think: The curious process of turning plants into vaccine “factories” | Plant Molecular Farming | Scoop.it
Scientists discovered a “cheap, easy, and quick” way to make polio vaccines using plant leaves.
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Plant‐expressed Fc‐fusion protein tetravalent dengue vaccine with inherent adjuvant properties

Plant‐expressed Fc‐fusion protein tetravalent dengue vaccine with inherent adjuvant properties | Plant Molecular Farming | Scoop.it
Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi‐Pasteur Denvaxia vaccine does not protect children under the age of nine an
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Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4

Transgenic Cavendish bananas with resistance to Fusarium wilt tropical race 4 | Plant Molecular Farming | Scoop.it

Banana (Musa spp.) is a staple food for more than 400 million people. Over 40% of world production and virtually all the export trade is based on Cavendish banana. However, Cavendish banana is under threat from a virulent fungus, Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) for which no acceptable resistant replacement has been identified. Here we report the identification of transgenic Cavendish with resistance to TR4. In our 3-year field trial, two lines of transgenic Cavendish, one transformed with RGA2, a gene isolated from a TR4-resistant diploid banana, and the other with a nematode-derived gene, Ced9, remain disease free. Transgene expression in the RGA2 lines is strongly correlated with resistance. Endogenous RGA2 homologs are also present in Cavendish but are expressed tenfold lower than that in our most resistant transgenic line. The expression of these homologs can potentially be elevated through gene editing, to provide non-transgenic resistance.

Ed Rybicki's insight:
The Man With the Golden Banana - sometime molecular farmer James Dale - and his team strike again! Good on yer, mates!
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