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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Scooped by Ed Rybicki
August 3, 2023 6:22 AM
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Frontiers | A plant-produced SARS-CoV-2 spike protein elicits heterologous immunity in hamsters

Frontiers | A plant-produced SARS-CoV-2 spike protein elicits heterologous immunity in hamsters | Plant Molecular Farming | Scoop.it
Molecular farming of vaccines has been heralded as a cheap, safe and scalable production platform. In reality, however, differences in the plant biosynthetic machinery, compared to mammalian cells, can complicate the production of viral glycoproteins. Remodelling the secretory pathway presents an opportunity to support key post-translational modifications, and to tailor aspects of glycosylation and glycosylation-directed folding. In this study, we applied an integrated host and glyco-engineering approach, NXS/T Generation™, to produce a SARS-CoV-2 prefusion spike trimer in Nicotiana benthamiana as a model antigen from an emerging virus. The size exclusion-purified protein exhibited a characteristic prefusion structure when viewed by transmission electron microscopy, and this was indistinguishable from the equivalent mammalian cell-produced antigen. The plant-produced protein was decorated with under-processed oligomannose N-glycans and exhibited a site occupancy that was comparable to the equivalent protein produced in mammalian cell culture. Complex-type glycans were almost entirely absent from the plant-derived material, which contrasted against the predominantly mature, complex glycans that were observed on the mammalian cell culture-derived protein. The plant-derived antigen elicited neutralizing antibodies against both the matched Wuhan and heterologous Delta SARS-CoV-2 variants in immunized hamsters, although titres were lower than those induced by the comparato
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January 5, 2023 5:49 AM
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Multiplexed promoter and gene editing in wheat using a virus-based guide RNA delivery system

The low efficiency of genetic transformation and gene editing across diverse cultivars hinder the broad application of CRISPR technology for crop improvement. The development of virus-based methods of CRISPR-Cas system delivery into the plant cells holds great promise to overcome these limitations. Here, we perform direct inoculation of wheat leaves with the barley stripe mosaic virus (BSMV) transcripts to deliver guide RNAs (sgRNA) into the Cas9-expressing wheat. We demonstrate that wheat inoculation with the pool of BSMV-sgRNAs could be used to generate heritable precise deletions in the promoter region of a transcription factor and to perform multiplexed editing of agronomic genes. We transfer the high-expressing locus of Cas9 into adapted spring and winter cultivars by marker-assisted introgression and use of the BSMV-sgRNAs to edit two agronomic genes. A strategy presented in our study could be applied to any adapted cultivar for creating new cis-regulatory diversity or large-scale editing of multiple genes in biological pathways or QTL regions, opening possibilities for the effective engineering of crop genomes, and accelerating gene discovery and trait improvement efforts.
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Scooped by Ed Rybicki
January 5, 2023 5:45 AM
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Artificial nanovesicles for dsRNA delivery in spray induced gene silencing for crop protection

Spray-Induced Gene Silencing (SIGS) is an innovative and eco-friendly technology where topical application of pathogen gene-targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of dsRNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross-kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for dsRNA encapsulation and control against the fungal pathogen, Botrytis cinerea. AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP, and DODMA, and examined for their ability to protect and deliver dsRNA. All three formulations enabled dsRNA delivery and uptake by B. cinerea. Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi-mediated protection against B. cinerea both on pre- and post-harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome dsRNA instability in SIGS for crop protection.
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December 15, 2022 7:36 AM
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A tetrapeptide from maize combined with probiotics exerted strong anti-inflammatory effects and modulated gut microbiota in DSS-induced colitis mice - Food & Function (RSC Publishing)

A tetrapeptide from maize combined with probiotics exerted strong anti-inflammatory effects and modulated gut microbiota in DSS-induced colitis mice - Food & Function (RSC Publishing) | Plant Molecular Farming | Scoop.it
Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by recurrent gastrointestinal inflammation caused by abnormal immune response, and patients usually have intestinal flora imbalance.
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November 18, 2022 8:17 AM
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Plants overcame bubbles to get to drier land

Plants overcame bubbles to get to drier land | Plant Molecular Farming | Scoop.it
Researchers have solved a 100-year-old mystery: How did ancient plants emerge from swamps and riverbanks to new habitats with less water?
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July 28, 2022 7:08 AM
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Augmenting glycosylation-directed folding pathways enhances the fidelity of HIV Env immunogen production in plants

Augmenting glycosylation-directed folding pathways enhances the fidelity of HIV Env immunogen production in plants | Plant Molecular Farming | Scoop.it
"Heterologous glycoprotein production relies on host glycosylation-dependent folding. When the biosynthetic machinery differs from the usual expression host, there is scope to remodel the assembly pathway to enhance glycoprotein production. Here we explore the integration of chaperone coexpression with glyco-engineering to improve the production of a model HIV-1 envelope antigen. Calreticulin was coexpressed to support protein folding together with Leishmania major STT3D oligosaccharyltransferase, to improve glycan occupancy, RNA interference to suppress the formation of truncated glycans, and Nicotiana benthamiana plants lacking α1,3-fucosyltransferase and β1,2-xylosyltransferase was used as an expression host to prevent plant-specific complex N-glycans forming. This approach reduced the formation of undesired aggregates, which predominated in the absence of glyco-engineering. The resulting antigen also exhibited increased glycan occupancy, albeit to a slightly lower level than the equivalent mammalian cell-produced protein. The antigen was decorated almost exclusively with oligomannose glycans, which were less processed compared with the mammalian protein. Immunized rabbits developed comparable immune responses to the plant-produced and mammalian cell-derived antigens, including the induction of autologous neutralizing antibodies when the proteins were used to boost DNA and modified vaccinia Ankara virus-vectored vaccines. This study demonstrates that engineering glycosylation-directed folding offers a promising route to enhance the production of complex viral glycoproteins in plants."
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July 28, 2022 6:55 AM
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Plant crude extracts containing oligomeric hemagglutinins protect chickens against highly Pathogenic Avian Influenza Virus after one dose of immunization

Plant crude extracts containing oligomeric hemagglutinins protect chickens against highly Pathogenic Avian Influenza Virus after one dose of immunization | Plant Molecular Farming | Scoop.it
"Highly pathogenic avian influenza viruses (HPAIV) have been responsible for causing several severe outbreaks across the world. To protect poultry farms and to prevent the possible spread of new influenza pandemics, vaccines that are both efficacious and low-cost are in high demand. We produced stable, large hemagglutinin H5 oligomers in planta by the specific interaction between S•Tag and S•Protein. H5 oligomers combined via S•Tag::S•Protein interaction in plant crude extracts induced strong humoral immune responses, strong neutralizing antibody responses, and resistance in chickens after challenge with a wild type HPAIV H5 virus strain. In all three parameters, plant crude extracts with H5 oligomers induced better responses than crude extracts containing trimers. The neutralizing antibodies induced by by two-dose and one dose immunization with an adjuvanted crude extract containing H5 oligomer protected vaccinated chickens from two lethal H5N1 virus strains with the efficiency of 92% and 100%, respectively. Following housing vaccinated chickens together with ten non-immunized chickens, only one of these chickens had detectable levels of the H5N1 virus. To facilitate the easy storage of a candidate vaccine, the H5 oligomer crude extracts were mixed with adjuvants and stored for 3.5 and 5.5 months at 4 °C, and chickens were immunized with these crude extracts. All these vaccinated chickens survived after a lethal H5N1 virus challenge. H5 oligomer crude extracts are comparable to commercial vaccines as they also induce strong virus-neutralizing immune responses following the administration of a single dose. The cost-effective production of plant crude extract vaccine candidates and the high stability after long-term storage will enable and encourage the further exploration of this technology for veterinary vaccine development."
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June 24, 2022 6:03 AM
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Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana

Production of Recombinant Active Human TGFβ1 in Nicotiana benthamiana | Plant Molecular Farming | Scoop.it

"The production of recombinant proteins in plant systems is receiving wider attention. Indeed, various plant-produced pharmaceuticals have been shown to be biologically active. However, the production of human growth factors and cytokines in heterologous systems is still challenging because they often act as complex forms, such as homo- or hetero-dimers, and their production is tightly regulated in vivo. In this study, we demonstrated that the mature form of human TGFβ1 produced and purified from Nicotiana benthamiana shows biological activity in animal cells. To produce the mature form of TGFβ1, various recombinant genes containing the mature form of TGFβ1 were generated and produced in N. benthamiana. Of these, a recombinant construct, BiP:M:CBM3:LAP[C33S]:EK:TGFβ1, was expressed at a high level in N. benthamiana. Recombinant proteins were one-step purified using cellulose-binding module 3 (CBM3) as an affinity tag and microcrystalline cellulose (MCC) beads as a matrix. The TGFβ1 recombinant protein bound on MCC beads was proteolytically processed with enterokinase to separate mature TGFβ1. The mature TGFβ1 still associated with Latency Associated Protein, [LAP(C33S)] that had been immobilized on MCC beads was released by HCl treatment. Purified TGFβ1 activated TGFβ1-mediated signaling in the A549 cell line, thereby inducing phosphorylation of SMAD-2, the expression of ZEB-2 and SNAIL1, and the formation of a filopodia-like structure. Based on these results, we propose that active mature TGFβ1, one of the most challenging growth factors to produce in heterologous systems, can be produced from plants at a high degree of purity via a few steps."

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June 24, 2022 5:19 AM
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Directed Biosynthesis of New to Nature Alkaloids in a Heterologous Nicotiana benthamiana Expression Host

Directed Biosynthesis of New to Nature Alkaloids in a Heterologous Nicotiana benthamiana Expression Host | Plant Molecular Farming | Scoop.it
Plants produce a wide variety of pharmacologically active molecules classified as natural products. Derivatization of these natural products can modulate or improve the bioactivity of the parent compound. Unfortunately, chemical derivatization of natural products is often difficult or impractical. Here we use the newly discovered biosynthetic genes for two monoterpene indole alkaloids, alstonine and stemmadenine acetate, to generate analogs of these compounds. We reconstitute these biosynthetic genes in the heterologous host Nicotiana benthamiana along with an unnatural starting substrate to produce the corresponding new-to-nature alkaloid product.
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June 20, 2022 5:35 AM
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Enhanced HIV SOSIP Envelope yields in plants through transient co-expression of peptidyl-prolyl isomerase B and calreticulin chaperones and ER targeting 

Enhanced HIV SOSIP Envelope yields in plants through transient co-expression of peptidyl-prolyl isomerase B and calreticulin chaperones and ER targeting  | Plant Molecular Farming | Scoop.it
High yield production of recombinant HIV SOSIP envelope (Env) trimers has proven elusive as numerous disulfide bonds, proteolytic cleavage and extensive glycosylation pose high demands on the host cell machinery and stress imposed by accumulation of misfolded proteins may ultimately lead to cellular toxicity. The present study utilized the Nicotiana benthamiana/p19 (N.b./p19) transient plant system to assess co-expression of two ER master regulators and 5 chaperones, crucial in the folding process, to enhance yields of three Env SOSIPs, single chain BG505 SOSIP.664 gp140, CH505TF.6R.SOSIP.664.v4.1 and CH848-10.17-DT9. Phenotypic changes in leaves induced by SOSIP expression were employed to rapidly identify chaperone-assisted improvement in health and expression. Up to 15-fold increases were obtained by co-infiltration of peptidylprolvl isomerase (PPI) and calreticulin (CRT) which were further enhanced by addition of the ER-retrieval KDEL tags to the SOSIP genes; levels depending on individual SOSIP type, day of harvest and chaperone gene dosage. Results are consistent with reducing SOSIP misfolding and cellular stress due to increased exposure to the plant host cell’s calnexin/calreticulin network and accelerating the rate-limiting cis–trans isomerization of Xaa-Pro peptide bonds respectively. Plant transient co-expression facilitates rapid identification of host cell factors and will be translatable to other complex glycoproteins and mammalian expression systems.
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April 29, 2022 7:55 AM
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 The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants

 The B1 Domain of Streptococcal Protein G Serves as a Multi-Functional Tag for Recombinant Protein Production in Plants | Plant Molecular Farming | Scoop.it
Plants have long been considered a cost-effective platform for recombinant production. A recently recognized additional advantage includes the low risk of contamination of human pathogens, such as viruses and bacterial endotoxins. Indeed, a great advance has been made in developing plants as a “factory” to produce recombinant proteins to use for biopharmaceutical purposes. However, there is still a need to develop new tools for recombinant protein production in plants. In this study, we provide data showing that the B1 domain of Streptococcal protein G (GB1) can be a multi-functional domain of recombinant proteins in plants. N-terminal fusion of the GB1 domain increased the expression level of various target proteins ranging from 1.3- to 3.1-fold at the protein level depending on the target proteins. GB1 fusion led to the stabilization of the fusion proteins. Furthermore, the direct detection of GB1-fusion proteins by the secondary anti-IgG antibody eliminated the use of the primary antibody for western blot analysis. Based on these data, we propose that the small GB1 domain can be used as a versatile tag for recombinant protein production in plants.
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April 4, 2022 9:08 AM
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Lettuce Could Protect Astronauts’ Bones on Mars Trip

Lettuce Could Protect Astronauts’ Bones on Mars Trip | Plant Molecular Farming | Scoop.it
Astronauts might one day grow and eat genetically modified plants to ward off disease associated with long spaceflights. Researchers at the University of California, Davis, College of Engineering have developed a transgenic, or genetically modified, lettuce producing a drug to protect against bone density loss in microgravity. The work will be presented March 22 at the spring meeting of the American Chemical Society in San Diego.
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April 1, 2022 2:37 AM
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Agrobacterium-Mediated Genetic Transformation of Embryogenic Callus in a Liriodendron Hybrid (L. Chinense × L. Tulipifera)

Agrobacterium-Mediated Genetic Transformation of Embryogenic Callus in a Liriodendron Hybrid (L. Chinense × L. Tulipifera) | Plant Molecular Farming | Scoop.it
A highly efficient genetic transformation system of Liriodendron hybrid embryogenic calli through Agrobacterium-mediated genetic transformation was established and optimized. The Agrobacterium tumefaciens strain EHA105, harboring the plasmid pBI121, which contained the ß-glucuronidase (GUS) gene and neomycin phosphotransferase II (npt II) gene under the control of the CaMV35S promoter, was used for transformation. Embryogenic calli were used as the starting explant to study several factors affecting the Agrobacterium-mediated genetic transformation of the Liriodendron hybrid, including the effects of various media, selection by different Geneticin (G418) concentrations, pre-culture period, Agrobacterium optical density, infection duration, co-cultivation period, and delayed selection. Transformed embryogenic calli were obtained through selection on medium containing 90 mg L−1 G418. Plant regeneration was achieved and selected via somatic embryogenesis on medium containing 15 mg L−1 G418. The optimal conditions included a pre-culture time of 2 days, a co-culture time of 3 days, an optimal infection time of 10 min, and a delayed selection time of 7 days. These conditions, combined with an OD600 value of 0.6, remarkably enhanced the transformation rate. The results of GUS chemical tissue staining, polymerase chain reaction (PCR), and southern blot analysis demonstrated that the GUS gene was successfully expressed and integrated into the Liriodendron hybrid genome.
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August 3, 2023 6:21 AM
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Chimaeric plant-produced bluetongue virus particles as potential vaccine candidates

Chimaeric plant-produced bluetongue virus particles as potential vaccine candidates | Plant Molecular Farming | Scoop.it
Bluetongue virus (BTV) causes bluetongue disease in ruminants and sheep. The current live attenuated and inactivated vaccines available for prevention pose several risks, and there is thus a need for vaccines that are safer, economically viable, and effective against multiple circulating serotypes. This work describes the development of recombinant virus-like particle (VLP) vaccine candidates in plants, which are assembled by co-expression of the four BTV serotype 8 major structural proteins. We show that substitution of a neutralising tip domain of BTV8 VP2 with that of BTV1 VP2 resulted in the assembly of VLPs that stimulated serotype-specific antibodies as well as virus-specific neutralising antibodies.
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January 5, 2023 5:48 AM
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SARS-CoV-2 spike trimer vaccine expressed in Nicotiana benthamiana adjuvanted with Alum elicits protective immune responses in mice

The ongoing coronavirus disease 2019 (COVID-19) pandemic has spurred rapid development of vaccines as part of the public health response. However, the general strategy used to construct recombinant trimeric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) proteins in mammalian cells is not completely adaptive to molecular farming. Therefore, we generated several constructs of recombinant S proteins for high expression in Nicotiana benthamiana. Intramuscular injection of N. benthamiana-expressed Sct vaccine (NSctVac) into Balb/c mice elicited both humoral and cellular immune responses, and booster doses increased neutralizing antibody titres. In human angiotensin-converting enzyme knock-in mice, two doses of NSctVac induced anti-S and neutralizing antibodies, which cross-neutralized Alpha, Beta, Delta and Omicron variants. Survival rates after lethal challenge with SARS-CoV-2 were up to 80%, without significant body weight loss, and viral titres in lung tissue fell rapidly, with no infectious virus detectable at 7-day post-infection. Thus, plant-derived NSctVac could be a candidate COVID-19 vaccine.
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January 5, 2023 5:41 AM
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Fabry Disease Market Size, Industry Key Players, & Scenario

Fabry Disease Market Size, Industry Key Players, & Scenario | Plant Molecular Farming | Scoop.it
The Fabry Disease Market growth at a CAGR of 7.30% & expected USD 1850.60 million by 2029. It is fragmented as type, diagnosis, treatment, route of administration, distribution channel and end-user.
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December 15, 2022 7:35 AM
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Experts in insect taxonomy “threatened by extinction” reveals the first European Red List of Taxonomists - News

Experts in insect taxonomy “threatened by extinction” reveals the first European Red List of Taxonomists - News | Plant Molecular Farming | Scoop.it
Expertise tends to be particularly poor in the countries with the richest biodiversity, while taxonomists are predominantly male and ageing.
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November 3, 2022 7:17 AM
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Transcriptional deregulation of stress-growth balance in Nicotiana benthamiana biofactories producing insect sex pheromones

Transcriptional deregulation of stress-growth balance in Nicotiana benthamiana biofactories producing insect sex pheromones | Plant Molecular Farming | Scoop.it

"Plant biofactories are a promising platform for sustainable production of high-value compounds, among which are insect sex pheromones, a green alternative to conventional insecticides in agriculture. Recently, we have constructed transgenic Nicotiana benthamiana plants (“Sexy Plants”, SxP) that successfully produce a blend of moth (Lepidoptera) sex pheromone compounds (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate. However, efficient biosynthesis of sex pheromones resulted in growth and developmental penalty, diminishing the potential for commercial use of SxP in biomanufacturing. To gain insight into the underlying molecular responses, we analysed the whole-genome transcriptome and evaluated it in relation to growth and pheromone production in low- and high-producing transgenic plants of v1.0 and v1.2 SxP lines. In our study, high-producing SxPv1.2 plants accumulated the highest amounts of pheromones but still maintained better growth compared to v1.0 high producers. For an in-depth biological interpretation of the transcriptomic data, we have prepared a comprehensive functional N. benthamiana genome annotation as well as gene translations to Arabidopsis thaliana, enabling functional information transfer by using Arabidopsis knowledge networks. Differential gene expression analysis, contrasting pheromone producers to wild-type plants, revealed that while only a few genes were differentially regulated in low-producing plants, high-producing plants exhibited vast transcriptional reprogramming. They showed signs of stress-like response, manifested as downregulation of photosynthesis-related genes and significant differences in expression of hormonal signalling and secondary metabolism-related genes, the latter presumably leading to previously reported volatilome changes. Further network analyses confirmed stress-like response with activation of jasmonic acid and downregulation of gibberellic acid signalling, illuminating the possibility that the observed growth penalty was not solely a consequence of a higher metabolic burden imposed upon constitutive expression of a heterologous biosynthetic pathway, but rather the result of signalling pathway perturbation. Our work presents an example of comprehensive transcriptomic analyses of disadvantageous stress signalling in N. benthamiana biofactory that could be applied to other bioproduction systems."

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July 28, 2022 6:58 AM
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The immunogenicity of plant-based COE-GCN4pII protein in pigs against the highly virulent porcine epidemic diarrhea virus strain from genotype 2

The immunogenicity of plant-based COE-GCN4pII protein in pigs against the highly virulent porcine epidemic diarrhea virus strain from genotype 2 | Plant Molecular Farming | Scoop.it
Porcine epidemic diarrhea virus (PEDV) is a serious infectious causative agent in swine, especially in neonatal piglets. PEDV genotype 2 (G2) strains, particularly G2a, were the primary causes of porcine epidemic diarrhea (PED) outbreaks in Vietnam. Here, we produced a plant-based CO-26K-equivalent epitope (COE) variant from a Vietnamese highly virulent PEDV strain belonging to genotype 2a (COE/G2a) and evaluated the protective efficacy of COE/G2a-GCN4pII protein (COE/G2a-pII) in piglets against the highly virulent PEDV G2a strain following passive immunity. The 5-day-old piglets had high levels of PEDV-specific IgG antibodies, COE-IgA specific antibodies, neutralizing antibodies, and IFN-γ responses. After virulent challenge experiments, all of these piglets survived and had normal clinical symptoms, no watery diarrhea in feces, and an increase in their body weight, while all of the negative control piglets died. These results suggest that the COE/G2a-pII protein produced in plants can be developed as a promising vaccine candidate to protect piglets against PEDV G2a infection in Vietnam.
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July 18, 2022 3:18 AM
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Plant-Derived Human Vaccines: Recent Developments

Plant-Derived Human Vaccines: Recent Developments | Plant Molecular Farming | Scoop.it
"The idea of producing vaccines in plants originated in the late 1980s. Initially, it was contemplated that this notion could facilitate the concept of edible vaccines, making them more cost effective and easily accessible. Initial studies on edible vaccines focussed on the use of a variety of different transgenic plant host species for the production of vaccine antigens. However, adequate expression levels of antigens, the difficulties predicted with administration of consistent doses, and regulatory rules required for growth of transgenic plants gave way to the development of vaccine candidates that could be purified and administered parenterally. The field has subsequently advanced with improved expression techniques including a shift from using transgenic to transient expression of antigens, refinement of purification protocols, a deeper understanding of the biological processes and a wealth of evidence of immunogenicity and efficacy of plant-produced vaccine candidates, all contributing to the successful practice of what is now known as biopharming or plant molecular farming. The establishment of this technology has resulted in the development of many different types of vaccine candidates including subunit vaccines and various different types of nanoparticle vaccines targeting a wide variety of bacterial and viral diseases. This has brought further acceptance of plants as a suitable platform for vaccine production and in this review, we discuss the most recent advances in the production of vaccines in plants for human use."
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June 24, 2022 6:01 AM
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Covering the Combinatorial Design Space of Multiplex CRISPR/Cas Experiments in Plants

Covering the Combinatorial Design Space of Multiplex CRISPR/Cas Experiments in Plants | Plant Molecular Farming | Scoop.it

"Over the past years, CRISPR/Cas-mediated genome editing has revolutionized plant genetic studies and crop breeding. Specifically, due to its ability to simultaneously target multiple genes, the multiplex CRISPR/Cas system has emerged as a powerful technology for functional analysis of genetic pathways. As such, it holds great potential for application in plant systems to discover genetic interactions and to improve polygenic agronomic traits in crop breeding. However, optimal experimental design regarding coverage of the combinatorial design space in multiplex CRISPR/Cas screens remains largely unexplored. To contribute to well-informed experimental design of such screens in plants, we first establish a representation of the design space at different stages of a multiplex CRISPR/Cas experiment. We provide two independent computational approaches yielding insights into the plant library size guaranteeing full coverage of all relevant multiplex combinations of gene knockouts in a specific multiplex CRISPR/Cas screen. These frameworks take into account several design parameters (e.g., the number of target genes, the number of gRNAs designed per gene, and the number of elements in the combinatorial array) and efficiencies at subsequent stages of a multiplex CRISPR/Cas experiment (e.g., the distribution of gRNA/Cas delivery, gRNA-specific mutation efficiency, and knockout efficiency). With this work, we intend to raise awareness about the limitations regarding the number of target genes and order of genetic interaction that can be realistically analyzed in multiplex CRISPR/Cas experiments with a given number of plants. Finally, we establish guidelines for designing multiplex CRISPR/Cas experiments with an optimal coverage of the combinatorial design space at minimal plant library size."

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June 24, 2022 5:15 AM
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Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity

Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity | Plant Molecular Farming | Scoop.it
The efficiency of the CRISPR/Cas9 genome editing system remains limited in many crops. Utilizing strong promoters to boost the expression level of Cas9 are commonly used to improve the editing efficiency. However, these strategies also increase the risk of off-target mutation. Here, we developed a new strategy to utilize intron-mediated enhancement (IME)-assisted 35S promoter to drive Cas9 and sgRNA in a single transcript, which escalates the editing efficiency by moderately enhancing the expression of both Cas9 and sgRNA. In addition, we developed another strategy to enrich cells highly expressing Cas9/sgRNA by co-expressing the developmental regulator gene GRF5, which has been proved to ameliorate the transformation efficiency, and the transgenic plants from these cells also exhibited enhanced editing efficiency. This system elevated the genome editing efficiency from 14–28% to 54–81% on three targets tested in lettuce (Lactuca sativa) without increasing the off-target editing efficiency. Thus, we established a new genome editing system with highly improved on-target editing efficiency and without obvious increasement in off-target effects, which can be used to characterize genes of interest in lettuce and other crops.
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May 23, 2022 11:22 AM
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Successful Production and Ligninolytic Activity of a Bacterial Laccase, Lac51, Made in Nicotiana benthamiana via Transient Expression

Successful Production and Ligninolytic Activity of a Bacterial Laccase, Lac51, Made in Nicotiana benthamiana via Transient Expression | Plant Molecular Farming | Scoop.it
Giant panda could have bamboo as their exclusive diet for about 2 million years because of the contribution of numerous enzymes produced by their gut bacteria, for instance laccases. Laccases are blue multi-copper oxidases that catalyze the oxidation of a broad spectrum of phenolic and aromatic compounds with water as the only byproduct. As a “green enzyme,” laccases have potential in industrial applications, for example, when dealing with degradation of recalcitrant biopolymers, such as lignin. In the current study, a bacterial laccase, Lac51, originating from Pseudomonas putida and identified in the gut microbiome of the giant panda’s gut was transiently expressed in the non-food plant Nicotiana benthamiana and characterized. Our results show that recombinant Lac51 exhibits bacterial laccase properties, with optimal pH and temperature at 7–8 and 40°C, respectively, when using syringaldazine as substrate. Moreover, we demonstrate the functional capability of the plant expressed Lac51 to oxidize lignin using selected lignin monomers that serve as substrates of Lac51. In summary, our study demonstrates the potential of green and non-food plants as a viable enzyme production platform for bacterial laccases. This result enriches our understanding of plant-made enzymes, as, to our knowledge, Lac51 is the first functional recombinant laccase produced in plants.
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April 29, 2022 7:36 AM
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Glyco-Engineering Plants to Produce Helminth Glycoproteins as Prospective Biopharmaceuticals: Recent Advances, Challenges and Future Prospects

Glyco-Engineering Plants to Produce Helminth Glycoproteins as Prospective Biopharmaceuticals: Recent Advances, Challenges and Future Prospects | Plant Molecular Farming | Scoop.it

Glycoproteins are the dominant category among approved biopharmaceuticals, indicating their importance as therapeutic proteins. Glycoproteins are decorated with carbohydrate structures (or glycans) in a process called glycosylation. Glycosylation is a post-translational modification that is present in all kingdoms of life, albeit with differences in core modifications, terminal glycan structures, and incorporation of different sugar residues. Glycans play pivotal roles in many biological processes and can impact the efficacy of therapeutic glycoproteins. The majority of biopharmaceuticals are based on human glycoproteins, but non-human glycoproteins, originating from for instance parasitic worms (helminths), form an untapped pool of potential therapeutics for immune-related diseases and vaccine candidates. The production of sufficient quantities of correctly glycosylated putative therapeutic helminth proteins is often challenging and requires extensive engineering of the glycosylation pathway. Therefore, a flexible glycoprotein production system is required that allows straightforward introduction of heterologous glycosylation machinery composed of glycosyltransferases and glycosidases to obtain desired glycan structures. The glycome of plants creates an ideal starting point for N- and O-glyco-engineering of helminth glycans. Plants are also tolerant toward the introduction of heterologous glycosylation enzymes as well as the obtained glycans. Thus, a potent production platform emerges that enables the production of recombinant helminth proteins with unusual glycans. In this review, we discuss recent advances in plant glyco-engineering of potentially therapeutic helminth glycoproteins, challenges and their future prospects."

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April 1, 2022 2:40 AM
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Modified Gene Editing Systems: Diverse Bioengineering Tools and Crop Improvement

Modified Gene Editing Systems: Diverse Bioengineering Tools and Crop Improvement | Plant Molecular Farming | Scoop.it
Gene-editing systems have emerged as bioengineering tools in recent years. Classical gene-editing systems include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9), and these tools allow specific sequences to be targeted and edited. Various modified gene-editing systems have been established based on classical gene-editing systems. Base editors (BEs) can accurately carry out base substitution on target sequences, while prime editors (PEs) can replace or insert sequences. CRISPR systems targeting mitochondrial genomes and RNA have also been explored and established. Multiple gene-editing techniques based on CRISPR/Cas9 have been established and applied to genome engineering. Modified gene-editing systems also make transgene-free plants more readily available. In this review, we discuss the modifications made to gene-editing systems in recent years and summarize the capabilities, deficiencies, and applications of these modified gene-editing systems. Finally, we discuss the future developmental direction and challenges of modified gene-editing systems.
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