ABSTRACT
In planta expression of recombinant antibodies has been proposed as a strategy for herbicide resistance but is not well advanced yet. Here, an atrazine nanobody gene fused with a green fluorescent protein tag was transformed to Arabidopsis thaliana, which was confirmed with PCR, ELISA, and immunoblotting. High levels of nanobody accumulation were observed in the nucleus, cytoderm, and cytosol. The nanobody expressed in the plant had similar affinity, sensitivity, and selectivity as that expressed in Escherichia coli. The T3 homozygous line showed resistance in a dose-dependent manner up to 380 g ai/ha of atrazine, which is approximately one-third of the recommended field application rate. This is the first report of utilizing a nanobody in plants against herbicides. The results suggest that utilizing a high-affinity herbicide nanobody gene rather than increasing the expression of nanobodies in plants may be a technically viable approach to acquire commercial herbicide-resistant crops and could be a useful tool to study plant physiology.
Subject(s)
Arabidopsis , Atrazine , Herbicide Resistance , Herbicides , Plants, Genetically Modified , Single-Domain Antibodies , Atrazine/pharmacology , Herbicides/pharmacology , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis/metabolism , Arabidopsis/drug effects , Herbicide Resistance/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Single-Domain Antibodies/genetics , Single-Domain Antibodies/pharmacology , Single-Domain Antibodies/immunologyABSTRACT
RNA silencing is an innate immune mechanism of plants against invasion by viral pathogens. Artificial microRNA (amiRNA) can be engineered to specifically induce RNA silencing against viruses in transgenic plants and has great potential for disease control. Here, we describe the development and application of amiRNA-based technology to induce resistance to soybean mosaic virus (SMV), a plant virus with a positive-sense single-stranded RNA genome. We have shown that the amiRNA targeting the SMV P1 coding region has the highest antiviral activity than those targeting other SMV genes in a transient amiRNA expression assay. We transformed the gene encoding the P1-targeting amiRNA and obtained stable transgenic Nicotiana benthamiana lines (amiR-P1-3-1-2-1 and amiR-P1-4-1-2-1). Our results have demonstrated the efficient suppression of SMV infection in the P1-targeting amiRNA transgenic plants in an expression level-dependent manner. In particular, the amiR-P1-3-1-2-1 transgenic plant showed high expression of amiR-P1 and low SMV accumulation after being challenged with SMV. Thus, a transgenic approach utilizing the amiRNA technology appears to be effective in generating resistance to SMV.
Subject(s)
Disease Resistance , MicroRNAs , Nicotiana , Plant Diseases , Plants, Genetically Modified , Potyvirus , MicroRNAs/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/virology , Plants, Genetically Modified/immunology , Nicotiana/genetics , Nicotiana/virology , Nicotiana/immunology , Plant Diseases/virology , Plant Diseases/genetics , Plant Diseases/immunology , Disease Resistance/genetics , Potyvirus/pathogenicity , Potyvirus/genetics , RNA Interference , Glycine max/genetics , Glycine max/virology , Glycine max/immunologyABSTRACT
Phytophthora blight of pepper, which is caused by the notorious oomycete pathogen Phytophthora capsici, is a serious disease in global pepper production regions. Our previous study had identified two WRKY transcription factors (TFs), CaWRKY01-10 and CaWRKY08-4, which are prominent modulators in the resistant pepper line CM334 against P. capsici infection. However, their functional mechanisms and underlying signaling networks remain unknown. Herein, we determined that CaWRKY01-10 and CaWRKY08-4 are localized in plant nuclei. Transient overexpression assays indicated that both CaWRKY01-10 and CaWRKY08-4 act as positive regulators in pepper resistance to P. capsici. Besides, the stable overexpression of CaWRKY01-10 and CaWRKY08-4 in transgenic Nicotiana benthamiana plants also significantly enhanced the resistance to P. capsici. Using comprehensive approaches including RNA-seq, CUT&RUN-qPCR, and dual-luciferase reporter assays, we revealed that overexpression of CaWRKY01-10 and CaWRKY08-4 can activate the expressions of the same four Capsicum annuum defense-related genes (one PR1, two PR4, and one pathogen-related gene) by directly binding to their promoters. However, we did not observe protein-protein interactions and transcriptional amplification/inhibition effects of their shared target genes when coexpressing these two WRKY TFs. In conclusion, these data suggest that both of the resistant line specific upregulated WRKY TFs (CaWRKY01-10 and CaWRKY08-4) can confer pepper's resistance to P. capsici infection by directly activating a cluster of defense-related genes and are potentially useful for genetic improvement against Phytophthora blight of pepper and other crops.
Subject(s)
Capsicum , Disease Resistance , Gene Expression Regulation, Plant , Phytophthora , Plant Diseases , Plant Proteins , Transcription Factors , Phytophthora/physiology , Plant Diseases/microbiology , Plant Diseases/genetics , Plant Diseases/immunology , Capsicum/genetics , Capsicum/microbiology , Capsicum/immunology , Disease Resistance/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/immunology , Transcription Factors/genetics , Transcription Factors/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/microbiology , Plants, Genetically Modified/immunologyABSTRACT
Calcium (Ca2+) is a second messenger in various physiological processes within plants. The significance of the Ca2+/H+ exchanger (CAX) has been established in facilitating Ca2+ transport in plants; however, disease resistance functions of the CAX gene remain elusive. In this study, we conducted sequence characterization and expression analysis for a sugarcane CAX gene, ScCAX4 (GenBank Accession Number: MW206380). In order to further investigate the disease resistance functions, this gene was then transiently overexpressed in Nicotiana benthamiana leaves, which were subsequently inoculated with Fusarium solani var. coeruleum. Results showed that ScCAX4 overexpression increased the susceptibility of N. benthamiana to pathogen infection by regulating the expression of genes related to salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways, suggesting its negative role in disease resistance. Furthermore, we genetically transformed the ScCAX4 gene into N. benthamiana and obtained three positive T2 generation lines. Interestingly, the symptomatology of transgenic plants was consistent with that of transient overexpression after pathogen inoculation. Notably, the JA content in transgenic overexpression lines was significantly higher than that in the wild-type. RNA-seq revealed that ScCAX4 could mediate multiple signaling pathways, and the JA signaling pathway played a key role in modulating disease resistance. Finally, a regulatory model was depicted for the increased susceptibility to pathogen infection conferred by the ScCAX4 gene. This study provides genetic resources for sugarcane molecular breeding and the research direction for plant CAX genes.
Subject(s)
Cyclopentanes , Disease Resistance , Fusarium , Gene Expression Regulation, Plant , Oxylipins , Plant Diseases , Plant Proteins , Saccharum , Salicylic Acid , Plant Diseases/microbiology , Plant Diseases/genetics , Disease Resistance/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/immunology , Saccharum/genetics , Saccharum/microbiology , Saccharum/metabolism , Saccharum/immunology , Fusarium/physiology , Oxylipins/metabolism , Salicylic Acid/metabolism , Cyclopentanes/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/microbiology , Plants, Genetically Modified/immunology , Plants, Genetically Modified/metabolism , Nicotiana/genetics , Nicotiana/microbiology , Nicotiana/metabolism , Nicotiana/immunology , Ethylenes/metabolismABSTRACT
The Thinopyrum elongatum Fhb7E locus has been proven to confer outstanding resistance to Fusarium Head Blight (FHB) when transferred into wheat, minimizing yield loss and mycotoxin accumulation in grains. Despite their biological relevance and breeding implications, the molecular mechanisms underlying the resistant phenotype associated with Fhb7E have not been fully uncovered. To gain a broader understanding of processes involved in this complex plant-pathogen interaction, we analysed via untargeted metabolomics durum wheat (DW) rachises and grains upon spike inoculation with Fusarium graminearum (Fg) and water. The employment of DW near-isogenic recombinant lines carrying or lacking the Th. elongatum chromosome 7E region including Fhb7E on their 7AL arm, allowed clear-cut distinction between differentially accumulated disease-related metabolites. Besides confirming the rachis as key site of the main metabolic shift in plant response to FHB, and the upregulation of defence pathways (aromatic amino acid, phenylpropanoid, terpenoid) leading to antioxidants and lignin accumulation, novel insights were revealed. Fhb7E conferred constitutive and early-induced defence response, in which specific importance of polyamine biosynthesis, glutathione and vitamin B6 metabolisms, along with presence of multiple routes for deoxynivalenol detoxification, was highlighted. The results suggested Fhb7E to correspond to a compound locus, triggering a multi-faceted plant response to Fg, effectively limiting Fg growth and mycotoxin production.
Subject(s)
Disease Resistance , Fusarium , Plant Diseases , Plants, Genetically Modified , Poaceae , Triticum , Poaceae/genetics , Metabolomics , Genetic Loci , Fusarium/growth & development , Triticum/genetics , Triticum/immunology , Triticum/microbiology , Disease Resistance/genetics , Plant Diseases/genetics , Plant Diseases/microbiology , Chromosomes, Plant , Polyamines/metabolism , Genetic Engineering , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Plants, Genetically Modified/microbiologyABSTRACT
Digestive stability of a food protein in simulated gastric fluid (SGF) continues to be considered a risk factor for allergy, even though the current science does not support this belief. Methodological shortcomings of the adaption of the SGF assay for use with purified proteins has been cited as a reason to discount results that do not conform to this belief. Missteps in conducting and interpreting the results of SGF assays are reviewed here. However, these methodological shortcomings do not invalidate the conclusion that allergenic proteins are not systematically more stable to digestion than non-allergens. The growing evidence for the dual allergen exposure hypothesis, whereby sensitization to food allergens is primarily caused by dermal and inhalation exposure to food dust, and tolerization against food allergy is primarily induced by gut exposure in food, likely explains why the digestive stability of a protein is not a risk factor for allergenicity.
Subject(s)
Allergens , Crops, Agricultural , Dietary Proteins , Digestion , Enzyme Assays , Food Hypersensitivity , Gastric Juice , Plants, Genetically Modified , Humans , Allergens/chemistry , Crops, Agricultural/adverse effects , Food Hypersensitivity/etiology , Plants, Genetically Modified/immunology , Gastric Juice/enzymology , Dietary Proteins/chemistry , Protein StabilityABSTRACT
The ongoing SARS-CoV-2 coronavirus pandemic of 2020-2021 underscores the need for manufacturing platforms that can rapidly produce monoclonal antibody (mAb) therapies. As reported here, a platform based on Nicotiana benthamiana produced mAb therapeutics with high batch-to-batch reproducibility and flexibility, enabling production of 19 different mAbs of sufficient purity and safety for clinical application(s). With a single manufacturing run, impurities were effectively removed for a representative mAb product (the ZMapp component c4G7). Our results show for the first time the reproducibility of the platform for production of multiple batches of clinical-grade mAb, manufactured under current Good Manufacturing Practices, from Nicotiana benthamiana. The flexibility of the system was confirmed by the results of release testing of 19 different mAbs generated with the platform. The process from plant infection to product can be completed within 10 days. Therefore, with a constant supply of plants, response to the outbreak of an infectious disease could be initiated within a matter of weeks. Thus, these data demonstrated that this platform represents a reproducible, flexible system for rapid production of mAb therapeutics to support clinical development.
Subject(s)
Antibodies, Monoclonal , Antibodies, Viral , COVID-19/immunology , Nicotiana , Plants, Genetically Modified , SARS-CoV-2/immunology , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Viral/biosynthesis , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Humans , Plants, Genetically Modified/chemistry , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/immunology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Nicotiana/chemistry , Nicotiana/genetics , Nicotiana/growth & development , Nicotiana/immunology , COVID-19 Drug TreatmentABSTRACT
Viral pathogens are a major threat to stable crop production. Using a backcross strategy, we find that integrating a dominant brown planthopper (BPH) resistance gene Bph3 into a high-yield and BPH-susceptible indica rice variety significantly enhances BPH resistance. However, when Bph3-carrying backcross lines are infested with BPH, these BPH-resistant lines exhibit sterile characteristics, displaying panicle enclosure and failure of seed production at their mature stage. As we suspected, BPH-mediated viral infections could cause the observed sterile symptoms, and we characterized rice-infecting viruses using deep metatranscriptomic sequencing. Our analyses revealed eight novel virus species and five known viruses, including a highly divergent virus clustered within a currently unclassified family. Additionally, we characterized rice plant antiviral responses using small RNA sequencing. The results revealed abundant virus-derived small interfering RNAs in sterile rice plants, providing evidence for Dicer-like and Argonaute-mediated immune responses in rice plants. Together, our results provide insights into the diversity of viruses in rice plants, and our findings suggest that multiple virus infections occur in rice plants.
Subject(s)
Hemiptera/virology , Oryza/virology , Plant Diseases/virology , RNA Viruses/genetics , RNA Viruses/isolation & purification , Animals , Disease Resistance , Hemiptera/physiology , Oryza/genetics , Oryza/immunology , Oryza/parasitology , Plant Diseases/parasitology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Plants, Genetically Modified/parasitology , Plants, Genetically Modified/virology , RNA Viruses/classification , Sequence Analysis, RNA , TranscriptomeABSTRACT
We have developed a new binary epitope-presenting CVP platform based on bamboo mosaic virus (BaMV) by using the sortase A (SrtA)-mediated ligation technology. The reconstructed BaMV genome harbors two modifications: 1) a coat protein (CP) with N-terminal extension of the tobacco etch virus (TEV) protease recognition site plus 4 extra glycine (G) residues as the SrtA acceptor; and 2) a TEV protease coding region replacing that of the triple-gene-block proteins. Inoculation of such construct, pKB5G, on Nicotiana benthamiana resulted in the efficient production of filamentous CVPs ready for SrtA-mediated ligation with desired proteins. The second part of the binary platform includes an expression vector for the bacterial production of donor proteins. We demonstrated the applicability of the platform by using the recombinant envelope protein domain III (rEDIII) of Japanese encephalitis virus (JEV) as the antigen. Up to 40% of the BaMV CP subunits in each CVP were loaded with rEDIII proteins in 1 min. The rEDIII-presenting BaMV CVPs (BJLPET5G) could be purified using affinity chromatography. Immunization assays confirmed that BJLPET5G could induce the production of neutralizing antibodies against JEV infections. The binary platform could be adapted as a useful alternative for the development and mass production of vaccine candidates.
Subject(s)
Aminoacyltransferases/metabolism , Antigens, Viral/administration & dosage , Bacterial Proteins/metabolism , Cysteine Endopeptidases/metabolism , Encephalitis Virus, Japanese/immunology , Encephalitis, Japanese/prevention & control , Endopeptidases/metabolism , Japanese Encephalitis Vaccines/administration & dosage , Potexvirus/enzymology , Virion/enzymology , Aminoacyltransferases/genetics , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antigens, Viral/genetics , Antigens, Viral/immunology , Bacterial Proteins/genetics , Cell Line , Cysteine Endopeptidases/genetics , Disease Models, Animal , Encephalitis Virus, Japanese/genetics , Encephalitis, Japanese/blood , Encephalitis, Japanese/immunology , Encephalitis, Japanese/virology , Endopeptidases/genetics , Escherichia coli/genetics , Escherichia coli/immunology , Escherichia coli/metabolism , Female , Genetic Vectors , Immunogenicity, Vaccine , Japanese Encephalitis Vaccines/genetics , Japanese Encephalitis Vaccines/immunology , Mice, Inbred BALB C , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Plants, Genetically Modified/metabolism , Potexvirus/genetics , Potexvirus/immunology , Nicotiana/genetics , Nicotiana/immunology , Nicotiana/metabolism , Virion/genetics , Virion/immunologyABSTRACT
Efficient accumulation of flavonoids is important for increased tolerance to biotic stress. Although several plant defense mechanisms are known, the roles of many pathways, proteins, and secondary metabolites in stress tolerance are unknown. We generated a flavanone 3-hydroxylase (F3H) overexpressor rice line and inoculated Xanthomonas Oryzae pv. oryzae and compared the control and wildtype inoculated plants. In addition to promoting plant growth and developmental maintenance, the overexpression of F3H increased the accumulation of flavonoids and increased tolerance to bacterial leaf blight (BLB) stress. Moreover, leaf lesion length was higher in the infected wildtype plants compared with infected transgenics. Kaempferol and quercetin, which scavenge reactive oxygen species, overaccumulated in transgenic lines compared with wildtypes in response to pathogenic infection, detected by scanning electron microscopy and spectrophotometry. The induction of F3H altered the antioxidant system and reduced the levels of glutathione peroxidase activity and malondialdehyde (MDA) contents in the transgenic lines compared with the wildtypes. Downstream gene regulation analysis showed that the expression of F3H increased the regulation of flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), and slender rice mutant (SLR1) during BLB stress. The analysis of SA and JA signaling revealed an antagonistic interaction between both hormones and that F3H induction significantly promoted SA and inhibited JA accumulation in the transgenic lines. SA-dependent nonexpressor pathogenesis-related (NPR1) and Xa1 showed significant upregulation in the infected transgenic lines compared with the infected control and wildtype lines. Thus, the overexpression of F3H was essential for increasing BLB stress tolerance.
Subject(s)
Antioxidants/metabolism , Disease Resistance/immunology , Flavonoids/metabolism , Hormones/metabolism , Mixed Function Oxygenases/metabolism , Oryza/immunology , Plant Diseases/immunology , Disease Resistance/genetics , Gene Expression Regulation, Plant , Mixed Function Oxygenases/genetics , Oryza/genetics , Oryza/metabolism , Oryza/microbiology , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Leaves/genetics , Plant Leaves/immunology , Plant Leaves/metabolism , Plant Leaves/microbiology , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/microbiology , Stress, Physiological , Xanthomonas/physiologyABSTRACT
Background: We previously developed a transgenic rice that contains seven linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens Cry j 1 and Cry j 2. Oral administration of 80 g of transgenic rice for 20 weeks suppressed allergen-specific T-cell proliferation in participants with JC pollinosis, but their clinical symptoms did not improve. Objective: We examined the clinical efficacy of low-dose (5 g and 20 g) intake of the transgenic rice administered for two successive seasons. Methods: In this randomized, double-blind, placebo controlled study, transgenic rice seeds (5 g or 20 g) were orally administered to the participants for 24 weeks in each of two successive JC pollen seasons. We analyzed T-cell proliferation and cytokine expression, and monitored symptom and medication scores during the pollen season. Quality of life (QOL) was evaluated by using the Japanese Allergic Rhinitis Quality of Life Standard Questionnaire (JRQLQ). Results: Specific T-cell proliferation after stimulation with 7Crp, Cry j 1, and Cry j 2 was significantly suppressed in the second JC pollen season. No significant differences were found among the three groups (5 g, 20 g, and placebo) with regard to clinical symptoms or medication scores in the first season. However, the medication scores and face scale for overall condition of JRQLQ improved in the 5-g transgenic rice group in the second season, although careful re-examination with a large sample size is necessary to confirm the results. Conclusion: Low-dose oral administration of transgenic rice that contains 7Crp significantly reduced allergen-specific T-cell responses and improved medication scores during the second season of administration. Thus, oral intake of the transgenic rice has the potential to induce immune tolerance to JC pollen allergens when administered for at least two successive seasons.
Subject(s)
Cryptomeria , Hypersensitivity , Oryza , Administration, Oral , Allergens , Antigens, Plant , Cryptomeria/immunology , Epitopes, T-Lymphocyte/genetics , Humans , Oryza/genetics , Oryza/immunology , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Pollen/immunology , Quality of LifeABSTRACT
An investigation of the potential allergenicity of newly expressed proteins in genetically modified (GM) crops comprises part of the assessment of GM crop safety. However, allergenicity is not completely predictable from a definitive assay result or set of protein characteristics, and scientific opinions regarding the data that should be used to assess allergenicity are continuously evolving. Early studies supported a correlation between the stability of a protein exposed to digestive enzymes such as pepsin and the protein's status as a potential allergen, but over time the conclusions of these earlier studies were not confirmed. Nonetheless, many regulatory authorities, including the European Food Safety Authority (EFSA), continue to require digestibility analyses as a component of GM crop risk assessments. Moreover, EFSA has recently investigated the use of mass spectrometry (MS), to make digestion assays more predictive of allergy risk, because it can detect and identify small undigested peptides. However, the utility of MS is questionable in this context, since known allergenic peptides are unlikely to exist in protein candidates intended for commercial development. These protein candidates are pre-screened by the same bioinformatics processes that are normally used to identify MS targets. Therefore, MS is not a standalone allergen identification method and also cannot be used to predict previously unknown allergenic epitopes. Thus, the suggested application of MS for analysis of digesta does not improve the poor predictive power of digestion assays in identifying allergenic risk.
Subject(s)
Allergens/isolation & purification , Crops, Agricultural/immunology , Mass Spectrometry , Plants, Genetically Modified/immunology , Allergens/adverse effects , Allergens/immunology , Crops, Agricultural/adverse effects , Crops, Agricultural/chemistry , Food Safety , Food, Genetically Modified/adverse effects , Humans , Plants, Genetically Modified/adverse effects , Plants, Genetically Modified/chemistryABSTRACT
Standard food safety assessments of genetically modified crops require a thorough molecular characterization of the novel DNA as inserted into the plant that is intended for commercialization, as well as a comparison of agronomic and nutritional characteristics of the genetically modified to the non-modified counterpart. These characterization data are used to identify any unintended changes in the inserted DNA or in the modified plant that would require assessment for safety in addition to the assessment of the intended modification. An unusual case of an unintended effect discovered from the molecular characterization of a genetically modified late blight resistant potato developed for growing in Bangladesh and Indonesia is presented here. Not only was a significant portion of the plasmid vector backbone DNA inserted into the plant along with the intended insertion of an R-gene for late blight resistance, but the inserted DNA was split into two separate fragments and inserted into two separate chromosomes. One fragment carries the R-gene and the other fragment carries the NPTII selectable marker gene and the plasmid backbone DNA. The implications of this for the food safety assessment of this late blight resistant potato are considered.
Subject(s)
Crops, Agricultural/genetics , Food Safety/methods , Phytophthora infestans/pathogenicity , Plant Diseases/genetics , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Solanum tuberosum/genetics , Chromosome Mapping , Crops, Agricultural/immunology , Crops, Agricultural/microbiology , DNA, Plant/genetics , Genetic Markers , Immunity, Innate , Plant Diseases/immunology , Plant Diseases/microbiology , Plants, Genetically Modified/immunology , Plants, Genetically Modified/microbiology , Solanum tuberosum/immunology , Solanum tuberosum/microbiologyABSTRACT
Newly expressed proteins in genetically engineered crops are evaluated for potential cross reactivity to known allergens as part of their safety assessment. This assessment uses a weight-of-evidence approach. Two key components of this allergenicity assessment include any history of safe human exposure to the protein and/or the source organism from which it was originally derived, and bioinformatic analysis identifying amino acid sequence relatedness to known allergens. Phosphomannose-isomerase (PMI) has been expressed in commercialized genetically engineered (GE) crops as a selectable marker since 2010 with no known reports of allergy, which supports a history of safe exposure, and GE events expressing the PMI protein have been approved globally based on expert safety analysis. Bioinformatic analyses identified an eight-amino-acid contiguous match between PMI and a frog parvalbumin allergen (CAC83047.1). While short amino acid matches have been shown to be a poor predictor of allergen cross reactivity, most regulatory bodies require such matches be assessed in support of the allergenicity risk assessment. Here, this match is shown to be of negligible risk of conferring cross reactivity with known allergens.
Subject(s)
Allergens/immunology , Computational Biology/methods , Food Hypersensitivity/immunology , Mannose-6-Phosphate Isomerase/immunology , Plant Proteins/immunology , Plants, Genetically Modified/immunology , Zea mays/immunology , Allergens/genetics , Amino Acid Sequence , Cross Reactions , Food Hypersensitivity/genetics , Humans , Mannose-6-Phosphate Isomerase/genetics , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Sequence Homology , Zea mays/geneticsABSTRACT
Phytophthora sojae is a pathogen that causes stem and root rot in soybean (Glycine max [L.] Merr.). We previously demonstrated that GmBTB/POZ, a BTB/POZ domain-containing nuclear protein, enhances resistance to P. sojae in soybean, via a process that depends on salicylic acid (SA). Here, we demonstrate that GmBTB/POZ associates directly with soybean LIKE HETEROCHROMATIN PROTEIN1 (GmLHP1) in vitro and in vivo and promotes its ubiquitination and degradation. Both overexpression and RNA interference analysis of transgenic lines demonstrate that GmLHP1 negatively regulates the response of soybean to P. sojae by reducing SA levels and repressing GmPR1 expression. The WRKY transcription factor gene, GmWRKY40, a SA-induced gene in the SA signaling pathway, is targeted by GmLHP1, which represses its expression via at least two mechanisms (directly binding to its promoter and impairing SA accumulation). Furthermore, the nuclear localization of GmLHP1 is required for the GmLHP1-mediated negative regulation of immunity, SA levels and the suppression of GmWRKY40 expression. Finally, GmBTB/POZ releases GmLHP1-regulated GmWRKY40 suppression and increases resistance to P. sojae in GmLHP1-OE hairy roots. These findings uncover a regulatory mechanism by which GmBTB/POZ-GmLHP1 modulates resistance to P. sojae in soybean, likely by regulating the expression of downstream target gene GmWRKY40.
Subject(s)
Chromosomal Proteins, Non-Histone/metabolism , Glycine max/microbiology , Phytophthora/pathogenicity , Plant Roots/microbiology , Plants, Genetically Modified/microbiology , Soybean Proteins/metabolism , BTB-POZ Domain , Chromosomal Proteins, Non-Histone/genetics , Gene Expression Regulation, Plant , Host-Pathogen Interactions , Phytophthora/immunology , Plant Roots/genetics , Plant Roots/immunology , Plant Roots/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Plants, Genetically Modified/metabolism , Proteolysis , Soybean Proteins/genetics , Glycine max/genetics , Glycine max/immunology , Glycine max/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , UbiquitinationABSTRACT
Sclerotinia Stem Rot (SSR) caused by the oxalic acid (OA)-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, causes significant yields losses in the crop Brassica sps. Oxalate oxidase (OxO) can metabolize OA to CO2 and H2O2. Degradation of OA during the early phase of fungal-host interaction can interfere with the fungal infection and establishment processes. The present study demonstrates the potential of barley oxalate oxidase (BOxO) gene in conferring stable resistance against stem rot in a productive and highly susceptible Brassica juncea cv Varuna under field conditions. Four stable, independent, single-copy transgenic lines (B16, B17, B18, and B53) exhibited a significant reduction in the rate of lesion expansion i.e. 11-26%, 39-47%, and 24-35% reproducibly over the three-generation i.e. T2, T3, and T4 respectively. The enhanced resistance in the transgenic lines correlated with high OxO activity, accumulation of higher levels of H2O2, and robust activation of defense responsive genes upon infection by S. sclerotiorum.
Subject(s)
Ascomycota/physiology , Brassica/immunology , Disease Resistance/immunology , Hordeum/enzymology , Oxidoreductases/metabolism , Plant Diseases/immunology , Plants, Genetically Modified/immunology , Brassica/growth & development , Brassica/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Oxidoreductases/genetics , Plant Diseases/microbiology , Plant Proteins/genetics , Plant Proteins/metabolism , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/metabolismABSTRACT
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.
Subject(s)
Antigens, Plant/genetics , CRISPR-Cas Systems/genetics , Glycine max/genetics , Soybean Proteins/genetics , Transgenes/genetics , Antigens, Plant/adverse effects , Antigens, Plant/immunology , Biolistics , Crops, Agricultural/genetics , Gene Editing , Genome, Plant , Humans , Mutagenesis, Site-Directed , Mutation/genetics , Plant Breeding , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/immunology , Soybean Proteins/adverse effects , Soybean Proteins/immunology , Glycine max/growth & development , Glycine max/immunology , Transgenes/immunologyABSTRACT
The bioinformatic criteria adopted by regulatory agencies to predict the potential cross reactivity between newly expressed proteins in genetically engineered crops and known allergens involves amino acid identity thresholds and was formulated nearly two decades ago based on the opinion of allergy experts. Over the subsequent years, empirical evidence has been developed indicating that better bioinformatic tools based on amino acid similarity are available to detect real allergen cross-reactive risk while substantially reducing false-positive detections. Although the formulation of safety regulations, in the absence of empirical evidence, may require reliance on expert opinion, such expert opinion should not trump empirical evidence once it becomes available. The failure of regulation to maintain consistency with the best available scientific evidence diminishes its value and creates arbitrary barriers to the use of beneficial technologies by society.
Subject(s)
Allergens/immunology , Computational Biology/methods , Plants, Genetically Modified/immunology , Allergens/genetics , Animals , Cross Reactions/drug effects , Cross Reactions/immunology , Empirical Research , Forecasting , Humans , Plants, Genetically Modified/geneticsABSTRACT
The most devastating diseases in rice (Oryza sativa) are sheath blight caused by the fungal necrotroph Rhizoctonia solani, rice blast by hemibiotrophic fungus Magnaporthe oryzae, and leaf blight by bacterial biotroph Xanthomonas oryzae (Xoo). It has been reported that the Class III acyl-CoA-binding proteins (ACBPs) such as those from dicots (Arabidopsis and grapevine) play a role in defence against biotrophic pathogens. Of the six Arabidopsis (Arabidopsis thaliana) ACBPs, AtACBP3 conferred protection in transgenic Arabidopsis against Pseudomonas syringae, but not the necrotrophic fungus, Botrytis cinerea. Similar to Arabidopsis, rice possesses six ACBPs, designated OsACBPs. The aims of this study were to test whether OsACBP5, the homologue of AtACBP3, can confer resistance against representative necrotrophic, hemibiotrophic and biotrophic phytopathogens and to understand the mechanisms in protection. Herein, when OsACBP5 was overexpressed in rice, the OsACBP5-overexpressing (OsACBP5-OE) lines exhibited enhanced disease resistance against representative necrotrophic (R. solani & Cercospora oryzae), hemibiotrophic (M. oryzae & Fusarium graminearum) and biotrophic (Xoo) phytopathogens. Progeny from a cross between OsACBP5-OE9 and the jasmonate (JA)-signalling deficient mutant were more susceptible than the wild type to infection by the necrotroph R. solani. In contrast, progeny from a cross between OsACBP5-OE9 and the salicylic acid (SA)-signalling deficient mutant was more susceptible to infection by the hemibiotroph M. oryzae and biotroph Xoo. Hence, enhanced resistance of OsACBP5-OEs against representative necrotrophs appears to be JA-dependent whilst that to (hemi)biotrophs is SA-mediated.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/immunology , Carrier Proteins/metabolism , Disease Resistance/immunology , Oryza/immunology , Plant Diseases/immunology , Plant Proteins/metabolism , Plants, Genetically Modified/immunology , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Botrytis/pathogenicity , Carrier Proteins/genetics , Fusarium/pathogenicity , Gene Expression Regulation, Plant , Oryza/genetics , Oryza/growth & development , Oryza/microbiology , Plant Diseases/microbiology , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Plants, Genetically Modified/microbiology , Rhizoctonia/pathogenicity , Salicylic Acid/metabolismABSTRACT
Plant-based products have expanded to include cancer immunotherapy, which has made great strides over recent years. Plants are considered inexpensive and facile production platforms for recombinant monoclonal antibody (mAb) due to the latest advancements and diversification of transgenic techniques. Current human biologics, including those based on mAbs produced by fermentation technologies using primarily mammalian cell cultures, have been replaced by plant-produced mAbs, which are cost effective, more scalable, speedy, versatile, and safer. Moreover, the use of animals for antibody production is always a question of ethical unambiguity, and the suitability of animal models for predicting the immunogenicity of therapeutic mAbs in humans and transposition of the immunogenic potential of therapeutic antibodies in animals to the human situation has no scientific rationale. Quite a few plant-based mAbs are approved for the treatment of cancer, ranging from tumors to hematological malignancies. This review focuses on the cutting-edge approaches for using plant-derived mAbs to suppress or prevent cancers. It also discusses the avenues taken to prevent infection by oncogenic viruses, solid tumors, lymphomas, and other cancerous conditions using mAbs. The review emphasizes the use of a plant-derived monoclonal antibody as a premier platform to combat cancer.