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Salinization is one of the leading causes of arable land shrinkage and rice yield decline, recently. Therefore, developing and utilizing salt-tolerant rice varieties have been seen as a crucial and urgent strategy to reduce the effects of saline intrusion and protect food security worldwide. In the current study, the CRISPR/Cas9 system was utilized to induce targeted mutations in the coding sequence of the OsDSG1, a gene involved in the ubiquitination pathway and the regulation of biochemical reactions in rice. The CRISPR/Cas9-induced mutations of the OsDSG1 were generated in a local rice cultivar and the mutant inheritance was validated at different generations. The OsDSG1 mutant lines showed an enhancement in salt tolerance compared to wild type plants at both germination and seedling stages indicated by increases in plant height, root length, and total fresh weight as well as the total chlorophyll and relative water contents under the salt stress condition. In addition, lower proline and MDA contents were observed in mutant rice as compared to wild type plants in the presence of salt stress. Importantly, no effect on seed germination and plant growth parameters was recorded in the CRISRP/Cas9-induced mutant rice under the normal condition. This study again indicates the involvement of the OsDSG1 gene in the salt resistant mechanism in rice and provides a potential strategy to enhance the tolerance of local rice varieties to the salt stress.
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Oryza , Tolerancia a la Sal , Tolerancia a la Sal/genética , Sistemas CRISPR-Cas , Oryza/metabolismo , Estrés Salino , MutaciónRESUMEN
In rice (Oryza sativa L.), crown roots (CRs) have many important roles in processes such as root system expansion, water and mineral uptake, and adaptation to environmental stresses. Phytohormones such as auxin, cytokinin, and ethylene are known to control CR initiation and development in rice. However, the role of jasmonic acid (JA) in CR development remained elusive. Here, we report that JA promotes CR development by regulating OsGER4, a rice Germin-like protein. Root phenotyping analysis revealed that exogenous JA treatment induced an increase in CR number in a concentration-dependent manner. A subsequent genome-wide association study and gene expression analyses pinpointed a strong association between the Germin-like protein OsGER4 and the increase in CR number under exogenous JA treatment. The ProGER4::GUS reporter line showed that OsGER4 is a hormone-responsive gene involved in various stress responses, mainly confined to epidermal and vascular tissues during CR primordia development and to vascular bundles of mature crown and lateral roots. Notable changes in OsGER4 expression patterns caused by the polar auxin transport inhibitor NPA support its connection to auxin signaling. Phenotyping experiments with OsGER4 knockout mutants confirmed that this gene is required for CR development under exogenous JA treatment. Overall, our results provide important insights into JA-mediated regulation of CR development in rice.
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Oryza , Oryza/metabolismo , Estudio de Asociación del Genoma Completo , Raíces de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ácidos Indolacéticos/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
BACKGROUND: Powdery mildew is a major disease that causes great losses in soybean yield and seed quality. Disease-resistant varieties, which are generated by reducing the impact of susceptibility genes through mutation in host plants, would be an effective approach to protect crops from this disease. The Mildew Locus O (MLO) genes are well-known susceptibility genes for powdery mildew in plant. In this study, we utilized the CRISPR/Cas9 system to induce targeted mutations in the soybean GmMLO genes to improve powdery mildew resistance. RESULTS: A dual-sgRNA CRISPR/Cas9 construct was designed and successfully transferred into the Vietnamese soybean cultivar DT26 through Agrobacterium tumefaciens-mediated transformation. Various mutant forms of the GmMLO genes including biallelic, chimeric and homozygous were found at the T0 generation. The inheritance and segregation of CRISPR/Cas9-induced mutations were confirmed and validated at the T1 and T2 generations. Out of six GmMLO genes in the soybean genome, we obtained the Gmmlo02/Gmmlo19/Gmmlo23 triple and Gmmlo02/Gmmlo19/Gmmlo20/Gmmlo23 quadruple knockout mutants at the T2 generation. When challenged with Erysiphe diffusa, a fungus that causes soybean powdery mildew, all mutant plants showed enhanced resistance to the pathogen, especially the quadruple mutant. The powdery mildew severity in the mutant soybeans was reduced by up to 36.4% compared to wild-type plants. In addition, no pleiotropic effect on soybean growth and development under net-house conditions was observed in the CRISPR/Cas9 mutants. CONCLUSIONS: Our results indicate the involvement of GmMLO02, GmMLO19, GmMLO20 and GmMLO23 genes in powdery mildew susceptibility in soybean. Further research should be conducted to investigate the roles of individual tested genes and the involvement of other GmMLO genes in this disease infection mechanism. Importantly, utilizing the CRISPR/Cas9 system successfully created the Gmmlo transgene-free homozygous mutant lines with enhanced resistance to powdery mildew, which could be potential materials for soybean breeding programs.
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Sistemas CRISPR-Cas , Glycine max , Glycine max/genética , ARN Guía de Sistemas CRISPR-Cas , Fitomejoramiento , Mutación , Hongos , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Resistencia a la Enfermedad/genéticaRESUMEN
MAIN CONCLUSION: Induced mutations in the SC-uORF of the tomato transcription factor gene SlbZIP1 by the CRISPR/Cas9 system led to the high accumulation of sugar and amino acid contents in tomato fruits. Tomato (Solanum lycopersicum) is one of the most popular and consumed vegetable crops in the world. Among important traits for tomato improvement such as yield, biotic and abiotic resistances, appearance, post-harvest shelf life and fruit quality, the last one seems to face more challenges because of its genetic and biochemical complexities. In this study, a dual-gRNAs CRISPR/Cas9 system was developed to induce targeted mutations in uORF regions of the SlbZIP1, a gene involved in the sucrose-induced repression of translation (SIRT) mechanism. Different induced mutations in the SlbZIP1-uORF region were identified at the T0 generation, stably transferred to the offspring, and no mutation was found at potential off-target sites. The induced mutations in the SlbZIP1-uORF region affected the transcription of SlbZIP1 and related genes in sugar and amino acid biosynthesis. Fruit component analysis showed significant increases in soluble solid, sugar and total amino acid contents in all SlbZIP1-uORF mutant lines. The accumulation of sour-tasting amino acids, including aspartic and glutamic acids, raised from 77 to 144%, while the accumulation of sweet-tasting amino acids such as alanine, glycine, proline, serine, and threonine increased from 14 to 107% in the mutant plants. Importantly, the potential SlbZIP1-uORF mutant lines with desirable fruit traits and no impaired effect on plant phenotype, growth and development were identified under the growth chamber condition. Our result indicates the potential utility of the CRISPR/Cas9 system for fruit quality improvement in tomato and other important crops.
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Solanum lycopersicum , Factores de Transcripción , Factores de Transcripción/genética , Aminoácidos/metabolismo , Azúcares/metabolismo , Solanum lycopersicum/genética , Sistemas CRISPR-Cas , Frutas/genética , Frutas/metabolismoRESUMEN
Overexpression of GA20 oxidase gene has been a recent trend for improving plant growth and biomass. Constitutive expression of GA20ox has successfully improved plant growth and biomass in several plant species. However, the constitutive expression of this gene causes side-effects, such as reduced leaf size and stem diameter, etc. To avoid these effects, we identified and employed different tissue-specific promoters for GA20ox overexpression. In this study, we examined the utility of At1g promoter to drive the expression of GUS (ß-glucuronidase) reporter and AtGA20ox genes in tobacco and Melia azedarach. Histochemical GUS assays and quantitative real-time-PCR results in tobacco showed that At1g was a root-preferential promoter whose expression was particularly strong in root tips. The ectopic expression of AtGA20ox gene under the control of At1g promoter showed improved plant growth and biomass of both tobacco and M. azedarach transgenic plants. Stem length as well as stem and root fresh weight increased by up to 1.5-3 folds in transgenic tobacco and 2 folds in transgenic M. azedarach. Both tobacco and M. azedarach transgenic plants showed increases in root xylem width with xylem to phloem ratio over 150-200% as compared to WT plants. Importantly, no significant difference in leaf shape and size was observed between At1g::AtGA20ox transgenic and WT plants. These results demonstrate the great utility of At1g promoter, when driving AtGA20ox gene, for growth and biomass improvements in woody plants and potentially some other plant species.
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Regulación de la Expresión Génica de las Plantas , Nicotiana , Biomasa , Glucuronidasa/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas/genética , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
This study was performed to investigate the effects of different supplemental light spectra and doses (duration and illuminance) on the essential oil of basil (Ocimum basilicum L.) cultivated in the net-house in Vietnam during four months. Ten samples of basil aerial parts were hydrodistilled to obtain essential oils which had the average yields from 0.88 to 1.30% (v/w, dry). The oils analyzed using GC-FID and GC-MS showed that the main component was methyl chavicol (87.4−90.6%) with the highest values found in the oils of basil under lighting conditions of 6 h/day and 150−200 µmol·m−2·s−1. Additional lighting conditions caused the significant differences (p < 0.001) in basil biomass and oil production with the highest values found in the oils of basil under two conditions of (1) 71% Red: 20% Blue: 9.0% UVA in at 120 µmol·m−2·s−1 in 6 h/day and (2) 43.5% Red: 43.5% Blue: 8.0% Green: 5.0% Far-Red at 100 µmol·m−2·s−1 in 6 h/day. The oils of basil in some formulas showed weak inhibitory effects on only the Bacillus subtilis strain. Different light spectra affect the biomass and essential oil production of basil, as well as the concentrations of the major components in the oil.
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Antiinfecciosos , Ocimum basilicum , Aceites Volátiles , Antiinfecciosos/farmacología , Cromatografía de Gases y Espectrometría de Masas , Aceites Volátiles/farmacología , Aceites de Plantas/farmacologíaRESUMEN
BACKGROUND: Multiple epiphyseal dysplasia (MED) is a common skeletal dysplasia that is characterized by variable degrees of epiphyseal abnormality primarily involving the hip and knee joints. Mutations in a gene encoding matrilin-3 (MATN3) have been reported as disease causing of autosomal dominant MED. The current study identified a novel c.572 C > A variant (p.A191D) in exon 2 of MATN3 in a Vietnamese family with MED. CASE PRESENTATION: A standard clinical tests and radiological examination were performed in an 8-year-old Vietnamese girl patient. The clinical examination showed that patient height was under average, with bent lower limbs, limited mobility and dislocation of the joints at both knees. Radiological documentation revealed abnormal cartilage development at the epiphysis of the femur and patella. The patient has a varus deformity of the lower limbs. The patient was diagnosed with autosomal dominant MED using molecular testing in the order of the coding sequences and flanking sequences of five genes: COMP (exons 8-19), MATN3 (exon 2), COL9A2 (exon 3), COL9A3 (exon 3), COL9A1 (exon 8) by Sanger sequencing. A novel heterozygous missense variant (c.572 C > A, p.A191D) in MATN3 was identified in this family, which were not inherited from parents. The p.A191D was predicted and classified as a pathogenic variant. When the two predicted structures of the wild type and mutant matrilin-3 were compared, the p.A191D substitution caused conformational changes near the substitution site, resulting in deformity of the ß-sheet of the single A domain of matrilin- 3. CONCLUSIONS: This is the first Vietnamese MED family attributed to p.A191D matrilin-3 variant, and our clinical, radiological and molecular data suggest that the novel de novo missense variant in MATN3 contributed to MED.
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Mutación Missense , Osteocondrodisplasias/genética , Pueblo Asiatico/genética , Niño , Exones/genética , Familia , Femenino , Humanos , Proteínas Matrilinas/genética , Osteocondrodisplasias/diagnóstico por imagen , Linaje , RadiografíaRESUMEN
The endophytic actinomycete strain YBQ59 was isolated from Cinnamomum cassia Prels in Yen Bai province (21°53'14â³N; 104°35'9â³E) of northern Vietnam. Based on analysis of morphological, physiological characteristics and 16S rRNA gene sequence (GenBank Acc. No. MF950891), the strain YBQ59 possessed high similarity to Streptomyces cavourensis subsp. cavourensis strain NRRL 2740, therefore assigned as S. cavourensis YBQ59. The ethyl acetate extract of the YBQ59 culture broth isolated eight pure secondary metabolites, identified as 1-monolinolein (1), bafilomycin D (2), nonactic acid (3), daidzein (4), 3'-hydroxydaidzein (5), 5,11-epoxy-10-cadinanol (6), prelactone B (7), and daucosterol (8). Compounds 1, 3-8 were reported for the first time from S. cavourensis. Compounds 1-5 exhibited antimicrobial activities against both methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA) and methicillin-resistant Staphylococcus epidermidis ATCC 35984 (MRSE) among which the compound 1 revealed the strongest effects with minimum inhibitory concentrations of 8.5 and 14.6 µg/mL, respectively. The compound 2 showed high potential effect against MRSA (MIC of 11.1 µg/mL) but less effect against MRSE (MIC of 30.3 µg/mL). The cytotoxicity of the compounds 1-7 was investigated against human lung adenocarcinoma EGFR-TKI-resistant cells, among which compounds 1, 2, and 5 exhibited the strong effect against A549 cells with IC50 values of 3.6, 6.7, and 7.8 µM, respectively. Taken together, the experimental findings in this study suggested that the compounds 1 and 2 could be reproducible metabolites applicable for inhibition of both drug-resistant bacteria and cancer cell lines.
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Antibacterianos/farmacología , Cinnamomum aromaticum/microbiología , Streptomyces/química , Streptomyces/aislamiento & purificación , Antibacterianos/química , Antibacterianos/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/crecimiento & desarrollo , Pruebas de Sensibilidad Microbiana , Filogenia , Streptomyces/clasificación , Streptomyces/genética , VietnamRESUMEN
Plant-based transient expression is an alternative platform to produce hemagglutinin-based subunit vaccines. This production system provides not only fast and effective response in the context of a pandemic but also enables the supply of big volume vaccines at low cost. Crude plant extracts containing influenza hemagglutinin are considered to use as vaccine sources because of avoidance of related purification steps resulting in low cost production allowing veterinary applications. Highly immunogenic influenza hemagglutinins are urgently required to meet these pre-conditions. Here, we present a new and innovative way to generate functional H5 oligomers from avian flu hemagglutinin in planta by the specific interaction of S·Tag and S·Protein. A S·Tag was fused to H5 trimers and this construct was transiently co-expressed in planta with S·Protein-TPs which was multimerized by disulfide bonds via cysteine residues in tailpiece sequences (TP) of IgM antibody. Multimerized S·Protein-TPs serve as bridges/molecular docks to combine S·Tag-fused hemagglutinin trimers to form very large hemagglutinin H5 oligomers. H5 oligomers in the plant crude extract were highly active in hemagglutination resulting in high titers. Immunization of mice with two doses of plant crude extracts containing H5 oligomers after storage for 1 week at 4 °C caused strong immune responses and induced neutralizing specific humoral immune responses in mice. These results allow for the development of cheap influenza vaccines for veterinary application in future.
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Hemaglutininas/metabolismo , Inmunidad/inmunología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/inmunología , Plantas Modificadas Genéticamente/metabolismo , Agrobacterium/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Hemaglutininas/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/prevención & control , Proteínas Recombinantes , Nicotiana/metabolismoRESUMEN
BACKGROUND: The continuing spread of the newly emerged H7N9 virus among poultry in China, as well as the possibility of human-to-human transmission, has attracted numerous efforts to develop an effective vaccine against H7N9. The use of nanoparticles in vaccinology is inspired by the fact that most pathogens have a dimension within the nano-size range and therefore can be processed efficiently by the immune system, which leads to a potent immune response. Herein, we report a facile approach to increase antigen size to achieve not only fast but also effective responses against the recombinant HA/H7N9 protein via a simple conjugation of the protein onto the surface of nanodiamond particles. RESULTS: In this study, trimeric Haemagglutinin (H7) that is transiently expressed in N. benthamiana was purified using affinity chromatography, and its trimeric state was revealed successfully by the cross-linking reaction. The trimeric H7 solution was subsequently mixed with a nanodiamond suspension in different ratios. The successful conjugation of the trimeric H7 onto the surface of nanodiamond particles was demonstrated by the changes in size and Zeta-potential of the particles before and after protein coating, Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western-blot analysis. Next, biofunction of the protein-nanodiamond conjugates was screened using a haemagglutination assay. A mixture containing 5 µg of trimeric H7 and 60 µg of nanodiamond corresponds to a ratio of 1:12 (w/w) of agglutinated chicken red blood cells at HA titer of 1024, which is 512-fold higher than the HA titer of free trimeric H7. After the 2nd and 3rd immunization in mice, ELISA and Western blot analyses demonstrated that the physical mixture of trimeric H7 protein and nanodiamond (1:12, w/w) elicited statistically significant stronger H7-specific-IgG response demonstrated by higher amounts of H7N9-specific IgG (over 15.4-fold with P < 0.05 after the second immunization). CONCLUSIONS: These results indicated a potential effect inherent to nanodiamond towards modulating immune systems, which should be further evaluated and broadly applied in nanovaccine development.
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Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H7N9 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Nanodiamantes , Infecciones por Orthomyxoviridae/prevención & control , Animales , Formación de Anticuerpos , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/uso terapéutico , Humanos , Inmunoglobulina G/inmunología , Vacunas contra la Influenza/química , Vacunas contra la Influenza/uso terapéutico , Gripe Humana/inmunología , Gripe Humana/prevención & control , Ratones , Ratones Endogámicos BALB C , Nanodiamantes/química , Nanodiamantes/uso terapéutico , Nanodiamantes/ultraestructura , Infecciones por Orthomyxoviridae/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéuticoRESUMEN
Rubella virus (RV) infection is an unresolved clinical complication that affects children in developing countries including Vietnam. RV infection during the first trimester of pregnancy causes severe birth defects known as congenital rubella syndrome. This study reports on the genomic characterization of RV strains circulating in northern Vietnam during 2011-2013. RV-IgM positive amniotic fluid specimens were collected from 38 women from northern Vietnam who presented with clinical rubella at the National Hospital of Obstetrics and Gynecology in Hanoi, Vietnam. The RV genes were determined by nested PCR with primers amplifying the 739-nucleotide coding region of the E1 gene. The sequences from the amplified DNA fragments were phylogenetically analyzed and compared to reference RV strains. Seventeen out of 38 samples are positive for RV detecting. All new RV isolates are clustered to genotype 2B. Eighteen amino acid mutations were found in the T and B cell epitopes. These results suggest that genotype 2B RV strains frequently circulate in northern Vietnam. These data describe the RV genotype in Vietnam with the aim of improving maternal and child health in this country.
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Complicaciones Infecciosas del Embarazo/virología , Virus de la Rubéola/clasificación , Virus de la Rubéola/genética , Rubéola (Sarampión Alemán)/virología , Análisis por Conglomerados , Femenino , Genotipo , Humanos , Epidemiología Molecular , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Embarazo , Complicaciones Infecciosas del Embarazo/epidemiología , Rubéola (Sarampión Alemán)/epidemiología , Virus de la Rubéola/aislamiento & purificación , Análisis de Secuencia de ADN , Vietnam/epidemiología , Proteínas del Envoltorio Viral/genéticaRESUMEN
Introduction: Patient-derived induced pluripotent stem cells (iPSCs) have been widely used as disease models to test new therapeutic strategies. Moreover, the regenerative potential of stem cells can be improved with the use of biologically active compounds. Our study was designed to explore the effect of honokiol, a small polyphenol molecule extracted from Magnolia officinalis, on the survival and culture time of iPSC-derived neurons from a sporadic Alzheimer's disease (AD) patient. This study aimed to generate iPSCs from peripheral blood mononuclear cells (PBMCs) of an AD patient using episomal plasmids with a nucleofector system and differentiate them into neurons. These iPSC-derived neurons were used to investigate the effect of honokiol extracted from M. officinalis on their survival and long-term cultures. Methods: IPSCs were generated from PBMCs of an AD patient by introducing Oct-3/4, Sox2, Klf4, L-Myc, and Lin28 using NucleofectorTM Technology. Differentiation of neurons derived from iPSCs was carried out using inducers and recognized by biomarkers. The viability of iPSC-derived neurons with the addition of honokiol extracted from the bark of M. officinalis was determined by the MTT analytical kit. Results: IPSCs were generated by reprogramming AD patient-derived PBMCs and subsequently converted into neurons. The survival and growth of iPSC-derived neurons were significantly enhanced by adding honokiol in the experiment conditions. Conclusion: AD iPSC-derived neurons had a high viability rate when cultured in the presence of honokiol. These results have shown that AD iPSC-derived neurons can be an excellent model for screening neurotrophic agents and improving the conditions for long-term cultures of human iPSC-derived neurons. Honokiol proves to be a potential candidate for cellular therapeutics against neurodegenerative disorders.
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Acropora is one of the most common coral genera found in Phu Quoc Islands, Vietnam. However, the presence of marine snails, such as the coralllivorous gastropod Drupella rugosa, was a potential threat to the survival of many scleractinian species, leading to changes in the health status and bacterial diversity of coral reefs in Phu Quoc Islands. Here, we describe the composition of bacterial communities associated with two species of Acropora (Acropora formosa and Acropora millepora) using the Illumina sequencing technology. This dataset includes 5 coral samples of each status (grazed or healthy), which were collected in Phu Quoc Islands (9°55'20.6â³N 104°01'16.4â³E) in May 2020. A total of 19 phyla, 34 classes, 98 orders, 216 families and 364 bacterial genera were detected from 10 coral samples. Overall, Proteobacteria and Firmicutes were the two most common bacterial phyla in all samples. Significant differences in the relative abundances of the genera Fusibacter, Halarcobacter, Malaciobacter, and Thalassotalea between grazed and healthy status were observed. However, there was no differences in alpha diversity indices between the two status. Furthermore, the dataset analysis also indicated that Vibrio and Fusibacter were core genera in the grazed samples, whereas Pseudomonas was the core genus in the healthy samples.
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Huperzine A (HupA) is an important drug for treating Alzheimer's disease (AD) and is primarily extracted from the Huperzia serrata (Lycopodiaceae). Failures in the chemical synthesis of Hup and in vitro culture have put H. serrata in danger of extinction, and there is a need for an extensive investigation of Hup from alternative perspectives. The aim of this study is to identify endophytic fungi that produce high Hup or simultaneously produce many types of Hup and have high genetic stability derived from other Lycopodiaceae species as a source of materials for natural Hup production. In this work, Hup-producing endophytic fungi were isolated from three species: Lycopodium clavatum, Phlegmariurus squarrosus, and P. phlegmaria. Of these, L. clavatum and P. squarrosus were confirmed as novel sources of Hup-producing fungi. Based on morphological characteristics and nuclear ribosomal DNA ITS sequences, four endophytic fungi Colletotrichum siamense THG1-17, Epicoccum sorghinum THG01-18, Phoma sp. TKH3-2, and Phyllosticta sp. THG2-27 were firstly isolated from these Lycopodiaceae plants, which were capable of simultaneously producing both HupA and HupB, as evidenced by high-performance liquid chromatography analysis. The four strains showed stability in Hup yield over 50 generations of culture with an in vitro storage period of 3 months. These isolated fungi will provide a new source of materials for further research to develop drugs containing HupA as well as HupB for AD treatment in the future.
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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 Sâ¢Tag and Sâ¢Protein. H5 oligomers combined via Sâ¢Tag::Sâ¢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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Subtipo H5N1 del Virus de la Influenza A , Vacunas contra la Influenza , Gripe Aviar , Animales , Hemaglutininas , Pollos , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Vacunación/veterinariaRESUMEN
This study was performed to evaluate the sequential transformation for soybean genome editing using the CRISPR/Cas9 system as well as to show a strategy for examining the activity of CRISPR/Cas9 constructs, especially the designed guide RNAs (gRNAs). The gRNAs for targeted mutations of an exogenous gene and multiple endogenous genes were constructed and transferred into a stably-overexpressed-Cas9 soybean line using Agrobacterium rhizogenes-mediated hairy root induction system. The targeted mutations were identified and characterized by the poly-acrylamide gel electrophoresis (PAGE) heteroduplex method and by sequencing. Induced mutations of the exogenous gene (gus) were observed in 57% of tested transgenic hairy roots, while 100% of the transgenic root lines showed targeted mutations of the endogenous (SACPD-C) gene. Multiple gRNAs targeting two endogenous genes (SACPD-C and SMT) induced mutation rates of 75% and 67%, respectively. Various indels including small and large deletions as well as insertions were found in target sites of the tested genes. This sequential transformation method could present the targeting efficacy of different gRNAs of each tested gene. Additionally, in this study differences in gRNA ratings were found between bioinformatics predictions and actual experimental results. This is the first successful application of the sequential transformation method for genome editing in soybean using the hairy root system. This method could be potentially useful for validating CRISPR/Cas9 constructs, evaluating gRNA targeting efficiencies, and could be applied for other research directions.
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Tobacco is an important commercial crop and a rich source of alkaloids for pharmaceutical and agricultural applications. However, its yield can be reduced by up to 70% due to virus infections, especially by a potyvirus Potato virus Y (PVY). The replication of PVY relies on host factors, and eukaryotic translation initiation factor 4Es (eIF4Es) have already been identified as recessive resistance genes against potyviruses in many plant species. To investigate the molecular basis of PVY resistance in the widely cultivated allotetraploid tobacco variety K326, we developed a dual guide RNA CRISPR/Cas9 system for combinatorial gene editing of two clades, eIF4E1 (eIF4E1-S and eIF4E1-T) and eIF4E2 (eIF4E2-S and eIF4E2-T) in the eIF4E gene family comprising six members in tobacco. We screened for CRISPR/Cas9-induced mutations by heteroduplex analysis and Sanger sequencing, and monitored PVYO accumulation in virus challenged regenerated plants by DAS-ELISA both in T0 and T1 generations. We found that all T0 lines carrying targeted mutations in the eIF4E1-S gene displayed enhanced resistance to PVYO confirming previous reports. More importantly, our combinatorial approach revealed that eIF4E1-S is necessary but not sufficient for complete PVY resistance. Only the quadruple mutants harboring loss-of-function mutations in eIF4E1-S, eIF4E1-T, eIF4E2-S and eIF4E2-T showed heritable high-level resistance to PVYO in tobacco. Our work highlights the importance of understanding host factor redundancy in virus replication and provides a roadmap to generate virus resistance by combinatorial CRISPR/Cas9-mediated editing in non-model crop plants with complex genomes.
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Potyvirus , Solanum tuberosum , Sistemas CRISPR-Cas , Mutación , Enfermedades de las Plantas , NicotianaRESUMEN
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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Type 2 diabetes mellitus (T2DM) is an important public health problem. The knowledge of bacterial communities in the gut of Vietnamese patients with T2DM and non diabetic controls is still insufficient. We report in this article the 16S rDNA amplicon data of the gut microbiomes of Vietnamese patients with T2DM and nondiabetic controls carried out using the Illumina sequencing. This work included 7 patients and 7 controls. A total of 1,627,646 reads were obtained and a total of 13 phyla, 25 classes, 94 genera were revealed. The top three dominant bacterial phyla in all subjects were Firmicutes, Bacteroidetes and Proteobacteria. Significant differences in the relative abundances of the phylum Firmicutes and class Clostridia between patients and controls were observed, suggesting that the reducing of phylum Firmicutes and class Clostridia in the gut may be linked to obesity and T2DM. All sequencing libraries were deposited in the NCBI SRA as BioProject PRJNA668251. The datasets are needed to determine the association between the bacterial composition of the gut and the pathogenesis of T2DM in Vietnamese patients.
RESUMEN
Hairy root induction system has been applied in various plant species as an effective method to study gene expression and function due to its fast-growing and high genetic stability. Recently, these systems have shown to be an effective tool to evaluate activities of CRISPR/Cas9 systems for genome editing. In this study, Rhizobium rhizogenes mediated hairy root induction was optimized to provide an effective tool for validation of plant transformation vector, CRISPR/Cas9 construct activities as well as selection of targeted gRNAs for gene editing in cucumber (Cucumis sativus L.). Under the optimized conditions including OD650 at 0.4 for infection and 5 days of co-cultivation, the highest hairy root induction frequency reached 100% for the cucumber variety Choka F1. This procedure was successfully utilized to overexpress a reporter gene (gus) and induce mutations in two Lotus japonicus ROOTHAIRLESS1 homolog genes CsbHLH66 and CsbHLH82 using CRISPR/Cas9 system. For induced mutation, about 78% of transgenic hairy roots exhibited mutant phenotypes including sparse root hair and root hair-less. The targeted mutations were obtained in individual CsbHLH66, CsbHLH82, or both CsbHLH66 and CsbHLH82 genes by heteroduplex analysis and sequencing. The hairy root transformation system established in this study is sufficient and potential for further research in genome editing of cucumber as well as other cucumis plants.