RESUMEN
Stomatal opening is largely promoted by light-activated plasma membrane-localized proton ATPases (PM H+-ATPases), while their closure is mainly modulated by abscisic acid (ABA) signaling during drought stress. It is unknown whether PM H+-ATPases participate in ABA-induced stomatal closure. We established that BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) interacts with, phosphorylates and activates the major PM Arabidopsis H+-ATPase isoform 2 (AHA2). Detached leaves from aha2-6 single mutant Arabidopsis thaliana plants lost as much water as bak1-4 single and aha2-6 bak1-4 double mutants, with all three mutants losing more water than the wild-type (Columbia-0 [Col-0]). In agreement with these observations, aha2-6, bak1-4, and aha2-6 bak1-4 mutants were less sensitive to ABA-induced stomatal closure than Col-0, whereas the aha2-6 mutation did not affect ABA-inhibited stomatal opening under light conditions. ABA-activated BAK1 phosphorylated AHA2 at Ser-944 in its C-terminus and activated AHA2, leading to rapid H+ efflux, cytoplasmic alkalinization, and reactive oxygen species (ROS) accumulation, to initiate ABA signal transduction and stomatal closure. The phosphorylation-mimicking mutation AHA2S944D driven by its own promoter could largely compensate for the defective phenotypes of water loss, cytoplasmic alkalinization, and ROS accumulation in both aha2-6 and bak1-4 mutants. Our results uncover a crucial role of AHA2 in cytoplasmic alkalinization and ABA-induced stomatal closure during the plant's response to drought stress.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Mutación/genética , Fosforilación , Estomas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Agua/metabolismoRESUMEN
BACKGROUND: Anthocyanins are water-soluble flavonoids in plants, which give plants bright colors and are widely used as food coloring agents, nutrients, and cosmetic additives. There are several limitations for traditional techniques of collecting anthocyanins from plant tissues, including species, origin, season, and technology. The benefits of using engineering microbial production of natural products include ease of use, controllability, and high efficiency. RESULTS: In this study, ten genes encoding enzymes involved in the anthocyanin biosynthetic pathway were successfully cloned from anthocyanin-rich plant materials blueberry fruit and purple round eggplant rind. The Yeast Fab Assembly technology was utilized to construct the transcriptional units of these genes under different promoters. The transcriptional units of PAL and C4H, 4CL and CHS were fused and inserted into Chr. XVI and IV of yeast strain JDY52 respectively using homologous recombination to gain Strain A. The fragments containing the transcriptional units of CHI and F3H, F3'H and DFR were inserted into Chr. III and XVI to gain Strain B1. Strain B2 has the transcriptional units of ANS and 3GT in Chr. IV. Several anthocyanidins, including cyanidin, peonidin, pelargonidin, petunidin, and malvidin, were detected by LC-MS/MS following the predicted outcomes of the de novo biosynthesis of anthocyanins in S. cerevisiae using a multi-strain co-culture technique. CONCLUSIONS: We propose a novel concept for advancing the heterologous de novo anthocyanin biosynthetic pathway, as well as fundamental information and a theoretical framework for the ensuing optimization of the microbial synthesis of anthocyanins.
Asunto(s)
Antocianinas , Arándanos Azules (Planta) , Saccharomyces cerevisiae , Antocianinas/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Arándanos Azules (Planta)/genética , Arándanos Azules (Planta)/metabolismo , Ingeniería Metabólica/métodos , Vías Biosintéticas , Redes y Vías Metabólicas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Anthocyanins are high-value natural compounds, but to date, their production still mainly relies on extraction from plants. A five-step metabolic pathway was constructed in probiotic Lactococcus lactis NZ9000 for rapid, stable, and glycosylated anthocyanin biosynthesis using chalcone as a substrate. The genes were cloned from anthocyanin-rich blueberry: chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDPG-flavonoid 3-O-glycosyltransferase (3GT). Using HR, the polysaccharide pellicle (PSP) segment of the cell wall polysaccharide synthesis (cwps) gene cluster from L. lactis NZ9000 was cloned into vector p15A-Cm-repDE. Then, CHI and F3H were placed sequentially under the control of NZProm 3 of this gene cluster in the vector, which was transformed into L. lactis NZ9000 to obtain Strain A. Furthermore, Strain B was constructed by placing F3H-DFR-ANS and 3GT under NZProm 2 and 3, respectively. Using LC-MS/MS analysis, several types of anthocyanins, including callistephin chloride, oenin chloride, malvidin O-hexoside, malvidin 3,5-diglucoside, and pelargonidin 3-O-malonyl-malonylhexoside, increased in the supernatant of the co-culture of Strains A and B compared to that of L. lactis NZ9000. This is the first time that a five-step metabolic pathway has been developed for anthocyanin biosynthesis in probiotic L. lactis NZ9000. This work lays the groundwork for novel anthocyanin production by a process involving the placement of several biosynthesis genes under the control of a gene cluster.
RESUMEN
The phytohormone abscisic acid (ABA) and the Polycomb group proteins have key roles in regulating plant growth and development; however, their interplay and underlying mechanisms are not fully understood. Here, we identified an Arabidopsis (Arabidopsis thaliana) nodulin homeobox (AtNDX) protein as a negative regulator in the ABA signaling pathway. AtNDX mutants are hypersensitive to ABA, as measured by inhibition of seed germination and root growth, and the expression of AtNDX is downregulated by ABA. AtNDX interacts with the Polycomb Repressive Complex1 (PRC1) core components AtRING1A and AtRING1B in vitro and in vivo, and together, they negatively regulate the expression levels of some ABA-responsive genes. We identified ABA-INSENSITIVE (ABI4) as a direct target of AtNDX. AtNDX directly binds the downstream region of ABI4 and deleting this region increases the ABA sensitivity of primary root growth. Furthermore, ABI4 mutations rescue the ABA-hypersensitive phenotypes of ndx mutants and ABI4-overexpressing plants are hypersensitive to ABA in primary root growth. Thus, our work reveals the critical functions of AtNDX and PRC1 in some ABA-mediated processes and their regulation of ABI4.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Plantas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Transducción de Señal , Ácido Abscísico/farmacología , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Secuencia de Bases , Proteínas Portadoras/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Germinación/efectos de los fármacos , Proteínas de Homeodominio/genética , Modelos Biológicos , Mutación/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Complejo Represivo Polycomb 1/genética , Unión Proteica/efectos de los fármacos , Plantones/efectos de los fármacos , Plantones/crecimiento & desarrollo , Transducción de Señal/efectos de los fármacosRESUMEN
REV7, also termed mitotic arrest-deficient 2-like 2 (MAD2L2 or MAD2B), acts as an interaction module in a broad array of cellular pathways, including translesion DNA synthesis, cell cycle control, and nonhomologous end joining. Numerous REV7 binding partners have been identified, including the human small GTPase Ras-associated nuclear protein (RAN), which acts as a potential upstream regulator of REV7. Notably, the Shigella invasin IpaB hijacks REV7 to disrupt cell cycle control to prevent intestinal epithelial cell renewal and facilitate bacterial colonization. However, the structural details of the REV7-RAN and REV7-IpaB interactions are mostly unknown. Here, using fusion protein and rigid maltose-binding protein tagging strategies, we determined the crystal structures of these two complexes at 2.00-2.35 Å resolutions. The structures revealed that both RAN and IpaB fragments bind the "safety belt" region of REV7, inducing rearrangement of the C-terminal ß-sheet region of REV7, conserved among REV7-related complexes. Of note, the REV7-binding motifs of RAN and IpaB each displayed some unique interactions with REV7 despite sharing consensus residues. Structural alignments revealed that REV7 has an adaptor region within the safety belt region that can rearrange secondary structures to fit a variety of different ligands. Our structural and biochemical results further indicated that REV7 preferentially binds GTP-bound RAN, implying that a GTP/GDP-bound transition of RAN may serve as the molecular switch that controls REV7's activity. These results provide insights into the regulatory mechanism of REV7 in cell cycle control, which may help with the development of small-molecule inhibitors that target REV7 activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Guanosina Difosfato/metabolismo , Proteínas Mad2/química , Proteínas Mad2/metabolismo , Shigella/metabolismo , Proteína de Unión al GTP ran/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Análisis Mutacional de ADN , Guanosina Trifosfato/metabolismo , Ligandos , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Proteína de Unión al GTP ran/químicaRESUMEN
In the early stage of virus infection, the pattern recognition receptor (PRR) signaling pathway of the host cell is activated to induce interferon production, activating interferon-stimulated genes (ISGs) that encode antiviral proteins that exert antiviral effects. Viperin is one of the innate antiviral proteins that exert broad-spectrum antiviral effects by various mechanisms. Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes huge losses to the pig industry. Research on early antiviral responses in the gastrointestinal tract is essential for developing strategies to prevent the spread of PEDV. In this study, we investigated the mechanisms of viperin in PEDV-infected IPEJ-C2 cells. Increased expression of interferon and viperin and decreased replication of PEDV with a clear reduction in the viral load were observed in PEDV-infected IPEC-J2 cells. Amino acids 1-50 of porcine viperin contain an endoplasmic reticulum signal sequence that allows viperin to be anchored to the endoplasmic reticulum and are necessary for its function in inhibiting PEDV proliferation. The interaction of the viperin S-adenosylmethionine domain with the N protein of PEDV was confirmed via confocal laser scanning microscopy and co-immunoprecipitation. This interaction might interfere with viral replication or assembly to reduce virus proliferation. Our results highlight a potential mechanism whereby viperin is able to inhibit PEDV replication and play an antiviral role in innate immunity.
Asunto(s)
Antivirales/metabolismo , Interacciones Microbiota-Huesped/fisiología , Proteínas de la Nucleocápside/fisiología , Virus de la Diarrea Epidémica Porcina/fisiología , Animales , Línea Celular , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/inmunología , Inmunidad Innata , Interferones/biosíntesis , Ratones , Ratones Endogámicos BALB C , Proteínas de la Nucleocápside/antagonistas & inhibidores , Proteínas de la Nucleocápside/química , Virus de la Diarrea Epidémica Porcina/inmunología , Virus de la Diarrea Epidémica Porcina/patogenicidad , Dominios y Motivos de Interacción de Proteínas , Proteínas/química , Proteínas/genética , Proteínas/fisiología , Interferencia de ARN , Porcinos , Replicación ViralRESUMEN
Although research has determined that reactive oxygen species (ROS) function as signaling molecules in plant development, the molecular mechanism by which ROS regulate plant growth is not well known. An aba overly sensitive mutant, abo8-1, which is defective in a pentatricopeptide repeat (PPR) protein responsible for the splicing of NAD4 intron 3 in mitochondrial complex I, accumulates more ROS in root tips than the wild type, and the ROS accumulation is further enhanced by ABA treatment. The ABO8 mutation reduces root meristem activity, which can be enhanced by ABA treatment and reversibly recovered by addition of certain concentrations of the reducing agent GSH. As indicated by low ProDR5:GUS expression, auxin accumulation/signaling was reduced in abo8-1. We also found that ABA inhibits the expression of PLETHORA1 (PLT1) and PLT2, and that root growth is more sensitive to ABA in the plt1 and plt2 mutants than in the wild type. The expression of PLT1 and PLT2 is significantly reduced in the abo8-1 mutant. Overexpression of PLT2 in an inducible system can largely rescue root apical meristem (RAM)-defective phenotype of abo8-1 with and without ABA treatment. These results suggest that ABA-promoted ROS in the mitochondria of root tips are important retrograde signals that regulate root meristem activity by controlling auxin accumulation/signaling and PLT expression in Arabidopsis.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Meristema/genética , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Genotipo , Intrones , Meristema/metabolismo , Fenotipo , Desarrollo de la Planta , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Regiones Promotoras Genéticas , Empalme del ARN , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
UNLABELLED: The human immunodeficiency virus type 1 (HIV-1)-encoded virion infectivity factor (Vif) is required to inactivate the host restriction factor APOBEC3 by engaging Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-ß) is a novel regulator of Vif-CRL5 function; as yet, its mechanism of regulation remains unclear. In the present study, we demonstrate that CBF-ß promotion of Vif-CRL5 assembly is independent of its influence on Vif stability and is also a conserved feature of primate lentiviral Vif proteins. Furthermore, CBF-ß is critical for the formation of the Vif-ElonginB/ElonginC-Cul5 core E3 ubiquitin ligase complex in vitro. CBF-ß from diverse vertebrate species supported HIV-1 Vif function, indicating the conserved nature of Vif-CBF-ß interfaces. Considering the importance of the interaction between Vif and CBF-ß in viral CRL5 function, disrupting this interaction represents an attractive pharmacological intervention against HIV-1. IMPORTANCE: HIV-1 encodes virion infectivity factor (Vif) to inactivate its host's antiviral APOBEC3 proteins. Vif triggers APOBEC3 degradation by forming Vif-Cullin 5 (Cul5)-RING ubiquitin ligase (CRL5). Core binding factor beta (CBF-ß) is a novel regulator of Vif-CRL5 function whose mechanism of regulation remains poorly defined. In the present study, we demonstrate that the promotion of Vif-CRL5 assembly by CBF-ß can be separated from its influence on Vif stability. The promotion of Vif-CRL5 assembly, but not the influence on Vif stability, is conserved among primate lentiviral Vif proteins: we found that CBF-ß from diverse vertebrate species supported HIV-1 Vif function. Considering the importance of the interaction between Vif and CBF-ß in viral CRL5 function and HIV-1 replication, disrupting this interaction is an attractive strategy against HIV-1.
Asunto(s)
Subunidad beta del Factor de Unión al Sitio Principal/genética , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Proteínas Cullin/metabolismo , Evolución Molecular , Infecciones por VIH/metabolismo , VIH-1/metabolismo , Virus de la Inmunodeficiencia de los Simios/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Subunidad beta del Factor de Unión al Sitio Principal/química , Proteínas Cullin/genética , Elonguina , Productos del Gen vif/química , Productos del Gen vif/genética , Productos del Gen vif/metabolismo , Infecciones por VIH/enzimología , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Alineación de Secuencia , Virus de la Inmunodeficiencia de los Simios/química , Virus de la Inmunodeficiencia de los Simios/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The plant hormone abscisic acid (ABA) regulates stomatal movement under drought stress, and this regulation requires hydrogen peroxide (H2O2). We isolated GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), which encodes a receptor-like kinase localized on the plasma membrane in Arabidopsis thaliana. ghr1 mutants were defective ABA and H2O2 induction of stomatal closure. Genetic analysis indicates that GHR1 is a critical early component in ABA signaling. The ghr1 mutation impaired ABA- and H2O2-regulated activation of S-type anion currents in guard cells. Furthermore, GHR1 physically interacted with, phosphorylated, and activated the S-type anion channel SLOW ANION CHANNEL-ASSOCIATED1 when coexpressed in Xenopus laevis oocytes, and this activation was inhibited by ABA-INSENSITIVE2 (ABI2) but not ABI1. Our study identifies a critical component in ABA and H2O2 signaling that is involved in stomatal movement and resolves a long-standing mystery about the differential functions of ABI1 and ABI2 in this process.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Peróxido de Hidrógeno/metabolismo , Fosfotransferasas/metabolismo , Estomas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Animales , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Células Cultivadas , Femenino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Oocitos/metabolismo , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Fosfotransferasas/genética , Estomas de Plantas/genética , Proteínas Quinasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Xenopus laevisRESUMEN
It is well known that abscisic acid (ABA) promotes reactive oxygen species (ROS) production through plasma membrane-associated NADPH oxidases during ABA signaling. However, whether ROS from organelles can act as second messengers in ABA signaling is largely unknown. Here, we identified an ABA overly sensitive mutant, abo6, in a genetic screen for ABA-mediated inhibition of primary root growth. ABO6 encodes a DEXH box RNA helicase that is involved in regulating the splicing of several genes of complex I in mitochondria. The abo6 mutant accumulated more ROS in mitochondria, as established using a mitochondrial superoxide indicator, circularly permuted yellow fluorescent protein. Two dominant-negative mutations in ABA insensitive1 (abi1-1) and abi2-1 greatly reduced ROS production in mitochondria. The ABA sensitivity of abo6 can also be compromised by the atrbohF mutation. ABA-mediated inhibition of seed germination and primary root growth in abo6 was released by the addition of reduced GSH and exogenous auxin to the medium. Expression of auxin-responsive markers ProDR5:GUS (for synthetic auxin response element D1-4 with site-directed mutants in the 5'-end from soybean):ß-glucuronidase) and Indole-3-acetic acid inducible2:GUS was greatly reduced by the abo6 mutation. Hence, our results provide molecular evidence for the interplay between ABA and auxin through the production of ROS from mitochondria. This interplay regulates primary root growth and seed germination in Arabidopsis thaliana.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/farmacología , ARN Helicasas/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ARN Helicasas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
The phytohormone abscisic acid (ABA) is an important regulator of plant development and response to environmental stresses. In this study, we identified two ABA overly sensitive mutant alleles in a gene encoding Auxin Response Factor2 (ARF2). The expression of ARF2 was induced by ABA treatment. The arf2 mutants showed enhanced ABA sensitivity in seed germination and primary root growth. In contrast, the primary root growth and seed germination of transgenic plants over-expressing ARF2 are less inhibited by ABA than that of the wild type. ARF2 negatively regulates the expression of a homeodomain gene HB33, the expression of which is reduced by ABA. Transgenic plants over-expressing HB33 are more sensitive, while transgenic plants reducing HB33 by RNAi are more resistant to ABA in the seed germination and primary root growth than the wild type. ABA treatment altered auxin distribution in the primary root tips and made the relative, but not absolute, auxin accumulation or auxin signal around quiescent centre cells and their surrounding columella stem cells to other cells stronger in arf2-101 than in the wild type. These results indicate that ARF2 and HB33 are novel regulators in the ABA signal pathway, which has crosstalk with auxin signal pathway in regulating plant growth.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Genes de Plantas/genética , Proteínas de Homeodominio/genética , Proteínas Represoras/metabolismo , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Inmunoprecipitación de Cromatina , Ciclina B/genética , Ciclina B/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Germinación/efectos de los fármacos , Glucuronidasa/metabolismo , Proteínas de Homeodominio/metabolismo , Ácidos Indolacéticos/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Interferencia de ARN/efectos de los fármacos , Proteínas Represoras/genética , Semillas/efectos de los fármacos , Semillas/genética , Semillas/crecimiento & desarrollo , Transducción de Señal/efectos de los fármacosRESUMEN
Infectious bursal disease (IBD), as a highly infectious immunosuppressive disease, causes severe economic losses in the poultry industry worldwide. Saccharomyces cerevisiae is an appealing vehicle used in oral vaccine formulations to safely and effectively deliver heterologous antigens. It can elicit systemic and mucosal responses. This study aims to explore the potential as oral an vaccine for S. cerevisiae expressing the capsid protein VP2 of IBDV. We constructed the recombinant S. cerevisiae, demonstrated that VP2 was displayed on the cell surface and had high immunoreactivity. By using the live ST1814G/Aga2-VP2 strain to immunize the mice, the results showed that recombinant S. cerevisiae significantly increased specific IgG and sIgA antibody titers, indicating the potential efficacy of vaccine-induced protection. These results suggested that the VP2 protein-expressing recombinant S. cerevisiae strain was a promising candidate oral subunit vaccine to prevent IBDV infection.
RESUMEN
Hydropericardium-hepatitis syndrome (HHS) caused by fowl adenovirus (FAdV) serotype 4 strains is a highly contagious disease that causes significant economic loss to the global poultry industry. However, subunit vaccine against FAdV-4 infection is not yet commercially available to date. This study aims to explore the potential for oral immunization of recombinant Saccharomyces cerevisiae expressing Fiber-2 of FAdV-4 as a subunit vaccine. Here, we constructed recombinant S. cerevisiae (ST1814G/Fiber-2) expressing recombinant Fiber-2 (rFiber-2), which was displayed on the cell surface. To evaluate the immune response and protective effect of live recombinant S. cerevisiae, chickens were orally immunized with the constructed live ST1814G/Fiber-2, three times at 5-day intervals, and then challenged with FAdV-4. The results showed that oral administration of live ST1814G/Fiber-2 could stimulate the production of humoral immunity, enhance the body's antiviral activity and immune regulation ability, improve the composition of gut microbiota, provide protection against FAdV-4 challenge, reduce viral load in the liver, and alleviate the pathological damage of heart, liver, and spleen for chicken. In addition, we found the synergistic effect in combining the ST1814G/Fiber-2 yeast and inactivated vaccine to trigger stronger humoral immunity and mucosal immunity. Our results suggest that oral live ST1814G/Fiber-2 is a potentially safer auxiliary preparation strategy in controlling FAdV-4 infection.
Asunto(s)
Infecciones por Adenoviridae , Aviadenovirus , Enfermedades de las Aves de Corral , Vacunas Virales , Adenoviridae , Infecciones por Adenoviridae/prevención & control , Infecciones por Adenoviridae/veterinaria , Animales , Anticuerpos Antivirales , Aviadenovirus/genética , Pollos , Inmunización/veterinaria , Saccharomyces cerevisiae/genética , Serogrupo , Vacunas de SubunidadRESUMEN
African swine fever virus (ASFV) infects domestic pigs and European wild boars with strong, hemorrhagic and high mortality. The primary cellular targets of ASFV is the porcine macrophages. Up to now, no commercial vaccine or effective treatment available to control the disease. In this study, three recombinant Saccharomyces cerevisiae (S. cerevisiae) strains expressing fused ASFV proteins-porcine Ig heavy chains were constructed and the immunogenicity of the S. cerevisiae-vectored cocktail ASFV feeding vaccine was further evaluated. To be specific, the P30-Fcγ and P54-Fcα fusion proteins displaying on surface of S. cerevisiae cells were produced by fusing the Fc fragment of porcine immunoglobulin IgG1 or IgA1 with p30 or p54 gene of ASFV respectively. The recombinant P30-Fcγ and P54-Fcα fusion proteins expressed by S. cerevisiae were verified by Western blotting, flow cytometry and immunofluorescence assay. Porcine immunoglobulin Fc fragment fused P30/P54 proteins elicited P30/P54-specific antibody production and induced higher mucosal immunity in swine. The absorption and phagocytosis of recombinant S. cerevisiae strains in IPEC-J2 cells or porcine alveolar macrophage (PAM) cells were significantly enhanced, too. Here, we introduce a kind of cheap and safe oral S. cerevisiae-vectored vaccine, which could activate the specific mucosal immunity for controlling ASFV infection.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Fiebre Porcina Africana/prevención & control , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/inmunología , Animales , Anticuerpos Antivirales , Formación de Anticuerpos , Fragmentos Fc de Inmunoglobulinas/genética , Leucocitos Mononucleares , Saccharomyces cerevisiae/genética , Sus scrofa , PorcinosRESUMEN
Elongator is a histone acetyl-transferase complex consisting of six subunits, and is highly conserved in eukaryotic organisms. Here, we isolated two novel mutants, elp2 and elp6, during a genetic screening for ABA-hypersensitive Arabidopsis mutants. Map-based cloning identified ELP2 and ELP6, which encode the orthologs of the yeast Elongator subunits, ELP2 and ELP6, respectively. Another Elongator subunit mutant, elp4/elo1, was obtained from the SALK T-DNA collection. The elp1/abo1/elo2 mutant was isolated in a previous study. All four of the Elongator mutants had narrow leaves, reduced root growth, ABA hypersensitivity and an increased accumulation of anthocyanins. Mutations in the core subcomplex subunits ELP1/ABO1 and ELP2, but not in the accessory subcomplex subunits ELP4/ELO1 and ELP6, caused stomatal closing to be supersensitive to ABA. In addition, the four mutants were all more resistant than the wild type to oxidative stress produced by methyl viologen, and to CsCl. Gene expression analysis indicated that the four mutants had increased transcript levels of CAT3 under normal conditions, increased transcript levels of ZAT10 when treated with ABA and reduced transcript levels of MYBL2, which encodes a single-repeat MYB protein, acting as a negative regulator of anthocyanin biosynthesis. Our results suggest that Elongator plays crucial roles in regulating plant responses to ABA, oxidative stress resistance and anthocyanin biosynthesis in Arabidopsis.
Asunto(s)
Ácido Abscísico/metabolismo , Antocianinas/biosíntesis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Histona Acetiltransferasas/metabolismo , Estrés Oxidativo , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Mapeo Cromosómico , Clonación Molecular , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Germinación , Histona Acetiltransferasas/genética , Mutagénesis Insercional , Mutación , ARN de Planta/genética , Semillas/genética , Semillas/crecimiento & desarrollo , Análisis de Secuencia de ADNRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes severe systemic inflammation. Based on transcriptome sequencing data, a new cold-inducible RNA-binding protein (CIRBP) was identified, and its upregulated expression was detected in PRRSV-infected porcine alveolar macrophages (PAMs). However, the immunoregulatoryeffect of CIRBP in PRRSV infection remains unclear. In this study, we found that CIRBP, as an RNA-binging protein, migrates to the cytoplasm from the nucleus and exists in cytoplasmic stress granules under PRRSV infection. In addition, as a new pro-inflammatory factor, the overexpression of CIRBP promotes the expression of inflammatory cytokines and oxidative stress as showing the production of iNOS and ROS in PRRSV-infected cells, which contributes to the inflammatory response via the NF-κB pathway. Our findings suggested that CIRBP is involved in the regulation of PRRSV-induced inflammatory response.
Asunto(s)
Inflamación/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Proteínas de Unión al ARN/inmunología , Animales , Células Cultivadas , Citocinas/metabolismo , Inflamación/complicaciones , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/ultraestructura , Macrófagos Alveolares/virología , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Porcinos , Regulación hacia ArribaRESUMEN
Small, secreted signaling peptides that are perceived by receptor-like kinases (RLKs) constitute an important regulatory mechanism in plant organ formation and stem cell maintenance. However, functional redundancy at the level of both ligand and receptor families often makes it difficult to clearly discern the role of individual members by a genetic approach. Here, we show that driven by a constitutive CaMV 35S promoter, a truncated BAM protein (BAMΔ) that lacks either the signal peptide (SP) or the cytoplasmic kinase (Ki) domain could cause defective shoot apical meristem (SAM) maintenance, which phenotypically resembled the triple bam mutant. Such a dominant-negative effect could also be achieved when the same transgene was driven by the native AtBAM1 promoter, but not by the CLV1 promoter. When introduced into a clv1-4 background, BAMΔ proteins abolished the typical clv phenotype by suppressing the transcriptional level of clv1-4. In addition to a clear reduction in root length and a decreased number of meristematic cells, the 35S:BAMΔ transgenic seedlings exhibited considerable resistance to CLE40p- but not to CLV3p-mediated root growth inhibition, implying that BAMs play key roles in the regulation of proximal meristem activity in root through CLE40 peptide. Findings present here not only provide evidence that truncated BAM proteins are strongly dominant negative in regulating apical meristem development but also propose that expression of a truncated version of plant LRR receptor kinase could potentially be used as a powerful tool to reveal its in vivo function in signal transduction.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Expresión Génica Ectópica , Regulación de la Expresión Génica de las Plantas , Brotes de la Planta/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Transducción de Señal , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Meristema/genética , Meristema/crecimiento & desarrollo , Meristema/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Populus/genética , Populus/crecimiento & desarrollo , Populus/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10h, which is a promising rapid method to detect Salmonella in emergency.
Asunto(s)
Contaminación de Alimentos , Microbiología de Alimentos/métodos , Separación Inmunomagnética/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Salmonella/aislamiento & purificación , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/metabolismo , Proteínas Bacterianas/inmunología , Carne/microbiología , Leche/microbiología , Salmonella/genética , Sensibilidad y EspecificidadRESUMEN
Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.
Asunto(s)
Animales , Salmonella/aislamiento & purificación , Contaminación de Alimentos , Separación Inmunomagnética/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Microbiología de Alimentos/métodos , Salmonella/genética , Proteínas Bacterianas/inmunología , Sensibilidad y Especificidad , Leche/microbiología , Carne/microbiología , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/metabolismoRESUMEN
Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.