RESUMEN
Vacuolar acidification is essential for vacuoles in diverse physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection remain unknown. Here, we show that Barley stripe mosaic virus (BSMV) replicase γa, but not its mutant γaR569A , directly blocks acidification of vacuolar lumen and suppresses autophagic degradation to promote viral infection in plants. These were achieved via molecular interaction between γa and V-ATPase catalytic subunit B2 (VHA-B2), leading to disruption of the interaction between VHA-B2 and V-ATPase catalytic subunit E (VHA-E), which impairs the membrane localization of VHA-B2 and suppresses V-ATPase activity. Furthermore, a mutant virus BSMVR569A with the R569A point mutation possesses less viral pathogenicity. Interestingly, multiple viral infections block vacuolar acidification. These findings reveal that functional vacuolar acidification is required for plant antiviral defense and disruption of vacuolar acidification could be a general viral counter-defense strategy employed by multiple viruses.
Asunto(s)
Nicotiana/virología , Virus de Plantas/patogenicidad , Vacuolas/metabolismo , Proteinas del Complejo de Replicasa Viral/metabolismo , Proteínas de Plantas/metabolismo , Virus de Plantas/fisiología , Unión Proteica , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/virología , Proteinas del Complejo de Replicasa Viral/química , Replicación ViralRESUMEN
Viral replication and movement are intimately linked; however, the molecular mechanisms regulating the transition between replication and subsequent movement remain largely unknown. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein promotes viral replication and movement by interacting with the αa replicase and TGB1 movement proteins. Here, we found that γb is palmitoylated at Cys-10, Cys-19, and Cys-60 in Nicotiana benthamiana, which supports BSMV infection. Intriguingly, non-palmitoylated γb is anchored to chloroplast replication sites and enhances BSMV replication, whereas palmitoylated γb protein recruits TGB1 to the chloroplasts and forms viral replication-movement intermediate complexes. At the late stages of replication, γb interacts with NbPAT15 and NbPAT21 and is palmitoylated at the chloroplast periphery, thereby shifting viral replication to intracellular and intercellular movement. We also show that palmitoylated γb promotes virus cell-to-cell movement by interacting with NbREM1 to inhibit callose deposition at the plasmodesmata. Altogether, our experiments reveal a model whereby palmitoylation of γb directs a dynamic switch between BSMV replication and movement events during infection.
Asunto(s)
Lipoilación , Virus de Plantas , Nicotiana/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γb directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γb predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γb binds to the surface of rod-shaped BSMV virions in a Zn2+-dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn2+-dependent physical association of γb with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus) and Benyviridae. Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus morphogenesis.
Asunto(s)
Virión , Zinc , Zinc/metabolismo , Virión/metabolismo , Proteínas de la Cápside/metabolismo , Ensamble de Virus/fisiología , Virus de Plantas/metabolismo , Virus de Plantas/fisiología , Enfermedades de las Plantas/virología , Cisteína/metabolismo , Proteínas Virales/metabolismo , MorfogénesisRESUMEN
Nonalcoholic fatty liver disease (NAFLD), especially nonalcoholic steatohepatitis (NASH), has emerged as a prevalent cause of liver cirrhosis and hepatocellular carcinoma, posing severe public health challenges worldwide. The incidence of NASH is highly correlated with an increased prevalence of obesity, insulin resistance, diabetes, and other metabolic diseases. Currently, no approved drugs specifically targeted for the therapies of NASH partially due to the unclear pathophysiological mechanisms. G protein-coupled estrogen receptor 1 (GPER1) is a membrane estrogen receptor involved in the development of metabolic diseases such as obesity and diabetes. However, the function of GPER1 in NAFLD/NASH progression remains unknown. Here, we show that GPER1 exerts a beneficial role in insulin resistance, hepatic lipid accumulation, oxidative stress, or inflammation in vivo and in vitro. In particular, we observed that the lipid accumulation, inflammatory response, fibrosis, or insulin resistance in mouse NAFLD/NASH models were exacerbated by hepatocyte-specific GPER1 knockout but obviously mitigated by hepatic GPER1 activation in female and male mice. Mechanistically, hepatic GPER1 activates AMP-activated protein kinase signaling by inducing cyclic AMP release, thereby exerting its protective effect. These data suggest that GPER1 may be a promising therapeutic target for NASH.
Asunto(s)
Diabetes Mellitus , Resistencia a la Insulina , Neoplasias Hepáticas , Enfermedad del Hígado Graso no Alcohólico , Animales , Femenino , Masculino , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Diabetes Mellitus/metabolismo , Modelos Animales de Enfermedad , Receptor alfa de Estrógeno/metabolismo , Proteínas de Unión al GTP/metabolismo , Lípidos/farmacología , Hígado/metabolismo , Neoplasias Hepáticas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Ratones Endogámicos C57BL , Estrógenos/deficiencia , Estrógenos/metabolismo , Dieta Alta en GrasaRESUMEN
Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of â¼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.
Asunto(s)
Ciclotidas , Insecticidas , Oldenlandia , Ciclotidas/genética , Ciclotidas/farmacología , Ciclotidas/química , Insecticidas/química , Insecticidas/farmacología , Leucina , Lisina/genética , Mutagénesis , Proteínas de Plantas/metabolismo , Oldenlandia/química , Estabilidad Proteica , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacosRESUMEN
The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH-dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC-mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2-Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC-mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.
Asunto(s)
Cloroplastos/metabolismo , Interacciones Huésped-Patógeno , Nicotiana/virología , Virus de Plantas/fisiología , Proteínas no Estructurales Virales/fisiología , Replicación Viral , Estrés Oxidativo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Virus de Plantas/genética , Plantas Modificadas Genéticamente/virología , Reductasa de Tiorredoxina-Disulfuro/genética , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Nicotiana/genéticaRESUMEN
Seed size and weight are important factors that influence soybean yield. Combining the weighted gene co-expression network analysis (WGCNA) of 45 soybean accessions and gene dynamic changes in seeds at seven developmental stages, we identified candidate genes that may control the seed size/weight. Among these, a PLATZ-type regulator overlapping with 10 seed weight QTLs was further investigated. This zinc-finger transcriptional regulator, named as GmPLATZ, is required for the promotion of seed size and weight in soybean. The GmPLATZ may exert its functions through direct binding to the promoters and activation of the expression of cyclin genes and GmGA20OX for cell proliferation. Overexpression of the GmGA20OX enhanced seed size/weight in soybean. We further found that the GmPLATZ binds to a 32-bp sequence containing a core palindromic element AATGCGCATT. Spacing of the flanking sequences beyond the core element facilitated GmPLATZ binding. An elite haplotype Hap3 was also identified to have higher promoter activity and correlated with higher gene expression and higher seed weight. Orthologues of the GmPLATZ from rice and Arabidopsis play similar roles in seeds. Our study reveals a novel module of GmPLATZ-GmGA20OX/cyclins in regulating seed size and weight and provides valuable targets for breeding of crops with desirable agronomic traits.
Asunto(s)
Glycine max , Transcriptoma , Glycine max/genética , Transcriptoma/genética , Fitomejoramiento , Sitios de Carácter Cuantitativo , Semillas/genéticaRESUMEN
Salicylic acid (SA) acts as a signaling molecule to perceive and defend against pathogen infections. Accordingly, pathogens evolve versatile strategies to disrupt the SA-mediated signal transduction, and how plant viruses manipulate the SA-dependent defense responses requires further characterization. Here, we show that barley stripe mosaic virus (BSMV) infection activates the SA-mediated defense signaling pathway and upregulates the expression of Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1). The γb protein interacts directly with NbTRXh1 in vivo and in vitro. The overexpression of NbTRXh1, but not a reductase-defective mutant, impedes BSMV infection, whereas low NbTRXh1 expression level results in increased viral accumulation. Similar with its orthologs in Arabidopsis (Arabidopsis thaliana), NbTRXh1 also plays an essential role in SA signaling transduction in N. benthamiana. To counteract NbTRXh1-mediated defenses, the BSMV γb protein targets NbTRXh1 to dampen its reductase activity, thereby impairing downstream SA defense gene expression to optimize viral cell-to-cell movement. We also found that NbTRXh1-mediated resistance defends against lychnis ringspot virus, beet black scorch virus, and beet necrotic yellow vein virus. Taken together, our results reveal a role for the multifunctional γb protein in counteracting plant defense responses and an expanded broad-spectrum antibiotic role of the SA signaling pathway.
Asunto(s)
Virus de Plantas , Ácido Salicílico , Oxidorreductasas/metabolismo , Enfermedades de las Plantas , Virus de Plantas/metabolismo , Ácido Salicílico/metabolismo , Tiorredoxina h/genética , Tiorredoxina h/metabolismo , Nicotiana/metabolismoRESUMEN
Pain is one of the most severe complications affecting the quality of life of cancer patients. Although substantial progress has been made in the diagnosis and treatment of cancer, the neurobiological mechanism of cancer pain is still unclear. In the present study, we identified the critical role of CXC chemokine 2 (CXCL2), released by Schwann cells after being activated by cancer cells, in maintaining cancer-induced macrophage infiltration and the resulting mechanical hypersensitivity and persistent spontaneous nociception. In vitro, Schwann cells cocultured with breast cancer cells exhibited a significant increase in CXCL2 expression; in addition, conditioned medium from Schwann cells activated by breast cancer cells had a similar effect to recombinant CXCL2 in terms of inducing macrophage migration. Targeting CXCL2 signaling by both CXC chemokine receptor 2 (CXCR2) antagonist pharmacological blockade and anti-CXCL2 mAb immunological blockade robustly prevented conditioned medium-induced macrophage migration. In vivo, both application of recombinant CXCL2 and perineural breast cancer cell implantation resulted in mechanical hypersensitivity and persistent spontaneous nociception in mice, along with increased macrophage infiltration into the sciatic nerves. Similar to the in vitro results, inhibition of CXCL2/CXCR2 signaling or conditional knockdown of CXCL2 in sciatic nerve Schwann cells effectively attenuated breast cancer cell-induced mechanical hypersensitivity, persistent spontaneous nociception, and macrophage recruitment in the sciatic nerve. Mechanistically, we found that redox effector factor-1 (Ref-1) secreted by breast cancer cells activated hypoxia inducible factor-1α (HIF-1α) expression and inhibited reactive oxygen species (ROS) production in Schwann cells, ultimately inducing CXCL2 expression in Schwann cells. In brief, the present study expands new insights into cancer pain mechanisms from promising animal models to provide new strategies for the control of cancer pain.
Asunto(s)
Dolor en Cáncer , Neoplasias , Ratones , Animales , Quimiocinas CXC/metabolismo , Dolor en Cáncer/metabolismo , Medios de Cultivo Condicionados/farmacología , Medios de Cultivo Condicionados/metabolismo , Calidad de Vida , Macrófagos/metabolismo , Factores Inmunológicos , Células de Schwann/metabolismo , Neoplasias/metabolismoRESUMEN
Synthetic phenolic antioxidants (SPAs) are an environmental concern due to their persistence nature and bioaccumulation. However, the hepatoxicity and mechanisms of SPAs in aquatic organisms remain poorly understood. In this study, grass carp were exposed to two representative SPAs (BHA and BHT) at environmentally relevant levels (0.1 µM) for 30 days. We observed that BHA and BHT exposure significantly increased the levels of serum aminotransferase (ALT) and aspartate aminotransferase (AST) in grass carp, accompanied by mild inflammatory cell infiltration and irregularity in the shape of hepatocytes. Dihydro ethylenediamine staining showed that BHA and BHT exposure resulted in elevated levels of superoxide levels, accompanied by increased antioxidant enzyme activities (T-AOC, SOD, CAT, GSH-PX) and MDA levels, which is suggestive of oxidative stress responses in the liver of grass carp. Besides, BHA and BHT could dock into the pocket of phosphatidylinositol 3-kinases (PI3K) and thereby inhibiting PI3K/mammalian target of rapamycin (mTOR)/protein kinase B (AKT) signaling cascades. Meanwhile, our results clarified that BHA and BHT could promote autophagosome production and increase the expression of key autophagy proteins, likely due to inhibition of PI3K/mTOR/AKT signaling pathway. Moreover, BHA and BHT could induce apoptotic process by upregulating the expression of Bax, Caspase3 and Caspase8 and downregulating Bcl2 expression. Notably, BHT exhibited more hepatoxicity on the indicators of the apoptosis and oxidative stress than BHA. In summary, our findings demonstrated that BHA and BHT exposure could induce liver damage induced via regulating ROS/PI3K-mediated autophagic hyperactivation, which is a crucial step in triggering hepatocyte death. This study provides novel insight into the potential mechanisms underlying liver damage caused by BHA and BHT in aquatic organisms, and offers a new theoretical basis for ecological risk assessment of SPAs.
Asunto(s)
Antioxidantes , Carpas , Animales , Antioxidantes/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Sirolimus , Carpas/metabolismo , Fenoles , Apoptosis , Autofagia , Serina-Treonina Quinasas TOR/metabolismo , Mamíferos/metabolismoRESUMEN
The magnetoelectric (ME) sensor is a new type of magnetic sensor with ultrahigh sensitivity that suitable for the measurement of low-frequency weak magnetic fields. In this study, a metglas/PZT-5B ME sensor with mechanical resonance frequency fres of 60.041 kHz was prepared. It is interesting to note that its magnetic field resolution reached 0.20 nT at fres and 0.34 nT under a DC field, respectively. In order to measure ultralow-frequency AC magnetic fields, a frequency up-conversion technique was employed. Using this technique, a limit of detection (LOD) under an AC magnetic field lower than 1 nT at 8 Hz was obtained, and the minimum LOD of 0.51 nT was achieved at 20 Hz. The high-resolution ME sensor at the sub-nT level is promising in the field of low-frequency weak magnetic field measurement technology.
RESUMEN
Seed weight is usually associated with seed size and is one of the important agronomic traits that determine yield. Understanding of seed weight control is limited, especially in soybean plants. Here we show that Glycine max JASMONATE-ZIM DOMAIN 3 (GmJAZ3), a gene identified through gene co-expression network analysis, regulates seed-related traits in soybean. Overexpression of GmJAZ3 promotes seed size/weight and other organ sizes in stable transgenic soybean plants likely by increasing cell proliferation. GmJAZ3 interacted with both G. max RESPONSE REGULATOR 18a (GmRR18a) and GmMYC2a to inhibit their transcriptional activation of cytokinin oxidase gene G. max CYTOKININ OXIDASE 3-4 (GmCKX3-4), which usually affects seed traits. Meanwhile, the GmRR18a binds to the promoter of GmMYC2a and activates GmMYC2a gene expression. In GmJAZ3-overexpressing soybean seeds, the protein contents were increased while the fatty acid contents were reduced compared to those in the control seeds, indicating that the GmJAZ3 affects seed size/weight and compositions. Natural variation in JAZ3 promoter region was further analyzed and Hap3 promoter correlates with higher promoter activity, higher gene expression and higher seed weight. The Hap3 promoter may be selected and fixed during soybean domestication. JAZ3 orthologs from other plants/crops may also control seed size and weight. Taken together, our study reveals a novel molecular module GmJAZ3-GmRR18a/GmMYC2a-GmCKXs for seed size and weight control, providing promising targets during soybean molecular breeding for better seed traits.
Asunto(s)
Glycine max , Semillas , Glycine max/metabolismo , Fenotipo , Semillas/genética , Semillas/metabolismo , Perfilación de la Expresión Génica , Ácidos Grasos/metabolismoRESUMEN
Nine genera of viruses in five different families use triple gene block (TGB) proteins for virus movement. The TGB modules fall into two classes: hordei-like and potex-like. Although TGB-mediated viral movement has been extensively studied, determination of the constituents of the viral ribonucleoprotein (vRNP) movement complexes and the mechanisms underlying their involvement in vRNP-mediated movement are far from complete. In the current study, immunoprecipitation of TGB1 protein complexes formed during Barley stripe mosaic virus (BSMV) infection revealed the presence of the γb protein in the products. Further experiments demonstrated that TGB1 interacts with γb in vitro and in vivo, and that γb-TGB1 localizes at the periphery of chloroplasts and plasmodesmata (PD). Subcellular localization analyses of the γb protein in Nicotiana benthamiana epidermal cells indicated that in addition to chloroplast localization, γb also targets the ER, actin filaments and PD at different stages of viral infection. By tracking γb localization during BSMV infection, we demonstrated that γb is required for efficient cell-to-cell movement. The N-terminus of γb interacts with the TGB1 ATPase/helicase domain and enhances ATPase activity of the domain. Inactivation of the TGB1 ATPase activity also significantly impaired PD targeting. In vitro translation together with co-immunoprecipitation (co-IP) analyses revealed that TGB1-TGB3-TGB2 complex formation is enhanced by ATP hydrolysis. The γb protein positively regulates complex formation in the presence of ATP, suggesting that γb has a novel role in BSMV cell-to-cell movement by directly promoting TGB1 ATPase-mediated vRNP movement complex assembly. We further demonstrated that elimination of ATPase activity abrogates PD and actin targeting of Potato virus X (PVX) and Beet necrotic yellow vein virus (BNYVV) TGB1 proteins. These results expand our understanding of the multifunctional roles of γb and provide new insight into the functions of TGB1 ATPase domains in the movement of TGB-encoding viruses.
Asunto(s)
Nicotiana/virología , Proteínas de Movimiento Viral en Plantas/metabolismo , Virus de Plantas/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Ensamble de Virus/fisiología , Adenosina Trifosfatasas/metabolismo , Potexvirus/fisiología , Ribonucleoproteínas/metabolismoRESUMEN
Posttranslational modifications (PTMs) play important roles in virus-host interplay. We previously demonstrated that Barley stripe mosaic virus (BSMV) γb protein is phosphorylated by different host kinases to support or impede viral infection. However, whether and how other types of PTMs participate in BSMV infection remains to be explored. Here, we report that S-adenosylmethionine decarboxylase 3 (SAMDC3) from Nicotiana benthamiana or wheat (Triticum aestivum) interacts with γb. BSMV infection induced SAMDC3 expression. Overexpression of SAMDC3 led to the destabilization of γb and reduction in viral infectivity, whereas knocking out NbSAMDC3 increased susceptibility to BSMV. NbSAMDC3 positively regulated the 26S proteasome-mediated degradation of γb via its PEST domain. Further mechanistic studies revealed that γb can be ubiquitinated in planta and that NbSAMDC3 promotes the proteasomal degradation of γb by increasing γb ubiquitination. We also found evidence that ubiquitination occurs at nonlysine residues (Ser-133 and Cys-144) within γb. Together, our results provide a function for SAMDC3 in defence against BSMV infection through targeting of γb abundance, which contributes to our understanding of how a plant host deploys the ubiquitin-proteasome system to mount defences against viral infections.
Asunto(s)
Hordeum , Virus de Plantas , Adenosilmetionina Descarboxilasa/metabolismo , Hordeum/metabolismo , Virus de Plantas/metabolismo , Ubiquitinación , Proteínas Virales/metabolismoRESUMEN
Protein phosphorylation is a common post-translational modification that frequently occurs during plant-virus interaction. Host protein kinases often regulate virus infectivity and pathogenicity by phosphorylating viral proteins. The Barley stripe mosaic virus (BSMV) γb protein plays versatile roles in virus infection and the coevolutionary arms race between plant defense and viral counter-defense. Here, we identified that the autophosphorylated cytosolic serine/threonine/tyrosine (STY) protein kinase 46 of Nicotiana benthamiana (NbSTY46) phosphorylates and directly interacts with the basic motif domain (aa 19-47) of γb in vitro and in vivo. Overexpression of wild-type NbSTY46, either transiently or transgenically, suppresses BSMV replication and ameliorates viral symptoms, whereas silencing of NbSTY46 leads to increased viral replication and exacerbated symptom. Moreover, the antiviral role of NbSTY46 requires its kinase activity, as the NbSTY46T436A mutant, lacking kinase activity, not only loses the ability to phosphorylate and interact with γb but also fails to impair BSMV infection when expressed in plants. NbSTY46 could also inhibit the replication of Lychnis ringspot virus, another chloroplast-replicating hordeivirus. In summary, we report a function of the cytosolic kinase STY46 in defending against plant viral infection by phosphorylating a viral protein in addition to its basal function in plant growth, development, and abiotic stress responses.
Asunto(s)
Nicotiana/inmunología , Proteínas de Plantas/genética , Virus de Plantas/fisiología , Proteínas Serina-Treonina Quinasas/genética , Virus ARN/fisiología , Fosforilación , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Nicotiana/genética , Nicotiana/virologíaRESUMEN
Omega-transaminases (ω-TAs) have attracted considerable interest for their use in asymmetric synthesis of chiral amines with a high degree of optical purity and yield. The rapid evaluation of the characteristics of newly identified or engineered ω-TAs is important for industrial applications. In this study, a visible spectrophotometric assay was developed for rapid quantitative determination of ω-TA activities based on the transamination of 2-(4-nitrophenyl)ethan-1-amine to generate a red product (E)-N-(4-nitrophenethyl)-2-(4-nitrophenyl)ethen-1-amine. After various co-solvents were evaluated, dimethyl sulfoxide (DMSO) was considered optimal because the red product exhibits good solubility and retains its original color. The red product dissolved in DMSO has its highest absorbance at 465 nm, and its concentration has a good linear relationship with the absorbance. A spectrophotometric assay was established and validated using conventional HPLC analysis (<10% divergence). This method was then used to characterize an ω-TA from thermophilic Geobacillus thermoleovorans and an ω-TA obtained from the metagenome of a soda lake. The results demonstrated that ω-transaminase enzymatic properties could be characterized simply, rapidly, and at low cost, using this newly established visible spectrophotometric assay method.
Asunto(s)
Dimetilsulfóxido , Transaminasas , Aminas , SolventesRESUMEN
Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, hampering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.
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Ciclotidas , Secuencia de Aminoácidos , Cristalografía , Cistina , Pliegue de ProteínaRESUMEN
Poa semilatent virus (PSLV), Lychnis ringspot virus (LRSV), and Barley stripe mosaic virus (BSMV) are members of the genus Hordeivirus in the family Virgaviridae. However, the biological properties and molecular genetics of PSLV have not been compared with other hordeiviruses. Here, we have constructed an infectious cDNA clone of the PSLV Canadian strain and provided evidence that PSLV differs from BSMV and LRSV. First, unlike the other two hordeiviruses that replicate in chloroplasts, PSLV induces dramatic structural changes in peroxisome during its infection in barley. The αa replication protein also localizes to peroxisomes, suggesting that PSLV replication occurs in peroxisomes. Second, PSLV encodes a γb protein that shares 19 to 23% identity with those of other hordeiviruses, and its activity as a viral suppressor of RNA (VSR) silencing is distinct from those of BSMV and LRSV. Substitution of the BSMV γb protein with that of PSLV or LRSV revealed a negative correlation between VSR activity and symptom severity of the recombinant BSMV derivatives. Intriguingly, the Ser-Lys-Leu (SKL) peroxisome-targeting signals differ among γb proteins of various hordeiviruses, including some BSMV strains. The presence of the C-terminal SKL motif in the γb protein impairs its silencing suppressor activity and influences symptoms. Finally, we developed a PSLV-based virus-induced gene silencing vector that induced strong and effective silencing phenotypes of endogenous genes in barley, wheat, and millet. Our results shed new light on hordeivirus pathogenesis and evolution, and provide an alternative tool for genomics studies of model hosts and economically important monocots.
Asunto(s)
Hordeum , Enfermedades de las Plantas , Virus de Plantas , Virus ARN , ARN Viral , Proteínas Virales , Canadá , ADN Complementario/genética , Hordeum/virología , Enfermedades de las Plantas/virología , Virus de Plantas/genética , Virus de Plantas/patogenicidad , Virus ARN/genética , Virus ARN/patogenicidad , Virulencia/genéticaRESUMEN
Chloroplasts are multifunctional organelles whose morphology is affected by environmental stresses. Although the three-dimensional (3D) architecture of thylakoid membranes has been reported previously, a 3D visualization of chloroplast under stress has not been explored. In this work, we used a positive-strand RNA ((+)RNA) virus, barley stripe mosaic virus (BSMV) to observe chloroplast structural changes during infection by electron tomography. The analyses revealed remodeling of the chloroplast membranes, characterized by the clustering of outer membrane-invaginated spherules in inner membrane-derived packets. Diverse morphologies of cytoplasmic invaginations (CIs) were evident with spherules at the periphery and different sized openings connecting the CIs to the cytoplasm. Immunoelectron microscopy of these viral components verified that the aberrant membrane structures were sites for BSMV replication. The BSMV αa replication protein localized at the surface of the chloroplasts and played a prominent role in eliciting chloroplast membrane rearrangements. In sum, our results have revealed the 3D structure of the chloroplasts induced by BSMV infection. These findings contribute to our understanding of chloroplast morphological changes under stress conditions and during assembly of plant (+)RNA virus replication complexes.