RESUMEN
The lipopeptide antibiotic daptomycin exhibits bactericidal activity against Gram-positive bacteria by forming a complex with phosphatidylglycerol (PG) and lipid II in the cell membrane, causing membrane perforation. With the emergence of daptomycin-resistant bacteria, understanding the mechanisms of bacterial resistance to daptomycin has gained great importance. In this study, we aimed to identify the genetic factors contributing to daptomycin resistance in Bacillus subtilis, a model Gram-positive bacterium. Our findings demonstrated that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis. Specifically, overexpression of ugtP resulted in increased levels of diglucosyldiacylglycerol (Glc2DAG) and decreased levels of acidic phospholipids cardiolipin and PG, as well as the basic phospholipid lysylphosphatidylglycerol. However, ugtP overexpression did not alter the cell surface charge and the susceptibility to the cationic antimicrobial peptide nisin or the cationic surfactant hexadecyltrimethylammonium bromide. Furthermore, by serial passaging in the presence of daptomycin, we obtained daptomycin-resistant mutants carrying ugtP mutations. These mutants showed increased levels of Glc2DAG and a >4-fold increase in the minimum inhibitory concentration of daptomycin. These results suggest that increased Glc2DAG levels, driven by ugtP overexpression, modify the phospholipid composition and confer daptomycin resistance in B. subtilis without altering the cell surface charge of the bacteria.IMPORTANCEDaptomycin is one of the last-resort drugs for the treatment of methicillin-resistant Staphylococcus aureus infections, and the emergence of daptomycin-resistant bacteria has become a major concern. Understanding the mechanism of daptomycin resistance is important for establishing clinical countermeasures against daptomycin-resistant bacteria. In the present study, we found that overexpression of ugtP, which encodes diglucosyldiacylglycerol synthase, induces daptomycin resistance in B. subtilis, a model Gram-positive bacteria. The overexpression of UgtP increased diglucosyldiacylglycerol levels, resulting in altered phospholipid composition and daptomycin resistance. These findings are important for establishing clinical strategies against daptomycin-resistant bacteria, including their detection and management.
RESUMEN
In heterotrophs, heme degradation produces bilirubin, a tetrapyrrole compound that has antioxidant activity. In plants, heme is degraded in plastids and is believed to be converted to phytochromobilin rather than bilirubin. Recently, we used the bilirubin-inducible fluorescent protein UnaG to reveal that plants produce bilirubin via a non-enzymatic reaction with NADPH. In the present study, we used an UnaG-based live imaging system to visualize bilirubin accumulation in Arabidopsis thaliana and Nicotiana benthamiana at the organelle and tissue levels. In chloroplasts, bilirubin preferentially accumulated in the stroma, and the stromal bilirubin level increased upon dark treatment. Investigation of intracellular bilirubin distribution in leaves and roots showed that it accumulated mostly in plastids, with low levels detected in the cytosol and other organelles, such as peroxisomes, mitochondria and the endoplasmic reticulum. A treatment that increased bilirubin production in chloroplasts decreased the bilirubin level in peroxisomes, implying that a bilirubin precursor is transported between the two organelles. At the cell and tissue levels, bilirubin showed substantial accumulation in the root elongation region but little or none in the root cap and guard cells. Intermediate bilirubin accumulation was observed in other shoot and root tissues, with lower levels in shoot tissues. Our data revealed the distribution of bilirubin in plants, which has implications for the transport and physiological function of tetrapyrroles.
Asunto(s)
Arabidopsis , Bilirrubina , Nicotiana , Raíces de Plantas , Arabidopsis/metabolismo , Nicotiana/metabolismo , Bilirrubina/metabolismo , Raíces de Plantas/metabolismo , Hojas de la Planta/metabolismo , Cloroplastos/metabolismo , Peroxisomas/metabolismoRESUMEN
When infecting plants, fungal pathogens secrete cell wall-degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsRMC binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsRMC to various CBM1-containing enzymes, suggesting that it has a general role in inhibiting the action of CBM1. OsRMC is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown and overexpression of OsRMC reduced and enhanced rice defense against M. oryzae, respectively, demonstrating that inhibition of CBM1-containing fungal enzymes by OsRMC is crucial for rice defense. We also identified additional CBM-interacting proteins (CBMIPs) from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.
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Arabidopsis , Oryza , Celulosa , Cisteína , Proteínas Fúngicas/genética , Oryza/genética , XilanosRESUMEN
The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Estrés Fisiológico , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Fosforilación , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Plant cells alter the intracellular positions of chloroplasts to ensure efficient photosynthesis, a process controlled by the blue light receptor phototropin. Chloroplasts migrate toward weak light (accumulation response) and move away from excess light (avoidance response). Chloroplasts are encircled by the endoplasmic reticulum (ER), which forms a complex network throughout the cytoplasm. To ensure rapid chloroplast relocation, the ER must alter its structure in conjunction with chloroplast relocation movement, but little is known about the underlying mechanism. Here, we searched for interactors of phototropin in the liverwort Marchantia polymorpha and identified a RETICULON (RTN) family protein; RTN proteins play central roles in ER tubule formation and ER network maintenance by stabilizing the curvature of ER membranes in eukaryotic cells. Marchantia polymorpha RTN1 (MpRTN1) is localized to ER tubules and the rims of ER sheets, which is consistent with the localization of RTNs in other plants and heterotrophs. The Mprtn1 mutant showed an increased ER tubule diameter, pointing to a role for MpRTN1 in ER membrane constriction. Furthermore, Mprtn1 showed a delayed chloroplast avoidance response but a normal chloroplast accumulation response. The live cell imaging of ER dynamics revealed that ER restructuring was impaired in Mprtn1 during the chloroplast avoidance response. These results suggest that during the chloroplast avoidance response, MpRTN1 restructures the ER network and facilitates chloroplast movement via an interaction with phototropin. Our findings provide evidence that plant cells respond to fluctuating environmental conditions by controlling the movements of multiple organelles in a synchronized manner.
Asunto(s)
Marchantia , Cloroplastos/metabolismo , Retículo Endoplásmico/metabolismo , Luz , Marchantia/fisiología , Fototropinas/metabolismoRESUMEN
KEY MESSAGE: By using the organelle glue technique, we artificially manipulated organelle interactions and controlled the plant metabolome at the pathway level. Plant cell metabolic activity changes with fluctuating environmental conditions, in part via adjustments in the arrangement and interaction of organelles. This hints at the potential for designing plants with desirable metabolic activities for food and pharmaceutical industries by artificially controlling the interaction of organelles through genetic modification. We previously developed a method called the organelle glue technique, in which chloroplast-chloroplast adhesion is induced in plant cells using the multimerization properties of split fluorescent proteins. Here, we generated transgenic Arabidopsis (Arabidopsis thaliana) plants in which chloroplasts adhere to each other and performed metabolome analysis to examine the metabolic changes in these lines. In plant cells expressing a construct encoding the red fluorescent protein mCherry targeted to the chloroplast outer envelope by fusion with a signal sequence (cTP-mCherry), chloroplasts adhered to each other and formed chloroplast aggregations. Mitochondria and peroxisomes were embedded in the aggregates, suggesting that normal interactions between chloroplasts and these organelles were also affected. Metabolome analysis of the cTP-mCherry-expressing Arabidopsis shoots revealed significantly higher levels of glycine, serine, and glycerate compared to control plants. Notably, these are photorespiratory metabolites that are normally transported between chloroplasts, mitochondria, and peroxisomes. Together, our data indicate that chloroplast-chloroplast adhesion alters organellar interactions with mitochondria and peroxisomes and disrupts photorespiratory metabolite transport. These results highlight the possibility of controlling plant metabolism at the pathway level by manipulating organelle interactions.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Células Vegetales/metabolismo , Cloroplastos/metabolismo , Peroxisomas/metabolismo , Proteínas de Arabidopsis/genética , MetabolomaRESUMEN
The flavonoid glycoside apiin (apigenin 7-O-[ß-D-apiosyl-(1â2)-ß-D-glucoside]) is abundant in apiaceous and asteraceous plants, including celery and parsley. Although several enzymes involved in apiin biosynthesis have been identified in celery, many of the enzymes in parsley (Petroselinum crispum) have not been identified. In this study, we identified parsley genes encoding the glucosyltransferase, PcGlcT, and the apiosyltransferase, PcApiT, that catalyze the glycosylation steps of apiin biosynthesis. Their substrate specificities showed that they were involved in the biosynthesis of some flavonoid 7-O-apiosylglucosides, including apiin. The expression profiles of PcGlcT and PcApiT were closely correlated with the accumulation of flavonoid 7-O-apiosylglucosides in parsley organs and developmental stages. These findings support the idea that PcGlcT and PcApiT are involved in the biosynthesis of flavonoid 7-O-apiosylglucosides in parsley. The identification of these genes will elucidate the physiological significance of apiin and the development of apiin production methods.
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Apium , Glicósidos Cardíacos , Glicósidos/química , Petroselinum/química , Glicosiltransferasas/genética , Flavonoides/químicaRESUMEN
Vancomycin resistance of Gram-positive bacteria poses a serious health concern around the world. In this study, we searched for vancomycin-tolerant mutants from a gene deletion library of a model Gram-positive bacterium, Bacillus subtilis, to elucidate the mechanism of vancomycin resistance. We found that knockout of ykcB, a glycosyltransferase that is expected to utilize C55-P-glucose to glycosylate cell surface components, caused reduced susceptibility to vancomycin in B. subtilis. Knockout of ykcB altered the susceptibility to multiple antibiotics, including sensitization to ß-lactams and increased the pathogenicity to silkworms. Furthermore, the ykcB-knockout mutant had (i) a decreased amount of lipoteichoic acid, (ii) decreased biofilm formation, and (iii) an increased content of diglucosyl diacylglycerol, a glycolipid that shares a precursor with C55-P-glucose. These phenotypes and vancomycin tolerance were abolished by knockout of ykcC, a gene in the same operon with ykcB probably involved in C55-P-glucose synthesis. Overexpression of ykcC enhanced vancomycin tolerance in both the parent strain and the ykcB-knockout mutant. These findings suggest that ykcB deficiency induces structural changes of cell surface molecules depending on the ykcC function, leading to reduced susceptibility to vancomycin, decreased biofilm formation, and increased pathogenicity to silkworms. IMPORTANCE Although vancomycin is effective against Gram-positive bacteria, vancomycin-resistant bacteria are a major public health concern. While the vancomycin-resistance mechanisms of clinically important bacteria such as Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae are well studied, they remain unclear in other Gram-positive bacteria. In the present study, we searched for vancomycin-tolerant mutants from a gene deletion library of a model Gram-positive bacterium, Bacillus subtilis, and found that knockout of a putative glycosyltransferase, ykcB, caused vancomycin tolerance in B. subtilis. Notably, unlike the previously reported vancomycin-resistant bacterial strains, ykcB-deficient B. subtilis exhibited increased virulence while maintaining its growth rate. Our results broaden the fundamental understanding of vancomycin-resistance mechanisms in Gram-positive bacteria.
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Antibacterianos , Bacillus subtilis , Vancomicina , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/genética , Glicosiltransferasas/genética , Pruebas de Sensibilidad Microbiana , Vancomicina/farmacología , Farmacorresistencia BacterianaRESUMEN
Resistance (R) genes encode intracellular nucleotide-binding/leucine-rich repeat-containing (NLR) family proteins that serve as critical plant immune receptors to induce effector-triggered immunity (ETI). NLR proteins possess a tripartite domain architecture consisting of an N-terminal variable region, a central nucleotide-binding domain, and a C-terminal leucine-rich repeat. N-terminal coiled-coil (CC) or Toll-interleukin 1 receptor (TIR) domains of R proteins appear to serve as platforms to trigger immune responses, because overexpression of the CC or TIR domain of some R proteins is sufficient to induce an immune response. Because direct downstream signaling molecules of R proteins remain obscure, the molecular mechanisms by which R proteins regulate downstream signaling are largely unknown. We reported previously that a rice R protein named Pit triggers ETI through a small GTPase, OsRac1, although how Pit activates OsRac1 is unclear. Here, we identified OsSPK1, a DOCK family guanine nucleotide exchange factor, as an interactor of Pit and activator for OsRac1. OsSPK1 contributes to signaling by two disease-resistance genes, Pit and Pia, against the rice blast fungus Magnaporthe oryzae and facilitates OsRac1 activation in vitro and in vivo. The CC domain of Pit is required for its binding to OsSPK1, OsRac1 activation, and the induction of cell death. Overall, we conclude that OsSPK1 is a direct and key signaling target of Pit-mediated immunity. Our results shed light on how R proteins trigger ETI through direct downstream molecules.
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Oryza/genética , Oryza/inmunología , Enfermedades de las Plantas/inmunología , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas , Magnaporthe , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genéticaRESUMEN
Perception of microbe-associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen-activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho-signaling transduction pathway from PRR-mediated pathogen recognition to MAPK activation in plants. We found that the receptor-like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1-LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin-induced MAPK activation and disease resistance to Alternaria brassicicola PBL27 phosphorylates MAPKKK5 in vitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin-induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Quitina/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Alternaria/inmunología , Alternaria/patogenicidad , Arabidopsis/enzimología , Arabidopsis/genética , Membrana Celular/metabolismo , Técnicas de Inactivación de Genes , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiologíaRESUMEN
Synaptotagmin 1 (SYT1) has been recognised as a tethering factor of plant endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (EPCSs) and partially localises to around plasmodesmata (PD). However, other components of EPCSs associated with SYT1 and functional links between the EPCSs and PD have not been identified. We explored interactors of SYT1 by immunoprecipitation and mass analysis. The dynamics, morphology and spatial arrangement of the ER in Arabidopsis mutants lacking the EPCS components were investigated using confocal microscopy and electron microscopy. PD permeability of EPCS mutants was assessed using a virus movement protein and free green fluorescent protein (GFP) as indicators. We identified two additional components of the EPCSs, SYT5 and SYT7, that interact with SYT1. The mutants of the three SYTs were defective in the anchoring of the ER to the PM. The ER near the PD entrance appeared to be weakly squeezed in the triple mutant compared with the wild-type. The triple mutant suppressed cell-to-cell movement of the virus movement protein, but not GFP diffusion. We revealed major additional components of EPCS associated with SYT1 and suggested that the EPCSs arranged around the PD squeeze the ER to regulate active transport via PD.
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Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Plasmodesmos/metabolismo , Sinaptotagmina IRESUMEN
Human papillomavirus (HPV) has been identified as a causative agent of cervical cancer and oropharyngeal cancer (OPC). Intriguingly, estrogen and HPV were shown to play synergistic roles in cervical carcinogenesis. We recently demonstrated that the apolipoprotein B mRNA-editing catalytic polypeptide 3 (APOBEC3, A3) family, which is inducible by estrogen, could lead to HPV DNA hypermutation and cause viral DNA integration. In the present study, we examined the relationships between estrogen-estrogen receptor α (ERα) and A3s in HPV-positive OPC. ERα expression was associated with HPV positivity in OPC biopsy samples using immunohistochemical analysis and reverse-transcription quantitative polymerase chain reaction. In addition, ERα was significantly associated with improved overall survival in HPV-positive OPC (hazard ratio, 0.26; p = 0.029). APOBEC3A (A3A) mRNA was induced by estrogen in HPV and ERα-positive OPC cells. Furthermore, A3A mRNA and protein expression were significantly higher in ERα-positive cases than in ERα-negative ones, among HPV-positive biopsy samples (p = 0.037 and 0.047). These findings suggest that A3A is associated with a good prognosis in ERα-positive OPC, and indicate the prognostic significance of ERα in HPV-positive OPC. This is the first study to demonstrate the prognostic role of ERα in HPV-positive OPC.
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Alphapapillomavirus/aislamiento & purificación , Receptor alfa de Estrógeno/metabolismo , Neoplasias Orofaríngeas/patología , Anciano , Línea Celular Tumoral , Citidina Desaminasa/genética , Citidina Desaminasa/metabolismo , Estrógenos/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Orofaríngeas/metabolismo , Neoplasias Orofaríngeas/virología , Pronóstico , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Plant virus movement proteins (MPs) localize to plasmodesmata (PD) to facilitate virus cell-to-cell movement. Numerous studies have suggested that MPs use a pathway either through the ER or through the plasma membrane (PM). Furthermore, recent studies reported that ER-PM contact sites and PM microdomains, which are subdomains found in the ER and PM, are involved in virus cell-to-cell movement. However, functional relationship of these subdomains in MP traffic to PD has not been described previously. We demonstrate here the intracellular trafficking of fig mosaic virus MP (MPFMV) using live cell imaging, focusing on its ER-directing signal peptide (SPFMV). Transiently expressed MPFMV was distributed predominantly in PD and patchy microdomains of the PM. Investigation of ER translocation efficiency revealed that SPFMV has quite low efficiency compared with SPs of well-characterized plant proteins, calreticulin and CLAVATA3. An MPFMV mutant lacking SPFMV localized exclusively to the PM microdomains, whereas SP chimeras, in which the SP of MPFMV was replaced by an SP of calreticulin or CLAVATA3, localized exclusively to the nodes of the ER, which was labeled with Arabidopsis synaptotagmin 1, a major component of ER-PM contact sites. From these results, we speculated that the low translocation efficiency of SPFMV contributes to the generation of ER-translocated and the microdomain-localized populations, both of which are necessary for PD localization. Consistent with this hypothesis, SP-deficient MPFMV became localized to PD when co-expressed with an SP chimera. Here we propose a new model for the intracellular trafficking of a viral MP. A substantial portion of MPFMV that fails to be translocated is transferred to the microdomains, whereas the remainder of MPFMV that is successfully translocated into the ER subsequently localizes to ER-PM contact sites and plays an important role in the entry of the microdomain-localized MPFMV into PD.
Asunto(s)
Arabidopsis/virología , Membrana Celular/virología , Retículo Endoplásmico/metabolismo , Proteínas de Movimiento Viral en Plantas/metabolismo , Plasmodesmos/virología , Virus del Mosaico del Tabaco/aislamiento & purificación , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/virología , Microdominios de Membrana/metabolismo , Microdominios de Membrana/virología , Microtúbulos/metabolismo , Microtúbulos/virología , Plasmodesmos/metabolismo , Transporte de Proteínas/fisiología , Nicotiana/virología , Virus del Mosaico del Tabaco/metabolismoRESUMEN
The plant endoplasmic reticulum (ER), which is morphologically divided into tubules and sheets, seems to flow continuously as a whole, but locally, mobile and immobile regions exist. In eukaryotes, the ER physically and functionally interacts with the plasma membrane (PM) at domains called ER-PM contact sites (EPCSs). Extended synaptotagmin family proteins are concentrated in the cortical ER to form one type of EPCS; however, it is unclear whether the localization of extended synaptotagmin corresponds to the EPCS and where in the cortical ER the EPCSs are formed. Here, we analyzed the spatiotemporal localization of SYNAPTOTAGMIN1 (SYT1), a synaptotagmin in Arabidopsis (Arabidopsis thaliana), to investigate the precise distribution of SYT1-associated EPCSs in the cortical ER. Three-dimensional imaging using superresolution confocal live imaging microscopy demonstrated that SYT1 was specifically localized to the ER-PM boundary. Time-lapse imaging revealed that SYT1 was distributed to immobile ER tubules, but not to mobile tubules. Moreover, SYT1 was frequently localized to the edges of ER sheets that were transformed into immobile ER tubules over time. A lower intracellular calcium ion concentration resulted in an increased EPCS area and disrupted the ER network. Finally, SYT1 deficiency caused a reduction of the immobile tubules and enlargement of the ER meshes. Taken together, our findings show that SYT1-associated EPCS are distributed to immobile tubules and play an important role in the formation of the tubular ER network. This provides important insight into the relationship between the function and dynamics/morphology of the cortical ER.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Sinaptotagmina I/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Sinaptotagmina I/genéticaRESUMEN
Nasopharyngeal carcinoma (NPC) is very common in southern China and Southeast Asia. In regions where NPC is endemic, undifferentiated subtypes constitute most cases and are invariably associated with Epstein-Barr virus (EBV) infection, whereas the differentiated subtype is more common in other parts of the world. Undifferentiated NPC is a unique malignancy with regard to its epidemiology, etiology, and clinical presentation. Clinically, NPC is highly invasive and metastatic, but sensitive to both chemotherapy and radiotherapy (RT). Overall prognosis has dramatically improved over the past three decades because of advances in management, including the improvement of RT technology, the broader application of chemotherapy, and more accurate disease staging. Despite the excellent local control with modern RT, distant failure remains a challenging problem. Advances in molecular technology have helped to elucidate the molecular pathogenesis of NPC. This article reviews the contribution of EBV gene products to NPC pathogenesis and the current management of NPC.
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Infecciones por Virus de Epstein-Barr/fisiopatología , Herpesvirus Humano 4/metabolismo , Neoplasias Nasofaríngeas/terapia , Neoplasias Nasofaríngeas/virología , Proteínas Virales/metabolismo , Infecciones por Virus de Epstein-Barr/virología , Humanos , Proteínas de la Matriz Viral/metabolismoRESUMEN
Latent membrane protein 1 (LMP1) is a primary oncogene encoded by the Epstein-Barr virus, and various portions of LMP1 are detected in nasopharyngeal carcinoma (NPC) tumor cells. LMP1 has been extensively studied since the discovery of its transforming property in 1985. LMP1 promotes cancer cell growth during NPC development and facilitates the interaction of cancer cells with surrounding stromal cells for invasion, angiogenesis, and immune modulation. LMP1 is detected in 100% of pre-invasive NPC tumors and in approximately 50% of advanced NPC tumors. Moreover, a small population of LMP1-expressing cells in advanced NPC tumor tissue is proposed to orchestrate NPC tumor tissue maintenance and development through cancer stem cells and progenitor cells. Recent studies suggest that LMP1 activity shifts according to tumor development stage, but it still has a pivotal role during all stages of NPC development.
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Carcinoma/patología , Infecciones por Virus de Epstein-Barr/metabolismo , Neoplasias Nasofaríngeas/patología , Proteínas de la Matriz Viral/metabolismo , Carcinoma/metabolismo , Carcinoma/virología , Proliferación Celular , Herpesvirus Humano 4/metabolismo , Humanos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/virología , Estadificación de Neoplasias , Células Madre Neoplásicas/metabolismo , Microambiente TumoralRESUMEN
One of the plant host resistance machineries to viruses is attributed to recessive alleles of genes encoding critical host factors for virus infection. This type of resistance, also referred to as recessive resistance, is useful for revealing plant-virus interactions and for breeding antivirus resistance in crop plants. Therefore, it is important to identify a novel host factor responsible for robust recessive resistance to plant viruses. Here, we identified a mutant from an ethylmethane sulfonate (EMS)-mutagenized Arabidopsis population which confers resistance to plantago asiatica mosaic virus (PlAMV, genus Potexvirus). Based on map-based cloning and single nucleotide polymorphism analysis, we identified a premature termination codon in a functionally unknown gene containing a GYF domain, which binds to proline-rich sequences in eukaryotes. Complementation analyses and robust resistance to PlAMV in a T-DNA mutant demonstrated that this gene, named Essential for poteXvirus Accumulation 1 (EXA1), is indispensable for PlAMV infection. EXA1 contains a GYF domain and a conserved motif for interaction with eukaryotic translation initiation factor 4E (eIF4E), and is highly conserved among monocot and dicot species. Analysis using qRT-PCR and immunoblotting revealed that EXA1 was expressed in all tissues, and was not transcriptionally responsive to PlAMV infection in Arabidopsis plants. Moreover, accumulation of PlAMV and a PlAMV-derived replicon was drastically diminished in the initially infected cells by the EXA1 deficiency. Accumulation of two other potexviruses also decreased in exa1-1 mutant plants. Our results provided a functional annotation to GYF domain-containing proteins by revealing the function of the highly conserved EXA1 gene in plant-virus interactions.
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Arabidopsis/metabolismo , Arabidopsis/virología , Enfermedades de las Plantas/virología , Virus de Plantas/patogenicidad , Arabidopsis/genética , Enfermedades de las Plantas/genéticaRESUMEN
UNLABELLED: Although many studies have demonstrated intracellular movement of viral proteins or viral replication complexes, little is known about the mechanisms of their motility. In this study, we analyzed the localization and motility of the nucleocapsid protein (NP) of Fig mosaic virus (FMV), a negative-strand RNA virus belonging to the recently established genus Emaravirus. Electron microscopy of FMV-infected cells using immunogold labeling showed that NPs formed cytoplasmic agglomerates that were predominantly enveloped by the endoplasmic reticulum (ER) membrane, while nonenveloped NP agglomerates also localized along the ER. Likewise, transiently expressed NPs formed agglomerates, designated NP bodies (NBs), in close proximity to the ER, as was the case in FMV-infected cells. Subcellular fractionation and electron microscopic analyses of NP-expressing cells revealed that NBs localized in the cytoplasm. Furthermore, we found that NBs moved rapidly with the streaming of the ER in an actomyosin-dependent manner. Brefeldin A treatment at a high concentration to disturb the ER network configuration induced aberrant accumulation of NBs in the perinuclear region, indicating that the ER network configuration is related to NB localization. Dominant negative inhibition of the class XI myosins, XI-1, XI-2, and XI-K, affected both ER streaming and NB movement in a similar pattern. Taken together, these results showed that NBs localize in the cytoplasm but in close proximity to the ER membrane to form enveloped particles and that this causes passive movements of cytoplasmic NBs by ER streaming. IMPORTANCE: Intracellular trafficking is a primary and essential step for the cell-to-cell movement of viruses. To date, many studies have demonstrated the rapid intracellular movement of viral factors but have failed to provide evidence for the mechanism or biological significance of this motility. Here, we observed that agglomerates of nucleocapsid protein (NP) moved rapidly throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses.
Asunto(s)
Citoplasma/virología , Retículo Endoplásmico/virología , Proteínas de la Nucleocápside/metabolismo , Virus de Plantas/fisiología , Multimerización de Proteína , Virus ARN/fisiología , Ficus , Microscopía Inmunoelectrónica , Transporte de Proteínas , NicotianaRESUMEN
Plant pathogens alter the course of plant developmental processes, resulting in abnormal morphology in infected host plants. Phytoplasmas are unique plant-pathogenic bacteria that transform plant floral organs into leaf-like structures and cause the emergence of secondary flowers. These distinctive symptoms have attracted considerable interest for many years. Here, we revealed the molecular mechanisms of the floral symptoms by focusing on a phytoplasma-secreted protein, PHYL1, which induces morphological changes in flowers that are similar to those seen in phytoplasma-infected plants. PHYL1 is a homolog of the phytoplasmal effector SAP54 that also alters floral development. Using yeast two-hybrid and in planta transient co-expression assays, we found that PHYL1 interacts with and degrades the floral homeotic MADS domain proteins SEPALLATA3 (SEP3), APETALA1 (AP1) and CAULIFLOWER (CAL). This degradation of MADS domain proteins was dependent on the ubiquitin-proteasome pathway. The expression of floral development genes downstream of SEP3 and AP1 was disrupted in 35S::PHYL1 transgenic plants. PHYL1 was genetically and functionally conserved among other phytoplasma strains and species. We designate PHYL1, SAP54 and their homologs as members of the phyllody-inducing gene family of 'phyllogens'.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Flores/metabolismo , Proteínas de Dominio MADS/metabolismo , Phytoplasma/metabolismo , Hojas de la Planta/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Flores/genética , Flores/ultraestructura , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Interacciones Huésped-Patógeno , Immunoblotting , Proteínas de Dominio MADS/genética , Microscopía Confocal , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Phytoplasma/genética , Hojas de la Planta/genética , Hojas de la Planta/ultraestructura , Plantas Modificadas Genéticamente , Unión Proteica , Proteolisis , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Systemic necrosis is one of the most severe symptoms caused by plant RNA viruses. Recently, systemic necrosis has been suggested to have similar features to a defense response referred to as the hypersensitive response (HR), a form of programmed cell death. In virus-infected plant cells, host intracellular membrane structures are changed dramatically for more efficient viral replication. However, little is known about whether this replication-associated membrane modification is the cause of the symptoms. In this study, we identified an amino-terminal amphipathic helix of the helicase encoded by Radish mosaic virus (RaMV) (genus Comovirus) as an elicitor of cell death in RaMV-infected plants. Cell death caused by the amphipathic helix had features similar to HR, such as SGT1-dependence. Mutational analyses and inhibitor assays using cerulenin demonstrated that the amphipathic helix-induced cell death was tightly correlated with dramatic alterations in endoplasmic reticulum (ER) membrane structures. Furthermore, the cell death-inducing activity of the amphipathic helix was conserved in Cowpea mosaic virus (genus Comovirus) and Tobacco ringspot virus (genus Nepovirus), both of which are classified in the family Secoviridae. Together, these results indicate that ER membrane modification associated with viral intracellular replication may be recognized to prime defense responses against plant viruses.