RESUMEN
We recently reported that the p28 auxiliary replication protein encoded by turnip crinkle virus (TCV) is also responsible for eliciting superinfection exclusion (SIE) against superinfecting TCV. However, it remains unresolved whether the replication function of p28 could be separated from its ability to elicit SIE. Here, we report the identification of two single amino acid mutations that decouple these two functions. Using an Agrobacterium infiltration-based delivery system, we transiently expressed a series of p28 deletion and point mutants, and tested their ability to elicit SIE against a cointroduced TCV replicon. We found that substituting alanine (A) for valine (V) and phenylalanine (F) at p28 positions 181 and 182, respectively, modestly compromised SIE in transiently expressed p28 derivatives. Upon incorporation into TCV replicons, V181A and F182A decoupled TCV replication and SIE diametrically. Although V181A impaired SIE without detectably compromising replication, F182A abolished TCV replication but had no effect on SIE once the replication of the defective replicon was restored through complementation. Both mutations diminished accumulation of p28 protein, suggesting that p28 must reach a concentration threshold in order to elicit a strong SIE. Importantly, the severe reduction of F182A protein levels correlated with a dramatic loss in the number of intracellular p28 foci formed by p28-p28 interactions. Together, these findings not only decouple the replication and SIE functions of p28 but also unveil a concentration dependence for p28 coalescence and SIE elicitation. These data further highlight the role of p28 multimerization in driving the exclusion of secondary TCV infections.
Asunto(s)
Carmovirus , Replicación Viral , Carmovirus/genética , Carmovirus/fisiología , Eliminación de Secuencia , Replicación Viral/genéticaRESUMEN
RNA-based silencing functions as an important antiviral immunity mechanism in plants. Plant viruses evolved to encode viral suppressors of RNA silencing (VSRs) that interfere with the function of key components in the silencing pathway. As effectors in the RNA silencing pathway, ARGONAUTE (AGO) proteins are targeted by some VSRs, such as that encoded by Turnip crinkle virus (TCV). A VSR-deficient TCV mutant was used to identify AGO proteins with antiviral activities during infection. A quantitative phenotyping protocol using an image-based color trait analysis pipeline on the PlantCV platform, with temporal red, green, and blue imaging and a computational segmentation algorithm, was used to measure plant disease after TCV inoculation. This process captured and analyzed growth and leaf color of Arabidopsis (Arabidopsis thaliana) plants in response to virus infection over time. By combining this quantitative phenotypic data with molecular assays to detect local and systemic virus accumulation, AGO2, AGO3, and AGO7 were shown to play antiviral roles during TCV infection. In leaves, AGO2 and AGO7 functioned as prominent nonadditive, anti-TCV effectors, whereas AGO3 played a minor role. Other AGOs were required to protect inflorescence tissues against TCV. Overall, these results indicate that distinct AGO proteins have specialized, modular roles in antiviral defense across different tissues, and demonstrate the effectiveness of image-based phenotyping to quantify disease progression.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Proteínas Argonautas/inmunología , Carmovirus/inmunología , Procesamiento de Imagen Asistido por Computador/métodos , Arabidopsis/genética , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Carmovirus/genética , Carmovirus/fisiología , Resistencia a la Enfermedad/genética , Resistencia a la Enfermedad/inmunología , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Mutación , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/virología , Unión Proteica , Interferencia de ARN/inmunologíaRESUMEN
Diverse animal and plant viruses block the re-infection of host cells by the same or highly similar viruses through superinfection exclusion (SIE), a widely observed, yet poorly understood phenomenon. Here we demonstrate that SIE of turnip crinkle virus (TCV) is exclusively determined by p28, one of the two replication proteins encoded by this virus. p28 expressed from a TCV replicon exerts strong SIE to a different TCV replicon. Transiently expressed p28, delivered simultaneously with, or ahead of, a TCV replicon, largely recapitulates this repressive activity. Interestingly, p28-mediated SIE is dramatically enhanced by C-terminally fused epitope tags or fluorescent proteins, but weakened by N-terminal modifications, and it inversely correlates with the ability of p28 to complement the replication of a p28-defective TCV replicon. Strikingly, p28 in SIE-positive cells forms large, mobile punctate inclusions that trans-aggregate a non-coalescing, SIE-defective, yet replication-competent p28 mutant. These results support a model postulating that TCV SIE is caused by the formation of multimeric p28 complexes capable of intercepting fresh p28 monomers translated from superinfector genomes, thereby abolishing superinfector replication. This model could prove to be applicable to other RNA viruses, and offer novel targets for antiviral therapy.
Asunto(s)
Carmovirus/fisiología , Sobreinfección/microbiología , Replicación Viral/fisiología , Immunoblotting , Microscopía Confocal , Enfermedades de las Plantas/virología , Nicotiana/virologíaRESUMEN
Small RNAs play essential regulatory roles in genome stability, development, and responses to biotic and abiotic stresses in most eukaryotes. In plants, the RNaseIII enzyme DICER-LIKE1 (DCL1) produces miRNAs, whereas DCL2, DCL3, and DCL4 produce various size classes of siRNAs. Plants also encode RNASE THREE-LIKE (RTL) enzymes that lack DCL-specific domains and whose function is largely unknown. We found that virus infection induces RTL1 expression, suggesting that this enzyme could play a role in plant-virus interaction. To first investigate the biochemical activity of RTL1 independent of virus infection, small RNAs were sequenced from transgenic plants constitutively expressing RTL1. These plants lacked almost all DCL2-, DCL3-, and DCL4-dependent small RNAs, indicating that RTL1 is a general suppressor of plant siRNA pathways. In vivo and in vitro assays revealed that RTL1 prevents siRNA production by cleaving dsRNA prior to DCL2-, DCL3-, and DCL4-processing. The substrate of RTL1 cleavage is likely long-perfect (or near-perfect) dsRNA, consistent with the RTL1-insensitivity of miRNAs, which derive from DCL1-processing of short-imperfect dsRNA. Virus infection induces RTL1 mRNA accumulation, but viral proteins that suppress RNA silencing inhibit RTL1 activity, suggesting that RTL1 has evolved as an inducible antiviral defense that could target dsRNA intermediates of viral replication, but that a broad range of viruses counteract RTL1 using the same protein toolbox used to inhibit antiviral RNA silencing. Together, these results reveal yet another level of complexity in the evolutionary battle between viruses and plant defenses.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/virología , Regulación de la Expresión Génica de las Plantas , Interacciones Huésped-Patógeno , Virus ARN/fisiología , ARN de Planta/antagonistas & inhibidores , ARN Interferente Pequeño/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Sustitución de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Carmovirus/fisiología , Biología Computacional/métodos , Cucumovirus/fisiología , Isoenzimas/genética , Isoenzimas/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/virología , Mutación Puntual , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Tobamovirus/fisiología , Tymovirus/fisiologíaRESUMEN
Members of the plant mitochondrial energy-dissipation pathway (MEDP) coordinate cellular energy metabolism, redox homeostasis and the balance of ROS production. However, the roles of MEDP members, particularly uncoupling protein (UCP), in resistance to turnip crinkle virus infection (TCV) are poorly understood. Here, we showed that disrupting some MEDP genes compromises plant resistance to TCV viral infection and this is partly associated with damaged photosynthetic characteristics, altered cellular redox and increased ROS production. Experiments using mutant plants with impaired cellular compartment redox poising further demonstrated that impaired chloroplast/mitochondria and cystosol redox increases the susceptibility of plants to viral infection. Our results illustrate a mechanism by which MEDP and cellular compartment redox act in concert to regulate plant resistance to viral infections.
Asunto(s)
Arabidopsis/fisiología , Arabidopsis/virología , Carmovirus/fisiología , Mitocondrias/virología , Enfermedades de las Plantas/virología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ácido Ascórbico/genética , Ácido Ascórbico/metabolismo , Cloroplastos/metabolismo , Cloroplastos/virología , Genes de Plantas , Glutatión/genética , Glutatión/metabolismo , Mitocondrias/metabolismo , Oxidación-Reducción , Fotosíntesis , Enfermedades de las Plantas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
KEY MESSAGE: A long intergenic noncoding RNA LINC - AP2 is upregulated and negatively correlated with AP2 gene expression with Turnip crinkle virus infection in Arabidopsis. Plant vegetative growth and floral reproductive structure were severely retarded and distorted in Turnip crinkle virus (TCV)-infected Arabidopsis thaliana. Compared to mock-inoculated plants, the stamen filaments were shorter in flowers of TCV-infected plants. However, TCV-infected plants can still produce normal seeds through artificial pollination, indicating both its pollen and stigma were biologically functional. From our high-throughput RNA-Seq transcriptome analysis, a floral structure-related APETALA2 (AP2) gene was found to be downregulated and its neighboring long intergenic noncoding RNAs (lincRNA), At4NC069370 (named LINC-AP2 in this study), were upregulated significantly in TCV-infected plants. This LINC-AP2 was further confirmed for its existence using 5'RACE technology. LINC-AP2 overexpression (LINC-AP2 OE) transgenic Arabidopsis plants were generated to compare with TCV-infected WT plants. TCV-infected LINC-AP2 OE plants which contained lower AP2 gene expression displayed more severe symptoms (including floral structure distortion) and higher TCV-CP gene transcript and coat protein levels. Furthermore, compared to TCV-infected WT plants, TCV-infected ap2 mutant plants failed to open their flower buds and displayed more severe viral symptoms. In conclusion, upregulation of LINC-AP2 is negatively correlated with AP2 gene expression with TCV infection in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/virología , Carmovirus/fisiología , Regulación de la Expresión Génica de las Plantas , Proteínas de Homeodominio/genética , Proteínas Nucleares/genética , Enfermedades de las Plantas/virología , ARN Largo no Codificante/genética , Regulación hacia Arriba/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/metabolismo , Flores/anatomía & histología , Perfilación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Mutación/genética , Proteínas Nucleares/metabolismo , Enfermedades de las Plantas/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Transcriptoma/genéticaRESUMEN
Melon necrotic spot virus (MNSV) (genus Carmovirus, family Tombusviridae) is a single-stranded, positive-sense RNA virus that has become an experimental model for the analysis of cell-to-cell virus movement and translation of uncapped viral RNAs, whereas little is known about its replication. Analysis of the cytopathology after MNSV infection showed the specific presence of modified organelles that resemble mitochondria. Immunolocalization of the glycine decarboxylase complex (GDC) P protein in these organelles confirmed their mitochondrial origin. In situ hybridization and immunolocalization experiments showed the specific localization of positive-sense viral RNA, capsid protein (CP), and double-stranded (ds)RNA in these organelles meaning that replication of the virus takes place in association with them. The three-dimensional reconstructions of the altered mitochondria showed the presence of large, interconnected, internal dilations which appeared to be linked to the outside cytoplasmic environment through pores and/or complex structures, and with lipid bodies. Transient expression of MNSV p29 revealed that its specific target is mitochondria. Our data document the extensive reorganization of host mitochondria induced by MNSV, which provides a protected environment to viral replication, and show that the MNSV p29 protein is the primary determinant of this effect in the host.
Asunto(s)
Carmovirus/fisiología , Cucurbitaceae/virología , Interacciones Huésped-Patógeno/fisiología , Mitocondrias/ultraestructura , Mitocondrias/virología , Replicación Viral/fisiología , Carmovirus/patogenicidad , Cucurbitaceae/citología , Cucurbitaceae/ultraestructura , Mitocondrias/química , Mitocondrias/metabolismo , Enfermedades de las Plantas , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The Pea enation mosaic virus (PEMV) 3' translational enhancer, known as the kissing-loop T-shaped structure (kl-TSS), binds to 40S subunits, 60S subunits, and 80S ribosomes, whereas the Turnip crinkle virus (TCV) TSS binds only to 60S subunits and 80S ribosomes. Using electrophoretic mobility gel shift assay (EMSA)-based competition assays, the kl-TSS was found to occupy a different site in the ribosome than the P-site-binding TCV TSS, suggesting that these two TSS employ different mechanisms for enhancing translation. The kl-TSS also engages in a stable, long-distance RNA-RNA kissing-loop interaction with a 12-bp 5'-coding-region hairpin that does not alter the structure of the kl-TSS as revealed by molecular dynamics simulations. Addition of the kl-TSS in trans to a luciferase reporter construct containing either wild-type or mutant 5' and 3' PEMV sequences suppressed translation, suggesting that the kl-TSS is required in cis to function, and both ribosome-binding and RNA interaction activities of the kl-TSS contributed to translational inhibition. Addition of the kl-TSS was more detrimental for translation than an adjacent eIF4E-binding 3' translational enhancer known as the PTE, suggesting that the PTE may support the ribosome-binding function of the kl-TSS. Results of in-line RNA structure probing, ribosome filter binding, and high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (hSHAPE) of rRNAs within bound ribosomes suggest that kl-TSS binding to ribosomes and binding to the 5' hairpin are compatible activities. These results suggest a model whereby posttermination ribosomes/ribosomal subunits bind to the kl-TSS and are delivered to the 5' end of the genome via the associated RNA-RNA interaction, which enhances the rate of translation reinitiation.
Asunto(s)
Regiones no Traducidas 5'/genética , Arabidopsis/metabolismo , Carmovirus/fisiología , Elementos de Facilitación Genéticos/genética , Virus del Mosaico/fisiología , Biosíntesis de Proteínas , ARN Viral/metabolismo , Ribosomas/metabolismo , Regiones no Traducidas 3'/genética , Arabidopsis/genética , Sitios de Unión , Ensayo de Cambio de Movilidad Electroforética , Factor 4E Eucariótico de Iniciación/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Saccharomyces cerevisiaeRESUMEN
The majority of the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) was previously identified as forming a highly interactive structure with a ribosome-binding tRNA-shaped structure (TSS) acting as a scaffold and undergoing a widespread conformational shift upon binding to RNA-dependent RNA polymerase (RdRp). Tertiary interactions in the region were explored by identifying two highly detrimental mutations within and adjacent to a hairpin H4 upstream of the TSS that reduce translation in vivo and cause identical structural changes in the loop of the 3' terminal hairpin Pr. Second-site changes that compensate for defects in translation/accumulation and reverse the structural differences in the Pr loop were found in the Pr stem, as well as in a specific stem within the TSS and within the capsid protein (CP) coding region, suggesting that the second-site changes were correcting a conformational defect and not restoring specific base pairing. The RdRp-mediated conformational shift extended upstream through this CP open reading frame (ORF) region after bypassing much of an intervening, largely unstructured region, supporting a connection between 3' elements and coding region elements. These data suggest that the Pr loop, TSS, and H4 are central elements in the regulation of translation and replication in TCV and allow for development of an RNA interactome that maps the higher-order structure of a postulated RNA domain within the 3' region of a plus-strand RNA virus.
Asunto(s)
Regiones no Traducidas 3' , Carmovirus/genética , Carmovirus/metabolismo , Biosíntesis de Proteínas , ARN Viral/química , Replicación Viral , Secuencia de Bases , Carmovirus/fisiología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación de Ácido NucleicoRESUMEN
While RNA silencing is a potent antiviral defense in plants, well-adapted plant viruses are known to encode suppressors of RNA silencing (VSR) that can neutralize the effectiveness of RNA silencing. As a result, most plant genes involved in antiviral silencing were identified by using debilitated viruses lacking silencing suppression capabilities. Therefore, it remains to be resolved whether RNA silencing plays a significant part in defending plants against wild-type viruses. We report here that, at a higher plant growth temperature (26°C) that permits rigorous replication of Turnip crinkle virus (TCV) in Arabidopsis, plants containing loss-of-function mutations within the Dicer-like 2 (DCL2), Argonaute 2 (AGO2), and HEN1 RNA methyltransferase genes died of TCV infection, whereas the wild-type Col-0 plants survived to produce viable seeds. To account for the critical role of DCL2 in ensuring the survival of wild-type plants, we established that higher temperature upregulates the activity of DCL2 to produce viral 22-nucleotide (nt) small interfering RNAs (vsRNAs). We further demonstrated that DCL2-produced 22-nt vsRNAs were fully capable of silencing target genes, but that this activity was suppressed by the TCV VSR. Finally, we provide additional evidence supporting the notion that TCV VSR suppresses RNA silencing through directly interacting with AGO2. Together, these results have revealed a specialized RNA silencing pathway involving DCL2, AGO2, and HEN1 that provides the host plants with a competitive edge against adapted viruses under environmental conditions that facilitates robust virus reproduction.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Carmovirus/fisiología , Proteínas de Ciclo Celular/inmunología , Enfermedades de las Plantas/virología , Proteínas de Unión al ARN/inmunología , Ribonucleasa III/inmunología , Arabidopsis/genética , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Proteínas Argonautas , Carmovirus/genética , Proteínas de Ciclo Celular/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Interferencia de ARN , Proteínas de Unión al ARN/genética , Ribonucleasa III/genética , Temperatura , Replicación ViralRESUMEN
Light harvested by plants is essential for the survival of most life forms. This light perception ability requires the activities of proteins termed photoreceptors. We report a function for photoreceptors in mediating resistance (R) protein-derived plant defense. The blue-light photoreceptors, cryptochrome (CRY) 2 and phototropin (PHOT) 2, are required for the stability of the R protein HRT, and thereby resistance to Turnip Crinkle virus (TCV). Exposure to darkness or blue-light induces degradation of CRY2, and in turn HRT, resulting in susceptibility. Overexpression of HRT can compensate for the absence of PHOT2 but not CRY2. HRT does not directly associate with either CRY2 or PHOT2 but does bind the CRY2-/PHOT2-interacting E3 ubiquitin ligase, COP1. Application of the proteasome inhibitor, MG132, prevents blue-light-dependent degradation of HRT, consequently these plants show resistance to TCV under blue-light. We propose that CRY2/PHOT2 negatively regulate the proteasome-mediated degradation of HRT, likely via COP1, and blue-light relieves this repression resulting in HRT degradation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Criptocromos/metabolismo , Proteínas Represoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Carmovirus/fisiología , Criptocromos/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Huésped-Patógeno , Inmunidad Innata/efectos de la radiación , Immunoblotting , Luz , Microscopía Confocal , Mutación , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ácido Salicílico/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Nicotiana benthamiana has been described as non-host for Melon necrotic spot virus (MNSV). We investigated the basis of this resistance using the unique opportunity provided by strain MNSV-264, a recombinant virus that is able to overcome the resistance. Analysis of chimeric MNSV mutants showed that virulence in N. benthamiana is conferred by a 49 nucleotide section of the MNSV-264 3'-UTR, which acts in this host as a cap-independent translational enhancer (3'-CITE). Although the 3'-CITE of non-adapted MNSV-Mα5 is active in susceptible melon, it does not promote efficient translation in N. benthamiana, thus preventing expression of proteins required for virus replication. However, MNSV-Mα5 gains the ability to multiply in N. benthamiana cells if eIF4E from a susceptible melon variety (Cm-eIF4E-S) is supplied in trans. These data show that N. benthamiana resistance to MNSV-Mα5 results from incompatibility between the MNSV-Mα5 3'-CITE and N. benthamiana eIF4E in initiating efficient translation of the viral genome. Therefore, non-host resistance conferred by the inability of a host susceptibility factor to support viral multiplication may be a possible mechanism for this type of resistance to viruses.
Asunto(s)
Carmovirus/genética , Factor 4E Eucariótico de Iniciación/genética , Inmunidad Innata , Nicotiana/virología , Enfermedades de las Plantas/virología , ARN Viral/genética , Regiones no Traducidas 3'/genética , Carmovirus/patogenicidad , Carmovirus/fisiología , Genoma Viral , Conformación de Ácido Nucleico , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Hojas de la Planta/genética , Hojas de la Planta/virología , Biosíntesis de Proteínas , Protoplastos/virología , Recombinación Genética , Nicotiana/genética , Virulencia , Replicación ViralRESUMEN
In both Hibiscus chlorotic ringspot virus (HCRSV)-infected and HCRSV coat protein (CP) agroinfiltrated plant leaves, we showed that sulfur metabolism pathway related genes-namely, sulfite oxidase (SO), sulfite reductase, and adenosine 5'-phosphosulfate kinase-were upregulated. It led us to examine a plausible relationship between sulfur-enhanced resistance (SED) and HCRSV infection. We broadened an established method to include different concentrations of sulfur (0S, 1S, 2S, and 3S) to correlate them to symptom development of HCRSV-infected plants. We treated plants with glutathione and its inhibitor to verify the SED effect. Disease resistance was induced through elevated glutathione contents during HCRSV infection. The upregulation of SO was related to suppression of symptom development induced by sulfur treatment. In this study, we established that HCRSV-CP interacts with SO which, in turn, triggers SED and leads to enhanced plant resistance. Thus, we have discovered a new function of SO in the SED pathway. This is the first report to demonstrate that the interaction of a viral protein and host protein trigger SED in plants. It will be interesting if such interaction applies generally to other host-pathogen interactions that will lead to enhanced pathogen defense.
Asunto(s)
Proteínas de la Cápside/genética , Carmovirus/fisiología , Hibiscus/inmunología , Enfermedades de las Plantas/inmunología , Sulfito-Oxidasa/genética , Azufre/metabolismo , Vías Biosintéticas , Proteínas de la Cápside/metabolismo , Carmovirus/genética , Cloroplastos/metabolismo , Cistina/análisis , Cistina/metabolismo , Regulación de la Expresión Génica de las Plantas , Glutatión/análisis , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Hibiscus/enzimología , Hibiscus/genética , Hibiscus/virología , Interacciones Huésped-Patógeno , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Peroxisomas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Enfermedades de las Plantas/virología , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/virología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes de Fusión , Plantones/enzimología , Plantones/genética , Plantones/inmunología , Plantones/virología , Sulfito-Oxidasa/metabolismo , Azufre/farmacología , Regulación hacia Arriba , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Plus-strand RNA viruses without 5' caps require noncanonical mechanisms for ribosome recruitment. A translational enhancer in the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. We now report that the 63-nucleotide (nt) 5' UTR of TCV contains a 19-nt pyrimidine-rich element near the initiation codon that supports translation of an internal open reading frame (ORF) independent of upstream 5' UTR sequences. Addition of 80S ribosomes to the 5' UTR reduced the flexibility of the polypyrimidine residues and generated a toeprint consistent with binding to this region. Binding of salt-washed 40S ribosomal subunits was reduced 6-fold when the pyrimidine-rich sequence was mutated. 40S subunit binding generated the same toeprint as 80S ribosomes but also additional ones near the 5' end. Generation of out-of-frame AUGs upstream of the polypyrimidine region reduced translation, which suggests that 5'-terminal entry of 40S subunits is followed by scanning and that the polypyrimidine region is needed for an alternative function that requires ribosome binding. No evidence for RNA-RNA interactions between 5' and 3' sequences was found, suggesting that TCV utilizes an alternative means for circularizing its genome. Combining 5' and 3' UTR fragments in vitro had no discernible effect on the structures of the RNAs. In contrast, when 80S ribosomes were added to both fragments, structural changes were found in the 5' UTR polypyrimidine tract that were not evident when ribosomes interacted with the individual fragments. This suggests that ribosomes can promote an interaction between the 5' and 3' UTRs of TCV.
Asunto(s)
Regiones no Traducidas 3' , Regiones no Traducidas 5' , Carmovirus/fisiología , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Ribosomas/metabolismo , Conformación de Ácido Nucleico , Proteínas Virales/biosíntesisRESUMEN
Resistance (R) protein-associated pathways are well known to participate in defense against a variety of microbial pathogens. Salicylic acid (SA) and its associated proteinaceous signaling components, including enhanced disease susceptibility 1 (EDS1), non-race-specific disease resistance 1 (NDR1), phytoalexin deficient 4 (PAD4), senescence associated gene 101 (SAG101), and EDS5, have been identified as components of resistance derived from many R proteins. Here, we show that EDS1 and SA fulfill redundant functions in defense signaling mediated by R proteins, which were thought to function independent of EDS1 and/or SA. Simultaneous mutations in EDS1 and the SA-synthesizing enzyme SID2 compromised hypersensitive response and/or resistance mediated by R proteins that contain coiled coil domains at their N-terminal ends. Furthermore, the expression of R genes and the associated defense signaling induced in response to a reduction in the level of oleic acid were also suppressed by compromising SA biosynthesis in the eds1 mutant background. The functional redundancy with SA was specific to EDS1. Results presented here redefine our understanding of the roles of EDS1 and SA in plant defense.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/inmunología , Proteínas de Unión al ADN/inmunología , Inmunidad Innata , Enfermedades de las Plantas/inmunología , Ácido Salicílico/inmunología , Ácido Salicílico/metabolismo , Transducción de Señal , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Carmovirus/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Enfermedades de las Plantas/virologíaRESUMEN
BACKGROUND: Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21-24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. RESULTS: We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analysed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. CONCLUSION: We have discovered and analysed a large number of conserved and melon-specific sRNAs, including miRNAs and their potential target genes. This provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melon-virus interactions.
Asunto(s)
Cucumis melo/genética , Secuenciación de Nucleótidos de Alto Rendimiento , ARN de Planta/genética , ARN Pequeño no Traducido/genética , Secuencia de Bases , Carmovirus/fisiología , Cotiledón/genética , Cotiledón/crecimiento & desarrollo , Cotiledón/inmunología , Cotiledón/virología , Cucumis melo/crecimiento & desarrollo , Cucumis melo/inmunología , Cucumis melo/virología , Resistencia a la Enfermedad/genética , Biblioteca de Genes , MicroARNs/genética , Fotosíntesis/genética , Polinización/genética , Potyvirus/fisiología , Especificidad de la Especie , TranscriptomaRESUMEN
The transcriptional mechanism utilized by turnip crinkle carmovirus to synthesize subgenomic (sg) mRNAs was investigated by analyzing viral RNAs and their associated regulatory RNA elements. In vivo analyses revealed the following: (i) that minus-strand sg RNAs are detectable in infections, (ii) that minus-strand sg RNA accumulation can be partially uncoupled from that of their plus-strand sg mRNA counterparts, and (iii) that an RNA secondary structure located upstream of the sg mRNA start site mediates transcription by functioning in the plus strand of the viral genome. Collectively, these observations are consistent with this carmovirus using a premature termination mechanism for sg mRNA transcription.
Asunto(s)
Brassica napus/virología , Carmovirus/fisiología , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Transcripción Genética , Replicación Viral , Secuencia de Bases , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Viral/química , ARN Viral/genética , Regiones Terminadoras GenéticasRESUMEN
Plant viruses hijack endogenous host transport machinery to aid their intracellular spread. Here, we study the localization of the p7B, the membrane-associated viral movement protein (MP) of the Melon necrotic spot virus (MNSV), and also the potential involvement of the secretory pathway on the p7B targeting and intra- and intercellular virus movements. p7B fused to fluorescent proteins was located throughout the endoplasmic reticulum (ER) at motile Golgi apparatus (GA) stacks that actively tracked the actin microfilaments, and at the plasmodesmata (PD). Hence, the secretory pathway inhibitor, Brefeldin A (BFA), and the overexpression of the GTPase-defective mutant of Sar1p, Sar1[H74L], fully retained the p7B within the ER, revealing that the protein is delivered to PD in a BFA-sensitive and COPII-dependent manner. Disruption of the actin cytoskeleton with latrunculin B led to the accumulation of p7B in the ER, which strongly suggests that p7B is also targeted to the cell periphery in an actin-dependent manner. Remarkably, the local spread of the viral infection was significantly restricted either with the presence of BFA or under the overexpression of Sar1[H74L], thus revealing the involvement of an active secretory pathway in the intracellular movement of MNSV. Overall, these findings support a novel route for the intracellular transport of a plant virus led by the GA.
Asunto(s)
Carmovirus/metabolismo , Proteínas de Movimiento Viral en Plantas/metabolismo , Vías Secretoras , Proteínas Virales/metabolismo , Transporte Biológico/efectos de los fármacos , Brefeldino A/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Carmovirus/genética , Carmovirus/fisiología , Cucurbitaceae/virología , Retículo Endoplásmico/metabolismo , Espacio Extracelular/virología , Aparato de Golgi/metabolismo , Interacciones Huésped-Patógeno , Espacio Intracelular/virología , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Mutación , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Proteínas de Movimiento Viral en Plantas/genética , Plasmodesmos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Tiazolidinas/farmacología , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/virología , Proteínas Virales/genéticaRESUMEN
Pelargonium flower break virus (PFBV) belongs to the genus Carmovirus (family Tombusviridae) and, as with the remaining members of the group, possesses a monopartite genome of single-stranded, positive-sense RNA that contains five ORFs. The two 5'-proximal ORFs (ORFs 1 and 2) encode two polypeptides of 27 and 86 kDa (p27 and p86), respectively, that show homology with replication proteins. The p27 does not present any motif to explain its presumed involvement in replication, while p86 has the motifs conserved in RNA-dependent RNA polymerases. In this work, we have confirmed the necessity of p27 and p86 for PFBV replication. To gain insights into the function(s) of p27, we have expressed and purified the protein from Escherichia coli and tested its ability to bind RNA in vitro. The results have shown that p27 is able to bind ssRNA with high affinity and in a cooperative fashion and that it is also capable of binding other types of nucleic acids, though to a lesser extent. Additionally, competition experiments suggest that p27 has a preference for PFBV-derived ssRNAs. Using truncated forms of p27, it can be concluded that several regions of the protein contribute to its RNA-binding properties and that this contribution is additive. This study is the first to show nucleic acid-binding ability of the ORF1 product of a carmovirus and the data obtained suggest that this product plays an essential role in selection and recruitment of viral RNA replication templates.
Asunto(s)
Carmovirus/enzimología , Pelargonium/virología , ARN Viral/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/metabolismo , Replicación Viral , Sitios de Unión , Carmovirus/fisiología , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Unión Proteica , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Eliminación de Secuencia , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
RNA secondary structures play diverse roles in positive-sense (+) RNA virus infections, but those located with the replication protein coding sequence can be difficult to investigate. Structures that regulate the translation of replication proteins pose particular challenges, as their potential involvement in post-translational steps cannot be easily discerned independent of their roles in regulating translation. In the current study, we attempted to overcome these difficulties by providing viral replication proteins in trans. Specifically, we modified the plant-infecting turnip crinkle virus (TCV) into variants that are unable to translate one (p88) or both (p28 and p88) replication proteins, and complemented their replication with the corresponding replication protein(s) produced from separate, non-replicating constructs. This approach permitted us to re-examine the p28/p88 coding region for potential RNA elements needed for TCV replication. We found that, while more than a third of the p88 coding sequence could be deleted without substantially affecting viral RNA levels, two relatively small regions, known as RSE and IRE, were essential for robust accumulation of TCV genomic RNA, but not subgenomic RNAs. In particular, the RSE element, found previously to be required for regulating the translational read-through of p28 stop codon to produce p88, contained sub-elements needed for efficient replication of the TCV genome. Application of this new approach in other viruses could reveal novel RNA secondary structures vital for viral multiplication.