RESUMEN
Changes in plant abiotic environments may alter plant virus epidemiological traits, but how such changes actually affect their quantitative relationships is poorly understood. Here, we investigated the effects of water deficit on Cauliflower mosaic virus (CaMV) traits (virulence, accumulation, and vectored-transmission rate) in 24 natural Arabidopsis thaliana accessions grown under strictly controlled environmental conditions. CaMV virulence increased significantly in response to water deficit during vegetative growth in all A. thaliana accessions, while viral transmission by aphids and within-host accumulation were significantly altered in only a few. Under well-watered conditions, CaMV accumulation was correlated positively with CaMV transmission by aphids, while under water deficit, this relationship was reversed. Hence, under water deficit, high CaMV accumulation did not predispose to increased horizontal transmission. No other significant relationship between viral traits could be detected. Across accessions, significant relationships between climate at collection sites and viral traits were detected but require further investigation. Interactions between epidemiological traits and their alteration under abiotic stresses must be accounted for when modelling plant virus epidemiology under scenarios of climate change.
Asunto(s)
Arabidopsis/crecimiento & desarrollo , Arabidopsis/virología , Caulimovirus/patogenicidad , Cambio Climático , Enfermedades de las Plantas/virología , Estrés Fisiológico , Virulencia , Agua , Animales , Áfidos/fisiología , Áfidos/virología , Arabidopsis/parasitología , AmbienteRESUMEN
Genetically engineered plants have varied applications in agriculture for enhancing the values of food and feed. Genetic engineering aims to introduce selected genetic regions with desirable traits into target plants for both spatial and temporal expressions. Promoters are the key elements responsible for regulating gene expressions by modulating the transcription factors (TFs) through recognition of RNA polymerases. Based on their recognition and expression, RNA polymerases were categorized into RNA pol II and pol III promoters. Promoter activity and specificity are the two prime parameters in regulating the transgene expression. Since the use of constitutive promoters like Cauliflower mosaic virus (CaMV) 35S may lead to adverse effects on nontarget organisms or ecosystem, inducible/tissue specific promoters and/or the RNA pol III promoters provide myriad opportunities for gene expressions with controlled regulation and with minimum adverse effects. Besides their role in transgene expression, their influence in synthetic biology and genome editing are also discussed. This review provides an update on the importance, current prospects, and insight into the advantages and disadvantages of promoters reported thus far would help to utilize them in the endeavour to develop nutritionally and agronomically improved transgenic crops for commercialization.
Asunto(s)
Plantas Modificadas Genéticamente/genética , ARN Polimerasa III/genética , ARN Polimerasa II/genética , Factores de Transcripción/genética , Caulimovirus/patogenicidad , Regulación de la Expresión Génica de las Plantas/genética , Ingeniería Genética/tendencias , Plantas/genética , Plantas/virología , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/virología , Regiones Promotoras Genéticas/genéticaRESUMEN
Cytosine methylation is an important defense against invasive DNAs. Here, cytosine methylation profiles of a plant pararetrovirus, Cauliflower mosaic virus (CaMV), were investigated. Nuclear CaMV DNA is highly methylated throughout the genome including at transcription regulatory regions, but the virion DNA is unmethylated. In vitro CG methylation of the viral 35S promoter reduces transcription from the downstream gene. Although nuclear CaMV DNA is highly methylated, its transcripts are accumulated in the nucleus. The data suggest that a small population of unmethylated viral genomes produced through reverse transcription are constantly delivered back to the nucleus. Small RNA profiles suggest that methylation of the CaMV DNA may be due to de novo methylation through 21-, 22-, and 24-nt small RNAs with adenines at their 5' terminus.
Asunto(s)
Caulimovirus/genética , Citosina/metabolismo , Genoma Viral , Adenina/metabolismo , Brassica rapa/virología , Caulimovirus/patogenicidad , Núcleo Celular/genética , Metilación de ADN , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno/fisiología , Hojas de la Planta/virología , Regiones Promotoras Genéticas , ARN ViralRESUMEN
BACKGROUND: Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS: Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS: This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform.
Asunto(s)
Caulimovirus/patogenicidad , Reacción en Cadena de la Polimerasa Multiplex/métodos , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/virología , Plantas Modificadas Genéticamente/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
During pathogenesis, viruses hijack the host cellular machinery to access molecules and sub-cellular structures needed for infection. We have evidence that the multifunctional viral translation transactivator/viroplasmin (TAV) protein from Cauliflower mosaic virus (CaMV) can function as a suppressor of nonsense-mediated mRNA decay (NMD). TAV interacts specifically with a scaffold protein of the decapping complex VARICOSE (VCS) in the yeast two-hybrid system, and co-localizes with components of the decapping complex in planta. Notably, plants transgenic for TAV accumulate endogenous NMD-elicited mRNAs, while decay of AU-rich instability element (ARE)-signal containing mRNAs are not affected. Using an agroinfiltration-based transient assay we confirmed that TAV specifically stabilizes mRNA containing a premature termination codon (PTC) in a VCS-dependent manner. We have identified a TAV motif consisting of 12 of the 520 amino acids in the full-length sequence that is critical for both VCS binding and the NMD suppression effect. Our data suggest that TAV can intercept NMD by targeting the decapping machinery through the scaffold protein VARICOSE, indicating that 5'-3' mRNA decapping is a late step in NMD-related mRNA degradation in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Caulimovirus/patogenicidad , Interacciones Huésped-Patógeno/fisiología , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas Virales/metabolismo , Arabidopsis/genética , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Caulimovirus/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hojas de la Planta/virología , Plantas Modificadas Genéticamente , Nicotiana/genética , Nicotiana/virología , Técnicas del Sistema de Dos HíbridosRESUMEN
The split GFP technique is based on the auto-assembly of GFP when two polypeptides-GFP1-10 (residues 1-214; the detector) and GFP11 (residues 215-230; the tag)-both non-fluorescing on their own, associate spontaneously to form a fluorescent molecule. We evaluated this technique for its efficacy in contributing to the characterization of Cauliflower mosaic virus (CaMV) infection. A recombinant CaMV with GFP11 fused to the viral protein P6 (a key player in CaMV infection and major constituent of viral factory inclusions that arise during infection) was constructed and used to inoculate transgenic Arabidopsis thaliana expressing GFP1-10. The mutant virus (CaMV11P6) was infectious, aphid-transmissible and the insertion was stable over many passages. Symptoms on infected plants were delayed and milder. Viral protein accumulation, especially of recombinant 11P6, was greatly decreased, impeding its detection early in infection. Nonetheless, spread of infection from the inoculated leaf to other leaves was followed by whole plant imaging. Infected cells displayed in real time confocal laser scanning microscopy fluorescence in wild type-looking virus factories. Thus, it allowed for the first time to track a CaMV protein in vivo in the context of an authentic infection. 11P6 was immunoprecipitated with anti-GFP nanobodies, presenting a new application for the split GFP system in protein-protein interaction assays and proteomics. Taken together, split GFP can be an attractive alternative to using the entire GFP for protein tagging.
Asunto(s)
Arabidopsis/virología , Caulimovirus/patogenicidad , Proteínas Fluorescentes Verdes/genética , Proteínas Virales/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Caulimovirus/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Confocal , Mutagénesis Sitio-Dirigida , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/virología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/metabolismoRESUMEN
Arabidopsis thaliana ecotype En-2 is resistant to several strains of Cauliflower mosaic virus (CaMV), including strain W260, but is susceptible to strain NY8153. Resistance in En-2 is conditioned by a single, semi-dominant gene called CAR1. We constructed several recombinant infectious clones between W260 and NY8153 and evaluated their capability to infect En-2. This analysis showed that the capacity of NY8153 to break resistance in En-2 was conditioned by mutations within the CaMV gene 1, a gene that encodes a protein dedicated to cell-to-cell movement (P1), and conversely, that P1 of W260 is responsible for eliciting the plant defense response. A previous study had shown that P6 of W260 was responsible for overcoming resistance in Arabidopsis ecotype Tsu-0 and that P6 of CaMV strain CM1841 was responsible for triggering resistance. The present study now shows that a second gene of CaMV is targeted by Arabidopsis for plant immunity.
Asunto(s)
Arabidopsis/genética , Caulimovirus/genética , Resistencia a la Enfermedad/genética , Interacciones Huésped-Patógeno , Inmunidad de la Planta/genética , Proteínas Virales/genética , Arabidopsis/inmunología , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Caulimovirus/metabolismo , Caulimovirus/patogenicidad , Regulación de la Expresión Génica , Ingeniería Genética , Genotipo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Recombinación Genética , Transducción de Señal , Proteínas Virales/metabolismoRESUMEN
RNA silencing is a common mechanism that plays a key role in antiviral defense. To overcome host defense responses, plant viruses encode silencing-suppressor proteins to target one or several key steps in the silencing machinery. Here, we report that the P6 protein encoded by Strawberry vein banding virus (SVBV) is an RNA silencing suppressor through Agrobacterium-mediated co-infiltration assays. SVBV P6 protein can suppress green fluorescent protein (GFP) gene silencing induced by single-stranded RNA but not by double-stranded RNA. The P6 protein can also inhibit systemic silencing of GFP through interfering the systemic spread of GFP silencing signal. Subcellular localization study indicated that P6 protein formed irregular bodies and distributed in both cytoplasm and nucleus of Nicotiana benthamiana cells. Furthermore, deletion analysis indicated that a nuclear localization signal (NLS, aa 402-426) in the P6 protein is responsible for the silencing suppression efficiency. In addition, expression of the P6 protein via a Potato virus X (PVX)-based vectors induced more severe mosaic symptoms in N. benthamiana leaves, and transgenic N. benthamiana plants expressing P6 showed obvious vein yellowing as well as severe mosaic symptoms in leaves. Taken together, our results demonstrates that SVBV P6 is a suppressor of RNA silencing, possibly acting at a upstream step for dsRNA generation.
Asunto(s)
Caulimovirus/genética , Proteínas de Unión al ADN/metabolismo , Enfermedades de las Plantas/virología , Interferencia de ARN , Proteínas Virales/metabolismo , Agrobacterium/genética , Caulimovirus/metabolismo , Caulimovirus/patogenicidad , Núcleo Celular/virología , Citoplasma/virología , Proteínas de Unión al ADN/genética , Silenciador del Gen , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Señales de Localización Nuclear/genética , Señales de Localización Nuclear/metabolismo , Hojas de la Planta/virología , Virus de Plantas/genética , Plantas Modificadas Genéticamente/virología , Potexvirus/genética , ARN/química , ARN/genética , Nicotiana/citología , Nicotiana/virología , Proteínas Virales/genéticaRESUMEN
Cauliflower mosaic virus (CaMV) TAV protein (TransActivator/Viroplasmin) plays a pivotal role during the infection cycle since it activates translation reinitiation of viral polycistronic RNAs and suppresses RNA silencing. It is also the major component of cytoplasmic electron-dense inclusion bodies (EDIBs) called viroplasms that are particularly evident in cells infected by the virulent CaMV Cabb B-JI isolate. These EDIBs are considered as virion factories, vehicles for CaMV intracellular movement and reservoirs for CaMV transmission by aphids. In this study, focused on different TAV mutants in vivo, we demonstrate that three physically separated domains collectively participate to the formation of large EDIBs: the N-terminal EKI motif, a sequence of the MAV domain involved in translation reinitiation and a C-terminal region encompassing the zinc finger. Surprisingly, EKI mutant TAVm3, corresponding to a substitution of the EKI motif at amino acids 11-13 by three alanines (AAA), which completely abolished the formation of large viroplasms, was not lethal for CaMV but highly reduced its virulence without affecting the rate of systemic infection. Expression of TAVm3 in a viral context led to formation of small irregularly shaped inclusion bodies, mild symptoms and low levels of viral DNA and particles accumulation, despite the production of significant amounts of mature capsid proteins. Unexpectedly, for CaMV-TAVm3 the formation of viral P2-containing electron-light inclusion body (ELIB), which is essential for CaMV aphid transmission, was also altered, thus suggesting an indirect role of the EKI tripeptide in CaMV plant-to-plant propagation. This important functional contribution of the EKI motif in CaMV biology can explain the strict conservation of this motif in the TAV sequences of all CaMV isolates.
Asunto(s)
Brassica napus/virología , Caulimovirus/metabolismo , Caulimovirus/patogenicidad , Transactivadores/química , Transactivadores/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Caulimovirus/ultraestructura , Cuerpos de Inclusión Viral/metabolismo , Cuerpos de Inclusión Viral/ultraestructura , Proteínas Mutantes/metabolismo , Fenotipo , Dominios Proteicos , Protoplastos/metabolismo , Transcripción Reversa/genética , Relación Estructura-Actividad , Virulencia , Replicación ViralRESUMEN
Virus interactions with plant silencing and innate immunity pathways can potentially alter the susceptibility of virus-infected plants to secondary infections with nonviral pathogens. We found that Arabidopsis plants infected with Cauliflower mosaic virus (CaMV) or transgenic for CaMV silencing suppressor P6 exhibit increased susceptibility to Pseudomonas syringae pv. tomato (Pst) and allow robust growth of the Pst mutant hrcC-, which cannot deploy effectors to suppress innate immunity. The impaired antibacterial defense correlated with the suppressed oxidative burst, reduced accumulation of the defense hormone salicylic acid (SA) and diminished SA-dependent autophagy. The viral protein domain required for suppression of these plant defense responses is dispensable for silencing suppression but essential for binding and activation of the plant target-of-rapamycin (TOR) kinase which, in its active state, blocks cellular autophagy and promotes CaMV translation. Our findings imply that CaMV P6 is a versatile viral effector suppressing both silencing and innate immunity. P6-mediated suppression of oxidative burst and SA-dependent autophagy may predispose CaMV-infected plants to bacterial infection.
Asunto(s)
Arabidopsis/inmunología , Arabidopsis/virología , Autofagia/efectos de los fármacos , Caulimovirus/fisiología , Pseudomonas syringae/crecimiento & desarrollo , Estallido Respiratorio , Ácido Salicílico/farmacología , Proteínas Virales/metabolismo , Arabidopsis/efectos de los fármacos , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Caulimovirus/efectos de los fármacos , Caulimovirus/patogenicidad , Silenciador del Gen/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/virología , Dominios Proteicos , Pseudomonas syringae/efectos de los fármacos , Estallido Respiratorio/efectos de los fármacos , Eliminación de Secuencia , Proteínas Virales/químicaRESUMEN
The plant pararetrovirus Cauliflower mosaic virus (CaMV) uses alternative splicing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5' region and suggested that the main role of CaMV splicing is to downregulate expression of open reading frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA.
Asunto(s)
Empalme Alternativo/genética , Caulimovirus/genética , Transcriptoma/genética , Secuencia de Aminoácidos , Secuencia de Bases , Caulimovirus/aislamiento & purificación , Caulimovirus/patogenicidad , Simulación por Computador , Secuencia Conservada/genética , Datos de Secuencia Molecular , Mutación/genética , Sitios de Empalme de ARN/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Cauliflower mosaic virus gene VI product (P6) is an essential protein that forms cytoplasmic, inclusion bodies (IBs). P6 contains four regions involved in self-association, termed D1-D4. D3 binds to D1, along with D4 and contains a spacer region (termed D3b) between two RNA-binding domains. Here we show D3b binds full-length P6 along with D1 and D4. Full-length P6s harboring single amino acid substitutions within D3b showed reduced binding to both D1 and D4. Full-length P6s containing D3b mutations and fused with green fluorescent protein formed inclusion-like bodies (IL-Bs) when expressed in Nicotiana benthamiana leaves. However, mutant P6s with reduced binding to D1 and D4, showed smaller IL-Bs, than wild type. Likewise, viruses containing these mutations showed a decrease in inoculated leaf viral DNA levels and reduced efficiency of systemic infection. These data suggest that mutations influencing P6 self-association alter IB formation and reduce virus infection.
Asunto(s)
Caulimovirus/metabolismo , Cuerpos de Inclusión Viral/metabolismo , Nicotiana/virología , Enfermedades de las Plantas/virología , Transactivadores/química , Transactivadores/genética , Caulimovirus/química , Caulimovirus/genética , Caulimovirus/patogenicidad , Cuerpos de Inclusión Viral/genética , Mutación , Estructura Terciaria de Proteína , Transactivadores/metabolismo , VirulenciaRESUMEN
The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Caulimovirus/metabolismo , Plasmodesmos/metabolismo , Proteínas Virales/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caulimovirus/patogenicidad , Membrana Celular/metabolismo , Técnicas de Silenciamiento del Gen , Interacciones Huésped-Patógeno , Cuerpos de Inclusión Viral/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Nicotiana/virología , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Virión/metabolismo , Proteína Fluorescente RojaRESUMEN
The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) µA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes.
Asunto(s)
Caulimovirus/metabolismo , Endosomas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Movimiento Viral en Plantas/metabolismo , Vesículas Transportadoras/metabolismo , Subunidades mu de Complejo de Proteína Adaptadora/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/metabolismo , Arabidopsis/virología , Brassica rapa/efectos de los fármacos , Brassica rapa/virología , Brefeldino A/farmacología , Caulimovirus/efectos de los fármacos , Caulimovirus/patogenicidad , Compartimento Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Membranas Intracelulares/efectos de los fármacos , Modelos Biológicos , Datos de Secuencia Molecular , Mutación/genética , Proteínas de Plantas/efectos de los fármacos , Proteínas de Plantas/metabolismo , Proteínas de Movimiento Viral en Plantas/química , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Protoplastos/efectos de los fármacos , Protoplastos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Nicotiana/metabolismo , Vesículas Transportadoras/efectos de los fármacos , Tirosina/metabolismo , Tirfostinos/farmacologíaRESUMEN
Plant virus genomes cross the barrier of the host cell wall and move to neighboring cells either in the form of nucleoprotein complex or encapsidated into virions. Virus transport is facilitated by virus-encoded movement proteins (MP), which are different from one another in number, size, sequence, and in the strategy used to overcome the size exclusion limit of plasmodesmata (PD). (1) A group of them forms tubules inside the lumen of highly modified PDs upon removal of the desmotubule. To date the molecular mechanism(s) and the host factors involved in the assembly of MP tubules as well as the mechanistic aspects of virus particle transport throughout them remain substantially unknown. In a recent study, we showed that Cauliflower mosaic virus (CaMV) MP traffics in the endocytic pathway with the help of 3 tyrosine-sorting signals, which are not required to target MP to the plasma membrane but are essential for tubule formation. (2) This evidence unravels a previously unknown connection between the plant endosomal system and tubule-mediated virus movement that is here supported by demonstration of hindrance of tubule assembly upon Brefeldin A (BFA) treatment. We discuss the implications of our data on the mechanisms of viral transport through tubules and draw parallels with plant mechanisms of polarized growth.
Asunto(s)
Brefeldino A/farmacología , Caulimovirus/patogenicidad , Membrana Celular/metabolismo , Nicotiana/metabolismo , Plasmodesmos/metabolismo , Proteínas Virales/metabolismo , Caulimovirus/metabolismo , Transporte de Proteínas , Nicotiana/efectos de los fármacos , Nicotiana/virologíaRESUMEN
Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.
Asunto(s)
Caulimovirus/patogenicidad , Estructura Terciaria de Proteína/genética , Interferencia de ARN/efectos de los fármacos , Ácido Salicílico/metabolismo , Transducción de Señal/efectos de los fármacos , Transactivadores/genética , Transactivadores/farmacología , Secuencia de Aminoácidos , Arabidopsis/virología , Caulimovirus/genética , Caulimovirus/metabolismo , Datos de Secuencia Molecular , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Eliminación de Secuencia , Transactivadores/química , Transactivadores/metabolismo , Replicación ViralRESUMEN
Cauliflower mosaic virus (CaMV) forms two types of inclusion bodies within infected plant cells: numerous virus factories, which are the sites for viral replication and virion assembly, and a single transmission body (TB), which is specialized for virus transmission by aphid vectors. The TB reacts within seconds to aphid feeding on the host plant by total disruption and redistribution of its principal component, the viral transmission helper protein P2, onto microtubules throughout the cell. At the same time, virions also associate with microtubules. This redistribution of P2 and virions facilitates transmission and is reversible; the TB reforms within minutes after vector departure. Although some virions are present in the TB before disruption, their subsequent massive accumulation on the microtubule network suggests that they also are released from virus factories. Using drug treatments, mutant viruses, and exogenous supply of viral components to infected protoplasts, we show that virions can rapidly exit virus factories and, once in the cytoplasm, accumulate together with the helper protein P2 on the microtubule network. Moreover, we show that during reversion of this phenomenon, virions from the microtubule network can either be incorporated into the reverted TB or return to the virus factories. Our results suggest that CaMV factories are dynamic structures that participate in vector transmission by controlled release and uptake of virions during TB reaction.
Asunto(s)
Áfidos/virología , Brassica rapa/virología , Caulimovirus/patogenicidad , Microtúbulos/virología , Enfermedades de las Plantas/virología , Protoplastos/virología , Replicación Viral , Animales , Áfidos/genética , Áfidos/metabolismo , Brassica rapa/genética , Brassica rapa/metabolismo , Técnica del Anticuerpo Fluorescente , Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas Virales , Virión/patogenicidadRESUMEN
The gene VI product, protein 6 (P6), of Cauliflower mosaic virus (CaMV) assembles into large, amorphous inclusion bodies (IBs) that are considered sites for viral protein synthesis and viral genome replication and encapsidation. P6 IBs align with microfilaments and require them for intracellular trafficking, a result implying that P6 IBs function to move virus complexes or virions within the cell to support virus physiology. Through a yeast two-hybrid screen we determined that CHUP1, a plant protein allowing chloroplast transport through an interaction with chloroplast and microfilament, interacts with P6. The interaction between CHUP1 and P6 was confirmed through colocalization in vivo and co-immunoprecipitation assays. A truncated CHUP1 fused with enhanced cyan fluorescent protein, unable to transport chloroplasts, inhibited intracellular movement of P6-Venus inclusions. Silencing of CHUP1 in N. edwardsonii impaired the ability of CaMV to infect plants. The findings suggest that CHUP1 supports CaMV infection through an interaction with P6.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Caulimovirus/patogenicidad , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Proteínas de Microfilamentos/metabolismo , Transactivadores/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Proteínas de Cloroplastos/genética , Cloroplastos/virología , Inmunoprecipitación , Proteínas de Microfilamentos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transactivadores/genética , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The transmission-virulence trade-off hypothesis is one of the few adaptive explanations of virulence evolution, and assumes that there is an overall positive correlation between parasite transmission and virulence. The shape of the transmission-virulence relationship predicts whether virulence should evolve toward either a maximum or to an intermediate optimum. A positive correlation between each of these traits and within-host growth is often suggested to underlie the relationship between virulence and transmission. There are few experimental tests of this hypothesis; this study reports on the first empirical test on a plant pathogen. We infected Brassica rapa plants with nine natural isolates of Cauliflower mosaic virus and then estimated three traits: transmission, virulence, and within-host viral accumulation. As predicted by the trade-off hypothesis, we observed a positive correlation between transmission and virulence, suggestive of the existence of an intermediate optimum. We discovered the unexpected existence of two groups of within-host accumulation, differing by at least an order of magnitude. When accumulation groups were not accounted for, within-host accumulation was correlated neither to virulence nor transmission, although our results suggest that within each group these correlations exist.
Asunto(s)
Brassica rapa/virología , Caulimovirus/patogenicidad , Caulimovirus/genética , Evolución Molecular , Interacciones Huésped-Patógeno/genética , Virulencia/genéticaRESUMEN
Many plant and animal viruses are spread by insect vectors. Cauliflower mosaic virus (CaMV) is aphid-transmitted, with the virus being taken up from specialized transmission bodies (TB) formed within infected plant cells. However, the precise events during TB-mediated virus acquisition by aphids are unknown. Here, we show that TBs react instantly to the presence of the vector by ultra-rapid and reversible redistribution of their key components onto microtubules throughout the cell. Enhancing or inhibiting this TB reaction pharmacologically or by using a mutant virus enhanced or inhibited transmission, respectively, confirming its requirement for efficient virus-acquisition. Our results suggest that CaMV can perceive aphid vectors, either directly or indirectly by sharing the host perception. This novel concept in virology, where viruses respond directly or via the host to the outside world, opens new research horizons, that is, investigating the impact of 'perceptive behaviors' on other steps of the infection cycle.DOI:http://dx.doi.org/10.7554/eLife.00183.001.