RESUMEN
The success of virus transmission by vectors relies on intricate trophic interactions between three partners, the host plant, the virus, and the vector. Despite numerous studies that showed the capacity of plant viruses to manipulate their host plant to their benefit, and potentially of their transmission, the molecular mechanisms sustaining this phenomenon has not yet been extensively analyzed at the molecular level. In this study, we focused on the deregulations induced in Arabidopsis thaliana by an aphid vector that were alleviated when the plants were infected with turnip yellows virus (TuYV), a polerovirus strictly transmitted by aphids in a circulative and nonpropagative mode. By setting up an experimental design mimicking the natural conditions of virus transmission, we analyzed the deregulations in plants infected with TuYV and infested with aphids by a dual transcriptomic and metabolomic approach. We observed that the virus infection alleviated most of the gene deregulations induced by the aphids in a noninfected plant at both time points analyzed (6 and 72 h) with a more pronounced effect at the later time point of infestation. The metabolic composition of the infected and infested plants was altered in a way that could be beneficial for the vector and the virus transmission. Importantly, these substantial modifications observed in infected and infested plants correlated with a higher TuYV transmission efficiency. This study revealed the capacity of TuYV to alter the plant nutritive content and the defense reaction against the aphid vector to promote the viral transmission.
Asunto(s)
Áfidos , Arabidopsis , Luteoviridae , Virus de Plantas , Animales , Enfermedades de las Plantas , Insectos Vectores , Arabidopsis/genética , Luteoviridae/fisiologíaRESUMEN
In plants and some animal lineages, RNA silencing is an efficient and adaptable defense mechanism against viruses. To counter it, viruses encode suppressor proteins that interfere with RNA silencing. Phloem-restricted viruses are spreading at an alarming rate and cause substantial reduction of crop yield, but how they interact with their hosts at the molecular level is still insufficiently understood. Here, we investigate the antiviral response against phloem-restricted turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana. Using a combination of genetics, deep sequencing, and mechanical vasculature enrichment, we show that the main axis of silencing active against TuYV involves 22-nt vsiRNA production by DCL2, and their preferential loading into AGO1. Moreover, we identify vascular secondary siRNA produced from plant transcripts and initiated by DCL2-processed AGO1-loaded vsiRNA. Unexpectedly, and despite the viral encoded VSR P0 previously shown to mediate degradation of AGO proteins, vascular AGO1 undergoes specific post-translational stabilization during TuYV infection. Collectively, our work uncovers the complexity of antiviral RNA silencing against phloem-restricted TuYV and prompts a re-assessment of the role of its suppressor of silencing P0 during genuine infection.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Argonautas/genética , Proteínas de Ciclo Celular/genética , Interacciones Huésped-Patógeno/genética , Luteoviridae/genética , Enfermedades de las Plantas/genética , Ribonucleasa III/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Arabidopsis/inmunología , Arabidopsis/virología , Proteínas de Arabidopsis/inmunología , Proteínas Argonautas/inmunología , Proteínas de Ciclo Celular/inmunología , Resistencia a la Enfermedad/genética , Regulación de la Expresión Génica , Genes Supresores , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno/inmunología , Luteoviridae/crecimiento & desarrollo , Luteoviridae/metabolismo , Floema/genética , Floema/inmunología , Floema/virología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/virología , Interferencia de ARN , Ribonucleasa III/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteínas Virales/metabolismoRESUMEN
RNA silencing is a major antiviral defense mechanism in plants and invertebrates. Plant ARGONAUTE1 (AGO1) is pivotal in RNA silencing, and hence is a major target for counteracting viral suppressors of RNA-silencing proteins (VSRs). P0 from Turnip yellows virus (TuYV) is a VSR that was previously shown to trigger AGO1 degradation via an autophagy-like process. However, the identity of host proteins involved and the cellular site at which AGO1 and P0 interact were unknown. Here we report that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8-Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing (PTGS) activity. Collectively, we identify ATI1 and ATI2 as components of an ER-associated AGO1 turnover and proper PTGS maintenance and further show how the VSR P0 manipulates this pathway.
Asunto(s)
Proteínas Argonautas/metabolismo , Autofagia , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Virales/metabolismo , Proteolisis , Vacuolas/metabolismoRESUMEN
In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing and is a key component in antiviral responses. The polerovirus F-box P0 protein triggers AGO1 degradation as a viral counterdefense. Here, we identified a motif in AGO1 that is required for its interaction with the S phase kinase-associated protein1-cullin 1-F-box protein (SCF) P0 (SCFP0) complex and subsequent degradation. The AGO1 P0 degron is conserved and confers P0-mediated degradation to other AGO proteins. Interestingly, the degron motif is localized in the DUF1785 domain of AGO1, in which a single point mutation (ago1-57, obtained by forward genetic screening) compromises recognition by SCFP0 Recapitulating formation of the RNA-induced silencing complex in a cell-free system revealed that this mutation impairs RNA unwinding, leading to stalled forms of AGO1 still bound to double-stranded RNAs. In vivo, the DUF1785 is required for unwinding perfectly matched siRNA duplexes, but is mostly dispensable for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing. Overall, our work sheds new light on the mode of AGO1 recognition by P0 and the in vivo function of DUF1785 in RNA silencing.
Asunto(s)
Arabidopsis/metabolismo , ARN Bicatenario/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Mutación Puntual/genética , Interferencia de ARNRESUMEN
Aphids are important pests which cause direct damage by feeding or indirect prejudice by transmitting plant viruses. Viruses are known to induce modifications of plant cues in ways that can alter vector behavior and virus transmission. In this work, we addressed whether the modifications induced by the aphid-transmitted Turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana also apply to the cultivated plant Camelina sativa, both belonging to the Brassicaceae family. In most experiments, we observed a significant increase in the relative emission of volatiles from TuYV-infected plants. Moreover, due to plant size, the global amounts of volatiles emitted by C. sativa were higher than those released by A. thaliana. In addition, the volatiles released by TuYV-infected C. sativa attracted the TuYV vector Myzus persicae more efficiently than those emitted by non-infected plants. In contrast, no such preference was observed for A. thaliana. We propose that high amounts of volatiles rather than specific metabolites are responsible for aphid attraction to infected C. sativa. This study points out that the data obtained from the model pathosystem A. thaliana/TuYV cannot be straightforwardly extrapolated to a related plant species infected with the same virus.
Asunto(s)
Áfidos/virología , Brassica/virología , Herbivoria , Insectos Vectores/virología , Enfermedades de las Plantas/virología , Virus de Plantas/aislamiento & purificación , Animales , Áfidos/fisiología , Arabidopsis/fisiología , Arabidopsis/virología , Brassica/fisiología , Insectos Vectores/fisiología , Compuestos Orgánicos Volátiles/análisis , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5' end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement.
Asunto(s)
Brassica napus/virología , Luteoviridae/genética , Luteovirus/genética , Sistemas de Lectura Abierta , Enfermedades de las Plantas/virología , Genoma Viral/inmunología , ARN Viral/genética , Alineación de SecuenciaRESUMEN
The long distance movement of potyviruses is a poorly understood step of the viral cycle. Only factors inhibiting this process, referred to as "Restricted TEV Movement" (RTM), have been identified in Arabidopsis thaliana. On the virus side, the potyvirus coat protein (CP) displays determinants required for long-distance movement and for RTM-based resistance breaking. However, the potyvirus CP was previously shown not to interact with the RTM proteins. We undertook the identification of Arabidopsis factors which directly interact with either the RTM proteins or the CP of lettuce mosaic virus (LMV). An Arabidopsis cDNA library generated from companion cells was screened with LMV CP and RTM proteins using the yeast two-hybrid system. Fourteen interacting proteins were identified. Two of them were shown to interact with CP and the RTM proteins suggesting that a multiprotein complex could be formed between the RTM proteins and virions or viral ribonucleoprotein complexes. Co-localization experiments in Nicotiana benthamiana showed that most of the viral and cellular protein pairs co-localized at the periphery of chloroplasts which suggests a putative role for plastids in this process.
Asunto(s)
Arabidopsis/virología , Proteínas de la Cápside/fisiología , Proteínas de Plantas/metabolismo , Potyvirus/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Regulación Viral de la Expresión Génica/fisiología , Microscopía Confocal , Floema/metabolismo , Floema/virología , Enfermedades de las Plantas/virología , Epidermis de la Planta/citología , Proteínas de Plantas/genética , Transporte de Proteínas , Nicotiana/fisiología , Nicotiana/virología , Técnicas del Sistema de Dos HíbridosRESUMEN
Posttranscriptional gene silencing (PTGS) mediated by siRNAs is an evolutionarily conserved antiviral defense mechanism in higher plants and invertebrates. In this mechanism, viral-derived siRNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. In Arabidopsis, a key component of RISC is ARGONAUTE1 (AGO1), which not only binds to siRNAs but also carries the RNA slicer activity. At present little is known about posttranslational mechanisms regulating AGO1 turnover. Here we report that the viral suppressor of RNA silencing protein P0 triggers AGO1 degradation by the autophagy pathway. Using a P0-inducible transgenic line, we observed that AGO1 degradation is blocked by inhibition of autophagy. The engineering of a functional AGO1 fluorescent reporter protein further indicated that AGO1 colocalizes with autophagy-related (ATG) protein 8a (ATG8a) positive bodies when degradation is impaired. Moreover, this pathway also degrades AGO1 in a nonviral context, especially when the production of miRNAs is impaired. Our results demonstrate that a selective process such as ubiquitylation can lead to the degradation of a key regulatory protein such as AGO1 by a degradation process generally believed to be unspecific. We anticipate that this mechanism will not only lead to degradation of AGO1 but also of its associated proteins and eventually small RNAs.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Autofagia , Proteolisis , Arabidopsis/genética , Arabidopsis/virología , Proteínas de Arabidopsis/genética , Proteínas Argonautas/genética , Silenciador del Gen , MicroARNs/genética , MicroARNs/metabolismo , Virus de Plantas/genética , Virus de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/virología , ARN de Planta/genética , ARN de Planta/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismo , Ubiquitinación/genéticaRESUMEN
Viral genomic RNA of the Turnip yellows virus (TuYV; genus Polerovirus; family Luteoviridae) is protected in virions formed by the major capsid protein (CP) and the minor component, the readthrough (RT*) protein. Long-distance transport, used commonly by viruses to systemically infect host plants, occurs in phloem sieve elements and two viral forms of transport have been described: virions and ribonucleoprotein (RNP) complexes. With regard to poleroviruses, virions have always been presumed to be the long-distance transport form, but the potential role of RNP complexes has not been investigated. Here, we examined the requirement of virions for polerovirus systemic movement by analysing CP-targeted mutants that were unable to form viral particles. We confirmed that TuYV mutants that cannot encapsidate into virions are not able to reach systemic leaves. To completely discard the possibility that the introduced mutations in CP simply blocked the formation or the movement of RNP complexes, we tested in trans complementation of TuYV CP mutants by providing WT CP expressed in transgenic plants. WT CP was able to facilitate systemic movement of TuYV CP mutants and this observation was always correlated with the formation of virions. This demonstrated clearly that virus particles are essential for polerovirus systemic movement.
Asunto(s)
Luteoviridae/fisiología , Virión/fisiología , Ensamble de Virus , Brassica napus/virología , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Hojas de la Planta/virología , Plantas Modificadas GenéticamenteRESUMEN
The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.
Asunto(s)
Nicotiana/virología , Enfermedades de las Plantas/virología , Virus de Plantas/fisiología , Proteínas Virales/genética , Secuencia de Aminoácidos , Beta vulgaris/virología , Nucléolo Celular/metabolismo , Chenopodium quinoa/virología , Citoplasma/metabolismo , Regulación de la Expresión Génica de las Plantas , Regulación Viral de la Expresión Génica , Proteínas Fluorescentes Verdes , Mutagénesis Sitio-Dirigida , Hojas de la Planta/ultraestructura , Hojas de la Planta/virología , Virus de Plantas/genética , Estabilidad Proteica , Transporte de Proteínas , Interferencia de ARN , Virus ARN/genética , Virus ARN/fisiología , ARN Interferente Pequeño , ARN Viral/genética , Proteínas Recombinantes de Fusión , Nicotiana/ultraestructura , Proteínas Virales/química , Proteínas Virales/metabolismo , Dedos de ZincRESUMEN
Plants employ post-transcriptional gene silencing (PTGS) as an antiviral defense response. In this mechanism, viral-derived small RNAs are incorporated into the RNA-induced silencing complex (RISC) to guide degradation of the corresponding viral RNAs. ARGONAUTE1 (AGO1) is a key component of RISC: it carries the RNA slicer activity. As a counter-defense, viruses have evolved various proteins that suppress PTGS. Recently, we showed that the Polerovirus P0 protein carries an F box motif required to form an SCF-like complex, which is also essential for P0's silencing suppressor function. Here, we investigate the molecular mechanism by which P0 impairs PTGS. First we show that P0's expression does not affect the biogenesis of primary siRNAs in an inverted repeat-PTGS assay, but it does affect their activity. Moreover, P0's expression in transformed Arabidopsis plants leads to various developmental abnormalities reminiscent of mutants affected in miRNA pathways, which is accompanied by enhanced levels of several miRNA-target transcripts, suggesting that P0 acts at the level of RISC. Interestingly, ectopic expression of P0 triggered AGO1 protein decay in planta. Finally, we provide evidence that P0 physically interacts with AGO1. Based on these results, we propose that P0 hijacks the host SCF machinery to modulate gene silencing by destabilizing AGO1.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/virología , Proteínas F-Box/metabolismo , Luteoviridae/fisiología , Interferencia de ARN , Proteínas Virales/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Modelos Biológicos , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/virología , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , Complejo Silenciador Inducido por ARN/fisiologíaRESUMEN
Members of the Luteoviridae family occupy a very special position among plant viruses. Unlike most plant viruses that can infect almost all cell types, these viruses exhibit a specific tropism restricted to vascular tissues. The infection of these tissues is maintained by a piercing-sucking insect vector, an aphid that promotes viral plant-to-plant transmission by feeding on phloem sap. This review focuses on the movement in the phloem of viruses belonging to the Luteoviridae family underlining the roles of viral proteins in this process. A second part is dedicated to the unique mode of action of the silencing suppressor of the Polerovirus genus (one of the three Luteoviridae genera). Finally, several hypotheses are discussed in order to explain the phloem restriction of these peculiar viruses.
RESUMEN
Plant viruses rely on both host plant and vectors for a successful infection. Essentially to simplify studies, transmission has been considered for decades as an interaction between two partners, virus and vector. This interaction has gained a third partner, the host plant, to establish a tripartite pathosystem in which the players can react with each other directly or indirectly through changes induced in/by the third partner. For instance, viruses can alter the plant metabolism or plant immune defence pathways to modify vector's attraction, settling or feeding, in a way that can be conducive for virus propagation. Such changes in the plant physiology can also become favourable to the vector, establishing a mutualistic relationship. This review focuses on the recent molecular data on the interplay between viral and plant factors that provide some important clues to understand how viruses manipulate both the host plants and vectors in order to improve transmission conditions and thus ensuring their survival.
Asunto(s)
Vectores de Enfermedades , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/virología , Virus de Plantas/fisiología , Animales , Resistencia a la Enfermedad , Regulación Viral de la Expresión Génica , Transporte de Proteínas , Transducción de Señal , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
During the process of virus acquisition by aphids, plants respond to both the virus and the aphids by mobilizing different metabolic pathways. It is conceivable that the plant metabolic responses to both aggressors may be conducive to virus acquisition. To address this question, we analyze the accumulation of the phloem-limited polerovirus Turnip yellows virus (TuYV), which is strictly transmitted by aphids, and aphid's life traits in six Arabidopsis thaliana mutants (xth33, ss3-2, nata1, myc234, quad, atr1D, and pad4-1). We observed that mutations affecting the carbohydrate metabolism, the synthesis of a non-protein amino acid and the glucosinolate pathway had an effect on TuYV accumulation. However, the virus titer did not correlate with the virus transmission efficiency. Some mutations in A.thaliana affect the aphid feeding behavior but often only in infected plants. The duration of the phloem sap ingestion phase, together with the time preceding the first sap ingestion, affect the virus transmission rate more than the virus titer did. Our results also show that the aphids reared on infected mutant plants had a reduced biomass regardless of the mutation and the duration of the sap ingestion phase.
Asunto(s)
Áfidos/fisiología , Arabidopsis/genética , Conducta Alimentaria , Luteoviridae/fisiología , Redes y Vías Metabólicas/genética , Mutación , Animales , Áfidos/virología , Femenino , Insectos Vectores/fisiología , Insectos Vectores/virología , Luteoviridae/genética , Floema/virología , Enfermedades de las Plantas/virologíaRESUMEN
Most phytoviruses rely on vectors for their spread and survival. Although a great variety of virus vectors have been described, there are relatively few different mechanisms mediating virus transmission by vectors: virions can either be internalized into vector cells where replication may or may not take place or they can simply be adsorbed on the vector's surface or cuticle. Virus transmission by vectors requires tight associations between viral proteins, generally capsid proteins, and vector compounds, usually referred to as receptors. This review will focus on the viral determinants involved in virus transmission. Only the best-known models for which molecular data are available are described.
Asunto(s)
Enfermedades de las Plantas/virología , Virus de Plantas/fisiología , Replicación Viral , Animales , Insectos Vectores , Insectos/virología , Virus de Plantas/patogenicidad , Fenómenos Fisiológicos de los VirusRESUMEN
The phloem-limited poleroviruses infect Arabidopsis thaliana without causing noticeable disease symptoms. In order to facilitate visual infection identification, we developed virus-induced gene silencing (VIGS) vectors derived from Turnip yellows virus (TuYV). Short sequences from the host gene AtCHLI1 required for chlorophyll biosynthesis [42 nucleotides in sense or antisense orientation or as an inverted-repeat (IR), or an 81 nucleotide sense fragment] were inserted into the 3' non-coding region of the TuYV genome to screen for the most efficient and robust silencing vector. All recombinant viruses produced a clear vein chlorosis phenotype on infected Arabidopsis plants due to the expression inhibition of the AtCHLI1 gene. The introduction of a sense-oriented sequence into TuYV genome resulted in a virus exhibiting a more sustainable chlorosis than the virus containing an IR of the same length. This observation was correlated with a higher stability of the sense sequence insertion in the viral genome. In order to evaluate the impact of the TuYV silencing suppressor P0 in the VIGS mechanism a P0 knock-out mutation was introduced into the recombinant TuYV viruses. They induced a similar but milder vein clearing phenotype due to lower viral accumulation. This indicates that P0 does not hinder the performances of the TuYV silencing effect and confirms that in the viral infection context, P0 has no major impact on the production, propagation and action of the short distance silencing signal in phloem cells. Finally, we showed that TuYV can be used to strongly silence the phloem specific AtRTM1 gene. The TuYV-derived VIGS vectors therefore represent powerful tools to easily detect and monitor TuYV in infected plants and conduct functional analysis of phloem-restricted genes. Moreover this example indicates the potential of poleroviruses for use in functional genomic studies of agronomic plants.
RESUMEN
A fluorescent viral clone of the polerovirus Turnip yellows virus (TuYV) was engineered by introducing the Enhanced Green Fluorescent Protein (EGFP) sequence into the non-structural domain sequence of the readthrough protein, a minor capsid protein. The resulting recombinant virus, referred to as TuYV-RTGFP, was infectious in several plant species when delivered by agroinoculation and invaded efficiently non-inoculated leaves. As expected for poleroviruses, which infect only phloem cells, the fluorescence emitted by TuYV-RTGFP was restricted to the vasculature of infected plants. In addition, TuYV-RTGFP was aphid transmissible and enabled the observation of the initial sites of infection in the phloem after aphid probing in epidermal cells. The aphid-transmitted virus moved efficiently to leaves distant from the inoculation sites and importantly retained the EGFP sequence in the viral genome. This work reports on the first engineered member in the Luteoviridae family that can be visualized by fluorescence emission in systemic leaves of different plant species after agroinoculation or aphid transmission.
Asunto(s)
Proteínas Fluorescentes Verdes/análisis , Luteoviridae/crecimiento & desarrollo , Enfermedades de las Plantas/virología , Coloración y Etiquetado/métodos , Agrobacterium/genética , Animales , Áfidos/virología , Proteínas Fluorescentes Verdes/genética , Insectos Vectores/virología , Luteoviridae/genética , Plantas/virología , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Transformación Genética , Proteínas Virales/genéticaRESUMEN
Interactions among plant pathogenic viruses in the family Luteoviridae and their plant hosts and insect vectors are governed by the topology of the viral capsid, which is the sole vehicle for long distance movement of the viral genome. Previous application of a mass spectrometry-compatible cross-linker to preparations of the luteovirid Potato leafroll virus (PLRV; Luteoviridae: Polerovirus) revealed a detailed network of interactions between viral structural proteins and enabled generation of the first cross-linking guided coat protein models. In this study, we extended application of chemical cross-linking technology to the related Turnip yellows virus (TuYV; Luteoviridae: Polerovirus). Remarkably, all cross-links found between sites in the viral coat protein found for TuYV were also found in PLRV. Guided by these data, we present two models for the TuYV coat protein trimer, the basic structural unit of luteovirid virions. Additional cross-links found between the TuYV coat protein and a site in the viral protease domain suggest a possible role for the luteovirid protease in regulating the structural biology of these viruses.
Asunto(s)
Proteínas de la Cápside/genética , Luteoviridae/genética , Luteoviridae/ultraestructura , Enfermedades de las Plantas/virología , Virus de Plantas/genética , Brassica/virología , Proteínas de la Cápside/metabolismo , Grano Comestible/virología , Genoma Viral/genética , Espectrometría de Masas , Modelos Moleculares , Unión Proteica , Saccharum/virología , Solanum tuberosum/virología , Glycine max/virología , Nicotiana/virologíaRESUMEN
Viral pathogenicity has often been correlated to the expression of the viral encoded-RNA silencing suppressor protein (SSP). The silencing suppressor activity of the P0 protein encoded by cereal yellow dwarf virus-RPV (CYDV-RPV) and -RPS (CYDV-RPS), two poleroviruses differing in their symptomatology was investigated. CYDV-RPV displays milder symptoms in oat and wheat whereas CYDV-RPS is responsible for more severe disease. We showed that both P0 proteins (P0(CY-RPV) and P0(CY-RPS)) were able to suppress local RNA silencing induced by either sense or inverted repeat transgenes in an Agrobacterium tumefaciens-mediated expression assay in Nicotiana benthamiana. P0(CY-RPS) displayed slightly higher activity. Systemic spread of the silencing signal was not impaired. Analysis of short-interfering RNA (siRNA) abundance revealed that accumulation of primary siRNA was not affected, but secondary siRNA levels were reduced by both CYDV P0 proteins, suggesting that they act downstream of siRNA production. Correlated with this finding we showed that both P0 proteins partially destabilized ARGONAUTE1. Finally both P0(CY-RPV) and P0(CY-RPS) interacted in yeast cells with ASK2, a component of an E3-ubiquitin ligase, with distinct affinities.
Asunto(s)
Luteoviridae/metabolismo , Nicotiana/genética , Enfermedades de las Plantas/virología , Interferencia de ARN , Proteínas Virales/metabolismo , Avena/genética , Avena/virología , Interacciones Huésped-Patógeno , Luteoviridae/genética , Enfermedades de las Plantas/genética , Nicotiana/virología , Triticum/genética , Triticum/virología , Proteínas Virales/genéticaRESUMEN
Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RTCter) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RTCter. Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells.