RESUMEN
Plant infection by poleroviruses is restricted to phloem tissues, preventing any classical leaf rub inoculation with viral RNA or virions. Efficient virus inoculation to plants is achieved by viruliferous aphids that acquire the virus by feeding on infected plants. The use of promoter-driven infectious cDNA is an alternative means to infect plants and allows reverse genetic studies to be performed. Using Beet mild yellowing virus isolate 2ITB (BMYV-2ITB), we produced a full-length infectious cDNA clone of the virus (named BMYV-EK) placed under the control of the T7 RNA polymerase and the Cauliflower mosaic virus 35S promoters. Infectivity of the engineered BMYV-EK virus was assayed in different plant species and compared with that of the original virus. We showed that in vitro- or in planta-derived transcripts were infectious in protoplasts and in whole plants. Importantly, the natural aphid vector Myzus persicae efficiently transmitted the viral progeny produced in infected plants. By comparing agroinoculation and aphid infection in a host range assay, we showed that the engineered BMYV-EK virus displayed a similar host range to BMYV-2ITB, except for Nicotiana benthamiana, which proved to be resistant to systemic infection with BMYV-EK. Finally, both the BMYV-EK P0 and the full-length clone were able to strongly interfere with post-transcriptional gene silencing.
Asunto(s)
Beta vulgaris/virología , ADN Complementario/genética , Especificidad del Huésped , Enfermedades de las Plantas/virología , Virus de Plantas/genética , Virus de Plantas/aislamiento & purificación , Animales , Áfidos/virología , Arabidopsis/virología , Secuencia de Bases , Células Clonales , Silenciador del Gen , Genoma Viral/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Protoplastos/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Virión/metabolismoRESUMEN
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
Beet necrotic yellow vein virus (BNYVV) is a multipartite RNA virus. BNYVV RNA3 does not accumulate in non-host transgenic Arabidopsis thaliana plants when expressed using a 35S promoter. However, a 3'-derivative species has been detected in transgenic plants and in transient expression assays conducted in Nicotiana benthamiana and Beta macrocarpa. The 3'-derivative species is similar to the previously reported subgenomic RNA3 produced during virus infection. 5' RACE revealed that the truncated forms had identical 5' ends. The 5' termini carried the coremin motif also present on BNYVV RNA5, beet soil-borne mosaic virus RNA3 and 4, and cucumber mosaic virus group 2 RNAs. This RNA3 species lacks a m(7)Gppp at the 5' end of the cleavage products, whether expressed transiently or virally. Mutagenesis revealed the importance of the coremin sequence for both long-distance movement and stabilization of the cleavage product in vivo and in vitro. The isolation of various RNA3 5'-end products suggests the existence of a cleavage between nt 212 and 1234 and subsequent exonucleolytic degradation, leading to the accumulation of a non-coding RNA. When RNA3 was incubated in wheatgerm extracts, truncated forms appeared rapidly and their appearance was protein- and divalent ion-dependent.
Asunto(s)
Genoma Viral , Virus de Plantas/patogenicidad , ARN no Traducido/metabolismo , ARN Viral/metabolismo , Arabidopsis/virología , Beta vulgaris/virología , Virus de Plantas/genética , Estabilidad del ARN , ARN Viral/genética , Nicotiana/virologíaRESUMEN
The RNA-3-encoded p25 protein was previously characterized as one of the major symptom determinants of the Beet necrotic yellow vein virus. Previous analyses reported the influence of the p25 protein in root proliferation phenotype observed in rhizomania disease on infected sugar beets (Beta vulgaris). A transgenic approach was developed, in which the p25 protein was constitutively expressed in Arabidopsis thaliana Columbia (Col-0) ecotype in order to provide new clues as to how the p25 protein might promote alone disease development and symptom expression. Transgenic plants were characterized by Southern blot and independent lines carrying single and multiple copies of the transgene were selected. Mapping of the T-DNA insertion was performed on the monocopy homozygote lines. P25 protein was localized both in the nucleus and in the cytoplasm of epidermal and root cells of transgenic plants. Although A. thaliana was not described as a susceptible host for BNYVV infection, abnormal root branching was observed on p25 protein-expressing A. thaliana plants. Moreover, these transgenic plants were more susceptible than wild-type plants to auxin analog treatment (2,4-D) but more resistant to methyl jasmonate (MeJA), abscisic acid (ABA) and to lesser extend to salicylic acid (SA). Hormonal content assays measuring plant levels of auxin (IAA), jasmonate (JA) and ethylene precursor (ACC) revealed major hormonal changes. Global transcript profiling analyses on roots displayed differential gene expressions that could corroborate root branching phenotype and stress signaling modifications.
Asunto(s)
Arabidopsis/metabolismo , Beta vulgaris/virología , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/fisiología , Plantas Modificadas Genéticamente/metabolismo , Virus ARN/metabolismo , Proteínas Virales/metabolismo , Arabidopsis/genética , Arabidopsis/virología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Ciclopentanos/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , ADN Bacteriano/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ácidos Indolacéticos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxilipinas/metabolismo , Fenotipo , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Raíces de Plantas/metabolismo , Raíces de Plantas/virología , Virus de Plantas/genética , Virus de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/virología , Virus ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/genéticaRESUMEN
A Beet necrotic yellow vein virus isolate containing a fifth RNA is present in the Pithiviers area of France. A full-length cDNA clone of RNA-5 was obtained and placed under the control of a T(7)-RNA-pol promoter that allowed the production of infectious transcripts. "Pithiviers" isolate-specific necrotic symptoms were obtained on Chenopodium quinoa when RNA-5-encoded p26 was expressed either from RNA-5 or from an RNA-3-derived replicon. By using haemagglutinin- and green fluorescent protein-tagged constructs, virally expressed p26-fusion proteins induced the same necrotic local lesions on host plants and were localized mainly in the nucleus of infected cells. Deletion mutagenesis permitted identification of two domains, responsible respectively for nuclear export and cytoplasmic retention of the p26 mutated proteins. By using a yeast two-hybrid system, Gal4DB-p26 protein self-activated transcription of the His3 reporter gene. The p26 transcription-activation domain was located within its first 55 aa and has been studied by alanine scanning. Resulting p26 mutants were tested for their capability to induce necrotic symptoms and to localize in the nuclear compartment.
Asunto(s)
Chenopodium quinoa/virología , Regulación Viral de la Expresión Génica , Enfermedades de las Plantas/virología , Virus ARN/patogenicidad , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Beta vulgaris/virología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Hojas de la Planta/virología , Virus de Plantas/genética , Virus de Plantas/patogenicidad , Virus ARN/genética , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/genéticaRESUMEN
The protein p25 encoded by beet necrotic yellow vein virus (BNYVV) RNA-3 is involved in symptom expression of infected plants. Confocal microscopy analysis of wild-type and mutated p25 fused to GFP and transiently expressed in BY-2 tobacco suspension cells identified a nuclear localization signal (NLS) in the N-terminal part of the protein. Functionality of the NLS was confirmed by pull-down assays using rice and pepper importin-alpha. Furthermore, it was demonstrated that p25 contains a nuclear export sequence sensitive to leptomycin B. The nuclear export signal (NES) was characterized by mutagenesis. A GFP-p25 fusion protein expressed during a BNYVV infection of Chenopodium quinoa leaves had the same subcellular localization as observed during transient expression in BY-2 cells. The symptom phenotype induced by expression of GFP-p25 during infection was similar to that induced by wild-type virus. Studies with mutated derivatives of GFP-p25 revealed that symptom phenotype was altered when the subcellular localization of GFP-p25 was modified.