RESUMEN
Citrus tristeza virus (CTV) has been studied intensively at the molecular level. However, knowledge regarding the dynamics of its evolution is practically non-existent. In the past, diverse authors have referred to CTV as a highly variable virus, implying rapid evolution. Others have, in recent times, referred to CTV as an exceptionally slowly evolving virus. In this work, we used the capsid protein (CP) gene to estimate the rate of evolution. This was obtained from a large set of heterochronous CP gene sequences using a bayesian coalescent approach. The best-fitting evolutionary and population models pointed to an evolutionary rate of 1.58×10(-4) nt per site year(-1) (95â% highest posterior density, 1.73×10(-5)-3.16×10(-4) nt per site year(-1)). For an unbiased comparison with other plant and animal viruses, the evolutionary rate of synonymous substitutions was considered. In a series of 88 synonymous evolutionary rates, ranging from 5.2×10(-6) to 6.2×10(-2) nt per site year(-1), CTV ranks in the 10th percentile, embedded among the slowest animal RNA viruses. At the time of citrus dissemination to Europe and the New World, the major clades that led to the current phylogenetic groups were already defined, which may explain the absence nowadays of geographical speciation.
Asunto(s)
Closterovirus/genética , Evolución Molecular , Américas , Sustitución de Aminoácidos , Proteínas de la Cápside/genética , Citrus/virología , Europa (Continente) , Mutación PuntualRESUMEN
At least five phylogenetic groups have been reported for Grapevine leafroll-associated virus 3 (GLRaV-3). The p19.7 protein encoded by the GLRaV-3 was previously identified as an RNA silencing suppressor. In this study, five constructs of p19.7 belonging to different groups were compared for their suppressing activity. For each p19.7 variant, the accumulation level of green fluorescent protein mRNA and specific siRNAs were determined using co-infiltration assays in transgenic 16C Nicotiana benthamiana. Differences in the suppressing activity were found among the variants assayed. Some constructs originated viral-like mosaic symptoms that evolved to necrosis. The intensity of these symptoms appeared to be related to the strength of the suppressor activity. A comparison of the protein sequences revealed a few amino acid substitutions that may be associated with the observed differences in the suppressing activity.
Asunto(s)
Closteroviridae/patogenicidad , Regulación Viral de la Expresión Génica , Interferencia de ARN , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Closteroviridae/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Datos de Secuencia Molecular , Mutación Missense , Enfermedades de las Plantas/virología , Plantas Modificadas Genéticamente/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Nicotiana/genética , Proteínas Virales/genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
The genetic variability and population structure of grapevine leafroll-associated virus 3 (GLRaV-3) variants were updated by examining the diversity within the viral coat protein (CP) gene among 174 isolates belonging to a collection of Vitis vinifera representing most of the Portuguese varieties. Phylogenetic analysis revealed the existence of five well-defined clusters. Three of these correspond to previously defined groups, another corresponds to variants from Chile for which only one sequence has been previously identified, and an additional new group includes only Portuguese variants. A typing tool based on asymmetric PCR-ELISA (APET) was developed within the frame of this population structure. This tool was used to assess the prevalence of each phylogenetic group among the infected grapevine varieties. Although most of the isolates harbour variants from groups 1 and 2, variants from the remaining three groups exist in a number of varieties, reinforcing the notion that they are genuine genomic variants and are not isolated, atypical cases.
Asunto(s)
Proteínas de la Cápside/genética , Closteroviridae/clasificación , Closteroviridae/genética , Variación Genética , Filogenia , ARN Viral/genética , Vitis/virología , Closteroviridae/aislamiento & purificación , Análisis por Conglomerados , Datos de Secuencia Molecular , Portugal , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
RNA silencing is an important defense mechanism in plants, yet several plant viruses encode proteins that suppress this mechanism. In this study, the genome of the Olive mild mosaic virus (OMMV) was screened for silencing suppressors. The full OMMV cDNA and 5 OMMV open reading frames (ORFs) were cloned into the Gateway binary vector pK7WG2, transformed into Agrobacterium tumefaciens, and agroinfiltrated into N. benthamiana 16C plants. CP and p6 showed suppressor activity, with CP showing significantly higher activity than p6, yet activity that was lower than the full OMMV, suggesting a complementary action of CP and p6. These viral suppressors were then used to induce OMMV resistance in plants based on RNA silencing. Two hairpin constructs targeting each suppressor were agroinfiltrated in N. benthamiana plants, which were then inoculated with OMMV RNA. When silencing of both suppressors was achieved, a significant reduction in viral accumulation and symptom attenuation was observed as compared to those of the controls, as well as to when each construct was used alone, proving them to be effective against OMMV infection. This is the first time that a silencing suppressor was found in a necrovirus, and that two independent proteins act as silencing suppressors in a virus member of the Tombusviridae family.
Asunto(s)
Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/virología , Interferencia de ARN , Tombusviridae/genética , Proteínas Virales/genética , Agrobacterium tumefaciens/genética , Clonación Molecular , Vectores Genéticos , Genoma Viral , Plantas Modificadas Genéticamente/virología , Nicotiana/virología , Proteínas Virales/metabolismoRESUMEN
To evaluate the dynamic role of the ferric-chelate reductase enzyme (FCR) and to identify possible pathways of regulation of its activity in different plant organs an investigation was conducted by virus-induced gene silencing (VIGS) using tobacco rattle virus (TRV) to silence the ferric reductase oxidase gene (FRO1) that encodes the FCR enzyme. Half of Nicotiana benthamiana plants received the VIGS vector and the rest remained as control. Four treatments were imposed: two levels of Fe in the nutrient solution (0 or 2.5 µM of Fe), each one with silenced or non-silenced (VIGS-0; VIGS-2.5) plants. Plants grown without iron (0; VIGS-0) developed typical symptoms of iron deficiency in the youngest leaves. To prove that FRO1 silencing had occurred, resupply of Fe (R) was done by adding 2.5 µM of Fe to the nutrient solution in a subset of chlorotic plants (0-R; VIGS-R). Twelve days after resupply, 0-R plants had recovered from Fe deficiency while plants containing the VIGS vector (VIGS-R) remained chlorotic and both FRO1 gene expression and FCR activity were considerably reduced, consequently preventing Fe uptake. With the VIGS technique we were able to silence the FRO1 gene in N. benthamiana and point out its importance in chlorophyll synthesis and Fe partition.
Asunto(s)
Hierro/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Clorofila/metabolismo , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de Plantas , Hidroponía , Hojas de la Planta/fisiología , Virus de Plantas/genéticaRESUMEN
ABSTRACT In situ reverse transcription-polymerase chain reaction (RT-PCR) was used in young leaves (from trees and in vitro shoots) and flower buds of almond (Prunus dulcis), a stone fruit, for cellular location of Prune dwarf virus (PDV, a member of the genus Ilarvirus). Sections obtained from samples fixed in formaldehyde and embedded in paraffin were refixed in formaldehyde to increase tissue preservation in the RT-PCR steps. The coat protein gene of PDV was used as the target to produce a cDNA copy that was amplified by PCR and visualized using a direct detection method with digoxigenin-labeled nucleotides. Protein digestion, PCR, and detection strategies were optimized for increased tissue preservation and signal intensity. PDV was found in infected samples within the vascular tissue of young leaves and flower buds as well as in the mesophyll in developing floral organs and in the generative and vegetative cells of pollen grains. PDV signals were observed in a ring surrounding the nucleus and spread in the cytoplasm. The results obtained are discussed in terms of the technique optimization and PDV distribution in tissues and transmission through pollen. The optimized protocol of in situ RT-PCR is a powerful technique to reveal low-abundant RNA species. Therefore, it is appropriate to study cell and subcellular distribution of RNA viruses in woody species.
RESUMEN
The genetic variability among 13 isolates of Olive mild mosaic virus (OMMV) and of 11 isolates of Tobacco necrosis virus D (TNV-D) recovered from Olea europaea L. samples from various sites in Portugal, was assessed through the analysis of the coat protein (CP) gene sequences. This gene was amplified through reverse transcriptase polymerase chain reaction (RT-PCR), cloned, and 5 clone sequences of each virus isolate, were analysed and compared, including sequences from OMMV and TNV-D isolates originally recovered from different hosts and countries and available in the GenBank, totalling 131 sequences. The encoded CP sequences consisted of 269 amino acids (aa) in OMMV and 268 in TNV-D. Comparison of the CP genomic and amino acid sequences of the isolates showed a very low variability among OMMV isolates, 0.005 and 0.007, respectively, as well as among TNV-D isolates, 0.006 and 0.008. The maximum nucleotide distances of OMMV and TNV-D sequences within isolates were also low, 0.013 and 0.031, respectively, and close to that found between isolates, 0.018 and 0.034, respectively. In some cases, less variability was found in clone sequences between isolates than in clone sequences within isolates, as also shown through phylogenetic analysis. CP aa sequence identities among OMMV and TNV-D isolates ranged from 84.3% to 85.8%. Comparison between the CP genomic sequences of the two viruses, showed a relatively low variability, 0.199, and a maximum nucleotide distance between isolates of 0.411. Analysis of comparative models of OMMV and TNV-D CPs, showed that naturally occurring substitutions in their respective sequences do not seem to cause significant alterations in the virion structure. This is consistent with a high selective pressure to preserve the structure of viral capsid proteins.
Asunto(s)
Variación Genética , Virus del Mosaico/genética , Olea/virología , Tombusviridae/genética , Secuencia de Aminoácidos , Proteínas de la Cápside/genética , Clonación Molecular , Evolución Molecular , Genes Virales , Genoma Viral , Geografía , Datos de Secuencia Molecular , Nucleótidos/química , Nucleótidos/genética , Filogenia , Portugal , Estructura Terciaria de Proteína , ARN Viral/genética , Homología de Secuencia de AminoácidoRESUMEN
The isolates of Citrus tristeza virus (CTV), the most destructive viral pathogen of citrus, display a high level of variability. As a result of genetic bottleneck induced by the bud-inoculation of CTV-infected material, inoculated seedlings of Citrus wilsonii Tanaka displayed different symptoms. All successfully grafted plants showed severe symptoms of stem pitting and seedling yellows, while plants in which inoculated buds died displayed mild symptoms. Since complex CTV population structure was detected in the parental host, the aim of this work was to investigate how it changed after the virus transmission, and to correlate it with observed symptoms. The coat protein gene sequence of the predominant genotype was identical in parental and grafted plants and clustered to the phylogenetic group 5 encompassing severe reference isolates. In seedlings displaying severe symptoms, the low-frequency variants clustering to other phylogenetic groups were detected, as well. Indicator plants were inoculated with buds taken from unsuccessfully grafted C. wilsonii seedlings. Surprisingly, they displayed no severe symptoms despite the presence of phylogenetic group 5 genomic variants. The results suggest that the appearance of severe symptoms in this case is probably induced by a complex CTV population structure found in seedlings displaying severe symptoms, and not directly by the predominant genomic variant.