RESUMEN

Resistance-breaking (RB) isolates of citrus tristeza virus (CTV) can replicate and move systemically in Poncirus trifoliata, a rootstock widely used for management of decline caused by CTV and other purposes. In Uruguay, severe CTV isolates are prevalent, and an RB isolate (designated as RB-UY1) was identified. In order to predict the implications of this genotype circulating in citrus crops grafted on trifoliate rootstocks, the aim of this work was to determine the biological and molecular characteristics of this isolate, the efficiency of its transmission by Toxoptera citricida, and its effects on plant growth performance of P. trifoliata. Our results show that RB-UY1 can be classified as a mild isolate, that it is phylogenetically associated with the RB1 group, and that it is efficiently transmitted by T. citrida. They also suggest that the RB-UY1 isolate should not affect the performance of citrus crops grafted on trifoliate rootstocks, although some growth parameters of P. trifoliata seedlings were affected four years after inoculation.

Asunto(s)

Citrus , Closterovirus , Poncirus , Poncirus/genética , Uruguay , Closterovirus/genética

RESUMEN

Citrus tristeza virus (CTV) is an important threat to the global citrus industry, causing severe economic losses worldwide. The disease management strategies are focused on vector control, tree culling, and the use of resistant varieties and rootstocks. Sweet orange (Citrus sinensis) trees showing either severe or mild CTV symptoms have been observed in orchards in Veracruz, Mexico, and were probably caused by different virus strains. To understand these symptomatic differences, transcriptomic analyses were conducted using asymptomatic trees. CTV was confirmed to be associated with infected plants, and mild and severe strains were successfully identified by a polymorphism in the coat protein (CP) encoding gene. RNA-Seq analysis revealed more than 900 significantly differentially expressed genes in response to mild and severe strains, with some overlapping genes. Importantly, multiple sequence reads corresponding to Citrus exocortis viroid and Hop stunt viroid were found in severe symptomatic and asymptomatic trees, but not in plants with mild symptoms. The differential gene expression profiling obtained in this work provides an overview of molecular behavior in naturally CTV-infected trees. This work may contribute to our understanding of citrus-virus interaction in more natural settings, which can help develop strategies for integrated crop management.

Asunto(s)

Citrus sinensis/virología , Closterovirus/patogenicidad , Perfilación de la Expresión Génica/métodos , Proteínas de Plantas/genética , Virus de Plantas/patogenicidad , Proteínas Virales/genética , Citrus sinensis/genética , Closterovirus/genética , Resistencia a la Enfermedad , Regulación de la Expresión Génica de las Plantas , Regulación Viral de la Expresión Génica , México , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Virus de Plantas/genética , RNA-Seq , Virulencia

RESUMEN

Proteomics is an excellent technique for detecting proteins involved in plant responses infected with the Citrus tristeza virus. Here we describe the process to extract proteins to obtain two-dimensional gels with a large number of spots, well distributed, and with good quality. The extraction process that associates successive washes with ultrasonication steps proved to be efficient for the detection of proteins. With this technique it was possible to obtain clear gels which were detected in about 600 spots distributed throughout the gel without the presence of drag.

Asunto(s)

Closterovirus/genética , Enfermedades de las Plantas/virología , Proteómica/métodos , Regulación de la Expresión Génica de las Plantas/genética , Interacciones Huésped-Patógeno

RESUMEN

High-throughput sequencing analysis detected a clostero-like virus from arracacha plants (Arracacia xanthorrhiza) in Brazil. The complete genome sequence, confirmed by RACE and Sanger sequencing, consists of 15,763 nucleotides with nine predicted open reading frames (ORFs) in a typical closterovirus genome organisation. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homologue (Hsp70h), and coat protein showed 55-65, 38-44, and 20-36% amino acid sequence identity, respectively, to the homologous proteins of known closteroviruses. Phylogenetic analysis of Hsp70h showed that this putative novel arracacha plant virus was related to members of the genus Closterovirus in the family Closteroviridae. These results suggest that this virus, tentatively named "arracacha virus 1" (AV-1), is a novel member of the genus Closterovirus. This is the first closterovirus identified in arracacha plants.

Asunto(s)

Apiaceae/virología , Closterovirus/aislamiento & purificación , Enfermedades de las Plantas/virología , Brasil , Closterovirus/clasificación , Closterovirus/genética , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Sistemas de Lectura Abierta , Filogenia , Proteínas Virales/genética

RESUMEN

Citrus tristeza virus (CTV) is a major pathogen affecting citrus trees worldwide. However, few studies have focused on CTV's evolutionary history and geographic behavior. CTV is locally dispersed by an aphid vector and long distance dispersion due to transportation of contaminated material. With the aim to delve deeper into the CTV-NC (New Clade) genotype evolution, we estimated an evolution rate of 1.19 × 10-3 subs/site/year and the most common recent ancestor in 1977. Furthermore, the place of origin of the genotype was in the United States, and a great expansion of the population was observed in Uruguay. This expansion phase could be a consequence of the increment in the number of naïve citrus trees in Uruguayan orchards encompassing citrus industry growth in the past years.

Asunto(s)

Citrus/virología , Closterovirus/genética , Evolución Molecular , Análisis Espacio-Temporal , Animales , Áfidos/virología , Closterovirus/patogenicidad , Genotipo , Filogeografía , Enfermedades de las Plantas/virología , Estados Unidos , Uruguay

RESUMEN

Citrus Tristeza disease, caused by CTV (Citrus tristeza virus), committs citrus plantations around the world and specifically attacks phloem tissues of the plant. The virus exists as a mixture of more or less severe variants, which may or may not cause symptoms of Tristeza. The objective of this study was to analyze the changes caused by CTV in the proteome of stems of sweet orange, as well as in the activity and gene expression of antioxidant enzymes. The CTV-infected sweet orange displayed mild symptoms, which were characterized by the presence of sparse stem pitting throughout their stems. The presence of virus was confirmed by RT-PCR. Proteomic analysis by 2DE-PAGE-MS / MS revealed the identity of 40 proteins differentially expressed between CTV- infected and -non-infected samples. Of these, 33 were up-regulated and 7 were down-regulated in CTV-infected samples. Among the proteins identified stands out a specific from the virus, the coat protein. Other proteins identified are involved with oxidative stress and for this their enzymatic activity was measured. The activity of superoxide dismutase (SOD) was higher in CTV-infected samples, as catalase (CAT) showed higher activity in uninfected samples. The activity of guaiacol peroxidase (GPX) did not vary significantly between samples. However, ascorbate peroxidase (APX) was more active in the infected samples. The relative expression of the genes encoding CAT, SOD, APX and GPX was analyzed by quantitative real time PCR (RT-qPCR). The CTV-infected samples showed greater accumulation of transcripts, except for the CAT gene. This gene showed higher expression in the uninfected samples. Taken together, it can be concluded that the CTV affects the protein profile and activity and gene expression of antioxidant enzymes in plants infected by this virus.

Asunto(s)

Citrus sinensis/virología , Closterovirus/fisiología , Enzimas/metabolismo , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Ascorbato Peroxidasas/genética , Ascorbato Peroxidasas/metabolismo , Catalasa/genética , Catalasa/metabolismo , Citrus sinensis/genética , Citrus sinensis/metabolismo , Closterovirus/genética , Electroforesis en Gel Bidimensional , Enzimas/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica de las Plantas , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno/genética , Peroxidasa/genética , Peroxidasa/metabolismo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Proteínas de Plantas/genética , Proteoma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Espectrometría de Masas en Tándem

RESUMEN

Citrus Tristeza Virus (CTV) is the most economically important virus of citrus worldwide. Genetic diversity and population structure of CTV isolates from all citrus growing areas from Uruguay were analyzed by RT-PCR and cloning of the three RNA silencing suppressor genes (p25, p20 and p23). Bayesian phylogenetic analysis revealed the circulation of three known genotypes (VT, T3, T36) in the country, and the presence of a new genetic lineage composed by isolates from around the world, mainly from South America. Nucleotide and amino acid identity values for this new genetic lineage were both higher than 97% for the three analyzed regions. Due to incongruent phylogenetic relationships, recombination analysis was performed using Genetic Algorithms for Recombination Detection (GARD) and SimPlot software. Recombination events between previously described CTV isolates were detected. High intra-sample variation was found, confirming the co-existence of different genotypes into the same plant. This is the first report describing: (1) the genetic diversity of Uruguayan CTV isolates circulating in the country and (2) the circulation of a novel CTV genetic lineage, highly present in the South American region. This information may provide assistance to develop an effective cross-protection program.

Asunto(s)

Citrus/virología , Closterovirus/genética , Genes Supresores , Filogenia , Enfermedades de las Plantas/virología , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Citrus/genética , Closterovirus/clasificación , Closterovirus/aislamiento & purificación , Variación Genética , Datos de Secuencia Molecular , Enfermedades de las Plantas/genética , Interferencia de ARN , América del Sur

RESUMEN

Citrus tristeza virus (CTV) is phloem restricted in natural citrus hosts. The 23-kDa protein (p23) encoded by the virus is an RNA silencing suppressor and a pathogenicity determinant. The expression of p23, or its N-terminal 157-amino-acid fragment comprising the zinc finger and flanking basic motifs, driven by the constitutive 35S promoter of cauliflower mosaic virus, induces CTV-like symptoms and other aberrations in transgenic citrus. To better define the role of p23 in CTV pathogenesis, we compared the phenotypes of Mexican lime transformed with p23-derived transgenes from the severe T36 and mild T317 CTV isolates under the control of the phloem-specific promoter from Commelina yellow mottle virus (CoYMV) or the 35S promoter. Expression of the constructs restricted to the phloem induced a phenotype resembling CTV-specific symptoms (vein clearing and necrosis, and stem pitting), but not the non-specific aberrations (such as mature leaf epinasty and yellow pinpoints, growth cessation and apical necrosis) observed when p23 was ectopically expressed. Furthermore, vein necrosis and stem pitting in Mexican lime appeared to be specifically associated with p23 from T36. Phloem-specific accumulation of the p23Δ158-209(T36) fragment was sufficient to induce the same anomalies, indicating that the region comprising the N-terminal 157 amino acids of p23 is responsible (at least in part) for the vein clearing, stem pitting and, possibly, vein corking in this host.

Asunto(s)

Citrus/virología , Closterovirus/genética , Floema/virología , Plantas Modificadas Genéticamente/virología , Closterovirus/patogenicidad , Regiones Promotoras Genéticas , Interferencia de ARN

RESUMEN

An assay to identify interactions between Citrus Dwarfing Viroid (CDVd) and Citrus Tristeza Virus (CTV) showed that viroid titer was enhanced by the coinfecting CTV in Mexican lime but not in etrog citron. Since CTV encodes three RNA silencing suppressors (RSSs), p23, p20 and p25, an assay using transgenic Mexican limes expressing each RSS revealed that p23 and, to a lesser extent, p25 recapitulated the effect observed with coinfections of CTV and CDVd.

Asunto(s)

Proteínas de la Cápside/metabolismo , Citrus/virología , Closterovirus/metabolismo , Coinfección/virología , Enfermedades de las Plantas/virología , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismo , Viroides/fisiología , Proteínas de la Cápside/genética , Citrus/genética , Closterovirus/genética , Coinfección/genética , Enfermedades de las Plantas/genética , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas Virales/genética , Viroides/genética

RESUMEN

Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Previously, we showed that Mexican lime transformed with an intron-hairpin construct including part of the gene p23 and the adjacent 3' untranslated region displays partial resistance to CTV, with a fraction of the propagations from some transgenic lines remaining uninfected. Here, we transformed Mexican lime with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20 and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Our results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appears critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded.

Asunto(s)

Citrus/virología , Closterovirus/genética , Resistencia a la Enfermedad , Genes Supresores , Enfermedades de las Plantas/genética , Interferencia de ARN , Citrus/genética , Closterovirus/patogenicidad , Vectores Genéticos , Intrones , Enfermedades de las Plantas/virología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/virología , ARN Catalítico/genética , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Proteínas Virales/genética

RESUMEN

The nucleotide sequence diversity of the CPm gene from 28 field isolates of Citrus tristeza virus (CTV) was assessed by SSCP and sequence analyses. These isolates showed two major shared haplotypes, which differed in distribution: A1 was the major haplotype in 23 isolates from different geographic regions, whereas R1 was found in isolates from a discrete region. Phylogenetic reconstruction clustered A1 within an independent group, while R1 was grouped with mild isolates T30 from Florida and T385 from Spain. Some isolates contained several minor haplotypes, which were very similar to, and associated with, the major haplotype.

Asunto(s)

Proteínas de la Cápside/genética , Citrus/virología , Closterovirus/genética , Variación Genética , Enfermedades de las Plantas/virología , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Closterovirus/clasificación , Colombia , Haplotipos , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADN

RESUMEN

The molecular characterization of isolates of citrus tristeza virus (CTV) from eight locations in Mexico was undertaken by analyzing five regions located at the opposite ends of the virus genome. Two regions have been previously used to study CTV variability (coat protein and p23), while the other three correspond to other genomic segments (p349-B, p349-C and p13). Our comparative nucleotide analyses included CTV sequences from different geographical origins already deposited in the GenBank databases. The largest nucleotide differences were located in two fragments located at the 5' end of the genome (p349-B and p349-C). Phylogenetic analyses on those five regions showed that the degree of nucleotide divergence among strains tended to correlate with their pathogenicity. Two main groups were defined: mild, with almost no noticeable effects on the indicator plants and severe, with drastic symptoms. Mild isolates clustered together in every analyzed ORF sharing a genetic distance below 0.022, in contrast with the severe isolates, which showed a more disperse distribution and a genetic distance of 0.276. Analyses of the p349-B and p349-C regions evidenced two lineages within the severe group: severe common subgroup (most of severe isolates) and severe divergent subgroup (T36-like isolates). This study represents the first attempt to analyze the genetic variability of CTV in Mexico by constructing phylogenetic trees based on new genomic regions that use group-specific nucleotide and amino acid sequences. These results may be useful to implement specific assays for strain discrimination. Moreover, it would be an excellent reference for the CTV situation in México to face the recent arrival of brown citrus aphid.

Asunto(s)

Citrus/virología , Closterovirus/genética , Closterovirus/patogenicidad , Sistemas de Lectura Abierta , Enfermedades de las Plantas/virología , Polimorfismo Genético , Closterovirus/aislamiento & purificación , Análisis por Conglomerados , México , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia , Virulencia

RESUMEN

BACKGROUND: Citrus sudden death (CSD), a disease that rapidly kills orange trees, is an emerging threat to the Brazilian citrus industry. Although the causal agent of CSD has not been definitively determined, based on the disease's distribution and symptomatology it is suspected that the agent may be a new strain of Citrus tristeza virus (CTV). CTV genetic variation was therefore assessed in two Brazilian orange trees displaying CSD symptoms and a third with more conventional CTV symptoms. RESULTS: A total of 286 RNA-dependent-RNA polymerase (RdRp) and 284 heat shock protein 70 homolog (HSP70h) gene fragments were determined for CTV variants infecting the three trees. It was discovered that, despite differences in symptomatology, the trees were all apparently coinfected with similar populations of divergent CTV variants. While mixed CTV infections are common, the genetic distance between the most divergent population members observed (24.1% for RdRp and 11.0% for HSP70h) was far greater than that in previously described mixed infections. Recombinants of five distinct RdRp lineages and three distinct HSP70h lineages were easily detectable but respectively accounted for only 5.9 and 11.9% of the RdRp and HSP70h gene fragments analysed and there was no evidence of an association between particular recombinant mosaics and CSD. Also, comparisons of CTV population structures indicated that the two most similar CTV populations were those of one of the trees with CSD and the tree without CSD. CONCLUSION: We suggest that if CTV is the causal agent of CSD, it is most likely a subtle feature of population structures within mixed infections and not merely the presence (or absence) of a single CTV variant within these populations that triggers the disease.

Asunto(s)

Citrus sinensis/virología , Closterovirus/genética , Genes Virales , Proteínas HSP70 de Choque Térmico/genética , Enfermedades de las Plantas/virología , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética , Brasil , Closterovirus/clasificación , Variación Genética , Recombinación Genética

RESUMEN

We studied the genetic variability of three genomic regions (p23, p25 and p27 genes) from 11 field Citrus tristeza virus isolates from the two main citrus growing areas of Argentina, a country where the most efficient vector of the virus, Toxoptera citricida, is present for decades. The pathogenicity of the isolates was determinated by biological indexing, single-strand conformation polymorphism analysis showed that most isolates contained high intra-isolate variability. Divergent sequence variants were detected in some isolates, suggesting re-infections of the field plants. Phylogenetic analysis of the predominant sequence variants of each isolate revealed similar grouping of isolates for genes p25 and p27. The analysis of p23 showed two groups contained the severe isolates. Our results showed a high intra-isolate sequence variability suggesting that re-infections could contribute to the observed variability and that the host can play an important role in the selection of the sequence variants present in these isolates.

Asunto(s)

Citrus/virología , Closterovirus/genética , Polimorfismo Conformacional Retorcido-Simple , Secuencia de Aminoácidos , Argentina , Secuencia de Bases , Clonación Molecular , Closterovirus/clasificación , Closterovirus/aislamiento & purificación , Closterovirus/patogenicidad , Variación Genética , Haplotipos , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/virología , Alineación de Secuencia

RESUMEN

Previously, we have shown that most Mexican limes (Citrus aurantifolia (Christ.) Swing.) expressing the p23 gene of Citrus tristeza virus (CTV) exhibit aberrations resembling viral leaf symptoms. Here we report that five independent transgenic lines having normal phenotype displayed characteristics typical of post-transcriptional gene silencing (PTGS): multiple copies of the transgene, low levels of the corresponding mRNA, methylation of the silenced transgene, and accumulation of p23-specific small interfering RNAs (siRNAs). When graft- or aphid-inoculated with CTV, some propagations of these silenced lines were immune: they neither expressed symptoms nor accumulated virions and viral RNA as estimated by DAS-ELISA and Northern blot hybridization, respectively. Other propagations were moderately resistant because they became infected later and showed attenuated symptoms compared to controls. The susceptible propagations, in addition to symptom expression and elevated virus titer, accumulated p23-specific siRNAs at levels significantly higher than immune or non-inoculated propagations, and showed transgene demethylation. This variable response among clonal transformants indicates that factors other than the genetic background of the transgenic plants play a key role in PTGS-mediated resistance.

Asunto(s)

Closterovirus/genética , Silenciador del Gen , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/genética , Proteínas Virales/genética , Northern Blotting , Southern Blotting , Metilación de ADN , Plantas Modificadas Genéticamente , ARN Interferente Pequeño/genética , Transgenes