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József Burgyán - One of the best experts on this subject based on the ideXlab platform.
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Transcriptome reprogramming in the shoot apical meristem of CymRSV-infected Nicotiana benthamiana plants associates with viral exclusion and the lack of recovery.
Molecular plant pathology, 2019Co-Authors: Anna Medzihradszky, György Szittya, Péter Gyula, Anita Sós-hegedűs, József BurgyánAbstract:In some plant-Virus interactions plants show a sign of healing from Virus infection, a phenomenon called symptom recovery. It is assumed that the meristem exclusion of the Virus is essential to this process. The discovery of RNA silencing provided a possible mechanism to explain meristem exclusion and recovery. Here we show evidence that silencing is not the reason for meristem exclusion in Nicotiana benthamiana plants infected with Cymbidium Ringspot Virus (CymRSV). Transcriptome analysis followed by in situ hybridization shed light on the changes in gene expression in the shoot apical meristem (SAM) on Virus infection. We observed the down-regulation of meristem-specific genes, including WUSCHEL (WUS). However, WUS was not down-regulated in the SAM of plants infected with meristem-invading Viruses such as turnip vein-clearing Virus (TVCV) and cucumber mosaic Virus (CMV). Moreover, there is no connection between loss of meristem function and fast shoot necrosis since TVCV necrotized the shoot while CMV did not. Our findings suggest that the observed transcriptional changes on Virus infection in the shoot are key factors in tip necrosis and symptom recovery. We observed a lack of GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (GAPDH) expression in tissues around the meristem, which likely stops Virus replication and spread into the meristem.
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Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants.
PLoS pathogens, 2016Co-Authors: Levente Kontra, Mario Tavazza, Alessandra Lucioli, Anna Medzihradszky, Tibor Csorba, Raffaela Tavazza, Simon Moxon, Viktória Tisza, Massimo Turina, József BurgyánAbstract:RNA silencing is one of the main defense mechanisms employed by plants to fight Viruses. In change, Viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine Virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of Virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from Virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium Ringspot Virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-Virus combination.
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2016Co-Authors: József Burgyán, Peter T D. Nagyt, Marcello RussoAbstract:A full-length DNA copy of Cymbidium Ringspot Virus RNA was cloned downstream of a phage T7 promoter. In vitro transcripts had no extra nucleotides at the 3' terminus, and a 5 ' end likely to be precisely as in genomic RNA. Transcripts were infective when inocu-lated into test plants. Northern blots from inoculated plants revealed the presence of genomic and subgeno-mic RNAs, but not of satellite RNA. Virus particles isolated from infected plants had the same outward aspect and size as those of the wild-type Virus and were decorated by an antiserum to CyRSV in immune electron microscopy tests. Cymbidium Ringspot Virus (CyRSV) is a member of the tombusVirus group (Martelli et al., 1989). The main features of expression of the genetic informatio
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RNA Silencing May Play a Role in but Is Not the Only Determinant of the Multiplicity of Infection
Journal of Virology, 2015Co-Authors: Livia Donaire, József Burgyán, Fernando García-arenalAbstract:ABSTRACT The multiplicity of infection (MOI), i.e., the number of viral genomes that infect a cell, is an important parameter in Virus evolution, which for each Virus and environment may have an optimum value that maximizes Virus fitness. Thus, the MOI might be controlled by Virus functions, an underexplored hypothesis in eukaryote-infecting Viruses. To analyze if the MOI is controlled by Virus functions, we estimated the MOI in plants coinfected by two genetic variants of Tomato bushy stunt Virus (TBSV); by TBSV and a TBSV-derived defective interfering RNA (DI-RNA); or by TBSV and a second tombusVirus, Cymbidium Ringspot Virus (CymRSV). The MOI was significantly larger in TBSV-CymRSV coinfections (∼4.0) than in TBSV-TBSV or TBSV–DI-RNA coinfections (∼1.7 to 2.2). Coinfections by CymRSV or TBSV with chimeras in which an open reading frame (ORF) of one Virus species was replaced by that of the other identified a role of viral proteins in determining the MOI, which ranged from 1.6 to 3.9 depending on the coinfecting genotypes. However, no Virus-encoded protein or genomic region was the sole MOI determinant. Coinfections by CymRSV and TBSV mutants in which the expression of the gene-silencing suppressor protein p19 was abolished also showed a possible role of gene silencing in MOI determination. Taken together, these results demonstrate that the MOI is a quantitative trait showing continuous variation and that as such it has a complex determination involving different Virus-encoded functions. IMPORTANCE The number of viral genomes infecting a cell, or the multiplicity of infection (MOI), is an important parameter in Virus evolution affecting recombination rates, selection intensity on viral genes, evolution of multipartite genomes, or hyperparasitism by satellites or defective interfering particles. For each Virus and environment, the MOI may have an optimum value that maximizes Virus fitness, but little is known about MOI control in eukaryote-infecting Viruses. We show here that in plants coinfected by two genotypes of Tomato bushy stunt Virus (TBSV), the MOI was lower than in plants coinfected by TBSV and Cymbidium Ringspot Virus (CymRSV). Coinfections by CymRSV or TBSV with TBSV-CymRSV chimeras showed a role of viral proteins in MOI determination. Coinfections by CymRSV and TBSV mutants not expressing the gene-silencing suppressor protein also showed a role of gene silencing in MOI determination. The results demonstrate that the MOI is a quantitative trait with a complex determination involving different viral functions.
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Az RNS silencing szerepe, mechanizmusa a vírus gazda kölcsönhatásban = The role and the mechanism of RNA silencing in the plant Virus interplay
2009Co-Authors: József Burgyán, György Szittya, Tibor Csorba, Endre Barta, Gábor Giczey, Angéla Judit Horkics, Tibor Nagy, Tamás B Pálfy, Gabor Toth, Anna VálócziAbstract:Az RNS silencing, egy geninaktivacios mechanizmus, amely szinte az osszes eukariota szervezetben műkodik. Kutatasaink soran feltartuk a Cymbidium Ringspot Virus genomrol keződő small interferalo (si) RNS eredetet nagy hatekonysagu 454 (Life Science) es Soplexa (illumina) szekvenalo rendszerek alkalmazasaval. A Virus genomrol szarmazo kis RNS-eket raterkepeztuk a Virus genomjara, amely alapjan "forro pontokat" tudtunk azonositani. Igazoltuk,hogy Virus siRNS-ek tulnyomo tobsege a Virus pozitiv szalarol szarmazik, es 21-22 nukleotid (nt) hosszu. Megallapitottuk, hogy Virus siRNS-ekkel toltott RISC (RNA Induced Silencing Complex) komplexek szekvenciaspecifikusan hasitjak a Virus genomot. Szamos silencing szupresszor feherje (p19, HC-Pro, es p122) reszletes analizisevel igazoltuk, hogy a noveny antiviralis valaszat, a Virus kodolta silencing szupresszorok hatekonyan gatoljak. Bizonyitottuk, hogy a siRNS-ek specifikus kotese es inaktivalasa a legelterjedtebb strategia a silencing szupresszor feherjek kozott. Feltartuk, hogy a silencing szuppresszor feherjek egy jelentős csoportja gatolja novenyek endogen siRNS es miRNS biogeneziset. A silencing szupressor feherjek interakcioja az endogen silencing utvonalakkal feltehetően a magyarazata a Virus okozta tunetek kialakulasanak, hiszen a szupresszor feherjek sulyosan zavarja noveny egyedfejlődeset. | RNA silencing is a gene inactivation mechanism, which is conserved in a broad range of eukaryotes. The central players in RNA-mediated gene silencing are the small 21-24 nucleotide long RNA molecules engaged in sequence-specific interactions to inhibit gene expression. RNA silencing fulfils fundamental regulatory roles, as well as antiviral functions. We profiled viral siRNAs using two different high-throughput sequencing platforms. Both deep sequencing techniques revealed a strong bias in viral siRNAs for the positive strand of the Virus and identified regions on the viral genome that produced viral siRNA in much higher abundance than other regions. We also analysed the viral RNA targeting by Virus induced gene silencing in tombusVirus infected plants, and we show evidence that antiviral response is based on viral RNA cleavage by RNA-induced silencing effector complex (RISC) programmed by Virus-specific siRNAs.. To counteract RNA silencing, Viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the RISC assembly. We demonstrated that these suppressors are able to interact with the endogenous silencing pathways suggesting that these interactions have an important role in the development of Virus-induced symptoms.
Marcello Russo - One of the best experts on this subject based on the ideXlab platform.
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2016Co-Authors: József Burgyán, Peter T D. Nagyt, Marcello RussoAbstract:A full-length DNA copy of Cymbidium Ringspot Virus RNA was cloned downstream of a phage T7 promoter. In vitro transcripts had no extra nucleotides at the 3' terminus, and a 5 ' end likely to be precisely as in genomic RNA. Transcripts were infective when inocu-lated into test plants. Northern blots from inoculated plants revealed the presence of genomic and subgeno-mic RNAs, but not of satellite RNA. Virus particles isolated from infected plants had the same outward aspect and size as those of the wild-type Virus and were decorated by an antiserum to CyRSV in immune electron microscopy tests. Cymbidium Ringspot Virus (CyRSV) is a member of the tombusVirus group (Martelli et al., 1989). The main features of expression of the genetic informatio
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A single amino acid substitution in the ORF1 of Cymbidium Ringspot Virus determines the accumulation of two satellite RNAs
Virus research, 2012Co-Authors: Luisa Rubino, Marcello RussoAbstract:TombusViruses may support the replication of satellite (sat) RNAs. In particular, two satRNAs, sat L and Cymsat RNAs, are replicated by carnation Italian Ringspot (CIRV) and tomato bushy stunt (TBSV) Virus, but not by Cymbidium Ringspot Virus (CymRSV) in vitro transcripts unless they contain a poly(A) tail at the 3' end. Conversely, the replication of both satRNAs was supported by Virus particles or viral RNA of the original CymRSV inoculum even in the absence of the poly(A) tail. Sequence and mutational analyses revealed that the full-length infectious CymRSV clone contains one relevant sequence variation in the ORF 1-encoded protein (p33) compared with the original inoculum, i.e. a Ser₁₉ TCC codon instead of a Phe₁₉ TTC codon, which inhibited the replication of sat L and Cymsat RNAs. It is suggested that this amino acid is contained in a domain essential for the replication of some subviral RNAs.
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Expression of the Cymbidium Ringspot Virus 33-kilodalton protein in Saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal
Journal of virology, 2004Co-Authors: Beatriz Navarro, Luisa Rubino, Marcello RussoAbstract:Open reading frame 1 in the viral genome of Cymbidium Ringspot Virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.
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Expression of tombusVirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA.
The Journal of general virology, 2004Co-Authors: Luisa Rubino, Vitantonio Pantaleo, Beatriz Navarro, Marcello RussoAbstract:Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian Ringspot Virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium Ringspot Virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.
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HOMOLOGY-DEPENDENT Virus RESISTANCE AGAINST Cymbidium Ringspot Virus IS INHIBITED BY POST-TRANSCRIPTIONAL GENE SILENCING SUPPRESSOR VirusES
Journal of Plant Pathology, 2002Co-Authors: F. Di Serio, Luisa Rubino, Marcello Russo, G. P. MartelliAbstract:Plants expressing viral sequences may show homology- dependent Virus resistance (HDR), which is characterized by specific degradation of transgenic RNA and viral RNAs homologous to the transgene. This resistance mechanism is a form of post-transcriptional gene silencing (PTGS), which can be suppressed by Virus-encoded proteins. We analysed the effect of Potato Virus Y, Cucumber mosaic Virus and Potato Virus X infections on HDR to Cymbidium Ringspot Virus (CymRSV) shown by Nicotiana benthamiana transgenic line 92KA1 expressing the full-length replicase gene of CymRSV. We observed breaking of resistance in transgenic plants doubly infected with PTGS suppressor Viruses and CymRSV. The need of considering the effect of PTGS suppressor Viruses on resistance or PTGS-dependent phenotype in transgenic crops is discussed.
Luisa Rubino - One of the best experts on this subject based on the ideXlab platform.
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A single amino acid substitution in the ORF1 of Cymbidium Ringspot Virus determines the accumulation of two satellite RNAs
Virus research, 2012Co-Authors: Luisa Rubino, Marcello RussoAbstract:TombusViruses may support the replication of satellite (sat) RNAs. In particular, two satRNAs, sat L and Cymsat RNAs, are replicated by carnation Italian Ringspot (CIRV) and tomato bushy stunt (TBSV) Virus, but not by Cymbidium Ringspot Virus (CymRSV) in vitro transcripts unless they contain a poly(A) tail at the 3' end. Conversely, the replication of both satRNAs was supported by Virus particles or viral RNA of the original CymRSV inoculum even in the absence of the poly(A) tail. Sequence and mutational analyses revealed that the full-length infectious CymRSV clone contains one relevant sequence variation in the ORF 1-encoded protein (p33) compared with the original inoculum, i.e. a Ser₁₉ TCC codon instead of a Phe₁₉ TTC codon, which inhibited the replication of sat L and Cymsat RNAs. It is suggested that this amino acid is contained in a domain essential for the replication of some subviral RNAs.
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GENERATION OF Virus-LIKE PARTICLES IN PLANTS EXPRESSING THE CAPSID PROTEIN OF Cymbidium Ringspot Virus
Journal of Plant Pathology, 2011Co-Authors: Luisa Rubino, Maria Giovanna Russo, A. De Stradis, G. P. MartelliAbstract:SUMMARY Agroinfiltration of Nicotiana benthamiana leaves with a construct expressing the coat protein of Cymbidium Ringspot Virus (CymRSV) resulted in the production of Virus-like particles (VLPs) which showed a preferential localization within mitochondria with an apparently intact bounding membrane. VLPs were either non penetrated or penetrated by the negative stain. The former had the same outward aspect and size of wild type CymRSV and were assumed to encapsidate the CP messanger RNA, which was recovered from Virus preparations purified from agroinfiltrated tissues. In addition, VLPs were shown to be able to encapsidate tombusviral satellite RNAs. Immunoblot analysis of dissociated VLPs showed that they were made up of a protein indistinguishable from the native CymRSV CP. A mitochondrial targeting signal was identified in the N-terminal region of CymRSV CP at amino acid (aa) position 10-27. The fusion protein CPMyc, containing the 10-aa Myc epitope fused to the CymRSV CP C-terminus, formed VLPs that were decorated by a Myc-specific antiserum.
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Analysis of sequences in the Cymbidium Ringspot Virus replicase protein P33 that promote targeting and anchoring to endoplasmic reticulum membranes.
Journal of Plant Pathology, 2008Co-Authors: Luisa Rubino, P. Vernile, Maria Giovanna RussoAbstract:SUMMARY RNA replication of Cymbidium Ringspot Virus (CymRSV) normally occurs in plant and yeast cells in association with membranes derived from peroxisomes. However, in yeast strains devoid of these organelles CymRSV RNA replication takes place on the perinuclear membrane and cytoplasmic endoplasmic reticulum (ER) strands. The association of the CymRSV RNA replication complex to ER is mediated by the ORF 1encoded protein p33. To analyze which part of this protein contains the information leading to localization to ER, a deletion analysis was carried out. The behaviour of deletion mutants was studied by confocal immunofluorescence microscopy and membrane floatation gradient analysis. It was shown that the signal targeting p33 to ER membranes largely coincides with the peroxisomal targeting signal, but contains additional elements in the first 87 amino acids.
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Expression of the Cymbidium Ringspot Virus 33-kilodalton protein in Saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal
Journal of virology, 2004Co-Authors: Beatriz Navarro, Luisa Rubino, Marcello RussoAbstract:Open reading frame 1 in the viral genome of Cymbidium Ringspot Virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.
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Expression of tombusVirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, RNA.
The Journal of general virology, 2004Co-Authors: Luisa Rubino, Vitantonio Pantaleo, Beatriz Navarro, Marcello RussoAbstract:Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian Ringspot Virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium Ringspot Virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.
M. Russo - One of the best experts on this subject based on the ideXlab platform.
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SHORT COMMUNICATION ANALYSIS OF SEQUENCES IN THE Cymbidium Ringspot Virus REPLICASE PROTEIN P33 THAT PROMOTE TARGETING AND ANCHORING TO ENDOPLASMIC RETICULUM MEMBRANES
2013Co-Authors: L. Rubino, P. Vernile, M. RussoAbstract:RNA replication of Cymbidium Ringspot Virus (CymRSV) normally occurs in plant and yeast cells in association with membranes derived from peroxisomes. However, in yeast strains devoid of these organelles CymRSV RNA replication takes place on the perinuclear membrane and cytoplasmic endoplasmic reticulum (ER) strands. The association of the CymRSV RNA replication complex to ER is mediated by the ORF 1-encoded protein p33. To analyze which part of this protein contains the information leading to localization to ER, a deletion analysis was carried out. The behaviour of deletion mutants was studied by confocal immunofluorescence microscopy and membrane floatation gradient analysis. It was shown that the signal targeting p33 to ER membranes largely coincides with the peroxisomal targeting signal, but contains additional elements in the first 87 amino acids. Key words: Cymbidium Ringspot Virus, RNA replication, Saccharomyces cerevisiae, cell membranes
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Properties of a novel satellite RNA associated with tomato bushy stunt Virus infections.
The Journal of general virology, 2010Co-Authors: L. Rubino, M. RussoAbstract:The biological and molecular properties of a novel satellite RNA (satRNA L) associated with tomato bushy stunt Virus (TBSV) are described. satRNA L consisted of a linear single-stranded RNA of 615 nt, lacked significant open reading frames (ORFs) and had no sequence identity with the helper genome other than in the 5'-proximal 7 nt and in a central region that is also conserved in all tombusVirus genomic, defective interfering and satellite RNAs. Secondary-structure analysis showed the presence of high-order domains similar to those described for other tombusVirus RNAs. Shorter-than-unit-length molecules were shown not to be related to a silencing mechanism. satRNA L did not modify the symptoms induced by TBSV under any of the temperature conditions tested. A full-length cDNA clone was constructed and used in co-inoculations with transcripts of carnation Italian Ringspot Virus (CIRV) and Cymbidium Ringspot Virus (CymRSV). CIRV, but not CymRSV, supported the replication of satRNA L. Using CIRV-CymRSV hybrid infectious clones, two regions were identified as possible determinants of the different ability to support satRNA L replication. The first region was in the 5'-untranslated region, which folds differently in CymRSV in comparison with CIRV and TBSV; the second region was in the ORF1-encoded protein where a more efficient satRNA L-binding domain is suggested to be present in CIRV.
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Immunodetection of the 33 K/92 K polymerase proteins in Cymbidium Ringspot Virus-infected and in transgenic plant tissue extracts
Archives of Virology, 1994Co-Authors: R. Lupo, L. Rubino, M. RussoAbstract:An antiserum was raised against the 33 K protein encoded by the 5′ proximal gene of Cymbidium Ringspot tombusVirus RNA. This antiserum reacts specifically with the 33 K and 92 K proteins, which constitute the viral replicase, in CyRSV-infected Nicotiana benthamiana plants and in transgenic plants transformed with the full-length replicase gene. In inoculated leaves of infected plants, synthesis of the 33 K/92 K proteins stops ten days after inoculation, whereas in newly produced systemically infected leaves there was continuous production of these proteins. In transgenic plants, both proteins were detected showing that readthrough of the termination codon of the 33 K protein does not depend on the presence of the replicating Virus. The subcellular localization of the 33 K/92 K proteins is similar in infected and transgenic plants. No correlation was found between the level of expression of integrated Virus gene and level of resistance to the challenging Virus.
James E Schoelz - One of the best experts on this subject based on the ideXlab platform.
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A survey of resistance to Tomato bushy stunt Virus in the genus Nicotiana reveals that the hypersensitive response is triggered by one of three different viral proteins.
Molecular plant-microbe interactions : MPMI, 2013Co-Authors: Carlos A. Angel, James E SchoelzAbstract:In this study, we screened 22 Nicotiana spp. for resistance to the tombusViruses Tomato bushy stunt Virus (TBSV), Cucumber necrosis Virus, and Cymbidium Ringspot Virus. Eighteen species were resistant, and resistance was manifested in at least two different categories. In all, 13 species responded with a hypersensitive response (HR)-type resistance, whereas another five were resistant but either had no visible response or responded with chlorotic lesions rather than necrotic lesions. Three different TBSV proteins were found to trigger HR in Nicotiana spp. in an agroinfiltration assay. The most common avirulence (avr) determinant was the TBSV coat protein P41, a protein that had not been previously recognized as an avr determinant. A mutational analysis confirmed that the coat protein rather than the viral RNA sequence was responsible for triggering HR, and it triggered HR in six species in the Alatae section. The TBSV P22 movement protein triggered HR in two species in section Undulatae (Nicotiana glutino...
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Comparative Analysis of the Capacity of TombusVirus P22 and P19 Proteins to Function as Avirulence Determinants in Nicotiana species
Molecular Plant-microbe Interactions, 2011Co-Authors: Carlos Ariel Angel, Yi-cheng Hsieh, James E SchoelzAbstract:We have used an agroinfiltration assay for a comparative study of the roles of tombusVirus P22 and P19 proteins in elicitation of hypersensitive response (HR)-like necrosis and the role of P19 in silencing suppression in Nicotiana species. The advantage of agroinfiltration rather than expression in plant Virus vectors is that putative viral avirulence proteins can be evaluated in isolation, eliminating the possibility of synergistic effects with other viral proteins. We found that tombusVirus P22 and P19 proteins elicited HR-like necrosis in certain Nicotiana species but, also, that Nicotiana species could recognize subtle differences in sequence between these proteins. Furthermore, Nicotiana species that responded with systemic necrosis to virion inoculations responded to agroinfiltration of tombusVirus P19 with a very weak and delayed necrosis, indicating that the rapid HR-like necrosis was associated with putative resistance genes and a plant defense response that limited the spread of the Virus. TombusVirus P19 proteins also appeared to differ in their effectiveness as silencing suppressors; in our assay, the P19 proteins of Cymbidium Ringspot Virus and Tomato bushy stunt Virus were stronger silencing suppressors than Cucumber necrosis Virus P20. Finally, we show that agroinfiltration can be used to track the presence of putative plant resistance genes in Nicotiana species that target either tombusVirus P19 or P22.
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Silencing of the N family of resistance genes in Nicotiana edwardsonii compromises the hypersensitive response to tombusViruses.
Molecular plant-microbe interactions : MPMI, 2007Co-Authors: Boovaraghan Balaji, Carlos A. Angel, John Cawly, Zhanyuan J. Zhang, Karuppaiah Palanichelvam, Anthony B. Cole, James E SchoelzAbstract:The nontarget effects associated with silencing of the N gene in Nicotiana edwardsonii, an amphidiploid species derived from N. glutinosa and N. clevelandii, have been characterized in this study. The N protein confers resistance to Tobacco mosaic Virus (TMV), and is representative of a family of nucleotide-binding site leucine-rich repeat proteins present in N. glutinosa. Previous studies have shown that silencing of the N gene or of other plant genes associated with N-mediated defenses abolishes host resistance to TMV, and this effect can be measured through enhancements in movement or replication of TMV in the N-silenced plants. However, the nontarget effects of gene silencing have not been investigated thoroughly. Notably, are the functions of other resistance (R) genes also affected in experiments designed to silence the N gene? To investigate whether heterologous sequences could silence the N gene, we selected an R gene homolog from N. glutinosa that differed from the N gene by approximately 17%, created a hairpin transgene, and developed transgenic N. edwardsonii plants. Expression of this hairpin in the transgenic N. edwardsonii plants compromised the hypersensitive response to TMV, demonstrating that a single hairpin transgene could silence a block of R genes related by sequence similarity. We then investigated whether the response of N-silenced plants to other Viruses would be altered, and found that the hypersensitive response triggered against the tombusViruses Tomato bushy stunt Virus and Cymbidium Ringspot Virus also was compromised. This study indicates that a TombusVirus R gene shares some homology with the N gene, which could facilitate the cloning of this gene.
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introgression of a tombusVirus resistance locus from nicotiana edwardsonii var columbia to n clevelandii
Phytopathology, 2006Co-Authors: James E Schoelz, Elizabeth B Wiggins, William M Wintermantel, Kathleen RossAbstract:Schoelz, J. E., Wiggins, B. E., Wintermantel, W. M., and Ross, K. 2006. Introgression of a tombusVirus resistance locus from Nicotiana edwardsonii var. Columbia to N. clevelandii. Phytopathology 96:453-459. A new variety of Nicotiana, N. edwardsonii var. Columbia, was evaluated for its capacity to serve as a new source for Virus resistance genes. Columbia was developed from a hybridization between N. glutinosa and N. clevelandii, the same parents used for the formation of the original N. edwardsonii. However, in contrast to the original N. edwardsonii, crosses between Columbia and either of its parents are fertile. Thus, the inheritance of Virus resistance genes present in N. glutinosa could be characterized by using Columbia as a bridge plant in crosses with the susceptible parent, N. clevelandii. To determine how Virus resistance genes would segregate in interspecific crosses between Columbia and N. clevelandii, we followed the fate of the N gene, a single dominant gene that specifies resistance to Tobacco mosaic Virus (TMV). Our genetic evidence indicated that the entire chromosome containing the N gene was introgressed into N. clevelandii to create an addition line, designated N. clevelandii line 19. Although line 19 was homozygous for resistance to TMV, it remained susceptible to Tomato bushy stunt Virus (TBSV) and Cauliflower mosaic Virus (CaMV) strain W260, indicating that resistance to these Viruses must reside on other N. glutinosa chromosomes. We also developed a second addition line, N. clevelandii line 36, which was homozygous for resistance to TBSV. Line 36 was susceptible to TMV and CaMV strain W260, but was resistant to other tombusViruses, including Cucumber necrosis Virus, Cymbidium Ringspot Virus, Lettuce necrotic stunt Virus, and Carnation Italian Ringspot Virus.
