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Juan Antonio García - One of the best experts on this subject based on the ideXlab platform.
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gene expression analysis of induced Plum Pox Virus sharka resistance in peach prunus persica by almond p dulcis grafting
International Journal of Molecular Sciences, 2021Co-Authors: Manuel Rubio, Bernardo Rodamilans, Juan Antonio García, F Dicenta, Pedro J Martinezgarcia, Azam Nikbakhtdehkordi, Angela S Prudencio, E M Gomez, Pedro MartinezgomezAbstract:No natural sources of resistance to Plum Pox Virus (PPV, sharka disease) have been identified in peach. However, previous studies have demonstrated that grafting a “Garrigues” almond scion onto “GF305” peach rootstock seedlings heavily infected with PPV can progressively reduce disease symptoms and Virus accumulation. Furthermore, grafting a “Garrigues” scion onto the “GF305” rootstock has been shown to completely prevent Virus infection. This study aims to analyse the rewiring of gene expression associated with this resistance to PPV transmitted by grafting through the phloem using RNA-Seq and RT-qPCR analysis. A total of 18 candidate genes were differentially expressed after grafting “Garrigues” almond onto healthy “GF305” peach. Among the up-regulated genes, a HEN1 homolog stands out, which, together with the differential expression of RDR- and DCL2-homologs, suggests that the RNA silencing machinery is activated by PPV infection and can contribute to the resistance induced by “Garrigues” almond. Glucan endo-1,3-beta D-glucosidase could be also relevant for the “Garrigues”-induced response, since its expression is much higher in “Garrigues” than in “GF305”. We also discuss the potential relevance of the following in PPV infection and “Garrigues”-induced resistance: several pathogenesis-related proteins; no apical meristem proteins; the transcription initiation factor, TFIIB; the speckle-type POZ protein; in addition to a number of proteins involved in phytohormone signalling.
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a functional link between rna replication and virion assembly in the potyVirus Plum Pox Virus
Journal of Virology, 2018Co-Authors: Araiz Gallo, Adrian Valli, Maria V Calvo, Juan Antonio GarcíaAbstract:Accurate assembly of viral particles in the potyVirus Plum Pox Virus (PPV) has been shown to depend on the contribution of the multifunctional viral protein HCPro. In this study, we show that other viral factors, in addition to the capsid protein (CP) and HCPro, are necessary for the formation of stable PPV virions. The CP produced in Nicotiana benthamiana leaves from a subviral RNA termed LONG, which expresses a truncated polyprotein that lacks P1 and HCPro, together with HCPro supplied in trans, was assembled into Virus-like particles and remained stable after in vitro incubation. In contrast, deletions in multiple regions of the LONG coding sequence prevented the CP stabilization mediated by HCPro. In particular, we demonstrated that the first 178 amino acids of P3, but not a specific nucleotide sequence coding for them, are required for CP stability and proper assembly of PPV particles. Using a sequential coagroinfiltration assay, we observed that the subviral LONG RNA replicates and locally spreads in N. benthamiana leaves expressing an RNA silencing suppressor. The analysis of the effect of both point and deletion mutations affecting RNA replication in LONG and full-length PPV demonstrated that this process is essential for the assembly of stable viral particles. Interestingly, in spite of this requirement, the CP produced by a nonreplicating viral RNA can be stably assembled into virions as long as it is coexpressed with a replication-proficient RNA. Altogether, these results highlight the importance of coupling encapsidation to other viral processes to secure a successful infection. IMPORTANCE Viruses of the family Potyviridae are among the most dangerous threats for basically every important crop, and such socioeconomical relevance has made them a subject of many research studies. In spite of this, very little is currently known about proteins and processes controlling viral genome encapsidation by the coat protein. In the case of Plum Pox Virus (genus PotyVirus), for instance, we have previously shown that the multitasking viral factor HCPro plays a role in the production of stable virions. Here, by using this potyVirus as a model, we move further to show that additional factors are also necessary for the efficient production of potyviral particles. More importantly, a comprehensive screening for such factors led us to the identification of a functional link between Virus replication and packaging, unraveling a previously unknown connection of these two key events of the potyviral infection cycle.
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Plum Pox Virus and sharka a model potyVirus and a major disease
Molecular Plant Pathology, 2014Co-Authors: Juan Antonio García, Miroslav Glasa, Mariano Cambra, Thierry CandresseAbstract:SummaryTaxonomic relationships Plum Pox Virus (PPV) is a member of the genus PotyVirus in the family Potyviridae. PPV diversity is structured into at least eight monophyletic strains. Geographical distribution First discovered in Bulgaria, PPV is nowadays present in most of continental Europe (with an endemic status in many central and southern European countries) and has progressively spread to many countries on other continents. Genomic structure Typical of potyViruses, the PPV genome is a positive-sense single-stranded RNA (ssRNA), with a protein linked to its 5′ end and a 3′-terminal poly A tail. It is encapsidated by a single type of capsid protein (CP) in flexuous rod particles and is translated into a large polyprotein which is proteolytically processed in at least 10 final products: P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIapro, NIb and CP. In addition, P3N-PIPO is predicted to be produced by a translational frameshift. Pathogenicity features PPV causes sharka, the most damaging viral disease of stone fruit trees. It also infects wild and ornamental Prunus trees and has a large experimental host range in herbaceous species. PPV spreads over long distances by uncontrolled movement of plant material, and many species of aphid transmit the Virus locally in a nonpersistent manner. Sources of resistance A few natural sources of resistance to PPV have been found so far in Prunus species, which are being used in classical breeding programmes. Different genetic engineering approaches are being used to generate resistance to PPV, and a transgenic Plum, ‘HoneySweet’, transformed with the viral CP gene, has demonstrated high resistance to PPV in field tests in several countries and has obtained regulatory approval in the USA.
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Transcriptomic Analysis of Prunus domestica Undergoing Hypersensitive Response to Plum Pox Virus Infection
PLoS ONE, 2014Co-Authors: Bernardo Rodamilans, Thierry Candresse, David San León, Louisa Mühlberger, Michael Neumüller, Juan Carlos Oliveros, Juan Antonio GarcíaAbstract:Plum Pox Virus (PPV) infects Prunus trees around the globe, posing serious fruit production problems and causing severe economic losses. One variety of Prunus domestica, named 'Jojo', develops a hypersensitive response to viral infection. Here we compared infected and non-infected samples using next-generation RNA sequencing to characterize the genetic complexity of the viral population in infected samples and to identify genes involved in development of the resistance response. Analysis of viral reads from the infected samples allowed reconstruction of a PPV-D consensus sequence. De novo reconstruction showed a second viral isolate of the PPV-Rec strain. RNA-seq analysis of PPV-infected 'Jojo' trees identified 2,234 and 786 unigenes that were significantly up- or downregulated, respectively (false discovery rate; FDR≤0.01). Expression of genes associated with defense was generally enhanced, while expression of those related to photosynthesis was repressed. Of the total of 3,020 differentially expressed unigenes, 154 were characterized as potential resistance genes, 10 of which were included in the NBS-LRR type. Given their possible role in plant defense, we selected 75 additional unigenes as candidates for further study. The combination of next-generation sequencing and a Prunus variety that develops a hypersensitive response to PPV infection provided an opportunity to study the factors involved in this plant defense mechanism. Transcriptomic analysis presented an overview of the changes that occur during PPV infection as a whole, and identified candidates suitable for further functional characterization.
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o glcnac modification of the coat protein of the potyVirus Plum Pox Virus enhances viral infection
Virology, 2013Co-Authors: Jose De Jesus Perez, Namrata D Udeshi, Jeffrey Shabanowitz, Sergio Ciordia, Silvia Juarez, Cheryl L Scott, Neil E Olszewski, Donald F Hunt, Juan Antonio GarcíaAbstract:Abstract O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum Pox Virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type Virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type Virus.
Thierry Candresse - One of the best experts on this subject based on the ideXlab platform.
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Plum Pox Virus potyviridae
2020Co-Authors: Miroslav Glasa, Thierry CandresseAbstract:Abstract Plum Pox Virus (PPV), the agent responsible for the Sharka disease of stone fruits and ornamental Prunus trees, belongs to the genus PotyVirus (family Potyviridae). The infection of susceptible genotypes results in characteristic foliar and fruit symptoms and premature fruit drop, making PPV the most detrimental Virus of these species. The geographical distribution covers the vast majority of Prunus producing area in the world. PPV is transmitted non-persistently by more than 20 aphid species, by grafting and vegetative multiplication of infected plants, but is not seed-borne. To date, at least 10 strains/molecular groups of PPV have been identified based on biological, serological and molecular properties (M, D, Rec, EA, T, W, C, CR, CV and An).
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Plum Pox Virus capsid protein suppresses plant pathogen‐associated molecular pattern (PAMP)‐triggered immunity
Molecular Plant Pathology, 2017Co-Authors: Valerie Nicaise, Thierry CandresseAbstract:Perception of pathogen-associated molecular patterns (PAMPs) by immune receptors launches defence mechanisms referred to as PAMP-triggered immunity (PTI). Successful pathogens must suppress PTI pathways via the action of effectors to efficiently colonize their hosts. So far, plant PTI has been reported to be active against most classes of pathogens, except Viruses, although this defence layer was recently hypothesized as an active part of antiviral immunity which needs to be suppressed by Viruses for infection success. Here, we report that Arabidopsis PTI genes are regulated upon infection by Viruses and contribute to plant resistance to Plum Pox Virus (PPV). Our experiments further show that PPV suppresses two early PTI responses, the oxidative burst and marker gene expression, during Arabidopsis infection. In planta expression of PPV capsid protein (CP) was found to strongly impair these responses in Nicotiana benthamiana and Arabidopsis, revealing its PTI-suppressor activity. In summary, we provide the first clear evidence that plant Viruses acquired the ability to suppress PTI mechanisms via the action of effectors, highlighting a novel strategy employed by Viruses to escape plant defences. This article is protected by copyright. All rights reserved.
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analysis of gene expression changes in peach leaves in response to Plum Pox Virus infection using rna seq
Molecular Plant Pathology, 2015Co-Authors: Manuel Rubio, Thierry Candresse, Anarosa Ballester, Manuel Castro De Moura, Luis Rodriguezmoreno, Claudio Bonghi, Pedro MartinezgomezAbstract:Summary Differences in gene expression were studied after Plum Pox Virus (PPV, sharka disease) infection in peach GF305 leaves with and without sharka symptoms using RNA-Seq. For each sample, more than 80% of 100-nucleotide paired-end (PE) Illumina reads were aligned on the peach reference genome. In the symptomatic sample, a significant proportion of reads were mapped to PPV reference genomes (1.04% compared with 0.00002% in non-symptomatic leaves), allowing for the ultra-deep assembly of the complete genome of the PPV isolate used (9775 nucleotides, missing only 11 nucleotides at the 5′ genome end). In addition, significant alternative splicing events were detected in 359 genes and 12 990 single nucleotide polymorphisms (SNPs) were identified, 425 of which could be annotated. Gene ontology annotation revealed that the high-ranking mRNA target genes associated with the expression of sharka symptoms are mainly related to the response to biotic stimuli, to lipid and carbohydrate metabolism and to the negative regulation of catalytic activity. A greater number of differentially expressed genes were observed in the early asymptomatic phase of PPV infection in comparison with the symptomatic phase. These early infection events were associated with the induction of genes related to pathogen resistance, such as jasmonic acid, chitinases, cytokinin glucosyl transferases and Lys-M proteins. Once the Virus had accumulated, the overexpression of Dicer protein 2a genes suggested a gene silencing plant response that was suppressed by the Virus HCPro and P1 proteins. These results illustrate the dynamic nature of the peach–PPV interaction at the transcriptome level and confirm that sharka symptom expression is a complex process that can be understood on the basis of changes in plant gene expression.
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Plum Pox Virus and sharka a model potyVirus and a major disease
Molecular Plant Pathology, 2014Co-Authors: Juan Antonio García, Miroslav Glasa, Mariano Cambra, Thierry CandresseAbstract:SummaryTaxonomic relationships Plum Pox Virus (PPV) is a member of the genus PotyVirus in the family Potyviridae. PPV diversity is structured into at least eight monophyletic strains. Geographical distribution First discovered in Bulgaria, PPV is nowadays present in most of continental Europe (with an endemic status in many central and southern European countries) and has progressively spread to many countries on other continents. Genomic structure Typical of potyViruses, the PPV genome is a positive-sense single-stranded RNA (ssRNA), with a protein linked to its 5′ end and a 3′-terminal poly A tail. It is encapsidated by a single type of capsid protein (CP) in flexuous rod particles and is translated into a large polyprotein which is proteolytically processed in at least 10 final products: P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIapro, NIb and CP. In addition, P3N-PIPO is predicted to be produced by a translational frameshift. Pathogenicity features PPV causes sharka, the most damaging viral disease of stone fruit trees. It also infects wild and ornamental Prunus trees and has a large experimental host range in herbaceous species. PPV spreads over long distances by uncontrolled movement of plant material, and many species of aphid transmit the Virus locally in a nonpersistent manner. Sources of resistance A few natural sources of resistance to PPV have been found so far in Prunus species, which are being used in classical breeding programmes. Different genetic engineering approaches are being used to generate resistance to PPV, and a transgenic Plum, ‘HoneySweet’, transformed with the viral CP gene, has demonstrated high resistance to PPV in field tests in several countries and has obtained regulatory approval in the USA.
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Transcriptomic Analysis of Prunus domestica Undergoing Hypersensitive Response to Plum Pox Virus Infection
PLoS ONE, 2014Co-Authors: Bernardo Rodamilans, Thierry Candresse, David San León, Louisa Mühlberger, Michael Neumüller, Juan Carlos Oliveros, Juan Antonio GarcíaAbstract:Plum Pox Virus (PPV) infects Prunus trees around the globe, posing serious fruit production problems and causing severe economic losses. One variety of Prunus domestica, named 'Jojo', develops a hypersensitive response to viral infection. Here we compared infected and non-infected samples using next-generation RNA sequencing to characterize the genetic complexity of the viral population in infected samples and to identify genes involved in development of the resistance response. Analysis of viral reads from the infected samples allowed reconstruction of a PPV-D consensus sequence. De novo reconstruction showed a second viral isolate of the PPV-Rec strain. RNA-seq analysis of PPV-infected 'Jojo' trees identified 2,234 and 786 unigenes that were significantly up- or downregulated, respectively (false discovery rate; FDR≤0.01). Expression of genes associated with defense was generally enhanced, while expression of those related to photosynthesis was repressed. Of the total of 3,020 differentially expressed unigenes, 154 were characterized as potential resistance genes, 10 of which were included in the NBS-LRR type. Given their possible role in plant defense, we selected 75 additional unigenes as candidates for further study. The combination of next-generation sequencing and a Prunus variety that develops a hypersensitive response to PPV infection provided an opportunity to study the factors involved in this plant defense mechanism. Transcriptomic analysis presented an overview of the changes that occur during PPV infection as a whole, and identified candidates suitable for further functional characterization.
Anna Sheveleva - One of the best experts on this subject based on the ideXlab platform.
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Sequence Analysis of Plum Pox Virus Strain C Isolates from Russia Revealed Prevalence of the D96E Mutation in the Universal Epitope and Interstrain Recombination Events
MDPI AG, 2018Co-Authors: Anna Sheveleva, Peter Ivanov, Tatiana Gasanova, Gennady Osipov, S N ChirkovAbstract:The understanding of genetic diversity, geographic distribution, and antigenic properties of Plum Pox Virus (PPV) is a prerequisite to improve control of sharka, the most detrimental viral disease of stone fruit crops worldwide. Forty new PPV strain C isolates were detected in sour cherry (Prunus cerasus) from three geographically distant (700–1100 km) regions of European Russia. Analysis of their 3’-terminal genomic sequences showed that nineteen isolates (47.5%) bear the D96E mutation in the universal epitope of the coat protein. Almost all of them cannot be detected by the monoclonal antibody 5B in triple antibody sandwich enzyme-linked immunosorbent assayand Western blot analysis that may potentially compromise serological PPV detection in cherries. Full-length genomes of seven PPV-C isolates were determined employing next-generation sequencing. Using the Recombination Detection Program (RDP4), the recombination event covering the region from (Cter)P1 to the middle of the HcPro gene was predicted in all the available PPV-C complete genomes. The isolates Tat-4, belonging to the strain CV, and RU-17sc (PPV-CR) were inferred as major and minor parents, respectively, suggesting possible pathways of evolution of the cherry-adapted strains. Downy cherry (P. tomentosa) was identified as the natural PPV-C host for the first time
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analysis of genetic diversity of russian sour cherry Plum Pox Virus isolates provides evidence of a new strain
Plant Disease, 2017Co-Authors: S N Chirkov, Anna Sheveleva, P. A. Ivanov, Alexander ZakubanskiyAbstract:Plum Pox Virus (PPV) exists as a complex of nine strains adapted to different Prunus hosts. Unusual PPV isolates that do not belong to the known cherry-adapted strains were discovered on sour cherry in Russia. Here, two complete genomes of isolates Tat-2 and Tat-4 were determined by sequencing on the Illumina HiSeq 2500 platform. Both were composed of 9,792 nucleotides, excluding the poly(A) tail, with the organization typical of PPV and had 99.4% and 99.7% identity between each other at the nucleotide and amino acid levels. The sequence identities between the Tat-2/Tat-4 and known PPV strains ranged from 77.6 to 83.3% for genomic RNA and from 80.0 to 93.8% for polyprotein. Phylogenetic analysis placed Tat-2 and Tat-4 in a separate clade, distinct from the C and CR strains. Three more Tat-2/Tat-4-like isolates were detected in local cherry plantings using the newly developed, specific RT-PCR assay. Based on the phylogenetic analysis, sequence identities and environmental distribution, the Tat-2, Tat-4 and...
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new highly divergent Plum Pox Virus isolates infecting sour cherry in russia
Virology, 2017Co-Authors: S N Chirkov, Anna Sheveleva, Alexander Zakubanskiy, Peter Ivanov, Gennady OsipovAbstract:Abstract Unusual Plum Pox Virus (PPV) isolates (named Tat isolates) were discovered on sour cherry ( Prunus cerasus ) in Russia. They failed to be recognized by RT-PCR using commonly employed primers specific to the strains C or CR (the only ones that proved able to infect sour cherry) as well as to the strains M and W. Some of them can be detected by RT-PCR using the PPV-D-specific primers P1/PD or by TAS-ELISA with the PPV-C-specific monoclonal antibody AC. Phylogenetic analysis of the 3′-terminal genomic region assigned the Tat isolates into the cluster of cherry-adapted strains. However, they grouped separately from the C and CR strains and from each other as well. The sequence divergence of the Tat isolates is comparable to the differences between the known PPV strains. They may represent new group(s) of cherry-adapted isolates which do not seem to belong to any known strain of the Virus.
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new highly divergent Plum Pox Virus isolates infecting sour cherry in russia
Virology, 2017Co-Authors: S N Chirkov, Anna Sheveleva, Alexander Zakubanskiy, P. A. Ivanov, Gennady OsipovAbstract:Abstract Unusual Plum Pox Virus (PPV) isolates (named Tat isolates) were discovered on sour cherry ( Prunus cerasus ) in Russia. They failed to be recognized by RT-PCR using commonly employed primers specific to the strains C or CR (the only ones that proved able to infect sour cherry) as well as to the strains M and W. Some of them can be detected by RT-PCR using the PPV-D-specific primers P1/PD or by TAS-ELISA with the PPV-C-specific monoclonal antibody AC. Phylogenetic analysis of the 3′-terminal genomic region assigned the Tat isolates into the cluster of cherry-adapted strains. However, they grouped separately from the C and CR strains and from each other as well. The sequence divergence of the Tat isolates is comparable to the differences between the known PPV strains. They may represent new group(s) of cherry-adapted isolates which do not seem to belong to any known strain of the Virus.
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detection and partial molecular characterization of atypical Plum Pox Virus isolates from naturally infected sour cherry
Archives of Virology, 2013Co-Authors: S N Chirkov, Peter Ivanov, Anna ShevelevaAbstract:Atypical isolates of Plum Pox Virus (PPV) were discovered in naturally infected sour cherry in urban ornamental plantings in Moscow, Russia. The isolates were detected by polyclonal double antibody sandwich ELISA and RT-PCR using universal primers specific for the 3′-non-coding and coat protein (CP) regions of the genome but failed to be recognized by triple antibody sandwich ELISA with the universal monoclonal antibody 5B and by RT-PCR using primers specific to for PPV strains D, M, C and W. Sequence analysis of the CP genes of nine isolates revealed 99.2-100 % within-group identity and 62-85 % identity to conventional PPV strains. Phylogenetic analysis showed that the atypical isolates represent a group that is distinct from the known PPV strains. Alignment of the N-terminal amino acid sequences of CP demonstrated their close similarity to those of a new tentative PPV strain, CR.
S N Chirkov - One of the best experts on this subject based on the ideXlab platform.
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Sequence Analysis of Plum Pox Virus Strain C Isolates from Russia Revealed Prevalence of the D96E Mutation in the Universal Epitope and Interstrain Recombination Events
MDPI AG, 2018Co-Authors: Anna Sheveleva, Peter Ivanov, Tatiana Gasanova, Gennady Osipov, S N ChirkovAbstract:The understanding of genetic diversity, geographic distribution, and antigenic properties of Plum Pox Virus (PPV) is a prerequisite to improve control of sharka, the most detrimental viral disease of stone fruit crops worldwide. Forty new PPV strain C isolates were detected in sour cherry (Prunus cerasus) from three geographically distant (700–1100 km) regions of European Russia. Analysis of their 3’-terminal genomic sequences showed that nineteen isolates (47.5%) bear the D96E mutation in the universal epitope of the coat protein. Almost all of them cannot be detected by the monoclonal antibody 5B in triple antibody sandwich enzyme-linked immunosorbent assayand Western blot analysis that may potentially compromise serological PPV detection in cherries. Full-length genomes of seven PPV-C isolates were determined employing next-generation sequencing. Using the Recombination Detection Program (RDP4), the recombination event covering the region from (Cter)P1 to the middle of the HcPro gene was predicted in all the available PPV-C complete genomes. The isolates Tat-4, belonging to the strain CV, and RU-17sc (PPV-CR) were inferred as major and minor parents, respectively, suggesting possible pathways of evolution of the cherry-adapted strains. Downy cherry (P. tomentosa) was identified as the natural PPV-C host for the first time
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analysis of genetic diversity of russian sour cherry Plum Pox Virus isolates provides evidence of a new strain
Plant Disease, 2017Co-Authors: S N Chirkov, Anna Sheveleva, P. A. Ivanov, Alexander ZakubanskiyAbstract:Plum Pox Virus (PPV) exists as a complex of nine strains adapted to different Prunus hosts. Unusual PPV isolates that do not belong to the known cherry-adapted strains were discovered on sour cherry in Russia. Here, two complete genomes of isolates Tat-2 and Tat-4 were determined by sequencing on the Illumina HiSeq 2500 platform. Both were composed of 9,792 nucleotides, excluding the poly(A) tail, with the organization typical of PPV and had 99.4% and 99.7% identity between each other at the nucleotide and amino acid levels. The sequence identities between the Tat-2/Tat-4 and known PPV strains ranged from 77.6 to 83.3% for genomic RNA and from 80.0 to 93.8% for polyprotein. Phylogenetic analysis placed Tat-2 and Tat-4 in a separate clade, distinct from the C and CR strains. Three more Tat-2/Tat-4-like isolates were detected in local cherry plantings using the newly developed, specific RT-PCR assay. Based on the phylogenetic analysis, sequence identities and environmental distribution, the Tat-2, Tat-4 and...
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new highly divergent Plum Pox Virus isolates infecting sour cherry in russia
Virology, 2017Co-Authors: S N Chirkov, Anna Sheveleva, Alexander Zakubanskiy, Peter Ivanov, Gennady OsipovAbstract:Abstract Unusual Plum Pox Virus (PPV) isolates (named Tat isolates) were discovered on sour cherry ( Prunus cerasus ) in Russia. They failed to be recognized by RT-PCR using commonly employed primers specific to the strains C or CR (the only ones that proved able to infect sour cherry) as well as to the strains M and W. Some of them can be detected by RT-PCR using the PPV-D-specific primers P1/PD or by TAS-ELISA with the PPV-C-specific monoclonal antibody AC. Phylogenetic analysis of the 3′-terminal genomic region assigned the Tat isolates into the cluster of cherry-adapted strains. However, they grouped separately from the C and CR strains and from each other as well. The sequence divergence of the Tat isolates is comparable to the differences between the known PPV strains. They may represent new group(s) of cherry-adapted isolates which do not seem to belong to any known strain of the Virus.
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new highly divergent Plum Pox Virus isolates infecting sour cherry in russia
Virology, 2017Co-Authors: S N Chirkov, Anna Sheveleva, Alexander Zakubanskiy, P. A. Ivanov, Gennady OsipovAbstract:Abstract Unusual Plum Pox Virus (PPV) isolates (named Tat isolates) were discovered on sour cherry ( Prunus cerasus ) in Russia. They failed to be recognized by RT-PCR using commonly employed primers specific to the strains C or CR (the only ones that proved able to infect sour cherry) as well as to the strains M and W. Some of them can be detected by RT-PCR using the PPV-D-specific primers P1/PD or by TAS-ELISA with the PPV-C-specific monoclonal antibody AC. Phylogenetic analysis of the 3′-terminal genomic region assigned the Tat isolates into the cluster of cherry-adapted strains. However, they grouped separately from the C and CR strains and from each other as well. The sequence divergence of the Tat isolates is comparable to the differences between the known PPV strains. They may represent new group(s) of cherry-adapted isolates which do not seem to belong to any known strain of the Virus.
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detection and partial molecular characterization of atypical Plum Pox Virus isolates from naturally infected sour cherry
Archives of Virology, 2013Co-Authors: S N Chirkov, Peter Ivanov, Anna ShevelevaAbstract:Atypical isolates of Plum Pox Virus (PPV) were discovered in naturally infected sour cherry in urban ornamental plantings in Moscow, Russia. The isolates were detected by polyclonal double antibody sandwich ELISA and RT-PCR using universal primers specific for the 3′-non-coding and coat protein (CP) regions of the genome but failed to be recognized by triple antibody sandwich ELISA with the universal monoclonal antibody 5B and by RT-PCR using primers specific to for PPV strains D, M, C and W. Sequence analysis of the CP genes of nine isolates revealed 99.2-100 % within-group identity and 62-85 % identity to conventional PPV strains. Phylogenetic analysis showed that the atypical isolates represent a group that is distinct from the known PPV strains. Alignment of the N-terminal amino acid sequences of CP demonstrated their close similarity to those of a new tentative PPV strain, CR.
Pedro Martinezgomez - One of the best experts on this subject based on the ideXlab platform.
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gene expression analysis of induced Plum Pox Virus sharka resistance in peach prunus persica by almond p dulcis grafting
International Journal of Molecular Sciences, 2021Co-Authors: Manuel Rubio, Bernardo Rodamilans, Juan Antonio García, F Dicenta, Pedro J Martinezgarcia, Azam Nikbakhtdehkordi, Angela S Prudencio, E M Gomez, Pedro MartinezgomezAbstract:No natural sources of resistance to Plum Pox Virus (PPV, sharka disease) have been identified in peach. However, previous studies have demonstrated that grafting a “Garrigues” almond scion onto “GF305” peach rootstock seedlings heavily infected with PPV can progressively reduce disease symptoms and Virus accumulation. Furthermore, grafting a “Garrigues” scion onto the “GF305” rootstock has been shown to completely prevent Virus infection. This study aims to analyse the rewiring of gene expression associated with this resistance to PPV transmitted by grafting through the phloem using RNA-Seq and RT-qPCR analysis. A total of 18 candidate genes were differentially expressed after grafting “Garrigues” almond onto healthy “GF305” peach. Among the up-regulated genes, a HEN1 homolog stands out, which, together with the differential expression of RDR- and DCL2-homologs, suggests that the RNA silencing machinery is activated by PPV infection and can contribute to the resistance induced by “Garrigues” almond. Glucan endo-1,3-beta D-glucosidase could be also relevant for the “Garrigues”-induced response, since its expression is much higher in “Garrigues” than in “GF305”. We also discuss the potential relevance of the following in PPV infection and “Garrigues”-induced resistance: several pathogenesis-related proteins; no apical meristem proteins; the transcription initiation factor, TFIIB; the speckle-type POZ protein; in addition to a number of proteins involved in phytohormone signalling.
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gene expression analysis of Plum Pox Virus sharka susceptibility resistance in apricot prunus armeniaca l
PLOS ONE, 2015Co-Authors: Manuel Rubio, Anarosa Ballester, Pedro Manuel Olivares, Manuel Castro De Moura, F Dicenta, Pedro MartinezgomezAbstract:RNA-Seq has proven to be a very powerful tool in the analysis of the Plum Pox Virus (PPV, sharka disease)/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, “Rojo Pasion” and “Z506-7”, resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the Virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925), which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene) or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein) PPVres region could also be involved in the resistance.
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analysis of gene expression changes in peach leaves in response to Plum Pox Virus infection using rna seq
Molecular Plant Pathology, 2015Co-Authors: Manuel Rubio, Thierry Candresse, Anarosa Ballester, Manuel Castro De Moura, Luis Rodriguezmoreno, Claudio Bonghi, Pedro MartinezgomezAbstract:Summary Differences in gene expression were studied after Plum Pox Virus (PPV, sharka disease) infection in peach GF305 leaves with and without sharka symptoms using RNA-Seq. For each sample, more than 80% of 100-nucleotide paired-end (PE) Illumina reads were aligned on the peach reference genome. In the symptomatic sample, a significant proportion of reads were mapped to PPV reference genomes (1.04% compared with 0.00002% in non-symptomatic leaves), allowing for the ultra-deep assembly of the complete genome of the PPV isolate used (9775 nucleotides, missing only 11 nucleotides at the 5′ genome end). In addition, significant alternative splicing events were detected in 359 genes and 12 990 single nucleotide polymorphisms (SNPs) were identified, 425 of which could be annotated. Gene ontology annotation revealed that the high-ranking mRNA target genes associated with the expression of sharka symptoms are mainly related to the response to biotic stimuli, to lipid and carbohydrate metabolism and to the negative regulation of catalytic activity. A greater number of differentially expressed genes were observed in the early asymptomatic phase of PPV infection in comparison with the symptomatic phase. These early infection events were associated with the induction of genes related to pathogen resistance, such as jasmonic acid, chitinases, cytokinin glucosyl transferases and Lys-M proteins. Once the Virus had accumulated, the overexpression of Dicer protein 2a genes suggested a gene silencing plant response that was suppressed by the Virus HCPro and P1 proteins. These results illustrate the dynamic nature of the peach–PPV interaction at the transcriptome level and confirm that sharka symptom expression is a complex process that can be understood on the basis of changes in plant gene expression.
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opportunities of marker assisted selection for Plum Pox Virus resistance in apricot breeding programs
Tree Genetics & Genomes, 2014Co-Authors: Manuel Rubio, Pedro Martinezgomez, David Ruiz, Jose A Egea, F DicentaAbstract:Evaluation of Plum Pox Virus (PPV) resistance is a laborious and expensive task, and the development of new accurate methods, including the use of molecular markers, would be very useful for breeding programs for resistance. In this work, the Plum Pox Virus resistance of 80 apricot genotypes of different genetic origins was evaluated in controlled greenhouse and natural field conditions. The genotypes for five simple sequence repeat (SSR) markers described as linked to PPV resistance were also determined. Depending on their behavior, cultivars were classified as resistant, susceptible, and uncertain, and the genotype was identified for each SSR linked to different phenotypes. Twenty genotypes were resistant and 37 susceptible in the greenhouse and in the field. However, 23 genotypes did not show clear behavior, probably due to the complex plant-Virus interaction, so they were classified as uncertain. In general, results showed a narrow relationship between the SSRs PGS1.21 and PGS1.24, and resistance to PPV, although some genotypes did not show this relationship. Most of the susceptible genotypes did not show the alleles of resistance. Therefore, in most cases, marker-assisted selection (MAS) could be used as a means of screening new seedlings for early selection, making it possible to remove those that are susceptible. However, in certain cases, MAS using these markers has not proven to be completely effective. The origin of such discrepancies could be the presence of a second locus involved in PPV resistance. In addition, other factors affecting efficiency of MAS discussed in the work are the presence of null alleles and recombinant events. Resistant seedlings would have to be evaluated in greenhouse and natural conditions to confirm their actual behavior against PPV. From the breeding point of view, the use of homozygous resistant parents for the SSR resistance alleles, with good agronomic characteristics, would increase the efficiency of breeding programs, since all seedlings would be resistant regardless of the other parent. Finally, new molecular markers should be developed to accurately select resistant seedlings regardless of the resistant progenitors involved.
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alteration in the chloroplastic metabolism leads to ros accumulation in pea plants in response to Plum Pox Virus
Journal of Experimental Botany, 2008Co-Authors: Pedro Diazvivancos, Juan Antonio García, Manuel Rubio, Pedro Martinezgomez, Enrique Olmos, Maria Jose Clementemoreno, Jose Antonio HernandezAbstract:In this work, a recombinant Plum Pox Virus (PPV, Sharka) encoding green fluorescent protein is used to study its effect on antioxidant enzymes and protein expression at the subcellular level in pea plants (cv. Alaska). PPV had produced chlorotic spots as well as necrotic spots in the oldest leaves at 13-15 d post-inoculation. At 15 d post-inoculation, PPV was present in the chlorotic and necrotic areas, as shown by the fluorescence signal produced by the presence of the green fluorescent protein. In the same areas, an accumulation of reactive oxygen species was noticed. Studies with laser confocal and electron microscopy demonstrated that PPV accumulated in the cytosol of infected cells. In addition, PPV infection produced an alteration in the chloroplast ultrastructure, giving rise to dilated thylakoids, an increase in the number of plastoglobuli, and a decreased amount of starch content. At 3 d post-inoculation, although no changes in the oxidative stress parameters were observed, an increase in the chloroplastic hydrogen peroxide levels was observed that correlated with a decrease in the enzymatic mechanisms involved in its elimination (ascorbate peroxidase and peroxidase) in this cell compartment. These results indicate that an alteration in the chloroplastic metabolism is produced in the early response to PPV. This oxidative stress is more pronounced during the development of the disease (15 d post-inoculation) judging from the increase in oxidative stress parameters as well as the imbalance in the antioxidative systems, mainly at the chloroplastic level. Finally, proteomic analyses showed that most of the changes produced by PPV infection with regard to protein expression at the subcellular level were related mainly to photosynthesis and carbohydrate metabolism. It seems that PPV infection has some effect on PSII, directly or indirectly, by decreasing the amount of Rubisco, oxygen-evolving enhancer, and PSII stability factor proteins. The results indicate that Sharka symptoms observed in pea leaves could be due to an imbalance in antioxidant systems as well as to an increased generation of reactive oxygen species in chloroplasts, induced probably by a disturbance of the electron transport chain, suggesting that chloroplasts can be a source of oxidative stress during viral disease development.