Plum pox virus (PPV) infects stone-fruit trees with important economical impact mainly in Europe ... more Plum pox virus (PPV) infects stone-fruit trees with important economical impact mainly in Europe and Mediterranean region. The data about PPV intra-species variability accumulated markedly in the last two decades. Six PPV strains have been recognized using different approaches including serology, protein analysis, specific amplification, and genome sequencing. Reliable and sensitive diagnostics is the most important requirement for application of early control and safety measures. Therefore, many techniques and their modifications have been adapted to detect PPV and its different forms. Here, we review the improvement of the PPV detection and variability analysis in the context of progress in laboratory methods since the virus discovery till today.
Plant viruses are a significant threat to a wide range of host species, causing substantial losse... more Plant viruses are a significant threat to a wide range of host species, causing substantial losses in agriculture. Particularly, Cucumber mosaic virus (CMV) evokes severe symptoms, thus dramatically limiting yield. Activation of plant defense reactions is associated with changes in the cellular proteome to ensure virus resistance. Herein, we studied two cultivars of cucumber (Cucumis sativus) resistant host Heliana and susceptible host Vanda. Plant cotyledons were mechanically inoculated with CMV isolate PK1, and systemic leaves were harvested at 33 days post-inoculation. Proteome was profiled by ultrahigh-performance liquid chromatography and comprehensively quantified by ion mobility enhanced mass spectrometry. From 1516 reproducibly quantified proteins using a label-free approach, 133 were differentially abundant among cultivars or treatments by strict statistic and effect size criteria. Pigments and hydrogen peroxide measurements corroborated proteomic findings. Comparison of both cultivars in the uninfected state highlighted more abundant photosynthetic and development-related proteins in resistant cucumber cultivar. Long-term CMV infection caused worse preservation of energy processes and less robust translation in the susceptible cultivar. Contrary, compatible plants had numerous more abundant stress and defense-related proteins. We proposed promising targets for functional validation in transgenic lines: A step toward durable virus resistance in cucurbits and other crops. SIGNIFICANCE: Sustainable production of crops requires an understanding of natural mechanisms of resistance/susceptibility to ubiquitous viral infections. We report original findings of comparative analysis of plant genotypes exposed to CMV. Deep discovery proteomics of resistant and susceptible cucumber cultivars, inoculated with widespread phytovirus, allowed to suggest several novel molecular targets for functional testing in plant protection strategies.
Sharka, caused by Plum pox virus (PPV), is the most detrimental viral disease of stone fruit tree... more Sharka, caused by Plum pox virus (PPV), is the most detrimental viral disease of stone fruit trees. First reported from Bulgaria in 1917, the virus is now widespread in Europe, the Mediterranean Basin, and Asia Minor and is sporadically present in North and South America. On the basis of molecular and serological properties, six PPV subgroups are recognized, from which PPV-D, PPV-M, and PPV-Rec are the most common (1,2). Several apricot trees (Prunus armeniaca) showing mild, pale green rings and diffuse chlorotic spots on leaves were found in a small orchard in the Baltistan District in northern Pakistan at approximately 2,400 m above sea level. Dried leaf samples from one symptomatic tree randomly selected from the orchard were positive for PPV using double-antibody sandwich enzyme-linked immunosorbent assay with antisera prepared in the laboratory, immunoblot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) targeting the capsid protein (CP) gene using standard procedures (1). To check the subgroup affiliation and evaluate the molecular variability, the 562-bp variable region spanning the C-terminus of NIb and the N-terminus of the CP was amplified, the RT-PCR product was cloned into the pGEM-T Easy vector (Promega, Madison, WI), and positive clones were analyzed by restriction and sequence analyses. Interestingly, sequence analysis of four clones revealed mixed infection, i.e., the presence of two different PPV isolates in the apricot sample. One isolate belonged to PPV-D (GenBank Accession No. DQ422147) and the other belonged to the PPV-Rec subgroup (GenBank Accession No. DQ422148). Multiple alignment of the sequenced genome portion of the Pakistan PPV-D isolate indicated 96 to 99% nt identity with various PPV-D isolates without unique, clear-cut differences. Similarly, the PPV-Rec isolate had 98 to 99% identity with European PPV-Rec isolates and retained the cross-over at nucleotide position 8450 in the 3′ terminus of NIb. This sequence had the amino acid signature at the N-terminus of the CP typical of the PPV-Rec subgroup (2). Moreover, no particular clustering of the Pakistan isolates within PPV-D and PPV-Rec could be observed after phylogenetic analysis. The DAG motif, essential for aphid transmission, was present in both sequences. To our knowledge, this is the first indication of PPV occurrence in Pakistan and first identification of the PPV-Rec isolate outside Europe. Together with previous reports on the PPV presence in China and Kazakhstan (3,4), this report indicates the need for more detailed epidemiological studies focusing the PPV spread and its molecular diversity in Asia. References: (1) T. Candresse et al. Phytopathology 88:198, 1998. (2) M. Glasa et al. J. Gen. Virol. 85:2671, 2004. (3) M. Navrátil et al. Plant Dis. 89:338, 2005. (4) S. Spiegel et al. Plant Dis. 88:973, 2004.
Six mouse monoclonal antibodies (MAbs) against potato virus A (PVA) were examined on their reacti... more Six mouse monoclonal antibodies (MAbs) against potato virus A (PVA) were examined on their reactivity with PVA and its denatured capsid protein (PVA-CP) bound to nitrocellulose membrane. Five MAbs reacted with native PVA, three of them also with PVA-CP. MAb 534 gave no reaction in dot-blot tests. In Western blot analysis only MAbs 151, 290, and 328 reacted with PVA-CP. Competition binding test data confirm mutual spatial proximity of epitopes corresponding to these MAbs. Pepscan (SPOTs tests) with overlapping octapeptides representing the sequence of the first 60 amino acids from the N terminus of PVA-CP showed that the epitopes detected by MAb 151, 328, and 634 are located in this region. MAb 534 was deduced to react with discontinuous CP epitope. Results of analogic peptide synthesis for the MAb 151 epitope indicate that two lysine residues are essential for binding of this MAb to its epitope. Our polyclonal antibodies against PVA reacted with six different regions in this part of the CP.
Closely related natural Plum pox virus (PPV) isolates derived from homologous RNA recombination b... more Closely related natural Plum pox virus (PPV) isolates derived from homologous RNA recombination between PPV-D and PPV-M have been recently identified and shown naturally spread in several European countries. As their serological properties were identical with those of conventional PPV-M isolates, they could be detected only by combined analysis of at least two different genome parts. To simplify the detection of such recombinants primers specific to PPV-M and PPV-D sequences with binding sites located on both sides of the recombination crossover situated in the C-terminal part of NIb were designed. They were used for direct differentiation of PPV-M, PPV-D and their recombinants by reverse transcription-polymerase chain reaction (RT-PCR). This method is convenient for identification of a recombinant PPV in single as well as mixed infection with PPV-M or PPV-D.
Plum pox virus (PPV) isolates of the strain PPV-M prevalently infect peaches under natural condit... more Plum pox virus (PPV) isolates of the strain PPV-M prevalently infect peaches under natural conditions in Middle Europe. Comparison of complete genome sequences obtained from subisolates of a PPV-M isolate maintained experimentally over a 6-year period in different Prunus host species and passaged in Nicotiana benthamiana was performed with the aim to highlight the mutations potentially connected with the virus-host adaptation. The results showed that the lowest number of non-silent mutations was accumulated in PPV-M maintained in peach (original host species), approximately two times higher diversity was recorded in plum, apricot and N. benthamiana, indicating the genetic determination of the PPV host preference. The sequence variability of Prunus subisolates was distributed more or less evenly along the PPV genome and no amino acid motif could be outlined as responsible for the host adaptation. In N. benthamiana the mutations were accumulated notably in the P1 and P3 genes indicating their non-essentiality in the infection of this experimental host plant.
ABSTRACT Plum pox virus (PPV) is the most important virus of stone fruit trees (Prunus spp.) worl... more ABSTRACT Plum pox virus (PPV) is the most important virus of stone fruit trees (Prunus spp.) worldwide. Three major PPV strains (PPV-D, PPV-Rec and PPV-M) are spread in the Central Europe, differing by genomic sequence and relative epidemiological impact in particular geographical regions. Although PPV strains could multiply in several Prunus species under experimental conditions, they showed a clear preference of natural host plant species. While PPV-M was usually isolated from peaches, PPV-D and PPV-Rec were predominantly found in plum trees. In our experiments the mixed strains of PPV revealed evidently higher accumulation of PPV-M in peach and apricot and PPV-D in plum, supporting the data of natural strain incidence. Screening of samples from an orchard with combined plum-peach planting showed presence of all three major strains in both species, often in double or triple mixtures. It is possible, that the spread of particular PPV strains in their atypical hosts could be accelerated by mixed infections with other („host-typical“) strains.
Plum pox virus (PPV) infects stone-fruit trees with important economical impact mainly in Europe ... more Plum pox virus (PPV) infects stone-fruit trees with important economical impact mainly in Europe and Mediterranean region. The data about PPV intra-species variability accumulated markedly in the last two decades. Six PPV strains have been recognized using different approaches including serology, protein analysis, specific amplification, and genome sequencing. Reliable and sensitive diagnostics is the most important requirement for application of early control and safety measures. Therefore, many techniques and their modifications have been adapted to detect PPV and its different forms. Here, we review the improvement of the PPV detection and variability analysis in the context of progress in laboratory methods since the virus discovery till today.
Plant viruses are a significant threat to a wide range of host species, causing substantial losse... more Plant viruses are a significant threat to a wide range of host species, causing substantial losses in agriculture. Particularly, Cucumber mosaic virus (CMV) evokes severe symptoms, thus dramatically limiting yield. Activation of plant defense reactions is associated with changes in the cellular proteome to ensure virus resistance. Herein, we studied two cultivars of cucumber (Cucumis sativus) resistant host Heliana and susceptible host Vanda. Plant cotyledons were mechanically inoculated with CMV isolate PK1, and systemic leaves were harvested at 33 days post-inoculation. Proteome was profiled by ultrahigh-performance liquid chromatography and comprehensively quantified by ion mobility enhanced mass spectrometry. From 1516 reproducibly quantified proteins using a label-free approach, 133 were differentially abundant among cultivars or treatments by strict statistic and effect size criteria. Pigments and hydrogen peroxide measurements corroborated proteomic findings. Comparison of both cultivars in the uninfected state highlighted more abundant photosynthetic and development-related proteins in resistant cucumber cultivar. Long-term CMV infection caused worse preservation of energy processes and less robust translation in the susceptible cultivar. Contrary, compatible plants had numerous more abundant stress and defense-related proteins. We proposed promising targets for functional validation in transgenic lines: A step toward durable virus resistance in cucurbits and other crops. SIGNIFICANCE: Sustainable production of crops requires an understanding of natural mechanisms of resistance/susceptibility to ubiquitous viral infections. We report original findings of comparative analysis of plant genotypes exposed to CMV. Deep discovery proteomics of resistant and susceptible cucumber cultivars, inoculated with widespread phytovirus, allowed to suggest several novel molecular targets for functional testing in plant protection strategies.
Sharka, caused by Plum pox virus (PPV), is the most detrimental viral disease of stone fruit tree... more Sharka, caused by Plum pox virus (PPV), is the most detrimental viral disease of stone fruit trees. First reported from Bulgaria in 1917, the virus is now widespread in Europe, the Mediterranean Basin, and Asia Minor and is sporadically present in North and South America. On the basis of molecular and serological properties, six PPV subgroups are recognized, from which PPV-D, PPV-M, and PPV-Rec are the most common (1,2). Several apricot trees (Prunus armeniaca) showing mild, pale green rings and diffuse chlorotic spots on leaves were found in a small orchard in the Baltistan District in northern Pakistan at approximately 2,400 m above sea level. Dried leaf samples from one symptomatic tree randomly selected from the orchard were positive for PPV using double-antibody sandwich enzyme-linked immunosorbent assay with antisera prepared in the laboratory, immunoblot analysis, and reverse transcription-polymerase chain reaction (RT-PCR) targeting the capsid protein (CP) gene using standard procedures (1). To check the subgroup affiliation and evaluate the molecular variability, the 562-bp variable region spanning the C-terminus of NIb and the N-terminus of the CP was amplified, the RT-PCR product was cloned into the pGEM-T Easy vector (Promega, Madison, WI), and positive clones were analyzed by restriction and sequence analyses. Interestingly, sequence analysis of four clones revealed mixed infection, i.e., the presence of two different PPV isolates in the apricot sample. One isolate belonged to PPV-D (GenBank Accession No. DQ422147) and the other belonged to the PPV-Rec subgroup (GenBank Accession No. DQ422148). Multiple alignment of the sequenced genome portion of the Pakistan PPV-D isolate indicated 96 to 99% nt identity with various PPV-D isolates without unique, clear-cut differences. Similarly, the PPV-Rec isolate had 98 to 99% identity with European PPV-Rec isolates and retained the cross-over at nucleotide position 8450 in the 3′ terminus of NIb. This sequence had the amino acid signature at the N-terminus of the CP typical of the PPV-Rec subgroup (2). Moreover, no particular clustering of the Pakistan isolates within PPV-D and PPV-Rec could be observed after phylogenetic analysis. The DAG motif, essential for aphid transmission, was present in both sequences. To our knowledge, this is the first indication of PPV occurrence in Pakistan and first identification of the PPV-Rec isolate outside Europe. Together with previous reports on the PPV presence in China and Kazakhstan (3,4), this report indicates the need for more detailed epidemiological studies focusing the PPV spread and its molecular diversity in Asia. References: (1) T. Candresse et al. Phytopathology 88:198, 1998. (2) M. Glasa et al. J. Gen. Virol. 85:2671, 2004. (3) M. Navrátil et al. Plant Dis. 89:338, 2005. (4) S. Spiegel et al. Plant Dis. 88:973, 2004.
Six mouse monoclonal antibodies (MAbs) against potato virus A (PVA) were examined on their reacti... more Six mouse monoclonal antibodies (MAbs) against potato virus A (PVA) were examined on their reactivity with PVA and its denatured capsid protein (PVA-CP) bound to nitrocellulose membrane. Five MAbs reacted with native PVA, three of them also with PVA-CP. MAb 534 gave no reaction in dot-blot tests. In Western blot analysis only MAbs 151, 290, and 328 reacted with PVA-CP. Competition binding test data confirm mutual spatial proximity of epitopes corresponding to these MAbs. Pepscan (SPOTs tests) with overlapping octapeptides representing the sequence of the first 60 amino acids from the N terminus of PVA-CP showed that the epitopes detected by MAb 151, 328, and 634 are located in this region. MAb 534 was deduced to react with discontinuous CP epitope. Results of analogic peptide synthesis for the MAb 151 epitope indicate that two lysine residues are essential for binding of this MAb to its epitope. Our polyclonal antibodies against PVA reacted with six different regions in this part of the CP.
Closely related natural Plum pox virus (PPV) isolates derived from homologous RNA recombination b... more Closely related natural Plum pox virus (PPV) isolates derived from homologous RNA recombination between PPV-D and PPV-M have been recently identified and shown naturally spread in several European countries. As their serological properties were identical with those of conventional PPV-M isolates, they could be detected only by combined analysis of at least two different genome parts. To simplify the detection of such recombinants primers specific to PPV-M and PPV-D sequences with binding sites located on both sides of the recombination crossover situated in the C-terminal part of NIb were designed. They were used for direct differentiation of PPV-M, PPV-D and their recombinants by reverse transcription-polymerase chain reaction (RT-PCR). This method is convenient for identification of a recombinant PPV in single as well as mixed infection with PPV-M or PPV-D.
Plum pox virus (PPV) isolates of the strain PPV-M prevalently infect peaches under natural condit... more Plum pox virus (PPV) isolates of the strain PPV-M prevalently infect peaches under natural conditions in Middle Europe. Comparison of complete genome sequences obtained from subisolates of a PPV-M isolate maintained experimentally over a 6-year period in different Prunus host species and passaged in Nicotiana benthamiana was performed with the aim to highlight the mutations potentially connected with the virus-host adaptation. The results showed that the lowest number of non-silent mutations was accumulated in PPV-M maintained in peach (original host species), approximately two times higher diversity was recorded in plum, apricot and N. benthamiana, indicating the genetic determination of the PPV host preference. The sequence variability of Prunus subisolates was distributed more or less evenly along the PPV genome and no amino acid motif could be outlined as responsible for the host adaptation. In N. benthamiana the mutations were accumulated notably in the P1 and P3 genes indicating their non-essentiality in the infection of this experimental host plant.
ABSTRACT Plum pox virus (PPV) is the most important virus of stone fruit trees (Prunus spp.) worl... more ABSTRACT Plum pox virus (PPV) is the most important virus of stone fruit trees (Prunus spp.) worldwide. Three major PPV strains (PPV-D, PPV-Rec and PPV-M) are spread in the Central Europe, differing by genomic sequence and relative epidemiological impact in particular geographical regions. Although PPV strains could multiply in several Prunus species under experimental conditions, they showed a clear preference of natural host plant species. While PPV-M was usually isolated from peaches, PPV-D and PPV-Rec were predominantly found in plum trees. In our experiments the mixed strains of PPV revealed evidently higher accumulation of PPV-M in peach and apricot and PPV-D in plum, supporting the data of natural strain incidence. Screening of samples from an orchard with combined plum-peach planting showed presence of all three major strains in both species, often in double or triple mixtures. It is possible, that the spread of particular PPV strains in their atypical hosts could be accelerated by mixed infections with other („host-typical“) strains.
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