Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.
- MeSH
- biotechnologie metody MeSH
- genetické inženýrství metody MeSH
- odolnost vůči nemocem * MeSH
- slivoň genetika virologie MeSH
- transgeny MeSH
- umlčování genů * MeSH
- virové plášťové proteiny genetika metabolismus MeSH
- virus šarky švestky genetika patogenita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
High-throughput sequencing technologies were used to identify plant viruses in cereal samples surveyed from 2012 to 2017. Fifteen genome sequences of a tenuivirus infecting wheat, oats, and spelt in Estonia, Norway, and Sweden were identified and characterized by their distances to other tenuivirus sequences. Like most tenuiviruses, the genome of this tenuivirus contains four genomic segments. The isolates found from different countries shared at least 92% nucleotide sequence identity at the genome level. The planthopper Javesella pellucida was identified as a vector of the virus. Laboratory transmission tests using this vector indicated that wheat, oats, barley, rye, and triticale, but none of the tested pasture grass species (Alopecurus pratensis, Dactylis glomerata, Festuca rubra, Lolium multiflorum, Phleum pratense, and Poa pratensis), are susceptible. Taking into account the vector and host range data, the tenuivirus we have found most probably represents European wheat striate mosaic virus first identified about 60 years ago. Interestingly, whereas we were not able to infect any of the tested cereal species mechanically, Nicotiana benthamiana was infected via mechanical inoculation in laboratory conditions, displaying symptoms of yellow spots and vein clearing evolving into necrosis, eventually leading to plant death. Surprisingly, one of the virus genome segments (RNA2) encoding both a putative host systemic movement enhancer protein and a putative vector transmission factor was not detected in N. benthamiana after several passages even though systemic infection was observed, raising fundamental questions about the role of this segment in the systemic spread in several hosts.
- MeSH
- genom virový * genetika MeSH
- Hemiptera virologie MeSH
- jedlá semena virologie MeSH
- nemoci rostlin virologie MeSH
- rostlinné viry * genetika MeSH
- viry mozaiky * genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Norsko MeSH
- Švédsko MeSH
Plants of the genus Pleione, originating from hobby growers in the Netherlands and in the Czech Republic, were conspicuous for viral infection, showing symptoms of leaf mosaic or flower breaking. Using Sanger and high throughput sequencing, the full genome sequence of a novel potyvirus was obtained from sequencing data. The genome sequence was annotated and compared to the genome of other potyviruses. The virus was experimentally transmitted by aphids into Pleione and Chenopodium quinoa plants. The name Pleione flower breaking virus (PlFBV) was suggested for the new virus. The presence of the virus was confirmed using RT-PCR, with newly designed primers targeting this new species. The incidence of the virus was contrasted between both countries and might have been influenced by the growth conditions and the exposure of the plants to aphids.
- MeSH
- anotace sekvence MeSH
- Chenopodium quinoa virologie MeSH
- hmyz - vektory MeSH
- incidence MeSH
- mšice MeSH
- nemoci rostlin virologie MeSH
- Orchidaceae virologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- Potyvirus klasifikace genetika izolace a purifikace MeSH
- přenos infekční nemoci MeSH
- sekvenční analýza DNA MeSH
- sekvenování celého genomu MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
- Nizozemsko MeSH
BACKGROUND: Methylation of cytosines is an evolutionarily conserved epigenetic mark that is essential for the control of chromatin activity in many taxa. It acts mainly repressively, causing transcriptional gene silencing. In plants, de novo DNA methylation is established mainly by RNA-directed DNA-methylation pathway. Even though the protein machinery involved is relatively well-described, the course of the initial phases remains covert. RESULTS: We show the first detailed description of de novo DNA-methylation dynamics. Since prevalent plant model systems do not provide the possibility to collect homogenously responding material in time series with short intervals, we developed a convenient system based on tobacco BY-2 cell lines with inducible production of siRNAs (from an RNA hairpin) guiding the methylation machinery to the CaMV 35S promoter controlling GFP reporter. These lines responded very synchronously, and a high level of promoter-specific siRNAs triggered rapid promoter methylation with the first increase observed already 12 h after the induction. The previous presence of CG methylation in the promoter did not affect the methylation dynamics. The individual cytosine contexts reacted differently. CHH methylation peaked at about 80% in 2 days and then declined, whereas CG and CHG methylation needed more time with CHG reaching practically 100% after 10 days. Spreading of methylation was only minimal outside the target region in accordance with the absence of transitive siRNAs. The low and stable proportion of 24-nt siRNAs suggested that Pol IV was not involved in the initial phases. CONCLUSIONS: Our results show that de novo DNA methylation is a rapid process initiated practically immediately with the appearance of promoter-specific siRNAs and independently of the prior presence of methylcytosines at the target locus. The methylation was precisely targeted, and its dynamics varied depending on the cytosine sequence context. The progressively increasing methylation resulted in a smooth, gradual inhibition of the promoter activity, which was entirely suppressed in 2 days.
- MeSH
- Caulimovirus genetika MeSH
- estradiol farmakologie MeSH
- malá interferující RNA genetika metabolismus MeSH
- metylace DNA * účinky léků MeSH
- plazmidy genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- RNA interference MeSH
- rostlinné buňky metabolismus MeSH
- tabák cytologie MeSH
- zelené fluorescenční proteiny antagonisté a inhibitory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY's flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA-coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein's amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.
- MeSH
- kapsida chemie metabolismus MeSH
- konformace proteinů * MeSH
- molekulární modely * MeSH
- nemoci rostlin virologie MeSH
- Potyvirus fyziologie ultrastruktura MeSH
- RNA virová chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- virion MeSH
- virové plášťové proteiny chemie metabolismus MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The genus Bromovirus currently contains six species whose members have relatively narrow host ranges. In the present work, a new bromovirus infecting elderberry (Sambucus nigra L.) is reported. dsRNA was purified and sequenced by next-generation sequencing, and with minimal additional completion by Sanger sequencing, the full tripartite genome was obtained. RNA1 is 3241 nt long and contains ORF1 (1a protein), RNA2 is 2810 nt long and contains ORF2 (2a protein), and RNA3 is 2244 nt long and contains ORF3a (movement protein) and ORF3b (coat protein, CP), separated by an intercistronic poly(A) stretch. Proteins 1a and 2a showed highest sequence identity (69.9% and 69.4%) to the corresponding proteins of melandrium yellow fleck virus. The coat protein showed highest sequence identity (67.9%) to that of brome mosaic virus. The genome shows a typical bromovirus organisation comprising of all the conserved protein domains within the genus. Phylogenetic analysis supports the assignment of this virus as a new member of the genus Bromovirus, for which the name "sambucus virus S" (SVS) is proposed.
- MeSH
- bez černý virologie MeSH
- Bromovirus klasifikace genetika izolace a purifikace MeSH
- fylogeneze MeSH
- genom virový * MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin virologie MeSH
- otevřené čtecí rámce MeSH
- RNA virová genetika MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Plum pox virus (PPV, family Potyviridae) is one of the most important viral pathogens of Prunus spp. causing considerable damage to stone-fruit industry worldwide. Among the PPV strains identified so far, only PPV-C, PPV-CR, and PPV-CV are able to infect cherries under natural conditions. Herein, we evaluated the pathogenic potential of two viral isolates in herbaceous host Nicotiana benthamiana. Significantly higher accumulation of PPV capsid protein in tobacco leaves infected with PPV-CR (RU-30sc isolate) was detected in contrast to PPV-C (BY-101 isolate). This result correlated well with the symptoms observed in the infected plants. To further explore the host response upon viral infection at the molecular level, a comprehensive proteomic profiling was performed. Using reverse-phase ultra-high-performance liquid chromatography followed by label-free mass spectrometry quantification, we identified 38 unique plant proteins as significantly altered due to the infection. Notably, the abundances of photosynthesis-related proteins, mainly from the Calvin-Benson cycle, were found more aggressively affected in plants infected with PPV-CR isolate than those of PPV-C. This observation was accompanied by a significant reduction in the amount of photosynthetic pigments extracted from the leaves of PPV-CR infected plants. Shifts in the abundance of proteins that are involved in stimulation of photosynthetic capacity, modification of amino acid, and carbohydrate metabolism may affect plant growth and initiate energy formation via gluconeogenesis in PPV infected N. benthamiana. Furthermore, we suggest that the higher accumulation of H2O2 in PPV-CR infected leaves plays a crucial role in plant defense and development by activating the glutathione synthesis.
- MeSH
- chlorofyl biosyntéza MeSH
- chromatografie s reverzní fází MeSH
- energetický metabolismus genetika MeSH
- fotosyntéza genetika MeSH
- genotyp MeSH
- glutathion biosyntéza MeSH
- hmotnostní spektrometrie MeSH
- interakce hostitele a patogenu genetika MeSH
- karotenoidy biosyntéza MeSH
- listy rostlin genetika metabolismus virologie MeSH
- nemoci rostlin genetika virologie MeSH
- oxidace-redukce MeSH
- peroxid vodíku metabolismus MeSH
- proteiny teplotního šoku klasifikace genetika metabolismus MeSH
- Prunus avium virologie MeSH
- regulace genové exprese u rostlin * MeSH
- rostlinné proteiny klasifikace genetika metabolismus MeSH
- slivoň švestka virologie MeSH
- tabák genetika metabolismus virologie MeSH
- virus šarky švestky klasifikace genetika růst a vývoj patogenita MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pea seed-borne mosaic virus (PSbMV) significantly reduces yields in a broad spectra of legumes. The eukaryotic translation initiation factor has been shown to confer resistance to this pathogen, thus implying that translation and proteome dynamics play a role in resistance. This study presents the results of a proteome-wide analysis of Pisum sativum L. response to PSbMV infection. LC-MS profiling of two contrasting pea cultivars, resistant (B99) and susceptible (Raman) to PSbMV infection, detected >2300 proteins, 116 of which responded to PSbMV ten and/or twenty days post-inoculation. These differentially abundant proteins are involved in number of processes that have previously been reported in the plant-pathogen response, including protein and amino acid metabolism, stress signaling, redox homeostasis, carbohydrate metabolism, and lipid metabolism. We complemented our proteome-wide analysis work with targeted analyses of free amino acids and selected small molecules, fatty acid profiling, and enzyme activity assays. Data from these additional experiments support our findings and validate the biological relevance of the observed proteome changes. We found surprising similarities in the resistant and susceptible cultivars, which implies that a seemingly unaffected plant, with no detectable levels of PSbMV, actively suppresses viral replication. BIOLOGICAL SIGNIFICANCE: Plant resistance to PSbMV is connected to translation initiation factors, yet the processes involved are still poorly understood at the proteome level. To the best of our knowledge, this is the first survey of the global proteomic response to PSbMV in plants. The combination of label-free LC-MS profiling and two contrasting cultivars (resistant and susceptible) provided highly sensitive snapshots of protein abundance in response to PSbMV infection. PSbMV is a member of the largest family of plant viruses and our results are in accordance with previously characterized potyvirus-responsive proteomes. Hence, the results of this study can further extend our knowledge about these pathogens. We also show that even though no viral replication is detected in the PSbMV-resistant cultivar B99, it is still significantly affected by PSbMV inoculation.
- MeSH
- chromatografie kapalinová MeSH
- eukaryotické iniciační faktory MeSH
- hrách setý virologie MeSH
- interakce hostitele a patogenu * MeSH
- nemoci rostlin virologie MeSH
- odolnost vůči nemocem genetika MeSH
- Potyvirus patogenita MeSH
- proteomika metody MeSH
- rostlinné viry MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plum pox virus (PPV), the agent responsible for sharka disease, is the most important viral pathogen of stone fruit trees. The fruits of these fruit species are widely used in the processing industry, thus being economically very attractive. This viral disease significantly reduces the vitality of the fruit trees and the quantity and quality of fruits. The present review describes recent methods used for the identification and characterization of economically important Plum pox virus. Understanding the diversity of plant viruses is an essential step to design efficient management strategies to eliminate economical losses.
