Clematis vitalba Is a Natural Host of the Novel Ilarvirus, Prunus Virus I.

Clematis vitalba L. is a climbing shrub and a pioneer plant in abandoned orchards or vineyards that are widespread in temperate climate zones. In past years, several viruses infecting the Clematis species have been identified, including different ilarviruses. Prunus virus I (PrVI) is a recently described ilarvirus, which has been shown to infect sweet cherries and peaches in Greece. Moreover, its presence has been detected in ornamental Clematis in Russia. In the present work, we analyzed the virome of wildly growing C. vitalba plants from Hungary, Slovakia and Croatia showing different kinds of symptoms using high-throughput sequencing (HTS) of small RNAs or ribodepleted RNAs. Applying HTS enabled us to identify the presence of PrVI in C. vitalba, and the bioinformatic analyses were further validated with RT-PCR using PrVI-specific primers and Sanger dideoxy sequencing. Nearly full genome sequences of all three viral RNAs of one Hungarian, two Slovak and one Croatian isolate were determined. Their phylogenetic analysis showed high similarity to each other and to other PrVI isolates described from Central Europe. As the sampled plants were co-infected with other viruses, it is not possible to determine a direct correlation between the infection with PrVI and the observed symptoms. Analyses of different Prunus species in stock collection showed infection of several peach and sweet cherry varieties in Hungary. Our results expand the knowledge on the natural host range of PrVI and highlight the necessity to evaluate alternative plant hosts (even non-Prunus) of PrVI and the role of the virus in the etiology of the potential diseases.

Molecular and Biological Characterization of Novel and Known Family Secoviridae Members Infecting Lettuce.

High-throughput sequencing of two lettuces showing virus-like symptoms in France provided evidence of infection by members of the family Secoviridae. One plant (JG1) had a complex mixed infection that involved, among others, a novel waikavirus (lettuce waikavirus 1) and two isolates of a sequivirus related to lettuce mottle virus (LeMoV). The second lettuce plant (JG2) was singly infected by LeMoV. Complete genomic sequences were obtained for all four isolates and, in addition, near complete genome sequences were obtained for other LeMoV or LeMoV-related isolates (from French cultivated and wild lettuces and from a Brazilian cultivated lettuce) and for two isolates of another family Asteraceae-infecting sequivirus, dandelion yellow mosaic virus (DaYMV). Analysis of these genomic sequences allows the proposal of tentative genome organization for the various viruses and clarification of their phylogenetic relationships. Sequence and host range comparisons point to significant differences between the two sequivirus isolates identified in the JG1 plant and LeMoV isolates from France and Brazil, suggesting they belong to a novel species for which the name lettuce star mosaic virus is proposed.

Analysis of Hop Stunt Viroid Diversity in Grapevine (Vitis vinifera L.) in Slovakia: Coexistence of Two Particular Genetic Groups.

The hop stunt viroid (HSVd) is a widespread subviral pathogen infecting a broad spectrum of plant hosts including grapevine (Vitis vinifera L.). Despite its omnipresence in virtually all grapevine growing areas around the world, molecular data characterizing HSVd populations are missing from Slovakia. Analysis of the complete nucleotide sequences of 19 grapevine variants revealed the existence of two genetic HSVd groups in Slovakia (internally named the "6A" and "7A" groups based on the particular stretch of adenines at nucleotide positions 39-44/45, respectively). Despite their sampling at different times in various unrelated vineyards, the 6A and 7A groups are characterized by low intra-group divergence (~0.3 and 0.2%, respectively). On the other hand, inter-group divergence reached 2.2% due to several mutations, seven of which were found to be group-specific and mainly (except for one) located in the region of the pathogenic domain. Interestingly, in addition to their frequent co-existence within the same geographical location, the mixed infection of the 6A and 7A type sequence variants was also unequivocally and repeatedly proven within single grapevine plants. The RNA secondary structure analysis of representative isolates from each of these two genetic groups indicated a potential compensatory explanation of such mutations. These group-specific sites could be pointing towards the evolutionary selection linked to the necessity of the viroid to retain its structural conformational integrity, crucial for its functional biochemical ability to interact with specific grapevine cellular host factors required for HSVd propagation.

A Novel and Highly Inclusive Quantitative Real-Time RT-PCR Method for the Broad and Efficient Detection of Grapevine Leafroll-Associated Virus 1.

Grapevine (Vitis vinifera L.) is one of the most important crops in the world due to its economic and social impact. Like many other crops, grapevine is susceptible to different types of diseases caused by pathogenic microorganisms. Grapevine leafroll-associated virus 1 (GLRaV-1) is a virus associated with grapevine leafroll disease and it is considered at the national and European level as a pathogen that must be absent in propagative plant material. For this reason, the availability of specific, sensitive and reliable detection techniques to ascertain the sanitary status of the plants is of great importance. The objective of this research was the development of a new GLRaV-1 detection method based on a TaqMan quantitative real-time RT-PCR targeted to the coat protein genomic region and including a host internal control in a duplex reaction. To this end, three new GLRaV-1 full genomes were recovered by HTS and aligned with all sequences available in the databases. The method has been validated following EPPO standards and applied for the diagnosis of field plant material and transmission vectors. The new protocol designed has turned out to be highly sensitive as well as much more specific than the current available methods for the detection and absolute quantitation of GLRaV-1 viral titer.

The Expanding Menagerie of Prunus-Infecting Luteoviruses.

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.

Weed Hosts Represent an Important Reservoir of Turnip Yellows Virus and a Possible Source of Virus Introduction into Oilseed Rape Crop.

Turnip yellows virus (TuYV) is one of the most important pathogens of oilseed rape worldwide. The virus has a large host range including many crop species (e.g., oilseed rape, pea, chickpea) and weeds from more than twenty plant families. Other than oilseed rape, we detected TuYV in many commonly grown weed species that share the fields and vegetation period together with canola crops in Czech and Slovak Republics. TuYV was detected by reverse-transcription polymerase chain reaction (RT-PCR) in at least 26 species including main crop hosts (oilseed rape), intercrops and weeds such as Amaranthus retroflexus, Atriplex patula (Amaranthaceae), Arctium lappa, Lactuca serriola, Taraxacum officinale, Tripleurospermum inodorum (Asteraceae), Phacelia tanacetifolia (Boraginaceae), Brassica napus, Capsella bursa-pastoris, Descurainia Sophia, Raphanus raphanistrum, Sinapis alba, Sisymbrium officinale, Thlaspi arvense (Brassicaceae), Silene alba, Stellaria media (Caryophyllaceae), Euphorbia helioscopia (Euphorbiaceae), Geranium rotundifolium (Geraniaceae), Lamium purpureum (Lamiaceae), Fumaria officinalis, Papaver rhoeas (Papaveraceae), Veronica persica (Plantaginaceae syn. Scrophulariaceae), Fallopia convolvulus (Polygonaceae), Solanum nigrum (Solanaceae), Urtica dioica (Urticaceae) and Viola arvensis (Violaceae). The detection of TuYV was further confirmed by RT-qPCR as well as Sanger sequencing of the PCR fragments. We discovered four new weed species as hosts of TuYV such as T. inodorum, S. alba, G. rotundifolium and E. helioscopia, representing their three respective plant families. The readthrough domain (RTD) gene sequence analysis of the Czech and Slovak TuYV isolates from oilseed rape and weed species showed similar within-group nucleotide divergence (7.1% and 5.6%, respectively) and the absence of geographical- or host-based phylogenetic clustering. The high-throughput sequencing of the P. rhoeas sample enabled the obtention of a nearly complete genome of TuYV and revealed the mixed infection of TuYV with turnip mosaic virus and cucumber mosaic virus. Our results thus show that weed species are an important TuYV reservoir and play a significant role in the spread and incidence of the disease in field crops such as oilseed rape.

High-Throughput Sequencing Discloses the Cucumber Mosaic Virus (CMV) Diversity in Slovakia and Reveals New Hosts of CMV from the Papaveraceae Family.

Cucumber mosaic virus (CMV; Cucumovirus, Bromoviridae) is an omnipresent virus characterized by a large host range and high genetic variability. Using high-throughput sequencing, we have characterized near complete genomes of 14 Slovak CMV variants from different plant hosts. Of these, three variants originated from the Papaveraceae species (oilseed poppy, common poppy and great celandine), previously poorly described as CMV natural hosts. Based on a BLAST search and phylogenetic analysis, the Slovak CMV isolates can be divided into two genetically different Groups, Ia and II, respectively. The SL50V variant, characterized by a divergent RNA2 sequence, potentially represents a reassortant variant. In four samples (T101, SL50V, CP2, MVU2-21), the presence of satellite CMV RNA was identified along with CMV. Although mechanically transmitted to experimental cucumber plants, the role of satellite RNA in the symptomatology observed could not be established due to a complex infection of original hosts with different viruses.

Evaluation of New Polyclonal Antibody Developed for Serological Diagnostics of Tomato Mosaic Virus.

Plant viruses threaten agricultural production by reducing the yield, quality, and economical benefits. Tomato mosaic virus (ToMV) from the genus Tobamovirus causes serious losses in the quantity and quality of tomato production. The management of plant protection is very difficult, mainly due to the vector-less transmission of ToMV. Resistant breeding generally has low effectiveness. The most practical approach is the use of a rapid diagnostic assay of the virus' presence before the symptoms occur in plants, followed by the eradication of virus-infected plants. Such approaches also include serological detection methods (ELISA and Western immunoblotting), where antibodies need to be developed for an immunochemical reaction. The development and characterization of polyclonal antibodies for the detection of ToMV with appropriate parameters (sensitivity, specificity, and cross-reactivity) were the subjects of this study. A new polyclonal antibody, AB-1, was developed in immunized rabbits using the modified oligopeptides with antigenic potential (sequences are revealed) derived from the coat protein of ToMV SL-1. the developed polyclonal antibody. AB-1, showed higher sensitivity when compared with commercially available analogs. It also detected ToMV in infected pepper and eggplant plants, and detected another two tobamoviruses (TMV and PMMoV) and ToMV in soil rhizosphere samples and root residues, even two years after the cultivation of the infected tomato plant.

Suitability of different plant species for experimental agroinfection with Plum pox virus-based expression vector for potential production of edible vaccines.

Nine herbaceous plant species were tested for susceptibility to Plum pox virus (PPV) by Agrobacterium-mediated delivery of its infectious cDNA clone. Two of them became infected, namely spinach (local infection) and oilseed poppy (systemic infection). As a control, PPV infection was successfully established in plum seedlings following agroinfiltration, thus providing the first report of agroinfection in Prunus species. According to our results, oilseed poppy can be considered as a candidate host for the production of edible vaccines by a PPV-derived expression vector. Keywords: agroinfiltration; virus host; poppy; spinach.

Molecular Characterization of Potato Virus Y (PVY) Using High-Throughput Sequencing: Constraints on Full Genome Reconstructions Imposed by Mixed Infection Involving Recombinant PVY Strains.

In recent years, high throughput sequencing (HTS) has brought new possibilities to the study of the diversity and complexity of plant viromes. Mixed infection of a single plant with several viruses is frequently observed in such studies. We analyzed the virome of 10 tomato and sweet pepper samples from Slovakia, all showing the presence of potato virus Y (PVY) infection. Most datasets allow the determination of the nearly complete sequence of a single-variant PVY genome, belonging to one of the PVY recombinant strains (N-Wi, NTNa, or NTNb). However, in three to-mato samples (T1, T40, and T62) the presence of N-type and O-type sequences spanning the same genome region was documented, indicative of mixed infections involving different PVY strains variants, hampering the automated assembly of PVY genomes present in the sample. The N- and O-type in silico data were further confirmed by specific RT-PCR assays targeting UTR-P1 and NIa genomic parts. Although full genomes could not be de novo assembled directly in this situation, their deep coverage by relatively long paired reads allowed their manual re-assembly using very stringent mapping parameters. These results highlight the complexity of PVY infection of some host plants and the challenges that can be met when trying to precisely identify the PVY isolates involved in mixed infection.

Efficient Confirmation of Plant Viral Proteins and Identification of Specific Viral Strains by nanoLC-ESI-Q-TOF Using Single-Leaf-Tissue Samples.

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456.

Diacylglycerol Acetyltransferase Gene Isolated from Euonymus europaeus L. Altered Lipid Metabolism in Transgenic Plant towards the Production of Acetylated Triacylglycerols.

Euonymus species from the Celastraceae family are considered as a source of unusual genes modifying the oil content and fatty acid composition of vegetable oils. Due to the possession of genes encoding enzyme diacylglycerol acetyltransferase (DAcT), Euonymus plants can synthesize and accumulate acetylated triacyglycerols. The gene from Euonymus europaeus (EeDAcT) encoding the DAcT was identified, isolated, characterized, and modified for cloning and genetic transformation of plants. This gene has a unique nucleotide sequence and amino acid composition, different from orthologous genes from other Euonymus species. Nucleotide sequence of original EeDAcT gene was modified, cloned into transformation vector, and introduced into tobacco plants. Overexpression of EeDAcT gene was confirmed, and transgenic host plants produced and accumulated acetylated triacylglycerols (TAGs) in immature seeds. Individual transgenic plants showed difference in amounts of synthesized acetylTAGs and also in fatty acid composition of acetylTAGs.

Plant Viruses Infecting Solanaceae Family Members in the Cultivated and Wild Environments: A Review.

Plant viruses infecting crop species are causing long-lasting economic losses and are endangering food security worldwide. Ongoing events, such as climate change, changes in agricultural practices, globalization of markets or changes in plant virus vector populations, are affecting plant virus life cycles. Because farmer's fields are part of the larger environment, the role of wild plant species in plant virus life cycles can provide information about underlying processes during virus transmission and spread. This review focuses on the Solanaceae family, which contains thousands of species growing all around the world, including crop species, wild flora and model plants for genetic research. In a first part, we analyze various viruses infecting Solanaceae plants across the agro-ecological interface, emphasizing the important role of virus interactions between the cultivated and wild zones as global changes affect these environments on both local and global scales. To cope with these changes, it is necessary to adjust prophylactic protection measures and diagnostic methods. As illustrated in the second part, a complex virus research at the landscape level is necessary to obtain relevant data, which could be overwhelming. Based on evidence from previous studies we conclude that Solanaceae plant communities can be targeted to address complete life cycles of viruses with different life strategies within the agro-ecological interface. Data obtained from such research could then be used to improve plant protection methods by taking into consideration environmental factors that are impacting the life cycles of plant viruses.

Comparative Transcriptome Analysis of Two Cucumber Cultivars with Different Sensitivity to Cucumber Mosaic Virus Infection.

Cucumber mosaic virus (CMV), with extremely broad host range including both monocots and dicots around the world, belongs to most important viral crop threats. Either natural or genetically constructed sources of resistance are being intensively investigated; for this purpose, exhaustive knowledge of molecular virus-host interaction during compatible and incompatible infection is required. New technologies and computer-based "omics" on various levels contribute markedly to this topic. In this work, two cucumber cultivars with different response to CMV challenge were tested, i.e., sensitive cv. Vanda and resistant cv. Heliana. The transcriptomes were prepared from both cultivars at 18 days after CMV or mock inoculation. Subsequently, four independent comparative analyses of obtained data were performed, viz. mock- and CMV-inoculated samples within each cultivar, samples from mock-inoculated cultivars to each other and samples from virus-inoculated cultivars to each other. A detailed picture of CMV-influenced genes, as well as constitutive differences in cultivar-specific gene expression was obtained. The compatible CMV infection of cv. Vanda caused downregulation of genes involved in photosynthesis, and induction of genes connected with protein production and modification, as well as components of signaling pathways. CMV challenge caused practically no change in the transcription profile of the cv. Heliana. The main differences between constitutive transcription activity of the two cultivars relied in the expression of genes responsible for methylation, phosphorylation, cell wall organization and carbohydrate metabolism (prevailing in cv. Heliana), or chromosome condensation and glucan biosynthesis (prevailing in cv. Vanda). Involvement of several genes in the resistant cucumber phenotype was predicted; this can be after biological confirmation potentially applied in breeding programs for virus-resistant crops.

The global phylogeny of Plum pox virus is emerging.

The 206 complete genomic sequences of Plum pox virus in GenBank (January 2019) were downloaded. Their main open reading frames (ORF)s were compared by phylogenetic and population genetic methods. All fell into the nine previously recognized strain clusters; the PPV-Rec and PPV-T strain ORFs were all recombinants, whereas most of those in the PPV-C, PPV-CR, PPV-CV, PPV-D, PPV-EA, PPV-M and PPV-W strain clusters were not. The strain clusters ranged in size from 2 (PPV-CV and PPV-EA) to 74 (PPV-D). The isolates of eight of the nine strains came solely from Europe and the Levant (with an exception resulting from a quarantine breach), but many PPV-D strain isolates also came from east and south Asia and the Americas. The estimated time to the most recent common ancestor (TMRCA) of all 134 non-recombinant ORFs was 820 (865-775) BCE. Most strain populations were only a few decades old, and had small intra-strain, but large inter-strain, differences; strain PPV-W was the oldest. Eurasia is clearly the 'centre of emergence' of PPV and the several PPV-D strain populations found elsewhere only show evidence of gene flow with Europe, so have come from separate introductions from Europe. All ORFs and their individual genes show evidence of strong negative selection, except the positively selected pipo gene of the recently migrant populations. The possible ancient origins of PPV are discussed.

High-Throughput Sequencing Reveals Bell Pepper Endornavirus Infection in Pepper (Capsicum annum) in Slovakia and Enables Its Further Molecular Characterization.

Ribosomal RNA-depleted total RNAs from a sweet pepper plant (Capsicum annuum, labelled as N65) grown in western Slovakia and showing severe virus-like symptoms (chlorosis, mottling and deformation of leaf lamina) were subjected to high-throughput sequencing (HTS) on an Illumina MiSeq platform. The de novo assembly of ca. 5.5 million reads, followed by mapping to the reference sequences, revealed the coinfection of pepper by several viruses; i.e., cucumber mosaic virus (CMV), watermelon mosaic virus (WMV), pepper cryptic virus 2 (PCV2) and bell pepper endornavirus (BPEV). A complete polyprotein-coding genomic sequence (14.6 kb) of BPEV isolate N65 was determined. A comparison of BPEV-N65 sequences with BPEV genomes available in GenBank showed 86.1% to 98.6% identity at the nucleotide level. The close phylogenetic relationship with isolates from India and China resulted in their distinct grouping compared to the other BPEV isolates. Further analysis has revealed the presence of BPEV in sweet or chili peppers obtained from various sources and locations in Slovakia (plants grown in gardens, greenhouse or retail shop). Additionally, the partial sequencing of two genomic portions from 15 BPEV isolates revealed that the Slovak isolates segregated into two molecular clusters, indicating a genetically distinct population (mean inter-group nucleotide divergence reaching 12.7% and 14.5%, respectively, based on the genomic region targeted). Due to the mix infections of BPEV-positive peppers by potato virus Y (PVY) and/or CMV, the potential role of individual viruses in the observed symptomatology could not be determined. This is the first evidence and characterization of BPEV from the central European region.

Grapevine virus T diversity as revealed by full-length genome sequences assembled from high-throughput sequence data.

RNASeq or double-stranded RNA based approaches allowed the reconstruction of a total of 9 full-length or near full-length genomes of the recently discovered grapevine virus T (GVT). In addition, datamining of publicly available grapevine RNASeq transcriptome data allowed the reconstruction of a further 14 GVT genomes from five grapevine sources. Together with four GVT sequences available in Genbank, these novel sequences were used to analyse GVT diversity. GVT shows a very limited amount of indels variation but a high level of nucleotide and aminoacid polymorphism. This level is comparable to that shown in the closely related grapevine rupestris stem pitting-associated virus (GRSPaV). Further analyses showed that GVT mostly evolves under conservative selection pressure and that recombination has contributed to its evolutionary history. Phylogenetic analyses allow to identify at least seven clearly separated groups of GVT isolates. Analysis of the only reported PCR GVT-specific detection primer pair indicates that it is likely to fail to amplify some GVT isolates. Taken together these results point at the distinctiveness of GVT but also at the many points it shares with GRSPaV. They constitute the first pan-genomic analysis of the diversity of this novel virus.

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.

Molecular and Biological Characterisation of Turnip mosaic virus Isolates Infecting Poppy (Papaversomniferum and P. rhoeas) in Slovakia.

In recent years, the accumulated molecular data of Turnip mosaic virus (TuMV) isolates from various hosts originating from different parts of the world considerably helped to understand the genetic complexity and evolutionary history of the virus. In this work, four complete TuMV genomes (HC9, PK1, MS04, MS15) were characterised from naturally infected cultivated and wild-growing Papaver spp., hosts from which only very scarce data were available previously. Phylogenetic analyses showed the affiliation of Slovak Papaver isolates to the world-B and basal-B groups. The PK1 isolate showed a novel intra-lineage recombination pattern, further confirming the important role of recombination in the shaping of TuMV genetic diversity. Biological assays indicated that the intensity of symptoms in experimentally inoculated oilseed poppy are correlated to TuMV accumulation level in leaves. This is the first report of TuMV in poppy plants in Slovakia.

Photosynthetic and Stress Responsive Proteins Are Altered More Effectively in Nicotiana benthamiana Infected with Plum pox virus Aggressive PPV-CR versus Mild PPV-C Cherry-Adapted Isolates.

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.