Elicitation of Hyoscyamine Production in Datura stramonium L. Plants Using Tobamoviruses.

Datura stramonium L. produces tropane alkaloids, and the hyoscyamine is dominant among them. Hyoscyamine is produced by hairy root cultures in vitro derived from native plants or plants with the genetically modified biosynthetic pathway for hyoscyamine. A common procedure is extraction from cultivated plants. Elicitors for increased production can be used in both cases. Live viruses are not well known for use as elicitors, therefore, D. stramonium plants grown in soil were artificially infected with the tobamoviruses Pepper mild mottle virus (PMMoV), Tomato mosaic virus (ToMV), and Tobacco mosaic virus (TMV). Differences in the content of hyoscyamine were between capsules and roots of infected and non-infected plants. Elicitation increased content of hyoscyamine in capsules 1.23-2.34 times, compared to the control. The most effective viruses were PMMoV and ToMV (isolate PV143), which increased content to above 19 mg/g of fresh weight of a capsule. The effect of each virus elicitor was expressed also in hyoscyamine content in roots. Elicited plants contained 5.41-16.54 times more hyoscyamine in roots compared to non-elicited plants. The most effective elicitor was ToMV SL-1, which raised production above 20 mg/g fresh weight of roots. It has been shown that tobamoviruses can be used as biotic elicitors.

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.

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.

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.

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.