The genus Armillaria (Basidiomycota, Agaricales, Physalacriaceae) comprises pathogenic fungi that cause root-rot disease in plants, as well as species with low pathogenicity, some of which are hosts of the fully mycoheterotrophic orchid plant Gastrodia elata (Orchidaceae). To investigate the mechanisms underlying such special interactions between Armillaria fungi and G. elata, it is crucial to establish genetic transformation platforms for the Armillaria fungi and G. elata. In this study, an Armillaria strain Arm37 was isolated from G. elata, which can form symbiosis with G. elata in axenic culture under laboratory conditions. A vector pYT-EV containing a cassette for hygromycin-resistance selection and a cassette for expressing or silencing target genes was constructed. An Agrobacterium tumefaciens-mediated transformation (ATMT) system for Arm37 was successfully developed and optimized to achieve a transformation efficiency of 32%. The ATMT system was successfully used to express the reporter genes eGFP encoding enhanced green fluorescent protein and GUS encoding β-glucuronidase and to effectively silence the endogenous gene URA3 encoding orotidine-5'-phosphate decarboxylase in Arm37. This ATMT system established for Arm37 provides an efficient genetic tool for exploring the Arm37 genes that are involved in the unique interaction between the Armillaria fungi and fully mycoheterotrophic plant G. elata.

• Klíčová slova
• Agrobacterium tumefaciens-mediated transformation, Armillaria, Gastrodia elata, Gene overexpression, RNA interference,
• Publikační typ
• časopisecké články MeSH

In recent years, there has been a growing interest in plant extracellular vesicles (pEVs) due to their immense potential for medical applications, particularly as carriers for drug delivery. To use the benefits of pEVs in the future, it is necessary to identify methods that facilitate their production in sufficient quantities while maintaining high quality. In this study, a comparative analysis of yields of tobacco pEV derived from apoplastic fluid, sterile calli, and suspension cultures, was performed to identify the most suitable plant material for vesicle isolation. Subsequent experiments focused on assessing the efficiency of small interfering RNA (siRNA) loading into callus-derived vesicles, employing various methods such as sonication, incubation, incubation supplemented with saponin, lipofection, and electroporation. Differences in loading efficiency among vesicles derived from apoplastic fluid, calli, and suspension cultures were observed. Moreover, our investigation extended to the presence of tobacco secondary metabolites, specifically anabasine and nicotine, within vesicles originating from three distinct tobacco sources. The outcomes of our study highlight variations not only in vesicle yields based on their source but also in their loadability and the presence of nicotine and anabasine. These findings contribute valuable insights into optimizing the production and application of pEVs for future medicinal purposes.

• MeSH
• buněčné kultury metody   MeSH
• extracelulární vezikuly * metabolismus   MeSH
• malá interferující RNA metabolismus   genetika   MeSH
• nikotin MeSH
• tabák * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• malá interferující RNA MeSH
• nikotin MeSH

The slug Arion vulgaris has attracted major attention as one of the worst invasive herbivore pests in Europe and is renowned for the stiff mucus it secretes for locomotion. In this study we focused on the isolation and characterisation of extracellular vesicles, specifically exosomes and exosome-like vesicles, from Arion secretions. We developed a method for slug mucus collection and subsequent vesicle isolation by ultracentrifugation. The isolated vesicles with an average diameter of ~ 100 nm carry abundant proteins and short RNAs, as well as adhesion molecules similar to mammalian galectins. We demonstrated that the slug extracellular vesicles are internalised by plant cells and human cancer cells in in vitro assays and are loadable by bioactive compounds, which makes them an interesting tool for utilisation in biotechnology.

• MeSH
• biotechnologie MeSH
• exozómy * metabolismus   MeSH
• hlen MeSH
• lidé MeSH
• plži * MeSH
• savci MeSH
• zavlečené druhy MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Plant extracellular vesicles (pEVs) derived from numerous edible sources gain a lot of attention in recent years, mainly due to the potential to efficiently carry bioactive molecules into mammalian cells. In the present study, we focus on isolation of PDNVs (plant-derived nanovesicles) and pEVs from callus culture and from BY-2 culture of Nicotiana tabacum (tobacco). Tobacco was selected as a source of plant vesicles, as it is commonly used by human, moreover it is a model organism with established techniques for cultivation of explant cultures in vitro. Explant cultures are suitable for the isolation of pEVs in large quantities, due to their fast growth in sterile conditions. As the efficiency of isolation methods varies, we were comparing two methods of isolation. We evaluated biophysical and biochemical properties of plant vesicles, as well as differences between isolates. We encountered difficulties in the form of vesicles aggregation, which is often described in publications focused on mammalian nanovesicles. In an effort to prevent vesicle aggregation, we used trehalose in different stages of isolation. We show tobacco-derived vesicles successfully enter tobacco and mesenchymal cell lines. We observed that tobacco-nanovesicles isolated by different methods incorporated fluorescent dye with different efficiency. The results of our study show tobacco-derived vesicles isolated by various isolation methods are able to enter plant, as well as mammalian cells.

• MeSH
• biologický transport MeSH
• extracelulární vezikuly * metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• mezenchymální kmenové buňky * MeSH
• rostliny metabolismus   MeSH
• savci MeSH
• tabák MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA interference (RNAi) mechanism has become one of the main areas of focus in plant-fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the V. nonalfalfae species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated V. nonalfalfae genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine V. nonalfalfae milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5' RLM-RACE approach.

• Klíčová slova
• RNA interference, Verticillium nonalfalfae, fungal pathogen, microRNA-like RNAs, plant-pathogen interactions,
• MeSH
• Ascomycota genetika   MeSH
• fungální RNA * MeSH
• fylogeneze MeSH
• genová ontologie MeSH
• interakce hostitele a patogenu MeSH
• konformace nukleové kyseliny MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• malá nekódující RNA genetika   MeSH
• mikro RNA genetika   MeSH
• nemoci rostlin mikrobiologie   MeSH
• regulace genové exprese u hub MeSH
• reprodukovatelnost výsledků MeSH
• stanovení celkové genové exprese MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fungální RNA * MeSH
• malá nekódující RNA MeSH
• mikro RNA MeSH

Communication shapes life on Earth. Transference of information has played a paramount role on the evolution of all living or extinct organisms since the appearance of life. Success or failure in this process will determine the prevalence or disappearance of a certain set of genes, the basis of Darwinian paradigm. Among different molecules used for transmission or reception of information, RNA plays a key role. For instance, the early precursors of life were information molecules based in primitive RNA forms. A growing field of research has focused on the contribution of small non-coding RNA forms due to its role on infectious diseases. These are short RNA species that carry out regulatory tasks in cis or trans. Small RNAs have shown their relevance in fine tuning the expression and activity of important regulators of essential genes for bacteria. Regulation of targets occurs through a plethora of mechanisms, including mRNA stabilization/destabilization, driving target mRNAs to degradation, or direct binding to regulatory proteins. Different studies have been conducted during the interplay of pathogenic bacteria with several hosts, including humans, animals, or plants. The sRNAs help the invader to quickly adapt to the change in environmental conditions when it enters in the host, or passes to a free state. The adaptation is achieved by direct targeting of the pathogen genes, or subversion of the host immune system. Pathogens trigger also an immune response in the host, which has been shown as well to be regulated by a wide range of sRNAs. This review focuses on the most recent host-pathogen interaction studies during bacterial infectious diseases, providing the perspective of the pathogen.

• Klíčová slova
• bacteria, host-pathogen interaction, infectious disease, information transfer, small RNA,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH