1001. A framework for the evaluation of biosecurity, commercial, regulatory, and scientific impacts of plant viruses and viroids identified by NGS technologiesSébastien Massart, Thierry Candresse, José Gil, Christophe Lacomme, Lukas Predajna, Maja Ravnikar, Jean-Sébastien Reynard, Artemis Rumbou, Pasquale Saldarelli, Dijana Škorić, Eeva J. Vainio, Jari P. T. Valkonen, Hervé Vanderschuren, Christina Varveri, Thierry Wetzel, 2017, original scientific article Abstract: Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.
Keywords: NGS, pest risk analysis, virus diseases, biological characterization, plant health, regulatory agencies
Published in DiRROS: 25.07.2024; Views: 287; Downloads: 155
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1002. Salicylic acid perturbs sRNA-gibberellin regulatory network in immune response of potato to Potato virus Y infectionMaja Križnik, Marko Petek, David Dobnik, Živa Ramšak, Špela Baebler, Stephan Pollmann, Jan F. Kreuze, Jana Žel, Kristina Gruden, 2017, original scientific article Abstract: Potato virus Y is the most economically important potato viral pathogen. We aimed at unraveling the roles of small RNAs (sRNAs) in the complex immune signaling network controlling the establishment of tolerant response of potato cv. Désirée to the virus. We constructed a sRNA regulatory network connecting sRNAs and their targets to link sRNA level responses to physiological processes. We discovered an interesting novel sRNAs-gibberellin regulatory circuit being activated as early as 3 days post inoculation (dpi) before viral multiplication can be detected. Two endogenous sRNAs, miR167 and phasiRNA931 were predicted to regulate gibberellin biosynthesis genes GA20-oxidase and GA3-oxidase. The increased expression of phasiRNA931 was also reflected in decreased levels of GA3-oxidase transcripts. Moreover, decreased concentration of gibberellin confirmed this regulation. The functional relation between lower activity of gibberellin signaling and reduced disease severity was previously confirmed in Arabidopsis-virus interaction using knockout mutants. We further showed that this regulation is salicylic acid-dependent as the response of sRNA network was attenuated in salicylic acid-depleted transgenic counterpart NahG-Désirée expressing severe disease symptoms. Besides downregulation of gibberellin signaling, regulation of immune receptor transcripts by miR6022 as well as upregulation of miR164, miR167, miR169, miR171, miR319, miR390, and miR393 in tolerant Désirée, revealed striking similarities to responses observed in mutualistic symbiotic interactions. The intertwining of different regulatory networks revealed, shows how developmental signaling, disease symptom development, and stress signaling can be balanced.
Keywords: gibberellin, miRNA/siRNA, plant immunity, potato, Potato virus Y, salicylic acid, symbiosis, tolerance
Published in DiRROS: 25.07.2024; Views: 353; Downloads: 174
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1003. Olfactory signaling of aggressive intent in male-male contests of cave crickets (Troglophilus neglectus; Orthoptera: Rhaphidophoridae)Nataša Stritih Peljhan, Alenka Žunič Kosi, 2017, original scientific article Abstract: In animal contests, communicating aggressive motivation is most often mediated by visual or acoustic signals, while chemical signals are not expected to serve such a function since they are less able to be modulated by the sender during the changing behavioral context. We describe a rare example of ephemeral olfactory signals in terrestrial animals, signals that are emitted via protrusive scent glands in male cave crickets Troglophilus neglectus (Orthoptera, Rhaphidophoridae) to reflect the state of the signaler’s aggression. We correlate the intensity of behaviorally expressed aggression of the individuals in dyadic contests with the frequency and extent of their gland tissue protrusion, the latter serving as an indication of the amount of released odor. We detected large amounts of odor release during brief gland protrusions, and the absence of its release during gland retraction. Males protruded the glands during and after encountering a rival, with the degree of protrusion increasing with the intensity of the signalers’ aggression. During the encounters, the degree of gland protrusion increased most strongly with the occurrence of the elevated body posture, directly preceding the attack. This degree was significantly higher in encounter winners than in losers displaying such posture, suggesting the highly important role of the released odor for contest resolution. After the encounters, glands were protruded almost exclusively by winners, apparently announcing victory. We tested for the function of the olfactory signals also directly, by preventing gland tissue protrusion in symmetric and asymmetric treatments of the contestants. Treating only the dominant individuals decreased the percentage of encounters they won by over 60%, while treating both contestants elicited a significant increase in the frequency and duration of fights. During contests, the olfactory signals of T. neglectus apparently function as a highly effective threat, which prevents maximal contest escalation and decreases the conflict-related costs.
Keywords: animal behaviour, bioacoustics, aggression
Published in DiRROS: 25.07.2024; Views: 283; Downloads: 189
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1004. ALF : a strategy for identification of unauthorized GMOs in complex mixtures by a GW-NGS method and dedicated bioinformatics analysisAlexandra Bogožalec Košir, Alfred J. Arulandhu, Marleen Voorhuijzen, Hongmei Xiao, Rico Hagelaar, Martijn Staats, Adalberto Costessi, Jana Žel, Esther Kok, Jeroen P. van Dijk, 2017, original scientific article Abstract: The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). In parallel, the number of reports of unauthorised GMOs (UGMOs) is gradually increasing. There is a lack of specific detection methods for UGMOs, due to the absence of detailed sequence information and reference materials. In this research, an adapted genome walking approach was developed, called ALF: Amplification of Linearly-enriched Fragments. Coupling of ALF to NGS aims for simultaneous detection and identification of all GMOs, including UGMOs, in one sample, in a single analysis. The ALF approach was assessed on a mixture made of DNA extracts from four reference materials, in an uneven distribution, mimicking a real life situation. The complete insert and genomic flanking regions were known for three of the included GMO events, while for MON15985 only partial sequence information was available. Combined with a known organisation of elements, this GMO served as a model for a UGMO. We successfully identified sequences matching with this organisation of elements serving as proof of principle for ALF as new UGMO detection strategy. Additionally, this study provides a first outline of an automated, web-based analysis pipeline for identification of UGMOs containing known GM elements.
Keywords: biotechnology, molecular biology
Published in DiRROS: 25.07.2024; Views: 245; Downloads: 211
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1005. Next generation sequencing for detection and discovery of plant viruses and viroids : comparison of two approachesAnja Pecman, Denis Kutnjak, Ion Gutiérrez-Aguirre, Ian Adams, Adrian Fox, Neil Boonham, Maja Ravnikar, 2017, original scientific article Abstract: Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of two RNA sequence inputs (small RNAs vs. ribosomal RNA depleted total RNA) for the detection of plant viruses by Illumina sequencing. This comparison includes several viruses, which differ in genome organization and viroids from both known families. The results demonstrate the ability for detection and identification of a wide array of known plant viruses/viroids in the tested samples by both approaches. In general, yield of viral sequences was dependent on viral genome organization and the amount of viral reads in the data. A putative novel Cytorhabdovirus, discovered in this study, was only detected by analysing the data generated from ribosomal RNA depleted total RNA and not from the small RNA dataset, due to the low number of short reads in the latter. On the other hand, for the viruses/viroids under study, the results showed higher yields of viral sequences in small RNA pool for viroids and viruses with no RNA replicative intermediates (single stranded DNA viruses).
Keywords: next generation sequencing, small RNA, ribosomal RNA depleted total RNA, detection, plant viruses, plant viroids
Published in DiRROS: 25.07.2024; Views: 290; Downloads: 182
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1006. Newly isolated bacteriophages from the Podoviridae, Siphoviridae, and Myoviridae families have variable effects on putative novel Dickeya spp.Špela Alič, Tina Mikuletič, Magda Tušek-Žnidarič, Maja Ravnikar, Nejc Rački, Matjaž Peterka, Tanja Dreo, 2017, original scientific article Abstract: Soft rot pathogenic bacteria from the genus Dickeya cause severe economic losses in orchid nurseries worldwide, and there is no effective control currently available. In the last decade, the genus Dickeya has undergone multiple changes as multiple new taxa have been described, and just recently a new putative Dickeya species was reported. This study reports the isolation of three bacteriophages active against putative novel Dickeya spp. isolates from commercially produced infected orchids that show variable host-range profiles. Bacteriophages were isolated through enrichment from Dickeya-infected orchid tissue. Convective interaction media monolith chromatography was used to isolate bacteriophages from wastewaters, demonstrating its suitability for the isolation of infective bacteriophages from natural sources. Based on bacteriophage morphology, all isolated bacteriophages were classified as being in the order Caudovirales, belonging to three different families, Podoviridae, Myoviridae, and Siphoviridae. The presence of three different groups of bacteriophages was confirmed by analyzing the bacteriophage specificity of bacterial hosts, restriction fragment length polymorphism and plaque morphology. Bacteriophage BF25/12, the first reported Podoviridae bacteriophage effective against Dickeya spp., was selected for further characterization. Its genome sequence determined by next-generation sequencing showed limited similarity to other characterized Podoviridae bacteriophages. Interactions among the bacteriophages and Dickeya spp. were examined using transmission electron microscopy, which revealed degradation of electron-dense granules in response to bacteriophage infection in some Dickeya strains. The temperature stability of the chosen Podoviridae bacteriophage monitored over 1 year showed a substantial decrease in the survival of bacteriophages stored at -20∘C over longer periods. It showed susceptibility to low pH and UV radiation but was stable in neutral and alkaline pH. Furthermore, the stability of the tested bacteriophage was also connected to the incubation medium and bacteriophage concentration at certain pH values. Finally, the emergence of bacteriophage-resistant bacterial colonies is highly connected to the concentration of bacteriophages in the bacterial environment. This is the first report on bacteriophages against Dickeya from the Podoviridae family to expand on potential bacteriophages to include in bacteriophage cocktails as biocontrol agents. Some of these bacteriophage isolates also showed activity against Dickeya solani, an aggressive strain that causes the soft rot of potatoes, which indicates their broad potential as biocontrol agents.
Keywords: bacteriophages, genome sequencing, resistance development, convective interaction media monolith chromatography
Published in DiRROS: 25.07.2024; Views: 309; Downloads: 218
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1007. Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCRAlexandra Bogožalec Košir, Carla Divieto, Jernej Pavšič, Stefano Pavarelli, David Dobnik, Tanja Dreo, Roberto Bellotti, Maria Paola Sassi, Jana Žel, 2017, original scientific article Abstract: Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.
Keywords: droplet digital PCR, droplet volume, DNA quantification, optical microscopy imaging
Published in DiRROS: 25.07.2024; Views: 306; Downloads: 235
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1008. RECQ1 helicase silencing decreases the tumour growth rate of U87 glioblastoma cell xenografts in zebrafish embryosMiloš Vittori, Barbara Breznik, Katja Hrovat, Saša Kenig, Tamara Lah Turnšek, 2017, original scientific article Abstract: RECQ1 helicase has multiple roles in DNA replication, including restoration of the replication fork and DNA repair, and plays an important role in tumour progression. Its expression is highly elevated in glioblastoma as compared to healthy brain tissue. We studied the effects of small hairpin RNA (shRNA)-induced silencing of RECQ1 helicase on the increase in cell number and the invasion of U87 glioblastoma cells. RECQ1 silencing reduced the rate of increase in the number of U87 cells by 30%. This corresponded with a 40% reduction of the percentage of cells in the G2 phase of the cell cycle, and an accumulation of cells in the G1 phase. These effects were confirmed in vivo, in the brain of zebrafish (Danio rerio) embryos, by implanting DsRed-labelled RECQ1 helicase-silenced and control U87 cells. The growth of resulting tumours was quantified by monitoring the increase in xenograft fluorescence intensity during a three-day period with fluorescence microscopy. The reduced rate of tumour growth, by approximately 30% in RECQ1 helicase-silenced cells, was in line with in vitro measurements of the increase in cell number upon RECQ1 helicase silencing. However, RECQ1 helicase silencing did not affect invasive behaviour of U87 cells in the zebrafish brain. This is the first in vivo confirmation that RECQ1 helicase is a promising molecular target in the treatment of glioblastoma.
Keywords: cancer, cell cycle, DNA damage, intravital imaging, RNA interference, theranostics
Published in DiRROS: 25.07.2024; Views: 305; Downloads: 208
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1009. Novel, male-produced aggregation pheromone of the cerambycid beetle Rosalia alpina, a priority species of European conservation concernAlenka Žunič Kosi, Yunfan Zou, Michal Hoskovec, Al Vrezec, Nataša Stritih Peljhan, Jocelyn G. Millar, 2017, original scientific article Abstract: Several recent studies have demonstrated the great potential for exploiting semiochemicals in ecology and conservation studies. The cerambycid beetle Rosalia alpina represents one of the flagship species of saproxylic insect biodiversity in Europe. In recent years its populations appear to have declined substantially, and its range has shrunk considerably as a result of forest management and urbanization. Here, we collected volatile chemicals released by males and females of R. alpina. Analyses of the resulting extracts revealed the presence of a single male-specific compound, identified as a novel alkylated pyrone structure. In field bioassays in Slovenia, traps baited with the synthesized pyrone captured both sexes of R. alpina, indicating that the pyrone functions as an aggregation pheromone. Our results represent the first example of a new structural class of pheromones within the Cerambycidae, and demonstrate that pheromone-baited traps can provide a useful tool for sampling R. alpina. This tool could be particularly useful in the ongoing development of conservation strategies for the iconic but endangered Alpine longicorn.
Keywords: pheromones, beetles, plants, endangered species, conservation science
Published in DiRROS: 25.07.2024; Views: 299; Downloads: 378
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1010. Development and inter-laboratory assessment of droplet digital PCR assays for multiplex quantification of 15 genetically modified soybean linesAlexandra Bogožalec Košir, Bjørn Spilsberg, Arne Holst-Jensen, Jana Žel, David Dobnik, 2017, original scientific article Abstract: Quantification of genetically modified organisms (GMOs) in food and feed products is often required for their labelling or for tolerance thresholds. Standard-curve-based simplex quantitative polymerase chain reaction (qPCR) is the prevailing technology, which is often combined with screening analysis. With the rapidly growing number of GMOs on the world market, qPCR analysis becomes laborious and expensive. Innovative cost-effective approaches are therefore urgently needed. Here, we report the development and inter-laboratory assessment of multiplex assays to quantify GMO soybean using droplet digital PCR (ddPCR). The assays were developed to facilitate testing of foods and feed for compliance with current GMO regulations in the European Union (EU). Within the EU, the threshold for labelling is 0.9% for authorised GMOs per ingredient. Furthermore, the EU has set a technical zero tolerance limit of 0.1% for certain unauthorised GMOs. The novel multiplex ddPCR assays developed target 11 GMO soybean lines that are currently authorised, and four that are tolerated, pending authorisation in the EU. Potential significant improvements in cost efficiency are demonstrated. Performance was assessed for the critical parameters, including limits of detection and quantification, and trueness, repeatability, and robustness. Inter-laboratory performance was also determined on a number of proficiency programme and real-life samples.
Keywords: droplet digital PCR, genetically modified organisms, soybean
Published in DiRROS: 25.07.2024; Views: 284; Downloads: 155
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