1. Plant molecular responses to potato virus Y : a continuum of outcomes from sensitivity and tolerance to resistanceŠpela Baebler, Anna Coll Rius, Kristina Gruden, 2020, review article Abstract: Potato virus Y (PVY) is the most economically important virus affecting potato production. PVY manipulates the plant cell machinery in order to successfully complete the infecting cycle. On the other side, the plant activates a sophisticated multilayer immune defense response to combat viral infection. The balance between these mechanisms, depending on the plant genotype and environment, results in a specific outcome that can be resistance, sensitivity, or tolerance. In this review, we summarize and compare the current knowledge on molecular events, leading to different phenotypic outcomes in response to PVY and try to link them with the known molecular mechanisms.
Keywords: potato virus Y, Potyviridae, potato, Solanum tuberosum, Solanaceae, plant immune signaling, plant hormones, tolerance, susceptibility, resistance
Published in DiRROS: 06.08.2024; Views: 41; Downloads: 25
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2. A primer on the analysis of high-throughput sequencing data for detection of plant virusesDenis Kutnjak, Lucie Tamisier, Ian Adams, Neil Boonham, Thierry Candresse, Michela Chiumenti, Kris De Jonghe, Jan F. Kreuze, Marie Lefebvre, Goncalo Silva, Martha Malapi-Wight, Paolo Margaria, Irena Mavrič Pleško, Sam McGreig, Laura Miozzi, Benoit Remenant, Jean-Sébastien Reynard, Johan Rollin, Mike Rott, Olivier Schumpp, Sébastien Massart, Annelies Haegeman, 2021, review article Abstract: High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.
Keywords: plant virus, high-throughput sequencing, bioinformatics, detection, discovery
Published in DiRROS: 05.08.2024; Views: 52; Downloads: 55
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3. Salicylic acid is an indispensable component of the Ny-1 resistance-gene-mediated response against Potato virus Y infection in potatoŠpela Baebler, Kamil Witek, Marko Petek, Katja Stare, Magda Tušek-Žnidarič, Maruša Pompe Novak, Jenny Renaut, K. Szajko, D. Strzelczyk-Żyta, W. Marczewski, Karolina Morgiewicz, Kristina Gruden, Jacek Hennig, 2014, original scientific article Abstract: The purpose of the study was to investigate the role of salicylic acid (SA) signalling in Ny-1-mediated hypersensitive resistance (HR) of potato (Solanum tuberosum L.) to Potato virus Y (PVY). The responses of the Ny-1 allele in the Rywal potato cultivar and transgenic NahG-Rywal potato plants that do not accumulate SA were characterized at the cytological, biochemical, transcriptome, and proteome levels. Analysis of noninoculated and inoculated leaves revealed that HR lesions started to develop from 3 d post inoculation and completely restricted the virus spread. At the cytological level, features of programmed cell death in combination with reactive oxygen species burst were observed. In response to PVY infection, SA was synthesized de novo. The lack of SA accumulation in the NahG plants led to the disease phenotype due to unrestricted viral spreading. Grafting experiments show that SA has a critical role in the inhibition of PVY spreading in parenchymal tissue, but not in vascular veins. The whole transcriptome analysis confirmed the central role of SA in orchestrating Ny-1-mediated responses and showed that the absence of SA leads to significant changes at the transcriptome level, including a delay in activation of expression of genes known to participate in defence responses. Moreover, perturbations in the expression of hormonal signalling genes were detected, shown as a switch from SA to jasmonic acid/ethylene signalling. Viral multiplication in the NahG plants was accompanied by downregulation of photosynthesis genes and activation of multiple energy-producing pathways.
Keywords: plant-pathogen interactions, Potato virus Y, salicylic acid, whole transcriptome analysis
Published in DiRROS: 01.08.2024; Views: 93; Downloads: 96
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4. The titre of the virus in the inoculum affects the titre of the viral RNA in the host plant and the occurrence of the disease symptomsMaruša Pompe Novak, Maja Križnik, Kristina Gruden, 2019, original scientific article Abstract: Potato virus Y (PVY) is the most economically important potato virus, therefore extensive research is focusing on elucidation of its interaction with the host. To obtain repeatable results, strict standardization of research methods is crucial. Mechanical inoculation by rubbing sap from a PVY infected plant onto the leaf surface together with a fine abrasive powder is the most convenient way of experimental transmission of PVY to host plants. However, factors determining reproducibility of this process need to be determined. In the present study, it was shown that higher titre of the virus in the inoculum resulted in faster increase of PVYNTN RNA titre in the inoculated leaves, as well as in faster translocation of PVYNTN from inoculated leaves into upper non-inoculated leaves. The final titre of PVYNTN RNA in upper non-inoculated leaves was independent of the virus titre in the inoculum. In addition, the occurrence of the disease symptoms was followed and the dependence to the titre of the virus in the inoculum was observed.
Keywords: krompir, virus krompirja Y, mehanska inokulacija, titer virusa, inokulum, Potato virus Y, PVY, potato, mechanical inoculation, inoculum, virus titre, symptoms
Published in DiRROS: 31.07.2024; Views: 81; Downloads: 63
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5. Bimodal dynamics of primary metabolism-related responses in tolerant potato-Potato virus Y interactionNeža Turnšek, Živa Ramšak, Katja Stare, Tjaša Stare, Dominik Vodnik, Andrej Blejec, Kristina Gruden, Wolfram Weckwerth, Stefanie Wienkoop, 2015, original scientific article Abstract: Background
Potato virus Y (PVY) is a major pathogen that causes substantial economic losses in worldwide potato production. Different potato cultivars differ in resistance to PVY, from severe susceptibility, through tolerance, to complete resistance. The aim of this study was to better define the mechanisms underlying tolerant responses of potato to infection by the particularly aggressive PVYNTN strain. We focused on the dynamics of the primary metabolism-related processes during PVYNTN infection.
Results
A comprehensive analysis of the dynamic changes in primary metabolism was performed, which included whole transcriptome analysis, nontargeted proteomics, and photosynthetic activity measurements in potato cv. Désirée and its transgenic counterpart depleted for accumulation of salicylic acid (NahG-Désirée). Faster multiplication of virus occurred in the NahG-Désirée, with these plants developing strong disease symptoms. We show that while the dynamics of responses at the transcriptional level are extensive and bimodal, this is only partially translated to the protein level, and to the final functional outcome. Photosynthesis-related genes are transiently induced before viral multiplication is detected and it is down-regulated later on. This is reflected as a deficiency of the photosynthetic apparatus at the onset of viral multiplication only. Interestingly, specific and constant up-regulation of some RuBisCO transcripts was detected in Désirée plants, which might be important, as these proteins have been shown to interact with viral proteins.
In SA-deficient and more sensitive NahG-Désirée plants, consistent down-regulation of photosynthesis-related genes was detected. A constant reduction in the photochemical efficiency from the onset of viral multiplication was identified; in nontransgenic plants this decrease was only transient. The transient reduction in net photosynthetic rate occurred in both genotypes with the same timing, and coincided with changes in stomatal conductivity.
Conclusions
Down-regulation of photosynthesis-related gene expression and decreased photosynthetic activity is in line with other studies that have reported the effects of biotic stress on photosynthesis. Here, we additionally detected induction of light-reaction components in the early stages of PVYNTN infection of tolerant interaction. As some of these components have already been shown to interact with viral proteins, their overproduction might contribute to the absence of symptoms in cv. Désirée.
Keywords: plant-pathogen interactions, Potato virus Y, potyviridae, salicylic acid, whole transcriptome analysis, shot-gun proteomics, photosynthetic parameters
Published in DiRROS: 29.07.2024; Views: 93; Downloads: 132
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6. Host range and symptomatology of Pepino mosaic virus strains occurring in EuropeDag-Ragnar Blystad, René van der Vlugt, Ana Alfaro-Fernández, María del Carmen Córdoba, Gábor Bese, Dimitrinka Hristova, Henryk Pospieszny, Nataša Mehle, Maja Ravnikar, Laura Tomassoli, Christina Varveri, Steen Lykke Nielsen, 2015, original scientific article Abstract: Pepino mosaic virus (PepMV) has caused great concern in the greenhouse tomato industry after it was found causing a new disease in tomato in 1999. The objective of this paper is to investigate alternative hosts and compare important biological characteristics of the three PepMV strains occurring in Europe when tested under different environmental conditions. To this end we compared the infectivity and symptom development of three, well characterized isolates belonging to three different PepMV strains, EU-tom, Ch2 and US1, by inoculating them on tomato, possible alternative host plants in the family Solanaceae and selected test plants. The inoculation experiments were done in 10 countries from south to north in Europe. The importance of alternative hosts among the solanaceous crops and the usefulness of test plants in the biological characterization of PepMV isolates are discussed. Our data for the three strains tested at 10 different European locations with both international and local cultivars showed that eggplant is an alternative host of PepMV. Sweet pepper is not an important host of PepMV, but potato can be infected when the right isolate is matched with a specific cultivar. Nicotiana occidentalis 37B is a useful indicator plant for PepMV studies, since it reacts with a different symptomatology to each one of the PepMV strains.
Keywords: Pepino mosaic virus, potexvirus, strains, host plants, test plants
Published in DiRROS: 26.07.2024; Views: 98; Downloads: 111
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7. Survey results on nucleic acid tests of infectious diseases : present status and need for rapid and near-patient diagnosticsJörg Neukammer, Martin Hussels, Andreas Kummrow, Alison S. Devonshire, Carole A. Foy, Jim F. Huggett, Helen C. Parkes, Jana Žel, Mojca Milavec, Heinz Schimmel, Wolfgang Unger, Müslüm Akgöz, Timothy D. McHugh, Viktorija Tomič, Hans-Peter Grunert, Heinz Zeichhardt, 2015, original scientific article Abstract: This survey discusses current and emerging isothermal and rapid polymerase chain reaction (PCR) based nucleic acid amplification methods for near-patient diagnostics.
To assess the clinical need of rapid diagnostics for infectious diseases based on nucleic acid tests (NATs) we performed and analysed a questionnaire among laboratories participating in corresponding INSTAND ring trials for external quality assurance. The questions concerning new amplification technologies like isothermal nucleic acid amplification, potentially suited to significantly decrease turnaround times, were complemented by questions to evaluate the present status of NATs. Besides end-users, companies were also addressed by sending out a manufacturer specific questionnaire.
Analysis of the answers from 48 laboratories in 14 European countries revealed that a much shorter turnaround time is requested for selected pathogens compared to about 2 h or longer when applying temperature cycling amplification, i.e. PCR. In this context, most frequently mentioned were methicillin-resistant Staphylococcus aureus (MRSA), norovirus, influenza A and B viruses, cytomegalovirus (CMV) as well as hepatitis B virus (HBV) and hepatitis C virus (HCV). At present, 8% of the laboratories having participated in this survey apply isothermal amplification of nucleic acids to identify infectious pathogens.
Keywords: nucleic acid tests, infectious diseases, virus detection, bacteria detection, isothermal nucleic acid amplification, status report, questionnaire, NAT, PCR
Published in DiRROS: 26.07.2024; Views: 103; Downloads: 261
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8. Methacrylate monolith chromatography as a tool for waterborne virus removalNejc Rački, Petra Kramberger, Andrej Steyer, Jernej Gašperšič, Aleš Štrancar, Maja Ravnikar, Ion Gutiérrez-Aguirre, 2015, original scientific article Abstract: Enteric viruses are commonly present in environmental waters and represent the major cause of waterborne infections and outbreaks. Since traditional wastewater treatments fail to remove enteric viruses in the water purification process, they are released daily into environmental waters. Monolithic supports have enabled chromatography to enter the field of virology. They have been successfully used in virus purification and concentration. In this work quaternary amine (QA) methacrylate monoliths were exploited to remove enteric viruses from wastewater treatment plant effluent. Expectedly, chromatographic processing of such a complex medium was troublesome, even for monoliths, characterized by extremely large pore dimensions. This problem was solved by introducing a pre-step chromatography using hydroxyl (OH) methacrylate monoliths. This way, molecules, that would hinder virus binding to the anion-exchanger monolith, were removed. As a result, the OH pre-column reduced backpressure increase on the subsequent anion-exchanger column, and increased both QA column binding capacity and life time. Wastewater effluent samples were successfully purified from five waterborne enteric viruses (rotavirus, norovirus genogroup I and II, astrovirus, sapovirus), below the detection limit of RT-qPCR. The breakthrough of the rotavirus binding capacity was not reached for concentrations that significantly exceeded those expected in effluent waters. The obtained results confirm that methacrylate monoliths can be a valuable tool for simultaneous removal of different waterborne viruses from contaminated water sources.
Keywords: monolith chromatography, waterborne, virus, removal, wastewater, qPCR
Published in DiRROS: 26.07.2024; Views: 110; Downloads: 55
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9. Solanum venturii, a suitable model system for virus-induced gene silencing studies in potato reveals StMKK6 as an important player in plant immunityDavid Dobnik, Ana Lazar, Tjaša Stare, Kristina Gruden, Vivianne G. A. A. Vleeshouwers, Jana Žel, 2016, original scientific article Abstract: Background
Virus-induced gene silencing (VIGS) is an optimal tool for functional analysis of genes in plants, as the viral vector spreads throughout the plant and causes reduced expression of selected gene over the whole plant. Potato (Solanum tuberosum) is one of the most important food crops, therefore studies performing functional analysis of its genes are very important. However, the majority of potato cultivars used in laboratory experimental setups are not well amenable to available VIGS systems, thus other model plants from Solanaceae family are used (usually Nicotiana benthamiana). Wild potato relatives can be a better choice for potato model, but their potential in this field was yet not fully explored. This manuscript presents the set-up of VIGS, based on Tobacco rattle virus (TRV) in wild potato relatives for functional studies in potato–virus interactions.
Results
Five different potato cultivars, usually used in our lab, did not respond to silencing of phytoene desaturase (PDS) gene with TRV-based vector. Thus screening of a large set of wild potato relatives (different Solanum species and their clones) for their susceptibility to VIGS was performed by silencing PDS gene. We identified several responsive species and further tested susceptibility of these genotypes to potato virus Y (PVY) strain NTN and N. In some species we observed that the presence of empty TRV vector restricted the movement of PVY. Fluorescently tagged PVYN-GFP spread systemically in only five of tested wild potato relatives. Based on the results, Solanum venturii (VNT366-2) was selected as the most suitable system for functional analysis of genes involved in potato–PVY interaction. The system was tested by silencing two different plant immune signalling-related kinases, StWIPK and StMKK6. Silencing of StMKK6 enabled faster spreading of the virus throughout the plant, while silencing of WIPK had no effect on spreading of the virus.
Conclusions
The system employing S. venturii (VNT366-2) and PVYN-GFP is a suitable method for fast and simple functional analysis of genes involved in potato–PVY interactions. Additionally, a set of identified VIGS responsive species of wild potato relatives could serve as a tool for general studies of potato gene function.
Keywords: potato, virus-induced gene silencing, VIGS, potato virus Y, PVY, Solanum venturii, StWIPK, StMKK6, TRV
Published in DiRROS: 25.07.2024; Views: 118; Downloads: 115
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10. Digital PCR for direct quantification of viruses without DNA extractionJernej Pavšič, Jana Žel, Mojca Milavec, 2016, original scientific article Abstract: DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.
Keywords: molecular diagnostics, direct quantification, viruses, virus reference materials, human cytomegalovirus, digital PCR
Published in DiRROS: 25.07.2024; Views: 101; Downloads: 64
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