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Potato (Solanum tuberosum) is the most popular tuber crop and a model organism. A variety of gene models for potato exist, and despite frequent updates, they are not unified. This hinders the comparison of gene models across versions, limits the ability to reuse experimental data without significant re-analysis, and leads to missing or wrongly annotated genes. Here, we unify the recent potato double monoploid v4 and v6 gene models by developing an automated merging protocol, resulting in a Unified poTato genome model (UniTato). We subsequently established an Apollo genome browser (unitato.nib.si) that enables public access to UniTato and further community-based curation. We demonstrate how the UniTato resource can help resolve problems with missing or misplaced genes and can be used to update or consolidate a wider set of gene models or genome information. The automated protocol, genome annotation files, and a comprehensive translation table are provided at github.com/NIB-SI/unitato.

In this white paper, we describe the founding of a new ELIXIR Community - the Systems Biology Community - and its proposed future contributions to both ELIXIR and the broader community of systems biologists in Europe and worldwide. The Community believes that the infrastructure aspects of systems biology - databases, (modelling) tools and standards development, as well as training and access to cloud infrastructure - are not only appropriate components of the ELIXIR infrastructure, but will prove key components of ELIXIR’s future support of advanced biological applications and personalised medicine. By way of a series of meetings, the Community identified seven key areas for its future activities, reflecting both future needs and previous and current activities within ELIXIR Platforms and Communities. These are: overcoming barriers to the wider uptake of systems biology; linking new and existing data to systems biology models; interoperability of systems biology resources; further development and embedding of systems medicine; provisioning of modelling as a service; building and coordinating capacity building and training resources; and supporting industrial embedding of systems biology. A set of objectives for the Community has been identified under four main headline areas: Standardisation and Interoperability, Technology, Capacity Building and Training, and Industrial Embedding. These are grouped into short-term (3-year), mid-term (6-year) and long-term (10-year) objectives.

Abstract Background Geminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV). Results We showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes. Conclusion We have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.

Summary: Insect physiology and reproduction depend on several terpenoid compounds, whose biosynthesis is mainly unknown. One enigmatic group of insect monoterpenoids are mealybug sex pheromones, presumably resulting from the irregular coupling activity of unidentified isoprenyl diphosphate synthases (IDSs). Here, we performed a comprehensive search for IDS coding sequences of the pest mealybug Planococcus citri. We queried the available genomic and newly generated short- and long-read P. citri transcriptomic data and identified 18 putative IDS genes, whose phylogenetic analysis indicates several gene family expansion events. In vitro testing confirmed regular short-chain coupling activity with five gene products. With the candidate with highest IDS activity, we also detected low amounts of irregular coupling products, and determined amino acid residues important for chain-length preference and irregular coupling activity. This work therefore provides an important foundation for deciphering terpenoid biosynthesis in mealybugs, including the sex pheromone biosynthesis in P. citri.

The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including ‘Candidiatus Phytoplasma solani’ are unknown. Six putative pathogenicity factors/effectors from six different strains of ‘Ca. P. solani’ were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate–glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate–glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.

Abstract We developed pISA-tree, a straightforward and flexible data management solution for organisation of life science project-associated research data and metadata. pISA-tree was initiated by end-user requirements thus its strong points are practicality and low maintenance cost. It enables on-the-fly creation of enriched directory tree structure (project/Investigation/Study/Assay) based on the ISA model, in a standardised manner via consecutive batch files. Templates-based metadata is generated in parallel at each level enabling guided submission of experiment metadata. pISA-tree is complemented by two R packages, pisar and seekr. pisar facilitates integration of pISA-tree datasets into bioinformatic pipelines and generation of ISA-Tab exports. seekr enables synchronisation with the FAIRDOMHub repository. Applicability of pISA-tree was demonstrated in several national and international multi-partner projects. The system thus supports findable, accessible, interoperable and reusable (FAIR) research and is in accordance with the Open Science initiative. Source code and documentation of pISA-tree are available at https://github.com/NIB-SI/pISA-tree .

In this white paper, we describe the founding of a new ELIXIR Community - the Systems Biology Community - and its proposed future contributions to both ELIXIR and the broader community of systems biologists in Europe and worldwide. The Community believes that the infrastructure aspects of systems biology - databases, (modelling) tools and standards development, as well as training and access to cloud infrastructure - are not only appropriate components of the ELIXIR infrastructure, but will prove key components of ELIXIR’s future support of advanced biological applications and personalised medicine. By way of a series of meetings, the Community identified seven key areas for its future activities, reflecting both future needs and previous and current activities within ELIXIR Platforms and Communities. These are: overcoming barriers to the wider uptake of systems biology; linking new and existing data to systems biology models; interoperability of systems biology resources; further development and embedding of systems medicine; provisioning of modelling as a service; building and coordinating capacity building and training resources; and supporting industrial embedding of systems biology. A set of objectives for the Community has been identified under four main headline areas: Standardisation and Interoperability, Technology, Capacity Building and Training, and Industrial Embedding. These are grouped into short-term (3-year), mid-term (6-year) and long-term (10-year) objectives.

Plant biofactories are a promising platform for sustainable production of high-value compounds, among which are insect sex pheromones, a green alternative to conventional insecticides in agriculture. Recently, we have constructed transgenic Nicotiana benthamiana plants (“Sexy Plants”, SxP) that successfully produce a blend of moth (Lepidoptera) sex pheromone compounds (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate. However, efficient biosynthesis of sex pheromones resulted in growth and developmental penalty, diminishing the potential for commercial use of SxP in biomanufacturing. To gain insight into the underlying molecular responses, we analysed the whole-genome transcriptome and evaluated it in relation to growth and pheromone production in low- and high-producing transgenic plants of v1.0 and v1.2 SxP lines. In our study, high-producing SxPv1.2 plants accumulated the highest amounts of pheromones but still maintained better growth compared to v1.0 high producers. For an in-depth biological interpretation of the transcriptomic data, we have prepared a comprehensive functional N. benthamiana genome annotation as well as gene translations to Arabidopsis thaliana, enabling functional information transfer by using Arabidopsis knowledge networks. Differential gene expression analysis, contrasting pheromone producers to wild-type plants, revealed that while only a few genes were differentially regulated in low-producing plants, high-producing plants exhibited vast transcriptional reprogramming. They showed signs of stress-like response, manifested as downregulation of photosynthesis-related genes and significant differences in expression of hormonal signalling and secondary metabolism-related genes, the latter presumably leading to previously reported volatilome changes. Further network analyses confirmed stress-like response with activation of jasmonic acid and downregulation of gibberellic acid signalling, illuminating the possibility that the observed growth penalty was not solely a consequence of a higher metabolic burden imposed upon constitutive expression of a heterologous biosynthetic pathway, but rather the result of signalling pathway perturbation. Our work presents an example of comprehensive transcriptomic analyses of disadvantageous stress signalling in N. benthamiana biofactory that could be applied to other bioproduction systems.

Abstract Background Rosaceae species are economically highly relevant crops. Their cultivation systems are constrained by phytopathogens causing severe losses. Plants respond to invading pathogens through signaling mechanisms, a component of which are of them being plant elicitor peptides (Peps). Exogenous application of Peps activates defense mechanisms and reduces the symptoms of pathogen infection in various pathosystems. We have previously identified the Rosaceae Peps and showed, in an ex vivo system, that their topical application efficiently enhanced resistance to the bacterial pathogen Xanthomonas arboricola pv. pruni (Xap). Results Here we demonstrate the effectiveness of Prunus persica peptides PpPep1 and PpPep2 in protecting peach plants in vivo at nanomolar doses, with 40% reduction of the symptoms following Xap massive infection. We used deep sequencing to characterize the transcriptomic response of peach plants to preventive treatment with PpPep1 and PpPep2. The two peptides induced highly similar massive transcriptomic reprogramming in the plant. One hour, 1 day and 2 days after peptide application there were changes in expression in up to 8% of peach genes. We visualized the transcriptomics dynamics in a background knowledge network and detected the minor variations between plant responses to PpPep1 and PpPep2, which might explain their slightly different protective effects. By designing a P. persica Pep background knowledge network, comparison of our data and previously published immune response datasets was possible. Conclusions Topical application of P. persica Peps mimics the PTI natural response and protects plants against massive Xap infection. This makes them good candidates for deployment of natural, targeted and environmental-friendly strategies to enhance resistance in Prunus species and prevent important biotic diseases.

TGA transcription factors are essential regulators of various cellular processes, their activity connected to different hormonal pathways, interacting proteins and regulatory elements. Belonging to the basic region leucine zipper (bZIP) family, TGAs operate by binding to their target DNA sequence as dimers through a conserved bZIP domain. Despite sharing the core DNA-binding sequence, the TGA paralogues exert somewhat different DNA-binding preferences. Sequence variability of their N- and C-terminal protein parts indicates their importance in defining TGA functional specificity through interactions with diverse proteins, affecting their DNA-binding properties. In this review, we provide a short and concise summary on plant TGA transcription factors from a structural point of view, including the relation of their structural characteristics to their functional roles in transcription regulation.

The use of more salt stress-tolerant vine rootstocks can be a sustainable strategy for adapting traditional grapevine cultivars to future conditions. However, how the new M1 and M4 rootstocks perform against salinity compared to conventional ones, such as the 1103-Paulsen, had not been previously assessed under real field conditions. Therefore, a field trial was carried out in a young ‘Tempranillo’ (Vitis vinifera L.) vineyard grafted onto all three rootstocks under a semi-arid and hot-summer Mediterranean climate. The vines were irrigated with two kinds of water: a non-saline Control with EC of 0.8 dS m–1 and a Saline treatment with 3.5 dS m–1. Then, various physiological parameters were assessed in the scion, and, additionally, gene expression was studied by high throughput sequencing in leaf and berry tissues. Plant water relations evidenced the osmotic effect of water quality, but not that of the rootstock. Accordingly, leaf-level gas exchange rates were also reduced in all three rootstocks, with M1 inducing significantly lower net photosynthesis rates than 1103-Paulsen. Nevertheless, the expression of groups of genes involved in photosynthesis and amino acid metabolism pathways were not significantly and differentially expressed. The irrigation with saline water significantly increased leaf chloride contents in the scion onto the M-rootstocks, but not onto the 1103P. The limitation for leaf Cl– and Na+ accumulation on the scion was conferred by rootstock. Few processes were differentially regulated in the scion in response to the saline treatment, mainly, in the groups of genes involved in the flavonoids and phenylpropanoids metabolic pathways. However, these transcriptomic effects were not fully reflected in grape phenolic ripeness, with M4 being the only one that did not cause reductions in these compounds in response to salinity, and 1103-Paulsen having the highest overall concentrations. These results suggest that all three rootstocks confer short-term salinity tolerance to the scion. The lower transcriptomic changes and the lower accumulation of potentially phytotoxic ions in the scion grafted onto 1103-Paulsen compared to M-rootstocks point to the former being able to maintain this physiological response in the longer term. Further agronomic trials should be conducted to confirm these effects on vine physiology and transcriptomics in mature vineyards.

Today, academic researchers benefit from the changes driven by digital technologies and the enormous growth of knowledge and data, on globalisation, enlargement of the scientific community, and the linkage between different scientific communities and the society. To fully benefit from this development, however, information needs to be shared openly and transparently. Digitalisation plays a major role here because it permeates all areas of business, science and society and is one of the key drivers for innovation and international cooperation. To address the resulting opportunities, the EU promotes the development and use of collaborative ways to produce and share knowledge and data as early as possible in the research process, but also to appropriately secure results with the European strategy for Open Science (OS). It is now widely recognised that making research results more accessible to all societal actors contributes to more effective and efficient science; it also serves as a boost for innovation in the public and private sectors. However for research data to be findable, accessible, interoperable and reusable the use of standards is essential. At the metadata level, considerable efforts in standardisation have already been made (e.g. Data Management Plan and FAIR Principle etc.), whereas in context with the raw data these fundamental efforts are still fragmented and in some cases completely missing. The CHARME consortium, funded by the European Cooperation in Science and Technology (COST) Agency, has identified needs and gaps in the field of standardisation in the life sciences and also discussed potential hurdles for implementation of standards in current practice. Here, the authors suggest four measures in response to current challenges to ensure a high quality of life science research data and their re-usability for research and innovation.

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.

Presently, pearl millet and wheat are belonging to highly important cereal crops. Pearl millet, however, is an under-utilized crop, despite its superior resilience to drought and heat stress in contrast to wheat. To investigate this in more detail, we performed comparative physiological screening and large scale proteomics of drought stress responses in drought-tolerant and susceptible genotypes of pearl millet and wheat. These chosen genotypes are widely used in breeding and farming practices. The physiological responses demonstrated large differences in the regulation of root morphology and photosynthetic machinery, revealing a stay-green phenotype in pearl millet. Subsequent tissue-specific proteome analysis of leaves, roots and seeds led to the identification of 12,558 proteins in pearl millet and wheat under well-watered and stress conditions. To allow for this comparative proteome analysis and to provide a platform for future functional proteomics studies we performed a systematic phylogenetic analysis of all orthologues in pearl millet, wheat, foxtail millet, sorghum, barley, brachypodium, rice, maize, Arabidopsis, and soybean. In summary, we define (i) a stay-green proteome signature in the drought-tolerant pearl millet phenotype and (ii) differential senescence proteome signatures in contrasting wheat phenotypes not capable of coping with similar drought stress. These different responses have a significant effect on yield and grain filling processes reflected by the harvest index. Proteome signatures related to root morphology and seed yield demonstrated the unexpected intra- and interspecies-specific biochemical plasticity for stress adaptation for both pearl millet and wheat genotypes. These quantitative reference data provide tissue- and phenotype-specific marker proteins of stress defense mechanisms which are not predictable from the genome sequence itself and have potential value for marker-assisted breeding beyond genome assisted breeding.

Today, academic researchers benefit from the changes driven by digital technologies and the enormous growth of knowledge and data, on globalisation, enlargement of the scientific community, and the linkage between different scientific communities and the society. To fully benefit from this development, however, information needs to be shared openly and transparently. Digitalisation plays a major role here because it permeates all areas of business, science and society and is one of the key drivers for innovation and international cooperation. To address the resulting opportunities, the EU promotes the development and use of collaborative ways to produce and share knowledge and data as early as possible in the research process, but also to appropriately secure results with the European strategy for Open Science (OS). It is now widely recognised that making research results more accessible to all societal actors contributes to more effective and efficient science; it also serves as a boost for innovation in the public and private sectors. However for research data to be findable, accessible, interoperable and reusable the use of standards is essential. At the metadata level, considerable efforts in standardisation have already been made (e.g. Data Management Plan and FAIR Principle etc.), whereas in context with the raw data these fundamental efforts are still fragmented and in some cases completely missing. The CHARME consortium, funded by the European Cooperation in Science and Technology (COST) Agency, has identified needs and gaps in the field of standardisation in the life sciences and also discussed potential hurdles for implementation of standards in current practice. Here, the authors suggest four measures in response to current challenges to ensure a high quality of life science research data and their re-usability for research and innovation.

Colorado potato beetle (CPB) is an agricultural pest of solanaceous crops, notorious for its rapid resistance development to chemical pesticides. Foliar spraying of dsRNA formulations is a promising innovative technology providing highly specific and environmentally acceptable option for CPB management. We designed dsRNA to silence CPB mesh gene (dsMESH) and performed laboratory feeding trials to assess impacts on beetle survival and development. We compared the effectiveness of in vivo and in vitro produced dsRNA in a series of laboratory experiments. We additionally performed a field trial in which the efficacy of dsRNA sprayed onto potato foliage was compared to a spinosad-based insecticide. We showed that dsMESH ingestion consistently and significantly impaired larval growth and decreased larval survival in laboratory feeding experiments. In vivo produced dsRNA performed similarly as in vitro synthesized dsRNA in laboratory settings. In the field trial, dsMESH was as effective in controlling CPB larvae as a commercial spinosad insecticide, its activity was however slower. We discuss limitations and benefits of a potential dsMESH-based CPB management strategy and list some important RNAi based CPB research topics, which will have to be addressed in future.

Potato virus Y (PVY), the type member of the genus Potyvirus (family Potyviridae), is the most widespread virus affecting potato and is included in the top five most economically detrimental plant viruses. Recently, the structure of the PVY virion has been determined by cryo-electron microscopy, which has opened the doors to functional studies that explore the involvement of selected amino acids in different stages of the viral cycle. The only way to functionally challenge in planta the role of particular amino acids in the coat protein of PVY, or in other viral proteins, is by using cDNA clones. The use and manipulation of PVY cDNA clones, unlike those of other potyviruses, has been traditionally impaired by the toxicity that certain sequences within the PVY genome pose to Escherichia coli. Here, we describe the use of a published PVY cDNA clone, which harbours introns that overcome the aforementioned toxicity, to explore the effects of different coat protein modifications on viral infection. The protocol includes manipulation of the cDNA clone in E. coli, biolistic inoculation of plants with the constructed clones, observation of the biological effects on plants, quantification of cDNA clones by reverse transcription quantitative PCR, and confirmation of virion formation by transmission electron microscopy. Future possibilities involve the use of PVY cDNA clones tagged with fluorescent protein reporters to allow further insights into the effects of coat protein mutations on the cell-to-cell movement of PVY virions.

Abstract Background Progress in high-throughput molecular methods accompanied by more complex experimental designs demands novel data visualisation solutions. To specifically answer the question which parts of the specifical biological system are responding in particular perturbation, integrative approach in which experimental data are superimposed on a prior knowledge network is shown to be advantageous. Results We have developed DiNAR, Differential Network Analysis in R, a user-friendly application with dynamic visualisation that integrates multiple condition high-throughput data and extensive biological prior knowledge. Implemented differential network approach and embedded network analysis allow users to analyse condition-specific responses in the context of topology of interest (e.g. immune signalling network) and extract knowledge concerning patterns of signalling dynamics (i.e. rewiring in network structure between two or more biological conditions). We validated the usability of software on the Arabidopsis thaliana and Solanum tuberosum datasets, but it is set to handle any biological instances. Conclusions DiNAR facilitates detection of network-rewiring events, gene prioritisation for future experimental design and allows capturing dynamics of complex biological system. The fully cross-platform Shiny App is hosted and freely available at https://nib-si.shinyapps.io/DiNAR. The most recent version of the source code is available at https://github.com/NIB-SI/DiNAR/ with a DOI 10.5281/zenodo.1230523 of the archived version in Zenodo.

Abstract Background Quantitative molecular biology remains a challenge for researchers due to inconsistent approaches for control of errors in the final results. Due to several factors that can influence the final result, quantitative analysis and interpretation of qPCR data are still not trivial. Together with the development of high-throughput qPCR platforms, there is a need for a tool allowing for robust, reliable and fast nucleic acid quantification. Results We have developed “quantGenius” ( http://quantgenius.nib.si ), an open-access web application for a reliable qPCR-based quantification of nucleic acids. The quantGenius workflow interactively guides the user through data import, quality control (QC) and calculation steps. The input is machine- and chemistry–independent. Quantification is performed using the standard curve approach, with normalization to one or several reference genes. The special feature of the application is the implementation of user-guided QC-based decision support system, based on qPCR standards, that takes into account pipetting errors, assay amplification efficiencies, limits of detection and quantification of the assays as well as the control of PCR inhibition in individual samples. The intermediate calculations and final results are exportable in a data matrix suitable for further statistical analysis or visualization. We additionally compare the most important features of quantGenius with similar advanced software tools and illustrate the importance of proper QC system in the analysis of qPCR data in two use cases. Conclusions To our knowledge, quantGenius is the only qPCR data analysis tool that integrates QC-based decision support and will help scientists to obtain reliable results which are the basis for biologically meaningful data interpretation.

Bois noir is the most widespread phytoplasma grapevine disease in Europe. It is associated with ‘Candidatus Phytoplasma solani’, but molecular interactions between the causal pathogen and its host plant are not well understood. In this work, we combined the analysis of high-throughput RNA-Seq and sRNA-Seq data with interaction network analysis for finding new cross-talks among pathways involved in infection of grapevine cv. Zweigelt with ‘Ca. P. solani’ in early and late growing seasons. While the early growing season was very dynamic at the transcriptional level in asymptomatic grapevines, the regulation at the level of small RNAs was more pronounced later in the season when symptoms developed in infected grapevines. Most differentially expressed small RNAs were associated with biotic stress. Our study also exposes the less-studied role of hormones in disease development and shows that hormonal balance was already perturbed before symptoms development in infected grapevines. Analysis at the level of communities of genes and mRNA-microRNA interaction networks revealed several new genes (e.g., expansins and cryptdin) that have not been associated with phytoplasma pathogenicity previously. These novel actors may present a new reference framework for research and diagnostics of phytoplasma diseases of grapevine.

Understanding temporal biological phenomena is a challenging task that can be approached using network analysis. Here, we explored whether network reconstruction can be used to better understand the temporal dynamics of bois noir, which is associated with ‘Candidatus Phytoplasma solani’, and is one of the most widespread phytoplasma diseases of grapevine in Europe. We proposed a methodology that explores the temporal network dynamics at the community level, i.e., densely connected subnetworks. The methodology offers both insights into the functional dynamics via enrichment analysis at the community level, and analyses of the community dissipation, as a measure that accounts for community degradation. We validated this methodology with cases on experimental temporal expression data of uninfected grapevines and grapevines infected with ‘Ca. P. solani’. These data confirm some known gene communities involved in this infection. They also reveal several new gene communities and their potential regulatory networks that have not been linked to ‘Ca. P. solani’ to date. To confirm the capabilities of the proposed method, selected predictions were empirically evaluated.

Biosensors are indispensable tools to understand a plant’s immunity as its spatiotemporal dimension is key in withstanding complex plant immune signaling. The diversity of genetically encoded biosensors in plants is expanding, covering new analytes with ever higher sensitivity and robustness, but their assortment is limited in some respects, such as their use in following biotic stress response, employing more than one biosensor in the same chassis, and their implementation into crops. In this review, we focused on the available biosensors that encompass these aspects. We show that in vivo imaging of calcium and reactive oxygen species is satisfactorily covered with the available genetically encoded biosensors, while on the other hand they are still underrepresented when it comes to imaging of the main three hormonal players in the immune response: salicylic acid, ethylene and jasmonic acid. Following more than one analyte in the same chassis, upon one or more conditions, has so far been possible by using the most advanced genetically encoded biosensors in plants which allow the monitoring of calcium and the two main hormonal pathways involved in plant development, auxin and cytokinin. These kinds of biosensor are also the most evolved in crops. In the last section, we examine the challenges in the use of biosensors and demonstrate some strategies to overcome them.

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.

Hypersensitive response (HR)-conferred resistance to viral infection restricts the virus spread and is accompanied by the induction of cell death, manifested as the formation of necrotic lesions. While it is known that salicylic acid is the key component in the orchestration of the events restricting viral spread in HR, the exact function of the cell death in resistance is still unknown. We show that potato virus Y (PVY) can be detected outside the cell death zone in Ny-1-mediated HR in potato plants (cv. Rywal), observed as individual infected cells or small clusters of infected cells outside the cell death zone. By exploiting the features of temperature dependent Ny-1-mediated resistance, we confirmed that the cells at the border of the cell death zone are alive and harbor viable PVY that is able to reinitiate infection. To get additional insights into this phenomenon we further studied the dynamics of both cell death zone expansion and occurrence of viral infected cell islands outside it. We compared the response of Rywal plants to their transgenic counterparts, impaired in SA accumulation (NahG-Rywal), where the lesions occur but the spread of the virus is not restricted. We show that the virus is detected outside the cell death zone in all lesion developmental stages of HR lesions. We also measured the dynamics of lesions expansion in both genotypes. We show that while rapid lesion expansion is observed in SA-depleted plants, virus spread is even faster. On the other hand the majority of analyzed lesions slowly expand also in HR-conferred resistance opening the possibility that the infected cells are eventually engulfed by cell death zone. Taken altogether, we suggest that the HR cell death is separated from the resistance mechanisms which lead to PVY restriction in Ny-1 genetic background. We propose that HR should be regarded as a process where the dynamics of events is crucial for effectiveness of viral arrest albeit the exact mechanism conferring this resistance remains unknown.

Background: In different human adult tissues and organs embryonic-like stem cells have already been found. A question is whether they are present also in the adult human ovary. In this study we tried to find whether embryonic-like stem cells are present in the ovarian surface epithe- lium of women with premature ovarian failure, and whether these cells can differentiate into oocytes by in vitro culture. Methods: In 10 women with premature ovarian failure the ovarian surface epithelium was laparo- scopically scraped. We tried to immunomagnetically isolate embryonic-like stem cells from the suspension of scraped cells and to confirm their existence immunocytochemically by the expression of the markers c-kit and surface antigen SSEA-4, characteristic of pluripotent stem cells. In the lab, we tried to differentiate stem cells into oocytes in a cell culture medium with added heterologous follicular fluid from the in vitro fertilization programme, which is rich in substances, and important for oocyte growth and maturation. The differentiation of oocyte-like cells was screened. Oocyte-like cells were isolated from the cell culture and im- munocytochemically stained for oocyte-specific markers (c-kit, VASA, ZP2-zona pellucida). Oocyte-like cells were analyzed for the expression of oocyte-specific genes (Oct-4A, ZP3, c-kit, SCP-3) by single-cell PCR. Results: From the ovarian surface epithelium of all patients small yellow coloured cells with diam- eters of 2 to 4 μm and positive for markers of pluripotent stem cells were isolated. In the lab, these cells were cultured into oocyte-like cells with diameter ranging from 60 to 80 μm. Some of oocyte-like cells were positive for oocyte-specific markers and expressed the genes, characteristic of oocytes, including meiotic genes. Conclusions: In patients with premature ovarian failure it is possible to culture oocyte-like cells from ovarian surface epithelium stem cells. Intense research is needed to evaluate the quality of oocyte-like cells developed in vitro and to estimate their potential use for in vitro fertilization in the far future.

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.

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.

Potato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 ± 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Désirée and NahG-Désirée and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild potato relatives. PVY N605-GFP is therefore a powerful tool for future studies of PVY-host interactions, such as functional analysis of viral and plant genes involved in viral movement.

Potato production is one of the most important agricultural sectors, and it is challenged by various detrimental factors, including virus infections. To control losses in potato production, knowledge about the virus-plant interactions is crucial. Here, we investigated the molecular processes in potato plants as a result of Potato virus Y (PVY) infection, the most economically important potato viral pathogen. We performed an integrative study that links changes in the metabolome and gene expression in potato leaves inoculated with the mild PVYN and aggressive PVYNTN isolates, for different times through disease development. At the beginning of infection (1 day post-inoculation), virus-infected plants showed an initial decrease in the concentrations of metabolites connected to sugar and amino-acid metabolism, the TCA cycle, the GABA shunt, ROS scavangers, and phenylpropanoids, relative to the control plants. A pronounced increase in those metabolites was detected at the start of the strong viral multiplication in infected leaves. The alterations in these metabolic pathways were also seen at the gene expression level, as analysed by quantitative PCR. In addition, the systemic response in the metabolome to PVY infection was analysed. Systemic leaves showed a less-pronounced response with fewer metabolites altered, while phenylpropanoid-associated metabolites were strongly accumulated. There was a more rapid onset of accumulation of ROS scavengers in leaves inoculated with PVYN than those inoculated with PVYNTN. This appears to be related to the lower damage observed for leaves of potato infected with the milder PVYN strain, and at least partially explains the differences between the phenotypes observed.

Current methods based on DNA targets for the detection, identification and quantification of genetically modified organisms (GMOs) involve extraction of the DNA. Different extraction procedures have been developed for the great variety of samples from food, feed, seeds and particular plant parts. This makes the operation of routine analytical laboratories complex and workloads heavy. Here we present a decision-making system, developed over many years of GMO testing on different samples, that result in the application of only a few extraction methods for the majority of samples. Developed decision-making system enables quicker and more cost effective testing of GMOs. In addition, the performance of DNA extraction resulting from the use of the selected extraction methods is presented for use in subsequent testing of GMOs by real time PCR methods. This approach can be used as a model for similar systems based on nucleic acid analysis in food, feed, seeds and plants.

BackgroundGlioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions.Experimental proceduresMicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits.ResultsWe identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients' survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM.ConclusionThe GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients.

Plant synthetic biology requires software tools to assist on the design of complex multi-genic expression plasmids. Here a vector design strategy to express genes in plants is formalized and implemented as a grammar in GenoCAD, a Computer-Aided Design software for synthetic biology. It includes a library of plant biological parts organized in structural categories and a set of rules describing how to assemble these parts into large constructs. Rules developed here are organized and divided into three main subsections according to the aim of the final construct: protein localization studies, promoter analysis and protein-protein interaction experiments. The GenoCAD plant grammar guides the user through the design while allowing users to customize vectors according to their needs. Therefore the plant grammar implemented in GenoCAD will help plant biologists take advantage of methods from synthetic biology to design expression vectors supporting their research projects.

In this white paper, we describe the founding of a new ELIXIR Community - the Systems Biology Community - and its proposed future contributions to both ELIXIR and the broader community of systems biologists in Europe and worldwide. The Community believes that the infrastructure aspects of systems biology - databases, (modelling) tools and standards development, as well as training and access to cloud infrastructure - are not only appropriate components of the ELIXIR infrastructure, but will prove key components of ELIXIR’s future support of advanced biological applications and personalised medicine. By way of a series of meetings, the Community identified seven key areas for its future activities, reflecting both future needs and previous and current activities within ELIXIR Platforms and Communities. These are: overcoming barriers to the wider uptake of systems biology; linking new and existing data to systems biology models; interoperability of systems biology resources; further development and embedding of systems medicine; provisioning of modelling as a service; building and coordinating capacity building and training resources; and supporting industrial embedding of systems biology. A set of objectives for the Community has been identified under four main headline areas: Standardisation and Interoperability, Technology, Capacity Building and Training, and Industrial Embedding. These are grouped into short-term (3-year), mid-term (6-year) and long-term (10-year) objectives.

Plant diseases caused by viral infection are affecting all major crops. Being an obligate intracellular organisms, chemical control of these pathogens is so far not applied in the field except to control the insect vectors of the viruses. Understanding of molecular responses of plant immunity is therefore economically important, guiding the enforcement of crop resistance. To disentangle complex regulatory mechanisms of the plant immune responses, understanding system as a whole is a must. However, integrating data from different molecular analysis (transcriptomics, proteomics, metabolomics, smallRNA regulation etc.) is not straightforward. We evaluated the response of potato (Solanum tuberosum L.) following the infection with potato virus Y (PVY). The response has been analyzed on two molecular levels, with microarray transcriptome analysis and mass spectroscopy-based proteomics. Within this report, we performed detailed analysis of the results on both levels and compared two different approaches for analysis of proteomic data (spectral count versus MaxQuant). To link the data on different molecular levels, each protein was mapped to the corresponding potato transcript according to StNIB paralogue grouping. Only 33% of the proteins mapped to microarray probes in a one-to-one relation and additionally many showed discordance in detected levels of proteins with corresponding transcripts. We discussed functional importance of true biological differences between both levels and showed that the reason for the discordance between transcript and protein abundance lies partly in complexity and structure of biological regulation of proteome and transcriptome and partly in technical issues contributing to it.

Cancer genome and transcriptome analyses advanced our understanding of cancer biology. We performed transcriptome analysis of all known genes of peptidases also called proteases and their endogenous inhibitors in glioblastoma multiforme (GBM), which is one of the most aggressive and deadly types of brain cancers, where unbalanced proteolysis is associated with tumor progression.Comparisons were performed between the transcriptomics of primary GBM tumors and unmatched non-malignant brain tissue, and between GBM cell lines (U87-MG and U373) and a control human astrocyte cell line (NHA). Publicly-available data sets and our own datasets were integrated and normalized using bioinformatics tools to reveal protease and protease inhibitor genes with deregulated expression in both malignant versus non-malignant tissues and cells.Of the 311 protease genes identified to be differentially expressed in both GBM tissues and cells, 5 genes were highly overexpressed, 2 genes coding for non-peptidase homologues transferrin receptor (TFRC) and G protein-coupled receptor 56 (GPR56), as well as 3 genes coding for the proteases endoplasmic reticulum aminopeptidase 2 (ERAP2), glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and cathepsin K (CTSK), whereas one gene, that of the serine protease carboxypeptidase E (CPE) was strongly reduced in expression. Seventy five protease inhibitor genes were differentially expressed, of which 3 genes were highly overexpressed, the genes coding for stefin B (CSTB), peptidase inhibitor 3 (PI3 also named elafin) and CD74. Seven out of 8 genes (except CSTB) were validated using RT-qPCR in GBM cell lines. CTSK overexpression was validated using RT-qPCR in GBM tissues as well. Cathepsin K immunohistochemical staining and western blotting showed that only proteolytically inactive proforms of cathepsin K were overexpressed in GBM tissues and cells.The presence of high levels of inactive proforms of cathepsin K in GBM tissues and cells indicate that in GBM the proteolytic/collagenolytic role is not its primary function but it plays rather a different yet unknown role.

Mitogen-activated protein kinase (MAPK) cascades have crucial roles in the regulation of plant development and in plant responses to stress. Plant recognition of pathogen-associated molecular patterns or pathogen-derived effector proteins has been shown to trigger activation of several MAPKs. This then controls defence responses, including synthesis and/or signalling of defence hormones and activation of defence related genes. The MAPK cascade genes are highly complex and interconnected, and thus the precise signalling mechanisms in specific plant-pathogen interactions are still not known. Here we investigated the MAPK signalling network involved in immune responses of potato (Solanum tuberosum L.) to Potato virus Y, an important potato pathogen worldwide. Sequence analysis was performed to identify the complete MAPK kinase (MKK) family in potato, and to identify those regulated in the hypersensitive resistance response to Potato virus Y infection. Arabidopsis has 10 MKK family members, of which we identified five in potato and tomato (Solanum lycopersicum L.), and eight in Nicotiana benthamiana. Among these, StMKK6 is the most strongly regulated gene in response to Potato virus Y. The salicylic acid treatment revealed that StMKK6 is regulated by the hormone that is in agreement with the salicylic acid-regulated domains found in the StMKK6 promoter. The involvement of StMKK6 in potato defence response was confirmed by localisation studies, where StMKK6 accumulated strongly only in Potato-virus-Y-infected plants, and predominantly in the cell nucleus. Using a yeast two-hybrid method, we identified three StMKK6 targets downstream in the MAPK cascade: StMAPK4_2, StMAPK6 and StMAPK13. These data together provide further insight into the StMKK6 signalling module and its involvement in plant defence.

In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.

Soil salinity and drought are among the most serious agricultural and environmental problems of today. Therefore, investigations of plant resistance to abiotic stress have received a lot of attention in recent years. In this study, we identified the complete coding sequence of a 3'-phosphoadenosine-5'-phosphatase protein, ApHal2, from the halotolerant yeast Aureobasidium pullulans. Expression of the ApHAL2 gene in a Saccharomyces cerevisiae hal2 mutant complemented the mutant auxotrophy for methionine, and rescued the growth of the hal2 mutant in media with high NaCl concentrations. A 21-amino-acids-long region of the ApHal2 enzyme was inserted into the Arabidopsis thaliana homologue of Hal2, the SAL1 phosphatase. The inserted sequence included the META motif, which has previously been implicated in increased sodium tolerance of the Hal2 homologue from a related fungal species. Transgenic Arabidopsis plants overexpressing this modified SAL1 (mSAL1) showed improved halotolerance and drought tolerance. In a medium with an elevated salt concentration, mSAL1-expressing plants were twice as likely to have roots in a higher length category in comparison with the wild-type Arabidopsis and with plants overexpressing the native SAL1, and had 5% to 10% larger leaf surface area under moderate and severe salt stress, respectively. Similarly, after moderate drought exposure, the mSAL1-expressing plants showed 14% increased dry weight after revitalisation, with no increase in dry weight of the wild-type plants. With severe drought, plants overexpressing native SAL1 had the worst rehydration success, consistent with the recently proposed role of SAL1 in severe drought. This was not observed for plants expressing mSAL1. Therefore, the presence of this fungal META motif sequence is beneficial under conditions of increased salinity and moderate drought, and shows no drawbacks for plant survival under severe drought. This demonstrates that adaptations of extremotolerant fungi should be considered as a valuable resource for improving stress-tolerance in plant breeding in the future.

In contrast to the application of human mesenchymal stem cells (hMSCs) in regenerative medicine, only a limited number of studies are addressing their use in anticancer therapy. As the latter may represent a new hope to improve the survival of patients with glioblastoma multiformae (GBM), the most common and malignant form of the brain tumors, we aimed to investigate the interactions of hMSCs and GBM cells under in vitro conditions. Four hMSC clones and three different GBM cell lines were used to study their mutual paracrine interactions in cocultures compared to their monocultures, where cells were grown under the same experimental conditions. The effects on cell growth, proliferation, and invasion in Matrigel were quantified. Further, bioinformatics tools were used to relate these results to the data obtained from cytokine macroarrays and cDNA microarrays that revealed proteins and genes significantly involved in cellular cross-talk. We showed that hMSCs are responsible for the impairment of GBM cell invasion and growth, possibly via induction of their senescence. On the other hand, GBM cells inversely affected some of these characteristics in hMSCs. We found CCL2/MCP-1 to be the most significantly regulated chemokine during hMSC and U87-MG paracrine signaling in addition to several chemokines that may account for changed cocultured cells' phenotype by affecting genes associated with proliferation ( Pmepa-1, NF -κ B, IL-6, IL-1b ), invasion ( EphB2, Sod2, Pcdh18, Col7A1, Gja1, Mmp1/2 ), and senescence ( Kiaa1199, SerpinB2 ). As we functionally confirmed the role of CCL2/MCP-1 in GBM cell invasion we thereby propose a novel mechanism of CCL2/MCP-1 antimigratory effects on GBM cells, distinct from its immunomodulatory role. Significant alterations of GBM phenotype in the presence of hMSCs should encourage the studies on the naive hMSC use for GBM treatment.

BACKGROUND: Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable. METHODOLOGY/PRINCIPAL FINDINGS: To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex "crossomics" analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4+T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset. CONCLUSIONS/SIGNIFICANCE: Our study shows that the intra-individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective.

Plant defence signalling response against various pathogens, including viruses, is a complex phenomenon. In resistant interaction a plant cell perceives the pathogen signal, transduces it within the cell and performs a reprogramming of the cell metabolism leading to the pathogen replication arrest. This work focuses on signalling pathways crucial for the plant defence response, i.e., the salicylic acid, jasmonic acid and ethylene signal transduction pathways, in the Arabidopsis thaliana model plant. The initial signalling network topology was constructed manually by defining the representation formalism, encoding the information from public databases and literature, and composing a pathway diagram. The manually constructed network structure consists of 175 components and 387 reactions. In order to complement the network topology with possibly missing relations, a new approach to automated information extraction from biological literature was developed. This approach, named Bio3graph, allows for automated extraction of biological relations from the literature, resulting in a set of (component1, reaction, component2) triplets and composing a graph structure which can be visualised, compared to the manually constructed topology and examined by the experts. Using a plant defence response vocabulary of components and reaction types, Bio3graph was applied to a set of 9,586 relevant full text articles, resulting in 137 newly detected reactions between the components. Finally, the manually constructed topology and the new reactions were merged to form a network structure consisting of 175 components and 524 reactions. The resulting pathway diagram of plant defence signalling represents a valuable source for further computational modelling and interpretation of omics data. The developed Bio3graph approach, implemented as an executable language processing and graph visualisation workflow, is publically available at http://ropot.ijs.si/bio3graph/and can be utilised for modelling other biological systems, given that an adequate vocabulary is provided.

To investigate the dynamics of the potato-Potato virus Y (PVY) compatible interaction in relation to salicylic acid-controlled pathways we performed experiments using non-transgenic potato cv. Désirée, transgenic NahG-Désirée, cv. Igor and PVY(NTN), the most aggressive strain of PVY. The importance of salicylic acid in viral multiplication and symptom development was confirmed by pronounced symptom development in NahG-Désirée, depleted in salicylic acid, and reversion of the effect after spraying with 2,6-dichloroisonicotinic acid (a salicylic acid-analogue). We have employed quantitative PCR for monitoring virus multiplication, as well as plant responses through expression of selected marker genes of photosynthetic activity, carbohydrate metabolism and the defence response. Viral multiplication was the slowest in inoculated potato of cv. Désirée, the only asymptomatic genotype in the study. The intensity of defence-related gene expression was much stronger in both sensitive genotypes (NahG-Désirée and cv. Igor) at the site of inoculation than in asymptomatic plants (cv. Désirée). Photosynthesis and carbohydrate metabolism gene expression differed between the symptomatic and asymptomatic phenotypes. The differential gene expression pattern of the two sensitive genotypes indicates that the outcome of the interaction does not rely simply on one regulatory component, but similar phenotypical features can result from distinct responses at the molecular level.

In this white paper, we describe the founding of a new ELIXIR Community - the Systems Biology Community - and its proposed future contributions to both ELIXIR and the broader community of systems biologists in Europe and worldwide. The Community believes that the infrastructure aspects of systems biology - databases, (modelling) tools and standards development, as well as training and access to cloud infrastructure - are not only appropriate components of the ELIXIR infrastructure, but will prove key components of ELIXIR’s future support of advanced biological applications and personalised medicine. By way of a series of meetings, the Community identified seven key areas for its future activities, reflecting both future needs and previous and current activities within ELIXIR Platforms and Communities. These are: overcoming barriers to the wider uptake of systems biology; linking new and existing data to systems biology models; interoperability of systems biology resources; further development and embedding of systems medicine; provisioning of modelling as a service; building and coordinating capacity building and training resources; and supporting industrial embedding of systems biology. A set of objectives for the Community has been identified under four main headline areas: Standardisation and Interoperability, Technology, Capacity Building and Training, and Industrial Embedding. These are grouped into short-term (3-year), mid-term (6-year) and long-term (10-year) objectives.