1. The StPti5 ethylene response factor acts as a susceptibility factorby negatively regulating the potato immune responseto pathogensAnna Coll Rius, Tjaša Lukan, Katja Stare, Maja Zagorščak, Tjaša Mahkovec Povalej, Špela Baebler, Salomé Prat, Núria Sánchez Coll, Marc Valls, Marko Petek, Kristina Gruden, 2024, original scientific article Abstract: Ethylene response factors (ERFs) have been associated with biotic stress in Arabidopsis, while their function in non-model plants is still poorly understood. Here we investigated the role of potato ERF StPti5 in plant immunity.
We show that StPti5 acts as a susceptibility factor. It negatively regulates potato immunity against potato virus Y and Ralstonia solanacearum, pathogens with completely different modes of action, and thereby has a different role than its orthologue in tomato. Remarkably, StPti5 is destabilised in healthy plants via the autophagy pathway and accumulates exclusively in the nucleus upon infection. We demonstrate that StEIN3 and StEIL1 directly bind the StPti5 promoter and activate its expression, while synergistic activity of the ethylene and salicylic acid pathways is required for regulated StPti expression.
To gain further insight into the mode of StPti5 action in attenuating potato defence responses, we investigated transcriptional changes in salicylic acid deficient potato lines with silenced StPti5 expression. We show that StPti5 regulates the expression of other ERFs and downregulates the ubiquitin-proteasome pathway as well as several proteases involved in directed proteolysis.
This study adds a novel element to the complex puzzle of immune regulation, by deciphering a two-level regulation of ERF transcription factor activity in response to pathogens. Keywords: ethylene response factor, immune signalling, potato virus Y, Pti5, Ralstonia solanacearum, Solanum tuberosum, susceptibility factor Published in DiRROS: 29.08.2024; Views: 48; Downloads: 565
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2. Challenges of implementing bridge weigh-in-motion on a century-old steel-riveted railway bridgeDoron Hekič, Mirko Kosič, Jan Kalin, Aleš Žnidarič, Andrej Anžlin, 2024, published scientific conference contribution Abstract: This study explores the challenges and methodologies involved in implementing bridge weigh-in-motion (B-WIM) system on a century-old steel riveted railway bridge. A unique aspect of this study, funded by the EU H2020 Shift2Rail Joint Undertaking, was the adaptation of B-WIM systems to the specific constraints of railway bridges since, traditionally, this technology is used to collect heavy gross vehicle loading data on road bridges. The paper details the experimental setup on an old steel-riveted railway bridge, including sensor placement and calibration processes. It highlights the complexities encountered, such as differences in bridge response due to passenger and other type of trains. It introduces the system calibration strategy using known axle loads from passenger locomotives. The study provides insights into the structural response of old steel bridges under traffic loads, contributing valuable data to the field of railway bridge monitoring and maintenance. Keywords: bridge, B-WIM, bridge Weigh-in-Motion system, numerical model updating, bridge response measurements, railways Published in DiRROS: 14.08.2024; Views: 122; Downloads: 114
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3. Load testing of the first stress ribbon bridge in SloveniaĐorđe Đukić, Doron Hekič, Mirko Kosič, Rok Vezočnik, Andrej Anžlin, Andrej Štrukelj, Marjan Pipenbaher, Tomaž Weingerl, 2024, published scientific conference contribution Abstract: Paper presents selected results of the diagnostic load testing of a newly-built footbridge in Novo Mesto, Slovenia. The bridge is significant as the first bridge in Slovenia being built with the stress ribbon technology. The diagnostic load testing took place in March 2023 and comprised of three-stage static load testing, dynamic testing with a light truck driving over artificial obstacles, and measurement of ambient vibrations. The consistency between the actual response of the bridge and the response of the finite element (FE) model, used in the design, was evaluated by comparing vertical displacements from the static load testing, natural frequencies and mode shapes. The comparison of the results indicates a satisfactory agreement between the measurements and the response of the FE model, confirming the appropriateness of the employed FE model. Keywords: bridge, bridge response measurements, stress ribon, load test Published in DiRROS: 14.08.2024; Views: 124; Downloads: 107
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4. Intertwined roles of reactive oxygen species and salicylic acid signaling are crucial for the plant response to biotic stressTjaša Lukan, Anna Coll Rius, 2022, review article Abstract: One of the earliest hallmarks of plant immune response is production of reactive oxygen species (ROS) in different subcellular compartments, which regulate plant immunity. A suitable equilibrium, which is crucial to prevent ROS overaccumulation leading to oxidative stress, is maintained by salicylic acid (SA), a chief regulator of ROS. However, ROS not only act downstream of SA signaling, but are also proposed to be a central component of a self-amplifying loop that regulates SA signaling as well as the interaction balance between different phytohormones. The exact role of this crosstalk, the position where SA interferes with ROS signaling and ROS interferes with SA signaling and the outcome of this regulation, depend on the origin of ROS but also on the pathosystem. The precise spatiotemporal regulation of organelle-specific ROS and SA levels determine the effectiveness of pathogen arrest and is therefore crucial for a successful immune response. However, the regulatory interplay behind still remains poorly understood, as up until now, the role of organelle-specific ROS and SA in hypersensitive response (HR)-conferred resistance has mostly been studied by altering the level of a single component. In order to address these aspects, a sophisticated combination of research methods for monitoring the spatiotemporal dynamics of key players and transcriptional activity in plants is needed and will most probably consist of biosensors and precision transcriptomics. Keywords: plant immune response, reactive oxygen species, ROS, salicylic acid, programmed cell death, biotic stress, biotechnology Published in DiRROS: 05.08.2024; Views: 156; Downloads: 79
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5. Potato Virus Y infection hinders potato defence response and renders plants more vulnerable to Colorado potato beetle attackMarko Petek, Ana Rotter, Polona Kogovšek, Špela Baebler, Axel Mithöfer, Kristina Gruden, 2014, original scientific article Abstract: In the field, plants are challenged by more than one biotic stressor at the same time. In this study, the molecular interactions between potato (Solanum tuberosum L.), Colorado potato beetle (Leptinotarsa decemlineata Say; CPB) and Potato virus YNTN (PVYNTN) were investigated through analyses of gene expression in the potato leaves and the gut of the CPB larvae, and of the release of potato volatile compounds. CPB larval growth was enhanced when reared on secondary PVYNTN-infected plants, which was linked to decreased accumulation of transcripts associated with the antinutritional properties of potato. In PVYNTN-infected plants, ethylene signalling pathway induction and induction of auxin response transcription factors were attenuated, while no differences were observed in jasmonic acid (JA) signalling pathway. Similarly to rearing on virus-infected plants, CPB larvae gained more weight when reared on plants silenced in JA receptor gene (coi1). Although herbivore-induced defence mechanism is regulated predominantly by JA, response in coi1-silenced plants only partially corresponded to the one observed in PVYNTN-infected plants, confirming the role of other plant hormones in modulating this response. The release of β-barbatene and benzyl alcohol was different in healthy and PVYNTN-infected plants before CPB larvae infestation, implicating the importance of PVYNTN infection in plant communication with its environment. This was reflected in gene expression profiles of neighbouring plants showing different degree of defence response. This study thus contributes to our understanding of plant responses in agro-ecosystems. Keywords: insect midgut transcriptional response, gene expression, plant defence, volatile organic compounds, potato Published in DiRROS: 02.08.2024; Views: 146; Downloads: 119
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6. Cell death is not sufficient for the restriction of potato virus Y spread in hypersensitive response-conferred resistance in potatoTjaša Lukan, Špela Baebler, Maruša Pompe Novak, Katja Guček, Maja Zagorščak, Anna Coll Rius, Kristina Gruden, 2018, original scientific article Abstract: 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. Keywords: potato virus Y, salicylic acid, hypersensitive response, programmed cell death, callose deposits, necrotic lesion Published in DiRROS: 24.07.2024; Views: 158; Downloads: 144
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7. Potato virus Y infection alters small RNA metabolism and immune response in tomatoMaria I. Prigigallo, Maja Križnik, Domenico De Paola, Domenico Catalano, Kristina Gruden, Mariella M. Finetti-Sialer, Fabrizio Cillo, 2019, original scientific article Abstract: Potato virus Y (PVY) isolate PVYC-to induces growth reduction and foliar symptoms in tomato, but new vegetation displays symptom recovery at a later stage. In order to investigate the role of micro(mi)RNA and secondary small(s)RNA-regulated mechanisms in tomato defenses against PVY, we performed sRNA sequencing from healthy and PVYC-to infected tomato plants at 21 and 30 days post-inoculation (dpi). A total of 792 miRNA sequences were obtained, among which were 123 canonical miRNA sequences, many isomiR variants, and 30 novel miRNAs. MiRNAs were mostly overexpressed in infected vs. healthy plants, whereas only a few miRNAs were underexpressed. Increased accumulation of isomiRs was correlated with viral infection. Among miRNA targets, enriched functional categories included resistance (R) gene families, transcription and hormone factors, and RNA silencing genes. Several 22-nt miRNAs were shown to target R genes and trigger the production of 21-nt phased sRNAs (phasiRNAs). Next, 500 phasiRNA-generating loci were identified, and were shown to be mostly active in PVY-infected tissues and at 21 dpi. These data demonstrate that sRNA-regulated host responses, encompassing miRNA alteration, diversification within miRNA families, and phasiRNA accumulation, regulate R and disease-responsive genes. The dynamic regulation of miRNAs and secondary sRNAs over time suggests a functional role of sRNA-mediated defenses in the recovery phenotype. Keywords: RNA silencing, plant defense response, Potato virus Y, molecular plant-virus interactions, microRNA, secondary small interfering RNA, small RNA sequencing, phasiRNA Published in DiRROS: 24.07.2024; Views: 698; Downloads: 149
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8. Chocolate and risk of chronic disease : a systematic review and dose-response meta-analysisJakub Morze, Carolina Schwedhelm, Aleksander Benčič, Georg Hofmann, Heiner Boeing, Katarzyna Przybylowicz, Lukas Schwingshackl, 2020, original scientific article Abstract: Purpose
Evidence for the association between chocolate intake and risk of chronic diseases is inconclusive. Therefore, we aimed to synthesize and evaluate the credibility of evidence on the dose-response association between chocolate consumption with risk of all-cause mortality, coronary heart disease (CHD), stroke, heart failure (HF), type 2 diabetes (T2D), colorectal cancer (CRC), and hypertension.
Methods
Prospective studies were searched until July 2018 in PubMed, Embase, and Web of Science. Random-effects meta-analyses comparing highest versus lowest intake categories, linear, and non-linear dose-response analyses were conducted. The credibility of evidence was evaluated with the NutriGrade scoring-system.
Results
Overall, 27 investigations were identified (n = 2 for all-cause mortality, n = 9 for CHD, n = 8 for stroke, n = 6 for HF, n = 6 for T2D, n = 2 for hypertension and CRC, respectively). No associations with HF (RR 0.99, 95% CI 0.94, 1.04) and T2D (RR 0.94, 95% CI 0.88, 1.01) per each 10 g/day increase in chocolate intake were observed in the linear dose-response meta-analyses. However, a small inverse association for each 10 g/daily increase could be shown for the risk of CHD (RR 0.96, 95% CI 0.93, 0.99), and stroke (RR 0.90, 95% CI 0.82, 0.98). The credibility of evidence was rated either very low (all-cause mortality, HF, T2D, CRC or hypertension) or low (CHD, stroke).
Conclusion
Chocolate consumption is not related to risk for several chronic diseases, but could have a small inverse association with CHD and stroke. Our findings are limited by very low or low credibility of evidence, highlighting important uncertainty for chocolate–disease associations. Keywords: chocolate, meta-analysis, dose-response, credibility of evidence, chronic diseases Published in DiRROS: 22.07.2024; Views: 154; Downloads: 124
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9. Precision transcriptomics of viral foci reveals the spatial regulation of immune-signaling genes and identifies RBOHD as an important player in the incompatible interaction between potato virus Y and potatoTjaša Lukan, Maruša Pompe Novak, Špela Baebler, Magda Tušek-Žnidarič, Aleš Kladnik, Maja Križnik, Andrej Blejec, Maja Zagorščak, Katja Stare, Barbara Dušak, Anna Coll Rius, Stephan Pollmann, Karolina Morgiewicz, Jacek Hennig, Kristina Gruden, 2020, original scientific article Abstract: Whereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA-deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA-seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling. Keywords: immune signaling network, NADPH oxidase RBOHD, reactive oxygen species, salicylic acid, Sola-num tuberosum (potato), spatiotemporal response analysis, virus resistance, Potyvirus Published in DiRROS: 22.07.2024; Views: 169; Downloads: 132
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10. CRISPR/Cas9-mediated fine-tuning of miRNA expression in tetraploid potatoTjaša Lukan, Florian Veillet, Maja Križnik, Anna Coll Rius, Tjaša Mahkovec Povalej, Karmen Pogačar, Katja Stare, Laura Chauvin, Jean-Eric Chauvin, Kristina Gruden, 2022, original scientific article Abstract: MicroRNAs (miRNAs) are small noncoding RNAs, which modulate the abundance and spatiotemporal accumulation of target mRNAs at transcriptional and post-transcriptional levels and through that play important roles in several biological processes in plants. Here we show that in polyploid species, CRISPR/Cas9 system can be used for fine-tuning of miRNA expression, which can have broader range of applications compared to knock-out mutants. We established the complete pipeline for CRISPR-Cas9-mediated modulation of miRNA expression in potato. It consists of (1) design and assembly of dual sgRNA CRISPR/Cas9 constructs, (2) transient transfection of protoplasts following fast and efficient screening by high resolution melting analysis to select functional sgRNAs, and (3) stable transformation of potato explants with functional sgRNAs and selection of regenerated transgenic lines with desired mutations and desired miRNA abundance based on sequencing and RT-qPCR. We show that miRNA-editing using dual sgRNA approach results in different types of mutations among transgenic lines but also in different alleles of the same plant, which are target site-dependent. The most frequent were short deletions, but we also detected 1-nt insertions (T or G), deletions between two sgRNAs and larger deletions. miRNA abundance correlates with the frequency and type of introduced mutations, as more extensive mutations in more alleles result in lower miRNA abundance. Interestingly, some mutated loci can generate alternative miRNAs, now novel targets were however predicted for those. In all transgenic lines with Cas9 expression, we detected mutations, suggesting high efficiency of Cas9-editing. We confirmed the miRNA-editing efficiency of our optimised approach in two different potato genotypes and three different loci. Keywords: CRISPR/Cas9, hypersensitive response (HR)-conferred resistance, immune signaling, live cell imaging, Solanum tuberosum (potato), spatiotemporal analysis, stromules, virus resistance Published in DiRROS: 17.07.2024; Views: 177; Downloads: 164
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