81. 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: 185; Downloads: 179
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82. Plant X-tender : an extension of the AssemblX system for the assembly and expression of multigene constructs in plantsTjaša Lukan, Fabian Machens, Anna Coll Rius, Špela Baebler, Katrin Messerschmidt, Kristina Gruden, 2018, original scientific article Abstract: 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.
Keywords: cloning, plasmid construction, polymerase chain reaction
Published in DiRROS: 24.07.2024; Views: 233; Downloads: 145
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83. Statistical modeling of long-term grapevine response to "Candidatus Phytoplasma solani" infection in the fieldAna Rotter, Petra Nikolić, Neža Turnšek, Polona Kogovšek, Andrej Blejec, Kristina Gruden, Marina Dermastia, 2018, original scientific article Abstract: Bois noir (BN) is the most widespread European grapevine yellows disease caused by ‘Candidatus Phytoplasma solani’. Although our knowledge of the mechanisms of interactions of this pathogenic bacteria with host is largely unknown, the plant-pathogen system of BN is commonly used as a model system for studying grapevine yellows diseases. We applied here a conceptual model of general plant pathology – a disease triangle for describing interactions among the host plant, the pathogen and the environment. We generated a proof-of-concept statistical model for disease triangle using original experimental data and different statistical and data mining approaches for a selected system of ‘Ca. P. solani’ infection of cv. ‘Chardonnay’ grapevine plants. We monitored individual plants from a single vineyard over a period of six years. Phytoplasma content, the expression of 21 selected grapevine genes and environmental conditions were recorded and related to disease severity. Our model predicts that in described conditions BN is a function of the expression of grapevine gene VvDMR6, summer rainfall and abundance of ‘Ca. P. solani’. The greatest impact among elements of the disease triangle is attributed to the pathogen, and is independent of the pathogen titer. We showed that this first de facto representation of the disease triangle is useful for showing disease dynamics over several years and could be applied to other plant-pathogen systems. The overall results of this study will contribute to understanding of ‘Ca. P. solani’ biology and its interactions with grapevine host.
Keywords: plant diseases, plant pathology
Published in DiRROS: 24.07.2024; Views: 396; Downloads: 195
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84. Structural basis for the multitasking nature of the potato virus Y coat proteinAndreja Kežar, Luka Kavčič, Martin Pólak, Jiři Nováček, Ion Gutiérrez-Aguirre, Magda Tušek-Žnidarič, Anna Coll Rius, Katja Stare, Kristina Gruden, Maja Ravnikar, David Pahovnik, Ema Žagar, Franci Merzel, Gregor Anderluh, Marjetka Podobnik, 2019, original scientific article Abstract: Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY’s flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA–coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein’s amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.
Keywords: plant pathogens, potato virus Y, viral RNA
Published in DiRROS: 23.07.2024; Views: 214; Downloads: 183
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85. Genotoxic effects of cylindrospermopsin, microcystin-LR and their binary mixture in human hepatocellular carcinoma (HepG2) cell lineLeticia Díez-Quijada, Klara Hercog, Martina Štampar, Metka Filipič, Ana M. Cameán, Angeles Jos, Bojana Žegura, 2020, original scientific article Abstract: Simultaneous occurrence of cylindrospermopsin (CYN) and microcystin-LR (MCLR) has been reported in the aquatic environment and thus human exposure to such mixtures is possible. As data on the combined effects of CYN/MCLR are scarce, we aimed to investigate the adverse effects related to genotoxic activities induced by CYN (0.125, 0.25 and 0.5 µg/mL) and MCLR (1 µg/mL) as single compounds and their combinations in HepG2 cells after 24 and 72 h exposure. CYN and CYN/MCLR induced DNA double-strand breaks after 72 h exposure, while cell cycle analysis revealed that CYN and CYN/MCLR arrested HepG2 cells in G0/G1 phase. Moreover, CYN and the combination with MCLR upregulated CYP1A1 and target genes involved in DNA-damage response (CDKN1A, GADD45A). Altogether, the results showed that after 72 h exposure genotoxic activity of CYN/MCLR mixture was comparable to the one of pure CYN. On the contrary, MCLR (1 µg/mL) had no effect on the viability of cells and had no influence on cell division. It did not induce DNA damage and did not deregulate studied genes after prolonged exposure. The outcomes of the study confirm the importance of investigating the combined effects of several toxins as the effects can differ from those induced by single compounds.
Published in DiRROS: 23.07.2024; Views: 178; Downloads: 164
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86. New alien Mediterranean biodiversity records 2020Michel Bariche, Sara A. A. Al-Mabruk, Maria A. Ateş, Adnan Büyük, Fabio Crocetta, Michail Dritsas, Diala Edde, Ana Fortič, Lovrenc Lipej, 2020, original scientific article Abstract: The current article presents 18 new records from seven Mediterranean countries. These records include one rhodophyte, four nudibranchs, two crustaceans, one stingray and 10 bony fishes. They are grouped by country as follows: Lebanon - first record of the Striped bass Morone saxatilis, the stingray Himantura leoparda, the Areolate grouper Epinephelus areolatus and the Spot-fin porcupinefish Diodon hystrix from various parts of the country; Turkey - first record of the invasive red alga Grateloupia turuturu from the sea of Marmara (region of Bandırma), the sea slug Goniobranchus obsoletus and the crab Arcania brevifrons from the Gulf of Antalya and the cladoceran Pleopis schmackeri from several locations along the Aegean Sea; Cyprus - first record of the alien sea slug Berthellina citrina from the region of Cape Greco and an observation of a butterflyfish Heniochus sp. from the northeastern side of the island; Greece - first record of the alien sea slug Anteaeolidiella lurana from the region of Heraklion in Crete and the record of the Atlantic spadefish Chaetodipterus faber and the Black surgeonfish Acanthurus cfr gahhm from Salamina Island; Slovenia - first record of the alien sea slug Thecacera pennigera from Izola; Italy - first record of the hybrid Striped bass (Morone saxatilis × Morone chrysops) from the northern Tyrrhenian Sea and a first record of the goldfish Carassius auratus from the region of Apulia; Libya - first record of the Red Sea goatfish Parupeneus forsskali and the African surgeonfish Acanthurus monroviae, respectively from the eastern (Al-Tamimi area) and the western shore (Al-Khums area).
Published in DiRROS: 23.07.2024; Views: 381; Downloads: 194
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87. Clinical outcomes in stage III non-small cell lung cancer patients treated with durvalumab after sequential or concurrent platinum-based chemoradiotherapy : single institute experienceMartina Vrankar, Karmen Stanič, Staša Jelerčič, Eva Ćirić, Ana Lina Vodušek, Jasna But-Hadžić, 2021, original scientific article Abstract: Chemoradiotherapy (ChT-RT) followed by 12-month durvalumab is the new standard treatment for unresectable stage III non-small cell lung cancer. Survival data for patients from everyday routine clinical practice is scarce, as well as potential impact on treatment efficacy of sequential or concomitant chemotherapy and the us-age of gemcitabine.Patients and methods. We retrospectively analysed unresectable stage III NSCLC patients who were treated with durvalumab after radical concurrent or sequential chemotherapy (ChT) from December 2017 and completed treat-ment until December 2020. We assessed progression free survival (PFS), overall survival (OS) and toxicity regarding baseline characteristic of patients.Results. Eighty-five patients with median age of 63 years of which 70.6% were male, 56.5% in stage IIIB and 58.8% with squamous cell carcinoma, were included in the analysis. Thirty-one patients received sequential ChT only, 51 patients received induction and concurrent ChT and 3 patients received concurrent ChT only. Seventy-nine patients (92.9%) received gemcitabine and cisplatin as induction chemotherapy and switched to etoposide and cisplatin during con-current treatment with radiotherapy (RT). Patients started durvalumab after a median of 57 days (range 12–99 days) from the end of the RT and were treated with the median of 10.8 (range 0.5–12 months) months. Forty-one patients (48.2%) completed treatment with planned 12-month therapy, 25 patients (29.4%) completed treatment early due to the toxicity and 16 patients (18.8%) due to the disease progression. Median PFS was 22.0 months, 12- and estimated 24-month PFS were 71% (95% CI: 61.2–80.8%) and 45.8% (95% CI: 32.7–58.9%). With the median follow-up time of 23 months (range 2–35 months), median OS has not been reached. Twelve- and estimated 24-month OS were 86.7% (95% CI: 79.5–93.9%) and 68.6% (95% CI: 57.2–79.9%).Conclusions. Our survival data are comparable with published research as well as with recently published real-world reports. Additionally, the regimen with gemcitabine and platinum-based chemotherapy as induction treatment was efficient and well tolerated.
Keywords: non-small cell lung cancer, stage III, chemoradiotherapy, durvalumab, acute toxicity
Published in DiRROS: 23.07.2024; Views: 229; Downloads: 50
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88. Non-indigenous species in the Mediterranean Sea : turning from pest to source by developing the 8Rs model, a new paradigm in pollution mitigationAna Rotter, Katja Klun, Janja Francé, Patricija Mozetič, Martina Orlando-Bonaca, 2020, original scientific article Abstract: For mitigation of the effects of pollution, the media, policy makers and, in turn, the scientific community and industry each provide contributions through development of a sense of urgency, and with guidelines and solutions. For non-indigenous species (NIS) that can frequently have negative impacts on the native biota, this is often conveyed in an emotive way to the general public, who are typically keen to help and to get personally involved. This might be through organization of cleaning campaigns, influence on the media, or collaboration with scientists, to inform them of the local presence and abundance of NIS. Alternatively, they might proactively develop technological solutions themselves. To assess the current state of affairs, we reviewed the presence and effects of NIS in the Mediterranean Sea. As so often, any well-planned and successful activity is directly linked to financing, or a lack thereof, and this leads to sometimes untargeted and sporadic measures that are developed within a project or over a limited timeframe, without any sustainability measures. Therefore, we also assessed the activities and strategies that have been financed in this area of NIS mitigation. Based on this review of the presence and impact of NIS, and previous and ongoing activities, we propose a new paradigm to mitigate such pollution: the 8Rs model (i.e., Recognize, Reduce, Replace, Reuse, Recycle, Recover/Restore, Remove, and Regulate). This model extends from the more traditional 3Rs model (i.e., Reduce, Reuse, and Recycle) that is often used and promoted for innovative waste management strategies. Importantly, the 8Rs model can be applied sequentially, for either prevention of NIS introduction, or preparation of mitigation measures. The 8Rs model was constructed based on Mediterranean NIS, although we believe it can be applied to other sources of pollution and other geographic areas. Importantly, the 8Rs model represents a general framework to organize and categorize future pollution mitigation strategies. This approach is essential for development of any action plan to influence the administrative and financial decision makers who essentially enable these activities, and therefore who have important roles in the guarantee of sustainability of these actions, and the creation of innovative societies.
Keywords: non-indigenous species, pollution mitigation, pollution mitigation, 8Rs model, quadruple helix, Mediterranean Sea, citizen science, communication, sustainability
Published in DiRROS: 22.07.2024; Views: 224; Downloads: 191
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89. A marine biodiversity observation network for genetic monitoring of hard-bottom communities (ARMS-MBON)Matthias Obst, Katrina Exter, A. Louise Allcock, Christos Arvanitidis, Alizz Axberg, Maria Bustamante, Ana Fortič, Borut Mavrič, Andreja Ramšak, 2020, original scientific article Abstract: Marine hard-bottom communities are undergoing severe change under the influence of multiple drivers, notably climate change, extraction of natural resources, pollution and eutrophication, habitat degradation, and invasive species. Monitoring marine biodiversity in such habitats is, however, challenging as it typically involves expensive, non-standardized, and often destructive sampling methods that limit its scalability. Differences in monitoring approaches furthermore hinders inter-comparison among monitoring programs. Here, we announce a Marine Biodiversity Observation Network (MBON) consisting of Autonomous Reef Monitoring Structures (ARMS) with the aim to assess the status and changes in benthic fauna with genomic-based methods, notably DNA metabarcoding, in combination with image-based identifications. This article presents the results of a 30-month pilot phase in which we established an operational and geographically expansive ARMS-MBON. The network currently consists of 20 observatories distributed across European coastal waters and the polar regions, in which 134 ARMS have been deployed to date. Sampling takes place annually, either as short-term deployments during the summer or as long-term deployments starting in spring. The pilot phase was used to establish a common set of standards for field sampling, genetic analysis, data management, and legal compliance, which are presented here. We also tested the potential of ARMS for combining genetic and image-based identification methods in comparative studies of benthic diversity, as well as for detecting non-indigenous species. Results show that ARMS are suitable for monitoring hard-bottom environments as they provide genetic data that can be continuously enriched, re-analyzed, and integrated with conventional data to document benthic community composition and detect non-indigenous species. Finally, we provide guidelines to expand the network and present a sustainability plan as part of the European Marine Biological Resource Centre (www.embrc.eu).
Keywords: benthic invertebrates, Marine Strategy Framework Directive (MSFD), Essential Biodiversity Variables (EBVs), Essential Ocean Variables (EOVs), European Marine Biological Resource Centre (EMBRC), non-indigenous species (NIS), genomic observatories, marine biodiversity assessment
Published in DiRROS: 22.07.2024; Views: 231; Downloads: 174
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90. New alien Mediterranean biodiversity records (October 2020)Michail Ragkousis, Nardjes Abdelali, Ernesto Azzurro, Ali Badreddine, Michel Bariche, Ghazi Bitar, Fabio Crocetta, Francesco Denitto, Ana Fortič, Borut Mavrič, 2020, original scientific article Abstract: This article includes 23 new records of alien and cryptogenic species in the Mediterranean Sea, belonging to 4 Phyla (Chordata, Echinodermata, Arthropoda and Mollusca), distributed from the Alboran to the Levantine Sea. Records are reported from eight countries listed from West to East as follows: Algeria: new records of the Atlantic blue crab Callinectes sapidus; Spain: further spread and establishment of the sea slug Lamprohaminoea ovalis in continental shores; Tunisia: first record of the Atlantic Blue Crab Callinectes sapidus in the Gulf of Gabes; Italy: a new occurrence of the pufferfish Lagocephalus sceleratus in Northern Ionian waters; first record of Cephalopholis taeniops in the Ionian Sea; first record of the redlip blenny, Ophioblennius atlanticus in the Ionian Sea; Slovenia: first record of the isopod Paranthura japonica in Slovenia; Greece: first record of the molluscs Eunaticina papilla, Plocamopherus ocellatus and the fish Cheilodipterus novemstriatus; first record of the ascidian Ecteinascidia turbinata in Kriti; the long-spined sea urchin Diadema setosum in the Ionian Sea; Turkey: first record of the sea spider Ammothea hilgendorfi; the stomatopod Cloridina cf. ichneumon; the fishes Pempheris rhomboidea from the Sea of Marmara and Paranthias furcifer from the Aegean Sea; Lebanon: new records of the fishes Arothron hispidus, Rachycentron canadum, Heniochus intermedius and Acanthurus monroviae; first record of Acanthostracion polygonius. The records of Cloridina cf. ichneumon from southern Turkey and the fish Acanthostracion polygonius from Lebanon, both being the first Mediterranean records, are noteworthy.
Published in DiRROS: 22.07.2024; Views: 326; Downloads: 174
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