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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: 130; Downloads: 136
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Abstract: Background: Plant responses to a wide range of stresses are known to be regulated by epigenetic mechanisms. Path-ogen-related investigations, particularly against RNA viruses, are however scarce. It has been demonstrated that Arabi-dopsis thaliana plants defective in some members of the RNA-directed DNA methylation (RdDM) or histone modi-fication pathways presented differential susceptibility to the turnip mosaic virus. In order to identify genes directly targeted by the RdDM-related RNA Polymerase V (POLV ) complex and the histone demethylase protein JUMONJI14 (JMJ14) during infection, the transcriptomes of infected mutant and control plants were obtained and integrated with available chromatin occupancy data for various epigenetic proteins and marks. Results: A comprehensive list of virus-responsive gene candidates to be regulated by the two proteins was obtained. Twelve genes were selected for further characterization, confirming their dynamic regulation during the course of infection. Several epigenetic marks on their promoter sequences were found using in silico data, raising confidence that the identified genes are actually regulated by epigenetic mechanisms. The altered expression of six of these genes in mutants of the methyltransferase gene CURLY LEAF and the histone deacetylase gene HISTONE DEACETYLASE 19 suggests that some virus-responsive genes may be regulated by multiple coordinated epigenetic complexes. A temporally separated multiple plant virus infection experiment in which plants were transiently infected with one virus and then infected by a second one was designed to investigate the possible roles of the identified POLV- and JMJ14-regulated genes in wild-type (WT ) plants. Plants that had previously been stimulated with viruses were found to be more resistant to subsequent virus challenge than control plants. Several POLV- and JMJ14-regulated genes were found to be regulated in virus induced resistance in WT plants, with some of them poisoned to be expressed in early infection stages. Conclusions: A set of confident candidate genes directly regulated by the POLV and JMJ14 proteins during virus infection was identified, with indications that some of them may be regulated by multiple epigenetic modules. A sub-set of these genes may also play a role in the tolerance of WT plants to repeated, intermittent virus infections.Keywords Biotic stress, Defense priming, Epigenetics, Histone modifications, Induced resistance, Potyvirus, RNA-directed DNA methylation.
Keywords: biotic stress, defense priming, epigenetics, histone modifications, induced resistance, Potyvirus, RNA-directed DNA methylation
Published in DiRROS: 17.05.2024; Views: 321; Downloads: 947
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Abstract: In order to calculate amount of interstitial condensation in a building envelope, water vapour resistance of each layer is of importance. Once having it, 1D calculation according to ISO 13788 with monthly average vapour pressures can be applied. In EN 14509 sandwich panels are considered to be impermeable for water vapour, thus (according to the standard) water vapour cannot enter from outside and condensate in the panels. But it is not always true for real sandwich panels, because joints between neighbouring panels can cause non-neglecting water vapour bridges. Although in measurements of linear water vapour transmittance of the joints (Ψv) stationary boundary vapour pressures can be applied, the measurements can be long lasting. We shortened time needed to get Ψv performing simulations in Delphin6.0. We simulated panels and steel sheets with joints using constant boundary vapour pressures and compared the results with the results of measurements on the equivalent systems. In systems under consideration a sealant in built-in-state, located at a joint of a sandwich panel, is a compressed EPDM tube. It is impossible to directly measure its effective μ according to ISO 12572. In the paper we study to which precision it is possible to determine it using measurements and simulations. Once having effective μ of the sealant (if all other necessary material parameters available) one can simulate condensation in envelopes including sandwich panels in 2D according to EN 15026 using hourly climatic data. Another goal of the study was determination of differences in resulting Ψv values when varying narrowest part of the gap dGAP at the joint in the panels without any sealant. Results confirm significant sensibility of Ψv to variations of dGAP.
Keywords: water vapour condensation, water vapour diffusion, numerical simulations, water vapour resistance, linear water vapour transmittance, sendwich panels
Published in DiRROS: 05.03.2024; Views: 336; Downloads: 222
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Keywords: Al-Ti-B grain-refiners, quality, TiB2 particles, Al3Ti particles, electrical resistance measurements
Published in DiRROS: 01.02.2024; Views: 356; Downloads: 172
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Abstract: As a surface-hardening technique, plasma nitriding is a common procedure for improving the properties of conventional Ni-based alloys. The diffusion of nitrogen hardens a layer on the surface of the alloy, leading to better wear resistance and a higher coefficient of friction, as well as a higher surface hardness. This study reports the effect of plasma nitriding on additive-manufactured (AM) Inconel 625 (IN625) compared to its conventional manufactured and nitrided counterparts. The samples produced with the laser powder-bed fusion (LPBF) process were subsequently plasma nitrided in the as-built condition, stress-relief annealed at 870 °C and solution treated at 1050 °C. The plasma nitridings were carried out at 430 °C and 500 °C for 15 h. The growth kinetics of the nitride layer of the AM samples depends on the prior heat treatments and is faster in the as-built state due to the specific cellular structure. The lower nitriding temperature leads to the formation of expanded austenite in the nitride layer, while at the higher nitriding temperature, the expanded austenite decomposes and CrN precipitation occurs. The XRD and SEM analyses confirmed the presence of two layers: the surface layer and the diffusion layer beneath. The lower nitriding temperature caused the formation of expanded austenite or a combination of expanded austenite and CrN. The higher nitriding temperature led to the decomposition of the expanded austenite and to the formation/precipitation of CrN. The higher nitriding temperature also decreased the corrosion resistance slightly due to the increased number of precipitated Cr-nitrides. On the other hand, the wear resistance was significantly improved after plasma nitriding and was much less influenced by the nitriding temperature.
Keywords: additive manufacturing, powder-bed fusion, plasma nitriding, expanded austenite, wear and corrosion resistance, Ni-based alloy
Published in DiRROS: 31.01.2024; Views: 418; Downloads: 154
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