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Povzetek: 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.
Ključne besede: plant immune response, reactive oxygen species, ROS, salicylic acid, programmed cell death, biotic stress, biotechnology
Objavljeno v DiRROS: 05.08.2024; Ogledov: 46; Prenosov: 19
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Povzetek: 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.
Ključne besede: CRISPR/Cas9, hypersensitive response (HR)-conferred resistance, immune signaling, live cell imaging, Solanum tuberosum (potato), spatiotemporal analysis, stromules, virus resistance
Objavljeno v DiRROS: 17.07.2024; Ogledov: 105; Prenosov: 120
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Povzetek: Electrochemotherapy (ECT) is a clinically acknowledged method that combines the use of anticancer drugs and electrical pulses. Electrochemotherapy with bleomycin (BLM) can induce immunogenic cell death (ICD) in certain settings. However, whether this is ubiquitous over different cancer types and for other clinically relevant chemotherapeutics used with electrochemotherapy is unknown. Here, we evaluated in vitro in the B16-F10, 4T1 and CT26 murine tumor cell lines, the electrochemotherapy triggered changes in the ICD-associated damage-associated molecular patterns (DAMPs): Calreticulin (CRT), ATP, High Mobility Group Box 1 (HMGB1), and four immunologically important cellular markers: MHCI, MHC II, PD-L1 and CD40. The changes in these markers were investigated in time up to 48 h after ECT. We showed that electrochemotherapy with all three tested chemotherapeutics induced ICD-associated DAMPs, but the induced DAMP signature was cell line and chemotherapeutic concentration specific. Similarly, electrochemotherapy with CDDP, OXA or BLM modified the expression of MHC I, MHC II, PD-L1 and CD40. The potential of electrochemotherapy to change their expression was also cell line and chemotherapeutic concentration specific. Our results thus put the electrochemotherapy with clinically relevant chemotherapeutics CDDP, OXA and BLM on the map of ICD inducing therapies.
Ključne besede: electrochemotherapy, cisplatin, immune response
Objavljeno v DiRROS: 21.03.2024; Ogledov: 335; Prenosov: 201
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Povzetek: Immunocastration is a sustainable alternative to piglet castration but faces limited market acceptance. The phenomenon of non-responders has not to date been examined in detail, but adverse and stressful housing conditions (e.g., mixing of groups) might impair the success of vaccinations. Therefore, we evaluated the influence of housing conditions on the immune response after two Improvac® vaccinations at an age of 12 and 22 weeks, respectively. Boars, immunocastrates and barrows (n = 48 each) were assigned to three different housing conditions (n = 36 enriched, n = 36 standard n = 72 repeated social mixing). Immune response was quantified by measuring GnRH-binding and its consequences for testosterone concentrations, development of the genital tract and boar taint. Growth performance was evaluated via average daily gain (ADG). GnRH-binding and testosterone levels revealed that immunocastration reliably suppressed testicular functions after the 2nd vaccination. Housing conditions did not modify testicular function but influenced ADG as animals under mixing grew slower than those under enriched conditions. Gonadal status had only a slight impact on ADG except in immunocastrates, which showed a temporarily higher ADG after the 2nd vaccination. The results show that immunocastration is a reliable procedure under different housing conditions and competitive in terms of growth performance.
Ključne besede: immunocastration, vaccination, Improvac, non-responder, immune response, housing conditions, surgical castration, boar taint, growth performance, genital tract
Objavljeno v DiRROS: 06.09.2022; Ogledov: 865; Prenosov: 289
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