1. Organelles and phytohormones : a network of interactions in plant stress responsesAndras Bittner, Agata Cieśla, Kristina Gruden, Tjaša Lukan, Sakil Mahmud, Markus Teige, Ute Vothknecht, Bernhard Wurzinger, 2022, pregledni znanstveni članek Povzetek: Phytohormones are major signaling components that contribute to nearly all aspects of plant life. They constitute an interconnected communication network to fine-tune growth and development in response to the ever-changing environment. To this end, they have to coordinate with other signaling components, such as reactive oxygen species and calcium signals. On the one hand, the two endosymbiotic organelles, plastids and mitochondria, control various aspects of phytohormone signaling and harbor important steps of hormone precursor biosynthesis. On the other hand, phytohormones have feedback actions on organellar functions. In addition, organelles and phytohormones often act in parallel in a coordinated matter to regulate cellular functions. Therefore, linking organelle functions with increasing knowledge of phytohormone biosynthesis, perception, and signaling will reveal new aspects of plant stress tolerance. In this review, we highlight recent work on organelle–phytohormone interactions focusing on the major stress-related hormones abscisic acid, jasmonates, salicylic acid, and ethylene. Ključne besede: abscisic acid (ABA), chloroplast, ethylene, mitochondria, jasmonates, plant organelles, phytohormones, salicylic acid (SA), plastids, stress signaling, retrograde signaling Objavljeno v DiRROS: 05.08.2024; Ogledov: 90; Prenosov: 116
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2. Intertwined roles of reactive oxygen species and salicylic acid signaling are crucial for the plant response to biotic stressTjaša Lukan, Anna Coll Rius, 2022, pregledni znanstveni članek 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: 113; Prenosov: 60
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3. CRISPR/Cas-mediated plant genome editing : outstanding challenges a decade after implementationTeodoro Cardi, Jana Murovec, Allah Bakhsh, Vladislava Galović, Tjaša Lukan, Kubilay Yıldırım, Milica Zlatković, Katrijn Van Laere, 2023, pregledni znanstveni članek Povzetek: The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants. Ključne besede: CRISPR applications, CRISPR platforms, gene regulations, mutant detection, plant regeneration Objavljeno v DiRROS: 05.08.2024; Ogledov: 85; Prenosov: 129
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4. 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, izvirni znanstveni članek Povzetek: 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. Ključne besede: potato virus Y, salicylic acid, hypersensitive response, programmed cell death, callose deposits, necrotic lesion Objavljeno v DiRROS: 24.07.2024; Ogledov: 107; Prenosov: 115
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5. 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, izvirni znanstveni članek Povzetek: 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. Ključne besede: cloning, plasmid construction, polymerase chain reaction Objavljeno v DiRROS: 24.07.2024; Ogledov: 159; Prenosov: 101
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6. 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, izvirni znanstveni članek Povzetek: 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. Ključne besede: immune signaling network, NADPH oxidase RBOHD, reactive oxygen species, salicylic acid, Sola-num tuberosum (potato), spatiotemporal response analysis, virus resistance, Potyvirus Objavljeno v DiRROS: 22.07.2024; Ogledov: 135; Prenosov: 115
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7. A mini-TGA protein modulates gene expression through heterogeneous association with transcription factorsŠpela Tomaž, Marko Petek, Tjaša Lukan, Karmen Pogačar, Katja Stare, Erica Teixeira Prates, Daniel A. Jacobson, Jan Zrimec, Gregor Bajc, Matej Butala, Maruša Pompe Novak, Ajda Taler-Verčič, Aleksandra Usenik, Dušan Turk, Anna Coll Rius, Kristina Gruden, 2022, izvirni znanstveni članek Povzetek: TGA (TGACG-binding) transcription factors, which bind their target DNA through a conserved basic region leucine zipper (bZIP) domain, are vital regulators of gene expression in salicylic acid (SA)-mediated plant immunity. Here, we investigated the role of StTGA2.1, a potato (Solanum tuberosum) TGA lacking the full bZIP, which we named a mini-TGA. Such truncated proteins have been widely assigned as loss-of-function mutants. We, however, confirmed that StTGA2.1 overexpression compensates for SA-deficiency, indicating a distinct mechanism of action compared with model plant species. To understand the underlying mechanisms, we showed that StTGA2.1 can physically interact with StTGA2.2 and StTGA2.3, while its interaction with DNA was not detected. We investigated the changes in transcriptional regulation due to StTGA2.1 overexpression, identifying direct and indirect target genes. Using in planta transactivation assays, we confirmed that StTGA2.1 interacts with StTGA2.3 to activate StPRX07, a member of class III peroxidases (StPRX), which are known to play role in immune response. Finally, via structural modeling and molecular dynamics simulations, we hypothesized that the compact molecular architecture of StTGA2.1 distorts DNA conformation upon heterodimer binding to enable transcriptional activation. This study demonstrates how protein truncation can lead to distinct functions and that such events should be studied carefully in other protein families. Objavljeno v DiRROS: 18.07.2024; Ogledov: 153; Prenosov: 75
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8. 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, izvirni znanstveni članek 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: 141; Prenosov: 143
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9. Studying cell death initiation using a digital microscopeTina Arnšek, Nuša Golob, Nastja Marondini, Maruša Pompe Novak, Kristina Gruden, Tjaša Lukan, 2023, izvirni znanstveni članek Povzetek: Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate. Ključne besede: digital microscope, cell death, inoculated leaves Objavljeno v DiRROS: 12.07.2024; Ogledov: 161; Prenosov: 88
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10. Candidate pathogenicity factor/effector proteins of ‘Candidatus Phytoplasma solani’ modulate plant carbohydrate metabolism, accelerate the ascorbate–glutathione cycle, and induce autophagosomesMarina Dermastia, Špela Tomaž, Rebeka Strah, Tjaša Lukan, Anna Coll Rius, Barbara Dušak, Timotej Čepin, Aleš Kladnik, Maja Zagorščak, Kristina Gruden, Maruša Pompe Novak, 2023, izvirni znanstveni članek Povzetek: 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. Ključne besede: autophagosome, effector, glycolysis, pathogenicity factor, StAMP Objavljeno v DiRROS: 24.08.2023; Ogledov: 734; Prenosov: 346
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