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The Role of selected exocyst subunits in response of plants to pathogen
Sabol, Peter ; Kulich, Ivan (advisor) ; Burketová, Lenka (referee) ; Dagdas, Yasin (referee)
In the recent years, there has been a growing number of publications indicating at the involvement of plant secretory pathway in defense against phytopathogens. Specifically, roles of plant exocyst complex have been explored in deeper detail in current research. Yet, exactly how exocyst- mediated exocytosis contributes to secretion of antimicrobials and cell wall-based defense remains unclear. In the presented Dissertation, I provide both experimental evidence and devise further hypotheses on selected exocyst's subunits in plant immune reactions. Particularly, I show that EXO70B1 exocyst subunit interacts with immunity-related RIN4 protein. Cleavage of RIN4 by AvrRpt2 Pseudomonas syringae effector protease releases both RIN4 fragments and EXO70B1 from the plasma membrane when transiently expressed in Nicotiana benthamiana leaves. I speculate on how this might have an implication in regulation of polarized callose deposition. In a co-authored opinion paper, we also hypothesize that EXO70B1-mediated autophagic degradation of TN2 resistance protein prevents its hyperactivation and lesion mimic phenotype development. In addition, in collaboration with my colleagues, I present data on EXO70H4's engagement in PMR4 callose synthase secretion, required for silica deposition. Representing a possible...
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Secretory pathway of plants in pathogene defence
Sabol, Peter ; Kulich, Ivan (advisor) ; Burketová, Lenka (referee)
Plants are sessile organisms that have to cope with the changes of their ambient environment. These changes include abiotic disturbances and stresses as well as biotic interactions with other organisms. In many of the biotic interactions, plant cells are hurt or damaged by various bacterial and fungal pathogens. Moreover, plants lack mobile immune cells, which would otherwise render them susceptible to most of these pathogens. Plants have, however, developed other mechanisms for suppressing the infection. Activating the components of the secretory pathway during resistance response is crucial step for suppressing the penetration and growth of pathogens. Fungal pathogens, such as powdery mildews, usually attempt to penetrate the cell wall in order to gain the access to protoplast providing nutrients and to exchange their virulence factors. On the other hand, pathogenic bacteria inhabit extracellular spaces of plant tissues. In both cases, however, the deposition of papilla into the plant cell wall was observed. Papilla thus seems to be the common defence mechanism. There is an increasing evidence confirming the indispensable role of polarized secretion in formation of papilla. Recently, SNARE proteins participating in papilla-associated defence have been described. PEN1 and SNAP33 syntaxins are of...
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The Effect of Asymmetrical Inclusion Arrangement on Concrete Specimens Subjected to Uniaxial Compression
Kováč, Maroš ; Sabol, Peter
Concrete is characterized by the diversity of its composition - heterogeneity. It is considered that the heterogeneous nature of concrete, in which coarse aggregate grains dominate, controls the process of crack propagation in concrete. In the case of normal concrete, coarse aggregate grains act as the significant stress concentrators, which determine the course of the force flow and thus influence the mechanical properties of the concrete. The aim of the research was to analyse the effect of the number of coarse aggregate grains (inclusions) in their asymmetrical arrangement on a series of test specimens of three different sizes. Attention was paid to the level of the ultimate stress value achieved by the specimens' failure under uniaxial compression.
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The role of exocyst subunit AtEXO70E2 in autophagy and secretion
Moulík, Michal ; Sabol, Peter (advisor) ; Janda, Martin (referee)
Exocyst is a protein complex composed of eight subunits, evolutionarily conserved in yeasts, animals, and plants. The main function of exocyst is to mediate the tethering of secretory vesicles to the plasma membrane. However, the involvement of exocyst in some other processes, especially in autophagy, has been recently discovered. Plant exocyst is specific because most of its subunits have multiple paralogs. The most diversified subunit is EXO70, which is encoded by 23 paralogous genes in Arabidopsis thaliana. In this thesis, I dealt with subunit AtEXO70E2 (AT5G61010), which has been localized to double-membrane compartments considerably reminiscent of autophagosomes. These compartments were named EXPOs (for exocyst-positive organelles) and described as a component of unconventional protein secretion pathways. There are also hints that EXO70E2 could play a role in autophagic processes. However, details of this relationship remained unexplored. For my experiments, I used stably transformed lines of A. thaliana and transiently transformed leaves of Nicotiana benthamiana. I performed numerous colocalization experiments, applied various pharmacological treatments to the studied lines, and analyzed a mutant line in the EXO70E2 gene. According to my observations, protein EXO70E2 is expressed especially...
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Use of transgenic plants in functional analysis of plant genes
Klincová, Martina ; Fischer, Lukáš (advisor) ; Sabol, Peter (referee)
Use of transgenic plants in functional analysis of plant genes Functional genomics makes it possible to detect the functions of uncharacterized genes. It allows you to understand not only the functions of individual genes, but also the relationships between them. The acquired knowledge can also be used in improving the properties of various cultivated plants. With decreasing cost and increasing sequencing efficiency, many plant genomes are sequenced, but without the associated function of individual genes. Reverse genetics approaches are used to characterize these genes. In the first place, these include approaches that include the analysis of insertional mutants or the use of RNA interference that inactivates or reduces the expression of the individual genes studied. We can also inactivate whether to specifically modify the function of a particular selected gene using site-specific endonucleases, which include meganucleases, zinc finger nucleases, TALEN (transcription activator-like effector nucleases) and CRISPR (clustered regularly interspaced short paindromic repeats). In contrast to inactivation approaches, there are approaches such as activating mutagenesis and ectopic expression, through which we increase the expression. In addition, gene expression and protein localization can be analyzed...
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Functional characterisation of AHL28 and its role in root system development
Škrabálková, Eliška ; Širl, Marek (advisor) ; Sabol, Peter (referee)
The root system architecture development is influenced by the interaction of numerous factors, one of which is the specific gene expression contributing to root development. The recently discovered AHL gene family, which, due to its characteristics act as transcriptional modulators, demonstrably comprises members taking part in regulation of root system development. Hereby we report the AHL28 gene that participates in this process. Its regulatory role corresponds with the expression pattern, regarding both the promotor activity and presence of nuclear localised protein. The expression can be observed in the cortex cells of transition zone of both primary and lateral roots. The phenothypic analysis of plants with modulated level of AHL28 transcription demonstrates its role in the primary root growth and differentiation, the apical meristem size and the number of lateral roots. Moreover, the gene also plays a role in vascular tissue patterning. Key words: AHL, nuclear protein, root system, apical meristem, lateral roots, xylem, Arabidopsis thaliana
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The Role of selected exocyst subunits in response of plants to pathogen
Sabol, Peter ; Kulich, Ivan (advisor) ; Burketová, Lenka (referee) ; Dagdas, Yasin (referee)
In the recent years, there has been a growing number of publications indicating at the involvement of plant secretory pathway in defense against phytopathogens. Specifically, roles of plant exocyst complex have been explored in deeper detail in current research. Yet, exactly how exocyst- mediated exocytosis contributes to secretion of antimicrobials and cell wall-based defense remains unclear. In the presented Dissertation, I provide both experimental evidence and devise further hypotheses on selected exocyst's subunits in plant immune reactions. Particularly, I show that EXO70B1 exocyst subunit interacts with immunity-related RIN4 protein. Cleavage of RIN4 by AvrRpt2 Pseudomonas syringae effector protease releases both RIN4 fragments and EXO70B1 from the plasma membrane when transiently expressed in Nicotiana benthamiana leaves. I speculate on how this might have an implication in regulation of polarized callose deposition. In a co-authored opinion paper, we also hypothesize that EXO70B1-mediated autophagic degradation of TN2 resistance protein prevents its hyperactivation and lesion mimic phenotype development. In addition, in collaboration with my colleagues, I present data on EXO70H4's engagement in PMR4 callose synthase secretion, required for silica deposition. Representing a possible...
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Role of exocyst at plant pathogen defense
Sabol, Peter ; Kulich, Ivan (advisor) ; Šašek, Vladimír Matěj (referee)
Exocyst is a protein complex conserved in yeast, animals and plants. It mediates tethering of a secretory vesicle to the plasma membrane in the semifinal step of exocytosis. Several roles of exocyst in the processes of cell polarization in plant cells have been implied, including polarized growth of polen tubes and root hairs, cytokinesis, deposition of seed coat pectin and possibly autophagy. One of the most recent roles of exocyst includes also a response to bacterial and fungal pathogens. Exo70B2 and Exo70H1 subunits were shown to play prominent roles in this respect, with Exo70H1 being responsible for mediating defense against bacterial (Pseudomonas syringae) and Exo70B2 defense against both bacterial and fungal (Blumeria graminis) pathogens. Recently, new data appeared indicating the interaction between Exo70B1 and RIN4 and Exo70A1 and NOI6, respectively. RPM-1 interacting protein 4 (RIN4) is a well known negative regulator of both basal and effector-triggered resistance. This thesis shows interaction between NOI6 and several exocyst subunits, confirming previous data. I show here that exocyst subunints interact specifically with N terminus of NOI6 protein and that this interaction is lost in the shorter version of NOI6 mimicking AvrRpt2 cleavage. Since AvrRpt2 is an effector protein from...
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Secretory pathway of plants in pathogene defence
Sabol, Peter ; Burketová, Lenka (referee) ; Kulich, Ivan (advisor)
Plants are sessile organisms that have to cope with the changes of their ambient environment. These changes include abiotic disturbances and stresses as well as biotic interactions with other organisms. In many of the biotic interactions, plant cells are hurt or damaged by various bacterial and fungal pathogens. Moreover, plants lack mobile immune cells, which would otherwise render them susceptible to most of these pathogens. Plants have, however, developed other mechanisms for suppressing the infection. Activating the components of the secretory pathway during resistance response is crucial step for suppressing the penetration and growth of pathogens. Fungal pathogens, such as powdery mildews, usually attempt to penetrate the cell wall in order to gain the access to protoplast providing nutrients and to exchange their virulence factors. On the other hand, pathogenic bacteria inhabit extracellular spaces of plant tissues. In both cases, however, the deposition of papilla into the plant cell wall was observed. Papilla thus seems to be the common defence mechanism. There is an increasing evidence confirming the indispensable role of polarized secretion in formation of papilla. Recently, SNARE proteins participating in papilla-associated defence have been described. PEN1 and SNAP33 syntaxins are of...
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