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Plant exocyst dependent secretory pathway and iron nutrition
Batík, Adam ; Žárský, Viktor (advisor) ; Vaňková, Radomíra (referee)
Previous results from our lab suggested possible phenotypic deviation of a double T-DNA knock- out mutant of an EXOCYST subunit EXO70E2 and vacuolar transmembrane protein LAZ1H1 upon iron starvation. To confirm these preliminary results, single mutants were obtained, with EXO70E2 knock-out mutant being produced here using CRISPR/Cas9 technology. Result did not confirm the previous findings in neither of the mutants tested (exo70e2, laz1h1, exo70e2/laz1h1 and exo70h4). This discrepancy is attributed to improper iron starvation protocol and different storage age of seeds used in the previously conducted experiments. Coumarins are synthetized and secreted from roots during iron starvation; protocol for their visualization was adopted and WT compared with mutants. No phenotypic deviations were observed in mutants. Pharmacological treatments of iron starved WT plants using BFA, wortmannin and concanamycin A did not suggest that endomembrane vesicle trafficking affect accumulation or secretion of coumarins. Previously published susceptibility of T-DNA knock-out mutant of EXO70E2 to drought stress was tested using CRISPR/Cas9 mutant produced here, the reported susceptibility was not confirmed. Upregulation of EXO70E2 mRNA upon UV exposure reported in eFP browser was also tested and not confirmed under...
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Analysis of the relationship between the function of the ARP2/3 protein complex and exocytosis in plant cells
Ničová, Klára ; Schwarzerová, Kateřina (advisor) ; Synek, Lukáš (referee)
Plant cells exhibit two types of growth: diffuse and apical. Mutation of the ARP2/3 complex, which is an actin nucleator, leads to phenotypic expression in both diffusely and apically growing cells. Many of these changes, such as impaired epidermal cell adhesion of hypocotyl cells or slower growth of pollen tubes, suggest that the observed phenotypes are cell wall related. Cell wall components are transported into the apoplastic space by exocytosis. Many factors are involved in controlled exocytosis, one of the most studied being the exocyst tethering complex. Mutants of the exocyst complex show phenotypes in both diffusely and apically growing cells, indicating that this complex is important for both types of growth. In addition, subunit EXO84b of the exocyst complex interacts with subunits of the ARP2/3 complex. In this thesis, I investigated the effect of mutation of ARP2/3 complex subunits on exocytosis of diffusely and apically growing cells by observing the localization and dynamics of the fluorescently labeled marker EXO84b-GFP. In epidermal hypocotyl cells (a model of diffuse growth), EXO84b-GFP had a shorter lifetime at the plasma membrane in ARP2/3 mutants compared to wild type. The pattern of its localization to the plasma membrane was also slightly different. It was not possible to...
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Vazba paralogů EXO70 na ATG8 a funkční rozdělení rodiny EXO70 dle účasti v autofagii (Arabidopsis thaliana).
Semerádová, Hana ; Kulich, Ivan (advisor) ; Motyka, Václav (referee)
The exocyst, an octameric protein complex conserved among all eukaryotes, mediates tethering of the vesicle prior to its fusion with the target membrane. Apart from the function of exocyst in exocytosis, new studies from both mammalian and plant fields report its involvement in the cellular self-eating process called autophagy. In land plants the number of paralogs of some exocyst subunits is extraordinarily large. There are 23 paralogs of Exo70 subunit in Arabidopsis thaliana. It is supposed that these paralogs have acquired functional specialization during the evolution - including involvement in autophagy. Using yeast two- hybrid assay it is shown here that Exo70B1 and Exo70B2, but not other Arabidopsis Exo70 paralogs interact with Atg8, an autophagosomal marker. The proximity of these two paralogs and Atg8 in vivo was confirmed by independent Förster resonance energy transfer (FRET) method. Interestingly, interaction of Atg8f with Exo70B2 paralog appears to be stronger than with Exo70B1. Exo70B1-mRUBY expressed under the natural promoter shows punctate membrane structures that are mostly static. That changes after the tunicamycin treatment - movement of some of these dots was induced. Homology modeling of Exo70B1 and Exo70B2 proteins tertiary structure in combination with bioinformatic prediction based...
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The role of the exocyst in development and maintaining of cell migration structures
Vaškovičová, Katarína ; Brábek, Jan (advisor) ; Žárský, Viktor (referee)
The exocyst is a hetero-octameric protein complex which mediates tethering secretory vesicles to specific sites of plasma membrane for polarized exocytosis. The exocyst was long known to contribute to processes such as yeast budding, cytokinesis, epithelia polarization and neurite outgrowth. Recently, the role of the exocyst in regulation of actin cytoskeleton and cell migration was discovered. It was shown, that the exocyst is important for formation of cell migration structures such as lamellipodia and filopodia in motile cells and invadopodia in invasive cancer cells. These structures are all actin-based membrane protrusions and the exocyst can through its Exo70 subunit interact with the Arp2/3 complex, the activator of actin nucleation. By binding and activating the Arp2/3 complex, the exocyst mediates actin polymerization resulting in formation of these membrane protrusions. Furthermore, the exocyst probably targets the Arp2/3 complex to specific sites of plasma membrane that are intended to become membrane protrusions. In addition, the exocyst mediates secretion of matrix metalloproteinases (MMPs) in invadopodia. MMPs are important for degradation of the extracellular matrix, an essential process in cancer cell invasion. The exocyst seems to be part of the cascade downstream of cytokines...
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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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Conventional and Novel Functions of the Exocyst Complex in Plants
Kulich, Ivan ; Žárský, Viktor (advisor) ; Baluška, František (referee) ; Hašek, Jiří (referee)
Exocyst is an octameric protein complex, conserved across all Eukaryotes. Its role, originally described in yeast, resides in a tethering of the secretory vesicles to the plasma membrane prior to the membrane fusion of the two membranes. Subunits SEC3 and EXO70 are believed to be spatial landmarks for the vesicles delivery. While yeast genome encodes single EXO70, we find dozens of them in land plants (23 in Arabidopsis). This work is focused at a role of the exocyst complex in plant cells. Its first part documents, that exocyst is essential for delivery of the cell wall components, namely pectins, but also for pathogen induced secondary cell wall thickening. Second part reveals an unconventional role of EXO70B1 subunit harboring exocyst subcomplex at an autophagic pathway to the vacuole and raises many questions about plant secretory pathway.
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Study of the interaction of proteins involved in the exocytosis in the plant defense against pathogens
Ortmannová, Jitka ; Pečenková, Tamara (advisor) ; Burketová, Lenka (referee)
Plant cells are mostly immobile, therefore it is crucial for them to distinguish a direction of the signals coming into the cell and on the other hand they have to precisely target their own signals. To achieve this communication, plant cells use endomembrane system and secretory vesicles, which are recruited to the specific membrane domains. This ability is important for the plant defense against pathogenic microorganisms and it even forms a part of the innate plant immunity. Two complexes, the exocyst and SNARE, play a prominent role in the process of polarized secretion. In this work, we focused on a possible interaction between these two complexes in preinvasive defense and particularly, we studied the exocyst subunit EXO70B2 and SNARE protein SYP121. We obtained double mutant plants of EXO70B2 and SYP121 by utilizing the reverse genetics approach. These mutant plants did not show any obvious phenotype under standard conditions in comparison with Wt plants. However, we observed marked defects of secretory pathway in double mutant exo70B2/syp121 after infection by pathogenic fungi Blumeria graminis f. sp. hordei. Using histochemical staining, we described problems with the deposition of defensive papilla and secretion of haustorial encasement. We prove that these defects are not connected with...
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Secretion and autophagy in plant defence against microbes
Dobešová, Karolína ; Žárský, Viktor (advisor) ; Burketová, Lenka (referee)
Plants are sessile organisms and when attacked by microbes, they cannot easily run away. For this reason, they have developed sophisticated defensive mechanisms, that allow them to defend themselves. Since plants, unlike mammals, do not have any special immune cells, their defense takes place in each cell separately. The key moment during a microbial infection is the recognition of the microbe by the plant through its released molecular patterns (mostly proteins) associated with microbes (MAMPs). MAMPs trigger signaling cascades that lead to the secretion of antimicrobial compounds to the site of an attack. The process of autophagy is also important in the defense against microbes, which not only maintains a cellular homeostasis and controls the level of phytohormones and defense proteins in the plant cytoplasm, but also participates in the secretory activity of the cell. Recent analyzes of plant secretome have shown that plants secrete many proteins (including defensive ones) independently of the signal peptide and compartments of a conventional secretion. During exocytosis a vesicle fuses with the cytoplasmic membrane. The octameric protein complex exocyst and SNARE proteins take part in this process. The exocyst complex is highly diversified in plants - especially it's EXO70 subunit, which is...
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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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Functional specialization of EXO70A and EXO70B paralogs of the EXO70 exocyst subunit in Arabidopsis.
Markovič, Vedrana ; Žárský, Viktor (advisor) ; Yalovsky, Shaul (referee) ; Ovečka, Miroslav (referee)
Many studies in different eukaryotes have shown the importance of the vesicle-tethering exocyst complex for cellular processes dependent on intensive polarized secretion. The plant exocyst complex is crucial for regulation of cell polarity, morphogenesis, and defence. In land plants, gene encoding the EXO70 exocyst subunit multiplied into many paralogs, but only a few of them have been functionally described. In this thesis, the EXO70A2 isoform, a member of the EXO70.1 subfamily, was found to be the main EXO70 exocyst subunit involved in the canonical function of the exocyst complex in Arabidopsis pollen. EXO70A2 is important for several stages of pollen development-pollen grain maturation, germination, and pollen tube growth. Pollen-expressed EXO70A2 was the only EXO70 isoform able to substitute for the function of EXO70A1 in the sporophyte, but not vice-versa. This indicates partial functional redundancy of these two closely related isoforms and a high specificity for pollen-related processes. The finding that the exocyst is targeted to the plasma membrane via EXO70A1 subunit is further elaborated in the thesis. EXO70A1 binds plasma membrane via interactions with specific phospholipids that form a unique plasma membrane-lipid signature in plants. Other isoform, EXO70B1 from the EXO70.2 subfamily,...
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