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Functional characterization of plant EXO70 exocyst subunit isoforms and their membrane targeting mechanisms
Sekereš, Juraj ; Potocký, Martin (advisor) ; Jaillais, Yvon (referee) ; Ischebeck, Till (referee)
Vesicle tethering complex exocyst is one of the key regulators of the cell polarity and morphogenesis in eukaryotes. The complex interacts with the secretory vesicle, as well as plasma membrane, and facilitates formation of cis SNARE complex leading into fusion of the vesicle with target destination. Two of the eight exocyst subunits, the SEC3 and EXO70 are known to bind plasma membrane via protein and lipid interactors in Opisthokont model organisms. Genomes of angiosperm plants encode a surprisingly wide repertoire of EXO70 isoforms with over 20 present in both Arabidopsis and diploid tobacco genome. It has been proposed that different EXO70 isoforms would form parts of functionally distinct subtypes of the plant exocyst complex driving membrane trafficking to various membrane domains. Specific interactions of peripheral membrane proteins with particular membrane phospholipids largely contribute to targeting of cellular components to subcellular compartments and membrane domains. This thesis focuses on role of protein-lipid interactions in regulation of plant cell polarity and contributes to functional analysis of the plant EXO70 family diversity. We introduce the topic with the theoretical reviews summarizing role of protein-lipid interactions in establishing plant cell membrane domains at...
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Cellular Protein Transport and Its Role in Patogenesis
Najdrová, Vladimíra ; Doležal, Pavel (advisor) ; Uzlíková, Magdalena (referee)
The main topic of this thesis are the protein secretion processes in several important human parasites - Toxoplasma gondii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp. and Giardia intestinalis. Described here are the parasite's and the host proteins which participate in the pathogenic processes involving the protein secretion. As shown here, the protein secretion into the host environment is one of key tools serving the parasite to survive within and manipulate the host organism. Interestingly, different parasitic organisms use functionally and evolutionary distinct strategies to fulfill this aim. Key words secretory pathway, translocon, signal sequence, Toxoplasma gongii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp., Giardia intestinalis
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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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Specificity of selected exocyst subunits in trichome development
Glanc, Matouš ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee)
Trichomes are fine epidermal outgrowths covering aerial organs of most land plants. Although unicellular trichomes of Arabidopsis thaliana have long been used as a model system in plant cell and developmental biology, surprisingly little is known about the processes involved in cell wall biogenesis during the last stage of trichome maturation. A role of EXO70H4, a putative subunit of the vesicle tethering complex exocyst, in trichome maturation has recently been identified in our laboratory. Image analysis, histochemical detection and FT-IR spectroscopy methods were used in this study to analyze cell wall defects of the exo70H4 LOF mutant, revealing the mutation causes altered deposition of pectins and possibly also lignins and hemicelluloses. Transgenic lines with EXO70 paralogues driven by the EXO70H4 promoter were prepared and their analysis revealed that the closest paralogue EXO70H3, unlike EXO70A1 and EXO70B1, can complement the exo70H4 mutation. Based on the results, questions concerning trichome cell wall composition, the role of EXO70H4 in trichome maturation and functions of the plant exocyst complex are discussed. Keywords: Arabidopsis, trichome, cell wall, secretory pathway, exocyst complex, EXO70H4, FT-IR spectroscopy
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Secretory pathway in plant cell wall polysaccharide biosynthesis and FT-IR spectroscopy methods in plant cell wall composition analysis
Glanc, Matouš ; Žárský, Viktor (advisor) ; Votrubová, Olga (referee)
All plant cells are encapsulated in a cell wall that determines the cells' shape and size and is essential to many of their vital processes. The cell wall of streptophyte plants is composed mainly of polysaccharides of high molecular weight. Cellulose, the main constituent of the plant cell wall, is synthesized by protein complexes bound to the PM, while hemicelluloses and pectins are synthesized in the Golgi apparatus. Several proteins that participate in the synthesis of cell wall polysaccharides have been identified, but hundreds of them remain to be discovered. Secretory pathway plays an important role in plant cell wall biogenesis as it transports cellulose synthase complexes and noncellulosic polysaccharide molecules to the cell surface. Some regulatory mechanisms that might be involved in cell wall material secretion, such as actin cytoskeleton, Ca2+ gradient or PIP kinases have been proposed; however, the regulation of this process is very complex and far from being understood. FT-IR spectroscopy is a method that can detect molecular vibrations and provide information about chemical composition of virtually all substances. It has been used successfully in screens for cell wall mutants, the study of interactions between cell wall polymers, as well as other areas of cell wall biology. FT-IR...
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Role of exocyst complex in growth and development of moss Physcomitrella patens
Rawat, Anamika Ashok ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee) ; Fendrych, Matyáš (referee)
During the course of evolution the early land plants gained extensive innovations that can be seen in modern day plants. The polar growth is an ancient feature of eukaryotic cells and is one of preadaptations that helped plants in successful colonization of land. The polar growth in plants regulates not only the direction of cell expansion and structural properties of cell wall but especially also the orientation of cell division, and is governed by various factors, including the exocyst complex. The exocyst is a well conserved vesicle tethering multi-subunit complex involved in tethering of secretory vesicles to the target membrane. The essential role of the exocyst complex in regulation of various cellular processes in Angiosperms is now well documented. Here I present results of a doctoral project that contributed to phylogenetic analyses of the land plant exocyst complex and especially to uncovering functions of three moss exocyst subunits, namely EXO70 (isoform PpEXO70.3d), SEC6 and SEC3 (isoforms PpSEC3A and PpSEC3B) in the model organism Physcomitrella patens. Various knock-out (KO) mutants in several moss exocyst subunits (Ppexo70.3d, Ppsec6, Ppsec3a and Ppsec3b) show pleiotropic defects directly or indirectly linked to the cell polarity regulation. Cell elongation and differentiation,...
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Functions of Exocyst Complex in the Regulation of Stomata Dynamics
Röder, Matěj ; Žárský, Viktor (advisor) ; Burketová, Lenka (referee)
Stomata are structures in plant epidermis which regulate contact between inner and outer environment of the plant by mediating their stomatal aperture. Many inner and outer signals contribute to the ontogenesis of the stomatal pattern. Guard cells undergo significant change of volume and surface during stomatal movement. This change of surface must be compensated by intracellular trafficking of membrane material because biological membrane has limited elasticity. Most of this trafficking takes place between plasma membrane and endosomal compartments. Complex exocyst is protein complex that ensures proper targeting of secretory vesicles to their destination on the plasma membrane. Function of this complex is essential for many cellular processes that require precise targeting of secretion. Mutation in gene Exo70B1 causes different development of the stomatal pattern. Plants with mutated Exo70B1 differ in stomatal size depending on the cultivation conditions more than wild type plant. Protein EXO70B1 is also directly involved in stomatal dynamics because mutants exo70B1 have retarded stomatal opening in response to light. This direct connection can be observed on the fluorescently labeled protein EXOB1 which significantly changes its localization during stomatal movements. None of these observed phenotypes is...
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Specificity of selected exocyst subunits in trichome development
Glanc, Matouš ; Žárský, Viktor (advisor) ; Binarová, Pavla (referee)
Trichomes are fine epidermal outgrowths covering aerial organs of most land plants. Although unicellular trichomes of Arabidopsis thaliana have long been used as a model system in plant cell and developmental biology, surprisingly little is known about the processes involved in cell wall biogenesis during the last stage of trichome maturation. A role of EXO70H4, a putative subunit of the vesicle tethering complex exocyst, in trichome maturation has recently been identified in our laboratory. Image analysis, histochemical detection and FT-IR spectroscopy methods were used in this study to analyze cell wall defects of the exo70H4 LOF mutant, revealing the mutation causes altered deposition of pectins and possibly also lignins and hemicelluloses. Transgenic lines with EXO70 paralogues driven by the EXO70H4 promoter were prepared and their analysis revealed that the closest paralogue EXO70H3, unlike EXO70A1 and EXO70B1, can complement the exo70H4 mutation. Based on the results, questions concerning trichome cell wall composition, the role of EXO70H4 in trichome maturation and functions of the plant exocyst complex are discussed. Keywords: Arabidopsis, trichome, cell wall, secretory pathway, exocyst complex, EXO70H4, FT-IR spectroscopy
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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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Cellular Protein Transport and Its Role in Patogenesis
Najdrová, Vladimíra ; Doležal, Pavel (advisor) ; Uzlíková, Magdalena (referee)
The main topic of this thesis are the protein secretion processes in several important human parasites - Toxoplasma gondii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp. and Giardia intestinalis. Described here are the parasite's and the host proteins which participate in the pathogenic processes involving the protein secretion. As shown here, the protein secretion into the host environment is one of key tools serving the parasite to survive within and manipulate the host organism. Interestingly, different parasitic organisms use functionally and evolutionary distinct strategies to fulfill this aim. Key words secretory pathway, translocon, signal sequence, Toxoplasma gongii, Plasmodium falciparum, Trypanosoma cruzi, Leishmania spp., Giardia intestinalis
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