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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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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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FUNCTIONAL ANAYSIS OF SELECTED EXO70 EXOCYST SUBUNITS IN PLANTS
Kubátová, Zdeňka ; Kulich, Ivan (advisor) ; Grossmann, Guido (referee) ; Lichtscheidl-Schultz, Irene (referee)
Arabidopsis thaliana trichomes are large unicellular epidermal outgrowths with a specific development and intriguing shape, which makes them an excellent cell type for our research of cell polarization mecha- nisms. Cell polarity is essential for plant development and the exocyst complex is one of its key regulators. It is an octameric protein complex that mediates polarized exocytosis and growth by targeted tethering of secretory vesicles to the plasma membrane. Its EXO70 subunit functions as a landmark for exocytosis site and physically binds the target membrane through interaction with phospholipids. A remarkable multipli- cation of EXO70 subunit paralogs in land plant genomes is well documented, but the functional diversity of these paralogs remains to be described. In trichomes we revealed the specific role of the EXO70H4 paralog in secondary cell wall deposi- tion, especially in callose synthase delivery. We documented formation of a thick secondary cell wall during the maturation phase of wild type trichome development and a lack of it in the exo70H4 mutant. Moreover, we showed evidence for silica deposition dependency on callose synthesis. Further, we unveiled the formation of apical and basal plasma membrane domains, which differ in their phospholipid compo- sition and ability to bind...
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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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