The initial interaction of vesicle and the target membrane prior to their fusion is called vesicle tethering, a process mediated by an octameric protein complex called the exocyst. The exocyst connects vesicles and binds them to phosphatidylinositol 4, 5-biphosphate (PI (4,5) P2), located on the plasma membrane. The exocyst complex is located at the target site, helping to prepare the soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) for docking and subsequent release of vesicular contents after fusion. The importance of the exocyst in cellular processes is inevitable since it performs central roles in exocytosis thereby inducing SNARE-mediated membrane fusion. The study presented here is concentrated on the role of exocyst genes during the defense response in Glycine max (soybean) against the plant- parasitic nematode Heterodera glycines known as the soybean cyst nematode (SCN). Using developmental genomics analysis, G. max root cells that have been induced by H. glycines through their pathogenic activities to develop into nurse cells known as a syncytium have been isolated by laser capture microdissection (LCM). RNA isolated from these cells undergoing resistant reactions in two different G. max genotypes have been used in gene expression profiling experiments that have led to the identification of the genes employed in this analysis. The results demonstrate the involvement of exocyst components in the defense process that G. max has toward H. glycines. Related studies also show the involvement of RPM1-INTERACTING PROTEIN 4 (RIN4) functioning in this defense process.

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...

Host colonization is mediated through the secretion of effector proteins in order to neutralize host immune responses. However, the mechanism of the effector delivery during biotrophic invasion is not well defined in M. oryzae. In this thesis, I define the role of the exocyst complex, an evolutionarily conserved octameric protein complex involved in vesicle docking to the plasma membrane (composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84), during infection-related development in M. oryzae. Like other filamentous fungi, M. oryzae, exocyst components localize to the vegetative hyphal tip distinct from the Spitzenkörper. However, at the initial stage of infection-related development all the exocyst components localise as a ring at the cortex of the appressorium and re-assembles around the appressorium pore in an actin-dependent manner in mature appressoria. I report that the septin network is required for the transition of exocyst ring from periphery to the appressorium pore. Deletion of Exo70 and Sec5 showed significant reduction in protein secretion and plant infection. I show that Sec6 is required for the exocyst assembly around the appressorium pore and effector secretion from the appressorium. I report that, during biotrophic invasion, effectors are secreted through a distinct pathway. Apoplastic effectors, Bas4 and Slp1 are secreted via a Golgi-dependent pathway while secretion of cytoplasmic effectors, Pwl2 and Bas1 meditates through a Golgi-independent pathway in which exocyst components Exo70 and Sec5 are involved.

The Exocyst is a hetero-octameric complex involved in tethering of post-Golgi vesicle transport to sites of membrane expansion. In budding yeast, the Exocyst targets vesicles to bud site resulting in bud emergence and abscission of the daughter cell. Mammalian Exocyst is recruited to developing lateral membranes after cadherin mediated adhesion and then is segregated to adherens junctional complexes (AJC). In polarized epithelia, the Exocyst is required for basal-lateral transport of LDL receptor. Additional Exocyst subunit localizations and functions have been identified. It is not known whether these supplementary roles can be attributed to the Exocyst or other unidentified Exocyst subcomplexes. Sec3, an Exocyst subunit, is hypothesized to be a landmark of polarization in yeast. In polarized epithelia, GFP tagged Sec3 remained cytosolic in polarized epithelia unlike Sec6/8. Sec3-GFP was recruited to lateral membranes only after dual over expression of heterologous GLYT1. Little is known about endogenous mammalian Sec3. Our work suggests Sec3 defines an Exocyst subcomplex that is required for desmosome integrity. Sec3 and additional subunits (Sec6, Sec8, Sec15, Exo70, and Exo84) were present at desmosomes, but Sec3 failed to localize to AJC. Only antibodies to Sec6 and Sec8 labeled AJC. Reduction of Sec3 protein expression resulted in the impairment of desmosome morphology and function with no detrimental effect on adherens junctions. These data suggest the existence of functionally different Exocyst subcomplexes. Sec3-exocyst recruited minus-end directed microtubule motor KifC3 to desmosomes. KifC3 was previously shown to be recruited with a microtubule anchoring complex to basal-lateral membrane. This suggests Sec3 may recruit KifC3 to organize microtubules at desmosomes. This would establish a pathway to efficiently transport newly synthesized basal-lateral cargo. These results suggest a novel mechanism of the Exocyst to regulate post-Golgi vesicular transport and intercellular adhesion.

HIV-1 Nef protein contributes to pathogenesis via multiple functions that include enhancement of viral replication and infectivity, alteration of intracellular trafficking, and modulation of cellular signaling pathways. Nef stimulates formation of tunnelling nanotubes and virological synapses, and is transferred to bystander cells via these intercellular contacts and secreted microvesicles. Nef associates with and activates Pak2, a kinase that regulates T-cell signaling and actin cytoskeleton dynamics, but how Nef promotes nanotube formation is unknown. In this dissertation, we developed and characterized a lentiviral vector-based system to express Nef in T-cell lines and primary human peripheral blood mononuclear cells, and then used this system to perform a proteomic screen to identify Nef-associated host cell factors and better understand how Nef hijacks the T-cell machinery to maximize HIV production and dissemination. Bioinformatic and cell-based analysis of the resulting host factors revealed a mechanism by which Nef enhances nanotube formation. To identify Nef binding partners involved in Pak2-association dependent Nef functions, we employed tandem mass spectrometry analysis of Nef immunocomplexes from Jurkat cells expressing wild-type Nef or Nef mutants defective for the ability to associate with Pak2 (F85L, F89H, H191F and A72P, A75P in NL4-3). Wild-type, but not mutant Nef, was associated with 5 components of the exocyst complex (EXOC1, EXOC2, EXOC3, EXOC4, and EXOC6), an octameric complex that tethers vesicles at the plasma membrane, regulates polarized exocytosis, and recruits membranes and proteins required for nanotube formation. Additionally, Pak2 kinase was associated exclusively with wild-type Nef. Association of EXOC1, EXOC2, EXOC3, and EXOC4 with wild-type, but not mutant Nef, was verified by co-immunoprecipitation assays in Jurkat cells. Furthermore, shRNA-mediated depletion of EXOC2 in Jurkat cells abrogated Nef-mediated enhancement of nanotube formation. Using bioinformatic tools, we visualized protein interaction networks that reveal functional linkages between Nef, the exocyst complex, and the cellular endocytic and exocytic trafficking machinery. Together, our findings identify the exocyst complex as a key effector of Nef-mediated enhancement of nanotube formation, and possibly microvesicle secretion. Furthermore, linkages revealed between Nef and the exocyst complex suggest a new paradigm of exocyst involvement in polarized targeting for intercellular transfer of viral proteins and viruses.

The exocyst is thought to tether secretory vesicles to specific sites on the plasma membrane. As a member of the exocyst, Sec5 is implicated in cell survival and membrane growth in Drosophila. Little is known of the exocyst function in mammals, with previous work suggesting involvement of exocyst in GTP-dependent exocytosis. Using RNA interference, we stably down-regulated Sec5 in PC12 cells. We found that these knockdown cells exhibit decreased GTP- and Ca2+-dependent exocytosis of dense-core vesicles (DCVs), and contain less proportion of docked vesicles. Expression of Sec6/8 is also slightly reduced in Sec5 knockdown cells. Our results suggest that Sec5 is involved in both GTP- and Ca2+-dependent exocytosis, possibly through the regulation of DCV docking. We also established doxycycline-inducible knockdown system for Sec5 in PC12 cells which may be more appropriate to study development-related proteins. Efforts were also made to re-introduce Sec5 into the Sec5 knockdown cells for rescue purposes.

The exocyst is thought to tether secretory vesicles to specific sites on the plasma membrane. As a member of the exocyst, Sec5 is implicated in cell survival and membrane growth in Drosophila. Little is known of the exocyst function in mammals, with previous work suggesting involvement of exocyst in GTP-dependent exocytosis. Using RNA interference, we stably down-regulated Sec5 in PC12 cells. We found that these knockdown cells exhibit decreased GTP- and Ca2+-dependent exocytosis of dense-core vesicles (DCVs), and contain less proportion of docked vesicles. Expression of Sec6/8 is also slightly reduced in Sec5 knockdown cells. Our results suggest that Sec5 is involved in both GTP- and Ca2+-dependent exocytosis, possibly through the regulation of DCV docking. We also established doxycycline-inducible knockdown system for Sec5 in PC12 cells which may be more appropriate to study development-related proteins. Efforts were also made to re-introduce Sec5 into the Sec5 knockdown cells for rescue purposes.

Le mode de coordination parmi les différentes molécules qui régulent la migration reste très peu connu. Ce travail traite de deux voies de transduction régulant la migration: la voie Rac1/WRC (Wave Regulatory Complex) qui contrôle la formation du réseau d'actine au front des cellules migrantes, et la voie RalB/exocyst, dont les mécanismes moléculaires de son implication dans la motilité cellulaire étaient inconnus au début de cette thèse. Rac1 et RalB sont des petites protéines G des familles Rho et Ras, respectivement. Les complexes WRC et exocyst sont leurs effecteurs directs.Au cours de la recherche de connexions entre l'exocyst et des régulateurs de la migration, nous avons trouvé que deux sous-unités de l'exocyst, Exo70 et Sec6, interagissent directement in vitro avec Abi et Cyfip, respectivement, deux sous unités du WRC. De plus, nous avons trouvé que les sous-unités de l'exocyst peuvent interagir in vitro avec le WRC entier. Nous avons également montré que ces deux complexes s'associent in vivo. Sur le plan fonctionnel, l'exocyst est requis pour le positionnement du complexe WRC au front des cellules migrantes. D'autre part, nous avons également trouvé que deux autres sous- unités de l'exocyst Sec8 et Exo84, interagissent avec SH3BP1 (une RhoGAP) en double hybride et en co-immunoprécipitation. SH3BP1 se localise au front des cellules migrantes, et cette localisation dépend de l'exocyst. De façon intéressante, in vivo, la voie RalB/exocyst/SH3BP1 cible spécifiquement Rac1, et non Cdc42. Grâce à plusieurs approches, nous concluons que SH3BP1 est requis pour inactiver Rac1 au front. Dans notre modèle nous proposons que RalB/exocyst règulerait la migration cellulaire en véhiculant au front de migration deux éléments majeurs de la signalisation de Rac1 : son complexe effecteur WRC, qui stimule la nucléation de filaments d'actine et son régulateur négatif SH3BP1, une GAP qui promeut l'inactivation et le cycle GDP/GTP de Rac1. En conclusion, ce travail fournit de nouvelles connexions moléculaires et fonctionnelles entre l'exocytose polarisée et la dynamique de l'actine au cours de la motilité cellulaire.

Plants have an artillery to defend themselves. The plant surface is protected by water- resistant cuticle and mechanically strong cell wall. Then each plant cell has tools to recognize and to answer to a pathogen threat. In an extreme case, the answer is programmed cell death. Plant immunity is a complex process integrating these passive and active mechanisms in an effort to overstay a pathogen attack. When the plant cell is attacked by a pathogen, the metabolic resources are redirected towards immunity reaction which results in growth restriction. Both the immunity reaction and the growth are dependent on the efficient polarized secretion of various cargoes. Exocyst complex mediates tethering of a secretory vesicle with a target membrane and SNARE complex orchestrates the subsequent steps of vesicle docking and fusion. Exocyst and SNAREs are regulated by various proteins. In my work, I focused on identifying the exocyst interaction partners in plant immunity. In cooperation with my colleagues, we found the direct association between Qa-SNARE SYP121 involved in plant penetration resistance and EXO70B2 exocyst subunit. Moreover, we confirmed the relevance of their interaction for the formation of epidermal defensive structures, papillae and haustorial encasements in plant defence against non-adapted...

Plants have an artillery to defend themselves. The plant surface is protected by water- resistant cuticle and mechanically strong cell wall. Then each plant cell has tools to recognize and to answer to a pathogen threat. In an extreme case, the answer is programmed cell death. Plant immunity is a complex process integrating these passive and active mechanisms in an effort to overstay a pathogen attack. When the plant cell is attacked by a pathogen, the metabolic resources are redirected towards immunity reaction which results in growth restriction. Both the immunity reaction and the growth are dependent on the efficient polarized secretion of various cargoes. Exocyst complex mediates tethering of a secretory vesicle with a target membrane and SNARE complex orchestrates the subsequent steps of vesicle docking and fusion. Exocyst and SNAREs are regulated by various proteins. In my work, I focused on identifying the exocyst interaction partners in plant immunity. In cooperation with my colleagues, we found the direct association between Qa-SNARE SYP121 involved in plant penetration resistance and EXO70B2 exocyst subunit. Moreover, we confirmed the relevance of their interaction for the formation of epidermal defensive structures, papillae and haustorial encasements in plant defence against non-adapted...