The functional and structural characterization of Xanthomonas campestris pv. campestris core effector XopP revealed a new kinase activity.

Exo70B1 is a protein subunit of the exocyst complex with a crucial role in a variety of cell mechanisms, including immune responses against pathogens. The calcium-dependent kinase 5 (CPK5) of Arabidopsis thaliana (hereafter Arabidopsis), phosphorylates AtExo70B1 upon functional disruption. We previously reported that, the Xanthomonas campestris pv. campestris effector XopP compromises AtExo70B1, while bypassing the host's hypersensitive response, in a way that is still unclear. Herein we designed an experimental approach, which includes biophysical, biochemical, and molecular assays and is based on structural and functional predictions, utilizing AplhaFold and DALI online servers, respectively, in order to characterize the in vivo XccXopP function. The interaction between AtExo70B1 and XccXopP was found very stable in high temperatures, while AtExo70B1 appeared to be phosphorylated at XccXopP-expressing transgenic Arabidopsis. XccXopP revealed similarities with known mammalian kinases and phosphorylated AtExo70B1 at Ser107, Ser111, Ser248, Thr309, and Thr364. Moreover, XccXopP protected AtExo70B1 from AtCPK5 phosphorylation. Together these findings show that XccXopP is an effector, which not only functions as a novel serine/threonine kinase upon its host target AtExo70B1 but also protects the latter from the innate AtCPK5 phosphorylation, in order to bypass the host's immune responses. Data are available via ProteomeXchange with the identifier PXD041405.

Exploitation of the host exocyst complex by bacterial pathogens.

Intracellular bacterial pathogens remodel the plasma membrane of eukaryotic cells in order to establish infection. A common and well-studied mechanism of plasma membrane remodelling involves bacterial stimulation of polymerization of the host actin cytoskeleton. Here, we discuss recent results showing that several bacterial pathogens also exploit the host vesicular trafficking pathway of 'polarized exocytosis' to expand and reshape specific regions in the plasma membrane during infection. Polarized exocytosis is mediated by an evolutionarily conserved octameric protein complex termed the exocyst. We describe examples in which the bacteria Listeria monocytogenes, Salmonella enterica serovar Typhimurium, and Shigella flexneri co-opt the exocyst to promote internalization into human cells or intercellular spread within host tissues. We also discuss results showing that Legionella pneumophila or S. flexneri manipulate exocyst components to modify membrane vacuoles to favour intracellular replication or motility of bacteria. Finally, we propose potential ways that pathogens manipulate exocyst function, discuss how polarized exocytosis might promote infection and highlight the importance of future studies to determine how actin polymerization and polarized exocytosis are coordinated to achieve optimal bacterial infection.

The exocyst complex regulates C. elegans germline stem cell proliferation by controlling membrane Notch levels.

The conserved exocyst complex regulates plasma membrane-directed vesicle fusion in eukaryotes. However, its role in stem cell proliferation has not been reported. Germline stem cell (GSC) proliferation in the nematode Caenorhabditis elegans is regulated by conserved Notch signaling. Here, we reveal that the exocyst complex regulates C. elegans GSC proliferation by modulating Notch signaling cell autonomously. Notch membrane density is asymmetrically maintained on GSCs. Knockdown of exocyst complex subunits or of the exocyst-interacting GTPases Rab5 and Rab11 leads to Notch redistribution from the GSC-niche interface to the cytoplasm, suggesting defects in plasma membrane Notch deposition. The anterior polarity (aPar) protein Par6 is required for GSC proliferation, and for maintaining niche-facing membrane levels of Notch and the exocyst complex. The exocyst complex biochemically interacts with the aPar regulator Par5 (14-3-3ζ) and Notch in C. elegans and human cells. Exocyst components are required for Notch plasma membrane localization and signaling in mammalian cells. Our study uncovers a possibly conserved requirement of the exocyst complex in regulating GSC proliferation and in maintaining optimal membrane Notch levels.

Listeria monocytogenes exploits host exocytosis to promote cell-to-cell spread.

The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.

Inhibition of the Exocyst Complex Attenuates the LRRK2 Pathological Effects.

Pathological mutations in leucine-rich repeat kinase 2 (LRRK2) gene are the major genetic cause of Parkinson's disease (PD). Multiple lines of evidence link LRRK2 to the control of vesicle dynamics through phosphorylation of a subset of RAB proteins. However, the molecular mechanisms underlying these processes are not fully elucidated. We have previously demonstrated that LRRK2 increases the exocyst complex assembly by Sec8 interaction, one of the eight members of the exocyst complex, and that Sec8 over-expression mitigates the LRRK2 pathological effect in PC12 cells. Here, we extend this analysis using LRRK2 drosophila models and show that the LRRK2-dependent exocyst complex assembly increase is downstream of RAB phosphorylation. Moreover, exocyst complex inhibition rescues mutant LRRK2 pathogenic phenotype in cellular and drosophila models. Finally, prolonged exocyst inhibition leads to a significant reduction in the LRRK2 protein level, overall supporting the role of the exocyst complex in the LRRK2 pathway. Taken together, our study suggests that modulation of the exocyst complex may represent a novel therapeutic target for PD.

Disrupted glucose homeostasis and skeletal-muscle-specific glucose uptake in an exocyst knockout mouse model.

Skeletal muscle is responsible for the majority of glucose disposal following meals, and this is achieved by insulin-mediated trafficking of glucose transporter type 4 (GLUT4) to the cell membrane. The eight-protein exocyst trafficking complex facilitates targeted docking of membrane-bound vesicles, a process underlying the regulated delivery of fuel transporters. We previously demonstrated the role of exocyst subunit EXOC5 in insulin-stimulated GLUT4 exocytosis and glucose uptake in cultured rat skeletal myoblasts. However, the in vivo role of EXOC5 in skeletal muscle remains unclear. Using mice with inducible, skeletal-muscle-specific knockout of exocyst subunit EXOC5 (Exoc5-SMKO), we examined how muscle-specific disruption of the exocyst would affect glucose homeostasis in vivo. We found that both male and female Exoc5-SMKO mice displayed elevated fasting glucose levels. Additionally, male Exoc5-SMKO mice had impaired glucose tolerance and lower serum insulin levels. Using indirect calorimetry, we observed that male Exoc5-SMKO mice have a reduced respiratory exchange ratio during the light period and lower energy expenditure. Using the hyperinsulinemic-euglycemic clamp method, we further showed that insulin-stimulated skeletal muscle glucose uptake is reduced in Exoc5-SMKO males compared with wild-type controls. Overall, our findings indicate that EXOC5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle and regulate glucose homeostasis in vivo.

Sec3 exocyst component knockdown inhibits axonal formation and cortical neuronal migration during brain cortex development.

Neurons are the largest known cells, with complex and highly polarized morphologies and consist of a cell body (soma), several dendrites, and a single axon. The establishment of polarity necessitates initial axonal outgrowth in concomitance with the addition of new membrane to the axon's plasmalemma. Axolemmal expansion occurs by exocytosis of plasmalemmal precursor vesicles primarily at the neuronal growth cone membrane. The multiprotein exocyst complex drives spatial location and specificity of vesicle fusion at plasma membrane. However, the specific participation of its different proteins on neuronal differentiation has not been fully established. In the present work we analyzed the role of Sec3, a prominent exocyst complex protein on neuronal differentiation. Using mice hippocampal primary cultures, we determined that Sec3 is expressed in neurons at early stages prior to neuronal polarization. Furthermore, we determined that silencing of Sec3 in mice hippocampal neurons in culture precluded polarization. Moreover, using in utero electroporation experiments, we determined that Sec3 knockdown affected cortical neurons migration and morphology during neocortex formation. Our results demonstrate that the exocyst complex protein Sec3 plays an important role in axon formation in neuronal differentiation and the migration of neuronal progenitors during cortex development.

The exocyst complex and Rab5 are required for abscission by localizing ESCRT III subunits to the cytokinetic bridge.

Cytokinesis is the final step of cell division following chromosome segregation that generates two daughter cells. The conserved exocyst complex is required for scission of the intercellular cytokinetic bridge, although the molecular mechanisms it employs in this process are unclear. We identify and validate the early endocytic GTPase Rab5 as interacting with the exocyst complex in mammalian cells. Rab5 localizes in the cytokinetic bridge and on the midbody ring in a manner similar to the exocyst complex. Depletion of Rab5 led to delayed abscission. Caenorhabditis elegans orthologs of both exocyst complex subunits and Rab5 localize along the cleavage furrow and are required for cytokinesis in early embryos. Cytokinetic cells depleted of either Rab5 or the exocyst subunits Exoc3 and Exoc4 showed impaired deposition of the endosomal sorting complexes required for transport (ESCRT) III subunits CHMP2B and/or CHMP4B near the midbody ring. The study reveals an evolutionarily conserved role for the early endocytic marker Rab5 in cytokinetic abscission. In addition, it uncovers a key requirement of the exocyst and Rab5 for the delivery of components of the membrane-severing ESCRT III machinery to complete cytokinesis.

CSI1, PATROL1, and exocyst complex cooperate in delivery of cellulose synthase complexes to the plasma membrane.

Cellulose synthesis occurs exclusively at the plasma membrane by cellulose synthase complexes (CSCs). Therefore, delivery of CSCs to discrete sites at the plasma membrane is critical for cellulose synthesis. Despite their significance, the delivery of CSCs is poorly understood. Here we used proteomics approaches, functional genetics, and live cell imaging to show that the de novo secretion of CSCs is mediated by cooperation among cellulose synthase interactive 1 (CSI1), the plant-specific protein PATROL1, and exocyst complex in Arabidopsis thaliana We propose that CSI1 plays a role in marking the docking site, which allows CSCs-containing vesicles access to the plasma membrane through its interaction with microtubules. PATROL1 assists in exocytosis by its interaction with multiple components, including CSI1, CSCs, and exocyst subunits. Both PATROL1 and the exocyst complex determine the rate of delivery of CSCs to the plasma membrane. By monitoring the exocyst complex, PATROL1, CSI1, and CSCs dynamics in real time, we present a timeline of events for exocytosis of CSCs. Our findings provide unique insights into the evolution of exocytosis in eukaryotes.

Exocyst Genes Are Essential for Recycling Membrane Proteins and Maintaining Slit Diaphragm in Drosophila Nephrocytes.

BACKGROUND: Studies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because Drosophila nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue. METHODS: We silenced genes encoding exocyst complex proteins specifically in Drosophila nephrocytes and studied the effects on protein reabsorption by lacuna channels and filtration by the slit diaphragm. We performed nephrocyte functional assays, carried out super-resolution confocal microscopy of slit diaphragm proteins, and used transmission electron microscopy to analyze ultrastructural changes. We also examined the colocalization of slit diaphragm proteins with exocyst protein Sec15 and with endocytosis and recycling regulators Rab5, Rab7, and Rab11. RESULTS: Silencing exocyst genes in nephrocytes led to profound changes in structure and function. Abolition of cellular accumulation of hemolymph proteins with dramatically reduced lacuna channel membrane invaginations offered a strong indication of reabsorption defects. Moreover, the slit diaphragm's highly organized surface structure-essential for filtration-was disrupted, and key proteins were mislocalized. Ultrastructural analysis revealed that exocyst gene silencing led to the striking appearance of novel electron-dense structures that we named "exocyst rods," which likely represent accumulated membrane proteins following defective exocytosis or recycling. The slit diaphragm proteins partially colocalized with Sec15, Rab5, and Rab11. CONCLUSIONS: Our findings suggest that the slit diaphragm of Drosophila nephrocytes requires balanced endocytosis and recycling to maintain its structural integrity and that impairment of the exocyst complex leads to disruption of the slit diaphragm and nephrocyte malfunction. This model may help identify therapeutic targets for treating kidney diseases featuring molecular defects in vesicle endocytosis, exocytosis, and recycling.

Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction, Photoreceptor Cell Degeneration, and Decreased Visual Function.

To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5-/- zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5-/- mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.

Functional Specialization within the EXO70 Gene Family in Arabidopsis.

Localized delivery of plasma-membrane and cell-wall components is a crucial process for plant cell growth. One of the regulators of secretory-vesicle targeting is the exocyst tethering complex. The exocyst mediates first interaction between transport vesicles and the target membrane before their fusion is performed by SNARE proteins. In land plants, genes encoding the EXO70 exocyst subunit underwent an extreme proliferation with 23 paralogs present in the Arabidopsis (Arabidopsis thaliana) genome. These paralogs often acquired specialized functions during evolution. Here, we analyzed functional divergence of selected EXO70 paralogs in Arabidopsis. Performing a systematic cross-complementation analysis of exo70a1 and exo70b1 mutants, we found that EXO70A1 was functionally substituted only by its closest paralog, EXO70A2. In contrast, none of the EXO70 isoforms tested were able to substitute EXO70B1, including its closest relative, EXO70B2, pointing to a unique function of this isoform. The presented results document a high degree of functional specialization within the EXO70 gene family in land plants.

Inhibition of Tunneling Nanotubes between Cancer Cell and the Endothelium Alters the Metastatic Phenotype.

The interaction of tumor cells with blood vessels is one of the key steps during cancer metastasis. Metastatic cancer cells exhibit phenotypic state changes during this interaction: (1) they form tunneling nanotubes (TNTs) with endothelial cells, which act as a conduit for intercellular communication; and (2) metastatic cancer cells change in order to acquire an elongated phenotype, instead of the classical cellular aggregates or mammosphere-like structures, which it forms in three-dimensional cultures. Here, we demonstrate mechanistically that a siRNA-based knockdown of the exocyst complex protein Sec3 inhibits TNT formation. Furthermore, a set of pharmacological inhibitors for Rho GTPase-exocyst complex-mediated cytoskeletal remodeling is introduced, which inhibits TNT formation, and induces the reversal of the more invasive phenotype of cancer cell (spindle-like) into a less invasive phenotype (cellular aggregates or mammosphere). Our results offer mechanistic insights into this nanoscale communication and shift of phenotypic state during cancer-endothelial interactions.

Inducible Exoc7/Exo70 knockout reveals a critical role of the exocyst in insulin-regulated GLUT4 exocytosis.

Insulin promotes glucose uptake by triggering the translocation of glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane through exocytosis. GLUT4 exocytosis is a vesicle fusion event involving fusion of GLUT4-containing vesicles with the plasma membrane. For GLUT4 vesicle fusion to occur, GLUT4 vesicles must first be tethered to the plasma membrane. A key tethering factor in exocytosis is a heterooctameric protein complex called the exocyst. The role of the exocyst in GLUT4 exocytosis, however, remains incompletely understood. Here we first systematically analyzed data from a genome-scale CRISPR screen in HeLa cells that targeted virtually all known genes in the human genome, including 12 exocyst genes. The screen recovered only a subset of the exocyst genes, including exocyst complex component 7 (Exoc7/Exo70). Other exocyst genes, however, were not isolated in the screen, likely because of functional redundancy. Our findings suggest that selection of an appropriate exocyst gene is critical for genetic studies of exocyst functions. Next we developed an inducible adipocyte genome editing system that enabled Exoc7 gene deletion in adipocytes without interfering with adipocyte differentiation. We observed that insulin-stimulated GLUT4 exocytosis was markedly inhibited in Exoc7 KO adipocytes. Insulin signaling, however, remained intact in these KO cells. These results indicate that the exocyst plays a critical role in insulin-stimulated GLUT4 exocytosis in adipocytes. We propose that the strategy outlined in this work could be instrumental in genetically dissecting other membrane-trafficking pathways in adipocytes.

The Arabidopsis exocyst subunits EXO70B1 and EXO70B2 regulate FLS2 homeostasis at the plasma membrane.

The plasma membrane (PM)-localized receptor kinase FLAGELLIN SENSING 2 (FLS2) recognizes bacterial flagellin or its immunogenic epitope flg22, and initiates microbe-associated molecular pattern-triggered immunity, which inhibits infection by bacterial pathogens. The localization, abundance and activity of FLS2 are under dynamic control. Here, we demonstrate that Arabidopsis thaliana EXO70B1, a subunit of the exocyst complex, plays a critical role in FLS2 signaling that is independent of the truncated Toll/interleukin-1 receptor-nucleotide binding sequence protein TIR-NBS2 (TN2). In the exo70B1-3 mutant, the abundance of FLS2 protein at the PM is diminished, consistent with the impaired flg22 response of this mutant. EXO70B1-GFP plants showed increased FLS2 accumulation at the PM and therefore enhanced FLS2 signaling. The EXO70B1-mediated trafficking of FLS2 to the PM is partially independent of the PENETRATION 1 (PEN1)-containing secretory pathway. In addition, EXO70B1 interacts with EXO70B2, a close homolog of EXO70B1, and both proteins associate with FLS2 and contribute to the accumulation of FLS2 at the PM. Taken together, our data suggest that the exocyst complex subunits EXO70B1 and EXO70B2 regulate the trafficking of FLS2 to the PM, which represents a new layer of regulation of FLS2 function in plant immunity.

RalGPS2 is involved in tunneling nanotubes formation in 5637 bladder cancer cells.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor (GEF) for RalA containing a PH domain and an SH3-binding region and it is involved in several cellular processes, such as cytokinesis, control of cell cycle progression, differentiation, cytoskeleton organization and rearrangement. Up to now, few data have been published regarding RalGPS2 role in cancer cells, and its involvement in bladder cancer is yet to be established. In this paper we demonstrated that RalGPS2 is expressed in urothelial carcinoma-derived 5637 cancer cells and is essential for cellular growth. These cells produces thin membrane protrusions that displayed the characteristics of actin rich tunneling nanotubes (TNTs) and here we show that RalGPS2 is involved in the formation of these cellular protrusions. In fact the overexpression of RalGPS2 or of its PH-domain increased markedly the number and the length of nanotubes, while the knock-down of RalGPS2 caused a strong reduction of these structures. Moreover, using a series of RalA mutants impaired in the interaction with different downstream components (Sec5, Exo84, RalBP1) we demonstrated that the interaction of RalA with Sec5 is required for TNTs formation. Furthermore, we found that RalGPS2 interacts with the transmembrane MHC class III protein leukocyte specific transcript 1 (LST1) and RalA, leading to the formation of a complex which promotes TNTs generation. These findings allow us to add novel elements to molecular models that have been previously proposed regarding TNTs formation.

InsP3R-SEC5 interaction on phagosomes modulates innate immunity to Candida albicans by promoting cytosolic Ca2+ elevation and TBK1 activity.

BACKGROUND: Candida albicans (C. albicans) invasion triggers antifungal innate immunity, and the elevation of cytoplasmic Ca2+ levels via the inositol 1,4,5-trisphosphate receptor (InsP3R) plays a critical role in this process. However, the molecular pathways linking the InsP3R-mediated increase in Ca2+ and immune responses remain elusive. RESULTS: In the present study, we find that during C. albicans phagocytosis in macrophages, exocyst complex component 2 (SEC5) promotes InsP3R channel activity by binding to its C-terminal α-helix (H1), increasing cytosolic Ca2+ concentrations ([Ca2+]c). Immunofluorescence reveals enriched InsP3R-SEC5 complex formation on phagosomes, while disruption of the InsP3R-SEC5 interaction by recombinant H1 peptides attenuates the InsP3R-mediated Ca2+ elevation, leading to impaired phagocytosis. Furthermore, we show that C. albicans infection promotes the recruitment of Tank-binding kinase 1 (TBK1) by the InsP3R-SEC5 interacting complex, leading to the activation of TBK1. Subsequently, activated TBK1 phosphorylates interferon regulatory factor 3 (IRF-3) and mediates type I interferon responses, suggesting that the InsP3R-SEC5 interaction may regulate antifungal innate immune responses not only by elevating cytoplasmic Ca2+ but also by activating the TBK1-IRF-3 pathway. CONCLUSIONS: Our data have revealed an important role of the InsP3R-SEC5 interaction in innate immune responses against C. albicans.

Arabidopsis Trichome Contains Two Plasma Membrane Domains with Different Lipid Compositions Which Attract Distinct EXO70 Subunits.

Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall-radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.

Solanaceous exocyst subunits are involved in immunity to diverse plant pathogens.

The exocyst, a multiprotein complex consisting of eight subunits, plays an essential role in many biological processes by mediating secretion of post-Golgi-derived vesicles towards the plasma membrane. In recent years, roles for plant exocyst subunits in pathogen defence have been uncovered, largely based on studies in the model plant Arabidopsis. Only a few studies have been undertaken to assign the role of exocyst subunits in plant defence in other plants species, including crops. In this study, predicted protein sequences from exocyst subunits were retrieved by mining databases from the Solanaceous plants Nicotiana benthamiana, tomato, and potato. Subsequently, their evolutionary relationship with Arabidopsis exocyst subunits was analysed. Gene silencing in N. benthamiana showed that several exocyst subunits are required for proper plant defence against the (hemi-)biotrophic plant pathogens Phytophthora infestans and Pseudomonas syringae. In contrast, some exocyst subunits seem to act as susceptibility factors for the necrotrophic pathogen Botrytis cinerea. Furthermore, the majority of the exocyst subunits were found to be involved in callose deposition, suggesting that they play a role in basal plant defence. This study provides insight into the evolution of exocyst subunits in Solanaceous plants and is the first to show their role in immunity against multiple unrelated pathogens.

Membrane targeting of the yeast exocyst complex.

The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits - Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP2) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP2. We found that PIP2 is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP2 in the adjacent leaflet. We further revealed that PIP2 is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP2 for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation.