Available online at www.sciencedirect.com

ScienceDirect Exocyst complexes multiple functions in plant cells secretory pathways Viktor Zˇa´rsky´1,2, Ivan Kulich1,2, Matya´sˇ Fendrych2,3 and Tamara Pecˇenkova´1,2 The exocyst is a complex of proteins mediating first contact (tethering) between secretory vesicles and the target membrane. Discovered in yeast as an effector of RAB and RHO small GTPases, it was also found to function in land plants. Plant cells and tissues rely on targeted exocytosis and this implies that the exocyst is involved in regulation of cell polarity and morphogenesis, including cytokinesis, plasma membrane protein recycling (including PINs, the auxin efflux carriers), cell wall biogenesis, fertilization, stress and biotic interactions including defence against pathogens. The dramatic expansion of the EXO70 subunit gene family, of which individual members are likely responsible for exocyst complex targeting, implies that there are specialized functions of different exocysts with different EXO70s. One of these functions comprises a role in autophagy-related Golgi independent membrane trafficking into the vacuole or apoplast. It is also possible, that some EXO70 paralogues have been recruited into exocyst independent functions. The exocyst has the potential to function as an important regulatory hub to coordinate endomembrane dynamics in plants. Addresses 1 Department of Experimental Plant Biology, Faculty of Sciences, Charles University, Vinicˇna´ 5, Prague 2, 128 43, Czech Republic 2 Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojova´ 263, Prague 6, 165 02, Czech Republic 3 Department of Plant Systems Biology, VIB, Technologiepark 927, 9052 Gent, Belgium Corresponding authors: Zˇa´rsky´, Viktor ([email protected])

Current Opinion in Plant Biology 2013, 16:726–733 This review comes from a themed issue on Cell biology Edited by David W Ehrhardt and Magdalena Bezanilla For a complete overview see the Issue and the Editorial Available online 15th November 2013 1369-5266/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pbi.2013.10.013

Introduction Due to their sessile life style, land plants rely on the highly regulated formation and reinforcement of cell walls and on the turgor pressure built up by the vacuole. Therefore an intricate coordination of plant endomembrane trafficking dynamics is expected. In this context it is surprising how little we know about the process of exocytosis in plants. In all transport steps within the endomembrane system of Current Opinion in Plant Biology 2013, 16:726–733

eukaryotic cells, the first contact of transport vesicles with the target endomembrane is mediated by specific tethering factors — long vesicle tethering proteins or extended rodshaped tethering complexes. In a genetic screen for secretion mutants in yeast, the exocyst was discovered as a tethering complex for exocytotic vesicles regulated by Rab and Rho GTPases and composed stoichiometrically of eight proteins (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p and Exo84p [1]; Exo70p and Exo84p were discovered later). The exocyst is also found in land plants. Already at this early stage of land plant exocyst exploration, it is becoming apparent that specific exocyst complexes (based on multiple isoforms of EXO70 subunit) will have multiple functions — not only in the regulation of exocytosis and plasma membrane (PM) recycling, but also in autophagy-related transport to the vacuole or possibly to the apoplast (Figure 1a).

EXO70 subunits extraordinary evolutionary dynamics Land plant genomes encode for a large number of EXO70 paralogues (23 in Arabidopsis and 47 in rice). This is unique to land plants because in other eukaryotes EXO70 is encoded by a single gene. Phylogenetic analyses [2,3,4] indicate that the common ancestor of land plants encoded three different EXO70s, which later diversified into many paralogues, all of which fit within three classes defined by the putative original paralogues. Interestingly, one whole class of EXO70s (EXO70.3) was lost early in clubmoss (Lycopodiaceae) evolution. Bewildering evolutionary dynamics is especially apparent in the grassspecific FX subfamily, where the speed and extent of EXO70 evolution indicates a possible link to stresses, biotic interactions, specifically a potential evolutionary race between grasses and plant pathogens. There are multiple, non-exclusive explanations for the EXO70 paralogue multiplicity within land plants: first, the need for cell-specific exocytosis within differentiating plant tissues [3,5,6]; second, need for differential exocytosis/trafficking pathways within a single cell, with more cortical recycling domains present [6], and as recently discovered to direct autophagy-related transport to the vacuole or apoplast [7,8,9] (Figure 1a); third, exocytosis related to stress responses such as pathogen attack [10] or abiotic stresses (e.g. copper stress [11]). It is highly probable that some EXO70s and other exocyst subunits will also have functions completely unrelated to www.sciencedirect.com

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Figure 1

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(a) Endomembrane trafficking pathways involving exocyst complexes. The two established pathways — exocytosis of TGN/EE produced vesicles mediated by EXO70A1 harbouring exocyst and EXO70B1 dependent autophagy related transport to the vacuole — are highlighted by solid arrows. Other putative pathways are marked by dashed arrows. A, autophagosome and autophagy related GA-independent traffic; CW, cell wall; ER, endoplasmic reticulum; GA, Golgi; IVB, intravacuolar bodies; MVB, multivesicular bodies; PM, cytoplasmic membrane; TGN/EE, trans-Golgi network/ early endosome; TN, tonoplast; V, vacuole. Exocyst complexes with different EXO70 subunits are symbolized by trapezoids connecting the transport containers to the target membranes. (b) Exocyst maxima at the initiation and insertion/maturation phases of cytokinesis visualized by EXO70A1-GFP; (c) exocyst in volcano cells wall biogenesis (pectin deposition in WT versus exo70a1 mutant) and (d) exocyst in the defence papilla formation upon the Blumeria graminis spore germination (WT versus exo70a1/exo70b2 double mutant).

the secretory pathway. In animals and fungi, exocyst subunits were observed to regulate the early secretory pathway at the endoplasmic reticulum (ER)-translocon, dynamics of cytoskeleton and splicing [12–14]. Recently functions for homo-oligomeric EXO70 complexes, in www.sciencedirect.com

negative curvature membrane formation and actin-independent stimulation of filopodia, that were independent of the exocyst complex were indicated in animal cells [15]. Very specific and strong transcriptional upregulation of some EXO70 isoforms in specialized cell types (e.g. Current Opinion in Plant Biology 2013, 16:726–733

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trichomes) also indicate the possibility of similar exocystindependent function for some plant EXO70s. It is therefore probable that not all EXO70 paralogues in land plants function as subunits of exocyst complexes but some were recruited to new exocyst unrelated roles. Also two or more different cellular functions for the same EXO70 paralogue may be expected. This question certainly is one of the most important challenges for future research.

Structure and interactions of exocyst subunits in Arabidopsis Homologous structural models of plant exocyst subunits indicate well conserved rod-like structural features of plant exocyst subunits, including putative phosphatidylinositol phosphate binding sites on the SEC3 and several EXO70 subunits — which are, along with RAB and RHO GTPases interactions, known to be crucial for the proper targeting of the exocyst to membranes [6,14] (Bloch et al., unpublished data). Overall conservation of the plant exocyst complex structure is supported by electron microscopy (EM) tomographic observations of exocyst-like structures during cytokinesis in Arabidopsis [16,17]. Interactions between exocyst subunits in the Arabidopsis complex have been studied mostly using yeast two hybrid or coimmunoprecipitation. It has also been possible to partially purify the complex [18–21]. Our current knowledge of demonstrated and putative exocyst interactions are summarized in Figure 2. These data suggest that the exocyst functions as an interaction hub — with many contacts mediated especially by different EXO70 subunits. Investigation of plant exocyst subunit composition with respect to EXO70 is in its infancy as currently there is information for only a few of the 23 EXO70 paralogues in Arabidopsis (Figure 2). Interaction of the exocyst with the SNARE machinery is consistent with a role in vesicle tethering (SEC1/KEULE [22], SNAP33 [10] and SYP23 [23]). The first interaction discovered between the exocyst subunit SEC3 and activated ROP GTPases in Arabidopsis was surprisingly indirect; it is mediated by the plant-specific adaptor/scaffold protein ICR1 [24]. Other interactors shown in Figure 2 likely reflect plantspecific roles of the exocyst: EXO70A1 and EXO70C1 bind to ROH1 (a homolog of BYPASS1) [20]; ROH1 is strongly expressed in pollen. EXO70H1 localizes to the nucleus ([10] and Pecˇenkova´, unpublished observations), and its interactors (identified in a high-throughput screen [25], Figure 2) include transcription factors, hinting at a novel, possibly non-trafficking related function of this exocyst subunit. Exocyst complex function relies on the intricately orchestrated hierarchy of interactions between exocyst and other proteins, but importantly also between exocyst and specific membrane lipids [14,26,27]. Recent work has shown that the Arabidopsis SEC3a subunit interacts with the membrane phospholipids (Bloch Current Opinion in Plant Biology 2013, 16:726–733

et al., unpublished data). Due to the multiplicity of EXO70s and their putative C-terminal lipid binding region it is likely that isoforms will have specific interactions with specific endomembrane lipids [6].

Exocyst complex dependent processes in plant cells include cell division, cell polarity and morphogenesis, cell wall biogenesis, integral PM proteins recycling and polarization At the onset of cytokinesis in land plants, the cell plate assembles from vesicles gathering in the equatorial plane in late anaphase [17]. Judging from the prominent localization of SEC3, SEC6, SEC8, SEC15b, EXO70A1 and EXO84b subunits at the initiation of cytokinesis [19,21] (Figure 1b), extensive initial fusion of these vesicles is likely to be stimulated by the exocyst complex. This is supported by the fact that the exo70A1 mutant has transient defects in cell plate assembly [19]. Later, the expanding cell plate possibly acquires a trans Golgi network (TGN) identity [28,29] and the exocyst GFP-signals are low in the centrifugally expanding cell plate [19,21]. However in tobacco cell culture the Arabidopsis SEC6 subunit decorated the margins of the expanding cell plate and co-localized with SEC1/KEULE [22]. This discrepancy might be a result of heterologous overexpression of SEC6. Co-localization of SEC6 with SEC1 is consistent with an interaction between these proteins, which was identified in yeast and is likely conserved in eukaryotes [27]. Finally, the exocyst strongly localizes to the cell plate at the moment of its insertion into the wall of the parental cell and persists during new cross-wall establishment (Figure 1b), playing a role in the fusion of the cell plate with the parental wall and new cell wall maturation [19,21,30] (Figure 2B). The sec6 and exo84 mutants show mild cytokinetic defects in leaf epidermal pavement cells and strong defects in the guard cell cytokinesis, both of which might result from the collapse of the immature post-cytokinetic cell walls [19,22]. The exocyst role in the initial fusion of vesicles during the onset of cell plate assembly implies post-TGN/EE (i.e. PM) identity of the cell plate initial as proposed by [31,17]. Defects in cytokinesis dynamics in Arabidopsis exocyst mutants partially explain meristem related defects (both shoot and root apical meristems) in whole plant development of these mutants [3,19]. Further dissecting the molecular roles of the exocyst in cell division plane establishment and maintenance requires more study. Cell plate biogenesis is an important example of directed secretion in plant cells. The exocyst has also been shown to be involved in other plant-specific cell polarization processes such as pollen tube germination and growth (our data indicate that, in contrast to [21], this is true also for Atsec3a mutant; Bloch et al., unpublished data), root hair growth, stigmatic papillae elongation and receptivity, secondary cell wall depositions in the tracheary elements www.sciencedirect.com

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The interaction network for the exocyst complex suggests that the exocyst complex is a central regulator of plant cell endomembrane dynamics. EXO70 paralogues likely act as mediators with different aspects of membrane trafficking. Interaction of the SEC3 subunit with membrane phospholipids was experimentally tested (Bloch et al., unpublished data) while dashed lines indicate interactions of EXO70s with phospholipids so far only computationally predicted. Not only proteins known to be involved in the regulation of cell polarity (ROP, GEF), vesicle trafficking (SEC1/KEULE, SNARES, membrane phospholipid phosphatase) or calcium signalling (calcium dependent kinase) are interacting, but also cytoskeletal protein (MAPS), E3 ubiquitin ligases (able to specifically target EXO70 subunits for degradation), putative autophagy ROS/senescence related protein or transcription regulators are indicated as known or putative interactors.

or polarized pectin accumulation in seed coat volcano cells [18,20,32] (Figure 1c). A mutant in the most abundant EXO70A1 paralogue has defects in recycling of integral PM proteins represented by PIN1/PIN2 and BRI1. The defects in PIN1 and PIN2 recycling lead to alterations in polar auxin transport — well documented in exo70A1 and sec8 mutants [33]. Importantly, ICR1 which mediates the interaction between SEC3 and activated ROP GTPases, is clearly an organizer of auxin transport and metabolism around the root quiescent centre, but also cell polarity and morphogenesis [24,34]. Many targets and pleiotropic consequences of exocyst disruption complicate interpretation of phenotypes of exocyst mutants — they need to be considered carefully. In this respect the recently proposed function of EXO70A1 exclusively in root xylem development and water transport is most www.sciencedirect.com

probably too narrow in relation to the above summarized defects of exo70A1 Arabidopsis mutant [32].

Exocyst dynamics at the PM — exocyst as a dynamic particle in exocytotic vesicles tethering? Super-sensitive and fast imaging methods [total internal reflection fluorescence microscopy/variable-angle epifluorescence microscopy (TIRFM/VAEM) or spinning disc confocal microscopy (SDCM)] have been used to monitor exocyst complex dynamics at the PM. SEC6, SEC8, EXO70A1 and EXO84b were examined in Arabidopsis by VAEM [35], SEC3 by SDCM [21], and in animal HeLa cells SEC8 was visualized by TIRFM [36]. The results of these three reports are remarkably similar. In all cases, the exocyst subunits appear at the PM as Current Opinion in Plant Biology 2013, 16:726–733

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non-motile transient puncta with median lifetimes of roughly 10 s. Some of these puncta represent vesicles that are tethered to the PM and eventually fuse with the PM. In HeLa cells, SEC8 partially co-localized with VAMP2 vesicles and a pHluorin-tagged cargo marker. In Arabidopsis, v-SNARE VAMP721 was used as the marker of secretory vesicles and partial co-localization with exocyst PM-localized puncta was reported. Moreover the SEC6, SEC8 and EXO84b subunits co-localized with each other, but were distinct from endocytic sites marked by DRP1C [35]. The number of exocyst puncta visualized at the Arabidopsis PM by TIRFM/VAEM is obviously distinctly higher than the expected number of vesicles docking and fusing at the PM, implying the possibility of the exocyst vesicle-independent dynamics at the PM; possibility supported by the fact that the exocyst still localizes to the PM when treated with brefeldin A (BFA) [35]. The inability of SEC3 to localize to the PM in the presence of BFA requires further investigation [21]. Overall the dynamics of exocyst at the PM supports the possibility that exocyst may function also as a dynamic particle in vesicle tethering [27,35,37,38]. Due to complex dynamic associations between subunits, it is also possible that the complex may be actively assembled and disassembled during a tethering event. These data are consistent with the endocytic patches in yeast where at any given time point components of the patches only look partially colocalized, but if the lifetime of a patch is taken into consideration, then the components are added to the patch in a very characteristic manner. Perhaps the exocyst in plants might show similar dynamics [39]. Functionality of the proteins analyzed is of central importance — only upon knocking down the endogenous SEC8 could the exogenous SEC8-tagRFP be incorporated into the complex in HeLa cells [36]; when the endogenous SEC8 was present, the tagged subunit clustered in aggregates detectable by confocal laser scanning microscopy. Similarly in Arabidopsis, the over-expressed EXO84b or some EXO70s subunits accumulated in PMproximal non-motile compartments, but were not present when the endogenous promoter was used [8,19].

Exocyst in biotic interactions — defence against pathogens and symbiosis EXO70s evolutionary dynamics as well as expression data showing that some Arabidopsis EXO70 isoforms are highly responsive to pathogens and their elicitors support a role for EXO70, and by extension the exocyst complex, in biotic, particularly pathogen interactions [10]. Analysis of knock-out mutants demonstrated that an EXO70B2 paralogue is involved in the defence response to three different pathogens tested: Pseudomonas syringae pv. maculicola, P. syringae pv. tomato and Hyaloperonospora parasitica [10,40]. In the EXO70B2 mutant non-host interaction with Blumeria graminis f. sp. hordei was also Current Opinion in Plant Biology 2013, 16:726–733

affected. Cell wall thickenings (papillae) on the contact site with germinating spores accumulated extensive vesicular compartments on the cytoplasmic side, suggesting a defect in vesicle tethering or fusion but without affecting the penetration efficiency of the fungus [10] (Figure 1d). However, when the same pathogen infects its host barley, the efficiency of its penetration was enhanced when the EXO70F1-like exocyst subunit was transiently silenced [41]. These observations imply that exocyst operates in general cellular defence against bacterial and fungal pathogens, including defensive papillae formation with a possible participation in multivesicular body (MVB)/exosome/paramural bodies delivery for papillae biogenesis. The exocyst functions also in the establishment of symbiotic relationship between plant hosts and mycorrhizal fungi, as documented by its localization to the perifungal membrane during the establishment of arbuscular mycorrhiza [42].

EXO70 subunits targeted degradation mediated by E3 ubiquitin ligases EXO70B2 was identified as a target of the plant U-boxtype ubiquitin ligase 22 (PUB22), which (together with PUB23 and PUB24) acts as a negative regulator of pathogen associated molecular patterns (PAMP) triggered responses, suggesting a role for EXO70B2 in PAMP signalling [40]. A similar mode of EXO70s regulation was observed in the pollen–stigma interaction; another Ubox ubiquitin E3-ligase ARC-1 (known component of pollen self-incompatibility pathway in Brassicaceae) ubiquitinated the EXO70A1 exocyst subunit resulting in its degradation and overall downregulation of stigmatic secretion and receptivity [43,44]. This mode of specific EXO70s downregulation by specific E3 ubiquitin ligases might contribute to very quick and dynamic adaptations of secretory pathways in plants in general. Exocyst complexes where specific EXO70 subunit are polyubiquitinated and degraded will be inactivated and eventually available to interact with other EXO70 isoforms to sustain a new secretory pathway (Figure 3).

Specific exocyst complexes also function in autophagy-related endomembrane trafficking In both animals and plants, an unexpected engagement of the exocyst complex in the autophagy pathway was recently discovered [7,8]. The small GTPase RalB activated by starvation in animal cells, localizes to the nascent autophagosome where it recruits an exocyst subcomplex via the EXO84 subunit, forming an assembly platform for proteins involved in autophagosome biogenesis [7]. Analysis of phenotypic deviations of the Arabidopsis exo70B1 mutant showed formation of low light dependent spontaneous lesions on leaves of mature plants and a large decrease in the autophagic compartments inside the vacuole and a reduction of anthocyanin www.sciencedirect.com

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Figure 3

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Possible general mechanism of exocyst downregulation by targeted EXO70 subunits polyubiquitination mediated by specific E3 ligases, resulting in degradation by proteasome. This might be also a mechanism allowing exchange of EXO70 isoforms within the core exocyst complex and its functional redirection after specific signalling events.

accumulation [8], which has been linked with Golgi-independent autophagic transport to the vacuole [45]. Cytological analysis uncovered co-localization of YFP-EXO70B1 with ATG8f-RFP positive compartments in the cytoplasm as well as in the vacuole after luminal alkalinization including co-localization with anthocyanincontaining compartments [8] (Figure 1a). Exosomes are apoplastic vesicles potentially derived from the autophagic process, as autophagy typically contributes also to MVB formation, and MVB fusion with the PM results in exosome vesicle release. In this context, secretion of EXO70s positive compartments from plant cells after the overexpression of several EX070s [9] may be interpreted as ectopic autophagosome formation and exosome release. It is possible that the formation and targeting of paramural bodies and exosomes release to the cell wall, known to contribute to defensive papillae formation against pathogen attack [46], might be dependent on some EXO70-specific form of the exocyst complex (Figure 2a).

Conclusions Comparison of exocyst subunits and functions across animals, fungi and plants clearly indicates that it was present in the last common eukaryotic ancestor (LECA) [47,48], and that in later land plant evolution, the exocyst adopted very specific functional specializations based on different EXO70 paralogues [4,5,6,8]. The list of prowww.sciencedirect.com

cesses involving the exocyst in Arabidopsis will continue to expand and will further underline that it participates in many aspects of the plant cell, including secretion, morphogenesis, defence against pathogens and stress reactions. It is however very well possible that some EXO70 paralogues acquired exocyst independent functions. Recent discoveries of exocyst involvement in autophagy in animals and plants [7,8] open a completely new perspective on exocyst roles – not only in the exocytosis to the PM, but also in the autophagy-related Golgi-independent delivery of endomembranes to the tonoplast/vacuole or apoplast. This gives the exocyst complex a regulatory position between the trafficking pathways with a potential to contribute significantly to the unexplored coordination of membrane and cargo delivery between the PM and the tonoplast.

Acknowledgements This work was supported by the Czech Science Foundation grant GACR P305/11/1629. Part of VZˇ’s income is from the project MSM0021620858 of the Czech Ministry of Education. Work on exocyst evolution is supported by the EU-ITN Grant No. 238640-PLANTORIGINS. We are grateful to our colleagues Michal Ha´la, Luka´sˇ Synek, Edita Jankova´ Drdova´, Marek Elia´sˇ, Martin Potocky´, Roman Pleskot and Fatima Cvrcˇkova´ from both the Institute of Experimental Botany ASCR and the Faculty of Science, Charles University in Prague for many important contributions and discussions; and our long-term colleagues from Oregon State University (OSU) at Corvallis in the USA, John Fowler and Rex Cole, for fruitful collaboration. Due to space limitation we were unable to refer to all relevant reports. Current Opinion in Plant Biology 2013, 16:726–733

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References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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Cvrcˇkova´ F, Grunt M, Bezvoda R, Ha´la M, Kulich I, Rawat A, Zˇarsky´ V: Evolution of the land plant exocyst complexes. Front Plant Sci 2012, 3:159. The phylogenetic history of all exocyst subunits in land plants is analyzed and discussed in the context of evolutionary adaptations and also whole genome duplications.

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Li S, van Os GMA, Ren S, Yu D, Ketelaar T, Emons AMC, Liu C-M: Expression and functional analyses of EXO70 genes in Arabidopsis implicate their roles in regulating cell typespecific exocytosis. Plant Physiol 2010, 154:1819-1830. Zˇarsky´ V, Cvrcˇkova´ F, Potocky´ M, Ha´la M: Exocytosis and cell polarity in plants — exocyst and recycling domains. New Phytol 2009, 183:255-272. Bodemann BO, Orvedahl A, Cheng T, Ram RR, Ou Y-H, Formstecher E, Maiti M, Hazelett CC, Wauson EM, Balakireva M et al.: RalB and the exocyst mediate the cellular starvation response by direct activation of autophagosome assembly. Cell 2011, 144:253-267.

Kulich I, Pecˇenkova´ T, Sekeresˇ J, Smetana O, Fendrych M, Foissner I, Ho¨ftberger M, Zˇa´rsky´ V: Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in the autophagy-related transport to the vacuole. Traffic 2013, 14:1155-1165. Exocyst, known as a tethering complex for exocytotic vesicles, is surprisingly discovered to be involved in the GA-independent autophagy-related import into the vacuole. This pathway is dependent on the specific subcomplex with the EXO70 isoform B1.

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10. Pecˇenkova´ T, Ha´la M, Kulich I, Kocourkova´ D, Drdova´ E,  Fendrych M, Toupalova´ H, Zˇa´rsky´ V: The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plantpathogen interaction. J Exp Bot 2011, 62:2107-2116. In this report the involvement of exocyst complexes with B2 and H1 EXO70 subunits in the defence against both bacterial and fungal pathogens is documented. 11. Lin C-Y, Trinh NN, Fu S-F, Hsiung Y-C, Chia L-C, Lin C-W,  Huang H-J: Comparison of early transcriptome responses to copper and cadmium in rice roots. Plant Mol Biol 2013, 81:507522. Participation of the exocyst complex in heavy-metal abiotic stress amelioration is documented. 12. Lipschutz JH, Lingappa VR, Mostov KE: The exocyst affects protein synthesis by acting on the translocation machinery of the endoplasmic reticulum. J Biol Chem 2003, 278:2095420960. 13. Dellago H, Lo¨scher M, Ajuh P, Ryder U, Kaisermayer C, GrillariVoglauer R, Fortschegger K, Gross S, Gstraunthaler A, Borth N et al.: Exo70, a subunit of the exocyst complex, interacts with SNEV(hPrp19/hPso4) and is involved in pre-mRNA splicing. Biochem J 2011, 438:81-91. Current Opinion in Plant Biology 2013, 16:726–733

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Exocyst complexes multiple functions in plant cells secretory pathways Kulich et al. 733

tracheary element development in Arabidopsis. Plant Cell 2013, 25:1774-1786. The most abundant Arabidopsis EXO70A1 isoform known to be involved in cytokinesis initiation, seed coat biogenesis, apical dominance, lateral root and root hair development, stigma receptivity and PINs recycling was shown to be crucial for root xylem differentiation. 33. Drdova´ EJ, Synek L, Pecˇenkova´ T, Ha´la M, Kulich I, Fowler JE,  Murphy AS, Zˇa´rsky´ V: The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis. Plant J Cell Mol Biol 2013, 73:709-719. The exocyst complex was described to be involved in the PM proteins’ recycling with consequences for polar auxin transport, as both PIN1 and PIN2 proteins were also affected. 34. Hazak O, Bloch D, Poraty L, Sternberg H, Zhang J, Friml J, Yalovsky S: A Rho scaffold integrates the secretory system with feedback mechanisms in regulation of auxin distribution. PLoS Biol 2010, 8:e1000282. 35. Fendrych M, Synek L, Pecˇenkova´ T, Drdova´ EJ, Sekeresˇ J,  Rycke R, de Nowack MK, Zˇa´rsky´ V: Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana. Mol Biol Cell 2013, 24:510-520. Using total internal reflection fluorescence microscopy, the exocyst complex dynamics at the PM was monitored for the first time. Frequency of co-localized subunit events of approximately 10 s life-time (stationary — without lateral movement) indicate the possibility of the exocystcomplex functioning as a particle. 36. Rivera-Molina F, Toomre D: Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion. J Cell Biol 2013, 201:673-680. 37. Bendezu´ FO, Vincenzetti V, Martin SG: Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles. PLoS ONE 2012, 7:e40248. 38. Wu H, Rossi G, Brennwald P: The ghost in the machine: small GTPases as spatial regulators of exocytosis. Trends Cell Biol 2008, 18:397-404. 39. Weinberg J, Drubin DG: Clathrin mediated endocytosis in budding yeast. Trends Cell Biol 2012, 22:1-13. 40. Stegmann M, Anderson RG, Ichimura K, Pecˇenkova´ T, Reuter P,  Zˇa´rsky V, McDowell JM, Shirasu K, Trujillo M: The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis. Plant Cell 2012, 24:4703-4716.

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The regulation of the exocyst functions in plant immunity against pathogens is determined by the U-BOX E3 polyubiquitin ligases. EXO70B2 is targeted for degradation. Likewise EXO70A1 was shown to be targeted for degradation by ARC1 E3-ligase in the self-incompatibility pollen rejection [43]. 41. Ostertag M, Stammler J, Douchkov D, Eichmann R,  Hu¨ckelhoven R: The conserved oligomeric Golgi complex is involved in penetration resistance of barley to the barley powdery mildew fungus. Mol Plant Pathol 2013, 14:230-240. EXO70 exocyst subunit from grasses-specific clade FX is shown to be necessary for resistance to Blumeria graminis in barley. 42. Genre A, Ivanov S, Fendrych M, Faccio A, Zˇa´rsky´ V, Bisseling T,  Bonfante P: Multiple exocytotic markers accumulate at the sites of perifungal membrane biogenesis in arbuscular mycorrhizas. Plant Cell Physiol 2012, 53:244-255. The exocyst is implied in the biogenesis of perifungal membranes and linked to the symbiotic fungal–root relationship establishment. 43. Samuel MA, Chong YT, Haasen KE, Aldea-Brydges MG, Stone SL, Goring DR: Cellular pathways regulating responses to compatible and self-incompatible pollen in Brassica and Arabidopsis stigmas intersect at Exo70A1, a putative component of the exocyst complex. Plant Cell 2009, 21:26552671. 44. Kitashiba H, Liu P, Nishio T, Nasrallah JB, Nasrallah ME: Functional test of Brassica self-incompatibility modifiers in Arabidopsis thaliana. Proc Natl Acad Sci USA 2011, 108:1817318178. 45. Pourcel L, Irani NG, Lu Y, Riedl K, Schwartz S, Grotewold E: The formation of anthocyanic vacuolar inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments. Mol Plant 2010, 3:78-90. 46. An Q, Hu¨ckelhoven R, Kogel K-H, van Bel AJE: Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus. Cell Microbiol 2006, 8:1009-1019. 47. Koumandou VL, Dacks JB, Coulson RMR, Field MC: Control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and SM proteins. BMC Evol Biol 2007, 7:29. 48. Vasˇkovicˇova´ K, Zˇa´rsky´ V, Ro¨sel D, Nikolicˇ M, Buccione R, Cvrcˇkova´ F, Bra´bek J: Invasive cells in animals and plants: searching for LECA machineries in later eukaryotic life. Biol Direct 2013, 8:8.

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