More Than Meets The Eye: Knowns And Unknowns Of The Trafficking Of Small Secreted Proteins In Arabidopsis.

Small proteins represent a significant portion of the cargo transported through plant secretory pathways, playing crucial roles in developmental processes, fertilization, and responses to environmental stresses. Despite their importance, substantial knowledge gaps persist regarding the regulatory mechanisms governing their trafficking along the secretory pathway, and ultimate localization/destination. To address these gaps, we conducted a comprehensive literature review, focusing particularly on trafficking and localization of small secreted proteins with potential biochemical and/or signaling roles in the extracellular space, typically within the size range of 101-200 amino acids. Our investigation reveals that while at least 6 members of the 21 mentioned families confirm extracellular localization, 8 of them exhibit intracellular localization (including cytoplasmic, nuclear, and chloroplastic locations, despite the presence of N-terminal signal peptides). Further investigation into the trafficking and secretion mechanisms of small protein cargo could not only deepen our understanding of plant cell biology and physiology but also provide a foundation for manipulation strategies leading to more efficient plant cultivation.

Small secreted proteins and exocytosis regulators: do they go along?

Small secreted proteins play an important role in plant development, as well as in reactions to changes in the environment. In Arabidopsis thaliana, they are predominantly members of highly expanded families, such as the pathogenesis-related (PR) 1-like protein family, whose most studied member PR1 is involved in plant defense responses by a so far unknown mechanism, or Clavata3/Endosperm Surrounding Region (CLE) protein family, whose members' functions in the development are well described. Our survey of the existing literature for the two families showed a lack of details on their localization, trafficking, and exocytosis. Therefore, in order to uncover the modes of their secretion, we tested the hypothesis that a direct link between the secreted cargoes and the secretion regulators such as Rab GTPases, SNAREs, and exocyst subunits could be established using in silico co-expression and clustering approaches. We employed several independent techniques to uncover that only weak co-expression links could be found for limited numbers of secreted cargoes and regulators. We propose that there might be particular spatio-temporal requirements for PR1 and CLE proteins to be synthesized and secreted, and efforts to experimentally cover these discrepancies should be invested along with functional studies.

Immunity functions of Arabidopsis pathogenesis-related 1 are coupled but not confined to its C-terminus processing and trafficking.

The pathogenesis-related 1 (PR1) proteins are members of the cross-kingdom conserved CAP superfamily (from Cysteine-rich secretory protein, Antigen 5, and PR1 proteins). PR1 mRNA expression is frequently used for biotic stress monitoring in plants; however, the molecular mechanisms of its cellular processing, localization, and function are still unknown. To analyse the localization and immunity features of Arabidopsis thaliana PR1, we employed transient expression in Nicotiana benthamiana of the tagged full-length PR1 construct, and also disrupted variants with C-terminal truncations or mutations. We found that en route from the endoplasmic reticulum, the PR1 protein transits via the multivesicular body and undergoes partial proteolytic processing, dependent on an intact C-terminal motif. Importantly, only nonmutated or processing-mimicking variants of PR1 are secreted to the apoplast. The C-terminal proteolytic cleavage releases a protein fragment that acts as a modulator of plant defence responses, including localized cell death control. However, other parts of PR1 also have immunity potential unrelated to cell death. The described modes of the PR1 contribution to immunity were found to be tissue-localized and host plant ontogenesis dependent.

Arabidopsis EXO70B2 exocyst subunit contributes to papillae and encasement formation in antifungal defence.

In the reaction to non-adapted Blumeria graminis f. sp. hordei (Bgh), Arabidopsis thaliana leaf epidermal cells deposit cell wall reinforcements called papillae or seal fungal haustoria in encasements, both of which involve intensive exocytosis. A plant syntaxin, SYP121/PEN1, has been found to be of key importance for the timely formation of papillae, and the vesicle tethering complex exocyst subunit EXO70B2 has been found to contribute to their morphology. Here, we identify a specific role for the EXO70B2-containing exocyst complex in the papillae membrane domains important for callose deposition and GFP-SYP121 delivery to the focal attack sites, as well as its contribution to encasement formation. The mRuby2-EXO70B2 co-localizes with the exocyst core subunit SEC6 and GFP-SYP121 in the membrane domain of papillae, and EXO70B2 and SYP121 proteins have the capacity to directly interact. The exo70B2/syp121 double mutant produces a reduced number of papillae and haustorial encasements in response to Bgh, indicating an additive role of the exocyst in SYP121-coordinated non-host resistance. In summary, we report cooperation between the plant exocyst and a SNARE protein in penetration resistance against non-adapted fungal pathogens.

Plasma membrane phospholipid signature recruits the plant exocyst complex via the EXO70A1 subunit.

Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid-protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3-SEC5-SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3-EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1-phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein-lipid code for the recruitment of peripheral membrane proteins.

Redundant and Diversified Roles Among Selected Arabidopsis thaliana EXO70 Paralogs During Biotic Stress Responses.

The heterooctameric vesicle-tethering complex exocyst is important for plant development, growth, and immunity. Multiple paralogs exist for most subunits of this complex; especially the membrane-interacting subunit EXO70 underwent extensive amplification in land plants, suggesting functional specialization. Despite this specialization, most Arabidopsis exo70 mutants are viable and free of developmental defects, probably as a consequence of redundancy among isoforms. Our in silico data-mining and modeling analysis, corroborated by transcriptomic experiments, pinpointed several EXO70 paralogs to be involved in plant biotic interactions. We therefore tested corresponding single and selected double mutant combinations (for paralogs EXO70A1, B1, B2, H1, E1, and F1) in their two biologically distinct responses to Pseudomonas syringae, root hair growth stimulation and general plant susceptibility. A shift in defense responses toward either increased or decreased sensitivity was found in several double mutants compared to wild type plants or corresponding single mutants, strongly indicating both additive and compensatory effects of exo70 mutations. In addition, our experiments confirm the lipid-binding capacity of selected EXO70s, however, without the clear relatedness to predicted C-terminal lipid-binding motifs. Our analysis uncovers that there is less of functional redundancy among isoforms than we could suppose from whole sequence phylogeny and that even paralogs with overlapping expression pattern and similar membrane-binding capacity appear to have exclusive roles in plant development and biotic interactions.

Three subfamilies of exocyst EXO70 family subunits in land plants: early divergence and ongoing functional specialization.

Localized delivery of plasma membrane and cell wall components is an essential process in all plant cells. The vesicle-tethering complex, the exocyst, an ancient eukaryotic hetero-octameric protein cellular module, assists in targeted delivery of exocytosis vesicles to specific plasma membrane domains. Analyses of Arabidopsis and later other land plant genomes led to the surprising prediction of multiple putative EXO70 exocyst subunit paralogues. All land plant EXO70 exocyst subunits (including those of Bryophytes) form three distinct subfamilies-EXO70.1, EXO70.2, and EXO70.3. Interestingly, while the basal well-conserved EXO70.1 subfamily consists of multiexon genes, the remaining two subfamilies contain mostly single exon genes. Published analyses as well as public transcriptomic and proteomic data clearly indicate that most cell types in plants express and also use several different EXO70 isoforms. Here we sum up recent advances in the characterization of the members of the family of plant EXO70 exocyst subunits and present evidence that members of the EXO70.2 subfamily are often recruited to non-canonical functions in plant membrane trafficking pathways. Engagement of the most evolutionarily dynamic EXO70.2 subfamily of EXO70s in biotic interactions and defence correlates well with massive proliferation and conservation of new protein variants in this subfamily.

Exocyst and autophagy-related membrane trafficking in plants.

Endomembrane traffic in eukaryotic cells functions partially as a means of communication; delivery of membrane in one direction has to be balanced with a reduction at the other end. This effect is typically the case during the defence against pathogens. To combat pathogens, cellular growth and differentiation are suppressed, while endomembrane traffic is poised towards limiting the pathogen attack. The octameric exocyst vesicle-tethering complex was originally discovered as a factor facilitating vesicle-targeting and vesicle-plasma membrane (PM) fusion during exocytosis prior to and possibly during SNARE complex formation. Interestingly, it was recently implicated both in animals and plants in autophagy membrane traffic. In animal cells, the exocyst is integrated into the mTOR-regulated energy metabolism stress/starvation pathway, participating in the formation and especially initiation of an autophagosome. In plants, the first functional link was to autophagy-related anthocyanin import to the vacuole and to starvation. In this concise review, we summarize the current knowledge of exocyst functions in autophagy and defence in plants that might involve unconventional secretion and compare it with animal conditions. Formation of different exocyst complexes during undisturbed cell growth, as opposed to periods of cellular stress reactions involving autophagy, might contribute to the coordination of endomembrane trafficking pathways.

Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae.

Background and Aims: Selected beneficial Pseudomonas spp. strains have the ability to influence root architecture in Arabidopsis thaliana by inhibiting primary root elongation and promoting lateral root and root hair formation. A crucial role for auxin in this long-term (1week), long-distance plant-microbe interaction has been demonstrated. Methods: Arabidopsis seedlings were cultivated in vitro on vertical plates and inoculated with pathogenic strains Pseudomonas syringae pv. maculicola (Psm) and P. syringae pv. tomato DC3000 (Pst), as well as Agrobacterium tumefaciens (Atu) and Escherichia coli (Eco). Root hair lengths were measured after 24 and 48h of direct exposure to each bacterial strain. Several Arabidopsis mutants with impaired responses to pathogens, impaired ethylene perception and defects in the exocyst vesicle tethering complex that is involved in secretion were also analysed. Key Results: Arabidopsis seedling roots infected with Psm or Pst responded similarly to when infected with plant growth-promoting rhizobacteria; root hair growth was stimulated and primary root growth was inhibited. Other plant- and soil-adapted bacteria induced similar root hair responses. The most compromised root hair growth stimulation response was found for the knockout mutants exo70A1 and ein2. The single immune pathways dependent on salicylic acid, jasmonic acid and PAD4 are not directly involved in root hair growth stimulation; however, in the mutual cross-talk with ethylene, they indirectly modify the extent of the stimulation of root hair growth. The Flg22 peptide does not initiate root hair stimulation as intact bacteria do, but pretreatment with Flg22 prior to Psm inoculation abolished root hair growth stimulation in an FLS2 receptor kinase-dependent manner. These early response phenomena are not associated with changes in auxin levels, as monitored with the pDR5::GUS auxin reporter. Conclusions: Early stimulation of root hair growth is an effect of an unidentified component of living plant pathogenic bacteria. The root hair growth response is triggered in the range of hours after bacterial contact with roots and can be modulated by FLS2 signalling. Bacterial stimulation of root hair growth requires functional ethylene signalling and an efficient exocyst-dependent secretory machinery.

Subcellular Localization of Arabidopsis Pathogenesis-Related 1 (PR1) Protein.

The Arabidopsisthaliana pathogenesis-related 1 (PR1) is an important defense protein, so far it has only been detected in extracellular space and its subcellular sorting and transport remain unexplained. Using a green fluorescent protein (GFP) tagged full length, as well as a C-terminus truncated version of PR1, we observed that when expressed ectopically in Nicotiana benthamiana leaves, PR1 co-localizes only partially with Golgi markers, and much more prominently with the late endosome (LE)/multivesicular body (MVB) FYVE marker. The C-truncated version PR1ΔC predominantly localized to the endoplasmic reticulum (ER). The same localizations were found for stable Arabidopsis transformants with expression of PR1 and PR1ΔC driven by the native promoter. We conclude that the A. thaliana PR1 (AtPR1) undergoes an unconventional secretion pathway, starting from the C-terminus-dependent sorting from the ER, and utilizing further transportation via phosphatidyl-inositol-3-phosphate (PI(3)P) positive LE/MVB-like vesicles. The homology model of the PR1 structure shows that the cluster of positively charged amino acid residues (arginines 60, 67, 137, and lysine 135) could indeed interact with negatively charged phospholipids of cellular membranes. It remains to be resolved whether Golgi and LE/MVB localization reflects an alternative sorting or trafficking succession, and what the role of lipid interactions in it will be.

Microtubule-dependent targeting of the exocyst complex is necessary for xylem development in Arabidopsis.

Cortical microtubules (MTs) play a major role in the patterning of secondary cell wall (SCW) thickenings in tracheary elements (TEs) by determining the sites of SCW deposition. The EXO70A1 subunit of the exocyst secretory vesicle tethering complex was implicated to be important for TE development via the MT interaction. We investigated the subcellular localization of several exocyst subunits in the xylem of Arabidopsis thaliana and analyzed the functional significance of exocyst-mediated trafficking in TE development. Live cell imaging of fluorescently tagged exocyst subunits in TE using confocal microscopy and protein-protein interaction assays were performed to describe the role of the exocyst and its partners in TE development. In TEs, exocyst subunits were localized to the sites of SCW deposition in an MT-dependent manner. We propose that the mechanism of exocyst targeting to MTs involves the direct interaction of exocyst subunits with the COG2 protein. We demonstrated the importance of a functional exocyst subunit EXO84b for normal TE development and showed that the deposition of SCW constituents is partially compromised, possibly as a result of the mislocalization of secondary cellulose synthase in exocyst mutants. We conclude that the exocyst complex is an important factor bridging the pattern defined by cortical MTs with localized secretion of the SCW in developing TEs.

The SBT6.1 subtilase processes the GOLVEN1 peptide controlling cell elongation.

The GOLVEN (GLV) gene family encode small secreted peptides involved in important plant developmental programs. Little is known about the factors required for the production of the mature bioactive GLV peptides. Through a genetic suppressor screen in Arabidopsis thaliana, two related subtilase genes, AtSBT6.1 and AtSBT6.2, were identified that are necessary for GLV1 activity. Root and hypocotyl GLV1 overexpression phenotypes were suppressed by mutations in either of the subtilase genes. Synthetic GLV-derived peptides were cleaved in vitro by the affinity-purified SBT6.1 catalytic enzyme, confirming that the GLV1 precursor is a direct subtilase substrate, and the elimination of the in vitro subtilase recognition sites through alanine substitution suppressed the GLV1 gain-of-function phenotype in vivo Furthermore, the protease inhibitor Serpin1 bound to SBT6.1 and inhibited the cleavage of GLV1 precursors by the protease. GLV1 and its homolog GLV2 were expressed in the outer cell layers of the hypocotyl, preferentially in regions of rapid cell elongation. In agreement with the SBT6 role in GLV precursor processing, both null mutants for sbt6.1 and sbt6.2 and the Serpin1 overexpression plants had shorter hypocotyls. The biosynthesis of the GLV signaling peptides required subtilase activity and might be regulated by specific protease inhibitors. The data fit with a model in which the GLV1 signaling pathway participates in the regulation of hypocotyl cell elongation, is controlled by SBT6 subtilases, and is modulated locally by the Serpin1 protease inhibitor.

Constitutive Negative Regulation of R Proteins in Arabidopsis also via Autophagy Related Pathway?

Even though resistance (R) genes are among the most studied components of the plant immunity, there remain still a lot of aspects to be explained about the regulation of their function. Many gain-of-function mutants of R genes and loss-of-function of their regulators often demonstrate up-regulated defense responses in combination with dwarf stature and/or spontaneous leaf lesions formation. For most of these mutants, phenotypes are a consequence of an ectopic activation of R genes. Based on the compilation and comparison of published results in this field, we have concluded that the constitutively activated defense phenotypes recurrently arise by disruption of tight, constitutive and multilevel negative control of some of R proteins that might involve also their targeting to the autophagy pathway. This mode of R protein regulation is supported also by protein-protein interactions listed in available databases, as well as in silico search for autophagy machinery interacting motifs. The suggested model could resolve some explanatory discrepancies found in the studies of the immunity responses of autophagy mutants.

Exocyst complexes multiple functions in plant cells secretory pathways.

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.

Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in autophagy-related transport to the vacuole.

Autophagic transport to the vacuole represents an endomembrane trafficking route, which is widely used in plants, not only during stress situations, but also for vacuole biogenesis and during developmental processes. Here we report a role in autophagic membrane transport for EXO70B1--one of 23 paralogs of Arabidopsis EXO70 exocyst subunits. EXO70B1 positive compartments are internalized into the central vacuole and co-localize with autophagosomal marker ATG8f. This internalization is boosted by induction of autophagy. Loss of function (LOF) mutations in exo70B1 cause reduction of internalized autopagic bodies in the vacuole. Mutant plants also show ectopic hypersensitive response (HR) mediated by salicylic acid (SA) accumulation, increased nitrogen starvation susceptibility and anthocyanin accumulation defects. Anthocyanin accumulation defect persists in npr1x exo70B1 double mutants with SA signaling compromised, while ectopic HR is suppressed. EXO70B1 interacts with SEC5 and EXO84 and forms an exocyst subcomplex involved in autophagy-related, Golgi-independent membrane traffic to the vacuole. We show that EXO70B1 is functionally completely different from EXO70A1 exocyst subunit and adopted a specific role in autophagic transport.

Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana.

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.

The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis.

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle-tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.

The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis.

Plant pathogens are perceived by pattern recognition receptors, which are activated upon binding to pathogen-associated molecular patterns (PAMPs). Ubiquitination and vesicle trafficking have been linked to the regulation of immune signaling. However, little information exists about components of vesicle trafficking involved in immune signaling and the mechanisms that regulate them. In this study, we identified Arabidopsis thaliana Exo70B2, a subunit of the exocyst complex that mediates vesicle tethering during exocytosis, as a target of the plant U-box-type ubiquitin ligase 22 (PUB22), which acts in concert with PUB23 and PUB24 as a negative regulator of PAMP-triggered responses. We show that Exo70B2 is required for both immediate and later responses triggered by all tested PAMPs, suggestive of a role in signaling. Exo70B2 is also necessary for the immune response against different pathogens. Our data demonstrate that PUB22 mediates the ubiquitination and degradation of Exo70B2 via the 26S Proteasome. Furthermore, degradation is regulated by the autocatalytic turnover of PUB22, which is stabilized upon PAMP perception. We therefore propose a mechanism by which PUB22-mediated degradation of Exo70B2 contributes to the attenuation of PAMP-induced signaling.

The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant-pathogen interaction.

Recently, the octameric vesicle-tethering complex exocyst was found in plants and its importance for Arabidopsis morphogenesis was demonstrated. Exo70 exocyst subunits in plants, unlike in yeasts and mammals, are represented by a multigene family, comprising 23 members in Arabidopsis. For Exo70B2 and Exo70H1 paralogues, transcriptional up-regulation was confirmed on treatment with an elicitor peptide, elf18, derived from the bacterial elongation factor. Their ability to participate in the exocyst complex formation was inferred by the interaction of both the Exo70s with several other exocyst subunits using the yeast two-hybrid system. Arabidopsis plants mutated in these two genes were used to analyse their local reaction upon inoculation with Pseudomonas syringae pv. maculicola and the fungal pathogen Blumeria graminis f. sp. hordei. The Pseudomonas sensitivity test revealed enhanced susceptibility for the two exo70B2 and one H1 mutant lines. After Blumeria inoculation, an increase in the proportion of abnormal papilla formation, with an unusual wide halo made of vesicle-like structures, was found in exo70B2 mutants. Intracellular localization of both Exo70 proteins was studied following a GFP fusion assay and Agrobacterium-mediated transient expression of the constructs in Nicotiana benthamiana leaf epidermis. GFP-Exo70H1 localizes in the vesicle-like structures, while GFP-Exo70B2 is localized mainly in the cytoplasm. It is concluded that both Exo70B2 and Exo70H1 are involved in the response to pathogens, with Exo70B2 having a more important role in cell wall apposition formation related to plant defence.

The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation.

Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein-tagged exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall.