EXO70 Genes Research Articles

Genome-Wide Analysis of Exocyst Complex Subunit Exo70 Gene Family in Cucumber.

Cucumber (Cucumis sativus L.) is an important vegetable worldwide, but its yield is affected by a wide range of pathogens and pests. As the major subunit of the exocyst complex, the roles of Exo70 members have been shown in Arabidopsis and rice, but their function are unknown in cucumber. Here, we identified 18 CsExo70 members in cucumber, which were divided into three groups (Exo70.1-Exo70.3) and nine subgroups (Exo70A-Exo70I) based on the phylogenetic tree. Subsequently, systematical analyses were performed, including collinearity, gene structure, cis-acting elements, conserved motifs, expression patterns, and subcellular localization. Our results showed that CsExo70 genes were generally expressed in all tissues, and CsExo70C1 and CsExo70C2 were highly expressed in the stamen. Moreover, the expression levels of most CsExo70 genes were induced by Pseudomonas syringae pv. lachrymans (Psl) and Fusarium oxysporum f. sp. cucumerinum Owen (Foc), especially CsExo70E2 and CsExo70H3. In addition, these CsExo70s displayed similar location patterns with discrete and punctate signals in the cytoplasm. Together, our results indicate that CsExo70 members may be involved in plant development and resistance, and provide a reference for future in-depth studies of Exo70 genes in cucumber.

Identification and Comprehensive Structural and Functional Analyses of the EXO70 Gene Family in Cotton.

The EXO70 gene is a vital component of the exocytosis complex and participates in biological processes ranging from plant cell division to polar growth. There are many EXO70 genes in plants and their functions are extensive, but little is known about the EXO70 gene family in cotton. Here, we analyzed four cotton sequence databases, identified 165 EXO70 genes, and divided them into eight subgroups (EXO70A–EXO70H) based on their phylogenetic relationships. EXO70A had the most exons (≥11), whereas the other seven each had only one or two exons. Hence, EXO70A may have many important functions. The 84 EXO70 genes in Asian and upland cotton were expressed in the roots, stems, leaves, flowers, fibers, and/or ovules. Full-length GhEXO70A1-A cDNA was homologously cloned from upland cotton (Gossypium hirsutum, G. hirsutum). Subcellular analysis revealed that GhEXO70A1-A protein was localized to the plasma membrane. A yeast two-hybrid assay revealed that GhEXO70A1-A interacted with GhEXO84A, GhEXO84B, and GhEXO84C. GhEXO70A1-A silencing significantly altered over 4000 genes and changed several signaling pathways related to metabolism. Thus, the EXO70 gene plays critical roles in the physiological functions of cotton.

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.

Genome-wide identification and expression analysis of the EXO70 gene family in grape (Vitis vinifera L).

EXO70 is the pivotal protein subunit of exocyst, which has a very crucial role in enhancing the shielding effect of the cell wall, resisting abiotic and hormonal stresses. This experiment aims to identify family members of the EXO70 gene family in grape and predict the characteristics of this gene family, so as to lay the foundation of further exploring the mechanism of resisting abiotic and hormone stresses of VvEXO70s. Therefore, the Vitis vinifera ‘Red Globe’ tube plantlet were used as materials. Bioinformatics was used to inquire VvEXO70 genes family members, gene structure, system evolution, cis-acting elements, subcellular and chromosomal localization, collinearity, selective pressure, codon bias and tissue expression. All of VvEXO70s had the conserved pfam03081 domain which maybe necessary for interacting with other proteins. Microarray analysis suggested that most genes expressed to varying degrees in tendrils, leaves, seeds, buds, roots and stems. Quantitative Real-Time PCR (qRT-PCR) showed that the expression levels of all genes with 5 mM salicylic acid (SA), 0.1 mM methy jasmonate (MeJA), 20% PEG6000 and 4 °C for 24 h were higher than for 12 h. With 20% PEG6000 treatment about 24 h, the relative expression of VvEXO70-02 was significantly up-regulated and 361 times higher than CK. All genes’ relative expression was higher at 12 h than that at 24 h after treatment with 7 mM hydrogen peroxide (H2O2) and 0.1 mM ethylene (ETH). In conclusion, the expression levels of 14 VvEXO70 genes are distinguishing under these treatments, which play an important role in the regulation of anti-stress signals in grape. All of these test results provide a reference for the future research on the potential function analysis and plant breeding of VvEXO70 genes.

SHORT-ROOT1 is critical to cell division and tracheary element development in rice roots.

The exocyst is a key factor in vesicle transport and is involved in cell secretion, cell growth, cell division and other cytological processes in eukaryotes. EXO70 is the key exocyst subunit. We obtained a gene, SHORT-ROOT1 (SR1), through map-based cloning and genetic complementation. SR1 is a conserved protein with an EXO70 domain in plants. SR1 mutation affected the whole root-development process: producing shorter radicles, adventitious roots and lateral roots, and demonstrating abnormal xylem development, resulting in dwarfing and reduced water potential and moisture content. SR1 was largely expressed in the roots, but only in developing root meristems and tracheary elements. The shortness of the sr1 mutant roots was caused by the presence of fewer meristem cells. The insitu histone H4 expression patterns confirmed that cell proliferation during root development was impaired. Tracheary element dysplasia was caused by marked decreases in the inner diameters of and distances between the perforations of adjacent tracheary elements. The membrane transport of sr1 mutants was blocked, affecting cell division in the root apical region and the development of root tracheary elements. The study of SR1 will deepen our understanding of the function of EXO70 genes in Oryza sativa (rice) and guide future studies on the molecular mechanisms involved in plant root development.

Overexpression of the dominant negative nbexo70d1 mutantionconfers tolerance to salt stress in transgenic tobacco

The vesicle trafficking process, which involves exocytotic and endocytotic pathways, has been reported to play a role in regulating plant responses to different environmental stresses. The Exo70 protein is important for the localization of the exocyst in the plasma membrane; however, its role in the physiology of stress tolerance is currently unclear. In this study, we characterized NbExo70D1, an Exo70 gene from tobacco (Nicotiana benthamiana). It was shown to have a role in the plant response to salt stress. More specifically, tolerance to salt stress is conferred by the overexpression of the dominant negative nbexo70d1 domain D mutation in transgenic tobacco. In addition, a reduced accumulation of reactive oxygen species (ROS) under salt treatment was observed in the transgenic lines compared to the wild type. Treatment with diphenylene iodonium, an NADPH oxidase inhibitor, resulted in a decrease in salt stress-triggered ROS production in the roots of both wild type tobacco and transgenic tobacco. Furthermore, there was a reduction in NADPH oxidase activity in the transgenic plants under salt treatment, which indicates NbExo70D1 is involved in NADPH oxidase-mediated ROS production. We also characterized the tissue-specific expression patterns of NbExo70D1 during salt stress response by expressing the ProNbExo70D1-β-glucuronidase reporter construct in plants. Importantly, the GFP-NbExo70D1 fusion protein was localized in both the plasma membrane and the cytoplasm; expressing the dominant negative mutation disrupted the interaction between NbExo70D1 protein and the plasma membrane. Overall, our study suggests that Exo70 plays an important role in regulating the production and transmission of ROS as part of a salt stress response in plants.

Identification and Characterization of the EXO70 Gene Family in Polyploid Wheat and Related Species.

The EXO70 gene family is involved in different biological processes in plants, ranging from plant polar growth to plant immunity. To date, analysis of the EXO70 gene family has been limited in Triticeae species, e.g., hexaploidy Triticum aestivum and its ancestral/related species. By in silico analysis of multiple Triticeae sequence databases, a total of 200 EXO70 members were identified. By homologue cloning approaches, 15 full-length cDNA of EXO70s were cloned from diploid Haynaldia villosa. Phylogenetic relationship analysis of 215 EXO70 members classified them into three groups (EXO70.1, EXO70.2, and EXO70.3) and nine subgroups (EXO70A to EXO70I). The distribution of most EXO70 genes among different species/sub-genomes were collinear, implying their orthologous relationship. The EXO70A subgroup has the most introns (at least five introns), while the remaining seven subgroups have only one intron on average. The expression profiling of EXO70 genes from wheat revealed that 40 wheat EXO70 genes were expressed in at least one tissue (leaf, stem, or root), of which 25 wheat EXO70 genes were in response to at least one biotic stress (stripe rust or powdery mildew) or abiotic stress (drought or heat). Subcellular localization analysis showed that ten EXO70-V proteins had distinct plasma membrane localization, EXO70I1-V showed a distinctive spotted pattern on the membrane. The 15 EXO70-V genes were differentially expressed in three tissue. Apart from EXO70D2-V, the remaining EXO70-V genes were in response to at least one stress (flg22, chitin, powdery mildew, drought, NaCl, heat, or cold) or phytohormones (salicylic acid, methyl jasmonate, ethephon, or abscisic acid) and hydrogen peroxide treatments. This research provides a genome-wide glimpse of the Triticeae EXO70 gene family and those up- or downregulated genes require further validation of their biological roles in response to biotic/abiotic stresses.

An Ancient Integration in a Plant NLR is Maintained as a Trans-species Polymorphism

The origin of genetic diversity in a species is a product of novel mutation and alleles maintained during speciation. Identification of alleles that are derived from a common ancestor is critical for understanding the selective processes experienced during speciation events. Here, we report on the inter- and intra-specific diversity of plant immune receptors in the Mla locus across Poaceae species. These immune receptors belong to the class of cytoplasmic-nuclear localized nucleotide-binding leucine-rich repeat (NLR) proteins. Plant immune receptors are under constant selective pressure to maintain resistance to plant pathogens, and novel mutations leading to improved immune responses are selected. We discovered a fusion of the plant immune receptor RGH2 with a component of the exocyst complex (Exo70). Phylogenetic analysis of Exo70 gene families from Poaceae species found that the integrated Exo70 was derived from Exo70F1. To identify the origin of this gene fusion, we assembled leaf transcriptomes from diverse Poaceae species. Inter-specific conservation in the RGH2- Exo70F1 gene fusion was found in several Pooideae species, whereas Brachypodium distachyon RGH2 is fused to a receptor-like kinase. To establish the time of gene fusion, we performed phylogenetic analysis using non-integrated and integrated Exo70F1. We found a diphyletic tree composed of non-integrated Exo70F1 and integrated Exo70F1, with Exo70F1 from B. distachyon and B. stacei forming an outgroup. This indicates that a single gene fusion event occurred after the speciation of Brachypodieae, but prior to Poeae-Triticeae radiation. These results, coupled with the observation of inter- and intra-specific variation in RGH2, demonstrate the maintenance of RGH2-Exo70F1 as a trans-species polymorphism over 24 My.

Expression and Functional Analysis of a Novel Group of Legume-specific WRKY and Exo70 Protein Variants from Soybean.

Legumes fix atmospheric nitrogen through symbiosis with microorganisms and contain special traits in nitrogen assimilation and associated processes. Recently, we have reported a novel WRKY-related protein (GmWRP1) and a new clade of Exo70 proteins (GmExo70J) from soybean with homologs found only in legumes. GmWRP1 and some of the GmExo70J proteins are localized to Golgi apparatus through a novel N-terminal transmembrane domain. Here, we report further analysis of expression and functions of the novel GmWRP1 and GmExo70J genes. Promoter-GUS analysis in Arabidopsis revealed distinct tissue-specific expression patterns of the GmExo70J genes not only in vegetative but also in reproductive organs including mature tissues, where expression of previously characterized Exo70 genes is usually absent. Furthermore, expression of some GmExo70J genes including GmExo70J1, GmExo70J6 and GmExo70J7 increases greatly in floral organ-supporting receptacles during the development and maturation of siliques, indicating a possible role in seed development. More importantly, suppression of GmWRP1, GmExo70J7, GmExo70J8 and GmExo70J9 expression in soybean using virus- or artificial microRNA-mediated gene silencing resulted in accelerated leaf senescence and reduced nodule formation. These results strongly suggest that legume-specific GmWRP1 and GmExo70J proteins play important roles not only in legume symbiosis but also in other processes critical for legume growth and development.

Chemical Biology And Endomembrane Trafficking In Plants

Endomembrane trafficking is essential for coordinated growth and development in plants and response to environment. In particular, key plasma membrane proteins such as PIN auxin transporters and the BRI1 brassinosteroid receptor translocate dynamically between the endosomes and plasma membrane. We utilized a previously published large‐scale chemical library screening approach in which we identified small molecules that inhibited pollen germination or tube growth. This tip growth requires exocytosis, endocytosis and other endomembrane trafficking processesWe then identified groups of small molecules that affect trafficking of PIN auxin transporters and other plasma membrane proteins in Arabidopsis roots. One of these compounds (ES2) targets the essential EXO70A1 component of the exocyst complex in Arabidopsis which is necessary for normal development. In Arabidopsis, there are 23 EXO70 genes (in mammals and yeast they are low copy number) where, among other roles, they appear to be involved in autophagy, cell division and pathogen responses. ES2 targets the exocyst complex in plants as well as mammals including humans where defective recycling/exocytosis have been correlated with disease. To our knowledge, ES2 is the first bioactive compound known to have this specificity. To examine exocytosis and its regulation in more detail, we have examined exo70 mutants, generated EXO70A1 structural data and are examining additional small molecules that may affect recycling/exocytosis.Support or Funding InformationAcknowledgement: This work was supported by the US Department of Energy DE‐FG02‐02ER15295 (GRH, NVR).

Disruption of OsEXO70A1 Causes Irregular Vascular Bundles and Perturbs Mineral Nutrient Assimilation in Rice.

Normal uptake, transportation, and assimilation of primary nutrients are essential to plant growth. Tracheary elements (TEs) are tissues responsible for the transport of water and minerals and characterized by patterned secondary cell wall (SCW) thickening. Exocysts are involved in the regulation of SCW deposition by mediating the targeted transport of materials and enzymes to specific membrane areas. EXO70s are highly duplicated in plants and provide exocysts with functional specificity. In this study, we report the isolation of a rice mutant rapid leaf senescence2 (rls2) that exhibits dwarfism, ferruginous spotted necrotic leaves, decreased hydraulic transport, and disordered primary nutrient assimilation. Histological analysis of rls2-1 mutants has indicated impaired cell expansion, collapsed vascular tissues, and irregular SCW deposition. Map-based cloning has revealed that RLS2 encodes OsEXO70A1, which is one of the 47 members of EXO70s in rice. RLS2 was widely expressed and spatially restricted in vascular bundles. Subcellular localization analysis demonstrated that RLS2 was present on both membrane and nuclear regions. Expression analysis revealed that mutations in rls2 triggers transcriptional fluctuation of orthologous EXO70 genes and affects genes involved in primary nutrient absorption and transport. In brief, our study revealed that RLS2 is required for normal vascular bundle differentiation and primary nutrient assimilation.

EXO70I Is Required for Development of a Sub-domain of the Periarbuscular Membrane during Arbuscular Mycorrhizal Symbiosis

In eukaryotic cells, polarized secretion mediated by exocytotic fusion of membrane vesicles with the plasma membrane is essential for spatially restricted expansion of the plasma membrane and for the delivery of molecules to specific locations at the membrane and/or cell surface. The EXOCYST complex is central to this process, and in yeast, regulation of the EXO70 subunit influences exocytosis and cargo specificity. In contrast to yeast and mammalian cells, plants have upwards of 23 EXO70 genes with largely unknown roles. During arbuscular mycorrhizal (AM) symbiosis, deposition of the plant periarbuscular membrane (PAM) around the fungal arbuscule creates an intracellular membrane interface between the symbionts. The PAM has two major membrane sub-domains, and symbiosis-specific transporter proteins are localized in the branch domain. Currently, the mechanisms and cellular machinery involved in biogenesis of the PAM are largely unknown. Here, we identify an EXO70I protein present exclusively in plants forming AM symbiosis. Medicago truncatula exo70i mutants are unable to support normal arbuscule development, and incorporation of two PAM-resident ABC transporters, STR and STR2, is limited. During arbuscule branching, EXO70I is located in spatially restricted zones adjacent to the PAM around the arbuscule hyphal tips where it interacts with Vapyrin, a plant-specific protein required for arbuscule development. We conclude that EXO70I provides a specific exocytotic capacity necessary for development of the main functional sub-domain of the PAM. Furthermore, in contrast to other eukaryotes, plant EXO70s have evolved distinct specificities and interaction partners to fulfill their specialized secretory requirements.

Comparison of <italic>EXO70</italic> duplication between <italic>Arabidopsis thaliana</italic> and <italic>Oryza sativa</italic>

EXO70, an important component of the exocyst complex, is encoded by a single gene in humans, mice and yeast. To understand EXO70 gene duplication in plant genomes, we identified and analyzed all OsEXO70 and AtEXO70 genes in Arabidopsis thaliana and Oryza sativa . A total of 23 different EXO70 genes were identified in the A. thaliana genome, of which 15 were present on the ‘-’ strand. We detected 27 different mature mRNAs of EXO70 arising from alternative splicing. In O. sativa , 38 OsEXO70 genes coding for proteins comprising more than 412 amino acids were found to be distributed across the 12 rice chromosomes. Of these genes, 29 were located on the ‘+’ strand. Interestingly, a product of large-scale genome duplication—an approximately 2-Mbp-long fragment including both OsEXO70H3 and OsEXO70H4 , was highly conserved between chromosomes 11 and 12. The gene duplication mode of OsEXO70 is more complex than that of AtEXO70 . Although synonymous codon bias exists in both AtEXO70 and OsEXO70 , codon preferences differ between them. All EXO70 proteins contain a pfam03081 domain, indicating that these proteins may have the same or similar functions. We conclude that EXO70 has been duplicated multiple times during the evolution of both A. thaliana and O. sativa , with more duplications occurring in O. sativa than in A. thaliana . Additionally, a greater number of gene duplication mechanisms are operative for OsEXO70 than for AtEXO70 . We also propose that the pfam03081 domain may play an essential role in EXO70 functional determination. Finally, many differences in synonymous codon preference exist between AtEXO70 and OsEXO70 .

Comparison of early transcriptome responses to copper and cadmium in rice roots

The phytotoxic effects of copper (Cu) and cadmium (Cd) on plant growth are well documented. However, Cu and Cd toxicity targets and the cellular systems contributing to acquisition of tolerance are not fully understood at the molecular level. We aimed to identify genes and pathways that discriminate the actions of Cu and Cd in rice roots (Oryza sativa L. cv. TN67). The transcripts of 1,450 and 1,172 genes were regulated after Cu and Cd treatments, respectively. We identified 882 genes specifically respond to Cu treatment, and 604 unique genes as Cd-responsive by comparison of expression profiles of these two regulated gene groups. Gene ontology analysis for 538 genes involved in primary metabolism, oxidation reduction and response to stimulus was changed in response to both metals. In the individual aspect, Cu specifically altered levels of genes involved in vesicle trafficking transport, fatty acid metabolism and cellular component biogenesis. Cd-regulated genes related to unfolded protein binding and sulfate assimilation. To further characterize the functions of vesicle trafficking transport under Cu stress, interference of excytosis in root tissues was conducted by inhibitors and silencing of Exo70 genes. It was demonstrated that vesicle-trafficking is required for mediation of Cu-induced reactive oxygen species (ROS) production in root tissues. These results may provide new insights into understanding the molecular basis of the early metal stress response in plants.

Expression and Functional Analyses ofEXO70Genes in Arabidopsis Implicate Their Roles in Regulating Cell Type-Specific Exocytosis

During exocytosis, Golgi-derived vesicles are tethered to the target plasma membrane by a conserved octameric complex called the exocyst. In contrast to a single gene in yeast and most animals, plants have greatly increased number of EXO70 genes in their genomes, with functions very much unknown. Reverse transcription-polymerase chain reactions were performed on all 23 EXO70 genes in Arabidopsis (Arabidopsis thaliana) to examine their expression at the organ level. Cell-level expression analyses were performed using transgenic plants carrying β-glucuronidase reporter constructs, showing that EXO70 genes are primarily expressed in potential exocytosis-active cells such as tip-growing and elongating cells, developing xylem elements, and guard cells, whereas no expression was observed in cells of mature organs such as well-developed leaves, stems, sepals, and petals. Six EXO70 genes are expressed in distinct but partially overlapping stages during microspore development and pollen germination. A mutation in one of these genes, EXO70C1 (At5g13150), led to retarded pollen tube growth and compromised male transmission. This study implies that multiplications of EXO70 genes may allow plants to acquire cell type- and/or cargo-specific regulatory machinery for exocytosis.

Characterization of the Arabidopsis thaliana exocyst complex gene families by phylogenetic, expression profiling, and subcellular localization studies

*The exocyst is a complex of eight proteins (Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p and Exo84p) involved in tethering vesicles to the plasma membrane during regulated or polarized secretion. Here, the plant exocyst complex was explored in phylogenetic, expression, and subcellular localization studies. *Evolutionary relationships of predicted exocyst subunits were examined in the complete genomes of Arabidopsis thaliana, Oryza sativa, Populus trichocarpa and Physcomitrella patens. Furthermore, detailed expression profiling of the A. thaliana microarray databases was performed and subcellular localization patterns were studied. *Several plant exocyst subunit genes appear to have undergone gene expansion in a common ancestor and subsequent duplication events in independent plant lineages. Expression profiling revealed that the A. thaliana Exo70 gene family exhibits dynamic expression patterns, while the remaining exocyst subunit genes displayed more static profiles. Subcellular localization patterns for A. thaliana exocyst subunits ranged from cytosolic to endosomal compartments (with enrichment in the early endosomes and the trans-Golgi network). Interestingly, two endosomal-localized AtExo70 proteins also recruited other exocyst subunits to these compartments. *Overall subcellular localization patterns were observed that were also found in yeast and animal cells, and this, coupled with the evolutionary relationships, suggests that the exocyst may perform similar conserved functions in plants.

AtEXO70A1, a member of a family of putative exocyst subunits specifically expanded in land plants, is important for polar growth and plant development

The exocyst is a hetero-oligomeric protein complex involved in exocytosis and has been extensively studied in yeast and animal cells. Evidence is now accumulating that the exocyst is also present in plants. Bioinformatic analysis of genes encoding plant homologs of the exocyst subunit, Exo70, revealed that three Exo70 subgroups are evolutionarily conserved among angiosperms, lycophytes and mosses. Arabidopsis and rice contain 22 and approximately 39 EXO70 genes, respectively, which can be classified into nine clusters considered to be ancient in angiosperms (one has been lost in Arabidopsis). We characterized two independent T-DNA insertional mutants of the AtEXO70A1 gene (exo70A1-1 and exo70A1-2). Heterozygous EXO70A1/exo70A1 plants appear to be normal and segregate in a 1:2:1 ratio, suggesting that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, both exo70A1-1 and exo70A1-2 homozygotes exhibit an array of phenotypic defects. The polar growth of root hairs and stigmatic papillae is disturbed. Organs are generally smaller, plants show a loss of apical dominance and indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that the putative exocyst subunit EXO70A1 is involved in cell and organ morphogenesis.

The exocyst complex in plants.

Plant cell morphogenesis requires precise regulation of localized cell expansion and cell division. Vectorial exocytosis is a major morphogenetic process in plant cells, intimately interwoven with the dynamics of the cytoskeleton. Small GTPases of the Ras superfamily act as molecular switches participating in the control of both vesicle trafficking (Rab GTPases) and cytoskeleton dynamics (Rho GTPases). In tip-growing plant cells, such as the pollen tubes, wall formation is confined to the tip. The cytoplasm is highly polarized and grows by exocytosis in the apex, which makes pollen tubes an excellent model to study a large variety of dynamic events: targeted vesicle transport and docking, cell wall formation, guidance and maintenance of polarity, organelle movement, membrane recycling. We have previously shown that Rab homologues (especially those involved in the delivery of secretory vesicles to the plasma membrane) are the most abundant small GTPases in tobacco pollen tubes (Zarsky and Cvrckova, 1997) and cloned a tobacco pollen Rop (Rho of plants) homologue (Cvrckova and Zarsky, 1999). We have also shown the importance of GTPases (possibly Rop) in polarized pollen tube growth by microinjection of GTP/GDP analogues into growing pollen tubes (Elias et al., 2001). Both Rab and Rho GTPases are potential interactors of the exocyst (Sec6/8) complex, a multisubunit protein assembly involved in exocytosis that has been characterized in Saccharomyces cerevisiae and mammalian cells (reviewed in Hsu et al., 1999). The exocyst is composed of eight distinct subunits, referred to in yeast as Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p and Exo84p. The mammalian exocyst contains homologues of all these proteins. The complex is critical for specification of the site of vesicle docking and fusion and probably acts as a tethering complex before the steps mediated by SNARE and associated proteins. The yeast exocyst is known to interact directly with both Rab (Sec4) and Rho GTPases. Importantly, the Rho family GTPases Rho1 and Cdc42 are involved in the regulation of intracellular localization of the exocyst (Guo et al., 2001). Since the exocyst seems to be well conserved among distinct lineages of eukaryotes, it is relevant to ask whether plants also contain a similar module involved in exocytosis and secretion. Attempts to clone plant genes homologous to SEC15 via complementation of a yeast mutant with an Arabidopsis cDNA library were unsuccessful (Matsuda and Nakano, 1998). However, our searches through public sequence databases did identify genes significantly similar to all exocyst subunits (Cvrckova et al., 2001, and our unpublished data). The genome of Arabidopsis thaliana seems to code for two highly similar genes homologous to SEC3, SEC5, one homologue of SEC6, SEC8 and SEC10 each, two paralogues related to SEC15 and three copies of a putative EXO84. Surprisingly, there are as many as 23 potential Arabidopsis genes related to EXO70, although Saccharomyces, Drosophila, Caenorhabditis and perhaps also mammals possess only one copy of the EXO70 gene. This suggests that the plant exocyst might have some special features compared to other eukaryotes (Fig. 1). We have started molecular characterization of the putative Arabidopsis exocyst subunits. First we isolated a cDNA clone of the AtSec15b gene using a PCR-based library screen, and obtained cDNA clones potentially containing complete coding sequence of other exocyst * Corresponding author. Tel.: +420-2-21953179; fax: +420-2-21953306. E-mail address: zarsky@ueb.cas.cz (V. Zarsky). Cell Biology International 27 (2003) 199–201 Cell Biology International