Tamara Pečenková

An Exocyst Complex Functions in Plant Cell Growth in Arabidopsis and Tobacco

The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis.

The Arabidopsis Exocyst Complex Is Involved in Cytokinesis and Cell Plate Maturation

The plant cell cytokinesis is driven from the onset by highly organized vesicle fusion resulting in cell plate and new cell wall formation separating daughter cells. The evolutionarily conserved exocyst complex regulating exocytic vesicle binding to the plasma membrane is involved in both the final separation of cells as in animals and also in the initiation of cell plate in plant cells. 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.

The Ubiquitin Ligase PUB22 Targets a Subunit of the Exocyst Complex Required for PAMP-Triggered Responses in Arabidopsis

Exo70B2, a subunit of the exocyst complex, which is involved in exocytosis, is targeted for degradation by the ubiquitin ligase PUB22 upon activation of the immune system. This work also reveals that Exo70B2 is required for the activation of PAMP-triggered responses. 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.

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.

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.

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.

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

The exocyst complex localizes to distinct foci at the plasma membrane of Arabidopsis thaliana cells. Their localization at the plasma membrane is insensitive to BFA treatment but is decreased in an exocyst-subunit mutant. In turn, exocyst-subunit mutants show decreased exocytosis.

Molecular Phylogeny of φ29-Like Phages and Their Evolutionary Relatedness to Other Protein-Primed Replicating Phages and Other Phages Hosted by Gram-Positive Bacteria

Abstract. The φ29-like phage genus of Podoviridae family contains phages B103, BS32, GA-1, M2, Nf, φ15, φ29, and PZA that all infect Bacillus subtilis. They have very similar morphology and their genomes consist of linear double-stranded DNA of approximately 20 kb. The nucleotide sequences of individual genomes or their parts determined thus far show that these phages evolved from a common ancestor. A terminal protein (TP) that is covalently bound to the DNA 5′-end primes DNA replication of these phages. The same mechanism of DNA replication is used by the Cp-1 related phages (also members of the Podoviridae family) and by the phage PRD1 (member of the Tectoviridae family). Based on the complete or partial genomic sequence data of these phages it was possible to analyze the evolutionary relationship within the φ29-like phage genus as well as to other protein-primed replicating phages. Noncoding regions containing origins of replication were used in the analysis, as well as amino acid sequences of DNA polymerases, and with the φ29-like phages also amino acid sequences of the terminal proteins and of the gene 17 protein product, an accessory component of bacteriophage DNA replicating machinery. Included in the analysis are also results of a comparison of these phage DNAs with the prophages present in the Bacillus subtilis genome. Based on this complex analysis we define and describe in more detail the evolutionary branches of φ29-like phages, one branch consisting of phages BS32, φ15, φ29, and PZA, the second branch composed of phages B103, M2, and Nf, and the third branch having phage GA-1 as its sole member. In addition, amino acid sequences of holins, proteins involved in phage lysis were used to extend the evolutionary study to other phages infecting Gram-positive bacteria. The analysis based on the amino acid sequences of holins showed several weak points in present bacteriophage classification.

The Rhodobacter capsulatus genome

The genome of Rhodobacter capsulatus has been completely sequenced. It consists of a single chromosome containing 3.5 Mb and a circular plasmid of 134 kb. This effort, started in 1992, began with a fine-structure restriction map of an overlapping set of cosmids that covered the genome. Cosmid sequencing led to a gapped genome that was filled by primer walking on the chromosome and by using lambda clones. Methods had to be developed to handle strong stops in the high GC (68%) inserts. Annotation was done with the ERGO system at Integrated Genomics, as was the reconstruction of the cells metabolism. It was possible to recognize 3709 orfs of which functional assignments could be made with high confidence to 2392 (65%). Unusual features include the presence of numerous cryptic phage genomes embedded in the chromosome.