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, Golgiindependent 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.
Plant cytokinesis is initiated in a transient membrane compartment, the cell plate, and completed by a process of maturation during which the cell plate becomes a cross wall. How the transition from juvenile to adult stages occurs is poorly understood. In this study, we monitor the Arabidopsis transport protein particle II (TRAPPII) and exocyst tethering complexes throughout cytokinesis. We show that their appearance is predominantly sequential, with brief overlap at the onset and end of cytokinesis. The TRAPPII complex is required for cell plate biogenesis, and the exocyst is required for cell plate maturation. The TRAPPII complex sorts plasma membrane proteins, including exocyst subunits, at the cell plate throughout cytokinesis. We show that the two tethering complexes physically interact and propose that their coordinated action may orchestrate not only plant but also animal cytokinesis.
Exocyst is an evolutionarily conserved vesicle tethering complex functioning especially in the last stage of exocytosis. Homologs of its eight canonical subunits – Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 – were found also in higher plants and confirmed to form complexes in vivo, and to participate in cell growth including polarized expansion of pollen tubes and root hairs. Here we present results of a phylogenetic study of land plant exocyst subunits encoded by a selection of completely sequenced genomes representing a variety of plant, mostly angiosperm, lineages. According to their evolution histories, plant exocyst subunits can be divided into several groups. The core subunits Sec6, Sec8, and Sec10, together with Sec3 and Sec5, underwent few, if any fixed duplications in the tracheophytes (though they did amplify in the moss Physcomitrella patens), while others form larger families, with the number of paralogs ranging typically from two to eight per genome (Sec15, Exo84) to several dozens per genome (Exo70). Most of the diversity, which can be in some cases traced down to the origins of land plants, can be attributed to the peripheral subunits Exo84 and, in particular, Exo70. As predicted previously, early land plants (including possibly also the Rhyniophytes) encoded three ancestral Exo70 paralogs which further diversified in the course of land plant evolution. Our results imply that plants do not have a single “Exocyst complex” – instead, they appear to possess a diversity of exocyst variants unparalleled among other organisms studied so far. This feature might perhaps be directly related to the demands of building and maintenance of the complicated and spatially diverse structures of the endomembranes and cell surfaces in multicellular land plants.
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.
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