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.

• Klíčová slova
• EXO70A1, cell polarity, exocyst, phospholipids, plasma membrane,
• MeSH
• Arabidopsis metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• exocytóza MeSH
• fosfolipidy metabolismus   MeSH
• polarita buněk MeSH
• proteiny huseníčku metabolismus   MeSH
• proteomika metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EXO70A1 protein, Arabidopsis MeSH Prohlížeč
• fosfolipidy MeSH
• proteiny huseníčku MeSH

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.

• Klíčová slova
• Arabidopsis, EXO70, EXO70A1, EXO70B1, exocyst complex, polar exocytosis,
• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• exocytóza MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• transportní vezikuly metabolismus   MeSH
• vezikulární transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny huseníčku MeSH
• vezikulární transportní proteiny MeSH

Pollen development, pollen grain germination, and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to ovules for fertilization. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in the male gametophyte, governing the conventional secretory function of the exocyst, analogous to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination, and pollen tube growth. GFP:EXO70A2 was localized to the nucleus and cytoplasm in developing pollen grains and later to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for EXO70A1 function in the sporophyte, but not vice versa, indicating partial functional redundancy of these two closely related isoforms and higher specificity of EXO70A2 for pollen development-related processes. Phylogenetic analysis revealed that the ancient duplication of EXO70A, one of which is always highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in Arabidopsis pollen that is required for efficient plant sexual reproduction.

• MeSH
• Arabidopsis genetika   růst a vývoj   MeSH
• exocytóza genetika   fyziologie   MeSH
• fylogeneze MeSH
• genetická variace MeSH
• genotyp MeSH
• pylová láčka genetika   růst a vývoj   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné geny MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

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.

• Klíčová slova
• Arabidopsis thaliana, EXO70, biotic stress, exocyst, gene expression, lipid binding, redundancy, root hairs,
• Publikační typ
• časopisecké články MeSH

Biogenesis of the plant secondary cell wall involves many important aspects, such as phenolic compound deposition and often silica encrustation. Previously, we demonstrated the importance of the exocyst subunit EXO70H4 for biogenesis of the trichome secondary cell wall, namely for deposition of the autofluorescent and callose-rich cell wall layer. Here, we reveal that EXO70H4-driven cell wall biogenesis is constitutively active in the mature trichome, but also can be activated elsewhere upon pathogen attack, giving this study a broader significance with an overlap into phytopathology. To address the specificity of EXO70H4 among the EXO70 family, we complemented the exo70H4-1 mutant by 18 different Arabidopsis (Arabidopsis thaliana) EXO70 paralogs subcloned under the EXO70H4 promoter. Only EXO70H4 had the capacity to rescue the exo70H4-1 trichome phenotype. Callose deposition phenotype of exo70H4-1 mutant is caused by impaired secretion of PMR4, a callose synthase responsible for the synthesis of callose in the trichome. PMR4 colocalizes with EXO70H4 on plasma membrane microdomains that do not develop in the exo70H4-1 mutant. Using energy-dispersive x-ray microanalysis, we show that both EXO70H4- and PMR4-dependent callose deposition in the trichome are essential for cell wall silicification.

• MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• buněčná stěna účinky léků   metabolismus   MeSH
• epidermis rostlin cytologie   účinky léků   metabolismus   MeSH
• fenotyp MeSH
• flagelin farmakologie   MeSH
• glukany MeSH
• glukosyltransferasy metabolismus   MeSH
• mutace genetika   MeSH
• oxid křemičitý metabolismus   MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteinové domény MeSH
• proteiny huseníčku chemie   metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• trichomy metabolismus   MeSH
• upregulace účinky léků   MeSH
• vezikulární transportní proteiny chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,3-beta-glucan synthase MeSH Prohlížeč
• callose MeSH Prohlížeč
• EXO70H4 protein, Arabidopsis MeSH Prohlížeč
• flagelin MeSH
• glukany MeSH
• glukosyltransferasy MeSH
• oxid křemičitý MeSH
• PMR4 protein, Arabidopsis MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny huseníčku MeSH
• vezikulární transportní proteiny MeSH

The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell.

• MeSH
• buněčná membrána metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• exocytóza genetika   MeSH
• fylogeneze MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• konfokální mikroskopie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteomika metody   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny klasifikace   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• tabák genetika   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein - isoformy MeSH
• rostlinné proteiny MeSH