Exocyst component of 70-kDa (EXO70) proteins are constituents of the exocyst complex implicated in vesicle tethering during exocytosis. MILDEW RESISTANCE LOCUS O (MLO) proteins are plant-specific calcium channels and some MLO isoforms enable fungal powdery mildew pathogenesis. We here detected an unexpected phenotypic overlap of Arabidopsis thaliana exo70H4 and mlo2 mlo6 mlo12 triple mutant plants regarding the biogenesis of leaf trichome secondary cell walls. Biochemical and Fourier transform infrared spectroscopic analyses corroborated deficiencies in the composition of trichome cell walls in these mutants. Transgenic lines expressing fluorophore-tagged EXO70H4 and MLO exhibited extensive colocalization of these proteins. Furthermore, mCherry-EXO70H4 mislocalized in trichomes of the mlo triple mutant and, vice versa, MLO6-GFP mislocalized in trichomes of the exo70H4 mutant. Expression of GFP-marked PMR4 callose synthase, a known cargo of EXO70H4-dependent exocytosis, revealed reduced cell wall delivery of GFP-PMR4 in trichomes of mlo triple mutant plants. In vivo protein-protein interaction assays in plant and yeast cells uncovered isoform-preferential interactions between EXO70.2 subfamily members and MLO proteins. Finally, exo70H4 and mlo6 mutants, when combined, showed synergistically enhanced resistance to powdery mildew attack. Taken together, our data point to an isoform-specific interplay of EXO70 and MLO proteins in the modulation of trichome cell wall biogenesis and powdery mildew susceptibility.

• MeSH
• Arabidopsis * metabolismus   MeSH
• buněčná stěna metabolismus   MeSH
• nemoci rostlin mikrobiologie   MeSH
• odolnost vůči nemocem genetika   MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• trichomy genetika   metabolismus   MeSH
• vezikulární transportní proteiny metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• EXO70H4 protein, Arabidopsis MeSH Prohlížeč
• protein - isoformy MeSH
• proteiny huseníčku * MeSH
• rostlinné proteiny MeSH
• vezikulární transportní proteiny 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

Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall-radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.

• Klíčová slova
• EXO70, cell wall, exocyst complex, phosphatidic acid, phosphatidylinositol 4,5-bisphosphate, phospholipids, plasma membrane domains, polar exocytosis, trichome,
• MeSH
• Arabidopsis chemie   genetika   MeSH
• buněčná membrána chemie   genetika   MeSH
• exocytóza genetika   MeSH
• fosfatidylinositol-4,5-difosfát chemie   metabolismus   MeSH
• fosfatidylseriny chemie   genetika   MeSH
• membránové lipidy genetika   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   MeSH
• trichomy chemie   genetika   MeSH
• vezikulární transportní proteiny chemie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• EXO70A1 protein, Arabidopsis MeSH Prohlížeč
• EXO70H4 protein, Arabidopsis MeSH Prohlížeč
• fosfatidylinositol-4,5-difosfát MeSH
• fosfatidylseriny MeSH
• membránové lipidy MeSH
• proteiny huseníčku MeSH
• vezikulární transportní proteiny 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 exocyst is a conserved vesicle-tethering complex with principal roles in cell polarity and morphogenesis. Several studies point to its involvement in polarized secretion during microbial pathogen defense. In this context, we have found an interaction between the Arabidopsis EXO70B1 exocyst subunit, a protein which was previously associated with both the defense response and autophagy, and RPM1 INTERACTING PROTEIN 4 (RIN4), the best studied member of the NOI protein family and a known regulator of plant defense pathways. Interestingly, fragments of RIN4 mimicking the cleavage caused by the Pseudomonas syringae effector protease, AvrRpt2, fail to interact strongly with EXO70B1. We observed that transiently expressed RIN4, but not the plasma membrane (PM) protein aquaporin PIP2, recruits EXO70B1 to the PM. Unlike EXO70B1, RIN4 does not recruit the core exocyst subunit SEC6 to the PM under these conditions. Furthermore, the AvrRpt2 effector protease delivered by P. syringae is able to release both RIN4 and EXO70B1 to the cytoplasm. We present a model for how RIN4 might regulate the localization and putative function of EXO70B1 and speculate on the role the AvrRpt2 protease might have in the regulation of this defense response.

• Klíčová slova
• Autophagy, EXO70B1, EXO70B2, RIN4, exocyst, plant immunity, secretion,
• MeSH
• akvaporiny genetika   metabolismus   MeSH
• Arabidopsis metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• buněčná membrána MeSH
• intracelulární signální peptidy a proteiny MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Pseudomonas syringae metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• vezikulární transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• akvaporiny MeSH
• avrRpt2 protein, Pseudomonas syringae MeSH Prohlížeč
• bakteriální proteiny MeSH
• EXO70B1 protein, Arabidopsis MeSH Prohlížeč
• intracelulární signální peptidy a proteiny MeSH
• plasma membrane intrinsic protein 2 Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• RIN4 protein, Arabidopsis MeSH Prohlížeč
• transportní proteiny MeSH
• vezikulární transportní proteiny MeSH

The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollen-specific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• konfokální mikroskopie MeSH
• kořeny rostlin genetika   metabolismus   MeSH
• mutace MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• pyl genetika   růst a vývoj   metabolismus   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• regulace genové exprese u rostlin * MeSH
• vezikulární transportní proteiny genetika   metabolismus   MeSH
• vývojová regulace genové exprese * MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• EXO70C2 protein, Arabidopsis MeSH Prohlížeč
• protein - isoformy MeSH
• proteiny huseníčku MeSH
• vezikulární transportní proteiny MeSH
• zelené fluorescenční 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

Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2 However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• časosběrné zobrazování metody   MeSH
• exocytóza * MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfatidylinositoly metabolismus   MeSH
• fylogeneze MeSH
• geneticky modifikované rostliny MeSH
• konfokální mikroskopie MeSH
• mutace MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteiny huseníčku klasifikace   genetika   metabolismus   MeSH
• pyl genetika   růst a vývoj   metabolismus   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• sekvenční homologie nukleových kyselin MeSH
• simulace molekulární dynamiky MeSH
• stanovení celkové genové exprese metody   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• vezikulární transportní proteiny klasifikace   genetika   metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• exocyst complex component sec3A, Arabidopsis MeSH Prohlížeč
• fosfatidylinositol-4,5-difosfát MeSH
• fosfatidylinositoly MeSH
• protein - isoformy MeSH
• proteiny huseníčku MeSH
• vezikulární transportní proteiny MeSH
• zelené fluorescenční proteiny MeSH