Although angiosperm plants generally react to immunity elicitors like chitin or chitosan by the cell wall callose deposition, this response in particular cell types, especially upon chitosan treatment, is not fully understood. Here we show that the growing root hairs (RHs) of Arabidopsis can respond to a mild (0.001%) chitosan treatment by the callose deposition and by a deceleration of the RH growth. We demonstrate that the glucan synthase-like 5/PMR4 is vital for chitosan-induced callose deposition but not for RH growth inhibition. Upon the higher chitosan concentration (0.01%) treatment, RHs do not deposit callose, while growth inhibition is prominent. To understand the molecular and cellular mechanisms underpinning the responses to two chitosan treatments, we analysed early Ca2+ and defence-related signalling, gene expression, cell wall and RH cellular endomembrane modifications. Chitosan-induced callose deposition is also present in the several other plant species, including functionally analogous and evolutionarily only distantly related RH-like structures such as rhizoids of bryophytes. Our results point to the RH callose deposition as a conserved strategy of soil-anchoring plant cells to cope with mild biotic stress. However, high chitosan concentration prominently disturbs RH intracellular dynamics, tip-localised endomembrane compartments, growth and viability, precluding callose deposition.

• Klíčová slova
• arabidopsis, cell wall, defence, gene expression, signalling,
• MeSH
• Arabidopsis * růst a vývoj   účinky léků   metabolismus   fyziologie   MeSH
• buněčná membrána metabolismus   MeSH
• buněčná stěna * metabolismus   MeSH
• chitosan * farmakologie   MeSH
• glukany * metabolismus   MeSH
• glukosyltransferasy metabolismus   MeSH
• kořeny rostlin * růst a vývoj   metabolismus   účinky léků   MeSH
• proteiny huseníčku * metabolismus   genetika   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• vápník metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• callose MeSH Prohlížeč
• chitosan * MeSH
• glukany * MeSH
• glukosyltransferasy MeSH
• proteiny huseníčku * MeSH
• vápník MeSH

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

In the evolution of land plants, the plant immune system has experienced expansion in immune receptor and signaling pathways. Lineage-specific expansions have been observed in diverse gene families that are potentially involved in immunity but lack causal association. Here, we show that Rps8-mediated resistance in barley to the pathogen Puccinia striiformis f. sp. tritici (wheat stripe rust) is conferred by a genetic module: Pur1 and Exo70FX12, which are together necessary and sufficient. Pur1 encodes a leucine-rich repeat receptor kinase and is the ortholog of rice Xa21, and Exo70FX12 belongs to the Poales-specific Exo70FX clade. The Exo70FX clade emerged after the divergence of the Bromeliaceae and Poaceae and comprises from 2 to 75 members in sequenced grasses. These results demonstrate the requirement of a lineage-specific Exo70FX12 in Pur1-mediated immunity and suggest that the Exo70FX clade may have evolved a specialized role in receptor kinase signaling.

• Publikační typ
• časopisecké články 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

Exocyst is a heterooctameric protein complex crucial for the tethering of secretory vesicles to the plasma membrane during exocytosis. Compared to other eukaryotes, exocyst subunit EXO70 is represented by many isoforms in land plants whose cell biological and biological roles, as well as modes of regulation remain largely unknown. Here, we present data on the phospho-regulation of exocyst isoform EXO70C2, which we previously identified as a putative negative regulator of exocyst function in pollen tube growth. A comprehensive phosphoproteomic analysis revealed phosphorylation of EXO70C2 at multiple sites. We have now performed localization and functional studies of phospho-dead and phospho-mimetic variants of Arabidopsis EXO70C2 in transiently transformed tobacco pollen tubes and stably transformed Arabidopsis wild type and exo70C2 mutant plants. Our data reveal a dose-dependent effect of AtEXO70C2 overexpression on pollen tube growth rate and cellular architecture. We show that changes of the AtEXO70C2 phosphorylation status lead to distinct outcomes in wild type and exo70c2 mutant cells, suggesting a complex regulatory pattern. On the other side, phosphorylation does not affect the cytoplasmic localization of AtEXO70C2 or its interaction with putative secretion inhibitor ROH1 in the yeast two-hybrid system.

• Klíčová slova
• Exo70, exocyst, membrane trafficking, phosphorylation, pollen tube, secretion inhibitor, tip-growth,
• 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

The collet (root-hypocotyl junction) region is an important plant transition zone between soil and atmospheric environments. Despite its crucial importance for plant development, little is known about how this transition zone is specified. Here we document the involvement of the exocyst complex in this process. The exocyst, an octameric tethering complex, participates in secretion and membrane recycling and is central to numerous cellular and developmental processes, such as growth of root hairs, cell expansion, recycling of PIN auxin efflux carriers and many others. We show that dark-grown Arabidopsis mutants deficient in exocyst subunits can form a hair-bearing ectopic collet-like structure above the true collet, morphologically resembling the true collet but also retaining some characteristics of the hypocotyl. The penetrance of this phenotypic defect is significantly influenced by cultivation temperature and carbon source, and is related to a defect in auxin regulation. These observations provide new insights into the regulation of collet region formation and developmental plasticity of the hypocotyl.

• Klíčová slova
• Arabidopsis thaliana, auxin, collet, etiolated hypocotyl, exocyst, root–hypocotyl junction, starch accumulation,
• MeSH
• Arabidopsis genetika   růst a vývoj   MeSH
• hypokotyl genetika   růst a vývoj   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny huseníčku 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