Kortikální cytoskelet, exocytický komplex exocyst a jejich role v morfogenzi rostlinných buněk

Plant cell morphogenesis is largely dependent on the coordination of cytoskeletal elements, plasma membrane, and vesicle trafficking. Formin proteins are nucleators of the actin cytoskeleton. Plant Class I family formins are integral membrane proteins and thus have the ability to coordinate cytoskeletal dynamics with the plasma membrane localization. We identified Arabidopsis thaliana formin AtFH4 as a microtubule associated protein. The binding is conferred by a novel domain located between the transmembrane domain and the formin homology 1 domain. The protein associated with actin in in vitro conditions. Overexpressed AtFH4 accumulated in the endoplasmic reticulum, and induced coalignment of endoplasmic reticulum membranes with microtubules. Together, these data suggest that the combination of plant-specific and conserved domains enables AtFH4 to function as an interface between membranes and both major cytoskeletal networks . Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plant cells. Prior to their fusion with the plasma membrane, secretory vesicles are tethered at exocytic sites by the exocyst, an octameric protein complex. We analysed the mutant in the EXO84b exocyst subunit, and discovered that the mutant plants were dwarfed and exhibited...Morfogeneze rostlinných buněk úzce souvisí s koordinací plasmatické membrány, cytoskeletu a exocytózy. Forminy jsou proteiny regulující tvorbu vláken aktinového cytoskeletu. Rostlinné forminy náležející do třídy I jsou integrální membránové proteiny, mohou tedy regulovat dynamiku cytoskeletu z pozice membránového proteinu. Zjistili jsme, že formin AtFH4 z Arabidopsis thaliana se váže na mikrotubulární cytoskelet. Doména zodpovědná za tuto vazbu se nachází mezi transmembránovou a "formin homology 1" doménou. V in vitro podmínkách vázal AtFH4 protein aktinová vlákna. AtFH4 se lokalizoval do membrán endoplasmatického retikula, když byl overexprimován v buňkách tabáku. Zároveň tato lokalizace způsobila rozmístění membrán endoplasmatického retikula podél mikrotubulárního cytoskeletu. Z těchto výsledků vyvozujeme, že AtFH4 je schopný být současně umístěn v membráně a svoji vazbou na aktin a mikrotubuly propojovat tyto dva typy cytoskeletu. Rostlinná cytokineze závisí na sekretorické dráze; na exocytóze i endocytóze. Před tím, než sekretorické váčky splynou s plasmatickou membránou, jsou k ní poutány "poutacím komplexem" zvaným exocyst, složeným z osmi podjednotek. Analyzovali jsme mutanty v jedné z podjednotek exocystu A. thaliana - EXO84b. Mutantní rostliny byly zakrslé, nejspíše kvůli porušené sekretorické...Department of Experimental Plant BiologyKatedra experimentální biologie rostlinFaculty of SciencePřírodovědecká fakult

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

The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering

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

AGC kinases and MAB4/MEL proteins maintain PIN polarity by limiting lateral diffusion in plant cells

Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into howthese factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. Wethus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.Plant science

Interaction of ectomycorrhizal and ericoid mycorrhizal host plants via ectomycorrhizal, ericoid mycorrhizal and pseudomycorrhizal fungi

Abstract 9. Abstract Roots of ectomycorrhizal and ericoid mycorrhizal plants are believed to be colonized by fungi belonging to different taxonomic groups. However, both frequent isolations of ericoid mycorrhizal fungi from ectomycorrhizal root tips and a few recent studies (Vrålstad et al. 2000, 2002b, Piercey et al. 2002, Hambleton & Sigler 2005) indicate that there is a group of mycobionts colonizing both types of roots. Ectomycorrhizal morphotype Piceirhiza bicolorata was shown to be induced by Meliniomyces sp. belonging to the Rhizoscyphus ericae aggregate (Vrålstad et al. 2000). The ability to colonize roots of potentially ectomycorrhizal and ericoid plants simultaneously was proven in in vitro experiments in the case of Rhizoscyphus ericae (Pirecey et al. 2002) and Cadophora finlandica (Villarreal­Ruiz et al. 2004). DSE fungi ("dark septate endophytes", formerly termed pseudomycorrhizal) represent another group of mycobionts colonizing both ericoid and potentially ectomycorrhizal plant roots. In the present work, we inoculated roots of ericoid (Vaccinium myrtillus) and potentially ectomycorrhizal plants (Picea abies, Pinus sylvestris and Betula nana) with typically ectomycorrhizal and ericoid mycorrhizal fungi and..

Kortikální cytoskelet, exocytický komplex exocyst a jejich role v morfogenzi rostlinných buněk

Plant cell morphogenesis is largely dependent on the coordination of cytoskeletal elements, plasma membrane, and vesicle trafficking. Formin proteins are nucleators of the actin cytoskeleton. Plant Class I family formins are integral membrane proteins and thus have the ability to coordinate cytoskeletal dynamics with the plasma membrane localization. We identified Arabidopsis thaliana formin AtFH4 as a microtubule associated protein. The binding is conferred by a novel domain located between the transmembrane domain and the formin homology 1 domain. The protein associated with actin in in vitro conditions. Overexpressed AtFH4 accumulated in the endoplasmic reticulum, and induced coalignment of endoplasmic reticulum membranes with microtubules. Together, these data suggest that the combination of plant-specific and conserved domains enables AtFH4 to function as an interface between membranes and both major cytoskeletal networks . Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plant cells. Prior to their fusion with the plasma membrane, secretory vesicles are tethered at exocytic sites by the exocyst, an octameric protein complex. We analysed the mutant in the EXO84b exocyst subunit, and discovered that the mutant plants were dwarfed and exhibited..

Interaction of ectomycorrhizal and ericoid mycorrhizal host plants via ectomycorrhizal, ericoid mycorrhizal and pseudomycorrhizal fungi

Abstract 9. Abstract Roots of ectomycorrhizal and ericoid mycorrhizal plants are believed to be colonized by fungi belonging to different taxonomic groups. However, both frequent isolations of ericoid mycorrhizal fungi from ectomycorrhizal root tips and a few recent studies (Vrålstad et al. 2000, 2002b, Piercey et al. 2002, Hambleton & Sigler 2005) indicate that there is a group of mycobionts colonizing both types of roots. Ectomycorrhizal morphotype Piceirhiza bicolorata was shown to be induced by Meliniomyces sp. belonging to the Rhizoscyphus ericae aggregate (Vrålstad et al. 2000). The ability to colonize roots of potentially ectomycorrhizal and ericoid plants simultaneously was proven in in vitro experiments in the case of Rhizoscyphus ericae (Pirecey et al. 2002) and Cadophora finlandica (Villarreal­Ruiz et al. 2004). DSE fungi ("dark septate endophytes", formerly termed pseudomycorrhizal) represent another group of mycobionts colonizing both ericoid and potentially ectomycorrhizal plant roots. In the present work, we inoculated roots of ericoid (Vaccinium myrtillus) and potentially ectomycorrhizal plants (Picea abies, Pinus sylvestris and Betula nana) with typically ectomycorrhizal and ericoid mycorrhizal fungi and..

Interaction of ectomycorrhizal and ericoid mycorrhizal host plants via ectomycorrhizal, ericoid mycorrhizal and pseudomycorrhizal fungi

Abstract 9. Abstract Roots of ectomycorrhizal and ericoid mycorrhizal plants are believed to be colonized by fungi belonging to different taxonomic groups. However, both frequent isolations of ericoid mycorrhizal fungi from ectomycorrhizal root tips and a few recent studies (Vrålstad et al. 2000, 2002b, Piercey et al. 2002, Hambleton & Sigler 2005) indicate that there is a group of mycobionts colonizing both types of roots. Ectomycorrhizal morphotype Piceirhiza bicolorata was shown to be induced by Meliniomyces sp. belonging to the Rhizoscyphus ericae aggregate (Vrålstad et al. 2000). The ability to colonize roots of potentially ectomycorrhizal and ericoid plants simultaneously was proven in in vitro experiments in the case of Rhizoscyphus ericae (Pirecey et al. 2002) and Cadophora finlandica (Villarreal­Ruiz et al. 2004). DSE fungi ("dark septate endophytes", formerly termed pseudomycorrhizal) represent another group of mycobionts colonizing both ericoid and potentially ectomycorrhizal plant roots. In the present work, we inoculated roots of ericoid (Vaccinium myrtillus) and potentially ectomycorrhizal plants (Picea abies, Pinus sylvestris and Betula nana) with typically ectomycorrhizal and ericoid mycorrhizal fungi and...Katedra experimentální biologie rostlinDepartment of Experimental Plant BiologyPřírodovědecká fakultaFaculty of Scienc

Auxin co-receptor IAA17/AXR3 controls cell elongation in Arabidopsis thaliana root solely by modulation of nuclear auxin pathway.

Publication status: PublishedThe nuclear TIR1/AFB-Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in Arabidopsis thaliana root development, response to auxin and gravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. We combined genetical and cell biological tools with transcriptomics and determination of auxin levels and employed live cell imaging and image analysis to address how the auxin response pathways influence the dynamics of root growth. We revealed that manipulations of the TIR1/AFB-Aux/IAA pathway rapidly modulate root cell elongation. While inducible overexpression of the AXR3-1 transcriptional inhibitor accelerated growth, overexpression of the dominant activator form of ARF5/MONOPTEROS inhibited growth. In parallel, AXR3-1 expression caused loss of auxin sensitivity, leading to transcriptional reprogramming, phytohormone signaling imbalance and increased levels of auxin. Furthermore, we demonstrated that AXR3-1 specifically perturbs nuclear auxin signaling, while the rapid auxin response remains functional. Our results shed light on the interplay between the nuclear and cytoplasmic auxin pathways in roots, revealing their partial independence but also the dominant role of the nuclear auxin pathway during the gravitropic response of Arabidopsis thaliana roots

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