scholarly journals Time-gated confocal microscopy reveals accumulation of exocyst subunits at the plant-pathogen interface

2019 ◽  
Author(s):  
Elysa J R Overdijk ◽  
Han Tang ◽  
Jan Willem Borst ◽  
Francine Govers ◽  
Tijs Ketelaar
Keyword(s):  
Plasma Membrane ◽  
Confocal Microscopy ◽  
Plant Development ◽  
Plant Pathogen ◽  
Protein Complex ◽  
Physcomitrella Patens ◽  
Phytophthora Capsici ◽  
Pathogen Invasion ◽  
Pathogen Attack ◽  
Polarized Exocytosis

Abstract Polarized exocytosis is essential for plant development and defence. The exocyst, an octameric protein complex that tethers exocytotic vesicles to the plasma membrane, targets exocytosis. Upon pathogen attack, secreted materials form papillae to halt pathogen penetration. To determine if the exocyst is directly involved in targeting exocytosis to infection sites, information about its localization is instrumental. Here, we investigated exocyst subunit localization in the moss Physcomitrella patens upon pathogen attack and infection by Phytophthora capsici. Time-gated confocal microscopy was used to eliminate autofluorescence of deposited material around infection sites allowing the visualization of the subcellular localization of exocyst subunits and of v-SNARE Vamp72A1-labeled exocytotic vesicles during infection. This showed that exocyst subunits Sec3a, Sec5b, Sec5d and Sec6 accumulated at sites of attempted pathogen penetration. Upon pathogen invasion, the exocyst subunits accumulated on the membrane surrounding papilla-like structures and hyphal encasements. Vamp72A1-labeled vesicles were found to localize in the cytoplasm around infection sites. The re-localization of exocyst subunits to infection sites suggests that the exocyst is directly involved in facilitating polarized exocytosis during pathogenesis.

scholarly journals The Small GTPase OsRac1 forms two distinct immune receptor complexes containing the PRR OsCERK1 and the NLR Pit

2020 ◽  
Author(s):  
Akira Akamatsu ◽  
Masayuki Fujiwara ◽  
Satoshi Hamada ◽  
Megumi Wakabayashi ◽  
Ai Yao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Complex ◽  
Small Gtpase ◽  
Surface Pattern ◽  
Transport Systems ◽  
Large Protein ◽  
Pathogen Invasion ◽  
Receptor Complexes ◽  
Immune Receptors ◽  
Different Types

AbstractPlants employ two different types of immune receptors, cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding and Leucine-rich repeat-containing proteins (NLRs), to cope with pathogen invasion. Both immune receptors often share similar downstream components and responses but it remains unknown whether a PRR and an NLR assemble into the same protein complex or two distinct receptor complexes. We have previously found that the small GTPase OsRac1 plays key roles in the signaling of OsCERK1, a PRR for fungal chitin, and of Pit, an NLR for rice blast fungus, and associates directly and indirectly with both of these immune receptors. In this study, using biochemical and bioimaging approaches, we reveal that OsRac1 formed two distinct receptor complexes with OsCERK1 and with Pit. Supporting this result, OsCERK1 and Pit utilized different transport systems for anchorage to the plasma membrane. Activation of OsCERK1 and Pit led to OsRac1 activation and, concomitantly, OsRac1 shifted from a small to a large protein complex fraction. We also found that the chaperone Hsp90 contributed to the proper transport of Pit to the plasma membrane and the immune induction of Pit. These findings illuminate how the PRR OsCERK1 and the NLR Pit orchestrate rice immunity through the small GTPase OsRac1.

scholarly journals The Small GTPase OsRac1 Forms Two Distinct Immune Receptor Complexes Containing the PRR OsCERK1 and the NLR Pit

2021 ◽  
Author(s):  
Akira Akamatsu ◽  
Masayuki Fujiwara ◽  
Satoshi Hamada ◽  
Megumi Wakabayashi ◽  
Ai Yao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Complex ◽  
Small Gtpase ◽  
Surface Pattern ◽  
Transport Systems ◽  
Large Protein ◽  
Pathogen Invasion ◽  
Receptor Complexes ◽  
Immune Receptors ◽  
Different Types

Abstract Plants employ two different types of immune receptors, cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding and leucine-rich repeat-containing proteins (NLRs), to cope with pathogen invasion. Both immune receptors often share similar downstream components and responses but it remains unknown whether a PRR and an NLR assemble into the same protein complex or two distinct receptor complexes. We have previously found that the small GTPase OsRac1 plays key roles in the signaling of OsCERK1, a PRR for fungal chitin, and of Pit, an NLR for rice blast fungus, and associates directly and indirectly with both of these immune receptors. In this study, using biochemical and bioimaging approaches, we revealed that OsRac1 formed two distinct receptor complexes with OsCERK1 and with Pit. Supporting this result, OsCERK1 and Pit utilized different transport systems for anchorage to the plasma membrane. Activation of OsCERK1 and Pit led to OsRac1 activation and, concomitantly, OsRac1 shifted from a small to a large protein complex fraction. We also found that the chaperone Hsp90 contributed to the proper transport of Pit to the plasma membrane and the immune induction of Pit. These findings illuminate how the PRR OsCERK1 and the NLR Pit orchestrate rice immunity through the small GTPase OsRac1.

Intracellular trafficking and cytoplasmic streaming under abiotic stress conditions

2019 ◽  
Vol 6 (04) ◽  
Author(s):  
JESHIMA KHAN YASIN ◽  
ANIL KUMAR SINGH
Keyword(s):  
Plasma Membrane ◽  
Abiotic Stress ◽  
Confocal Microscopy ◽  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Cytoplasmic Streaming ◽  
Living Cells ◽  
Stress Conditions ◽  
Vesicle Formation ◽  
External Stimuli

Cytoplasmic streaming is one among the vital activities of the living cells. In plants cytolplasmic streaming could clearly be seen in hypocotyls of growing seedlings. To observe cytoplsmic streaming and its correlated intracellular trafficking an investigation was conducted in legumes in comparison with GFP-AtRab75 and 35S::GFP:δTIP tonoplast fusion protein expressing arabidopsis lines. These seedlings were observed under confocal microscopy with different buffer incubation treatments and under different stress conditions. GFP expressing 35S::GFP:δTIP tonoplast lines were looking similar to the control lines and differ under stress conditions. Movement of cytoplasmic invaginations within the tonoplast and cytoplasmic sub vesicle or bulb budding during cytoplasmic streaming was observed in hypocotyls of At-GFP tonoplast plants. We found the cytoplasmic bulbs/ vesicles or sub vesicle formation from the plasma membrane. The streaming speed also depends on the incubation medium in which the specimen was incubated, indicating that the external stimuli as well as internal stimuli can alter the speed of streaming

scholarly journals Plant Pathogen Invasion Modifies the Eco-Evolutionary Host Plant Interactions of an Endangered Checkerspot Butterfly

Insects ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 246
Author(s):  
Paul M. Severns ◽  
Melinda Guzman-Martinez
Keyword(s):  
Pacific Northwest ◽  
Plant Pathogen ◽  
Plantago Lanceolata ◽  
Larval Feeding ◽  
Plant Interactions ◽  
Larval Food ◽  
Disease Symptoms ◽  
Pathogen Invasion ◽  
The Pacific ◽  
Germination Timing

New plant pathogen invasions typified by cryptic disease symptoms or those appearing sporadically in time and patchily in space, might go largely unnoticed and not taken seriously by ecologists. We present evidence that the recent invasion of Pyrenopeziza plantaginis (Dermateaceae) into the Pacific Northwest USA, which causes foliar necrosis in the fall and winter on Plantago lanceolata (plantain), the primary (non-native) foodplant for six of the eight extant Taylor’s checkerspot butterfly populations (Euphydryas editha taylori, endangered species), has altered eco-evolutionary foodplant interactions to a degree that threatens butterfly populations with extinction. Patterns of butterfly, larval food plant, and P. plantaginis disease development suggested the ancestral relationship was a two-foodplant system, with perennial Castilleja spp. supporting oviposition and pre-diapause larvae, and the annual Collinsia parviflora supporting post-diapause larvae. Plantain, in the absence of P. plantaginis disease, provided larval food resources throughout all butterfly life stages and may explain plantain’s initial adoption by Taylor’s checkerspot. However, in the presence of severe P. plantaginis disease, plantain-dependent butterfly populations experience a six-week period in the winter where post-diapause larvae lack essential plantain resources. Only C. parviflora, which is rare and competitively inferior under present habitat conditions, can fulfill the post-diapause larval feeding requirements in the presence of severe P. plantaginis disease. However, a germination timing experiment suggested C. parviflora to be suitably timed for only Washington Taylor’s checkerspot populations. The recent invasion by P. plantaginis appears to have rendered the ancestrally adaptive acquisition of plantain by Taylor’s checkerspot an unreliable, maladaptive foodplant interaction.

scholarly journals Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation

2018 ◽  
Vol 115 (51) ◽  
pp. E11914-E11923 ◽  
Author(s):  
Asit Manna ◽  
Huaying Zhao ◽  
Junya Wada ◽  
Lakshmi Balagopalan ◽  
Harichandra D. Tagad ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
T Cell ◽  
Protein Complex ◽  
Antigen Receptor ◽  
Receptor Activation ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Phospholipase Cγ1 ◽  
Quaternary Complex

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy–entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.

scholarly journals Dynamic accumulation of a helper NLR at the plant-pathogen interface underpins pathogen recognition

2021 ◽  
Author(s):  
Cian Duggan ◽  
Eleonora Moratto ◽  
Zachary Savage ◽  
Eranthika Hamilton ◽  
Hiroaki Adachi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Immune Responses ◽  
Subcellular Localization ◽  
Plant Pathogen ◽  
Coiled Coil ◽  
Dynamic Changes ◽  
Pathogen Effectors ◽  
Mediate Response ◽  
Potato Famine ◽  
Irish Potato Famine

Plants employ sensor-helper pairs of NLR immune receptors to recognize pathogen effectors and activate immune responses. Yet the subcellular localization of NLRs pre- and post- activation during pathogen infection remains poorly known. Here we show that NRC4, from the 'NRC' solanaceous helper NLR family, undergoes dynamic changes in subcellular localization by shuttling to and from the plant-pathogen haustorium interface established during infection by the Irish potato famine pathogen Phytophthora infestans. Specifically, prior to activation, NRC4 accumulates at the extra-haustorial membrane (EHM), presumably to mediate response to perihaustorial effectors, that are recognized by NRC4-dependent sensor NLRs. However not all NLRs accumulate at the EHM, as the closely related helper NRC2, and the distantly related ZAR1, did not accumulate at the EHM. NRC4 required an intact N- terminal coiled coil domain to accumulate at the EHM, whereas the functionally conserved MADA motif implicated in cell death activation and membrane insertion was dispensable for this process. Strikingly, a constitutively autoactive NRC4 mutant did not accumulate at the EHM and showed punctate distribution that mainly associated with the plasma membrane, suggesting that post-activation, NRC4 probably undergoes a conformation switch to form clusters that do not preferentially associate with the EHM. When NRC4 is activated by a sensor NLR during infection however, NRC4 formed puncta mainly at the EHM and to a lesser extent at the plasma membrane. We conclude that following activation at the EHM, NRC4 may spread to other cellular membranes from its primary site of activation to trigger immune responses.

scholarly journals Cell patterning by secretion-induced plasma membrane flows

2020 ◽  
Author(s):  
Veneta Gerganova ◽  
Iker Lamas ◽  
David M. Rutkowski ◽  
Aleksandar Vještica ◽  
Daniela Gallo Castro ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Negative Feedback ◽  
Reaction Diffusion ◽  
Cell Patterning ◽  
Yeast Cells ◽  
Membrane Insertion ◽  
Gtpase Activating Proteins ◽  
Associated Proteins ◽  
Bulk Membrane ◽  
Polarized Exocytosis

AbstractCells self-organize using reaction-diffusion and fluid-flow principles. Whether bulk membrane flows contribute to cell patterning has not been established. Here, using mathematical modelling, optogenetics and synthetic probes, we show that polarized exocytosis causes lateral membrane flows away from regions of membrane insertion. Plasma membrane-associated proteins with sufficiently low diffusion and/or detachment rates couple to the flows and deplete from areas of exocytosis. In rod-shaped fission yeast cells, zones of Cdc42 GTPase activity driving polarized exocytosis are limited by GTPase activating proteins (GAPs). We show that membrane flows pattern the GAP Rga4 distribution and coupling of a synthetic GAP to membrane flows is sufficient to establish the rod shape. Thus, membrane flows induced by Cdc42-dependent exocytosis form a negative feedback restricting the zone of Cdc42 activity.One Sentence SummaryExocytosis causes bulk membrane flows that drag associated proteins and form a negative feedback restricting the exocytic site.

scholarly journals A 36 kDa monomeric protein and its complex with a 10 kDa protein both isolated from bovine aorta are calpactin-like proteins that differ in their Ca2+-dependent calmodulin-binding and actin-severing properties

1988 ◽  
Vol 251 (3) ◽  
pp. 777-785 ◽  
Author(s):  
F Martin ◽  
J Derancourt ◽  
J P Capony ◽  
A Watrin ◽  
J C Cavadore
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein Complex ◽  
Actin Filaments ◽  
Thin Filament ◽  
Partial Amino Acid Sequence ◽  
Calmodulin Binding

Interaction of plasma membrane with the cytoskeleton involves a large number of proteins, among them a 36 kDa protein that was found to be involved in the interaction with actin filaments. We have isolated a 36 kDa protein from bovine aorta as a monomer and in a complex with a 10 kDa protein. Partial amino acid sequence determinations show that the 36 kDa and 10 kDa proteins isolated from bovine aorta are analogous to or identical with corresponding proteins purified from bovine intestine already described by Kristensen, Saris, Hunter, Hicks, Noonan, Glenney & Tack [(1986) Biochemistry 25, 4497-4503]. We report here that the association of the 10 kDa protein with the 36 kDa protein confers specific calmodulin-binding and actin-severing properties on the complex that are not possessed by the 36 kDa monomer alone. These findings suggest that the protein complex could be involved in thin-filament-related structures or could modulate some Ca2+-regulated events mediated by calmodulin.

scholarly journals The cell wall regulates dynamics and size of plasma-membrane nanodomains inArabidopsis

2019 ◽  
Vol 116 (26) ◽  
pp. 12857-12862 ◽  
Author(s):  
J. F. McKenna ◽  
D. J. Rolfe ◽  
S. E. D. Webb ◽  
A. F. Tolmie ◽  
S. W. Botchway ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Confocal Microscopy ◽  
Particle Tracking ◽  
Single Particle ◽  
Cluster Size ◽  
Particle Dynamics ◽  
Single Particle Tracking ◽  
Signaling Cascade ◽  
Plant Plasma Membrane

Plant plasma-membrane (PM) proteins are involved in several vital processes, such as detection of pathogens, solute transport, and cellular signaling. For these proteins to function effectively there needs to be structure within the PM allowing, for example, proteins in the same signaling cascade to be spatially organized. Here we demonstrate that several proteins with divergent functions are located in clusters of differing size in the membrane using subdiffraction-limited Airyscan confocal microscopy. Single particle tracking reveals that these proteins move at different rates within the membrane. Actin and microtubule cytoskeletons appear to significantly regulate the mobility of one of these proteins (the pathogen receptor FLS2) and we further demonstrate that the cell wall is critical for the regulation of cluster size by quantifying single particle dynamics of proteins with key roles in morphogenesis (PIN3) and pathogen perception (FLS2). We propose a model in which the cell wall and cytoskeleton are pivotal for regulation of protein cluster size and dynamics, thereby contributing to the formation and functionality of membrane nanodomains.

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