scholarly journals Barley ROP-INTERACTIVE PARTNER-a organizes into RAC1- and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1-dependent membrane domains

2019 ◽  
Author(s):  
Caroline Hoefle ◽  
Christopher McCollum ◽  
Ralph Hückelhoven
Keyword(s):  
Fungal Pathogens ◽  
Structural Integrity ◽  
Lateral Diffusion ◽  
Effector Proteins ◽  
Cell Periphery ◽  
Membrane Domains ◽  
Gtpase Activating Protein ◽  
Powdery Mildew Fungus ◽  
Membrane Heterogeneity ◽  
Rop Gtpase

AbstractSmall ROP (also called RAC) GTPases are key factors in polar cell development and in interaction with the environment. ROP-Interactive Partner (RIP) proteins are predicted scaffold or ROP-effector proteins, which function downstream of activated GTP-loaded ROP proteins in establishing membrane heterogeneity and cellular organization. Grass ROP proteins function in cell polarity, resistance and susceptibility to fungal pathogens but grass RIP proteins are little understood.We found that the barley (Hordeum vulgare L.) RIPa protein can interact with barley ROPs in yeast. Fluorescent-tagged RIPa, when co-expressed with the constitutively activated ROP protein CA RAC1, accumulates at the cell periphery or plasma membrane. Additionally, RIPa, locates into membrane domains, which are laterally restricted by microtubules, when co-expressed with RAC1 and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. Both structural integrity of MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1 and microtubule stability are key to maintenance of RIPa-labeled membrane domains. In this context, RIPa also accumulates at the interface of barley and invading hyphae of the powdery mildew fungus Blumeria graminis f.sp. hordei.Data suggest that barley RIPa interacts with barley ROPs and specifies RAC1 activity-associated membrane domains with potential signaling capacity. Lateral diffusion of this RAC1 signaling capacity is restricted the resulting membrane heterogeneity requires intact microtubules and MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN 1. Focal accumulation of RIPa at sites of fungal attack may indicate locally restricted ROP activity at sites of fungal invasion.

scholarly journals A Barley ROP GTPase ACTIVATING PROTEIN Associates with Microtubules and Regulates Entry of the Barley Powdery Mildew Fungus into Leaf Epidermal Cells

2011 ◽  
Vol 23 (6) ◽  
pp. 2422-2439 ◽  
Author(s):  
Caroline Hoefle ◽  
Christina Huesmann ◽  
Holger Schultheiss ◽  
Frederik Börnke ◽  
Götz Hensel ◽  
...  
Keyword(s):  
Powdery Mildew ◽  
Epidermal Cells ◽  
Gtpase Activating Protein ◽  
Powdery Mildew Fungus ◽  
Barley Powdery Mildew ◽  
Rop Gtpase

The organization of cell surface membrane domains

1989 ◽  
Vol 47 ◽  
pp. 804-805
Author(s):  
Michael Edidin
Keyword(s):  
Cell Surface ◽  
Membrane Lipids ◽  
Surface Membrane ◽  
Lateral Diffusion ◽  
Cell Types ◽  
Membrane Domains ◽  
Membrane Biology ◽  
Morphological Polarity ◽  
Discrete Domains ◽  
Membrane Components

Cell surface membranes are based on a fluid lipid bilayer and models of the membranes' organization have emphasised the possibilities for lateral motion of membrane lipids and proteins within the bilayer. Two recent trends in cell and membrane biology make us consider ways in which membrane organization works against its inherent fluidity, localizing both lipids and proteins into discrete domains. There is evidence for such domains, even in cells without obvious morphological polarity and organization [Table 1]. Cells that are morphologically polarised, for example epithelial cells, raise the issue of membrane domains in an accute form.The technique of fluorescence photobleaching and recovery, FPR, was developed to measure lateral diffusion of membrane components. It has also proven to be a powerful tool for the analysis of constraints to lateral mobility. FPR resolves several sorts of membrane domains, all on the micrometer scale, in several different cell types.

scholarly journals Disruption of barley immunity to powdery mildew by an in-frame Lys-Leu deletion in the essential protein SGT1

Genetics ◽  
2020 ◽  
Vol 217 (2) ◽  
Author(s):  
Antony V E Chapman ◽  
Matthew Hunt ◽  
Priyanka Surana ◽  
Valeria Velásquez-Zapata ◽  
Weihui Xu ◽  
...  
Keyword(s):  
Powdery Mildew ◽  
Fungal Pathogens ◽  
Novel Mutation ◽  
Exome Capture ◽  
Resistance Locus ◽  
Hordeum Vulgare L ◽  
Powdery Mildew Fungus ◽  
Pathogen Effectors ◽  
Cell Processes ◽  
Disease Resistances

Abstract Barley (Hordeum vulgare L.) Mla (Mildew resistance locus a) and its nucleotide-binding, leucine-rich-repeat receptor (NLR) orthologs protect many cereal crops from diseases caused by fungal pathogens. However, large segments of the Mla pathway and its mechanisms remain unknown. To further characterize the molecular interactions required for NLR-based immunity, we used fast-neutron mutagenesis to screen for plants compromised in MLA-mediated response to the powdery mildew fungus, Blumeria graminis f. sp. hordei. One variant, m11526, contained a novel mutation, designated rar3 (required for Mla6 resistance3), that abolishes race-specific resistance conditioned by the Mla6, Mla7, and Mla12 alleles, but does not compromise immunity mediated by Mla1, Mla9, Mla10, and Mla13. This is analogous to, but unique from, the differential requirement of Mla alleles for the co-chaperone Rar1 (required for Mla12 resistance1). We used bulked-segregant-exome capture and fine mapping to delineate the causal mutation to an in-frame Lys-Leu deletion within the SGS domain of SGT1 (Suppressor of G-two allele of Skp1, Sgt1ΔKL308–309), the structural region that interacts with MLA proteins. In nature, mutations to Sgt1 usually cause lethal phenotypes, but here we pinpoint a unique modification that delineates its requirement for some disease resistances, while unaffecting others as well as normal cell processes. Moreover, the data indicate that the requirement of SGT1 for resistance signaling by NLRs can be delimited to single sites on the protein. Further study could distinguish the regions by which pathogen effectors and host proteins interact with SGT1, facilitating precise editing of effector incompatible variants.

scholarly journals The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

2021 ◽  
Vol 7 (2) ◽  
pp. 86
Author(s):  
Bilal Ökmen ◽  
Daniela Schwammbach ◽  
Guus Bakkeren ◽  
Ulla Neumann ◽  
Gunther Doehlemann
Keyword(s):  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Expression System ◽  
Pathogenic Fungi ◽  
Effector Proteins ◽  
Mating Partner ◽  
Fungal Virulence ◽  
Functional Studies ◽  
Infection Structures ◽  
Ustilago Hordei

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

scholarly journals Stabilizing membrane domains antagonizes n-alcohol anesthesia

2016 ◽  
Author(s):  
B.B. Machta ◽  
E. Grey ◽  
M. Nouri ◽  
N.L.C. McCarthy ◽  
E.M. Gray ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Critical Temperature ◽  
Hydrostatic Pressure ◽  
General Anesthesia ◽  
Membrane Domains ◽  
Strongly Correlated ◽  
Membrane Heterogeneity ◽  
Anesthetic Potency ◽  
Better Than

AbstractDiverse molecules induce general anesthesia with potency strongly correlated both with their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma membrane derived vesicles by lowering the critical temperature (Tc) for phase separation. Here we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on Tc. First we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described ‘intoxication reversers’ raise Tc and counter ethanol’s effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that hydrostatic pressure, long known to reverse anesthesia, also raises Tc in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together,these results demonstrate that ΔTc predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.

scholarly journals Rabenosyn-5, a Novel Rab5 Effector, Is Complexed with Hvps45 and Recruited to Endosomes through a Fyve Finger Domain

2000 ◽  
Vol 151 (3) ◽  
pp. 601-612 ◽  
Author(s):  
Erik Nielsen ◽  
Savvas Christoforidis ◽  
Sandrine Uttenweiler-Joseph ◽  
Marta Miaczynska ◽  
Frederique Dewitte ◽  
...  
Keyword(s):  
Early Endosome ◽  
Effector Proteins ◽  
Endosomal Trafficking ◽  
Membrane Domains ◽  
Phosphatidylinositol 3 Kinase ◽  
Early Endosomes ◽  
Site Of Action ◽  
Endosomal Membranes ◽  
Novel Protein ◽  
Phosphatidylinositol 3

Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase–dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

scholarly journals Nonhost Resistance in Arabidopsis-Colletotrichum Interactions Acts at the Cell Periphery and Requires Actin Filament Function

2006 ◽  
Vol 19 (3) ◽  
pp. 270-279 ◽  
Author(s):  
Chiyumi Shimada ◽  
Volker Lipka ◽  
Richard O'Connell ◽  
Tetsuro Okuno ◽  
Paul Schulze-Lefert ◽  
...  
Keyword(s):  
Actin Filament ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Nonhost Resistance ◽  
Cell Periphery ◽  
Colletotrichum Species ◽  
Fungal Plant Pathogens

Pathogenesis of nonadapted fungal pathogens is often terminated coincident with their attempted penetration into epidermal cells of nonhost plants. The genus Colletotrichum represents an economically important group of fungal plant pathogens that are amenable to molecular genetic analysis. Here, we investigated interactions between Arabidopsis and Colletotrichum to gain insights in plant and pathogen processes activating nonhost resistance responses. Three tested nonadapted Colletotrichum species differentiated melanized appressoria on Arabidopsis leaves but failed to form intracellular hyphae. Plant cells responded to Colletotrichum invasion attempts by the formation of PMR4/GSL5-dependent papillary callose. Appressorium differentiation and melanization were insufficient to trigger this localized plant cell response, but analysis of nonpathogenic C. lagenarium mutants implicates penetration-peg formation as the inductive cue. We show that Arabidopsis PEN1 syntaxin controls timely accumulation of papillary callose but is functionally dispensable for effective preinvasion (penetration) resistance in nonhost interactions. Consistent with this observation, green fluorescent protein-tagged PEN1 did not accumulate at sites of attempted penetration by either adapted or nonadapted Colletotrichum species, in contrast to the pronounced focal accumulations of PEN1 associated with entry of powdery mildews. We observed extensive reorganization of actin microfilaments leading to polar orientation of large actin bundles towards appressorial contact sites in interactions with the nonadapted Colletotrichum species. Pharmacological inhibition of actin filament function indicates a functional contribution of the actin cytoskeleton for both preinvasion resistance and papillary callose formation. Interestingly, the incidence of papilla formation at entry sites was greatly reduced in interactions with C. higginsianum isolates, indicating that this adapted pathogen may suppress preinvasion resistance at the cell periphery.

scholarly journals Lateral diffusion of the PH-20 protein on guinea pig sperm: evidence that barriers to diffusion maintain plasma membrane domains in mammalian sperm

1987 ◽  
Vol 104 (4) ◽  
pp. 917-923 ◽  
Author(s):  
AE Cowan ◽  
DG Myles ◽  
DE Koppel
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Domain Boundary ◽  
Lateral Diffusion ◽  
Fold Increase ◽  
Membrane Domains ◽  
Mammalian Sperm ◽  
Acrosomal Membrane ◽  
Percent Recovery ◽  
Lipid Probe

PH-20 protein on the plasma membrane (PH-20PM) is restricted to the posterior head of acrosome-intact guinea pig sperm. During the exocytotic acrosome reaction the inner acrosomal membrane (IAM) becomes continuous with the posterior head plasma membrane, and PH-20PM migrates to the IAM. There it joins a second population of PH-20 protein localized to this region of the acrosomal membrane (PH-20AM) (Cowan, A.E., P. Primakoff, and D.G. Myles, 1986, J. Cell Biol. 103:1289-1297). To investigate how the localized distributions of PH-20 protein are maintained, the lateral mobility of PH-20 protein on these different membrane domains was determined using fluorescence redistribution after photobleaching. PH-20PM on the posterior head of acrosome-intact sperm was found to be mobile, with a diffusion coefficient and percent recovery typical of integral membrane proteins (D = 1.8 X 10(-10) cm2/s; %R = 73). This value of D was some 50-fold lower than that found for the lipid probe 1,1-ditetradecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate (C14diI) in the same region (D = 8.9 X 10(-9) cm2/s). After migration to the IAM of acrosome-reacted sperm, this same population of molecules (PH-20PM) exhibited a 30-fold increase in diffusion rate (D = 4.9 X 10(-9) cm2/s; %R = 78). This rate was similar to diffusion of the lipid probe C14diI in the IAM (D = 5.4 X 10(-9) cm2/s). The finding of free diffusion of PH-20PM in the IAM of acrosome-reacted sperm supports the proposal that PH-20 is maintained within the IAM by a barrier to diffusion at the domain boundary. The slower diffusion of PH-20PM on the posterior head of acrosome-intact sperm is also consistent with localization by barriers to diffusion, but does not rule out alternative mechanisms.

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