scholarly journals Necrosis-Inducing Proteins of Rhynchosporium commune, Effectors in Quantitative Disease Resistance

2012 ◽  
Vol 25 (10) ◽  
pp. 1314-1325 ◽  
Author(s):  
S. Kirsten ◽  
A. Navarro-Quezada ◽  
D. Penselin ◽  
C. Wenzel ◽  
A. Matern ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Quantitative Polymerase Chain Reaction ◽  
Fungal Spores ◽  
Growth Patterns ◽  
Effector Proteins ◽  
Defense Gene Expression ◽  
Host Genotype ◽  
Barley Cultivars ◽  
Green Fluorescent ◽  
The Impact

The barley pathogen Rhynchosporium commune secretes necrosis-inducing proteins NIP1, NIP2, and NIP3. Expression analysis revealed that NIP1 transcripts appear to be present in fungal spores already, whereas NIP2 and NIP3 are synthesized after inoculation of host plants. To assess the contribution of the three effector proteins to disease development, deletion mutants were generated. The development of these fungal mutants on four barley cultivars was quantified in comparison with that of the parent wild-type strain and with two fungal strains failing to secrete an “active” NIP1 avirulence protein, using quantitative polymerase chain reaction as well as microscopic imaging after fungal green fluorescent protein tagging. The impact of the three deletions varied quantitatively depending on the host genotype, suggesting that the activities of the fungal effectors add up to produce stronger growth patterns and symptom development. Alternatively, recognition events of differing intensities may be converted into defense gene expression in a quantitative manner.

scholarly journals Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts

2021 ◽  
Author(s):  
Johannes W. Debler ◽  
Bernadette M. Henares ◽  
Robert C. Lee
Keyword(s):  
Broad Spectrum ◽  
Faba Bean ◽  
Fluorescent Protein ◽  
Transient Gene Expression ◽  
Low Frequency ◽  
Effector Proteins ◽  
Field Pea ◽  
Grain Legume ◽  
Pathogen Effectors ◽  
Green Fluorescent

Abstract Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

Microscopic Observation and Processing Validation of Fruit Sanitizing Treatments for the Enhanced Microbiological Safety of Fresh Orange Juice†

2001 ◽  
Vol 64 (3) ◽  
pp. 310-314 ◽  
Author(s):  
STEVEN PAO ◽  
CRAIG L. DAVIS ◽  
MICKEY E. PARISH
Keyword(s):  
Fluorescent Protein ◽  
Citrus Fruit ◽  
Microbial Reduction ◽  
Hot Water ◽  
Near Surface ◽  
E Coli ◽  
Surface Areas ◽  
Orange Fruit ◽  
Green Fluorescent ◽  
The Impact

Studies were conducted to evaluate the infiltration of dye and bacteria into the interior of orange fruit and the impact of possible infiltration on achieving a 5-log microbial reduction during fresh juice processing. Fresh orange fruit were treated at the stem end area with dye and either Salmonella Rubislaw or Escherichia coli strains expressing green fluorescent protein. Microscopic images showed that bacterial contaminants localized at the surface or near surface areas that may be sanitized by surface treatments. Dye infiltration was not a reliable indicator of bacterial penetration in citrus fruit. To quantify the reduction of bacterial contamination, orange fruit were inoculated with E. coli and processed with and without hot water treatments. Greater than 5-log reductions were achieved in juice extracted from fruit immersed in hot water for 1 or 2 min at 80°C, in comparison to the E. coli level detected in the control juice obtained by homogenization of inoculated fruit.

scholarly journals Analyzing Defects in the Caenorhabditis elegans Nervous System Using Organismal and Cell Biological Approaches

2002 ◽  
Vol 1 (1) ◽  
pp. 18-25 ◽  
Author(s):  
Megan Guziewicz ◽  
Toni Vitullo ◽  
Bethany Simmons ◽  
Rebecca Eustance Kohn
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Synaptic Vesicle ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Vesicle Transport ◽  
Laboratory Exercise ◽  
Cellular Analysis ◽  
Green Fluorescent ◽  
The Impact

The goal of this laboratory exercise is to increase student understanding of the impact of nervous system function at both the organismal and cellular levels. This inquiry-based exercise is designed for an undergraduate course examining principles of cell biology. After observing the movement of Caenorhabditis elegans with defects in their nervous system, students examine the structure of the nervous system to categorize the type of defect. They distinguish between defects in synaptic vesicle transport and defects in synaptic vesicle fusion with membranes. The synaptic vesicles are tagged with green fluorescent protein (GFP), simplifying cellular analysis. The expected outcome of this experiment is that students will better understand the concepts of vesicle transport, neurotransmitter release, GFP, and the relation between the nervous system and behavior.

scholarly journals Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein

2009 ◽  
Vol 191 (9) ◽  
pp. 3120-3131 ◽  
Author(s):  
Joanne E. Morello ◽  
Alan Collmer
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Effector Proteins ◽  
Type Iii ◽  
Native Promoter ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.

Cytokine receptor cluster size impacts its endocytosis and signaling

2021 ◽  
Vol 118 (37) ◽  
pp. e2024893118
Author(s):  
Laura Salavessa ◽  
Thibault Lagache ◽  
Valérie Malardé ◽  
Alexandre Grassart ◽  
Jean-Christophe Olivo-Marin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Fluorescent Protein ◽  
Interleukin 2 ◽  
Lateral Diffusion ◽  
Close Correlation ◽  
Cytokine Receptor ◽  
Cholesterol Depletion ◽  
Green Fluorescent ◽  
The Impact

The interleukin-2 receptor (IL-2R) is a cytokine receptor essential for immunity that transduces proliferative signals regulated by its uptake and degradation. IL-2R is a well-known marker of clathrin-independent endocytosis (CIE), a process devoid of any coat protein, raising the question of how the CIE vesicle is generated. Here, we investigated the impact of IL-2Rγ clustering in its endocytosis. Combining total internal reflection fluorescence (TIRF) live imaging of a CRISPR-edited T cell line endogenously expressing IL-2Rγ tagged with green fluorescent protein (GFP), with multichannel imaging, single-molecule tracking, and quantitative analysis, we were able to decipher IL-2Rγ stoichiometry at the plasma membrane in real time. We identified three distinct IL-2Rγ cluster populations. IL-2Rγ is secreted to the cell surface as a preassembled small cluster of three molecules maximum, rapidly diffusing at the plasma membrane. A medium-sized cluster composed of four to six molecules is key for IL-2R internalization and is promoted by interleukin 2 (IL-2) binding, while larger clusters (more than six molecules) are static and inefficiently internalized. Moreover, we identified membrane cholesterol and the branched actin cytoskeleton as key regulators of IL-2Rγ clustering and IL-2–induced signaling. Both cholesterol depletion and Arp2/3 inhibition lead to the assembly of large IL-2Rγ clusters, arising from the stochastic interaction of receptor molecules in close correlation with their enhanced lateral diffusion at the membrane, thus resulting in a default in IL-2R endocytosis. Despite similar clustering outcomes, while cholesterol depletion leads to a sustained IL-2–dependent signaling, Arp2/3 inhibition prevents signal initiation. Taken together, our results reveal the importance of cytokine receptor clustering for CIE initiation and signal transduction.

scholarly journals Construction of Self-Transmissible Green Fluorescent Protein-Based Biosensor Plasmids and Their Use for Identification of N-Acyl Homoserine-Producing Bacteria in Lake Sediments

2010 ◽  
Vol 76 (18) ◽  
pp. 6119-6127 ◽  
Author(s):  
Putthapoom Lumjiaktase ◽  
Claudio Aguilar ◽  
Tom Battin ◽  
Kathrin Riedel ◽  
Leo Eberl
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Natural Habitat ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Broad Host Range ◽  
Construction Of Self ◽  
Green Fluorescent ◽  
The Impact

ABSTRACT Many bacteria utilize quorum sensing (QS) systems to communicate with each other by means of the production, release, and response to signal molecules. N-Acyl homoserine lactone (AHL)-based QS systems are particularly widespread among the Proteobacteria, in which they regulate various functions. It has become evident that AHLs can also serve as signals for interspecies communication. However, knowledge on the impact of AHLs for the ecology of bacteria in their natural habitat is scarce, due mainly to the lack of tools that allow the study of QS in bacterial communities in situ. Here, we describe the construction of self-mobilizable green fluorescent protein (GFP)-based AHL sensors that utilize the conjugation and replication properties of the broad-host-range plasmid RP4. We show that these novel AHL sensor plasmids can be easily transferred to different bacterial species by biparental mating and that they give rise to green fluorescent cells in case the recipient is an AHL producer. We also demonstrate that these sensor plasmids are capable of self-spreading within mixed biofilms and are a suitable tool for the identification of AHL-producing bacteria in lake sediment.

scholarly journals Impact of signaling microcompartment geometry on GPCR dynamics in live retinal photoreceptors

2012 ◽  
Vol 140 (3) ◽  
pp. 249-266 ◽  
Author(s):  
Mehdi Najafi ◽  
Mohammad Haeri ◽  
Barry E. Knox ◽  
William E. Schiesser ◽  
Peter D. Calvert
Keyword(s):  
Green Fluorescent Protein ◽  
Molecular Diffusion ◽  
Fluorescent Protein ◽  
Lateral Diffusion ◽  
Live Cell ◽  
Confocal Imaging ◽  
Slow Phase ◽  
Protein Diffusion ◽  
Green Fluorescent ◽  
The Impact

G protein–coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell–imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti–sapphire laser focused to the diffraction limit, which produced small sub–cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient Dlat = 0.130 ± 0.012 µm2s−1. Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.

scholarly journals pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

2015 ◽  
Vol 28 (2) ◽  
pp. 107-121 ◽  
Author(s):  
Xiaoyan Gong ◽  
Oscar Hurtado ◽  
Baohua Wang ◽  
Congqing Wu ◽  
Mihwa Yi ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Effector Proteins ◽  
Fungal Transformation ◽  
In Planta ◽  
Green Fluorescent

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their “directly fused” counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

Detection and localization of the endophyte Undifilum oxytropis in locoweed tissues

Botany ◽  
2012 ◽  
Vol 90 (12) ◽  
pp. 1229-1236 ◽  
Author(s):  
Roxanna Reyna ◽  
Peter Cooke ◽  
Daniel Grum ◽  
Daniel Cook ◽  
Rebecca Creamer
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Protein ◽  
Growth Patterns ◽  
Host Cells ◽  
Economic Losses ◽  
Thin Sections ◽  
Oxytropis Sericea ◽  
Pathogenic Interaction ◽  
Green Fluorescent ◽  
Detection And Localization

Poisoning of livestock owing to grazing on locoweeds results in significant economic losses in the western United States. Some Oxytropis spp. locoweeds contain a seed-transmitted endophytic fungus, Undifilum oxytropis, which produces the toxic alkaloid swainsonine. We sought to localize and characterize growth patterns of the fungus within leaves and petioles of Oxytropis lambertii Pursh and Oxytropis sericea Nutt. to help define the types of interactions between the fungus and its hosts. Vegetative hyphae were observed within locoweed tissues using integrated imaging. Topographical images from scanning electron microscopy revealed the presence of the endophyte in the pith tissue of petioles. The fungus was identified between plant cells but did not appear to penetrate host cells. Transmission electron microscopy images of thin sections revealed that hyphae were closely associated with host cell walls. Oxytropis sericea was innoculated with green fluorescent protein-transformed U. oxytropis and observed by confocal microscopy, confirming the presence of the endophyte hyphae in leaves and petioles. The fungus was identified only in the pith of petioles using fluorescence and in the vascular bundle throughout extracellular spaces in leaves. These results revealed no signs of a pathogenic interaction between plant and fungus and support the hypothesis of a mutualistic or commensal relationship.

scholarly journals Phytobacterial Type III Effectors HopX1, HopAB1 and HopF2 Enhance Sense-Post-Transcriptional Gene Silencing Independently of Plant R Gene-Effector Recognition

2011 ◽  
Vol 24 (8) ◽  
pp. 907-917 ◽  
Author(s):  
Panagiotis F. Sarris ◽  
Shang Gao ◽  
Konstantinos Karademiris ◽  
Hailing Jin ◽  
Kriton Kalantidis ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Pathogenic Bacteria ◽  
Fluorescent Protein ◽  
Effector Proteins ◽  
Transcriptional Gene Silencing ◽  
Type Iii ◽  
Post Transcriptional Gene Silencing ◽  
Rna Pathways ◽  
Green Fluorescent ◽  
Effector Recognition

Plant- and animal-pathogenic bacteria deploy a variable arsenal of type III effector proteins (T3EP) to manipulate host defense. Specific biochemical functions and molecular or subcellular targets have been demonstrated or proposed for a growing number of T3EP but remain unknown for the majority of them. Here, we show that transient expression of genes coding certain bacterial T3EP (HopAB1, HopX1, and HopF2), which did not elicit hypersensitive response (HR) in transgenic green fluorescent protein (GFP) Nicotiana benthamiana 16C line, enhanced the sense post-transcriptional gene silencing (S-PTGS) triggered by agrodelivery of a GFP-expressing cassette and the silencing enhancement could be blocked by two well-known viral silencing suppressors. Further analysis using genetic truncations and site-directed mutations showed that the receptor recognition domains of HopAB1 and HopX1 are not involved in enhancing silencing. Our studies provide new evidence that phytobacterial pathogen T3EP manipulate the plant small interfering RNA pathways by enhancing silencing efficiency in the absence of effector-triggered immunity signaling and suggest that phytopathogenic bacterial effectors affect host RNA silencing in yet other ways than previously described.

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