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.

Efficient fluorescence recovery using antifade reagents in correlative light and electron microscopy

Microscopy ◽  
2019 ◽  
Vol 68 (5) ◽  
pp. 417-421
Author(s):  
Kiminori Toyooka ◽  
Naeko Shinozaki-Narikawa
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Protein ◽  
Light And Electron Microscopy ◽  
Ultrastructural Level ◽  
Fluorescence Decay ◽  
Fluorescence Recovery ◽  
Recovery Method ◽  
Thin Sections ◽  
Electron Microscopies ◽  
Green Fluorescent

Abstract Correlative light and electron microscopy (CLEM) enables ultrastructural-level analysis of fluorescence-labeled proteins by combining images obtained from both fluorescence and electron microscopies. A technical challenge with the CLEM method is the effective detection of fluorescence from samples embedded in resins, which generally cause fluorescence decay. To overcome this issue, we developed a method for fluorescence recovery of green fluorescent protein (GFP) in resin-embedded semi-thin sections using commercially available antifade reagents. By applying this method, we successfully obtained CLEM images using field-emission scanning electron microscopy with moderately enhanced GFP signals, demonstrating the efficacy of this simple fluorescence recovery method.

Visualizing Green Fluorescent Protein and Fluorescence Associated with a Gold Conjugate in Thin Sections with Correlative Confocal and Electron Microscopy

2004 ◽  
Vol 10 (S02) ◽  
pp. 156-157 ◽  
Author(s):  
Paul. A Sims ◽  
Jeff Hardin
Keyword(s):  
Electron Microscopy ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Thin Sections ◽  
Gold Conjugate ◽  
Green Fluorescent ◽  
Confocal And Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

scholarly journals Nature and consequences of interactions between Salmonella enterica serovar Dublin and host cells in cattle

2019 ◽  
Vol 50 (1) ◽  
Author(s):  
Prerna Vohra ◽  
Christina Vrettou ◽  
Jayne C. Hope ◽  
John Hopkins ◽  
Mark P. Stevens
Keyword(s):  
Lymph Nodes ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Large Animal ◽  
Ileal Mucosa ◽  
Zoonotic Infections ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Salmonella Enterica Serovar Dublin

AbstractSalmonella enterica is a veterinary and zoonotic pathogen of global importance. While murine and cell-based models of infection have provided considerable knowledge about the molecular basis of virulence of Salmonella, relatively little is known about salmonellosis in naturally-affected large animal hosts such as cattle, which are a reservoir of human salmonellosis. As in humans, Salmonella causes bovine disease ranging from self-limiting enteritis to systemic typhoid-like disease and exerts significant economic and welfare costs. Understanding the nature and consequences of Salmonella interactions with bovine cells will inform the design of effective vaccines and interventions to control animal and zoonotic infections. In calves challenged orally with S. Dublin expressing green fluorescent protein (GFP) we observed that the bacteria were predominantly extracellular in the distal ileal mucosa and within gut-associated lymph nodes 48 h post-infection. Intracellular bacteria, identified by flow cytometry using the GFP signal, were predominantly within MHCII+ macrophage-like cells. In contrast to observations from murine models, these S. Dublin-infected cells had elevated levels of MHCII and CD40 compared to both uninfected cells from the same tissue and cells from the cognate tissue of uninfected animals. Moreover, no gross changes of the architecture of infected lymph nodes were observed as was described previously in a mouse model. In order to further investigate Salmonella-macrophage interactions, net replication of S. enterica serovars that differ in virulence in cattle was measured in bovine blood-derived macrophages by enumeration of gentamicin-protected bacteria and fluorescence dilution, but did not correlate with host-specificity.

scholarly journals Localization of GFP in Frozen Sections from Unfixed Mouse Tissues: Immobilization of a Highly Soluble Marker Protein by Formaldehyde Vapor

2003 ◽  
Vol 51 (3) ◽  
pp. 401-404 ◽  
Author(s):  
Harald Jockusch ◽  
Sylvana Voigt ◽  
Daniel Eberhard
Keyword(s):  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Striated Muscle ◽  
Host Cells ◽  
Frozen Sections ◽  
Mouse Tissues ◽  
Transplantation Experiment ◽  
Green Fluorescent ◽  
Cryostat Sections ◽  
Unfixed Cryostat

Green fluorescent protein (GFP) and its variants, such as enhanced GFP (EGFP), have been introduced into mammalian cells by transgenes, e.g., to distinguish donor from host cells after transplantation. Free GFP is extremely soluble and leaks out from liquid-covered cryostat sections so that fixation of whole organs before sectioning has been mandatory. This precludes the analysis of serial sections with respect to fixation-sensitive enzyme activities and antigens. We describe here a vapor fixation for sections from unfixed cryostat blocks of tissue that allows unrestricted enzyme and immunohistochemistry on adjacent sections, as demonstrated for cross-striated muscle and other tissues from EGFP transgenic “green mice” and for a transplantation experiment.

scholarly journals A C-Terminal Domain Targets the Pseudomonas aeruginosa Cytotoxin ExoU to the Plasma Membrane of Host Cells

2006 ◽  
Vol 74 (5) ◽  
pp. 2552-2561 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Jeffrey L. Veesenmeyer ◽  
Kathryn T. Bieging ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Type Iii ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a phospholipase injected into host cells by the type III secretion system of Pseudomonas aeruginosa, leads to rapid cytolytic cell death. Although the importance of ExoU in infection is well established, the mechanism by which this toxin kills host cells is less clear. To gain insight into how ExoU causes cell death, we examined its subcellular localization following transfection or type III secretion/translocation into HeLa cells. Although rapid cell lysis precluded visualization of wild-type ExoU by fluorescence microscopy, catalytically inactive toxin was readily detected at the periphery of HeLa cells. Biochemical analysis confirmed that ExoU was targeted to the membrane fraction of transfected cells. Visualization of ExoU peptides fused with green fluorescent protein indicated that the domain responsible for this targeting was in the C terminus of ExoU, between residues 550 and 687. Localization to the plasma membrane occurred within 1 h of expression, which is consistent with the kinetics of cytotoxicity. Together, these results indicate that a domain between residues 550 and 687 of ExoU targets this toxin to the plasma membrane, a process that may be important in cytotoxicity.

scholarly journals 2A Protease Is Not a Prerequisite for Poliovirus Replication

2010 ◽  
Vol 84 (12) ◽  
pp. 5947-5957 ◽  
Author(s):  
Hiroko Igarashi ◽  
Yasuko Yoshino ◽  
Miwako Miyazawa ◽  
Hitoshi Horie ◽  
Seii Ohka ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Rna Replication ◽  
Full Length ◽  
Host Cells ◽  
Progeny Virus ◽  
Coding Sequence ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Replication Activity ◽  
Efficient Viral Replication

ABSTRACT Poliovirus (PV) 2Apro has been considered important for PV replication and is known to be toxic to host cells. A 2Apro-deficient PV would potentially be less toxic and ideal as a vector. To examine whether 2Apro is needed to form progeny virus, a full-length cDNA of dicistronic (dc) PV with (pOME) or without (pOMEΔ2A) 2Apro was constructed in the strain PV1(M)OM. RNAs of both pOME and pOMEΔ2A were capable of forming progeny viruses, called OME and OMEΔ2A, respectively. In their ability to induce a cytopathic effect (CPE), the strains ranked as OMEΔ2A < OME ≒ PV1(M)OM. These results suggest that 2Apro is not essential for full-length dc PV to form progeny virus and that it contributes to the efficient viral replication and/or induction of a CPE. To clarify whether 2Apro is essential for P1-null (lacking the entire coding sequence for capsid proteins) PV, the RNA replication activity of P1-null PV (pOMΔP1) or P1-null PV without 2Apro (pOMΔP1Δ2A) or without both 2Apro and 2B (pOMΔP1Δ2AΔ2B) was examined. The RNAs of pOMΔP1 and pOMΔP1Δ2A could replicate and form progeny viruses under a trans supply of P1 protein, whereas the RNA of pOMΔP1Δ2AΔ2B could not. These results suggest that 2Apro is not needed for the replication of P1-null PV, although it is important for PV RNA replication and inducing a CPE. To know whether a 2Apro-deficient PV can be used as a vector, a P1-null PV containing the enhanced green fluorescent protein (EGFP) coding sequence with or without 2Apro was examined. It expressed fluorescent protein. This result suggests that 2Apro-deficient PV can express foreign genes.

scholarly journals Development of a Novel Avian Vaccine Vector Derived From the Emerging Fowl Adenovirus 4

2021 ◽  
Vol 12 ◽  
Author(s):  
Qing Pan ◽  
Yu Zhang ◽  
Aijing Liu ◽  
Hongyu Cui ◽  
Yulong Gao ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Virus Genome ◽  
Open Reading Frames ◽  
Vaccine Vector ◽  
Economic Losses ◽  
Foreign Gene ◽  
The Novel ◽  
Hydropericardium Syndrome ◽  
Fowl Adenovirus ◽  
Green Fluorescent

Severe hepatitis-hydropericardium syndrome (HHS) associated with a novel viral genotype, fowl adenovirus 4 (FAdV-4), has emerged and widely spread in China since 2015, causing severe economic losses to the poultry industry. We previously reported that the hexon gene is responsible for pathogenicity and obtained a non-pathogenic hexon-replacement rHN20 strain; however, the lack of information about the non-essential regions for virus replication limits the development of a FAdV-4 vector. This study first established an enhanced green fluorescent protein (EGFP)-indicator virus based on the FAdV-4 reverse genetic technique, effective for batch operations in the virus genome. Based on this, 10 open reading frames (ORFs) at the left end and 13 ORFs at the right end of the novel FAdV-4 genome were deleted separately and identified as non-essential genes for viral replication, providing preliminary insertion sites for foreign genes. To further improve its feasibility as a vaccine vector, seven combinations of ORFs were successfully replaced with EGFP without affecting the immunogenicity of the vector backbone. Finally, a recombinant rHN20-vvIBDV-VP2 strain, expressing the VP2 protein of very virulent infectious bursa disease virus (vvIBDV), was rescued and showed complete protection against FAdV-4 and vvIBDV. Thus, the novel FAdV-4 vector could provide sufficient protection for HHS and efficient exogenous gene delivery. Overall, our findings systemically identified 23 non-essential ORFs for FAdV-4 replication and seven foreign gene insertion regions, providing valuable information for an in-depth understanding of the novel FAdV-4 pathogenesis and development of multivalent vaccines.

Immunoprobe Localization by Correlative Microscopy: How Much Resolution is Enough?

2000 ◽  
Vol 6 (S2) ◽  
pp. 320-321
Author(s):  
Patricia G. Calarco
Keyword(s):  
Electron Microscopy ◽  
Fluorescent Protein ◽  
Near Field ◽  
Freeze Substitution ◽  
Biological Molecules ◽  
Correlative Microscopy ◽  
Classical Electron ◽  
Green Fluorescent ◽  
Near Field Scanning ◽  
Made In

With the advent of new ways to image biological material, to tag biological molecules, and to prepare samples, much progress has been made in understanding cellular and subcellular function without resorting to classical electron microscopy. Newer ways to image include the use of microscopies such as: differential interference, video, confocal, near field scanning optical, and magnetic resonance imaging. The use of tags such as cell-permeant organelle-specific markers and green fluorescent protein has further increased our ability to vision subcellular events in living cells, providing us with functional correlations of gene activity and cellular function in a number of biological systems. Techniques such as high pressure freezing coupled with freeze substitution have expanded the range of tissues and organisms that can be optimally preserved and are changing our understanding of cellular fine structure. However, even when electron microscopy is mandated, 1-2 angstrom resolution is rarely indicated except for investigations of molecular structure.

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