scholarly journals The cucumber mosaic virus 1a protein regulates interactions between the 2b protein and ARGONAUTE 1 while maintaining the silencing suppressor activity of the 2b protein

2020 ◽  
Vol 16 (12) ◽  
pp. e1009125
Author(s):  
Lewis G. Watt ◽  
Sam Crawshaw ◽  
Sun-Ju Rhee ◽  
Alex M. Murphy ◽  
Tomás Canto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Bimolecular Fluorescence Complementation ◽  
Confocal Laser ◽  
Protein Molecules ◽  
2B Protein ◽  
Argonaute 1

The cucumber mosaic virus (CMV) 2b viral suppressor of RNA silencing (VSR) is a potent counter-defense and pathogenicity factor that inhibits antiviral silencing by titration of short double-stranded RNAs. It also disrupts microRNA-mediated regulation of host gene expression by binding ARGONAUTE 1 (AGO1). But in Arabidopsis thaliana complete inhibition of AGO1 is counterproductive to CMV since this triggers another layer of antiviral silencing mediated by AGO2, de-represses strong resistance against aphids (the insect vectors of CMV), and exacerbates symptoms. Using confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation assays we found that the CMV 1a protein, a component of the viral replicase complex, regulates the 2b-AGO1 interaction. By binding 2b protein molecules and sequestering them in P-bodies, the 1a protein limits the proportion of 2b protein molecules available to bind AGO1, which ameliorates 2b-induced disease symptoms, and moderates induction of resistance to CMV and to its aphid vector. However, the 1a protein-2b protein interaction does not inhibit the ability of the 2b protein to inhibit silencing of reporter gene expression in agroinfiltration assays. The interaction between the CMV 1a and 2b proteins represents a novel regulatory system in which specific functions of a VSR are selectively modulated by another viral protein. The finding also provides a mechanism that explains how CMV, and possibly other viruses, modulates symptom induction and manipulates host-vector interactions.

scholarly journals The 28 Ser Amino Acid of Cucumber Mosaic Virus Movement Protein Has a Role in Symptom Formation and Plasmodesmata Localization

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 222
Author(s):  
Réka Sáray ◽  
Attila Fábián ◽  
László Palkovics ◽  
Katalin Salánki
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Current Knowledge ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chenopodium Murale ◽  
Symptom Formation

Cucumber mosaic virus (CMV, Cucumovirus, Bromoviridae) is an economically significant virus infecting important horticultural and field crops. Current knowledge regarding the specific functions of its movement protein (MP) is still incomplete. In the present study, potential post-translational modification sites of its MP were assayed with mutant viruses: MP/S28A, MP/S28D, MP/S120A and MP/S120D. Ser28 was identified as an important factor in viral pathogenicity on Nicotiana tabacum cv. Xanthi, Cucumis sativus and Chenopodium murale. The subcellular localization of GFP-tagged movement proteins was determined with confocal laser-scanning microscopy. The wild type movement protein fused to green fluorescent protein (GFP) (MP-eGFP) greatly colocalized with callose at plasmodesmata, while MP/S28A-eGFP and MP/S28D-eGFP were detected as punctate spots along the cell membrane without callose colocalization. These results underline the importance of phosphorylatable amino acids in symptom formation and provide data regarding the essential factors for plasmodesmata localization of CMV MP.

scholarly journals Virulence and Differential Local and Systemic Spread of Cucumber mosaic virus in Tobacco are Affected by the CMV 2b Protein

2002 ◽  
Vol 15 (7) ◽  
pp. 647-653 ◽  
Author(s):  
Avril J. Soards ◽  
Alex M. Murphy ◽  
Peter Palukaitis ◽  
John P. Carr
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Wild Type Virus ◽  
Virus Spread ◽  
Wild Type ◽  
Mesophyll Cells ◽  
Systemic Spread ◽  
2B Protein

A mutant of the Cucumber mosaic virus subgroup IA strain Fny (Fny-CMV) lacking the gene encoding the 2b protein (Fny-CMVΔ2b) induced a symptomless systemic infection in tobacco. Both the accumulation of Fny-CMVΔ2b in inoculated tissue and the systemic movement of the virus appeared to proceed more slowly than for wild-type Fny-CMV. The influence of the 2b protein on virus movement in the inoculated leaf was examined using viral constructs derived from Fny-CMV and Fny-CMVΔ2b expressing the green fluorescent protein. Laser scanning confocal microscopy was used to visualize the movement of these viruses. Whereas the wild-type virus spread between the epidermal cells as well as the mesophyll cells, the mutant virus spread less efficiently through the epidermal layer and moved preferentially through the mesophyll. Thus, the 2b protein of Fny-CMV influences the dynamics of movement of the virus both within the inoculated leaf and through the whole plant. We propose that this altered movement profile of Fny-CMVΔ2b results in the absence of disease symptoms in tobacco.

scholarly journals Genetic and Physiological Effects of Noncoherent Visible Light Combined with Hydrogen Peroxide on Streptococcus mutans in Biofilm

2008 ◽  
Vol 52 (7) ◽  
pp. 2626-2631 ◽  
Author(s):  
Doron Steinberg ◽  
Daniel Moreinos ◽  
John Featherstone ◽  
Moshe Shemesh ◽  
Osnat Feuerstein
Keyword(s):  
Gene Expression ◽  
Hydrogen Peroxide ◽  
Visible Light ◽  
Streptococcus Mutans ◽  
Laser Scanning ◽  
Antibacterial Effect ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Planktonic Bacteria ◽  
Deep Layers

ABSTRACT Oral biofilms are associated with the most common infections of the oral cavity. Bacteria embedded in the biofilms are less sensitive to antibacterial agents than planktonic bacteria are. Recently, an antibacterial synergic effect of noncoherent blue light and hydrogen peroxide (H2O2) on planktonic Streptococcus mutans was demonstrated. In this study, we tested the effect of a combination of light and H2O2 on the vitality and gene expression of S. mutans embedded in biofilm. Biofilms of S. mutans were exposed to visible light (wavelengths, 400 to 500 nm) for 30 or 60 s (equivalent to 34 or 68 J/cm2) in the presence of 3 to 300 mM H2O2. The antibacterial effect was assessed by microbial counts of each treated sample compared with that of the control. The effect of light combined with H2O2 on the different layers of the biofilm was evaluated by confocal laser scanning microscopy. Gene expression was determined by real-time reverse transcription-PCR. Our results show that noncoherent light, in combination with H2O2, has a synergistic antibacterial effect through all of the layers of the biofilm. Furthermore, this treatment was more effective against bacteria in biofilm than against planktonic bacteria. The combined light and H2O2 treatment up-regulated the expression of several genes such as gtfB, brp, smu630, and comDE but did not affect relA and ftf. The ability of noncoherent visible light in combination with H2O2 to affect bacteria in deep layers of the biofilm suggests that this treatment may be applied in biofilm-related diseases as a minimally invasive antibacterial procedure.

scholarly journals Studying the Function of Phytoplasma Effector Proteins Using a Chemical-Inducible Expression System in Transgenic Plants

2021 ◽  
Vol 22 (24) ◽  
pp. 13582
Author(s):  
Keziah M. Omenge ◽  
Florian Rümpler ◽  
Subha Suvetha Kathalingam ◽  
Alexandra C. U. Furch ◽  
Günter Theißen
Keyword(s):  
Gene Expression ◽  
Laser Scanning ◽  
Expression System ◽  
Inducible Expression ◽  
Effector Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Protein Accumulation ◽  
Disease Phenotypes ◽  
The Impact

Phytoplasmas are bacterial pathogens that live mainly in the phloem of their plant hosts. They dramatically manipulate plant development by secreting effector proteins that target developmental proteins of their hosts. Traditionally, the effects of individual effector proteins have been studied by ectopic overexpression using strong, ubiquitously active promoters in transgenic model plants. However, the impact of phytoplasma infection on the host plants depends on the intensity and timing of infection with respect to the developmental stage of the host. To facilitate investigations addressing the timing of effector protein activity, we have established chemical-inducible expression systems for the three most well-characterized phytoplasma effector proteins, SECRETED ASTER YELLOWS WITCHES’ BROOM PROTEIN 11 (SAP11), SAP54 and TENGU in transgenic Arabidopsis thaliana. We induced gene expression either continuously, or at germination stage, seedling stage, or flowering stage. mRNA expression was determined by quantitative reverse transcription PCR, protein accumulation by confocal laser scanning microscopy of GFP fusion proteins. Our data reveal tight regulation of effector gene expression and strong upregulation after induction. Phenotypic analyses showed differences in disease phenotypes depending on the timing of induction. Comparative phenotype analysis revealed so far unreported similarities in disease phenotypes, with all three effector proteins interfering with flower development and shoot branching, indicating a surprising functional redundancy of SAP54, SAP11 and TENGU. However, subtle but mechanistically important differences were also observed, especially affecting the branching pattern of the plants.

scholarly journals Replicase-Mediated Resistance to Cucumber Mosaic Virus Does Not Inhibit Localization and/or Trafficking of the Viral Movement Protein

1999 ◽  
Vol 12 (8) ◽  
pp. 743-747 ◽  
Author(s):  
Tomas Canto ◽  
Peter Palukaitis
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Microscopic Analysis ◽  
Potato Virus X ◽  
Confocal Laser ◽  
Viral Movement Protein ◽  
Green Fluorescent

Replicase-mediated resistance to cucumber mosaic virus (CMV) affects CMV replication and restricts CMV movement. Confocal laser scanning microscopic analysis of resistant plants inoculated with either CMV or potato virus X, expressing the CMV movement protein (MP) fused to the green fluorescent protein (GFP), showed that the CMV MP was not inhibited from either plasmodesmal association or trafficking in the CMV-resistant plants. CMV expressing free GFP was able to move to adjacent cells, demonstrating that replicase-mediated resistance did not directly block the trafficking of CMV RNA.

Ephrin-B1, -B2, and -B4 Expression is Decreased in Developing Diaphragms and Lungs of Fetal Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia

2018 ◽  
Vol 29 (01) ◽  
pp. 113-119
Author(s):  
Toshiaki Takahashi ◽  
Florian Friedmacher ◽  
Julia Zimmer ◽  
Prem Puri
Keyword(s):  
Gene Expression ◽  
Congenital Diaphragmatic Hernia ◽  
Diaphragmatic Hernia ◽  
Laser Scanning ◽  
Newborn Infants ◽  
Branching Morphogenesis ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Distal Airway ◽  
Lung Branching

Introduction Congenital diaphragmatic hernia (CDH) is assumed to originate from a malformation of the amuscular mesenchymal component of the primordial diaphragm. Mutations in ephrin-B1, a membrane protein that is expressed by mesenchymal cells, have been found in newborn infants with CDH and associated pulmonary hypoplasia (PH), highlighting its important role during diaphragmatic and airway development. Ephrin-B1, -B2, and -B4 are expressed in fetal rat lungs and have been identified as key players during lung branching morphogenesis. We hypothesized that diaphragmatic and pulmonary expression of ephrin-B1, -B2, and -B4 is decreased in the nitrofen-induced CDH model. Materials and Methods Time-mated rats received nitrofen or vehicle on day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Ephrin-B1, -B2, and -B4 gene expression was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for ephrin-B1, -B2, and -B4 was combined with mesenchymal and epithelial markers (Gata-4/Fgf-10 and calcitonin gene-related peptide) to evaluate protein expression/localization. Results Ephrin-B1, -B2, and -B4 gene expression was significantly reduced in pleuroperitoneal folds/primordial lungs (D13), developing diaphragms/lungs (D15), and fully muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal laser scanning microscopy demonstrated markedly diminished ephrin-B1 immunofluorescence in diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15, and D18 compared with controls, whereas ephrin-B2 and -B4 expression was mainly decreased in distal airway epithelium. Conclusion Decreased ephrin-B1, -B2, and -B4 expression may disrupt diaphragmatic development and lung branching morphogenesis by interfering with epithelial–mesenchymal interactions, thus causing diaphragmatic defects and PH.

scholarly journals Following Pathogen Development and Gene Expression in a Food Ecosystem: the Case of a Staphylococcus aureus Isolate in Cheese

2014 ◽  
Vol 80 (16) ◽  
pp. 5106-5115 ◽  
Author(s):  
Isabelle Fleurot ◽  
Marina Aigle ◽  
Renaud Fleurot ◽  
Claire Darrigo ◽  
Jacques-Antoine Hennekinne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Staphylococcus Aureus ◽  
Laser Scanning ◽  
Public Health Problem ◽  
Laser Scanning Microscopy ◽  
Solid Matrix ◽  
Confocal Laser ◽  
Natural Food ◽  
Content Type ◽  
Food Isolate

ABSTRACTHuman intoxication or infection due to bacterial food contamination constitutes an economic challenge and a public health problem. Information on thein situdistribution and expression of pathogens responsible for this risk is to date lacking, largely because of technical bottlenecks in detecting signals from minority bacterial populations within a complex microbial and physicochemical ecosystem. We simulated the contamination of a real high-risk cheese with a natural food isolate ofStaphylococcus aureus, an enterotoxin-producing pathogen responsible for food poisoning. To overcome the problem of a detection limit in a solid matrix, we chose to work with a fluorescent reporter (superfolder green fluorescent protein) that would allow spatiotemporal monitoring ofS. aureuspopulations and targeted gene expression. The combination of complementary techniques revealed thatS. aureuslocalizes preferentially on the cheese surface during ripening. Immunochemistry and confocal laser scanning microscopy enabled us to visualize, in a single image, dairy bacteria and pathogen populations, virulence gene expression, and the toxin produced. This procedure is readily applicable to other genes of interest, other bacteria, and different types of food matrices.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

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