scholarly journals Potato virus Y HCPro Localization at Distinct, Dynamically Related and Environment-Influenced Structures in the Cell Cytoplasm

2014 ◽  
Vol 27 (12) ◽  
pp. 1331-1343 ◽  
Author(s):  
Francisco del Toro ◽  
Fátima Tena Fernández ◽  
Jens Tilsner ◽  
Kathryn M. Wright ◽  
Francisco Tenllado ◽  
...  
Keyword(s):  
Potato Virus ◽  
Potato Virus Y ◽  
Fluorescent Protein ◽  
Biological Activities ◽  
Virus Transmission ◽  
Multifunctional Protein ◽  
Aphid Vectors ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Potyvirus HCPro is a multifunctional protein that, among other functions, interferes with antiviral defenses in plants and mediates viral transmission by aphid vectors. We have visualized in vivo the subcellular distribution and dynamics of HCPro from Potato virus Y and its homodimers, using green, yellow, and red fluorescent protein tags or their split parts, while assessing their biological activities. Confocal microscopy revealed a pattern of even distribution of fluorescence throughout the cytoplasm, common to all these modified HCPros, when transiently expressed in Nicotiana benthamiana epidermal cells in virus-free systems. However, in some cells, distinct additional patterns, specific to some constructs and influenced by environmental conditions, were observed: i) a small number of large, amorphous cytoplasm inclusions that contained α-tubulin; ii) a pattern of numerous small, similarly sized, dot-like inclusions distributing regularly throughout the cytoplasm and associated or anchored to the cortical endoplasmic reticulum and the microtubule (MT) cytoskeleton; and iii) a pattern that smoothly coated the MT. Furthermore, mixed and intermediate forms from the last two patterns were observed, suggesting dynamic transports between them. HCPro did not colocalize with actin filaments or the Golgi apparatus. Despite its association with MT, this network integrity was required neither for HCPro suppression of silencing in agropatch assays nor for its mediation of virus transmission by aphids.

scholarly journals Fluorescently Tagged Potato virus Y: A Versatile Tool for Functional Analysis of Plant-Virus Interactions

2015 ◽  
Vol 28 (7) ◽  
pp. 739-750 ◽  
Author(s):  
Matevz Rupar ◽  
Florence Faurez ◽  
Michel Tribodet ◽  
Ion Gutiérrez-Aguirre ◽  
Agnès Delaunay ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Potato Virus ◽  
Plant Virus ◽  
Potato Virus Y ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Cell Movement ◽  
Long Distance ◽  
Green Fluorescent

Potato virus Y (PVY) is an economically important plant virus that infects Solanaceous crops such as tobacco and potato. To date, studies into the localization and movement of PVY in plants have been limited to detection of viral RNA or proteins ex vivo. Here, a PVY N605 isolate was tagged with green fluorescent protein (GFP), characterized and used for in vivo tracking. In Nicotiana tabacum cv. Xanthi, PVY N605-GFP was biologically comparable to nontagged PVY N605, stable through three plant-to-plant passages and persisted for four months in infected plants. GFP was detected before symptoms and fluorescence intensity correlated with PVY RNA concentrations. PVY N605-GFP provided in vivo tracking of long-distance movement, allowing estimation of the cell-to-cell movement rate of PVY in N. tabacum cv. Xanthi (7.1 ± 1.5 cells per hour). PVY N605-GFP was adequately stable in Solanum tuberosum cvs. Désirée and NahG-Désirée and able to infect S. tuberosum cvs. Bintje and Bea, Nicotiana benthamiana, and wild potato relatives. PVY N605-GFP is therefore a powerful tool for future studies of PVY-host interactions, such as functional analysis of viral and plant genes involved in viral movement.

scholarly journals Comparative assessment of fluorescent proteins forin vivoimaging in an animal model system

2016 ◽  
Author(s):  
Jennifer K Heppert ◽  
Daniel J Dickinson ◽  
Ariel M Pani ◽  
Christopher D Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
C Elegans ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamicsin vivo. Recent advances in genome editing have enabled precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential ofin vivoimaging experiments, and they facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been madein vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap we quantitatively assessed fluorescent protein propertiesin vivoin an animal model system. We generated transgenicC. elegansstrains expressing green, yellow, or red fluorescent proteins in embryos, and we imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not brightin vivoas predicted based onin vitrodata, but that mNeonGreen is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imagingin vivoinC. elegansembryos, and they suggest good candidate fluorescent proteins to test in other animal model systems.

scholarly journals Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system

2016 ◽  
Vol 27 (22) ◽  
pp. 3385-3394 ◽  
Author(s):  
Jennifer K. Heppert ◽  
Daniel J. Dickinson ◽  
Ariel M. Pani ◽  
Christopher D. Higgins ◽  
Annette Steward ◽  
...  
Keyword(s):  
Animal Model ◽  
In Vivo Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Model System ◽  
Red Fluorescent Proteins ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments.

The Effect of Fluorescent Protein Tags on Phosphoglycerate Kinase Stability Is Nonadditive

2016 ◽  
Vol 120 (11) ◽  
pp. 2878-2885 ◽  
Author(s):  
Kapil Dave ◽  
Hannah Gelman ◽  
Chu Thi Hien Thu ◽  
Drishti Guin ◽  
Martin Gruebele
Keyword(s):  
Fluorescent Protein ◽  
Phosphoglycerate Kinase ◽  
Fluorescent Protein Tags ◽  
Protein Tags

scholarly journals Optimal fluorescent protein tags for quantifying protein oligomerization in living cells

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Valentin Dunsing ◽  
Madlen Luckner ◽  
Boris Zühlke ◽  
Roberto A. Petazzi ◽  
Andreas Herrmann ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Living Cells ◽  
Protein Oligomerization ◽  
Fluorescent Protein Tags ◽  
Protein Tags

Seasonal Abundance of Aphid Vectors of Potato Virus Y in the Red River Valley of Minnesota and North Dakota

1997 ◽  
Vol 90 (3) ◽  
pp. 824-831 ◽  
Author(s):  
Christina D. Difonzo ◽  
David W. Ragsdale ◽  
Edward B. Radcliffe ◽  
Neil C. Gudmestad ◽  
Gary A. Secor
Keyword(s):  
North Dakota ◽  
Potato Virus ◽  
Potato Virus Y ◽  
River Valley ◽  
Red River ◽  
Seasonal Abundance ◽  
Red River Valley ◽  
Aphid Vectors

scholarly journals Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y.

1997 ◽  
Vol 78 (12) ◽  
pp. 3141-3145 ◽  
Author(s):  
G Jakab ◽  
E Droz ◽  
P Malno√´ ◽  
G Brigneti ◽  
D Baulcombe
Keyword(s):  
Potato Virus ◽  
Cdna Clone ◽  
Potato Virus Y ◽  
Full Length ◽  
Full Length Cdna ◽  
In Vitro Transcripts

SOME EFFECTS OF FORMALDEHYDE ON POTATO VIRUS Y IN VITRO, AND ABILITY OF APHIDS TO TRANSMIT THE VIRUS WHEN THEIR STYLETS ARE TREATED WITH FORMALDEHYDE

1955 ◽  
Vol 1 (9) ◽  
pp. 783-793 ◽  
Author(s):  
R. H. E. Bradley ◽  
R. Y. Ganong
Keyword(s):  
Potato Virus ◽  
Myzus Persicae ◽  
Potato Virus Y ◽  
Tobacco Plants ◽  
Considerable Distance ◽  
Formaldehyde Treatment ◽  
Aphid Vectors ◽  
Infected Tobacco

Potato virus Y (PVY) was made noninfective by incubation with formaldehyde in vitro. Yet this noninfective virus reacted with PVY antiserum and caused antibodies to be produced in rabbits as readily as infective PVY. A method is described for baring the stylets of living aphids beyond the end of the labium, which normally encloses the stylets. Specimens of Myzus persicae (Sulz.) infective with PVY were made noninfective by treating the stylets for 30 sec. with concentrations of formaldehyde as low as 0.03%; and 0.25% formaldehyde caused the same effect in five seconds. Aphids were also made noninfective when the proboscis with the tip of the stylets bared was treated with formaldehyde, even after the stylets had been inserted a considerable distance into infected tobacco plants. By contrast, aphids usually remained infective when the proboscis with the stylets enclosed in the labium was treated with similar concentrations of formaldehyde. However, formaldehyde treatment of the stylets did not affect the ability of aphids immediately thereafter to acquire and transmit PVY. These results are compatible with the hypothesis that viruses transmitted like PVY are carried by the stylets of their aphid vectors.

scholarly journals Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in C. elegans

2018 ◽  
Author(s):  
Gregoriy A. Dokshin ◽  
Krishna S. Ghanta ◽  
Katherine M. Piscopo ◽  
Craig C. Mello
Keyword(s):  
Green Fluorescent Protein ◽  
Cost Effectiveness ◽  
Genome Editing ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
C Elegans ◽  
Multiple Loci ◽  
Green Fluorescent ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCRISPR-based genome editing using ribonucleoprotein (RNP) complexes and synthetic single stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs – such as green fluorescent protein (GFP) tags remains challenging in many systems. Here we describe a streamlined and optimized editing protocol for the nematode C. elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging all twelve of the Worm-specific Argonaute (WAGO) proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based partially single-stranded “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.

scholarly journals Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

2017 ◽  
Author(s):  
Stephen D. Carter ◽  
Shrawan K. Mageswaran ◽  
Zachary J. Farino ◽  
João I. Mamede ◽  
Catherine M. Oikonomou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Organic Dyes ◽  
Fluorescent Proteins ◽  
Secretory Granules ◽  
Light And Electron Microscopy ◽  
Low Contrast ◽  
Fluorescent Protein Tags ◽  
Protein Tags

AbstractCryogenic correlated light and electron microscopy (cryo-CLEM) is a valuable tool for studying biological processes in situ. In cryo-CLEM, a target protein of interest is tagged with a fluorophore and the location of the corresponding fluorescent signal is used to identify the structure in low-contrast but feature-rich cryo-EM images. To date, cryo-CLEM studies of mammalian cells have relied on very bright organic dyes or fluorescent protein tags concentrated in virus particles. Here we describe a method to expand the application of cryo-CLEM to cells harboring genetically-encoded fluorescent proteins. We discovered that a variety of mammalian cells exhibit strong punctate autofluorescence when imaged under cryogenic conditions (80K). Compared to fluorescent protein tags, these sources of autofluorescence exhibit a broader spectrum of fluorescence, which we exploited to develop a simple, robust approach to discriminate between the two. We validate this method in INS-1 E cells using a mitochondrial marker, and apply it to study the ultrastructural variability of secretory granules in a near-native state within intact INS-1E pancreatic cells by high-resolution 3D electron cryotomography.

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