scholarly journals An Improved Transformation System for Phytophthora cinnamomi Using Green Fluorescent Protein

2021 ◽  
Vol 12 ◽  
Author(s):  
Tingting Dai ◽  
Yue Xu ◽  
Xiao Yang ◽  
Binbin Jiao ◽  
Min Qiu ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Phytophthora Cinnamomi ◽  
Transformation Method ◽  
Transformation System ◽  
Wild Type ◽  
Green Fluorescence ◽  
Type Isolate ◽  
Green Fluorescent ◽  
Transformation Systems

Phytophthora cinnamomi is a destructive pathogen causing root rot and dieback diseases on hundreds of economically and ecologically important plant species. Effective transformation systems enable modifications of candidate genes to understand the pathogenesis of P. cinnamomi. A previous study reported a polyethylene glycol and calcium dichloride (PEG/CaCl2)-mediated protoplast transformation method of P. cinnamomi. However, the virulence of the transformants was compromised. In this study, we selected ATCC 15400 as a suitable wild-type isolate for PEG/CaCl2 transformation using the green fluorescent protein after screening 11 P. cinnamomi isolates. Three transformants, namely, PcGFP-1, PcGFP-3, and PcGFP-5, consistently displayed a green fluorescence in their hyphae, chlamydospores, and sporangia. The randomly selected transformant PcGFP-1 was as virulent as the wild-type isolate in causing hypocotyl lesions on lupines. Fluorescent hyphae and haustoria were observed intracellularly and intercellularly in lupine tissues inoculated with PcGFP-1 zoospores. The potential application of this improved transformation system for functional genomics studies of P. cinnamomi is discussed.

scholarly journals In Vivo Tracking of Campylobacter jejuni by Using a Novel Recombinant Expressing Green Fluorescent Protein

2003 ◽  
Vol 69 (5) ◽  
pp. 2864-2874 ◽  
Author(s):  
Philip F. Mixter ◽  
John D. Klena ◽  
Gary A. Flom ◽  
Amy M. Siegesmund ◽  
Michael E. Konkel
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Campylobacter Jejuni ◽  
Fluorescent Protein ◽  
Developed Countries ◽  
Wild Type ◽  
Reporter System ◽  
Type Isolate ◽  
Green Fluorescent

ABSTRACT Campylobacter jejuni is a leading cause of food-borne disease in developed countries. The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study of C. jejuni in a variety of niches. C. jejuni transformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry. Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally with C. jejuni harboring the gfp-containing vector. Four hours after injection of the mice, flow cytometry analyses determined that C. jejuni synthesizing GFP were predominantly associated with granulocytes. More specifically, the proportion of CD11b+ Gr-1+ lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b+ Gr-1− lavage macrophages ranged from 77.0 to 80.0%. In contrast, few CD11b− CD45R+ B lymphocytes from the lavage of the C. jejuni-injected mice were associated with green-fluorescent C. jejuni (proportions ranged from 0.75 to 0.77%). Cell-free C. jejuni was recovered from tissue homogenates after intraperitoneal injection. Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of the C. jejuni F38011 wild-type isolate. In vivo this variant displayed a phenotype identical to that of the wild-type isolate. In summary, we demonstrate that C. jejuni associates with marker-defined cellular subsets in vivo with a novel gfp reporter system and that C. jejuni genotypic variants can be isolated from both in vitro and in vivo systems.

scholarly journals Recombinant Lassa Virus Expressing Green Fluorescent Protein as a Tool for High-Throughput Drug Screens and Neutralizing Antibody Assays

Viruses ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 655 ◽  
Author(s):  
Yíngyún Caì ◽  
Masaharu Iwasaki ◽  
Brett Beitzel ◽  
Shuīqìng Yú ◽  
Elena Postnikova ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Neutralizing Antibody ◽  
Quantitative Detection ◽  
Lassa Virus ◽  
Wild Type ◽  
Green Fluorescent

Lassa virus (LASV), a mammarenavirus, infects an estimated 100,000–300,000 individuals yearly in western Africa and frequently causes lethal disease. Currently, no LASV-specific antivirals or vaccines are commercially available for prevention or treatment of Lassa fever, the disease caused by LASV. The development of medical countermeasure screening platforms is a crucial step to yield licensable products. Using reverse genetics, we generated a recombinant wild-type LASV (rLASV-WT) and a modified version thereof encoding a cleavable green fluorescent protein (GFP) as a reporter for rapid and quantitative detection of infection (rLASV-GFP). Both rLASV-WT and wild-type LASV exhibited similar growth kinetics in cultured cells, whereas growth of rLASV-GFP was slightly impaired. GFP reporter expression by rLASV-GFP remained stable over several serial passages in Vero cells. Using two well-characterized broad-spectrum antivirals known to inhibit LASV infection, favipiravir and ribavirin, we demonstrate that rLASV-GFP is a suitable screening tool for the identification of LASV infection inhibitors. Building on these findings, we established a rLASV-GFP-based high-throughput drug discovery screen and an rLASV-GFP-based antibody neutralization assay. Both platforms, now available as a standard tool at the IRF-Frederick (an international resource), will accelerate anti-LASV medical countermeasure discovery and reduce costs of antiviral screens in maximum containment laboratories.

Limitations on the use of fused green fluorescent protein to investigate structure–function relationships for the cauliflower mosaic virus movement protein

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1851-1855 ◽  
Author(s):  
Carole L. Thomas ◽  
Andrew J. Maule
Keyword(s):  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Cauliflower Mosaic Virus ◽  
Virus Movement ◽  
Wild Type ◽  
Tubule Formation ◽  
Mouth Disease Virus ◽  
Green Fluorescent

To investigate the process of tubule formation for the cauliflower mosaic virus movement protein (CaMV MP), the green fluorescent protein (GFP) was fused to the MP to provide a vital marker for MP location after expression in insect cells. In contrast to the long tubular structures seen previously following baculovirus-based expression of the wild-type MP, the fusion protein produced only aggregates of fluorescing material in the cytoplasm. However, by co-expressing wild-type MP and GFP–MP, or by engineering their co-accumulation by introducing a foot-and-mouth disease virus 2A cleavage sequence between GFP and MP, long GFP-fluorescing tubules were formed. The experiments suggest that the presence of GFP at the N or C terminus of the tubule-forming domain of the CaMV MP places steric constraints upon the aggregation of the MP into a tubule but that this can be overcome by providing wild-type protein for inclusion in the aggregate.

scholarly journals A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants

2003 ◽  
Vol 373 (2) ◽  
pp. 403-408 ◽  
Author(s):  
Nadya G. GURSKAYA ◽  
Arkady F. FRADKOV ◽  
Natalia I. POUNKOVA ◽  
Dmitry B. STAROVEROV ◽  
Maria E. BULINA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Random Mutagenesis ◽  
Wild Type ◽  
Green Luminescence ◽  
Mammalian Cell Lines ◽  
Biological Tool ◽  
Fluorescent Pattern ◽  
Green Fluorescent

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu222→Gly (E222G) substitution, which removed the evolutionarily invariant Glu222. Re-introduction of Glu222 into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of ‘humanized’ aceGFP and β-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.

scholarly journals Analysis of Localization of Mutated Tissue-Nonspecific Alkaline Phosphatase Proteins Associated with Neonatal Hypophosphatasia Using Green Fluorescent Protein Chimeras1

1998 ◽  
Vol 83 (11) ◽  
pp. 3936-3942
Author(s):  
Guiming Cai ◽  
Toshimi Michigami ◽  
Takehisa Yamamoto ◽  
Natsuo Yasui ◽  
Kenichi Satomura ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Enzymatic Activity ◽  
Fluorescent Protein ◽  
Clinical Severity ◽  
Wild Type ◽  
Neonatal Patient ◽  
Tissue Nonspecific Alkaline Phosphatase ◽  
Green Fluorescent

Hypophosphatasia is associated with a defect of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The onset and clinical severity are usually correlated in hypophosphatasia; patients with perinatal hypophosphatasia die approximately at the time of birth. In contrast, we describe a male neonatal patient with hypophosphatasia who had no respiratory problems and survived. He was compound heterozygous for the conversion of Phe to Leu at codon 310 (F310L) and the deletion of a nucleotide T at 1735 (delT1735), causing the frame shift with the result of the addition of 80 amino acids at the C-terminal of the protein. Because the C-terminal portion of TNSALP is known to be important for TNSALP to bind to the plasma membrane, the localization of wild-type and mutated TNSALP proteins was analyzed using green fluorescent protein chimeras. The expression vectors containing the complementary DNA of fusion proteins consisting of signal peptide, green fluorescent protein, and wild-type or mutated TNSALP, caused by delT1735 or F310L mutation, were introduced transiently or stably in Saos-2 cells. The delT1735 mutant failed to localize at the cell surface membrane, whereas the wild-type and the F310L mutants were located in the plasma membrane and cytoplasm. The assay for enzymatic activity of TNSALP revealed that the delT1735 mutant lost the activity and that the F310L mutant exhibited an enzymatic activity level that was 72% of the normal level. The F310L mutation was also detected in another neonatal patient with relatively mild (nonlethal) hypophosphatasia (reported in J Clin Endocrinol Metab, 81:4458–4461, 1996), suggesting that residual ALP activity of the F310L mutant contributes to the less severe phenotype. The patient is unique, with respect to a discrepancy between onset and clinical severity in hypophosphatasia.

scholarly journals A fine structure study of the anthocodium in Renilla mülleri. Evidence for the existence of a bioluminescent organelle, the luminelle.

1975 ◽  
Vol 64 (1) ◽  
pp. 15-28 ◽  
Author(s):  
B O Spurlock ◽  
M J Cormier
Keyword(s):  
Fine Structure ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Structure Study ◽  
Green Fluorescence ◽  
Subcellular Structure ◽  
Subcellular Organelle ◽  
Green Fluorescent ◽  
Structural Entity

A fine structure study of the anthocodium of the sea pansy, Renilla mülleri, was undertaken. The anthocodium, a known site of bioluminescence, was selected in order to determine whether a structural entity could be found which would satisfy the biochemical and physiological features associated with the known sites of bioluminescence in this animal. These sites, termed lumisomes, have previously been shown to be small (0.1-0.2 mum), membrane-enclosed vesicles which contain all the proteins necessary for bioluminescence and its immediate control. One of the lumisomal proteins is an intensely green fluorescent protein and has been used as a probe for the detection of the cellular sites of bioluminescence. This green fluorescence was associated only with gastrodermal cells. We report the identification of a unique morphological entity, restricted to the cells of the gastrodermis, which satisfies the biochemical and physiological requirements for bioluminescence in Renilla. It is a large (4-6 mum), membrane-bounded subcellular organelle comparable in size to a subcellular structure whose green fluorescence is typically associated with the in vivo bioluminescence. Furthermore, it is filled with smaller membrane-bounded vesicles which have the same size and shape as the lumisomes. We suggest that the organelle identified in this study be termed a luminelle.

scholarly journals SLAM (CD150)-Independent Measles Virus Entry as Revealed by Recombinant Virus Expressing Green Fluorescent Protein

2002 ◽  
Vol 76 (13) ◽  
pp. 6743-6749 ◽  
Author(s):  
Koji Hashimoto ◽  
Nobuyuki Ono ◽  
Hironobu Tatsuo ◽  
Hiroko Minagawa ◽  
Makoto Takeda ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
Infected Individual ◽  
Lymphocyte Activation ◽  
Wild Type ◽  
Low Efficiency ◽  
Histopathological Studies ◽  
Green Fluorescent

ABSTRACT Wild-type measles virus (MV) strains use human signaling lymphocyte activation molecule (SLAM) as a cellular receptor, while vaccine strains such as the Edmonston strain can use both SLAM and CD46 as receptors. Although the expression of SLAM is restricted to cells of the immune system (lymphocytes, dendritic cells, and monocytes), histopathological studies with humans and experimentally infected monkeys have shown that MV also infects SLAM-negative cells, including epithelial, endothelial, and neuronal cells. In an attempt to explain these findings, we produced the enhanced green fluorescent protein (EGFP)-expressing recombinant MV (IC323-EGFP) based on the wild-type IC-B strain. IC323-EGFP showed almost the same growth kinetics as the parental recombinant MV and produced large syncytia exhibiting green autofluorescence in SLAM-positive cells. Interestingly, all SLAM-negative cell lines examined also showed green autofluorescence after infection with IC323-EGFP, although the virus hardly spread from the originally infected individual cells and thus did not induce syncytia. When the number of EGFP-expressing cells after infection was taken as an indicator, the infectivities of IC323-EGFP for SLAM-negative cells were 2 to 3 logs lower than those for SLAM-positive cells. Anti-MV hemagglutinin antibody or fusion block peptide, but not anti-CD46 antibody, blocked IC323-EGFP infection of SLAM-negative cells. This infection occurred under conditions in which entry via endocytosis was inhibited. These results indicate that MV can infect a variety of cells, albeit with a low efficiency, by using an as yet unidentified receptor(s) other than SLAM or CD46, in part explaining the observed MV infection of SLAM-negative cells in vivo.

The effect of pressure on the excited-state proton transfer in the wild-type green fluorescent protein

2008 ◽  
Vol 455 (4-6) ◽  
pp. 303-306 ◽  
Author(s):  
Pavel Leiderman ◽  
Dan Huppert ◽  
S. James Remington ◽  
Laren M. Tolbert ◽  
Kyril M. Solntsev
Keyword(s):  
Excited State ◽  
Green Fluorescent Protein ◽  
Proton Transfer ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Effect Of Pressure ◽  
Excited State Proton Transfer ◽  
In The Wild ◽  
Green Fluorescent
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