scholarly journals GFP transformation sheds more light on a widespread mycoparasitic interaction

2019 ◽  
Author(s):  
Márk Z. Németh ◽  
Alexandra Pintye ◽  
Áron N. Horváth ◽  
Pál Vági ◽  
Gábor M. Kovács ◽  
...  
Keyword(s):  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Environmental Fate ◽  
Molecular Genetic ◽  
Powdery Mildews ◽  
Before And After ◽  
Mycoparasitic Fungi ◽  
Green Fluorescent ◽  
First Time ◽  
Plant Surfaces

ABSTRACTPowdery mildews (PMs), ubiquitous obligate biotrophic plant pathogens, are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. Some Ampelomyces strains are commercialized biocontrol agents of crop pathogenic PMs. Using Agrobacterium tumefaciens-mediated transformation (ATMT), we produced stable Ampelomyces transformants that constitutively expressed the green fluorescent protein (GFP), to (i) improve the visualization of the PM-Ampelomyces interaction; and (ii) decipher the environmental fate of Ampelomyces before and after acting as a mycoparasite. Detection of Ampelomyces structures, and especially hyphae, was greatly enhanced when diverse PM, leaf and soil samples containing GFP transformants were examined with fluorescence microscopy compared to brightfield and DIC optics. We showed for the first time that Ampelomyces can persist up to 21 days on PM-free host plant surfaces, where it can attack PM structures as soon as these appear after this period. As a saprobe in decomposing, PM-infected leaves on the ground, and also in autoclaved soil, Ampelomyces developed new hyphae, but did not sporulate. These results indicate that Ampelomyces occupies a niche in the phyllosphere where it acts primarily as a mycoparasite of PMs. Our work has established a framework for a molecular genetic toolbox for Ampelomyces using ATMT.

scholarly journals Green Fluorescent Protein Transformation Sheds More Light on a Widespread Mycoparasitic Interaction

2019 ◽  
Vol 109 (8) ◽  
pp. 1404-1416
Author(s):  
Márk Z. Németh ◽  
Alexandra Pintye ◽  
Áron N. Horváth ◽  
Pál Vági ◽  
Gábor M. Kovács ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Powdery Mildew ◽  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Environmental Fate ◽  
Molecular Genetic ◽  
Powdery Mildews ◽  
Before And After ◽  
Mycoparasitic Fungi ◽  
Green Fluorescent

Powdery mildews, ubiquitous obligate biotrophic plant pathogens, are often attacked in the field by mycoparasitic fungi belonging to the genus Ampelomyces. Some Ampelomyces strains are commercialized biocontrol agents of crop pathogenic powdery mildews. Using Agrobacterium tumefaciens-mediated transformation (ATMT), we produced stable Ampelomyces transformants that constitutively expressed green fluorescent protein (GFP) to (i) improve the visualization of the mildew–Ampelomyces interaction and (ii) decipher the environmental fate of Ampelomyces fungi before and after acting as a mycoparasite. Detection of Ampelomyces structures, and especially hyphae, was greatly enhanced when diverse powdery mildew, leaf, and soil samples containing GFP transformants were examined with fluorescence microscopy compared with brightfield and differential interference contrast optics. We showed for the first time, to our knowledge, that Ampelomyces strains can persist up to 21 days on mildew-free host plant surfaces, where they can attack powdery mildew structures as soon as these appear after this period. As saprobes in decomposing, powdery mildew-infected leaves on the ground and also in autoclaved soil, Ampelomyces strains developed new hyphae but did not sporulate. These results indicate that Ampelomyces strains occupy a niche in the phyllosphere where they act primarily as mycoparasites of powdery mildews. Our work has established a framework for a molecular genetic toolbox for the genus Ampelomyces using ATMT.

scholarly journals Changes in the Min Oscillation Pattern before and after Cell Birth

2010 ◽  
Vol 192 (16) ◽  
pp. 4134-4142 ◽  
Author(s):  
Jennifer R. Juarez ◽  
William Margolin
Keyword(s):  
Cell Division ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
The Other ◽  
Cell Pole ◽  
Daughter Cells ◽  
Division Site ◽  
Before And After ◽  
Dividing Cells ◽  
Green Fluorescent

ABSTRACT The Min system regulates the positioning of the cell division site in many bacteria. In Escherichia coli, MinD migrates rapidly from one cell pole to the other. In conjunction with MinC, MinD helps to prevent unwanted FtsZ rings from assembling at the poles and to stabilize their positioning at midcell. Using time-lapse microscopy of growing and dividing cells expressing a gfp-minD fusion, we show that green fluorescent protein (GFP)-MinD often paused at midcell in addition to at the poles, and the frequency of midcell pausing increased as cells grew longer and cell division approached. At later stages of septum formation, GFP-MinD often paused specifically on only one side of the septum, followed by migration to the other side of the septum or to a cell pole. About the time of septum closure, this irregular pattern often switched to a transient double pole-to-pole oscillation in the daughter cells, which ultimately became a stable double oscillation. The splitting of a single MinD zone into two depends on the developing septum and is a potential mechanism to explain how MinD is distributed equitably to both daughter cells. Septal pausing of GFP-MinD did not require MinC, suggesting that MinC-FtsZ interactions do not drive MinD-septal interactions, and instead MinD recognizes a specific geometric, lipid, and/or protein target at the developing septum. Finally, we observed regular end-to-end oscillation over very short distances along the long axes of minicells, supporting the importance of geometry in MinD localization.

scholarly journals Generation of iPSCs from Jaw Periosteal Cells Using Self-Replicating RNA

2019 ◽  
Vol 20 (7) ◽  
pp. 1648 ◽  
Author(s):  
Felix Umrath ◽  
Heidrun Steinle ◽  
Marbod Weber ◽  
Hans-Peter Wendel ◽  
Siegmar Reinert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Osteogenic Potential ◽  
Safety Standards ◽  
Stem Cell Source ◽  
Periosteal Cells ◽  
Induced Pluripotent ◽  
Green Fluorescent ◽  
First Time

Jaw periosteal cells (JPCs) represent a suitable stem cell source for bone tissue engineering (BTE) applications. However, challenges associated with limited cell numbers, stressful cell sorting, or the occurrence of cell senescence during in vitro passaging and the associated insufficient osteogenic potential in vitro of JPCs and other mesenchymal stem/stromal cells (MSCs) are main hurdles and still need to be solved. In this study, for the first time, induced pluripotent stem cells (iPSCs) were generated from human JPCs to open up a new source of stem cells for BTE. For this purpose, a non-integrating self-replicating RNA (srRNA) encoding reprogramming factors and green fluorescent protein (GFP) as a reporter was used to obtain JPC-iPSCs with a feeder- and xeno-free reprogramming protocol to meet the highest safety standards for future clinical applications. Furthermore, to analyze the potential of these iPSCs as a source of osteogenic progenitor cells, JPC-iPSCs were differentiated into iPSC-derived mesenchymal stem/stromal like cells (iMSCs) and further differentiated to the osteogenic lineage under xeno-free conditions. The produced iMSCs displayed MSC marker expression and morphology as well as strong mineralization during osteogenic differentiation.

scholarly journals Nonhost Resistance in Arabidopsis-Colletotrichum Interactions Acts at the Cell Periphery and Requires Actin Filament Function

2006 ◽  
Vol 19 (3) ◽  
pp. 270-279 ◽  
Author(s):  
Chiyumi Shimada ◽  
Volker Lipka ◽  
Richard O'Connell ◽  
Tetsuro Okuno ◽  
Paul Schulze-Lefert ◽  
...  
Keyword(s):  
Actin Filament ◽  
Fungal Pathogens ◽  
Plant Pathogens ◽  
Fluorescent Protein ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Nonhost Resistance ◽  
Cell Periphery ◽  
Colletotrichum Species ◽  
Fungal Plant Pathogens

Pathogenesis of nonadapted fungal pathogens is often terminated coincident with their attempted penetration into epidermal cells of nonhost plants. The genus Colletotrichum represents an economically important group of fungal plant pathogens that are amenable to molecular genetic analysis. Here, we investigated interactions between Arabidopsis and Colletotrichum to gain insights in plant and pathogen processes activating nonhost resistance responses. Three tested nonadapted Colletotrichum species differentiated melanized appressoria on Arabidopsis leaves but failed to form intracellular hyphae. Plant cells responded to Colletotrichum invasion attempts by the formation of PMR4/GSL5-dependent papillary callose. Appressorium differentiation and melanization were insufficient to trigger this localized plant cell response, but analysis of nonpathogenic C. lagenarium mutants implicates penetration-peg formation as the inductive cue. We show that Arabidopsis PEN1 syntaxin controls timely accumulation of papillary callose but is functionally dispensable for effective preinvasion (penetration) resistance in nonhost interactions. Consistent with this observation, green fluorescent protein-tagged PEN1 did not accumulate at sites of attempted penetration by either adapted or nonadapted Colletotrichum species, in contrast to the pronounced focal accumulations of PEN1 associated with entry of powdery mildews. We observed extensive reorganization of actin microfilaments leading to polar orientation of large actin bundles towards appressorial contact sites in interactions with the nonadapted Colletotrichum species. Pharmacological inhibition of actin filament function indicates a functional contribution of the actin cytoskeleton for both preinvasion resistance and papillary callose formation. Interestingly, the incidence of papilla formation at entry sites was greatly reduced in interactions with C. higginsianum isolates, indicating that this adapted pathogen may suppress preinvasion resistance at the cell periphery.

scholarly journals Switching from a Unicellular to Multicellular Organization in an Aspergillus niger Hypha

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Robert-Jan Bleichrodt ◽  
Marc Hulsman ◽  
Han A. B. Wösten ◽  
Marcel J. T. Reinders
Keyword(s):  
Aspergillus Niger ◽  
Fluorescent Protein ◽  
Stress Conditions ◽  
Physical Contact ◽  
Effective Control ◽  
Mobility Rate ◽  
Content Type ◽  
Green Fluorescent ◽  
First Time ◽  
Compound Concentration

ABSTRACT Pores in fungal septa enable cytoplasmic streaming between hyphae and their compartments. Consequently, the mycelium can be considered unicellular. However, we show here that Woronin bodies close ~50% of the three most apical septa of growing hyphae of Aspergillus niger. The incidence of closure of the 9th and 10th septa was even ≥94%. Intercompartmental streaming of photoactivatable green fluorescent protein (PA-GFP) was not observed when the septa were closed, but open septa acted as a barrier, reducing the mobility rate of PA-GFP ~500 times. This mobility rate decreased with increasing septal age and under stress conditions, likely reflecting a regulatory mechanism affecting septal pore diameter. Modeling revealed that such regulation offers effective control of compound concentration between compartments. Modeling also showed that the incidence of septal closure in A. niger had an even stronger impact on cytoplasmic continuity. Cytoplasm of hyphal compartments was shown not to be in physical contact when separated by more than 4 septa. Together, data show that apical compartments of growing hyphae behave unicellularly, while older compartments have a multicellular organization. IMPORTANCE The hyphae of higher fungi are compartmentalized by porous septa that enable cytosolic streaming. Therefore, it is believed that the mycelium shares cytoplasm. However, it is shown here that the septa of Aspergillus niger are always closed in the oldest part of the hyphae, and therefore, these compartments are physically isolated from each other. In contrast, only part of the septa is closed in the youngest part of the hyphae. Still, compartments in this hyphal part are physically isolated when separated by more than 4 septa. Even open septa act as a barrier for cytoplasmic mixing. The mobility rate through such septa reduces with increasing septal age and under stress conditions. Modeling shows that the septal pore width is set such that its regulation offers maximal control of compound concentration levels within the compartments. Together, we show for the first time that Aspergillus hyphae switch from a unicellular to multicellular organization.

scholarly journals Actin Filaments and Myosin I Alpha Cooperate with Microtubules for the Movement of Lysosomes

2001 ◽  
Vol 12 (12) ◽  
pp. 4013-4029 ◽  
Author(s):  
Marie-Neige Cordonnier ◽  
Daniel Dauzonne ◽  
Daniel Louvard ◽  
Evelyne Coudrier
Keyword(s):  
Long Range ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Myosin I ◽  
Latrunculin A ◽  
Living Mouse ◽  
Green Fluorescent ◽  
First Time

An earlier report suggested that actin and myosin I alpha (MMIα), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIα were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIα. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIα impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIα contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors.

scholarly journals Ecological Basis of the Interaction betweenPseudozyma flocculosaand Powdery Mildew Fungi

2010 ◽  
Vol 77 (3) ◽  
pp. 926-933 ◽  
Author(s):  
Walid Hammami ◽  
Candy Quiroga Castro ◽  
Wilfried Rémus-Borel ◽  
Caroline Labbé ◽  
Richard R. Bélanger
Keyword(s):  
Powdery Mildew ◽  
Fluorescent Protein ◽  
Close Relative ◽  
Tomato Plants ◽  
Powdery Mildews ◽  
Biocontrol Activity ◽  
Cellobiose Lipids ◽  
Powdery Mildew Fungi ◽  
Green Fluorescent ◽  
Ecological Basis

ABSTRACTIn this work, we sought to understand how glycolipid production and the availability of nutrients could explain the ecology ofPseudozyma flocculosaand its biocontrol activity. For this purpose, we compared the development ofP. flocculosato that of a close relative, the plant pathogenUstilago maydis, under different environmental conditions. This approach was further supported by measuring the expression ofcyp1, a pivotal gene in the synthesis of unique antifungal cellobiose lipids of both fungi. On healthy cucumber and tomato plants, the expression ofcyp1remained unchanged over time inP. flocculosaand was undetected inU. maydis. At the same time, green fluorescent protein (GFP) strains of both fungi showed only limited green fluorescence on control leaves. On powdery mildew-infected cucumber leaves,P. flocculosainduced a complete collapse of the pathogen colonies, but glycolipid production, as studied bycyp1expression, was still comparable to that of controls. In complete contrast,cyp1was upregulated nine times whenP. flocculosawas applied toBotrytis cinerea-infected leaves, but the biocontrol fungus did not develop very well on the pathogen. Analysis of the possible nutrients that could stimulate the growth ofP. flocculosaon powdery mildew structures revealed that the complex Zn/Mn played a key role in the interaction. Other related fungi such asU. maydisdo not appear to have the same nutritional requirements and hence lack the ability to colonize powdery mildews. Whether production of antifungal glycolipids contributes to the release of nutrients from powdery mildew colonies is unclear, but the specificity of the biocontrol activity ofP. flocculosatoward Erysiphales does appear to be more complex than simple antibiosis.

scholarly journals The Dynamics of Postmitotic Reassembly of the Nucleolus

2000 ◽  
Vol 150 (3) ◽  
pp. 433-446 ◽  
Author(s):  
Miroslav Dundr ◽  
Tom Misteli ◽  
Mark O.J. Olson
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Fluorescent Protein ◽  
Living Cells ◽  
M Phase ◽  
Specific Proteins ◽  
Distribution Of Components ◽  
Green Fluorescent ◽  
Related Proteins ◽  
First Time ◽  
Rrna Sequences

Mammalian cell nucleoli disassemble at the onset of M-phase and reassemble during telophase. Recent studies showed that partially processed preribosomal RNA (pre-rRNA) is preserved in association with processing components in the perichromosomal regions (PRs) and in particles called nucleolus-derived foci (NDF) during mitosis. Here, the dynamics of nucleolar reassembly were examined for the first time in living cells expressing fusions of the processing-related proteins fibrillarin, nucleolin, or B23 with green fluorescent protein (GFP). During telophase the NDF disappeared with a concomitant appearance of material in the reforming nuclei. Prenucleolar bodies (PNBs) appeared in nuclei in early telophase and gradually disappeared as nucleoli formed, strongly suggesting the transfer of PNB components to newly forming nucleoli. Fluorescence recovery after photobleaching (FRAP) showed that fibrillarin-GFP reassociates with the NDF and PNBs at rapid and similar rates. The reentry of processing complexes into telophase nuclei is suggested by the presence of pre-rRNA sequences in PNBs. Entry of specific proteins into the nucleolus approximately correlated with the timing of processing events. The mitotically preserved processing complexes may be essential for regulating the distribution of components to reassembling daughter cell nucleoli.

scholarly journals Monitoring disulfide bond formation in the eukaryotic cytosol

2004 ◽  
Vol 166 (3) ◽  
pp. 337-345 ◽  
Author(s):  
Henrik Østergaard ◽  
Christine Tachibana ◽  
Jakob R. Winther
Keyword(s):  
Fluorescent Protein ◽  
Redox Status ◽  
Reduced And Oxidized Glutathione ◽  
Cytosolic Glutathione ◽  
Green Fluorescent ◽  
Genetically Manipulated ◽  
First Time ◽  
Glutathione Redox

Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein–based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be −289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.

scholarly journals Functional Characterization of Candida albicans ABC Transporter Cdr1p

2003 ◽  
Vol 2 (6) ◽  
pp. 1361-1375 ◽  
Author(s):  
Suneet Shukla ◽  
Preeti Saini ◽  
Smriti ◽  
Sudhakar Jha ◽  
Suresh V. Ambudkar ◽  
...  
Keyword(s):  
Binding Sites ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Functional Characterization ◽  
Drug Binding ◽  
Transmembrane Segment ◽  
Green Fluorescent ◽  
Substrate Binding Sites ◽  
First Time

ABSTRACT In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and ΔF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (ΔF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ΔF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).

Sign in / Sign up

Export Citation Format

Share Document