scholarly journals The Molecular Biology of the Interactions Between Trichoderma spp., Phytopathogenic Fungi, and Plants

2006 ◽  
Vol 96 (2) ◽  
pp. 181-185 ◽  
Author(s):  
S. L. Woo ◽  
F. Scala ◽  
M. Ruocco ◽  
M. Lorito
Keyword(s):  
Fluorescent Protein ◽  
Phytopathogenic Fungi ◽  
Microbial Pathogens ◽  
Heterologous Proteins ◽  
Trichoderma Spp ◽  
Resistance Proteins ◽  
Functional Studies ◽  
Green Fluorescent

Trichoderma-based biofungicides are a reality in agriculture, with more than 50 formulations today available as registered products worldwide. Several strategies have been applied to identify the main genes and compounds involved in this complex, three-way cross-talk between the fungal antagonist, the plant, and microbial pathogens. Proteome and genome analysis have greatly enhanced our ability to conduct holistic and genome-based functional studies. We have identified and determined the role of a variety of novel genes and gene-products, including ABC transporters, enzymes and other proteins that produce or act as novel elicitors of induced resistance, proteins responsible for a gene-for-gene avirulent interaction between Trichoderma spp. and plants, mycoparasitism-related inducers, plant proteins specifically induced by Trichoderma, etc. We have transgenically demonstrated the ability of Trichoderma spp. to transfer heterologous proteins into plant during root colonization, and have used green fluorescent protein and other markers to study the interaction in vivo and in situ between Trichoderma spp. and the fungal pathogen or the plant.

scholarly journals Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

1998 ◽  
Vol 18 (11) ◽  
pp. 6805-6815 ◽  
Author(s):  
Jens Solsbacher ◽  
Patrick Maurer ◽  
F. Ralf Bischoff ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Wild Type ◽  
Importin Α ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

scholarly journals MukEF Is Required for Stable Association of MukB with the Chromosome

2007 ◽  
Vol 189 (19) ◽  
pp. 7062-7068 ◽  
Author(s):  
Weifeng She ◽  
Qinhong Wang ◽  
Elena A. Mordukhova ◽  
Valentin V. Rybenkov
Keyword(s):  
Fluorescent Protein ◽  
Cell Length ◽  
Stable Complex ◽  
Macromolecular Assembly ◽  
Stable Association ◽  
Structural Maintenance Of Chromosome ◽  
Green Fluorescent

ABSTRACT MukB is a bacterial SMC(structural maintenance of chromosome) protein required for correct folding of the Escherichia coli chromosome. MukB acts in complex with the two non-SMC proteins, MukE and MukF. The role of MukEF is unclear. MukEF disrupts MukB-DNA interactions in vitro. In vivo, however, MukEF stimulates MukB-induced DNA condensation and is required for the assembly of MukB clusters at the quarter positions of the cell length. We report here that MukEF is essential for stable association of MukB with the chromosome. We found that MukBEF forms a stable complex with the chromosome that copurifies with nucleoids following gentle cell lysis. Little MukB could be found with the nucleoids in the absence or upon overproduction of MukEF. Similarly, overproduced MukEF recruited MukB-green fluorescent protein (GFP) from its quarter positions, indicating that formation of MukB-GFP clusters and stable association with the chromosome could be mechanistically related. Finally, we report that MukE-GFP forms foci at the quarter positions of the cell length but not in cells that lack MukB or overproduce MukEF, suggesting that the clusters are formed by MukBEF and not by its individual subunits. These data support the view that MukBEF acts as a macromolecular assembly, a scaffold, in chromosome organization and that MukEF is essential for the assembly of this scaffold.

scholarly journals Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p.

1996 ◽  
Vol 7 (6) ◽  
pp. 917-934 ◽  
Author(s):  
A L Goldstein ◽  
C A Snay ◽  
C V Heath ◽  
C N Cole
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Main Body ◽  
The Novel ◽  
Conditional Allele ◽  
Green Fluorescent ◽  
Nucleocytoplasmic Export ◽  
In Cells

In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC). The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein. In cells containing either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A)+ RNA accumulates rapidly in nuclei after a shift from 23 degrees C to 37 degrees C. Under these same conditions, rapid fragmentation of the nucleolus was also observed. At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE. NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact. The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nup120p. In contrast,Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes. The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.

scholarly journals The Yeast Synaptojanin-like Proteins Control the Cellular Distribution of Phosphatidylinositol (4,5)-Bisphosphate

2002 ◽  
Vol 13 (2) ◽  
pp. 542-557 ◽  
Author(s):  
Christopher J. Stefan ◽  
Anjon Audhya ◽  
Scott D. Emr
Keyword(s):  
Fluorescent Protein ◽  
Pleckstrin Homology Domain ◽  
Mutant Form ◽  
Actin Organization ◽  
Conditional Allele ◽  
Intracellular Compartments ◽  
Green Fluorescent ◽  
Phosphatidylinositol 4

Phosphoinositides (PI) are synthesized and turned over by specific kinases, phosphatases, and lipases that ensure the proper localization of discrete PI isoforms at distinct membranes. We analyzed the role of the yeast synaptojanin-like proteins using a strain that expressed only a temperature-conditional allele of SJL2. Our analysis demonstrated that inactivation of the yeast synaptojanins leads to increased cellular levels of phosphatidylinositol (3,5)-bisphosphate and phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2), accompanied by defects in actin organization, endocytosis, and clathrin-mediated sorting between the Golgi and endosomes. The phenotypes observed in synaptojanin-deficient cells correlated with accumulation of PtdIns(4,5)P2, because these effects were rescued by mutations in MSS4or a mutant form of Sjl2p that harbors only PI 5-phosphatase activity. We utilized green fluorescent protein-pleckstrin homology domain chimeras (termed FLAREs for fluorescent lipid-associated reporters) with distinct PI-binding specificities to visualize pools of PtdIns(4,5)P2 and phosphatidylinositol 4-phosphate in yeast. PtdIns(4,5)P2 localized to the plasma membrane in a manner dependent on Mss4p activity. On inactivation of the yeast synaptojanins, PtdIns(4,5)P2 accumulated in intracellular compartments, as well as the cell surface. In contrast, phosphatidylinositol 4-phosphate generated by Pik1p localized in intracellular compartments. Taken together, our results demonstrate that the yeast synaptojanins control the localization of PtdIns(4,5)P2 in vivo and provide further evidence for the compartmentalization of different PI species.

scholarly journals Mutant Brucella abortus Membrane Fusogenic Protein Induces Protection against Challenge Infection in Mice

2015 ◽  
Vol 83 (4) ◽  
pp. 1458-1464 ◽  
Author(s):  
Job Alves de Souza Filho ◽  
Vicente de Paulo Martins ◽  
Priscila Carneiro Campos ◽  
Juliana Alves-Silva ◽  
Nathalia V. Santos ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Economic Losses ◽  
Macrophage Infection ◽  
Content Type ◽  
Live Vaccines ◽  
Interferon Regulatory Factor 1 ◽  
Potential Vaccine ◽  
Green Fluorescent

Brucellaspecies can cause brucellosis, a zoonotic disease that causes serious livestock economic losses and represents a public health threat. The mechanism of virulence ofBrucellaspp. is not yet fully understood. Therefore, it is crucial to identify new molecules that serve as virulence factors to better understand this host-pathogen interplay. Here, we evaluated the role of theBrucellamembrane fusogenic protein (Mfp) and outer membrane protein 19 (Omp19) in bacterial pathogenesis. In this study, we showed thatB. abortusΔmfp::kanand Δomp19::kandeletion mutant strains have reduced persistencein vivoin C57BL/6 and interferon regulatory factor 1 (IRF-1) knockout (KO) mice. Additionally, 24 h after macrophage infection with a Δmfp::kanor Δomp19::kanstrain expressing green fluorescent protein (GFP) approximately 80% or 65% ofBrucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP-1, respectively, whereas around 60% of BCVs containing wild-type S2308 were found in LAMP-1-negative compartments.B. abortusΔomp19::kanwas attenuatedin vivobut had a residual virulence in C57BL/6 and IRF-1 KO mice, whereas the Δmfp::kanstrain had a lower virulence in these same mouse models. Furthermore, Δmfp::kanand Δomp19::kanstrains were used as live vaccines. Challenge experiments revealed that in C57BL/6 and IRF-1 KO mice, the Δmfp::kanstrain induced greater protection than the vaccine RB51 and protection similar that of vaccine S19. However, a Δomp19::kanstrain induced protection similar to that of RB51. Thus, these results demonstrate thatBrucellaMfp and Omp19 are critical for full bacterial virulence and that the Δmfp::kanmutant may serve as a potential vaccine candidate in future studies.

scholarly journals Oxygen Limitation Contributes to Antibiotic Tolerance of Pseudomonas aeruginosa in Biofilms

2004 ◽  
Vol 48 (7) ◽  
pp. 2659-2664 ◽  
Author(s):  
Giorgia Borriello ◽  
Erin Werner ◽  
Frank Roe ◽  
Aana M. Kim ◽  
Garth D. Ehrlich ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorescent Protein ◽  
Oxygen Limitation ◽  
Anaerobic Conditions ◽  
Air Interface ◽  
Green Fluorescent ◽  
Local Oxygen

ABSTRACT The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro. Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h. It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions. Oxygen penetrated about 50 μm into the biofilms, which averaged 210 μm thick. The region of active protein synthesis was visualized by using an inducible green fluorescent protein. This zone was also limited to a narrow band , approximately 30 μm wide, adjacent to the air interface of the biofilm. The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h−1. These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state. In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air. When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically. Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested. Nitrate amendment stimulated the growth of untreated control P. aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics. Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P. aeruginosa biofilms causing infections in vivo.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

2021 ◽  
Author(s):  
Noemi Ruiz-Lopez ◽  
Jessica Pérez-Sancho ◽  
Alicia Esteban del Valle ◽  
Richard P Haslam ◽  
Steffen Vanneste ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Abiotic Stress ◽  
Fluorescent Protein ◽  
Mechanical Damage ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Contact ◽  
Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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.

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