scholarly journals Interspecific Competition for Colonization of Maize Plants Between Fusarium proliferatum and Fusarium verticillioides

Plant Disease ◽  
2020 ◽  
Vol 104 (8) ◽  
pp. 2102-2110
Author(s):  
A. Reyes Gaige ◽  
T. Todd ◽  
J. P. Stack
Keyword(s):  
Interspecific Competition ◽  
Fluorescent Protein ◽  
Fusarium Verticillioides ◽  
Fusarium Proliferatum ◽  
The Other ◽  
Real Time Quantitative Pcr ◽  
Seedborne Pathogens ◽  
Maize Plants ◽  
Green Fluorescent ◽  
Stem Segments

Fusarium proliferatum and F. verticillioides are mycotoxin-producing, seedborne pathogens of maize. They are often asymptomatic in seed, eluding symptom-based detection. Experiments were conducted in nonsterile soil to determine whether interspecific competition influenced establishment in maize plants of an introduced isolate of F. proliferatum or F. verticillioides. Hygromycin-resistant, green fluorescent protein (GFP) transformed (GFP-tagged) F. proliferatum (F. proliferatum-green) and hygromycin-resistant, monomeric red fluorescent protein (mRFP) transformed (mRFP-tagged) F. verticillioides (F. verticillioides-red) strains were developed to provide molecular markers to track fungal establishment. Heat-killed Fusarium-free maize seed, colonized with F. proliferatum-green or F. verticillioides-red by immersion in a spore suspension for 16 h, served as the source of inoculum. The ability of F. proliferatum-green and F. verticillioides-red to colonize viable maize plants already colonized by the other species was determined. Maize plants were retrieved from soil after 14 days and DNA was extracted from three consecutive root segments and three consecutive stem segments. A TaqMan multiplex real-time quantitative PCR protocol was developed to identify and quantify F. proliferatum-green and F. verticillioides-red from each plant segment from each treatment; the experiment was repeated three times. This experiment confirmed that F. proliferatum-green and F. verticillioides-red effectively colonized roots and stems of the maize plant already colonized with the other species. Prior colonization of maize tissues by F. verticillioides-red (P = 0.6749) and other seedborne microorganisms (P = 0.1910) reduced but did not prevent subsequent colonization by F. proliferatum-green. Similarly, prior colonization of maize tissues by F. proliferatum-green (P = 0.7032) and other seedborne microorganisms (P = 0.1447) reduced but did not prevent subsequent colonization by F. verticillioides-red.

scholarly journals Sec1p Binds to SNARE Complexes and Concentrates at Sites of Secretion

1999 ◽  
Vol 146 (2) ◽  
pp. 333-344 ◽  
Author(s):  
Chavela M. Carr ◽  
Eric Grote ◽  
Mary Munson ◽  
Frederick M. Hughson ◽  
Peter J. Novick
Keyword(s):  
Fluorescent Protein ◽  
Snare Complex ◽  
The Other ◽  
Complex Assembly ◽  
Vesicle Docking ◽  
Neuronal Protein ◽  
The Core ◽  
Target Membrane ◽  
Green Fluorescent ◽  
And Function

Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.

Dynamic localization of penicillin-binding proteins during spore development in Bacillus subtilis

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 999-1012 ◽  
Author(s):  
Dirk-Jan Scheffers
Keyword(s):  
Bacillus Subtilis ◽  
Green Fluorescent Protein ◽  
Binding Proteins ◽  
Cytoplasmic Membrane ◽  
Fluorescent Protein ◽  
Spore Formation ◽  
The Other ◽  
Spore Development ◽  
Penicillin Binding Proteins ◽  
Green Fluorescent

During Bacillus subtilis spore formation, many membrane proteins that function in spore development localize to the prespore septum and, subsequently, to the outer prespore membrane. Recently, it was shown that the cell-division-specific penicillin-binding proteins (PBPs) 1 and 2b localize to the asymmetric prespore septum. Here, the author studied the localization of other PBPs, fused to green fluorescent protein (GFP), during spore formation. Fusions to PBPs 4, 2c, 2d, 2a, 3, H, 4b, 5, 4a, 4* and X were expressed during vegetative growth, and their localization was monitored during sporulation. Of these PBPs, 2c, 2d, 4b and 4* have been implicated as having a function in sporulation. It was found that PBP2c, 2d and X changed their localization, while the other PBPs tested were not affected. The putative endopeptidase PbpX appears to spiral out in a pattern that resembles FtsZ redistribution during sporulation, but a pbpX knockout strain had no distinguishable phenotype. PBP2c and 2d localize to the prespore septum and follow the membrane during engulfment, and so are redistributed to the prespore membrane. A similar pattern was observed when GFP–PBP2c was expressed in the mother cell from a sporulation-specific promoter. This work shows that various PBPs known to function during sporulation are redistributed from the cytoplasmic membrane to the prespore.

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 Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Gaétan Bader ◽  
Ludovic Enkler ◽  
Yuhei Araiso ◽  
Marine Hemmerle ◽  
Krystyna Binko ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Gene ◽  
The Self ◽  
The Other ◽  
Green Fluorescent Protein Fusion ◽  
Mitochondrial Localization ◽  
Protein Fusion ◽  
Argonaute 2 ◽  
New Type ◽  
Green Fluorescent

A single nuclear gene can be translated into a dual localized protein that distributes between the cytosol and mitochondria. Accumulating evidences show that mitoproteomes contain lots of these dual localized proteins termed echoforms. Unraveling the existence of mitochondrial echoforms using current GFP (Green Fluorescent Protein) fusion microscopy approaches is extremely difficult because the GFP signal of the cytosolic echoform will almost inevitably mask that of the mitochondrial echoform. We therefore engineered a yeast strain expressing a new type of Split-GFP that we termed Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). Because one moiety of the GFP is translated from the mitochondrial machinery while the other is fused to the nuclear-encoded protein of interest translated in the cytosol, the self-reassembly of this Bi-Genomic-encoded Split-GFP is confined to mitochondria. We could authenticate the mitochondrial importability of any protein or echoform from yeast, but also from other organisms such as the human Argonaute 2 mitochondrial echoform.

scholarly journals Characterization of the Resistance Mechanism and Risk of Fusarium verticillioides to the Myosin Inhibitor Phenamacril

2020 ◽  
Vol 110 (4) ◽  
pp. 790-794 ◽  
Author(s):  
Weichao Ren ◽  
Na Liu ◽  
Yiping Hou ◽  
Baohua Li ◽  
Mingguo Zhou ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fusarium Species ◽  
Fusarium Verticillioides ◽  
Single Point ◽  
Resistance Mechanism ◽  
Point Mutations ◽  
Binding Mode ◽  
Ear Rot ◽  
Green Fluorescent ◽  
High Level

Fusarium verticillioides is a major pathogen of maize that causes ear rot and produces mycotoxins. Phenamacril is a novel cyanoacrylate fungicide that exhibits favorable activity against Fusarium species. In this study, the phenamacril-resistant mutants of F. verticillioides were obtained by ultraviolet mutagenesis. Single point mutations of S73L or E276K in the myosin-1 FvMyo1 were proven to be responsible for the high-level resistance of F. verticillioides to phenamacril. Phenamacril had a significant impact on the localization of the wild-type FvMyo1 (FvMyo1WT-green fluorescent protein [GFP]), but not on the mutated FvMyo1 (FvMyo1S73L-GFP and FvMyo1E276K-GFP) at the hyphal tips. Molecular docking analysis suggested that mutation (S73L or E276K) in FvMyo1 altered the binding mode and decreased the binding affinity between phenamacril and myosin-1. There was no significant fitness penalty in mycelial growth, conidiation, and virulence of F. verticillioides associated with resistance to phenamacril. The results will enhance our understanding of the resistance mechanism of F. verticillioides to phenamacril and provide new reference data for the management of maize ear rot.

239 MAC-T CELLS AS A TOOL TO EXAMINE GENOME EDITING USING CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)

2016 ◽  
Vol 28 (2) ◽  
pp. 251
Author(s):  
S. N. Lotti ◽  
I. Tasan ◽  
H. Zhao ◽  
M. B. Wheeler
Keyword(s):  
T Cells ◽  
Milk Production ◽  
Fluorescent Protein ◽  
The United States ◽  
The Other ◽  
Total Efficiency ◽  
Guide Rnas ◽  
Exon 1 ◽  
Green Fluorescent ◽  
A Site

In 2050, the expected size of the human population is 9 billion, the demand for food will increase, and the demand for milk will increase along with it. Genetically modifying animals is a tool that can be used to meet this growing demand. In the United States, Holstein is the leading breed for milk production and Holsteins produce on average 24 291 pounds of milk per year, whereas Jerseys, the other major dairy breed, produce on average 16 997 pounds. Their ability to produce large quantities of milk is linked to 2 mutations. These mutations are on the α-lactalbumin (α-lac) gene; the α-lac exon (+1) corresponds to the transcription start point of α-lac, (+15) and (–1689) are the positions corresponding to the single nucleotide polymorphism associated with increased milk production. Holstein cows have an adenine at both of these positions in contrast to the other cattle breeds with lower milk production, which have either a cytosine or guanine at either position. Inserting an adenine at position (+15) and (–1689) in cows without this mutation could lead to increased milk production and a better response to market demands. The purpose of this experiment was to test the cutting efficiency of candidate clustered regularly interspaced short palindromic repeats (CRISPR) that will later be used in knock-in experiments. CRISPRs were used because the CRISPR-Cas9 system is inexpensive, easily programmed, and efficient. In this preliminary study, we worked with Holstein MAC-T cells, which already contain the mutation at both positions. CRISPRs were used on this cell line to cut the DNA at a site near the mutation. Based on the genomic DNA sequence of these MAC-T cells, 3 guide RNAs were designed. Cells were then transfected with the designed CRISPRs by a variety of transfection methods, including Fugene™ (Promega, Madison, WI, USA), electroporation, and Lipofectamine (ThermoFisher Scientific, Waltham, MA, USA). Green fluorescent protein was used to determine the efficiency of transfection; 30% efficiency was seen for Fugene™, whereas electroporation and Lipofectamine™ had 70% efficiency. To select for successfully transfected cells, puromycin selection was applied. The DNA was later extracted and sent in for sequencing. Next, the website TIDE was used to compare the transfected MAC-T cells to normal MAC-T cells. The TIDE software measures the editing efficiency and looks for major insertions or deletions in pools of DNA by comparing 2 sequences to quantify the editing efficacy of CRISPR-Cas9. Our results showed that CRISPRs successfully cut the DNA near the α-lac mutation region with a total efficiency of 13.8%. The desired next step will be to insert a single-strand oligonucleotide (ssODN) donor to make a single basepair mutation. The ultimate aim of this research would be to insert these mutations into other cattle species in order to increase their milk production.

scholarly journals Head-to-tail oligomerization of calsequestrin

2001 ◽  
Vol 154 (3) ◽  
pp. 525-534 ◽  
Author(s):  
Giuliana Gatti ◽  
Sara Trifari ◽  
Nasrin Mesaeli ◽  
J.M. Robert Parker ◽  
Marek Michalak ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Striated Muscle ◽  
Smooth Muscles ◽  
Direct Interaction ◽  
Amino Acid Sequences ◽  
The Other ◽  
Heterogeneous Distribution ◽  
New Process ◽  
Green Fluorescent ◽  
Terminal Cisternae

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.

Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens

2006 ◽  
Vol 52 (7) ◽  
pp. 623-626 ◽  
Author(s):  
Weiguo Fang ◽  
Yan Pei ◽  
Michael J Bidochka
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Metarhizium Anisopliae ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Pathogenic Fungus ◽  
Single Copy ◽  
Transformation Method ◽  
The Other ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 ± 1.4 transformants per plate could be obtained using conidial concentrations of 106 conidia/mL and a 2 day co-cultivation in the presence of 200 µmol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.Key words: entomopathogenic fungi, Metarhizium anisopliae, Agrobacterium tumefaciens, genetic transformation.

scholarly journals Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units

2007 ◽  
Vol 88 (10) ◽  
pp. 2710-2718 ◽  
Author(s):  
Linda J. Rennick ◽  
W. Paul Duprex ◽  
Bert K. Rima
Keyword(s):  
Gene Expression ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
The Other ◽  
Green Fluorescent Protein Gene ◽  
Conserved Gene ◽  
Green Fluorescent ◽  
Single Living Cells ◽  
Cell Variation ◽  
Viral Sequences

Transcription from morbillivirus genomes commences at a single promoter in the 3′ non-coding terminus, with the six genes being transcribed sequentially. The 3′ and 5′ untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5′ UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5′ UTRs. Insertions into the 5′ UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5′ UTR, govern this decreased expression from TU2.

Sign in / Sign up

Export Citation Format

Share Document