Evaluation of resistance to aflatoxin contamination in kernels of maize genotypes using a GFP-expressing Aspergillus flavus strain

2013 ◽  
Vol 6 (2) ◽  
pp. 151-158 ◽  
Author(s):  
K. Rajasekaran ◽  
C.M. Sickler ◽  
R.L. Brown ◽  
J.W. Cary ◽  
D. Bhatnagar
Keyword(s):  
Fungal Infection ◽  
Aspergillus Flavus ◽  
Fluorescent Protein ◽  
Aflatoxin Contamination ◽  
Clear Correlation ◽  
Aleurone Layer ◽  
Screening Assay ◽  
Maize Germplasm ◽  
Antifungal Proteins ◽  
Green Fluorescent

Resistance or susceptibility of maize inbreds to infection by Aspergillus flavus was evaluated by the kernel screening assay. A green fluorescent protein-expressing strain of A. flavus was used to measure fungal spread and aflatoxin levels in real-time following fungal infection of kernels. Among the four inbreds tested, MI82 showed the most resistance and Ga209 the least. TZAR101 was also resistant to fungal infection, whereas Va35 was susceptible to fungal infection. However, Va35 produced lower aflatoxin levels compared to the susceptible line Ga209. Fluorescence microscopy indicated that the site of entry of the fungus into the kernel was consistently through the pedicel. Entry through the pericarp was never observed in undamaged kernels. In view of these results, incorporation or overexpression of antifungal proteins should be targeted to the pedicel and basal endosperm region in developing kernels. Once the fungus has entered through the pedicel, it spreads quickly through the open spaces between the pericarp and the aleurone layer, ultimately colonising the endosperm and scutellum and, finally, the embryo. A clear correlation was established between fungal fluorescence and aflatoxin levels. This method provides a quick, reliable means of evaluating resistance to A. flavus in undamaged kernels and provides breeders with a rapid method to evaluate maize germplasm.

scholarly journals Advances in the Development of Host Resistance in Corn to Aflatoxin Contamination by Aspergillus flavus

1999 ◽  
Vol 89 (2) ◽  
pp. 113-117 ◽  
Author(s):  
R. L. Brown ◽  
Z.-Y. Chen ◽  
T. E. Cleveland ◽  
J. S. Russin
Keyword(s):  
Fungal Infection ◽  
Aspergillus Flavus ◽  
Host Resistance ◽  
Animal Health ◽  
Fungal Growth ◽  
Aflatoxin Contamination ◽  
Genetically Engineered ◽  
Natural Resistance ◽  
Ear Rot ◽  
Screening Assay

Aflatoxins are toxic, highly carcinogenic secondary metabolites of Aspergillus flavus and A. parasiticus, which when produced during fungal infection of a susceptible crop in the field or after harvest contaminate food and feed and threaten human and animal health. Although there are several management strategies that may reduce aflatoxin contamination of corn, the preeminent strategy for elimination of aflatoxin is to develop preharvest host resistance to aflatoxin accumulation. This strategy has gained even greater prominence due to recent discoveries of natural resistance in corn that can be exploited in plant-breeding strategies. The ability to identify resistant corn genotypes has been enhanced by the development of a laboratory kernel-screening assay and by a strain of A. flavus genetically engineered to produce β-glucuronidase, an enzyme whose activity can be monitored to assess the degree of fungal infection in kernels. Investigations of resistant corn genotypes have associated kernel pericarp wax characteristics with resistance, identified kernel proteins associated with resistance to and inhibition of fungal growth or aflatoxin biosynthesis, and identified chromosome regions associated with resistance to Aspergillus ear rot and aflatoxin production. Such research advances could lead, in the near future, to commercially available, agronomically acceptable corn lines with multiple preharvest resistances to aflatoxin contamination.

scholarly journals A High-Content Screening Assay for Small Molecules That Stabilize Mutant Triose Phosphate Isomerase (TPI) as Treatments for TPI Deficiency

2021 ◽  
pp. 247255522110181
Author(s):  
Andreas Vogt ◽  
Samantha L. Eicher ◽  
Tracey D. Myers ◽  
Stacy L. Hrizo ◽  
Laura L. Vollmer ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Large Scale ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Triose Phosphate Isomerase ◽  
Pharmacological Intervention ◽  
Screening Assay ◽  
Triose Phosphate ◽  
The Common ◽  
Green Fluorescent

Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the “common” TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose–response, by limited structure–activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.

scholarly journals Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages

2015 ◽  
Vol 60 (1) ◽  
pp. 640-645 ◽  
Author(s):  
Flavia Sorrentino ◽  
Ruben Gonzalez del Rio ◽  
Xingji Zheng ◽  
Jesus Presa Matilla ◽  
Pedro Torres Gomez ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Screening Assay ◽  
Human Macrophages ◽  
High Throughput Screening Assay ◽  
Primary Identification ◽  
Green Fluorescent ◽  
Development And Validation ◽  
New Compounds

ABSTRACTHere we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.

scholarly journals Image-Based Screening for the Identification of Novel Proteasome Inhibitors

2007 ◽  
Vol 12 (2) ◽  
pp. 203-210 ◽  
Author(s):  
Linda Rickardson ◽  
Malin Wickström ◽  
Rolf Larsson ◽  
Henrik Lövborg
Keyword(s):  
Cell Line ◽  
Fluorescent Protein ◽  
Proteasome Inhibitors ◽  
Myeloma Cell ◽  
Cancer Drug ◽  
Screening Assay ◽  
Hek 293 ◽  
Compound Libraries ◽  
Human Embryo Kidney ◽  
Green Fluorescent

The proteasome is a new, interesting target in cancer drug therapy, and the proteasome inhibitor bortezomib has shown an effect in myeloma patients. It is of interest to efficiently discover and evaluate new proteasome inhibitors. The authors describe the development of an image-based screening assay for the identification of compounds with proteasome-inhibiting activity. The stably transfected human embryo kidney cell line HEK 293 ZsGreen Proteasome Sensor Cell Line expressing the ZsProSensor-1 fusion protein was used for screening and evaluation of proteasome inhibitors. Inhibition of the proteasome leads to accumulation of the green fluorescent protein ZsGreen, which is measured in the ArrayScan® High Content Screening system, in which cell morphology is studied simultaneously. When screening the LOPAC1280 substance library, several compounds with effect on the proteasome were found; among the hits were disulfiram and ammonium pyrrolidinedithiocarbamate (PDTC). Cytotoxic analysis of disulfiram and PDTC showed that the compounds induced cytotoxicity in the myeloma cell line RPMI 8226. The average Z' value for the assay was 0.66. The results indicate that the assay rapidly identifies new proteasome-inhibiting substances, and it will be further used as a tool for image-based screening of other chemically diverse compound libraries.

scholarly journals A Fluorescent-Based High-Throughput Screening Assay for Small Molecules That Inhibit the Interaction of MdmX with p53

2012 ◽  
Vol 18 (2) ◽  
pp. 191-198 ◽  
Author(s):  
Keiko Tsuganezawa ◽  
Yukari Nakagawa ◽  
Miki Kato ◽  
Shigenao Taruya ◽  
Fumio Takahashi ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Diffusion Time ◽  
Correlation Spectroscopy ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Green Fluorescent ◽  
Fluorescence Quencher

A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC50 values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.

scholarly journals Construction and use of aCupriavidus necatorH16 soluble hydrogenase promoter (PSH) fusion togfp(green fluorescent protein)

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2269 ◽  
Author(s):  
Bat-Erdene Jugder ◽  
Jeffrey Welch ◽  
Nady Braidy ◽  
Christopher P. Marquis
Keyword(s):  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Small Scale ◽  
Growth Media ◽  
Reporter System ◽  
Screening Assay ◽  
Fluorescent Reporter ◽  
Fusion Construct ◽  
Green Fluorescent

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2is a soluble [Ni–Fe] uptake hydrogenase (SH) produced byCupriavidus necatorH16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSHpromoter activity using several gene cloning approaches. A PSHpromoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSHpromoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinantC. necatorH16 cells. Here we report the first successful fluorescent reporter system to study PSHpromoter activity inC. necatorH16. The fusion construct allowed for the design of a simple screening assay to evaluate PSHactivity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

Resistance to Aflatoxin Accumulation in Kernels of Maize Inbreds Selected for Ear Rot Resistance in West and Central Africa

2001 ◽  
Vol 64 (3) ◽  
pp. 396-400 ◽  
Author(s):  
ROBERT L. BROWN ◽  
ZHI-YUAN CHEN ◽  
ABEBE MENKIR ◽  
THOMAS E. CLEVELAND ◽  
KITTY CARDWELL ◽  
...  
Keyword(s):  
Fungal Infection ◽  
Natural Infection ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Central Africa ◽  
Aflatoxin Contamination ◽  
Ear Rot ◽  
Screening Assay ◽  
West And Central Africa ◽  
Aflatoxin Accumulation

Thirty-six inbred lines selected in West and Central Africa for moderate to high resistance to maize ear rot under conditions of severe natural infection were screened for resistance to aflatoxin contamination using the previously established kernel screening assay. Results showed that more than half the inbreds accumulated aflatoxins at levels as low as or lower than the resistant U.S. lines GT-MAS:gk or MI82. In 10 selected aflatoxin-resistant or aflatoxin-susceptible inbreds, Aspergillus flavus growth, which was quantified using an A. flavus transformant containing a GUS-β-tubulin reporter gene construct, was, in general, positively related to aflatoxin accumulation. However, one aflatoxin-resistant inbred supported a relatively high level of fungal infection, whereas two susceptibles supported relatively low fungal infection. When kernels of the 10 tested lines were profiled for proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, significant variations from protein profiles of U.S. lines were observed. Confirmation of resistance in promising African lines in field trials may significantly broaden the resistant germplasm base available for managing aflatoxin contamination through breeding approaches. Biochemical resistance markers different from those being identified and characterized in U.S. genotypes, such as ones inhibitory to aflatoxin biosynthesis rather than to fungal infection, may also be identified in African lines. These discoveries could significantly enhance the host resistance strategy of pyramiding different traits into agronomically useful maize germplasm to control aflatoxin contamination.

scholarly journals Interaction of the Mite Aceria mangiferae with Fusarium mangiferae, the Causal Agent of Mango Malformation Disease

2009 ◽  
Vol 99 (2) ◽  
pp. 152-159 ◽  
Author(s):  
E. Gamliel-Atinsky ◽  
S. Freeman ◽  
A. Sztejnberg ◽  
M. Maymon ◽  
R. Ochoa ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fungal Infection ◽  
Fluorescent Protein ◽  
Infection Process ◽  
Apical Bud ◽  
Apical Buds ◽  
Green Fluorescent ◽  
Mango Malformation ◽  
Fusarium Mangiferae

The role of the mango bud mite, Aceria mangiferae, in carrying conidia of Fusarium mangiferae, vectoring them into potential infection sites, and assisting fungal infection and dissemination was studied. Following the mite's exposure to a green fluorescent protein-marked isolate, conidia were observed clinging to the mite's body. Agar plugs bearing either bud mites or the pathogen were placed on leaves near the apical buds of potted mango plants. Conidia were found in bud bracts only when both mites and conidia were co-inoculated on the plant, demonstrating that the mite vectored the conidia into the apical bud. Potted mango plants were inoculated with conidia in the presence or absence of mites. Frequency and severity of infected buds were significantly higher in the presence of mites, revealing their significant role in the fungal infection process. Conidia and mite presence were monitored with traps in a diseased orchard over a 2-year period. No windborne bud mites bearing conidia were found; however, high numbers of windborne conidia were detected in the traps. These results suggest that A. mangiferae can carry and vector conidia between buds and assist in fungal penetration but does not play a role in the aerial dissemination of conidia between trees.

scholarly journals Hemagglutinin-Pseudotyped Green Fluorescent Protein-Expressing Influenza Viruses for the Detection of Influenza Virus Neutralizing Antibodies

2009 ◽  
Vol 84 (4) ◽  
pp. 2157-2163 ◽  
Author(s):  
Luis Martínez-Sobrido ◽  
Richard Cadagan ◽  
John Steel ◽  
Christopher F. Basler ◽  
Peter Palese ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Green Fluorescent Protein ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Influenza Viruses ◽  
Serum Samples ◽  
Screening Assay ◽  
Ha Gene ◽  
Green Fluorescent ◽  
Gene Expressing

ABSTRACT Influenza virus is a highly contagious virus that causes yearly epidemics and occasional pandemics of great consequence. Influenza virus neutralizing antibodies (NAbs) are promising prophylactic and therapeutic reagents. Detection of NAbs in serum samples is critical to evaluate the prevalence and spread of new virus strains. Here we describe the development of a simple, sensitive, specific, and safe screening assay for the rapid detection of NAbs against highly pathogenic influenza viruses under biosafety level 2 (BSL-2) conditions. This assay is based on the use of influenza viruses in which the hemagglutinin (HA) gene is replaced by a gene expressing green fluorescent protein (GFP). These GFP-expressing influenza viruses replicate to high titers in HA-expressing cell lines, but in non-HA-expressing cells, their replication is restricted to a single cycle.

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