Project description:Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and A. parasiticus. In order to better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. A. flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B1 and B2 biosynthesis, while A. parasiticus cultures had significantly increased B1 and G1 biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed seventy seven genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis. Keywords: Aflatoxin, Aspergillus, flavus, Amnio Acids, Tryptophan
Project description:The molecular mechanisms underlying aflatoxin production have been well-studied in strains of the fungus Aspergillus flavus (A. flavus) under artificial conditions. However, aflatoxin biosynthesis has rarely been studied in natural isolates of A. flavus strains. In the present study, tandem mass tag (TMT) labeling and high-performance liquid chromatography (HPLC) coupled with tandem-mass spectrometry analysiswere used for proteomic quantification in natural isolates of high- and low-aflatoxin-yield A. flavus strains.
Project description:We identified a 14-3-3 homolog (maf1) in an EST library made under conditions conducive to aflatoxin biosynthesis. Disruption of this gene in A. flavus by site directed mutagenesis abolished aflatoxin production. The maf1 mutant was morphologically similar to wild type but had reduced conidiation and growth on some media. DNA expression analysis of the wild type and maf1 mutant revealed that the mutation affected the expression profile of a set of genes associated with aflatoxin production. Keywords = Aspergillus Keywords = aflatoxin Keywords = 14-3-3
Project description:Aspergillus flavus is a pathogen of corn, peanut, and other crops which produces carcinogenic mycotoxins known as aflatoxins. Previous studies have shown that drought stress results in exacerbated aflatoxin production. Drought-associated oxidative stress caused by reactive oxygen species (ROS) is suspected to contribute to increased aflatoxin production during infection. Here, the responses of field isolates of A. flavus with varying degrees of aflatoxin production capability to H2O2-derived oxidative stress were examined using iTRAQ proteomics. Three isolates: AF13 (highly toxigenic), NRRL3357 (moderately toxigenic), and K54A (atoxigenic), were cultures in aflatoxin conducive yeast extract-sucrose (YES) medium amended with 0, 10, or 20/25mM of H2O2. Identified differentially expressed proteins were used for functional prediction, cellular localization, and pathway analyses to identify molecular mechanisms involved in A. flavus oxidative stress responses relative to aflatoxin production capability. Correlative analyses with previously obtained transcriptome data for the same isolates under the same experimental conditions was also performed with a low degree of correlation (r = 0.1114) observed between the protein and transcript data suggesting possible post-transcriptional regulation of oxidative stress responses. The identified stress responsive mechanisms provide a basis of investigating novel approaches of enhancing host resistance against aflatoxin contamination.
Project description:We identified a 14-3-3 homolog (maf1) in an EST library made under conditions conducive to aflatoxin biosynthesis. Disruption of this gene in A. flavus by site directed mutagenesis abolished aflatoxin production. The maf1 mutant was morphologically similar to wild type but had reduced conidiation and growth on some media. DNA expression analysis of the wild type and maf1 mutant revealed that the mutation affected the expression profile of a set of genes associated with aflatoxin production. Keywords = Aspergillus Keywords = aflatoxin Keywords = 14-3-3 Keywords: repeat sample
Project description:Gene expression analysis of A. parasiticus grown under conditions conducive and nonconductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. A complex regulatory network governs the biosynthesis of aflatoxin. While several genes involved in aflatoxin production are known, their action alone cannot account for its regulation. Arrays of clones from an Aspergillus flavus cDNA library and glass slide microarrays of ESTs were screened to identify additional genes. An initial screen of the cDNA clone arrays lead to the identification of 753 unique ESTs. Many showed sequence similarity to known metabolic and regulatory genes; however, no function could be ascribed to over 50% of the ESTs. Gene expression analysis of Aspergillus parasiticus grown under conditions conducive and non-conductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. Twenty-four genes were more highly expressed during aflatoxin biosynthesis and 18 genes were more highly expressed prior to aflatoxin biosynthesis. No predicted function could be ascribed to 18 of the 24 genes whose elevated expression was associated with aflatoxin biosynthesis. Keywords = Aspergillus Keywords = aflatoxin Keywords: time-course
Project description:Aflatoxins are carcinogenic fungal secondary metabolites. Levels of aflatoxins in agricultural commodities are stringently regulated by many countries. A cluster of genes is responsible for aflatoxin biosynthesis by Aspergillus flavus and other closely related species. Expression of the clustered aflatoxin genes is governed by a complex network of regulatory mechanisms. To better understand the molecular events that are associated with aflatoxin production, transcription profiling by microarray analyses which compared three independent aflatoxigenic A. flavus strains to individual isogenic progenies that no longer produced aflatoxins after serial transfers was carried out. Twenty-two significantly differentially expressed features were identified. After physical mapping using the A. oryzae genome sequence as the reference, the number of unique genes was reduced to 16. Compared to the parental strains, changes in the aflatoxin gene expression levels in the progenies were not significant, which suggests that the inability to produce aflatoxins is not caused by decreased expression. The only gene showing higher expression levels in the progenies is homologous to glutathione S-transferease genes. Overexpression of this gene, named hcc, at six- to nine-fold in an aflatoxigenic A. flavus did not cause discernible changes in colony morphology or aflatoxin production. Loss of aflatoxin production after serial transfers may not result from a single event but caused by multiple factors. Keywords: Compartiave hybridization toxigenic and atoxigenic lines of Aspergillus Aspergillus flavus NRRL 29459, NRRL 29474, and NRRL 29490 are aflatoxigenic strains originated from soil collection in a peanut field (Terrell Co., Georgia, USA). Strains 459B-20-2, 474A-20, and 499A-20 were nonaflatoxigenic isolates obtained after 20 serial transfers of the parental strains on potato dextrose agar slants (Horn and Dorner 2002). Comparsions in each experiment consisted of one aflatoxigenic parental strain and one nonaflatoxigenic progeny, compared after 48- or 72-hr growth. Each comparison was repeated with duplicate dye-flip.
Project description:Gene expression analysis of A. parasiticus grown under conditions conducive and nonconductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. A complex regulatory network governs the biosynthesis of aflatoxin. While several genes involved in aflatoxin production are known, their action alone cannot account for its regulation. Arrays of clones from an Aspergillus flavus cDNA library and glass slide microarrays of ESTs were screened to identify additional genes. An initial screen of the cDNA clone arrays lead to the identification of 753 unique ESTs. Many showed sequence similarity to known metabolic and regulatory genes; however, no function could be ascribed to over 50% of the ESTs. Gene expression analysis of Aspergillus parasiticus grown under conditions conducive and non-conductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. Twenty-four genes were more highly expressed during aflatoxin biosynthesis and 18 genes were more highly expressed prior to aflatoxin biosynthesis. No predicted function could be ascribed to 18 of the 24 genes whose elevated expression was associated with aflatoxin biosynthesis. Keywords = Aspergillus Keywords = aflatoxin Keywords: time-course