Quantitative proteomic analysis for high- and low-aflatoxin-yield Aspergillus flavus strains isolated from natural environments
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus, Q Exactive
ORGANISM(S): Aspergillus Flavus (strain Atcc 200026 / Fgsc A1120 / Nrrl 3357 / Jcm 12722 / Srrc 167)
TISSUE(S): Cell Culture
SUBMITTER: 
涛 李
LAB HEAD: Rui Hu
PROVIDER: PXD027517 | Pride | 2022-02-17
REPOSITORIES: Pride
Publications
Quantitative Proteomic Analysis for High- and Low-Aflatoxin-Yield <i>Aspergillus flavus</i> Strains Isolated From Natural Environments.
Li Tao T Zhang Zhaowei Z Wang Yu Y Li Ying Y Zhu Jiang J Hu Rui R Yang Yunhuang Y Liu Maili M
Frontiers in microbiology 20210921
The molecular mechanisms underlying aflatoxin production have been well-studied in strains of the fungus <i>Aspergillus flavus</i> (<i>A. flavus</i>) under artificial conditions. However, aflatoxin biosynthesis has rarely been studied in <i>A. flavus</i> strains isolated from field conditions with different aflatoxin-producing ability. In the present study, tandem mass tag (TMT) labeling and high-performance liquid chromatography (HPLC) coupled with tandem-mass spectrometry analysis were used fo ...[more]
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Project description:Aspergillus flavus is one of the major fungal molds that colonize peanut in the field and during storage. The impact to human and animal health and to economy in agriculture and commerce are significant since this mold produces the most potent natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect to inhibit aflatoxin formation through down-regulating aflatoxin pathway gene expression in A. flavus as demonstrated by genechip analysis in liquid medium and peanuts. The results showed that aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98%) inhibited by B. megaterium. The expression of many of the aflatoxin biosynthetic genes in the fungus was confirmed to be turned down. Some of the target genes down-regulated by B. megaterium within the whole genome and within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX) were identified. These target genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was found to be significantly down-regulated. The effect of B. megaterium on aflatoxin biosynthesis and genes expression of pathogen was firstly tested in potato dextrose broth (PDB) and glucose minimal salts medium (MM). The cell suspension of B. megaterium (concentration in PDB and MM was finally adjusted to 108 CFU/ml) or sterile distilled water as a control was added into the 100 ml beaker flask containing 15 ml PDB or MM, respectively. Then 100 M-NM-<l of spore suspension (5 M-CM-^W 106 spores/ml) of A. flavus were added into each beaker flask. After 48 h of incubation at 28M-BM-0C at 200 rpm, mycelia were collected, fresh frozen with liquid nitrogen, ground to a fine powder in liquid nitrogen, and stored at -80M-BM-0C for further analysis. The effect of B. megaterium on aflatoxin biosynthesis and genes expression in the A. flavus fungal pathogen was also tested in two types of peanut kernels, UF 715133-1 and Jinhua 1012, respectively. Peanut kernels were wounded (6 mm diameter and approximately 3 mm deep) using a sterile borer and then 20 M-NM-<l of 1 M-CM-^W 108 CFU/ml cell suspension of B. megaterium was inoculated on wounded peanut kernels respectively. Sterile distilled water was also used for inoculation as control. Two hours after bacterial inoculation, 10 M-NM-<l A. flavus spore suspension was inoculated into each wound at a concentration of 106 spores/ml. The kernels were placed in artificial weather chamber to maintain high humidity (85%) and incubated at 28M-BM-0C for 7 days. Each treatment was replicated three times with 20 peanut kernels in each test. The mycelia on kernels were harvested at day 7 and fresh frozen immediately in liquid nitrogen, ground into powder, and stored at -80M-BM-0C for further analysis.
2013-10-22 | E-GEOD-51469 | biostudies-arrayexpress
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.
2010-05-26 | E-GEOD-8185 | biostudies-arrayexpress
Project description:Aspergillus flavus is the major producer of carcinogenic aflatoxins in crops worldwide. Natural populations of A. flavus show tremendous variation in aflatoxin production some of which can be attributed to extreme environmental conditions (e.g., drought), differential regulation of the aflatoxin biosynthetic pathway, missing cluster genes or loss-of-function mutations. Understanding the evolutionary processes that generate genetic diversity in A. flavus may also explain quantitative and qualitative differences in aflatoxigenicity. Several population studies provide indirect evidence of recombination in the aflatoxin gene cluster and genome-wide, using multilocus genealogical approaches. More recently A. flavus has been shown to be functionally heterothallic and capable of sexual reproduction in laboratory crosses. In the present study, we characterize the progeny from nine A. flavus crosses and show that crossovers in the aflatoxin cluster coincide with inferred recombination blocks and hotspots in natural populations, which suggests that recombination in the cluster is primarily driven by sex. Moreover, we show that a single crossover event in the cluster can restore aflatoxigenicity, which is significant as mycotoxin production in A. flavus is highly heritable. aCGH was used to corroborate inferences from cluster-based MLSTs and to possibly identify additional crosovers within the cluster. aCGH comparison between 3 strains of A. flavus: 2 parental (P) and 1 progeny (F1) analyzed at the probe level. A total of 9 trio comparisons were made from a total of 18 isolates analyzed by aCGH. Trio comparisons are as follows: IC278 (P), IC1179 (P) and IC1650 (F1); IC201 (P), IC310 (P) and IC1719 (F1); IC307 (P), IC308 (P) and IC1751 (F1); IC277 (P), IC311 (P) and IC1766 (F1); IC277 (P), IC311 (P) and IC1775 (F1); IC244 (P), IC277 (P) and IC2205 (F1); IC244 (P), IC277 (P) and IC2207 (F1); IC301 (P), IC1179 (P) and IC2171 (F1); and finally IC244 (P), IC277 (P) and IC2209 (F1).
2012-03-07 | E-GEOD-27484 | biostudies-arrayexpress
Project description:Aspergillus flavus contaminates crops during preharvest and post-harvest periods and produce carcinogenic mycotoxin aflatoxins posing severe threat to food safety and human health. Lysine 2-hydroxyisobutyrylation is one of the most important reversible post-translational modifications and plays a vital regulatory role in various cellular processes. In order to explore the potential roles of lysine 2-hydroxyisobutyrylation in aflatoxin biosynthesis, protein 2-hydroxyisobutyrylation analysis of A. flavus was performed, and a total of 7156 2-hydroxyisobutyrylation sites in 1473 proteins were identified.
2022-06-06 | PXD030912 | Pride
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.
2018-02-27 | PXD007164 | Pride
Project description:Aflatoxin contamination caused by the opportunistic pathogen A. flavus is a major concern in maize production prior to harvest and during storage. Previous studies indicate that both constitutive and induced resistance are involved in maize kernel defense against A. flavus infection, little is known about molecular mechanisms of mature kernels in response to fungal infection. The purpose of this study is to determine gene expression differences in maize kernels between resistant and susceptible lines in response to A. flavus challenge. To avoid the environmental effects in the field inoculation, a laboratory based inoculation technique Kernel Screening Assay (KSA) was used to challenge kernels with A. flavus. After 72 hours incubation of inoculated and noninculated mature kernels, gene expression profiles of two Near Isogenic Lines (NIL) of Eyl25 (A. flavus resistant) and Eyl31 (A. flavus susceptible) were compared using oligonucleotide array. Direct comparisons were designed. The comparisons of NIL Eyl25 and Eyl31 include: Treated/Control, Control/Control and Treated/Treated. After 72 hours incubation under KSA conditions, forty seeds used in each A. flavus inoculated and noninculated group were bulked to extract total RNA. Four technical replications were performed in each comparison including two dye-swaps, total 16 hybridization reactions.
2010-10-01 | E-GEOD-19883 | biostudies-arrayexpress
Project description:Aspergillus flavus is the major producer of carcinogenic aflatoxins in crops worldwide. Natural populations of A. flavus show tremendous variation in aflatoxin production some of which can be attributed to extreme environmental conditions (e.g., drought), differential regulation of the aflatoxin biosynthetic pathway, missing cluster genes or loss-of-function mutations. Understanding the evolutionary processes that generate genetic diversity in A. flavus may also explain quantitative and qualitative differences in aflatoxigenicity. Several population studies provide indirect evidence of recombination in the aflatoxin gene cluster and genome-wide, using multilocus genealogical approaches. More recently A. flavus has been shown to be functionally heterothallic and capable of sexual reproduction in laboratory crosses. In the present study, we characterize the progeny from nine A. flavus crosses and show that crossovers in the aflatoxin cluster coincide with inferred recombination blocks and hotspots in natural populations, which suggests that recombination in the cluster is primarily driven by sex. Moreover, we show that a single crossover event in the cluster can restore aflatoxigenicity, which is significant as mycotoxin production in A. flavus is highly heritable. aCGH was used to corroborate inferences from cluster-based MLSTs and to possibly identify additional crosovers within the cluster.
2012-03-07 | GSE27484 | GEO
Project description:Aspergillus flavus contaminates crops during preharvest and post-harvest periods and produce carcinogenic mycotoxin aflatoxins posing severe threat to food safety and human health. To identify potential targets to tackle aflatoxin contamination, we have characterized a novel Afper1, sharing sequence identity with the yeast protein per1, regulating cell development and secondary metabolism in A. flavus. Notably, proteomics analysis revealed that the enzymes involved in extracellular hydrolases, conidial development, ER homeostasis, and aflatoxin biosynthesis significantly varied. Unexpectedly, enzymes participated in scavenging ROS including catA, catB, cpeB, and sodM were significantly downregulated and the accumulated ROS and sensitivity to hydrogen peroxide were confirmed by experiments. Surprisingly, Afper1 deletion significantly unregulated heterochromatin protein (HepA) and downregulated GNAT family acetyltransferase involved in heterochromatin formation. Accompanying the accumulated ROS and chromatin remodelling, enzymes participating in aflatoxins, ustiloxin B, and gliotoxin were impaired. These results implied that Afper1 affected chromatin remodelling and disturbed ER homeostasis leading to the accumulation of ROS, and ultimately resulted in growth defect and impaired secondary metabolites biosynthesis.
2022-07-13 | PXD030015 | Pride
Project description:Aspergillus flavus and A. parasiticus are two of the most important aflatoxin-producing species that contaminate agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here, we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O-methylsterigmatocystin, but not CPA. Only four out of forty-five attempted interspecific crosses between compatible mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were isolated and were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of putative allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important species.
2015-10-01 | GSE61981 | GEO
Project description:Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understand host response to infection by the fungus, transcription of approximately 9,000 maize genes were monitored during the host-pathogen interaction with a custom-designed Affymetrix GeneChip® DNA array. More than 1,000 maize genes were found differentially expressed at a fold change of 2 or greater. This included the up regulation of defense-related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development. Maize kernels were mock inoculated at the blister (R2) or dough (R4) stage or inoculated with A. flavus at the blister (R2), milk (R3), dough (R4), or dent (R5) stage, and harvested 4 days later. Each treatment consisted of three biological replications. For each biological replication, 8 kernels were ground and RNA was isolated and further processed.
2014-05-14 | E-GEOD-57629 | biostudies-arrayexpress