Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic profiling of Aspergillus flavus in response to 5-azacytidine & gallic acid

ABSTRACT: Aspergillus flavus is a common saprophyte and opportunistic pathogen producing aflatoxin (AF) and many other secondary metabolites. 5-Azacytidine (5-AC), a derivative of nucleoside cytidine, is widely used for studies in epigenetics and cancer biology as an inactivator of DNA methyltransferase and is also used for studying secondary metabolism in fungi. Our previous studies showed that 5-AC affects development and inhibits AF production in A. flavus, and that A. flavus lacks DNA methylation. How this common DNA methyltransferase inhibitor affects development and AF production is not clear. In this study, we applied an RNA-Seq approach to elucidate the mechanism of 5-ACM-bM-^@M-^Ys effect on A. flavus. In our current study, we identified 240 significantly differently expressed (Q-value<0.05) genes after 5-AC treatment, including two backbone genes in secondary metabolite clusters #27 and #35, which are involved in development or survival of sclerotia. With 5-AC treatment, about three quarters of the genes in the AF biosynthetic gene cluster in A. flavus were down-regulated to a certain degree. Strikingly, at least two genes aflI and aflLa, were completely inhibited. Interestingly, several genes involved in fungal development were down-regulated, especially veA, which is a gene that encodes protein bridges VelB and LaeA. This result supports the hypothesis that 5-AC affects development and AF production through weakening or even interrupting the connection between VelB and LaeA and then causing dysregulation of the expression pattern of genes involved in development and secondary metabolism. Our results improved the A. flavus genome annotation, provided a comprehensive view of the transcriptome of A. flavus responding to 5-AC and confirmed that fungal development and secondary metabolism are co-regulated. In additon, the RNA-Seq data of another sample treated with gallic acid was used to improve A. flavus genome annotation. mRNA of Aspergillus flavus cultured in three different culture media PDB, PDB+5-AC(5-Azacytidine),and PDB+GA(gallic acid) was subjected to sequence independently.

ORGANISM(S): Aspergillus flavus

SUBMITTER: Zhumei He

PROVIDER: E-GEOD-40202 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Transcriptomic profiling of Aspergillus flavus in response to 5-azacytidine.

Lin Jian-Qing JQ Zhao Xi-Xi XX Zhi Qing-Qing QQ Zhao Ming M He Zhu-Mei ZM

Fungal genetics and biology : FG & B 20130501

Aspergillus flavus is a common saprophyte and opportunistic pathogen producing aflatoxin (AF) and many other secondary metabolites. 5-Azacytidine (5-AC), a derivative of the nucleoside cytidine, is widely used for studies in epigenetics and cancer biology as an inactivator of DNA methyltransferase and is also used for studying secondary metabolism in fungi. Our previous studies showed that 5-AC affects development and inhibits AF production in A. flavus, and that A. flavus lacks DNA methylation. ...[more]

PMID: 23644151

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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.

Project description:Diverse expression patterns for secondary metabolism gene clusters from Aspergillus flavus under different environmental conditions and in genetic mutants: Insights into regulation of cyclopiazonic acid along with aflatoxin Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis predicts that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in A. flavus. Presumably, secondary metabolites play important roles in the ecology of the producing species, but functions for most secondary metabolites remain unknown. Only three metabolic pathways have been associated with the predicted clusters in A. flavus. These include aflatoxin, cyclopiazonic acid (CPA), and aflatrem. To gain insight into the regulation of, and infer ecological significance for the 55 secondary metabolite gene clusters predicted in A. flavus, we examined their expression over 28 diverse conditions. Variables included culture media and temperature, fungal development, colonization of developing maize seeds, and misexpression of laeA, the global regulator of secondary metabolism. Hierarchical clustering analysis of expression profiles allowed us to categorize the gene clusters into four distinct clades. Gene clusters for the production of aflatoxins, CPA, and seven other unknown compound(s) were identified as belonging to one clade. To further explore the relationships found by gene expression analysis, aflatoxin and CPA production were quantified under five different cell culture environments known to be conducive or non-conducive for aflatoxin biosynthesis and during colonization of developing maize seeds. Results from these studies showed that secondary metabolism gene clusters have distinctive gene expression profiles. Aflatoxin and CPA were found to have unique regulation but similar enough that they would be expected to co-occur in commodities colonized with A. flavus.

Dataset Information

Transcriptomic profiling of Aspergillus flavus in response to 5-azacytidine & gallic acid

Publications

Transcriptomic profiling of Aspergillus flavus in response to 5-azacytidine.

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