Project description: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.
Project description: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-AC’s 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.
Project description:Aspergillus flavus first gained scientific attention for its production of aflatoxin, the most potent naturally occurring toxin and hepatocarcinogenic secondary metabolite. For several decades, The DNA methylation status of A. flavus remains to be controversial. We first applied bisulfite sequencing, the gold standard at present, in conjunction with a biological replicate strategy to investigate the DNA methylation profiling of A. flavus genome. Our results reveal that the DNA methylation level of this fungus turns out to be negligible, comparable to the unmethylated lambda DNA we set as the false positive control of our bisulfite experiments. When comparing the DNA methyltransferase homolog of A. flauvs with that from several selected hypermethylated speices, we find that the DNA methyltransferase homolog of A.flavus as well as the other Aspergillus members groups closely with the RID from Neurospora crassa and Masc1 from Ascobolus immerses, which has been reported as DMT-incapable, but it diverges distantly from the other capable DNA methyltransferases. We observe significant depletion of repeat components within the A. flavus, which may possibly explain the lack of DNA methylation in this fungus. What's more, the RIP-index of the repeat of A. flavus turns out to be higher than the fungi without RID-like enzyme, suggesting this asexual fungus may possibly possess RIP process during the obscure sexual-stage which is very evanescent and may potentially related to DNA methylation. This work contributes to our understanding on the DNA methylation status of A. flavus. Also, it reinforces our views on the DNA methylation in fungal species. What's more, our strategy of applying bisulfite sequencing to DNA methylation detection on species with low DNA methylation may serve as a reference for later scientific investigations on other hypomethylated species. Two replicates were subjected to bisulfite conversion independently, unmethylated lambda DNA as a false positive control is added to both replicates.
Project description:Dimethyl sulfoxide (DSMO) is a simple molecule widely used because of its great solvating ability. Beyond its physico-chemical properties, it is also biologically active, including on fungal species. Aspergillus flavus is a saprophytic and famous pathogenic fungus able to produce Aflatoxin B1 (AFB1), a potent carcinogenic mycotoxin which may contaminate many food crops. The aim of this study was to characterize the effect of DMSO on A. flavus transcriptome profile using high-throughput RNA-sequencing assay.
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.
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.
Project description:Objective: Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of GO analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. This study aimed to investigate the integrative function of the aflR gene in A. flavus. Design: In this study, we used Aspergillus flavus NRRL3357 as a wild-type strain (WT) and constructed a knockout strain of A. flavus ΔaflR by homologous recombination. Based on the transcriptomics technology, we investigated the metabolic effects of aflR gene on growth, development and toxin synthesis of A. flavus, and discussed the overall regulation mechanism of aflR gene on A. flavus at the transcriptional level. Results: The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. In addition, disrupted strains of the aflR gene produced relatively sparse conidia and a very small number of sclerotia. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly down-regulated. Meanwhile, the ΔaflR strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT strain. Conclusions: The results showed that the A. flavus aflR gene also played a positive role in the growth and development of fungi.