Project description:RNA-seq was used to compare differential gene expressions for Aspergillus flavus wild type strain and ASPES transcription factor deletion strains.The goals of this study are to explore the aflatoxin regulation pathway in A. flavus.
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: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.
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:The experiment consisted of a field design containing two maize inbred lines Va35 and Mp313E. Va35 has yellow kernels and is susceptible to kernel infection by Aspergillus flavus (A. flavus). Mp313E is a white dent maize inbred line and was released primarily as a source of resistance to kernel infection by A. flavus. The test ears were inoculated with Aspergillus flavus and collected two days after inoculation. Non-inoculated ears collected 16 days after pollination were also used as a control. The microarray experimental design was a randomized complete block design with three replications. One microarray slide was used for each field plot. Each slide contained the inoculated and non-inoculated sub-treatments. A second slide for one replication of each genotype contained a dye swap for the inoculation treatment and three subsamples (dots) within the slide represented each contig. Keywords: Direct comparison
Project description:Mycotic leratitis is the corneal inflammation predominantly caused by Fusarium and Aspergillus species. Corneal epithelium is the earliest cell type encounter the invading pathogen. The innate immune responses of human corneal epithelial cells against Aspergillus flavus is not known. Here we studied the role of human corneal epithelial cells against Aspergillus flavus infection. The results showed that corneal epithelial cells internalized Aspergillus flavus conidia through actin mediated polymerization surrounding the conidia. Further the actin inhibitor cytochalasin D treatment reduced the formation actin ring around the conidia. The engulfed conidia acquired endosomal proteins as revealed by immunofluorescence analsyisis. Mass spectromtery of phagosomal proteins confirmed the recruitment of endosomal proteins and other proteins involved in phagocytosis. These results show the involvement of corneal epithelial cells in anti fungal defense.
Project description:Frequently observed in tropical and sub-tropical regions, crops contamination by aflatoxin B1 (AFB1) produced by Aspergillus flavus, is emerging in Europe, due to climate change. Many alternative methods are currently developed to reduce the use of chemical inputs to prevent mycotoxin contamination, such as biocontrol agents (BCAs). Actinobacteria are known to produce many bioactive compounds and some of them are able to reduce in vitro AFB1 concentration. In this context, the present study aims to analyze the effect of a cell free supernatant (CFS) from Streptomyces roseolus liquid culture on A. flavus development, as well as on its transcriptome profile using microarray assay and its impact on AFB1 concentration. To study the impact of Streptomyces roseolus cell free supernatant on global transcriptome of Aspergillus flavus we have employed whole genome microarray expression profiling.
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.
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.