Dataset Information

Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus.

ABSTRACT: Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen.

SUBMITTER: Ren S

PROVIDER: S-EPMC5036175 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus.

Ren Silin S Yang Mingkun M Li Yu Y Zhang Feng F Chen Zhuo Z Zhang Jia J Yang Guang G Yue Yuewei Y Li Siting S Ge Feng F Wang Shihua S

Scientific reports 20160926

Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A ...[more]

PMID: 27667718

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Project description:BACKGROUND: An available whole genome sequence for Aspergillus flavus provides the opportunity to characterize factors involved in pathogenicity and to elucidate the regulatory networks involved in aflatoxin biosynthesis. Functional analysis of genes within the genome is greatly facilitated by the ability to disrupt or mis-express target genes and then evaluate their result on the phenotype of the fungus. Large-scale functional analysis requires an efficient genetic transformation system and the ability to readily select transformants with altered expression, and usually requires generation of double (or multi) gene deletion strains or the use of prototrophic strains. However, dominant selectable markers, an efficient transformation system and an efficient screening system for transformants in A. flavus are absent. RESULTS: The efficiency of the genetic transformation system for A. flavus based on uracil auxotrophy was improved. In addition, A. flavus was shown to be sensitive to the antibiotic, phleomycin. Transformation of A. flavus with the ble gene for resistance to phleomycin resulted in stable transformants when selected on 100 mug/ml phleomycin. We also compared the phleomycin system with one based on complementation for uracil auxotrophy which was confirmed by uracil and 5-fluoroorotic acid selection and via transformation with the pyr4 gene from Neurospora crassa and pyrG gene from A. nidulans in A. flavus NRRL 3357. A transformation protocol using pyr4 as a selectable marker resulted in site specific disruption of a target gene. A rapid and convenient colony PCR method for screening genetically altered transformants was also developed in this study. CONCLUSION: We employed phleomycin resistance as a new positive selectable marker for genetic transformation of A. flavus. The experiments outlined herein constitute the first report of the use of the antibiotic phleomycin for transformation of A. flavus. Further, we demonstrated that this transformation protocol could be used for directed gene disruption in A. flavus. The significance of this is twofold. First, it allows strains to be transformed without having to generate an auxotrophic mutation, which is time consuming and may result in undesirable mutations. Second, this protocol allows for double gene knockouts when used in conjunction with existing strains with auxotrophic mutations. To further facilitate functional analysis in this strain we developed a colony PCR-based method that is a rapid and convenient method for screening genetically altered transformants. This work will be of interest to those working on molecular biology of aflatoxin metabolism in A. flavus, especially for functional analysis using gene deletion and gene expression.

Project description:Aspergillus flavus produces carcinogenic and mutagenic aflatoxins, which cause economic losses and risk of food safety by contaminating grains, food and feed. In this study, we characterized two bZIP transcription factors, AflatfA and AflatfB, and their genetic interaction. Compared to the wild type (WT), AflatfA deletion and AflatfA and AflatfB double deletion both caused retarded vegetative growth of mycelia. Relative to WT, the AflatfA deletion strain (ΔAflatfA) and AflatfA and AflatfB double deletion strain (ΔAflatfAΔAflatfB) produced more sclerotia, whereas the AflatfB deletion strain (ΔAflatfB) produced less sclerotia. After 4 °C preservation and incubation at 50 °C, conidia viability dramatically decreased in the ΔAflatfA and ΔAflatfAΔAflatfB but ΔAflatfB mutants, whereas conidia viability of the ΔAflatfAΔAflatfB strain was higher after storage at 4 °C than in AflatfA mutant. Conidia of ΔAflatfA, ΔAflatfB and ΔAflatfAΔAflatfB strains significantly increased in sensitivity to H2O2 in comparison with WT. Compared to WT, the mycelium of ΔAflatfA and ΔAflatfB strains were more sensitive to H2O2; conversely, the ΔAflatfAΔAflatfB strain showed less sensitivity to H2O2. ΔAflatfA and ΔAflatfAΔAflatfB strains displayed less sensitivity to the osmotic reagents NaCl, KCl and Sorbitol, in comparison with WT and ΔAflatfB strains. When on YES medium and hosts corn and peanut, ΔAflatfA and ΔAflatfAΔAflatfB strains produced less aflatoxin B1 (AFB1) than ΔAflatfB, and the AFB1 yield of ΔAflatfB was higher than that of WT. When WT and mutants were inoculated on corn and peanut, the ΔAflatfA and ΔAflatfAΔAflatfB but not ΔAflatfB mutants produced less conidia than did WT. Taken together, this study reveals that AflatfA controls more cellular processes, and the function of AflatfA is stronger than that of AflatfB when of the same process is regulated, except the response to H2O2, which might result from the effect of AflatfA on the transcriptional level of AflatfB.

Dataset Information

Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus.

Publications

Global Phosphoproteomic Analysis Reveals the Involvement of Phosphorylation in Aflatoxins Biosynthesis in the Pathogenic Fungus Aspergillus flavus.

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