Dataset Information

Molecular and structural basis of nucleoside diphosphate kinase-mediated regulation of spore and sclerotia development in the fungus Aspergillus flavus.

ABSTRACT: The fundamental biological function of nucleoside diphosphate kinase (NDK) is to catalyze the reversible exchange of the ?-phosphate between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP). This kinase also has functions that extend beyond its canonically defined enzymatic role as a phosphotransferase. However, the role of NDK in filamentous fungi, especially in Aspergillus flavus (A. flavus), is not yet known. Here we report that A. flavus has two NDK-encoding gene copies as assessed by qPCR. Using gene-knockout and complementation experiments, we found that AfNDK regulates spore and sclerotia development and is involved in plant virulence as assessed in corn and peanut seed-based assays. An antifungal test with the inhibitor azidothymidine suppressed AfNDK activity in vitro and prevented spore production and sclerotia formation in A. flavus, confirming AfNDK's regulatory functions. Crystallographic analysis of AfNDK, coupled with site-directed mutagenesis experiments, revealed three residues (Arg-104, His-117, and Asp-120) as key sites that contribute to spore and sclerotia development. These results not only enrich our knowledge of the regulatory role of this important protein in A. flavus, but also provide insights into the prevention of A. flavus infection in plants and seeds, as well as into the structural features relevant for future antifungal drug development.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6699835 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Molecular and structural basis of nucleoside diphosphate kinase-mediated regulation of spore and sclerotia development in the fungus Aspergillus flavus.

Wang Yu Y Wang Sen S Nie Xinyi X Yang Kunlong K Xu Peng P Wang Xiuna X Liu Mengxin M Yang Yongshuai Y Chen Zhuo Z Wang Shihua S

The Journal of biological chemistry 20190626 33

The fundamental biological function of nucleoside diphosphate kinase (NDK) is to catalyze the reversible exchange of the γ-phosphate between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP). This kinase also has functions that extend beyond its canonically defined enzymatic role as a phosphotransferase. However, the role of NDK in filamentous fungi, especially in Aspergillus flavus (A. flavus), is not yet known. Here we report that A. flavus has two NDK-encodin ...[more]

PMID: 31243100

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Project description:Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.

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.

Dataset Information

Molecular and structural basis of nucleoside diphosphate kinase-mediated regulation of spore and sclerotia development in the fungus Aspergillus flavus.

Publications

Molecular and structural basis of nucleoside diphosphate kinase-mediated regulation of spore and sclerotia development in the fungus <i>Aspergillus flavus</i>.

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