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Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus.

ABSTRACT: 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.

SUBMITTER: He ZM

PROVIDER: S-EPMC2212646 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus.

He Zhu-Mei ZM Price Michael S MS Obrian Gregory R GR Georgianna D Ryan DR Payne Gary A GA

BMC microbiology 20071126

<h4>Background</h4>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 ...[more]

PMID: 18039373

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Project description:Reversible phosphorylation by protein kinases and phosphatases plays a central role in regulating cellular processes. However, knowledge of the functions of protein phosphatase 2C (PP2C) S/T phosphatases in Aspergillus flavus has been unreported until now. Here, we have identified seven members of the PP2C family of protein phosphatases in A. flavus. Evolutionary and functional analyses indicated that two redundant PP2C phosphatases, Ptc1 and Ptc2, are highly conserved and regulate conidia development, aflatoxin synthesis, seed infection, and autophagic vesicle formation. The cytoplasmic proteins Ptc1 and Ptc2 exhibit nuclear infiltration after DNA damage-induced autophagy. Their degradation is closely linked to autophagy induction. The Asp residue coordinated with Mg2+ is critical for phosphatase Ptc1 and Ptc2 activity, thermal stability, and biofunction in A. flavus. An immunoprecipitation-mass spectrometry proteomic investigation indicated that 133 proteins co-interact with Ptc1 and Ptc2. Among these proteins, phosphoglycerate kinase 1 (PGK1) interacts with Ptc1 and Ptc2 and shows high levels of phosphorylation in Δptc1, Δptc2, and Δptc1/ptc2 mutants. Furthermore, PGK1 S203 phosphorylation levels are associated with aflatoxin synthesis and autophagic vesicle formation. Overall, these findings contribute to our understanding of the roles and mechanisms of PP2C family phosphatases in A. flavus and highlight their importance in various cellular processes. Furthermore, it reveals a new regulation model in A. flavus, where Ptc1 and Ptc2 activate autophagy and aflatoxin synthesis by regulating PGK1. IMPORTANCE Aspergillus flavus is a model filamentous fungus that can produce aflatoxins when it infects agricultural crops. This study evaluated the protein phosphatase 2C (PP2C) family as a potential drug target with important physiological functions and pathological significance in A. flavus. We found that two redundant PP2C phosphatases, Ptc1 and Ptc2, regulate conidia development, aflatoxin synthesis, autophagic vesicle formation, and seed infection. The target protein phosphoglycerate kinase 1 (PGK1) that interacts with Ptc1 and Ptc2 is essential to regulate metabolism and the autophagy process. Furthermore, Ptc1 and Ptc2 regulate the phosphorylation level of PGK1 S203, which is important for influencing aflatoxin synthesis. Our results provide a potential target for interdicting the toxicity of A. flavus.

Dataset Information

Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus.

Publications

Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus.

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