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CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum.

ABSTRACT: Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, we developed a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two different strategies, homology-independent targeted integration (HITI) and homology-dependent recombination integration (HDRI). The HITI strategy was able to facilitate integration of a large DNA fragment, ?8 kb in length, into the genome of F. oxysporum at the sgRNA cleavage site, and was used to insert a C-terminal 3×sGFP tag to the FoCHS5 gene and a N-terminal mCherry tag to the FoSSO2 gene. The HDRI strategy was used to tag the paralogous gene, FoSSO1, with a C-terminal mCherry marker. FoChs5-3×sGFP localized to conidia, some septa, and fungal tips. A majority of the FoSso1-mCherry was distributed in the conidia, septum, and hyphae that were distal from the fungal tips. While FoSso1-mCherry showed a very weak fluorescent signal at the fungal tips, mCherry-FoSso2 accumulated in the plasma membrane of conidia, germlings, fungal tips, hyphae, and phialides, suggesting FoSSO1 and FoSSO2 are regulated differently during fungal development. These results indicate this EGT system is efficient and can be another molecular tool to resolve the function(s) of proteins and infection strategies of F. oxysporum.

SUBMITTER: Wang Q

PROVIDER: S-EPMC6456373 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum.

Wang Qiang Q Coleman Jeffrey J JJ

Fungal genetics and biology : FG & B 20190206

Fusarium oxysporum is an economically important pathogen that widely exists in the environment and is capable of causing serious problems in crop production and animal/human health. One important step for characterization of a fungal protein with an unknown function is to determine its subcellular localization within the cell. To facilitate the study of important functional regulators or key virulence factors, we developed a CRISPR/Cas9-mediated endogenous gene tagging (EGT) system based on two ...[more]

PMID: 30738140

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Project description:BackgroundGene editing using CRISPR/Cas9 is a widely used tool for precise gene modification, modulating gene expression and introducing novel proteins, and its use has been reported in various filamentous fungi including the genus Fusarium. The aim of this study was to optimise gene editing efficiency using AMA1 replicator vectors for transient expression of CRISPR constituents in Fusarium venenatum (A3/5), used commercially in the production of mycoprotein (Quorn™).ResultsWe present evidence of CRISPR/Cas9 mediated gene editing in Fusarium venenatum, by targeting the endogenous visible marker gene PKS12, which encodes a polyketide synthase responsible for the synthesis of the pigment aurofusarin. Constructs for expression of single guide RNAs (sgRNAs) were cloned into an AMA1 replicator vector incorporating a construct for constitutive expression of cas9 codon-optimised for Aspergillus niger or F. venenatum. Vectors were maintained under selection for transient expression of sgRNAs and cas9 in transformed protoplasts. 100% gene editing efficiency of protoplast-derived isolates was obtained using A. niger cas9 when sgRNA transcription was regulated by the F. venenatum 5SrRNA promoter. In comparison, expression of sgRNAs using a PgdpA-ribozyme construct was much less effective, generating mutant phenotypes in 0-40% of isolates. Viable isolates were not obtained from protoplasts transformed with an AMA1 vector expressing cas9 codon-optimised for F. venenatum.ConclusionsUsing an AMA1 replicator vector for transient expression of A. niger cas9 and sgRNAs transcribed from the native 5SrRNA promoter, we demonstrate efficient gene editing of an endogenous marker gene in F. venenatum, resulting in knockout of gene function and a visible mutant phenotype in 100% of isolates. This establishes a platform for further development of CRISPR/Cas technology in F. venenatum for use as a research tool, for understanding the controls of secondary metabolism and hyphal development and validating prototypes of strains produced using traditional methods for strain improvement.

Dataset Information

CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum.

Publications

CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum.

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