Dataset Information

On Variant Discovery in Genomes of Fungal Plant Pathogens.

ABSTRACT: Comparative genome analyses of eukaryotic pathogens including fungi and oomycetes have revealed extensive variability in genome composition and structure. The genomes of individuals from the same population can exhibit different numbers of chromosomes and different organization of chromosomal segments, defining so-called accessory compartments that have been shown to be crucial to pathogenicity in plant-infecting fungi. This high level of structural variation confers a methodological challenge for population genomic analyses. Variant discovery from population sequencing data is typically achieved using established pipelines based on the mapping of short reads to a reference genome. These pipelines have been developed, and extensively used, for eukaryote genomes of both plants and animals, to retrieve single nucleotide polymorphisms and short insertions and deletions. However, they do not permit the inference of large-scale genomic structural variation, as this task typically requires the alignment of complete genome sequences. Here, we compare traditional variant discovery approaches to a pipeline based on de novo genome assembly of short read data followed by whole genome alignment, using simulated data sets with properties mimicking that of fungal pathogen genomes. We show that the latter approach exhibits levels of performance comparable to that of read-mapping based methodologies, when used on sequence data with sufficient coverage. We argue that this approach further allows additional types of genomic diversity to be explored, in particular as long-read third-generation sequencing technologies are becoming increasingly available to generate population genomic data.

SUBMITTER: Potgieter L

PROVIDER: S-EPMC7176817 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

On Variant Discovery in Genomes of Fungal Plant Pathogens.

Potgieter Lizel L Feurtey Alice A Dutheil Julien Y JY Stukenbrock Eva H EH

Frontiers in microbiology 20200416

Comparative genome analyses of eukaryotic pathogens including fungi and oomycetes have revealed extensive variability in genome composition and structure. The genomes of individuals from the same population can exhibit different numbers of chromosomes and different organization of chromosomal segments, defining so-called accessory compartments that have been shown to be crucial to pathogenicity in plant-infecting fungi. This high level of structural variation confers a methodological challenge f ...[more]

PMID: 32373089

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Project description:Magnaporthe oryzae and Ustilaginoidea virens are two filamentous fungal pathogens that threaten rice production worldwide. Genetic tools that permit fast gene deletion and silencing are of great interest for functional genomics of fungal pathogens. As a revolutionary genome editing tool, clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) enable many innovative applications. Here, we developed a CRISPR interference (CRISPRi) toolkit using nuclease activity dead Cas9 (dCas9) to silence genes of interest in M. oryzae and U. virens. We optimized the components of CRISPRi vectors, including transcriptional repression domains, dCas9 promoters, and guide RNA (gRNA) promoters. The CRISPRi tool was tested using nine gRNAs to target the promoters of MoATG3, MoATG7, and UvPal1. The results indicated that a single gRNA could direct the dCas9-fused transcriptional repression domain to efficiently silence the target gene in M. oryzae and U. virens. In both fungi, the target genes were repressed >100-fold, and desired phenotypes were observed in CRISPRi strains. Importantly, we showed that multiple genes could be easily silenced using polycistronic tRNA-gRNA in CRISPRi. Furthermore, gRNAs that bind different promoter regions displayed variable repression levels of target genes, highlighting the importance of gRNA design for CRISPRi efficiency. Together, this study provides an efficient and robust CRISPRi tool for targeted gene silencing in M. oryzae and U. virens. Owing to its simplicity and multiplexity, CRISPRi will be a useful tool for gene function discovery in fungal pathogens. IMPORTANCE Many devastating plant diseases are caused by fungal pathogens that evolve rapidly to adapt to host resistance and environmental changes. Therefore, genetic tools that enable fast gene function discovery are needed to study the pathogenicity and stress adaptation of fungal pathogens. In this study, we adopted the CRISPR/Cas9 system to silence genes in Magnaporthe oryzae and Ustilaginoidea virens, which are two dominant fungal pathogens that threaten rice production worldwide. We present a versatile and robust CRISPRi toolkit that represses target gene expression >100-fold using a single gRNA. We also demonstrated that CRISPRi could simultaneously silence multiple genes using the tRNA-gRNA strategy. The CRISPRi technologies described in this study would accelerate the functional genomics of fungal pathogens.

Dataset Information

On Variant Discovery in Genomes of Fungal Plant Pathogens.

Publications

On Variant Discovery in Genomes of Fungal Plant Pathogens.

Similar Datasets

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