Browsing by Author "Johnson LJ"

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    Complete Genome Sequence of Paenibacillus sp. Strain E222, a Bacterial Symbiont of an Epichloë Fungal Endophyte of Ryegrass.
    (American Society for Microbiology, 2020-10-08) Bastías DA; Jauregui R; Applegate ER; Altermann E; Card SD; Johnson LJ; Stajich JE
    We report on the whole-genome sequence of Paenibacillus sp. strain E222, a bacterium isolated from a fresh culture of Epichloë festucae var. lolii, a mutualistic fungal endophyte of perennial ryegrass. The genome has a size of 7.8 Mb and a G+C content of 46% and encodes 6,796 putative protein-coding genes.
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    CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis
    (Elsevier BV on behalf of the British Mycological Society, 2022-01) Rocafort M; Arshed S; Hudson D; Sidhu JS; Bowen JK; Plummer KM; Bradshaw RE; Johnson RD; Johnson LJ; Mesarich CH; Brown NA
    Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.