Targeted Gene Mutations in the Forest Pathogen Dothistroma septosporum Using CRISPR/Cas9

Abstract

Dothistroma needle blight, caused by Dothistroma septosporum, has increased in incidence and severity over the last few decades and is now one of the most important global diseases of pines. Disease resistance breeding could be accelerated by knowledge of pathogen virulence factors and their host targets. However, this is hindered due to inefficient targeted gene disruption in D. septosporum, which is required for virulence gene characterisation. Here we report the first successful application of CRISPR/Cas9 gene editing to a Dothideomycete forest pathogen, D. septosporum. Disruption of the dothistromin pathway regulator gene AflR, with a known phenotype, was performed using nonhomologous end-joining repair with an efficiency of >90%. Transformants with a range of disruption mutations in AflR were produced. Disruption of Ds74283, a D. septosporum gene encoding a secreted cell death elicitor, was also achieved using CRISPR/Cas9, by using a specific donor DNA repair template to aid selection where the phenotype was unknown. In this case, 100% of screened transformants were identified as disruptants. In establishing CRISPR/Cas9 as a tool for gene editing in D. septosporum, our research could fast track the functional characterisation of candidate virulence factors in D. septosporum and helps set the foundation for development of this technology in other forest pathogens.

Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing

“…Aspergillus is a genetically tractable species and when this CRISPR-Cas9 system was developed and optimised for this specific species, the gene inactivation rate was highly efficient (75%-100%) although was dependent on the guide and gene targeted ( Song et al., 2018 ; Kun et al., 2021 ). A similar gene inactivation rate was observed in the Dothistroma study (90%-100%), which was again gene and guide dependent ( McCarthy et al., 2022 ). In contrast, Venturia is a genetically intractable species and when this CRISPR-Cas9 system (not developed or optimised for this specific species) was used, the gene inactivation rate dropped to 0%-16.7%, dependent on the guide.…”

“…The CRISPR-Cas9 delivery system developed by Song et al. (2018) has now been successfully used in four different filamentous fungal species; Aspergillus niger ( Song et al., 2018 ; Kun et al., 2021 ), Venturia ( Rocafort et al., 2022 ), Dothistroma septosporum ( McCarthy et al., 2022 ), and Epichloë sp. Lp TG-3 strain AR37 (this study) to inactivate a range of genes.…”

Beyond the genomes of Fulvia fulva (syn. Cladosporium fulvum) and Dothistroma septosporum: New insights into how these fungal pathogens interact with their host plants

“…For instance, through continued genome sequencing of isolates collected from around the world, and in conjunction with gene editing techniques involving CRISPR‐Cas technology, it may be possible to identify a repertoire of core effector genes from F. fulva and D. septosporum that is essential for host colonization. Crucially, as a starting point for this analysis, CRISPR‐Cas technology has already been applied to D. septosporum (McCarthy et al, 2022 ). Such technology may also provide insights into the role(s) of the dispensable chromosome in F. fulva .…”

Characterization of two conserved cell death elicitor families from the Dothideomycete fungal pathogens Dothistroma septosporum and Fulvia fulva (syn. Cladosporium fulvum)

“…Therefore, it will be interesting to create strains in which genes corresponding to several or all family members are deleted to assess whether fungal virulence, particularly in the necrotrophic infection stage, is compromised. Recently, we reported the first successful application of CRISPR/Cas9 genome editing to D. septosporum by disrupting a gene encoding a secreted protein that elicited cell death in N. benthamiana and pine ( McCarthy et al, 2022 ), which will ultimately help with creating such strains.…”