Agrobacterium-meditated gene disruption using split-marker in Grosmannia clavigera, a mountain pine beetle associated pathogen
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Grosmannia clavigera is a fungal pathogen associated with the mountain pine beetle (Dendroctonus ponderosae) which is devastating large areas of western Canada’s conifer forests. This fungus also produces a dark melanin pigment that discolors pine sapwood. We have generated the draft genome of G. clavigera. However, functional characterization of genes identified in the genome sequence requires an efficient gene disruption method. In this work, we report a gene replacement strategy for G. clavigera using the Agrobacterium-mediated transformation in conjunction with linear or split-marker deletion cassettes. In addition, we used long flanking regions up to 3 kb from both sides of the targeted genes in our deletion cassettes. We assessed this gene disruption method with two genes from the melanin biosynthesis pathway that produce easily detectable white and red/brown mutant phenotypes: polyketide synthase and scytalone dehydratase. The approach yielded G. clavigera gene replacements with homologous recombination rates between 65 and 82%. For filamentous fungi, this is the first report showing that split-markers can be used with Agrobacterium-mediated transformation to generate appropriate mutants. This method can now be applied to efficiently identify genes involved in G. clavigera fungal pathogenicity and will facilitate understanding how the fungus overcomes the host defence system.
KeywordsOphiostomatoid Ascomycete Conifer Split-marker Co-transformation Melanin biosynthesis
We would like to thank Philippe Tanguay (Laurentian Forestry Centre, Québec) for discussion about AMT, Gordon Robertson (B.C. Cancer Agency Genome Sciences Centre) for suggestions that improved the manuscript, and Huang-Ju Chen for technical assistance. This work was supported by grants to CB and JB from the Natural Sciences and Engineering Research Council of Canada (NSERC) and funds to CB and JB for the Tria Project (http://www.thetriaproject.ca) provided by the Province of British Columbia through Genome British Columbia, by the Government of Alberta through Genome Alberta, and by Genome Canada.
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