International Symposium on Cereal Leaf Blights 2019 | University College Dublin, Ireland | 22-24 May 2019
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The significant threat that necrotrophic pathogens pose to global wheat production has driven an increased interest in better understanding the genetic background of these fungi. Early genetic studies in these pathogens were hampered by a lack of efficient gene targeting techniques. CRISPR/Cas9, a new genome-editing tool, has revolutionized reverse genetics by providing an efficient method for targeted mutations. This technique deploys Cas9 endonuclease and a 20bp sequence, gRNA which identifies a 3bp sequence, protospacer adjacent motif in genome, resulting in binding of Cas9gRNA complex with target gene where Cas9 initiates gene editing.
Despite the effectiveness of CRISPR/Cas9 tool, its application has only been limited to a few model fungal strains. Therefore, we tested the efficacy of CRISPR/Cas9 approach in two necrotrophic fungi; Zymoseptoria tritici and Parastagonospora nodorum. We studied the delivery of CRISPR/Cas9 in the form of a preassembled protein-RNA complex, Cas9-RNP, for initiating targeted editing of marker genes. The RNP technique is a preferred approach for Cas9 delivery as the protein complex is quickly degraded by protein degradation pathways once inside the cell minimising off-target mutations.
We demonstrate that transformation of the RNP complex with a selectable marker harbouring flanking DNA homologous to target resulted in homologous recombination efficiencies exceeding 70% in P. nodorum. We further demonstrated that homologous recombination was effective, albeit at lower efficiencies, when using 50bp flanks. In contrast, we were unable to generate any CRISPR/Cas9 mutants in Z. tritici using the RNP approach. We further attempted genome editing by constitutively expressing Cas9 within Z. tritici but again was unsuccessful suggesting that the genome editing approach is ineffective in Septoria tritici blotch pathogen. These data highlight the significant potential that CRISPR/Cas9 has in improving reverse genetics in plant pathogenic fungi but also that its efficacy is dependent on the targeted pathogen.