CRISPR-Cas9 Knockout of the Epichloë Festucae Antifungal Protein Gene.

Strong creeping red fescue (Festuca rubra subsp. rubra) is a commercially important low-maintenance turfgrass and is often naturally infected with the fungal endophyte Epichloë festucae. Endophyte-mediated disease resistance to the fungal pathogen Sclerotinia homoeocarpa F.T. Bennett, the causal agent of dollar spot disease, has been observed in the field. In a previous quantitative transcriptome study of the Epichloë festucae–strong creeping red fescue interaction, the second most abundant fungal transcript was found to encode a protein similar to antifungal proteins from Penicillium and Aspergillus species. This E. festucae gene is designated Efe-afpA and the protein is designated Efe-AfpA. The protein has been shown to inhibit the growth of S. homoeocarpa in plate assays. To further study the role of this protein in the endophyte-mediated disease resistance, we generated a gene knockout of Efe-afpA using the CRISPR-Cas9 system. This gene was disrupted by an insertion of a fragment from the Cas9 vector at the cutting site of Cas9 endonuclease. This was confirmed by PCR and sequencing. Next, the knockout isolate will be reintroduced into endophyte-free strong creeping red fescue to evaluate susceptibility of the host plant to dollar spot disease using an established greenhouse assay.