CRISPR/CAS9 mediated gene knockout in the brown rot fungus Monilinia fructicola

M. fructicola is one of the most important causal agents of brown rot causing blossom and twig blight, as well as pre- and post-harvest fruit rot with severe yield losses, especially on stone fruit. In this work, a simply and efficient method to generate targeted gene disruption mediated by a CRISPR/Cas9 ribonucleoprotein complex coupled with PEG-mediated transformation was applied to the reference strain Mfrc123 of M. fructicola. Based on genomic and transcriptomic data, the endo-β-1,4-xylanase (xyn11A-like) gene predicted as an effector candidate and the polyketide synthase (pks) in a gene cluster putatively involved in chloromonilicin biosynthesis were selected as genes of interest. Chloromonilicin has been described as a non-phytotoxic compound showing a strong antimicrobial activity. The recombinant Cas9 protein was extracted and purified from Escherichia coli RosettaTM (DE3) strain transformed with the pHis-parallel1-NLSH2BCas9 plasmid, and single guide RNAs (sgRNAs) designed and synthesized for the two genes. The ability of the purified Cas9 RNPs complex to efficiently cleave at the specified target sites was firstly confirmed in in vitro assays. No mutants for the putative effector gene of M. fructicola were obtained, while eight mutants were generated by targeting the pks gene. For all of them, PCR-based molecular analysis confirmed single, double or multiple insertions of the hygromycin cassette used as selective marker at the target site. Further molecular and phenotypic characterization of the obtained mutants is in progress. This will provide new knowledge of the functional role of a BGC potentially involved in the chloromonilicin biosynthesis in M. fructicola. This study was carried out within the Agritech National Research Center and received funding from the European Union Next-GenerationEU (PIANO NAZIONALE DI RIPRESA E RESILIENZA (PNRR) – MISSIONE 4 COMPONENTE 2, INVESTIMENTO 1.4 – D.D. 1032 17/06/2022, CN00000022). This manuscript reflects only the authors’ views and opinions, neither the European Union nor the European Commission can be considered responsible for them.