CRISPR/CAS9 mediated knockout of the pks1 gene in the chloromonilicin biosynthetic gene cluster of the brown rot fungus Monilinia fructicola

Monilinia fructicola is a major pathogen responsible for brown rot leading to blossom and twig blight, as well as pre- and post-harvest fruit rot that cause heavy yield losses, especially on stone fruit. Analysis of the M. fructicola genome sequence (Genbank accession number GCA_008692225.1) revealed a type I polyketide synthase gene cluster, named BGC-10, putatively involved in chloromonilicin biosynthesis. This gene cluster, ∼55 kb in length, consists of twenty-four genes, including one polyketide synthase (pks1), one flavin-dependent halogenase, two N-acetyltransferases and three methyltransferases, six oxidoreduction-related genes, one scytalone dehydratase and two NAD-dependent epimerases, one lactamase-like protein gene, two transcription factors, and three transporter protein genes. Chloromonilicin has been described as a nonphytotoxic compound showing a strong antimicrobial activity against bacteria, yeasts, and plant pathogenic fungi. To investigate the function of BGC-10, a simple and efficient method for targeted gene disruption was performed by CRISPR/Cas9 ribonucleoprotein complex coupled with polyethylene glycol (PEG)-mediated transformation to the reference strain Mfrc123 of M. fructicola. Mutants targeting the pks1 gene were characterized by sequence analysis at the target cleavage, digital droplet PCR assays to determine the copy number of hygromycin insertion and gene expression analysis. Phenotypic characterization of the mutants revealed no statistical differences between mutants and the wild-type strain in conidia production and germination rate, colony growth, and virulence on artificially inoculated peach fruits. Moreover, metabolomic analysis using UHPLC-MS/MS showed that production of chloromonilicin and other metabolites putatively associated with BGC-10 (i.e., chloromonilinic acid A and B, and 4-chloropinselin) significantly increased in wild-type strains co-cultured with Penicillium expansum compared with monoculture and was very low after inoculation on fruits. Mutants did not produce detectable amounts of these metabolites in all tested growing conditions.