Characterization of the Chloromonilicin Biosynthetic Gene Cluster in the Brown Rot Fungus Monilinia fructicola by a CRISPR/Cas9 Transformation System

Monilinia fructicola is a major pathogen responsible for brown rot, causing substantial yield losses, particularly in stone fruit. Genomic analysis identified a type I polyketide synthase gene cluster, approximately 55 kb in length and consisting of twenty-four genes, putatively involved in chloromonilicin biosynthesis. These include a polyketide synthase (PKS), flavin-dependent halogenase, N- acetyltransferases, methyltransferases, a lactamase-like protein, scytalone dehydratase, and genes related to oxidoreduction, transcription factors, and transporters. To elucidate the product of the cluster, targeted gene disruption was achieved using a CRISPR/Cas9 ribonucleoprotein complex and polyethylene glycol-mediated transformation in the reference strain Mfrc123. Mutants with disruption in the ChmN core gene (encoding the PKS) were analysed by sequencing, digital droplet PCR to assess the insertion copy number of the hygromycin resistance cassette, and gene expression profiling. Phenotypic analysis indicated no significant differences between mutants and the WT strain in conidia production, germination rate, colony growth, or virulence on inoculated fruits. Metabolomic profiling using UHPLC-MS/MS demonstrated that the mutants did not produce chloromonilicin or other cluster-associated metabolites, such as chloromonilinic acids C and D, and 4-chloropinselin, indicating that the gene cluster is essential for their biosynthesis. In the WT strain, production of these metabolites increased following co-culture with Penicillium expansum compared to monoculture and remained low after fruit inoculation, suggesting a role in competition rather than pathogenesis. Gene expression analysis further showed upregulation of ChmN in response to various plant-associated microbes, but not to other Monilinia species.