Characterization of the chloromonilicin biosynthetic gene cluster in the brown rot fungus Monilinia fructicola

Monilinia fructicola is a major plant pathogen responsible for brown rot leading to blossom and twig blight and pre- and post-harvest fruit rot that cause significant 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 relevant antimicrobial activity against bacteria, yeasts, and plant pathogenic fungi. Functional analysis of BGC-10 was performed by CRISPR/Cas9-mediated targeted gene disruption. Mutants targeting the pks1 gene were characterized by sequence analysis at the target cleavage site, ddPCR assays for copy number determination of hygromicine insertion, and gene expression analysis. There were no statistical differences between mutants and the wild-type strain in conidia production and germination rate, colony growth, and virulence on artificially inoculated fruits. Moreover, metabolomic analysis using UHPLC-MS/MS showed that chloromonilicin production 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.