Development of a Protoplast-Mediated TransformationSystem for Monilinia fructicola: A Tool for FunctionalGenomics

Abstract

Monilinia fructicola is a major causal agent of brown rot in stone fruit and is recognised as the most aggressive species within thegenus Monilinia. Despite its economic impact, the molecular basis of its pathogenicity remains poorly understood, due to the lackof efficient genetic transformation tools. This study aimed to develop and optimise a robust protoplast-mediated transformationprotocol for M. fructicola, providing a foundational platform for functional genomics in this pathogen. To achieve this, differentculture media and enzymatic combinations were tested to maximise protoplast yield and viability. Best results were obtainedusing hyphae grown in Murashige and Skoog (MS) medium supplemented with 0.1 M fructose, and enzymatic digestion with acombination of VinoTaste Pro and Yatalase, yielding up to 3 × 10 7 protoplasts g−1 of fresh mycelium. Regeneration rates reachedup to 60% under optimal conditions. Competent protoplasts were transformed with a self-propagating, unstable telomeric plas-mid conferring fenhexamid resistance, achieving a transformation efficiency of 38.8%, within or above the range reported forother filamentous fungi. Upon subculturing transformants under nonselective conditions, the resistance marker was lost infewer than 50% of cases, suggesting that plasmid persistence in M. fructicola is higher than expected. Although M. fructicola canbe transformed efficiently using circular DNA, the limited rate of plasmid loss may require further optimisation to enable fullutility in marker-free genome editing strategies. This work provides an essential step forward in enabling genetic manipulationin M. fructicola, opening the door to advanced tools such as CRISPR/Cas9-mediated genome editing.