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Summary Metschnikowia pulcherrima synthesises the pigment pulcherrimin, from cyclodileucine (cyclo(Leu‐Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi. This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity of M. pulcherrima, we compared a wild‐type strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wild‐type and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator gene SNF2 in the mutant. Complementation of the mutant strain with the wild‐type SNF2 gene restored pigmentation and recovered the strong antifungal activity. Mass spectrometry (UPLC HR HESI‐MS) proved the presence of the pulcherrimin precursors cyclo(Leu‐Leu) and pulcherriminic acid and identified new precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wild‐type and complemented strain, but were undetectable in the pigmentless snf2 mutant strain. These results thus identify Snf2 as a regulator of antifungal activity and pulcherriminic acid biosynthesis in M. pulcherrima and provide a starting point for deciphering the molecular functions underlying the antagonistic activity of this yeast., Metschnikowia pulcherrima is a strong antifungal yeast and a promising species for biocontrol applications. This multidisciplinary study on the M. pulcherrima mode of action compared a wild‐type isolate with a pigmentless mutant exhibiting reduced antifungal activity. The transcriptional regulator Snf2 was identified as an important regulator of antifungal activity of M. pulcherrima via PUL gene transcription, cyclodipeptide synthesis and additional, yet uncharacterised mechanisms.

Metschnikowia pulcherrimasynthesizes the red pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi such asBotrytisandGibberella(i.e.,Fusarium). This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity ofM. pulcherrima, we compared a wildtype strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wildtype and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulatorSNF2in the mutant strain. Complementation of thesnf2mutant strain with the wildtypeSNF2gene restored pigmentation and recovered the strong antifungal activity ofM. pulcherrimaagainst plant pathogensin vitroand on cherries. Ultra-performance liquid chromatography-high resolution heated electrospray ionization mass spectrometry (UPLC HR HESI-MS) proved the presence and structure of the pulcherrimin precursors cyclo(Leu-Leu) and pulcherriminic acid and also identified new compounds that likely represented an additional precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wildtype and complemented strain, but were undetectable in the pigmentlesssnf2mutant strain. These results thus identifySNF2as a regulator of antifungal activity and pulcherriminic acid biosynthesis inM. pulcherrimaand provide a starting point for deciphering the molecular functions underlying the antagonistic activity of this yeast.Significance statementMetschnikowia pulcherrimais a strongly antifungal yeast and a most promising species for the control of notorious plant diseases. This multidisciplinary study on theM. pulcherrimamode of action compared a wildtype isolate with a pigmentless mutant exhibiting reduced antifungal activity. The transcriptional regulator Snf2 was identified as a “biocontrol regulator” controlling antifungal activity ofM. pulcherrimaviaPULgene transcription, cyclodipeptide synthesis and additional, yet uncharacterized mechanisms. The identification of cyclo(Leu-Leu), pulcherriminic acid, as well as novel precursor and degradation products of pulcherrimin, opens up new avenues for research on the metabolism and functions of pulcherrimin. Overall, this works establishesM. pulcherrimaas a genetically tractable model and will benefit the development of biocontrol solutions for important plant diseases.