The Dual O-Methyltransferase and FAD-Dependent Monooxygenase <i>ESCB3</i> Is Essential for Elsinochrome Biosynthesis and Virulence in <i>Elsinoë arachidis</i>

The filamentous fungus <i>Elsinoë arachidis</i> is a major foliar pathogen responsible for peanut scab, which is a significant disease affecting commercial peanut cultivation. Elsinochrome (ESC), produced by numerous phytopathogenic <i>Elsinoë</i> species, is a non-host-selective polyketide phytotoxin with strong photosensitive activity and plays a crucial role in pathogenesis on host plants. In <i>E. arachidis</i>, a dual-domain enzyme encoded by the gene <i>ESCB3</i>, containing both O-methyltransferase and a FAD-dependent monooxygenase domain, has been identified. To elucidate the role of <i>ESCB3</i>, the biological function, expression pattern of the ESC biosynthesis gene cluster, and associated metabolomics analyses were investigated in the present study. An <i>ESCB3</i> deletion mutant (Δ<i>ESCB3</i>) was created by targeted gene disruption. Notably, ESC production was completely blocked in the Δ<i>ESCB3</i> mutant, and the expression of ESC biosynthetic genes, except for the polyketide synthase gene <i>ESCB1</i>, was significantly downregulated. Additionally, the Δ<i>ESCB3</i> exhibited heightened sensitivity to multiple stress tolerance compared with the wild type, especially oxidative stress/H₂O₂, highlighting the crucial role of <i>ESCB3</i> in growth, development, and ESC biosynthesis in <i>E. arachidis</i>. Pathogenicity assays revealed a significant reduction in the pathogenicity of the Δ<i>ESCB3</i> mutant, suggesting a possible correlation with the suppressed biosynthesis of ESC. Metabolomic analyses further confirmed that <i>ESCB3</i> is indispensable for the ESC biosynthetic process and acts as a key regulatory factor. Collectively, the results of this study provide significant insights into the molecular mechanisms governing <i>ESCB3</i>-mediated virulence and ESC production in <i>E. arachidis</i>, offering potential targets for disease control strategies in peanut scab. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.