Field-derived insights into rice bakanae management: A novel transcription factor RARF1 regulates resistance to respiration-inhibiting fungicides in Fusarium fujikuroi.

A comprehensive survey of field-isolated Fusarium fujikuroi populations was conducted to evaluate their sensitivity to the quinone outside inhibitor (QoI) orysastrobin. The assessment revealed widespread insensitivity to this fungicide, whereas canonical resistance-conferring mutations in the target cytochrome b (cytb) gene were absent in the representative isolates examined. To clarify the non-target site resistance (NTSR) mechanisms driving this phenomenon, we identified a novel Zn2Cys6 transcription factor, FFUJ_00276 (designated as RARF1, Respiration Associated Regulatory Factor 1), which was transcriptionally induced upon orysastrobin exposure. Functional characterization via CRISPR-Cpf1-mediated gene deletion demonstrated that Δrarf1 mutants exhibited increased susceptibility to both QoI and succinate dehydrogenase inhibitor (SDHI) fungicides. In planta assays indicated that while RARF1 is dispensable for fungal pathogenicity, it is required to sustain orysastrobin resistance during host infection. Comparative transcriptomic analysis revealed that the loss of RARF1 resulted in the coordinated downregulation of genes governing ribosome biogenesis and mitochondrial oxidative phosphorylation. Simultaneously, orysastrobin treatment triggered the upregulation of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters. These results establish RARF1 as a key regulatory component governing fundamental cellular bioenergetics under fungicidal stress. Importantly, this study provides a pioneering framework for exploring QoI NTSR mechanisms and highlights RARF1 as a promising molecular target for managing resistance in F. fujikuroi.