Vacuolar H ⁺ -ATPase subunit a was identified as the target protein of the oomycete inhibitor fluopicolide

Approximately 240 fungicides are currently in use. However, only a few direct targets have been identified, which limits the development of fungicides and rapid resistance monitoring. Fluopicolide, which is an excellent oomycete inhibitor, is classified as delocalization of spectrin-like proteins inhibitors by FRAC. In the current study, a Pcα-actinin knockout had no effect on the sensitivity of Phytophthora capsici to fluopicolide. The vacuolar H + -ATPase subunit a (PcVHA-a) was identified using a BSA-seq and DARTS assay. Four kinds of point mutations in PcVHA-a that cause fluopicolide resistance in P . capsici were confirmed using site-directed mutagenesis. The results of MST, molecular docking, and a DARTS assay indicated that PcVHA-a could bind fluopicolide. Sequence analysis and a molecular docking assay proved the specificity of fluopicolide to oomycetes or fish. Our results suggest that PcVHA-a is the target of fluopicolide, and H + -ATPase could be used as a novel target for the development of new fungicides.