A novel fungicide aminopyrifen inhibits GWT-1 protein in glycosylphosphatidylinositol-anchor biosynthesis in Neurospora crassa

Aminopyrifen, 4-phenoxybenzyl 2-amino-6-methylnicotinate, strongly inhibited the mycelial growth of a wild type Neurospora crassa strain on Vogel's minimal medium containing 1.2% sucrose, with a 0.001 mg/L concentration required for 50% growth inhibition. Similar to micafungin, an inhibitor of beta-1, 3-glucan synthetase, aminopyrifen further inhibited the growth of N. crassa deletion mutants of MAP kinase cascade genes, such as mak-1 and mak-2, than the wild-type strain, suggesting that aminopyrifen perturbs cell wall-related processes. Furthermore, we found that three chitin synthase gene mutants (chs-1, chs-5, and chs-7) were highly sensitive to both chemicals; however, aminopyrifen, but not micafungin, induced a swollen germ tube from the conidia of chs-5 and chs-7 mutants on Vogel's medium containing 1.2% sucrose. To elucidate the target protein of aminopyrifen, we isolated mutants resistant to aminopyrifen after UV treatment of conidia of the wild-type strain or the chs-5 strain. The resistance mutations were localized to the gwt-1 gene that encodes an acyltransferase, GWT-1, which participates in the biosynthesis of the glycosylphosphatidylinositol (GPI) precursor, and were found to result in S180F and V178A alterations in the protein. These results strongly suggest that aminopyrifen works as an inhibitor targeting GWT-1, a protein involved in GPI-anchor biosynthesis.