Induction of OS-2 phosphorylation in Neurospora crassa by treatment with phenylpyrrole fungicides and osmotic stress

Neurospora crassa is sensitive to phenylpyrrole fungicides and to high osmolarity. By contrast, the osmosensitive mutant os-2 is resistant to this class of fungicides. To understand the relationship between the phenylpyrroles fungitoxicity and the osmoregulatory pathway, we investigated the regulation of the mitogen-activated protein kinase (MAPK) OS-2 in Neurospora. The proteomic tools were used for the characterization of the OS-2 activation and for the measurement of the MAPK cascade induction in situ. Finally, the identification of the genes controlled by os-2 was attempted by comparing the proteomes of the wild type and the os-2 mutant strains. The results indicated that OS-2 is regulated by a dual phosphorylation on a Thr-X-Tyr motif. Furthermore, OS-2 undergoes a rapid phosphorylation after treatment with phenylpyrroles (fenpiclonil and fludioxonil) and under elevated salt concentrations in the surrounding medium. Finally, different enzymes involved in the metabolism of sugars and amino acids were identified as os-2-dependent.