Results The conserved domains of CaNik1p were essential for the susceptibility of S. cerevisiae transformants to antifungals After alignment with other HKs, in CaNik1p histidine 510 and aspartate 924 were identified as the essential residues for the HisKA and the

REC domains respectively [17] and asparagine 627 for the N-box of the ATP-binding domain. Hence, to inhibit the conserved phosphorylation reactions within CaNik1p, mutant genes were generated, in which either Asn627 from the HATPase_c domain was substituted by aspartate (N627D), His510 by glutamine (H510Q) or Asp924 by asparagine (D924N). S. cerevisiae was transformed with the plasmids carrying the mutated CaNIK1 genes, and the resultant transformants were treated with the antifungals fludioxonil, MCC950 order iprodione

and ambruticin VS3. As shown in Figure 2, the strain EPZ5676 purchase YES transformed with the empty vector was resistant to all fungicides, while the strain NIK was susceptible to the studied antifungals. The H510Q and D924N point Selleck Rabusertib mutations in the HisKA and REC domains respectively, led to complete loss of susceptibility, while the N627D substitution in the HATPase_c domain only decreased the susceptibility to the fungicides in comparison to the strain NIK. Figure 2 The conserved domains of CaNik1p were essential for the susceptibility to the fungicides. The phenylpyrrole fludioxonil, the dicarboximide iprodione and the myxobacterial secondary metabolite ambruticin VS3 were used as representative PIK3C2G antifungal compounds targeting fungal group III histidine kinases. Error bars represent the standard deviation from three independent experiments. His510 and Asp924 are the conserved phosphate-accepting residues in the HisKA and the REC domains, respectively, which are required for kinase function of hybrid HKs. They are phosphorylated by the histidine kinase activity of the protein (His510) and the subsequent phosphate-transfer to the REC domain within the same protein (Asp924). Loss of fungicide susceptibility of the respective mutants suggested that the functionality

of both the HisKA and the REC domain was essential for the antifungal activity. Probably the N627D mutation did not completely prevent ATP binding to the HATPase_c domain and as a result only a partial effect was obtained. Functional HisKA, HATPase_c and REC domains were essential for the phosphorylation of Hog1p after fludioxonil treatment Treatment with fludioxonil led to phosphorylation of the MAPK Hog1p, i.e. to the activation of the HOG pathway, in S. cerevisiae transformed with full-length and truncated forms of CaNIK1[25]. Therefore, phosphorylation of Hog1p was also analyzed after fludioxonil-treatment of S. cerevisiae transformed with CaNIK1 carrying the H510Q, N627D and D924N point mutations.