Thus, this website activation of Hog1p correlated with the inhibition of the yeast’s growth by fludioxonil and both effects required the functionality of the domains that are essential for the histidine kinase function of the protein, which involves
phosphorylation of both His510 and Asp924 of CaNik1p. Figure 3 Hog1p phosphorylation after fludioxonil treatment was dependent on the functionality of conserved domains of CaNik1p. The phosphorylation of Hog1p (upper panel, Hog1-P) was detected by Western blot after treatment of the strains YES, NIK, N627, D924 and H510 with fludioxonil (10 μg/ml) and sorbitol (1 M), respectively, for 15 min. The presence of Hog1p in all strains was proven (lower panel, Hog1). Hog1p appeared at approximately 50 kDa. Since high concentrations of sorbitol activate the HOG pathway via inhibition of the HK Sln1p, treatment of the transformants with 1 M sorbitol was used as a positive CP673451 cell line control. Normal growth of the yeast was inhibited upon expression of CaNik1pΔHAMP and was restored by inhibition of the HOG pathway Previous work had shown that deletion of single and
double pairs of HAMP domains of CaNik1p affected the susceptibility of the resultant mutants Selleckchem SGC-CBP30 to the fungicides , and for the HK DhNik1 it was described that deletion of four out of five amino acid repeats generated a constitutively active HK, which could not be inhibited by fludioxonil . Thus we decided to delete all HAMP domains from CaNIK1p. Transforming S. cerevisiae with a plasmid carrying a truncated version of CaNIK1, in which all HAMP domains were deleted from the protein, resulted
in the ΔHa and ΔHb strains (Table 1). These strains were able to grow on SD-ura agar plates, where expression of CaNIK1ΔHAMP was not induced. Surprisingly no growth was observed on SG-ura plates, where galactose induced the expression of CaNIK1ΔHAMP (Figure 4). This indicated that the presence of CaNIK1ΔHAMP had inhibitory effects on the growth of the S. cerevisiae LY294002 transformant, whereas deletion of up to two pairs of HAMP domains did not affect growth of the transformed strain ΔH3H4  (Figure 4A). Simultaneous inactivation of the HisKA domain by the H510Q point mutation restored normal growth of the resultant transformed strains ΔHaH510 and ΔHbH510 (Figure 4). Figure 4 CaNIK1ΔHAMP expression led to growth inhibition that was dependent on His510 (A) and a functional HOG pathway (B). (A) Strains BWG1-7a, YES, NIK, ΔHa, ΔHaH510 and ΔH3H4 were streaked on SD-ura and SG-ura agar plates and incubated at 30°C for 4 days. Strain BWG1-7a was the parent strain which is auxotrophic for uracil. (B) Strains BY4741, ΔHbΔhog, ΔHbΔpbs2, ΔHbΔssk1, ΔHbH510 and ΔHb were streaked on SD-ura and SG-ura agar plates and incubated at 30°C for 4 days. BY4741 was the parent strain of the single gene deletion mutants, which is auxotrophic for uracil.