Characterization of mutations in the two-component histidine kinase gene that confer fludioxonil resistance and osmotic sensitivity in the os-1 mutants of Neurospora crassa

Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resist ance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have p reviously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which a re highly resistant to iprodione and fludioxonil but moderately sensitive t o osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resist ance to these fungicides, we cloned and sequenced the mutant os-1 genes tha t encode putative osmo-sensing histidine kinase. Within the os-1 gene produ ct (Os1p), the type I strains, NM233t and Y256M209, carried a stop codon at amino acid position 308 and a frameshift at amino acid position 294, respe ctively. These mutation sites were located on the upstream of histidine kin ase and the response regulator domains of Os1p, strongly suggesting that ty pe I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for t hese fungicides to express their antifungal activity. The amino acid change s in Os1p, (625)Pro from Leu, (578)Val from Ala, and (580)Arg from Gly were found in the type II strains, M16, M155-1 and P5990, respectively. Os1p is novel in having six tandem repeats of 90 amino acids in the N terminal. Ea ch amino acid change of the type II strains was located on the fifth unit o f six tandem repeats. Type II strains with single amino acid changes were m ore sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important funct ion in osmo-regulation. (C) 2001 Society of Chemical Industry.