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

Abstract

Osmotic-sensitive (os-1) mutant alleles in Neurospora crassa exhibit resistance to dicarboximides, aromatic hydrocarbons and phenylpyrroles. We have previously reported that the os-1 mutants can be classified into two groups based on their resistance to fungicides and osmotic stress: type I, which are highly resistant to iprodione and fludioxonil but moderately sensitive to osmotic stress, and type II, which are highly sensitive to osmotic stress but moderately resistant to fungicides. To explain the mechanism of resistance to these fungicides, we cloned and sequenced the mutant os-1 genes that encode putative osmo-sensing histidine kinase. Within the os-1 gene product (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, respectively. These mutation sites were located on the upstream of histidine kinase and the response regulator domains of Os1p, strongly suggesting that type I strains are null mutants. The null mutants, NM233t and Y256M209, were highly resistant to iprodione and fludioxonil; thus Os1p is essential for these fungicides to express their antifungal activity. The amino acid changes in Os1p, 625Pro from Leu, 578Val from Ala, and 580Arg 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. Each amino acid change of the type II strains was located on the fifth unit of six tandem repeats. Type II strains with single amino acid changes were more sensitive to osmotic stress than the null mutants (type I), indicating that the amino acid repeats of Os1p were responsible for an important function in osmo-regulation.

The Response Regulator RRG-1 Functions Upstream of a Mitogen-activated Protein Kinase Pathway Impacting Asexual Development, Female Fertility, Osmotic Stress, and Fungicide Resistance inNeurospora crassa

“…Further analysis showed that the os-1 mutant did not produce viable ascospores 10 d postfertilization (data not shown). The Y256M209 os-1 mutant allele was generated using UV light and contains a frameshift mutation at amino acid 294 (Ochiai et al, 2001). The difference in phenotypes observed for ⌬rrg-1 and os-2 versus os-1 mutants supports the hypothesis that the OS-2 MAPK pathway is regulated by multiple histidine kinases under different environmental conditions (e.g., osmotic stress vs. nitrogen starvation) in Neurospora.…”

“…For example, macroconidia from ⌬rrg-1 mutants develop a deep orange pigment and lyse easily and ⌬rrg-1 strains grow poorly in high concentrations of NaCl, KCl, and sorbitol. We determined that ⌬rrg-1 mutants were resistant to certain fungicides, a characteristic also associated with os mutants (Fujimura et al, 2000a,b;Ochiai et al, 2001;Zhang et al, 2002). We uncovered evidence suggesting that RRG-1 is upstream of the OS-4/OS-5/OS-2 MAPK pathway, in that OS-2-phosphate levels were greatly reduced in ⌬rrg-1 mutants relative to wild type.…”

“…Subsequent work demonstrated that nik-1 is allelic with os-1 (Schumacher et al, 1997) and that three additional os genes (os-4 MAPKKK, os-5 MAPKK, and os-2 MAPK) comprise an MAPK pathway in Neurospora whose members are similar to those of the S. cerevisiae Hog1p system (Zhang et al, 2002;Fujimura et al, 2003). Mutation of os genes also leads to increased resistance to phenylpyrrole and dicarboximide fungicides (Fujimura et al, 2000a,b;Ochiai et al, 2001;Zhang et al, 2002). Of interest, wild-type S. cerevisiae strains are resistant to these fungicides (Zhang et al, 2002;Motoyama et al, 2005), but they are rendered sensitive by introduction of an OS-1/NIK-1-related histidine kinase gene from the filamentous fungus Magnaporthe grisea (Motoyama et al, 2005).…”

Interactions among HAMP Domain Repeats Act as an Osmosensing Molecular Switch in Group III Hybrid Histidine Kinases from Fungi

“…NIK1 orthologs from filamentous fungi are directly linked with the resistance to few important fungicidal agents used in agriculture. Point mutations in different HAMP domain repeats in Nik1p or its orthologs from B. fuckeliana and C. heterostrophus could confer varying degrees of drug resistance and osmosensitivity to the host (17,27,41). Thus, the structural insights about the HAMP domain repeats will not only help to understand these interesting mutants but also will aid in developing novel fungicides in the future.…”

Role of MAP Kinase Signaling in Secondary Metabolism and Adaptation to Abiotic/Fungicide Stress in Fusarium

“…In silico analysis of HKs by reciprocal BLASTP searches in Fusarium genome sequences led to the identification of Fhk1 (FOXG_01684) orthologues both in F. verticillioides (hypothetical protein FVEG_08048) and F. graminearum (hypothetical protein FGSG_07118) (Nagygyörgy and Ádám, unpublished). HKs have five HAMP (histidine kinase, adenylate cyclase, methyl binding protein and phosphatase) repeats: mutations in these sequences are responsible for the increased osmosensitivity and fungicide resistance of N. crassa, C. heterostrophus, Alternaria brassicicola and Botrytis cinerea (Ochiai et al, 2001;Yoshimi et al, 2004;Motoyama et al, 2005;Viaud et al, 2006). Recent microarray analyses have further shown that the transcriptional response to fludioxonil depends on a Hog1 orthologue in A. nidulans.…”