殺菌剤ジエトフェンカルブの選択的殺菌メカニズムとβ-チューブリンのアミノ酸置換に関する研究

博士論文

Two carbendazim(MBC)-resistant mutants of Neurospora crassa, F914 and F939, were sensitive to diethofencarb at the concentration of 0.1 µg/ml, while the wild-type strain and other MBC-resistant mutants showed resistance to diethofencarb at the concentration of 100 µg/ml. Genetic analysis indicated that the mutations in these two strains were closely linked to the βml locus which codes for β-tubulin. When the wild-type strain was transformed by the cloned β-tubulin gene of the strain F914, the transformants showed MBC resistance as well as diethofencarb sensitivity in the presence of MBC. On the other hand, the diethofencarb sensitivity of the F914 strain was cancelled by transformation with the wild-type β-tubulin gene. DNA sequencing of the F914 β-tubulin gene revealed that glutamic acid at position 198 of β-tubulin in the wild-type strain was substituted by glycine in the F914 strain. Therefore, a single base change in the β-tubulin gene was proved to confer both the MBC resistance and diethofencarb sensitivity.In the presence of 0.5 µg/ml diethofencarb, conidia of a enzimidazole-resistant mutant of N. crassa, F914, germinated with distorted and swollen germ tubes. Diethofencarb induced scattering of nuclei and inhibited mitotic nuclear division in the F914 strain. The morphological abnormality was quite similar to the one observed in the wild-type strain treated with MBC. Diethofencarb, however, did not morphologically affect the germ tubes and nuclei of the wild-type strain. Diethofencarb formed a complex in vitro with a protein present in the mycelial extracts of the F914 strain, but such a complex was not bserved in the extracts or the wild type strain. The binding protein of the F914 strain was retained on a DEAE-Sephadex A-50 column and eluted with 0.5 M KCl. The molecular weight of the binding protein was estimated to be 105,000 by gel filtration on Sephacryl S-200. The data coincide with those on the MBC-binding protein in the wild-type strain, suggesting that diethofencarb was selectively toxic to the benzimidazole-resistant strain by binding to the mutated tubulin.After UV-treatment of the F914 strain, revertants resistant to diethofencarb were isolated and characterized. Two revertant strains FR422 and FR424 showed high resistance to N-phenylcarbamates and sensitivity to benzimidazoles, as can be seen in the wild-type strain. 0ther five revertants, FR511, FR513, FR521, FR421 and FRB3, were more resistant to diethofencarb than the strain F914 but less resistant to diethofencarb than the wild-type strain. These moderately resistant strains were more resistant to benzimidazoles than the parent strain F914. Among the strains, FR42l and FR521 were resistant to griseofulvin as well. All the diethofencarb-resistant mutations were linked to the Bml (β-tubulin) locus. To identify the changes in theβ-tubulin genes in the diethofencarb-resistant revertants, gene cloning and sequencing were carried out. The results showed that the amino acid changes, 250Leu to Phe, 165Ala to Val, 237Thr to Ala and 198Gly to Lys, were responsible for revertant phenotypes in the strains FR511, FR513, FR421 and FRB3, respectively.The amino acid substitution (165Ala to Val) of the β-tubulin in the FR513 strain was same to that of the thiabendazole-resistant mutant (benA-16) of Aspergillus nidulans, and this amino acid position was close to that of the mutation (167Phe to Tyr) of the benomyl-resistant mutant (Bml 511) of N. crassa. Furthermore, the position of amino acid substitution (237Thr to Ala) in FR421 strain was close to that of the mutation (241Arg tO His) of a benomyl-resistant mutant (Ben-R) of Saccharomyces cerevisiae, suggesting that diethofencarb shares the binding sites on β-tubulin with benzimidazoles. In strains FRB3, F914 and wild-type, the amino acid at position 198 were Lys, Gly and Glu, respectively. A new β-tubulin gene with Ala at position 198 was constructed by site-directed mutagenesis. This gene transformed the wild-type strain to MBC resistance, while transformation of F914 strain with this gene resulted in growth on a medium containing 0.5 µg/ml diethofencarb but not on that containing 75µg/ml diethofencarb. The F914 strain transformed by β-tubulin genes of the wild-type strain and the FRB3 strain grew even on a medium containing 75 µg/ml diethofencarb. These data suggest that the Glu at position 198 in β-tubulin is responsible for MBC sensitivity. Different amino acid substitutions at position 198 confer different sensitivities to diethofencarb and the order of amino acids that results in increasing sensitivity is Glu, Ala and Gly (most sensitive).Based on these data, a model for binding mode of diethofencarb and MBC to β-tubulin is described. These approaches should work in clarifying the function of β-tubulin and also aid in designing new potent fungicides to circumbate the drug resistance problem.

指導教授 : 山口勇