Isolation and characterization of polyene-resistant mutants from the maize smut pathogen, Ustilago maydis, defective in ergosterol biosynthesis

Ustilago maydis mutants resistant to polyene antibiotics were screened for defects in ergosterol biosynthesis. Slow-growing mutants recovered after selecting for amphotericin B resistance were devoid of ergosterol and accumulated the methylated sterols, 14 alpha-methylfecosterol, obtusifoliol and eburicol, indicating that these isolates were impaired in C-14 sterol demethylation and were similar to the Erg40 mutant of U. maydis. By contrast, nystatin- and pimaricin-resistant isolates which exhibited reduced growth rates showed a dysfunction in C-8 sterol isomerization. In these mutants (Erg2) ergosterol was replaced by the delta 8-sterols, ergosta-5,8,22-trienol, ergosta-8,22-dienol, fecosterol and ergost-8-enol. Analysis of a random sample of polyene-resistant isolates that grew normally revealed that although most retained a typical wild-type sterol profile, two of the isolates failed to accumulate ergosterol. The major sterols detected in these isolates were ergosta-7,22-dienol and ergosta-7-enol, suggesting a lesion in C-5 sterol desaturation in these mutants (Erg3). Of four Erg2 mutants recovered, one mutant, selected on nystatin, contained low but detectable amounts of ergosterol. Ergosterol was not detected immediately after selection in the three other pimaricin-resistant Erg2 mutants. Although the growth of three of the Erg2 mutants remained unchanged during non-selective culture, one mutant reverted and began to grow at a greater rate than the rest; analysis of the sterols produced by this strain revealed that ergosterol was now present, but at lower concentrations than those in the wild-type strain. No changes in the type of sterol formed were observed in the other slower-growing Erg2 mutants even after prolonged culture. AH the Erg2 mutants exhibited morphological abnormalities; sporidia were swollen and distorted, and the inability of sporidia to separate after cell division led to the development of highly branched, multicellular groups of cells.