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

Abstract

Ustilago maydis mutants resistant to polyene antibiotics were screened for defects in ergosterol biosynthesis. Slowgrowing mutants recovered after selecting for amphotericin B resistance were devoid of ergosterol and accumulated the methylated sterols, 14a-methylfecosterol, obtusifoliol and eburid, indicating that these isolates were impaired in C-14 sterol demethylation and were similar to the Erg40 mutant of U. m y & 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 A*-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 msjor sterols detected in these isolates were ergosta-7,22-dienol and ergosta-7-eno1, 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. All 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.

Alteration of Cell Wall Composition Leads to Amphotericin B Resistance in Aspergillus flavus

“…Most of the fungal strains resistant to polyene antibiotics have been obtained using mutagenic agents (11,22,28,36). However, Mazumder et al (29) selected nystatin-resistant Aspergillus niger by serial transfer in increasing concentrations of AmB, and Grunwald-Raij and Margalith (20) isolated nystatin-resistant strains of Saccharomyces cerevisiae by similar methods.…”

“…Reduced growth rate in polyeneresistant mutants of Candida albicans (36) and S. cerevisiae (3) has been reported previously. James et al (22) described AmB-resistant isolates of Ustilago maydis that exhibited reduced rates of growth and contained no ergosterol. Actually, ergosterol deficiency was thought to be the main cause of fungal growth delay (26,34).…”

ABotrytis cinereaEmopamil Binding Domain Protein, Required for Full Virulence, Belongs to a Eukaryotic Superfamily Which Has Expanded in Euascomycetes

“…Ergosterol is an essential structural element of the cell membranes, as well as a regulatory component (58). Therefore, an ergosterol-deficient mutant would be expected to grow more slowly than the wild type and exhibit abnormally branched morphology, as previously shown with Saccharomyces cerevisiae, Ustilago maydis, and Magnaporthe grisea mutants (1,29,33).…”

“…It is tempting to suggest that ⌬ 8 -⌬ 7 sterol isomerization is catalyzed by the B. cinerea ortholog of the yeast ERG2 protein, similar to that in Magnaporthe grisea and Ustilago maydis (29,33). A putative ortholog of the ERG2 gene was found in the B. cinerea genome (corresponding to Broad Institute protein identification no.…”

“…BcPIE3, the first protein of this clade to be characterized, was shown to be dispensable for ergosterol biosynthesis in B. cinerea. If the SI activity in fungi is indeed encoded by ERG2 orthologs, as shown for the euascomycete Magnaporthe grisea and the basidiomycete Ustilago maydis (29,33), then fungal EBP genes may have acquired different functions.…”