Pisatin resistance in Dictyostelium discoideum and Neurospora crassa: comparison of mutant phenotypes

Abstract

The pea phytoalexin pisatin, at its inhibitory concentration, was shown to have two distinct inhibitory effects on amoebae of the cellular slime mould Dictyostelium discoideum. One effect was cytolytic and was demonstrable even in non-growing cells whereas the second effect was observed only under conditions favourable to growth. Pretreatment with a sublethal concentration of pisatin induced the amoebae to acquire resistance to both these effects. Mutations in nysC that alter membrane sterols and confer resistance to the polyene antibiotics nystatin and pimaricin blocked resistance to the growth-associated inhibitory effect but did not affect acquisition of resistance to the cytolytic effect. The nysB sunD double mutant HK412 displayed a partially constitutive resistance to the cytolytic effect but, like the nysC mutants, was blocked in the acquisition of resistance to the growth-associated inhibitory effect. Pisatin-treated cells incubated in pisatin-free medium lost their ability to grow on pisatin-containing medium much more rapidly than they lost resistance to the cytolytic effect of pisatin. These results suggest that the induction of pisatin resistance may involve the turning-on of independent resistance mechanisms against the two inhibitory effects of pisatin. This could account for our inability to isolate pisatin-resistant mutants in a single step. The Neurospora crassa erg1 and erg3 mutants that have altered membrane sterols and are nystatin resistant displayed sensitivity to pisatin. The pisatin-sensitivity phenotype of the erg mutants was used in selections to identify complementing plasmids from an ordered Neurospora genomic library. The association of pisatin sensitivity with membrane sterol alterations in both D. discoideum and N. crassa supports the hypothesis that mechanisms underlying nondegradative pisatin resistance are evolutionarily conserved.