Alteration of different domains in AFLR affects aflatoxin pathway metabolism in Aspergillus parasiticus transformants

AFLR, a zinc binuclear cluster DNA-binding protein, is required for activation of genes comprising the aflatoxin biosynthetic pathway in Aspergillus spp. Transformation of Aspergillus parasiticus with plasmids containing the intact aflR gene gave clones that produced fivefold more aflatoxin pathway metabolites than did the untransformed strain. When a 13-bp region in the aflR promoter (positions -102 to -115 with respect to the ATG) was deleted, including a portion of a palindromic site previously shown to bind recombinant AFLR, metabolite production was 40% that of transformants with intact aflR. This result provides further evidence that this site may be involved in the autoregulation of aflR, Overexpression of pathway genes could also result from increased quantities of AFLR titrating out a putative repressor protein. In AFLR, a 20-amino-acid acidic region near its carboxy-terminus resembles the region in yeast GAL4 required for GAL80 repressor binding. When 3 of the acidic amino acids in this region were deleted, levels of metabolites were even higher than those produced by transformants with intact aflR, as would be expected if repressor binding was suppressed in transformants containing this altered protein. Transformation with plasmids mutated at the AFLR zinc cluster (Cys to Trp at amino acid position 49) or at a putative nuclear localization signal region (RRARK deleted) gave clones with one-fifth the metabolite production of the untransformed fungus in spite of the transformants making the same or more aflR mRNA. Since these transformants retained a copy of intact aflR, the latter results can be explained best by assuming that AFLR activates genes involved in aflatoxin production as a dimeric protein and that heterodimers containing both mutant and intact AFLR strands are inactive. (C) 1998 Academic Press.