5.5.13
ABC TRANSPORTERS IN ASPERGILLUS NIDULANS

AC ANDRADE1, X XUEI2, G DEL SORBO3, PL SKATRUD2 and MA DE WAARD1

1Department of Phytopathology, Wageningen Agricultural University, Binnenhaven 9, 6700 EE Wageningen, The Netherlands; 2 Eli Lilly and Company, Indianapolis, IN 46285, USA; 3 Dipartimento di Arboricultura, Botanica e Patologia Vegetale, Via Universitá 100, 80055 Portici (NA), Italy.

Background and objectives
ATP-Binding Cassette (ABC) transporters constitute a large protein superfamily with members involved in the multi-drug resistance (MDR) phenomena in a variety of organisms, ranging from bacteria to humans. These proteins are transport ATPases which may have a wide range of substrates such as natural products (e.g. antibiotics or mating pheromones) or xenobiotics (e.g. drugs or fungicides). We use the saprophytic wild type fungus Aspergillus nidulans and laboratory-generated mutants selected for resistance to azoles, displaying an MDR phenomena, to study the role of ABC transporters in fungicide sensitivity and MDR.

Results and Conclusions
Four single copy genes, designated atrA (ABC transporter A), atrB, atrC and atrD were isolated, cloned and characterised. The deduced proteins contain two groups of six transmembrane regions (TM6), conserved Walker A and B motifs and two nucleotide binding folds (NBF). The topology of AtrAp and AtrBp is (NBF-TM6)2 whereas that of AtrCp and AtrDp (TM6-NBF)2. The function of these genes has been studied by analysis of the transcript levels in the wild-type strain (003) and azole resistant mutants (130 , 146 and 264) upon pre-treatment of mycelium with fungicides, plant defence compounds and secondary fungal metabolites in northern analysis experiments. A number of compounds were identified that upregulate the expression of specific atr genes. We have also found a differential effect of these compounds on transcription of the atr genes among the isolates tested. The results indicate that ABC transporters can play an important role in protection of the fungus against naturally toxic compounds and xenobiotics. Current research focuses on further analysis of the genes by construction of knock-out mutants followed by phenotypic characterisation.

References
1. Del Sorbo G et al., 1997. Mol Gen Genet 254, 417-426.
2. De Waard MA, 1997. Pestic Sci. 51, 271-275.