5.5.7
STUDIES ON THE ROLE OF ABC TRANSPORTERS IN MULTIDRUG RESISTANCE AND PATHOGENICITY OF FILAMENTOUS FUNGI

G DEL SORBO1, M RUOCCO1, AC ANDRADE2, H SCHOONBEEK2, M LORITO1, A ZOINA1, MA DE WAARD2 and F SCALA1

1Dipartimento di Arboricoltura Botanica e Patologia Vegetale, Sezione di Patologia Vegetale, UniversitÓ di Napoli and CETELOBI, 80055 Portici (Naples), Italy, 2Department of Phytopathology, Wageningen Agricultural University, P.O. Box 6025, 6700EE Wageningen, The Netherlands

Background and objectives
ATP-binding cassette (ABC) transporters are ATP-dependent permeases which mediate the transport of a number of substrates including small organic molecules, peptides, sugars, heavy metal cations etc. over biological membranes. Overexpression of some ABC transporters determines simultaneous resistance to a series of chemically unrelated compounds, a phenomenon known as multidrug resistance (MDR). Other ABC transporters mediate important physiological functions such as secretion of mating factors or ability to import and utilize nutrients. We hypothesize that ABC transporters of filamentous fungi have an important role in a number of processes such as resistance to environmental toxicants either produced by soil microflora or introduced by human activities (e.g. fungicides, heavy metals, pollutants), secretion of factors necessary for the establishment of a compatible interaction with a host plant or for creation of a favourable microenvironment. In addition, ABC transporter-dependent secretion could protect pathogens against accumulation of defense compounds (phytoncides, phytoalexins) released or produced de-novo by the host plant upon pathogenic attack.

Results and conclusions
We cloned and sequenced several genes encoding putative ABC transporters from the saprophytic model fungus Aspergillus nidulans [1] and from the plant pathogen Botrytis cinerea. The gene atrB (ABC transporter B) of Aspergillus nidulans is constitutively expressed at low level. Its transcription rate increases dramatically within a few minutes upon treatment with several fungicides, antibiotics or plant phytoalexins. Functional expression of the cDNA of this gene in Saccharomyces cerevisiae determines restoration of normal levels of sensitivity towards a number of compounds, including cycloheximide, chloramphenicol, triazole fungicides and some plant defense products, in mutants lacking the gene PDR5, which encodes the major yeast multidrug resistance pump. The expression patterns and functional analysis of three more genes (atrA, atrC and atrD) encoding putative ABC transporters is being investigated.
In B. cinerea (anamorph of Botryotinia fuckeliana), two genes, coded Bc-atrA and Bc-atrB encoding typical ABC transporter have been isolated and sequenced. Bc-atrA is constitutively expressed at a low level. Treatment of germlings of B. cinerea with cycloheximide determines a strong and rapid increase in the level of Bc-atrA transcript. The same effect is observed, to a lower extent, by some other antibiotics, triazole fungicides and plant defense compounds. In order to study the role of Bc-atrA in MDR and pathogenesis, we generated knock out mutants by transformation-mediated gene disruption. The absence of intact copies of Bc-atrA does not cause changes in colony morphology, sporulation, ability to form sclerotia and growth rate on common growth media. Bc-atrA disruptants display a slight increase in sensitivity to triazole fungicides and to the fluorescent dye rhodamine 6G but not to cycloheximide, chloramphenicol, cadmium sulphate and the grape phytoalexin resveratrol. This evidence indicate a role of Bc-atrAp in protection of the fungus against toxicants. Virulence and saprophytic fitness of disruptants are being investigated.

References
1. Del Sorbo G, Andrade AC, Van Nistelrooy JGM, Van Kan JAL, Balzi E, De Waard MA, 1997. Mol. Gen. Genet. 254, 417-26.