5.5.31
ATP-BINDING CASSETTE TRANSPORTERS INVOLVED IN MULTIDRUG RESISTANCE IN THE PLANT PATHOGENIC FUNGI

T HIBI1, R NAKAUNE1, K ADACHI1, O NAWATA1, M NAKAJIMA2, H HAMAMOTO1, YJ LEE1, Y MAKIZUMI1, M TOMIYAMA3 and K AKUTSU2

1Department of Agricultural and Environmental Biology, The University of Tokyo, Bunkyo-ku, Tokyo 1 1 3-8657, Japan; 2 Faculty of Agriculture, Ibaraki University, Ami-machi, Ibaraki 300-3, Japan; 3Department of Biotechnology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305, Japan

Background and objectives
Many demethylation inhibitor's (DMIs)-resistant field strains have developed recently in various plant pathogenic fungi. However, the resistant mechanism has not been clear. In the yeasts and the human cells, the multidrug resistance genes encoding ATP-binding cassette (ABC) transporters (P-glycoproteins) were demonstrated to confer the resistance against many kinds of drug. Previously, DMIs-resistant strains of the plant pathogenic fungi Penicillium digitatum were shown to be resistant simultaneously to cycloheximide, 4-nitr,oquinoline-N-oxide (4NQO) and achfiavine [1]. Therefore, we intended to detect, clone and sequence the ABC-transporter gene of P. digitatum and analysed the roles of the gene in relation to the DMIs-resistance. The homologs of the ABC-transporter gene were also detected in the other several species of the plant pathogenic fungi.

Results and conclusions
PMR1,(Penicillium multidrug resistance 1), gene encoding a ABC-transporter was cloned from a genomic DNA library of a DMIs-resistant strain of P.digitatum by heterologous hybridization with a DNA fragment including ABC of Saccharomyces cerevisiae. Sequence analysis revealed the significant amino acid homology among the primary structures of PMR1(protein encoded by PMR1 gene) and ABCtransporters of the yeasts and Aspergillus nidulans. Disruption of PMR1 gene of P.digitatum DMIs-reistant strain LC2 demonstrated that PMR1 was an important determinant of the resistance to DMIs. Northern blot analysis indicated that several-fold more PMR1 transcripts were constitutively accumulated in the DMIs-resistant strains as compared with those in DMIs-sensitive strains in the absence of fungicide. In both DMIs-resistant and -sensitive strains, the transcription of PMR1 was strongly enhanced within 10 min after treatment with a DMI fungicide triflumizole. These results suggested that the toxicant efflux system comprised of PMR1 participate directly in the DMI-resistance of the fungus and that PMR1 in resistant strains is constitutively more active than that in sensitive strains and immediately operative upon contact with fungicides. The homologes of PMR1 were detected, cloned and sequenced also from the other plant pathogenic fungi, including Magnaporthe grisea and Botrytis cinerea.

References
1. Nakaune R, Adachi K, Nawata 0, Tomiyama M, Akutsu K, Hibi T, Applied and Environmental Microbiology, submitted.