Cloning and functional characterization of BcatrA, a gene encoding an ABC transporter of the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea) 5 Giovanni DEL SORBO a , Michelina RUOCCO b, *, Henk-jan SCHOONBEEK c , Felice SCALA a , Catello PANE a , Francesco VINALE a , Marteen A. DE WAARD c a Department Ar.Bo.Pa.Ve. – Section of Plant Pathology, University of Naples ‘‘Federico II’’, Via Universita ` 100, 80055 Portici (Naples), Italy b Institute for Plant Protection (CNR-IPP), Via Universita ` 130, 80055 Portici (Naples), Italy c Laboratory of Phytopathology, University of Wageningen, Binnenhaven 5, NL-6709 PD Wageningen, The Netherlands article info Article history: Received 9 August 2007 Received in revised form 23 November 2007 Accepted 10 January 2008 Corresponding Editor: Gareth W. Griffith Keywords: Gene expression Heterologous expression Multidrug resistance PDR5 Protein encoding abstract BcatrA was cloned from the plant pathogenic fungus Botryotinia fuckeliana (Botrytis cinerea) and sequenced. Sequence analysis revealed that BcatrA encodes a protein composed of 1562 amino acid residues displaying high similarity with various fungal ATP-binding cas- sette (ABC) transporters having the (NBF-TM 6 ) 2 topology. Expression of BcatrA is barely detectable during normal vegetative growth in liquid substrates. Transcript levels of BcatrA are enhanced in a dose- and time-dependent manner after treatment with cycloheximide or catechol, but not by a number of other drugs or fungicides, including fludioxonil, fenar- imol, imazalil, and the plant defense compounds pisatin and resveratrol. Quantitative analysis of BcatrA during the synchronized infection of bean leaves revealed an overaccu- mulation of the gene transcript at 6, 12 and 24 h post-inoculation, suggesting an involve- ment of the gene in the first steps of pathogenesis. Functional analysis of BcatrA was performed by targeted gene replacement in a wild-type strain of the fungus, and by over- expression in a mutant of Saccharomyces cerevisiae carrying multiple non-functional multi- drug-resistance genes. BcatrA replacement mutants did not show any significant increase in sensitivity to drugs, including inducers of BcatrA transcription, and displayed an unal- tered virulence on several common host plants of B. cinerea. However, when expressed in the heterologous system, BcatrA reduced sensitivity to cycloheximide and catechol, thus indicating the ability of the BcatrA product to function as a multidrug transporter. ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction ATP-binding cassette (ABC) transporters are polytopic membrane proteins utilizing the energy derived from ATP hydrolysis to mediate secretion of a number of endogenous or exogenous metabolites and synthetic toxicants. ABC transporters include a wide range of substrates, including peptides, sugars, steroids, hydrophobic drugs, and inorganic ions. These data indicate that ABC permeases are significant in a number of physiological processes, including those related to human health and explain why research on ABC transporters has attracted much attention in recent years. 5 The present paper is dedicated to the memory of Professor Giovanni Del Sorbo. * Corresponding author. Tel.: þ39 081 2539337; fax: þ39 081 2539339. E-mail address: miruocco@unina.it journal homepage: www.elsevier.com/locate/mycres mycological research 112 (2008) 737–746 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.01.005