RESEARCH ARTICLE Mary S. Connolly Æ Yasuko Sakihama Vipaporn Phuntumart Æ Yinjun Jiang Franklin Warren Æ Lindsay Mourant Paul F. Morris Heterologous expression of a pleiotropic drug resistance transporter from Phytophthora sojae in yeast transporter mutants Received: 24 July 2005 / Revised: 24 July 2005 / Accepted: 28 July 2005 / Published online: 5 November 2005 Ó Springer-Verlag 2005 Abstract A system for the expression of an ATP binding cassette (ABC) transporter from the soybean pathogen Phytophthora sojae is described. Pdr1, an ABC trans- porter with homology to the pleiotropic drug resistance (PDR) family of transporters, was cloned by primer walking from a P. sojae genomic library. Reverse transcriptase PCR assays showed that the transcript disappeared after encystment of zoospores and was not detected in hyphal germlings in dilute salts, in hyphae growing in liquid V8 media, or in tissue extracts from infected hypocotyls. BLAST analysis of Pdr1 against the P. sojae EST database also revealed that this gene was present only in zoospore libraries. Comparison of the number of hits to Pdr1 with that of a set of house- keeping genes revealed that Pdr1 was expressed at rates two- to threefold higher than other transcripts. To test the hypothesis that Pdr1p functions as a broad substrate membrane transporter, Pdr1 was transformed into yeast mutants deficient in several drug resistance transporters. Yeast mutants transformed with Pdr1 possessed partial drug resistance against only 5 of 17 chemically distinct compounds. Thus, when expressed in yeast, this trans- porter has a significantly narrower substrate specificity in comparison to the yeast transporters, Pdr5p, Yorlp, and Snq2p. Keywords ABC transporter Æ Zoospores Æ Oomycetes Introduction Phytophthora species are oomycetes grouped with other heterokonts (having two different flagella) in the Kingdom Stramenopila (Van de Peer and De Wachter 1997; Baldauf 2003). Oomycetes grow in the soil and infect plants as hyphae, but are distinct from fungi in having diploid nuclei and coenocytic cells with cellu- losic rather than chitinaceous cell walls (Erwin and Ribeiro 1996). Oomycete pathogens attack a wide variety of plant species, some causing significant eco- nomic damage to multiple agricultural species, while others have a narrow host range (Erwin and Ribeiro 1996). Phytophthora sojae is a soil-borne pathogen of soybeans that has a significant economic impact in the United States (Wrather et al. 2001; Dorrance et al. 2003) and Canada (Anderson and Buzzell 1992). Under flooding conditions, vegetative hyphae produce vertical branches with terminal sporangia. Each sporan- gia release 20–30 single-celled asexual zoospores. In agricultural fields, the zoospore is a particularly effective dispersal agent (Carlile 1983). Zoospores use two flagella to swim at 125–150 lms 1 (Ho and Hickman 1967) through the surface films of water in the soil, and are rapidly dispersed by water movements in soils (Carlile 1983). Both the zoospores and hyphae are chemotactically attracted by isoflavones released from soybean roots (Morris and Ward 1992; Morris et al. 1998). Upon reaching the root the zoospores encyst, a developmental process that results in the loss of flagella and formation of a cell wall (Carlile 1983). The cyst germinates and hyphae ramify through the host tissues. When the nutrients are exhausted, the hyphae form sexual gametangia. The fer- tilized oospore has a thick cell wall and enables the pathogen to remain dormant in the soil overwinter (Erwin and Ribeiro 1996). Communicated by S. Hohmann Present address: M. S. Connolly Department of Biology, University of Dayton, Dayton, OH 45469, USA Present address: Y. Sakihama Laboratory of Ecological Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan Present address: Y. Jiang Department of Experimental Radiation Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA V. Phuntumart Æ F. Warren Æ L. Mourant Æ P. F. Morris (&) M. S. Connolly Æ Y. Sakihama Æ Y. Jiang Biology Sciences, Bowling Green State University, Bowling Green, OH 4340, USA E-mail: pmorris@bgnet.bgsu.edu. Tel.: +1-419-3720481 Fax: +1-416-3722024 Curr Genet (2005) 48: 356–365 DOI 10.1007/s00294-005-0015-4