Expression and functional characterization of plant pleiotropic drug resistance transporters in heterologous and homologous hosts

Abstract

ABC transporters mediate ATP-dependent translocation of a wide range of structurally and functionally unrelated compounds across biological membranes. In our laboratory, we have identified plasma membrane ABC transporters belonging to the pleiotropic drug resistance (PDR) subfamily in the plant species Nicotiana plumbaginifolia and Nicotiana tabacum. Analysis of transgenic plants silenced for NpPDR1 and NtPDR3 expression indicated that they are involved in the plant response to fungal attack and to iron deficiency, respectively. However, identification of their substrates is required to fully understand their physiological roles. For that purpose, this work aimed at setting up a convenient expression system for plant PDR transporters. First, NpPDR1 and NtPDR3 were expressed in a heterologous system, the yeast Saccharomyces cerevisiae. Even though they did not properly localize to the plasma membrane, phenotypic characterization indicated that these plant PDR transporters are functional in yeast. Growth tests in the presence of toxic concentrations of putative substrates showed that NpPDR1 probably transports antifungal diterpenes while NtPDR3 seems to transport molecules structurally related to phenolic compounds that help to solubilize iron in the soil. Second, NtPDR3 expression in N. tabacum BY2 suspension cells resulted in its localization to the plasma membrane. Growth tests and direct transport assays confirmed phenolic compounds as substrates. Finally, expression of a 10-His tagged NtPDR3 led to its solubilization and partial purification by Ni-chromatography. Altogether, these data allowed us to identify NtPDR3 substrates and indicate that N. tabacum BY2 suspension cells are an interesting expression system for transport assays of plant PDR and possibly other ABC transporters.(AGRO 3) -- UCL, 201