A Plant Plasma Membrane ATP Binding Cassette–Type Transporter Is Involved in Antifungal Terpenoid Secretion

Abstract

ATP binding cassette (ABC) transporters, which are found in all species, are known mainly for their ability to confer drug resistance. To date, most of the ABC transporters characterized in plants have been localized in the vacuolar membrane and are considered to be involved in the intracellular sequestration of cytotoxins. Working on the assumption that certain ABC transporters might be involved in defense metabolite secretion and their expression might be regulated by the concentration of these metabolites, we treated a Nicotiana plumbaginifolia cell culture with sclareolide, a close analog of sclareol, an antifungal diterpene produced at the leaf surface of Nicotiana spp; this resulted in the appearance of a 160-kD plasma membrane protein, which was partially sequenced. The corresponding cDNA ( NpABC1 ) was cloned and shown to encode an ABC transporter. In vitro and in situ immunodetection showed NpABC1 to be localized in the plasma membrane. Under normal conditions, expression was found in the leaf epidermis. In cell culture and in leaf tissues, NpABC1 expression was strongly enhanced by sclareolide and sclareol. In parallel with NpABC1 induction, cells acquired the ability to excrete a labeled synthetic sclareolide derivative. These data suggest that NpABC1 is involved in the secretion of a secondary metabolite that plays a role in plant defense. ń INTRODUCTIONThe ATP binding cassette (ABC) superfamily of membrane transporters is found in all prokaryotic and eukaryotic species. All members of this family are involved in the active transport of many chemically and structurally unrelated compounds and use ATP hydrolysis as a source of energy (Higgins, 1992). Several of these proteins have been identified by their ability to confer drug resistance, hence their designation as multidrug resistance (MDR) or pleiotropic drug resistance (PDR) proteins. For example, the human MDR1 (Chen et al., 1986), which is also designated P-glycoprotein, is implicated in resistance of cell lines to antitumor drugs (Ueda et al., 1987), PDR5 is a multidrug transporter found in Saccharomyces cerevisiae (Balzi et al., 1994), and Candida PDR1 (CDR1) and CDR2, identified in the yeast Candida albicans , contribute to fungicide resistance (Prasad et al., 1995;Sanglard et al., 1997). An analog of human MDR1 also has been found in prokaryotes (van Veen et al., 1996). Other ABC transporters were characterized by their ability to secrete internal compounds; this is the case with the S. cerevisiae protein STE6, which is involved in the transport of a mating pheromone peptide (Kuchler et al., 1989), and mammalian MDR2, which acts as a phospholipid translocator (Ruetz and Gros, 1994).Multidrug resistance-related proteins (MRPs) are ABC proteins that can be distinguished from the proteins described above by their possession of an N-terminal hydrophobic extension and an internal regulatory domain. In mammals, the MRPs HmMRP1 (Cole et al., 1992) and cMOAT (Buchler et al., 1996) transport glutathione S -conjugates and are implicated in the de...

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

“…749 and 750), that implies that the diffusion of xenobiotics through phospholipid bilayers in intact cells is normally negligible. It is now clear that transporters enhance (and are probably required for) the transmembrane transport even of hydrophobic molecules such as alkanes, 1316–1321 terpenoids, 1319,1322,1323 long-chain, 1324–1328 and short-chain 1329–1332 fatty acids, and even CO 2 . 1333,1334 This may imply a significantly enhanced role for transporter engineering in whole cell biocatalysis.…”

How drugs get into cells: tested and testable predictions to help discriminate between transporter-mediated uptake and lipoidal bilayer diffusion

“…While other mechanisms are also possible (Dikicioglu et al, 2014), the ABC transporter (Sá-Correia et al, 2009) Pdr18 (Teixeira et al, 2012) and the glyceroaquaporin Fps1 (Teixeira et al, 2009) have properties consistent with such a role as ethanol transporters in yeast, a fact of considerable biotechnological relevance (Dunlop et al, 2011). In the context of biofuels production (and ethanol is a biofuel), and based on similar strategies of toxicity resistance to the one that we exploited earlier (Lanthaler et al, 2011), we now also know them for a variety of other rather lipophilic substances such as alkanes (Tsukagoshi and Aono, 2000; Fernandes et al, 2003; Ankarloo et al, 2010; Chen et al, 2013; Doshi et al, 2013; Foo and Leong, 2013; Ling et al, 2013; Nishida et al, 2013), arenes (Kieboom et al, 1998b), terpenoids (Jasiński et al, 2001; Yazaki, 2006; Foo and Leong, 2013), long-chain fatty acids (Wu et al, 2006a,b; Khnykin et al, 2011; Lin and Khnykin, 2014; Villalba and Alvarez, 2014), short-chain fatty acids (Gimenez et al, 2003; Islam et al, 2008; Moschen et al, 2012; Sá-Pessoa et al, 2013), etc. These are all substances for which bilayer lipoidal diffusion was “once widely assumed” (and presumably still is in some quarters).…”

Beyond cellular detoxification: a plethora of physiological roles for MDR transporter homologs in plants

“…All PDRs characterized so far are plasma membrane transporters (Kang et al, 2011), and the first to be identified in plants, the Spirodella polyrhiza SpTUR2 and the Nicotiana plumbaginifolia NpPDR1, mediate the transport of terpenes (Jasinski et al, 2001; van den Brule et al, 2002). SpTUR2 expression in Arabidopsis confers resistance to the diterpenoid sclareol, as does the Arabidopsis AtABCG40 (Campbell et al, 2003) later shown to function in cellular uptake of the sesquiterpenoid ABA (Kang et al, 2010).…”

“…Another PDR transporter, AtABCG36, is involved in cadmium (Kim et al, 2007) and sodium toxicity (Kim et al, 2010) resistance. Following the findings that NpPDR1 secretes antifungal terpenoids (Jasinski et al, 2001) and contributes to basal plant defense (Stukkens et al, 2005), AtABCG36 was identified as a key factor in the resistance to fungal and bacterial pathogens (Kobae et al, 2006; Stein et al, 2006; Underwood and Somerville, 2013; Xin et al, 2013). Moreover, both AtABCG36 and AtABCG37 excrete a range of synthetic auxins, including 2,4-D, and indole-3-butyric acid (IBA), the natural IAA precursor (Ito and Gray, 2006; Strader and Bartel, 2009; Ruzicka et al, 2010).…”