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The ascomycete Grosmannia clavigera (Gc) is a fungal pathogen of pine trees (Pinus spp.) and is vectored by the mountain pine beetle (Dendroctonus ponderosae, MPB). MPB, Gc and other vectored microorganisms form an interactive biological complex that has caused a rapid, large-scale decline of lodgepole pine (Pinus contorta) in western North America (Lee et al., 2005). Grosmannia clavigera can kill trees and stain the sapwood blue or black when it is inoculated manually into trees at a certain density; such discoloration reduces the commercial value of lumber. In British Columbia alone, the MPB epidemic has already killed over 16 million hectares of lodgepole pine forests. With the recent spread of the MPB epidemic into forests east of the Rocky Mountains and an expansion of its host range into Jack pine (Pinus banksiana) (Cullingham et al., 2011), the MPB–microbial complex now threatens the Canadian boreal forest. This large-scale disturbance has caused massive economic losses to forest-based industries, and has important implications for forest ecosystem stability and global atmospheric carbon balance (Kurz et al., 2008).
While the MPB–Gc complex can successfully colonize > 20 different pine species, its preferred host is P. contorta (Safranyik et al., 2010). Like all conifers, the pine hosts of the MPB epidemic have complex oleoresin-based chemical defences that protect these trees against most potential pests and pathogens (Keeling & Bohlmann, 2006a,b; Boone et al., 2011; Bohlmann, 2012). The oleoresin of most conifers consists predominantly of monoterpenes and diterpene resin acids, with smaller amounts of sesquiterpenes. These terpenes can be fungistatic or fungicidal. Small lipophilic monoterpenes diffuse easily into and through eukaryotic cell membranes, interact with membranes and membrane-bound enzymes, and can change membrane fluidity and ultrastructure (Parveen et al., 2004; Bakkali et al., 2008; Witzke et al., 2010). They can also cause fungal cells to swell, shrink and vacuolize (Soylu et al., 2006). While antimicrobial properties of monoterpenes are documented, little is known about the mechanisms used by some microorganisms, particularly fungi that colonize conifers, to survive and grow in the presence of monoterpenes. The highly specialized MPB–Gc complex, which colonizes the monoterpene-rich environment of pine phloem and sapwood, requires mechanisms to overcome host defence chemicals. To discover mechanisms involved in the ability of Gc to cope with host terpenes, we analysed the Gc genome and transcriptome to identify genes that are differentially expressed in response to terpene treatments (DiGuistini et al., 2011). We noted that a gene annotated as an ATP-binding cassette (ABC) transporter, GcABC-G1 (previously reported as GLEAN_8030), was highly up-regulated in the terpene-induced Gc transcriptome responses (DiGuistini et al., 2011).
Fungal plant pathogens evolve a combination of strategies to colonize and survive in the unfavourable conditions occurring in their living environment, the host. These strategies include transporting toxic chemicals out of the cell or sequestrating them in cellular organelles, detoxifying host defence compounds by converting or modifying them and interfering with host signalling (Morrissey & Osbourn, 1999). Mechanisms can involve enzymes such as cytochrome P450s and membrane proteins such as ABC or MFS transporters (Han et al., 2001; Coleman & Mylonakis, 2009). Fungal ABC transporters are well known for their roles in the secretion of harmful chemicals (Sipos & Kuchler, 2006; Coleman & Mylonakis, 2009; Coleman et al., 2011). Typical ABC transporters consist of two transmembrane domains (TMDs) and two nucleotide-binding folds (NBFs); ‘half-transporters’ contain only one TMD and one NBF. ABC transporters are classified into subfamilies according to sequence homology and domain topology (Sipos & Kuchler, 2006; Lamping et al., 2010). In eukaryotes, eight major subfamilies have been defined: ABC-A to ABC-H (Dean & Allikmets, 2001; Verrier et al., 2008). Among these, full-size ABC-B, ABC-C and ABC-G are, respectively, referred to as multidrug resistance (MDR), multidrug resistance-associated protein (MRP) and pleiotropic drug resistance (PDR) (Paumi et al., 2009; Kovalchuk & Driessen, 2010). Fungal PDR transporters are located in the cytoplasmic membrane and function as efflux pumps, contributing to drug resistance, chemical sensitivity and cellular detoxification (Coleman & Mylonakis, 2009; Lamping et al., 2010).
Here, we characterized the responses of Gc to a range of individual terpenes and terpene mixtures. We identified and classified all putative ABC transporter genes in the Gc genome. We then profiled their expression and characterized the function of GcABC-G1, which was the gene most strongly induced by terpenes. Deleting this gene in Gc, and expressing it heterologously in Saccharomyces cerevisiae demonstrated that GcABC-G1 confers tolerance to monoterpenes. GcABC-G1 appears to be specific to monoterpenes and to function as an efflux ABC transporter. Transcripts of GcABC-G1 were detected in stem tissues of young lodgepole pine trees inoculated with Gc. The development of the symptoms in young trees that were inoculated with the deletion mutant was delayed relative to trees inoculated with the wild-type Gc, suggesting that GcABC-G1 contributes to the fungus' ability to overcome host defence chemicals and survive in an environment that is highly unfavourable to most organisms. Plant terpenes, beyond being important in conifer defence (Keeling & Bohlmann, 2006a,b) and in the interactions of plants with other organisms (Gershenzon & Dudareva, 2007), are also being actively explored for metabolic engineering of biofuels and bioproducts in microbial hosts (Bohlmann & Keeling, 2008; Peralta-Yahya et al., 2011). The discovery of a role of GcABC-G1 in tolerance to monoterpenes may lead to applications for improved microbial production systems for terpenoids (Dunlop et al., 2011; Ignea et al., 2011).
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- Materials and Methods
- Supporting Information
To survive and become established in a pine tree, the MPB symbiont Gc has to overcome preformed or induced host defence chemicals (Bohlmann, 2012). Terpenoids, and specifically monoterpenes, are among the most abundant antimicrobial pine defence chemicals. In previous work, we reported that terpenes induce a stress response and activate a cluster of Gc genes that may be involved in detoxification or tolerance of host terpenes, and that monoterpenes can serve as a sole carbon source for Gc (DiGuistini et al., 2011). In the present work, we demonstrated a role for GcABC-G1 in tolerance to certain monoterpenes, using a combination of growth experiments with a genetic deletion in Gc and heterologous expression in S. cerevisiae. We also inoculated young pine trees with Gc and the Δgcabc-g1 mutant to compare their pathogenicity and their expression of GcABC-G1 in the host. We propose that Gc employs a combination of mechanisms to cope with host defence monoterpenes. The pathogen may control intracellular levels of monoterpenes by the induced expression of an efflux ABC transporter GcABC-G1, and it can metabolize monoterpenes and use them as a carbon source.
The Gc genome contains all ABC transporter subfamilies found in closely related species. As indicators of Gc having mechanisms for processing xenobiotics, the Gc ABC transporter subfamilies include the ABC-B, C and G subfamilies, whose members confer drug resistance in other fungi. RNA-seq expression analysis showed that transcript levels of members of the GcABC-F and GcABC-G subfamilies were upregulated when Gc was exposed to terpenes or grown in the presence of monoterpenes as the sole carbon source, but not under other stress conditions tested. The ABC-F subfamily has been reported as essential for cell viability and involved in ribosome biogenesis and translation, which would be activated while the fungus responded to sudden exposure to toxic compounds (Kovalchuk & Driessen, 2010). There are three ABC-G group I transporters in Gc (GcABC-G1, GcABC-G2 and GcABC-G3) and they are all upregulated in Gc in response to terpenes; GcABC-G1 was the most strongly induced in response to terpenes (i.e. > 100-fold change) while G2 (fourfold changes) and G3 (eightfold change) were expressed at a much lower level. GcABC-G2 has orthologues that are described as pathogenicity factors in M. grisea (MGG13624), Gibberella pulicaris (GpABC1), and N. haematococca (NECHADRAFT_63178) (Urban et al., 1999; Fleissner et al., 2002; Coleman et al., 2011), but its function remains to be tested in Gc in the future. The GcABC-G1 protein sequence had the full (NBF-TMD)2 domain organization that is common in PDR efflux transporters, which are localized in the cytoplasmic membrane in other fungal species; for example, the camalexin exporter BcatrB in B. cinierea and the pisatin exporter NhABC1 in N. haematococca (Stefanato et al., 2009; Coleman et al., 2011). The literature suggests that fungal PDRs evolved broad substrate specificities to export diverse antifungal compounds, including compounds derived from plants (Sipos & Kuchler, 2006; de Waard et al., 2006; Cannon et al., 2009). However, GcABC-G1 conferred resistance to monoterpenes but not to typical PDR substrates such as azoles, antibiotics, flavonoids, phytoalexins or simple phenolics. GcABC-G1 also had no close orthologues in the large set of ascomycete sequences analysed in this work, suggesting a specialized function as opposed to general xenobiotic transport. When GcABC-G1 was deleted, the mutant's transcriptome showed an elevated stress response to monoterpenes that included upregulation of other ABC transporters. However, none of these were upregulated to the same extent as GcABC-G1 in Gc, and none appeared to substitute functionally for GcABC-G1 in the mutant.
GcABC-G1 appears to have evolved as a specialized monoterpene transporter that may allow Gc to better colonize a unique ecological niche: the monoterpene-rich tissues of living pine hosts. As Witzke et al. (2010) has shown that limonene can freely diffuse into biological membranes, it is plausible that monoterpenes can enter fungal cells by diffusion. While measuring both this volatile monoterpene and the fungal biomass were challenging, we were able to detect (+)-limonene in the young germinating spores of Gc or its mutant when exposed to this compound for 1 h, which is too short an exposure time for the fungus to have produced ABC transporter proteins. However, after 18 h, while the level of (+)-limonene remained high in the cells of the mutant, we detected only trace amounts of this monoterpene in Gc (Table S3). The data at 1 h are inconsistent with the ABC transporter importing monoterpenes, while the data at 18 h are consistent with induced GcABC-G1 playing a role in reducing the concentration of monoterpene in the cells. We suggest that a similar process for controlling cellular monoterpene levels would occur in the pine phloem when the MPB vector disperses and so exposes fungal spores and young colonizing mycelia to monoterpenes, where strong inducible expression of GcABC-G1 should provide an adaptive advantage in survival and growth.
ATP-binding cassette transporters, and particularly the ABC-G group I (PDR) transporters, play a role in plant infection by fungal pathogens by protecting pathogens from exogenous toxic compounds produced by hosts (Urban et al., 1999). Given this, we compared the pathogenicity of Gc and its mutant on young pine trees, and measured the transcript abundance of GcABC-G1 in pine stem tissue inoculated with Gc or the mutant. While both Gc and the mutant affected the health of young pine trees, symptoms were delayed by 3 d for the mutant, and the survival rate was higher for the mutant than for Gc. These results suggest that Gc survives in and colonizes pine trees more efficiently than the mutant, which lacks the ABC transporter. It should not be surprising that the difference in effects caused by Gc or its mutant was not more pronounced, given that pine trees combat fungi with a range of chemical defences (Kolosova & Bohlmann, 2012). The results are consistent with our in vitro data for monoterpene treatments on MEA, in which the GcABC-G1 mutant survived the treatment but required 2–3 d more than Gc to adapt to the chemical, and then grew more slowly than Gc. A role for the GcABC-G1 transporter in pine colonization is further supported by the induction profile of the fungal GcABC-G1 gene in young pine trees inoculated with Gc. The absence of transporter expression in young pine inoculated with the deletion mutant, and the early and peak expression at 7 d of the GcABC-G1 gene, indicate that this transporter plays a role in the early phase of fungal colonization.
The unique ecological pine host niche colonized by Gc has high levels of monoterpenes and so would be unsuitable to most microorganisms. For example, in the broad range of niches in which Sc strains are found in nature, including grapevine berries, concentrations of terpenes are typically low, and, to this point, no mechanisms for coping with high concentrations of monoterpenes have been reported for Sc. The heterologous expression of GcABC-G1 in Sc conferred increased resistance to monoterpenes, consistent with this transporter potentially being an efflux pump that may remove toxic monoterpenes from cells. In the current work, the four monoterpenes assessed were far more toxic to Sc than to Gc mycelia or germinating Gc spores. When exposed for shorter periods of time to certain monoterpenes, more cells survived for Sc transformed with GcABC-G1 than for Sc transformed with only the vector. Metabolic engineering of Sc and other microorganisms is being actively pursued for the production of monoterpenes and other terpenoids of plant origin as high-value bioproducts and advanced biofuels (Kirby & Keasling, 2009; Fischer et al., 2011; Zerbe et al., 2012). While many plant species, and in particular the resin-producing conifers, develop specialized anatomical structures for sequestering large amounts of low molecular weight terpenoids (Bohlmann & Keeling, 2008), in engineered single-cell production systems, the toxicity of monoterpenes and biofuels may limit yield and performance (Dunlop et al., 2011). As GcABC-G1 may be the first reported eukaryotic ABC transporter with a role in enhanced tolerance against monoterpenes, this gene may be of interest for protein and metabolic engineering geared towards improved terpenoid production in Sc and other systems.
Finally, we also demonstrated that Gc could process monoterpenes as a carbon source for growth. While conversion of monoterpenoids and diterpenoids into nontoxic compounds has been shown for Penicillium caseifulvum, Aspergillus niger and Botryococcus braunii (de Carvalho & da Fonseca, 2006), to our knowledge, fungal utilization of monoterpenes as carbon sources has not been reported in filamentous fungi. It is important to note that the GcABC-G1 mutant did not grow at very low dosages of monoterpenes but was able to survive. Except for GcABC-G1, when Gc and the mutant were grown on MEA with monoterpenes, RNA-seq data showed that similar genes were upregulated (for example, acetyl-CoA-acyltransferase, alcohol dehydrogenase and genes involved in fatty acid metabolism). This suggests that the same enzyme-mediated metabolism pathways were induced in both Gc and its mutant. In ongoing work we are creating additional mutants in order to characterize the functions of these genes and their roles in monoterpene utilization.