FgABC1 (FGSG_04580) is predicted to encode a pleiotropic drug resistance class ABC transporter in Fusarium graminearum, a globally important pathogen of wheat. Deletion mutants of FgABC1 showed reduced virulence towards wheat in crown and root infection assays but were unaltered in infectivity on barley. Expression of FgABC1 during head blight and crown rot disease increases during the necrotrophic phases of infection suggestive of a role for FgABC1 in late infection stages in different tissue types. Deletion of FgABC1 also led to increased sensitivity of the fungus to the antifungal compound benalaxyl in culture, but the response to known cereal defence compounds, gramine, 2-benzoxazalinone and tryptamine was unaltered. FgABC1 appears to have a role in protecting the fungus from antifungal compounds and is likely to help combat as yet unidentified wheat defence compounds during disease development.
The pathogen Fusarium graminearum causes Fusarium head blight (FHB) disease in wheat and other small grain crops (Goswami & Kistler, 2004; Walter et al., 2010; Kazan et al., 2012). Fusarium crown rot (FCR) and Fusarium root rot (FRR) are also important diseases of wheat that can be caused by Fusarium pathogens, including F. graminearum (Mudge et al., 2006; Dyer et al., 2009). The symptoms of FCR consist of a spreading dark necrotic lesion at the stem base and crown. As the plant matures, the development of white heads with empty grain is observed (Burgess et al., 2001). FRR causes necrosis on roots with the fungus found in the cortex but not in the stele after artificial inoculation (Beccari et al., 2011). In general, there is much less known about host–pathogen interactions associated with FCR and FRR than for FHB. Trichothecene toxins are well known as virulence factors for FHB facilitating the spread of the pathogen through the infected head (Jansen et al., 2005) and are also produced at high concentrations during FCR where they contribute to stem colonization (Mudge et al., 2006). Infection of the stem base following inoculation with conidia of F. graminearum has suggested that there is phasic infection during FCR reflected in biomass differences and differences in pathogen gene expression profiles (Stephens et al., 2008). Using the Affymetrix global gene expression analysis arrays that are available for F. graminearum (Güldener et al., 2006b), many fungal genes were identified that were expressed in much higher levels during the infection process when compared with the expression in axenic culture (Stephens et al., 2008). Several genes that are highly expressed in planta had putative detoxification functions and this included the putative ATP-binding cassette (ABC) transporter gene (FGSG_04580), which we have termed FgABC1. The recently sequenced genome of F. pseudograminearum has an almost identical copy of FgABC1, termed FpABC1 (Gardiner et al., 2012).
Many studies have shown that ABC transporters, particularly those with multidrug resistance (MDR) or pleiotropic drug resistance (PDR) domains, are important in the resistance of fungal pathogens to xenobiotics (Kretschmer et al., 2009). Transporters in fungi have recently been reviewed (Coleman & Mylonakis, 2009), but for the majority of those encoded in fungal genomes, the specific substrate(s) is/are unknown. Transporters responsible for efflux of plant-derived defence compounds have been characterized in a small number of species. These include the following: the Botrytis cinerea BcatrB which transports camalexin from Arabidopsis and resveratrol from grape, NhABC1 from F. solani f. sp. pisi which transports pisatin from pea, the F. sambucinum GpABC1 which exports rishitin from potato and GcABC-G1 which transports terpenes from pine in Grosmannia clavigera (Schoonbeek et al., 2001; Fleißner et al., 2002; Stefanato et al., 2009; Coleman et al., 2011; Wang et al., 2012). Other ABC transporters have roles in virulence and PDR, but specific substrates related to their hosts are yet to be identified. These include the Magnaporthe oryzae transporters ABC1 and ABC4 that impart resistance to phytoalexins isolated from nonhost species, suggesting a role in MDR and potential export of unknown rice phytoalexins (Urban et al., 1999; Gupta & Chattoo, 2008). Likewise, in F. culmorum and Mycosphaerella graminicola, ABC transporters are involved in virulence towards wheat (Stergiopoulos et al., 2003; Skov et al., 2004). In F. solani f. sp pisi, NhABC1 acts with another detoxification mechanism (chemical modification) to provide tolerance to the pea phytoalexin pisatin (Coleman et al., 2011). If similar multiple-faceted detoxification systems exist in other fungi, identification of specific plant-derived defence chemicals that are substrates for these transporters may be difficult. A recent analysis of the F. graminearum genome has identified 62 potential ABC transporter genes (Kovalchuk & Driessen, 2010). Only one of these (FgABC2) has been studied functionally, and it does not have a role in FHB of wheat (Goswami et al., 2006). In the present study, we have investigated the function of FgABC1 using knockout mutants, chemical screening assays and heterologous expression in yeast. This work aimed to investigate the function of FgABC1 in protecting against antifungal compounds and in virulence towards wheat during root and crown infection.
Materials and methods
Isolates and FgABC1 deletion
The Australian F. graminearum isolate CS3005 was used (Akinsanmi et al., 2006). The FgABC1 gene replacement was created by the PCR split-marker method using PCR primers shown in Supporting Information, Table S1 (Fairhead et al., 1996; de Hoogt et al., 2000). The strategy and primer-binding positions are shown in Fig. 1. All fungal transformations were completed according to Desmond et al. (2008b). Hygromycin-resistant transformants were screened by multiplex PCR amplification (Table S1).
Phenotype microarrays were performed as previously described in defined minimal media with 5 mM glutamine as the nitrogen source and 3% sucrose as the carbon source (Gardiner et al., 2009). In total, 96 different compounds were screened in PM plates PM21A, PM22, PM24A and PM25 from Biolog (CA). Full tables of compounds present in these plates can be found at www.biolog.com. Macroconidia for inoculation were produced as described by Stephens et al. (2008). OD600 was used as a measure of fungal growth.
Chemical sensitivity assays
Benalaxyl, BOA (2-benzoxazolinone), gramine and tryptamine were purchased from Sigma. Compounds were dissolved in dimethyl sulfoxide (benylazyl, BOA and tryptamine) or ethanol (gramine) at a concentration of 50 mg mL−1. Half-strength potato dextrose agar plates containing the test compounds were prepared on the day of use from fresh preparations of the compounds added just prior to pouring. Final concentrations were 0.2 and 0.5 mg mL−1 with DMSO or ethanol added to control plates at the same final concentration in all treatments. Plates were spot-inoculated with 5 μL of spore suspensions prepared at 106, 105, 104 and 103 spores mL−1 using every second tip of an eight channel pipette. Parental and mutant strains were inoculated on opposite halves of the same plate. Plates were allowed to dry in a laminar flow cabinet, sealed with sealing film (PhytoTechnology Laboratories) and maintained in the dark at room temperature. Each assay was performed in triplicate and photographed 3 days postinoculation. Assays with benalaxyl were performed on two separate occasions with similar results.
Root rot assays were performed and scored as previously described for F. pseudograminearum (Gardiner et al., 2012). Wheat cultivar Kennedy and barley cultivar Golden Promise were used. Both are highly susceptible to stem base infection. Stem base (crown rot) assays were conducted as previously described (Mitter et al., 2006) in a constant temperature (24 °C) walk in growth chamber using fluorescent lighting (16/8 h day/night) at the CSIRO Plant Industry containment facility. The susceptible wheat cultivar Kennedy was used. Barley was not tested as a host in these assays.
Gene expression analysis
Publically available gene expression data were accessed and analysed at www.plexdb.org. Experiments FG1, FG12, FG15 and FG19 (Güldener et al., 2006b; Stephens et al., 2008; Lysøe et al., 2011; Zhang et al., 2012) were used. ABC-G subfamily members (19 in total) gene identifiers were extracted from the analysis conducted by Kovalchuk & Driessen (2010) and converted to probe set identifiers corresponding to the F. graminearum Affymetrix GeneChip using the mappings available from the F. graminearum database (FGDB) hosted by the Munich Information Center for Protein Sequences (Güldener et al., 2006a). The expression data were extracted from the analysis at www.plexdb.org and exported to Microsoft Excel. Presented data represent the log-transformed average of biological replicates. Heatmaps were generated using the conditional formatting options in Microsoft Excel and exported to Adobe Illustrator CS5.1 for annotation with the most recent gene identifier entries in FGDB.
Expression of FpABC1 in yeast
The FpABC1 cDNA was amplified from wheat root samples infected with F. pseudograminearum isolate CS3427 using primers FpABC1f and FpABC1r (Table S1) using Phusion DNA polymerase (Finnzymes). The PCR product was cloned into the HindIII and XbaI sites of the yeast expression plasmid pYES2 (Life Technologies). Yeast strains used were BY4743 (purchased from Life Technologies) and AD12345678, which lacks eight different PDR transporters (MATα, PDR1–3, ura3, his1, Δsnq2::hisG, Δpdr5::hisG, Δpdr10::hisG, Δpdr11::hisG, Δycf1::hisG, Δpdr3::hisG, Δpdr15::hisG; Decottignies et al., 1998). AD12345678 has previously been used to characterize fungal ABC transporters (Zwiers et al., 2003; Del Sorbo et al., 2008). Strains were transformed as previously described (Gietz & Schiestl, 2007). Xenobiotic sensitivity assays were performed by growing overnight cultures of the yeast strains either containing an empty vector (pYES2) or FpABC1 construct in synthetic dropout media (Sigma) lacking uracil with glucose as a carbon source. Sensitivity to benalaxyl was assessed in BY4743 and other compounds in BY4743 and AD12345678. Overnight cultures were centrifuged (5000 g 5 min), washed twice in sterile water and resuspended to a final OD600 of 0.05 units in synthetic dropout media lacking uracil with galactose as the carbon source. Compounds were dissolved in DMSO at either 50 (benalaxyl) or 100 mg mL−1 (BOA, gramine, tryptamine). Dilution series of compounds were created in a 96-well microtitre plates by consecutive 1.5-fold dilutions across the plates leaving the final column as DMSO control. Concentrations at which there was approximately 90% inhibition of growth of the vector alone containing strains compared with DMSO control were used to determine whether the FpABC1 could impart resistance to the test compound. Each well of the microtitre plate contained 200 μL of media with DMSO at a final concentration of 4% (v/v). Plates were incubated at 28 °C for 3 (BOA, gramine and tryptamine) or 4 (benalaxyl) days prior to reading OD600 using an iEMS plate reader (Thermo Fisher).
Results and discussion
FgABC1 is a member of an ABC transporter family that in other Fusaria is important for virulence
FgABC1 encodes a 1489 amino acid protein that contains the conserved domains characteristic of the PDR family of ABC transporters. In the Human Genome Organisation scheme for classification of ABC transporters, FgABC1 belongs to the ABC-G subfamily, a class often associated with pleiotropic drug resistance (Kovalchuk & Driessen, 2010). blastp searches (Altschul et al., 1997) of the nonredundant database of the National Center for Biotechnology Information (NCBI) using FgABC1 as a query indicated that FgABC1 shares 90% amino acid sequence identity with the predicted protein Gpabc1 of F. sambucinum, which has a role in virulence on potato and resistance to the phytoalexin rishitin (Fleißner et al., 2002) and 80% amino acid sequence identity with NhABC1 from F. solani that is required for virulence on pea and tolerance to the phytoalexin pisitin (Coleman et al., 2011). FgABC1 also has 62% amino acid sequence identity to the ABC1 transporter of Magnaporthe grisea, which is required for pathogenicity on rice (Urban et al., 1999). However, there are matches to ABC transporters of nonpathogens with similar levels of sequence identity. A region of FgABC1 (aa residues 1123–1487) has 95% amino acid sequence identity to a partial protein sequence from F. culmorum that corresponds to a partially characterized gene termed FcABC1, which has a role in aggressiveness in FHB (Skov et al., 2004). As the complete sequence of FcABC1 is not available, the full extent of amino acid sequence identity to FgABC1 cannot be determined. Nonetheless, taken together, these sequence comparisons would suggest that FgABC1 falls within a group of transporter genes related in sequence, some of which have demonstrated roles in virulence towards plants. Coleman et al. (2011) also demonstrated that FgABC1, GpABC1 and NhABC1 form a Fusarium-specific clade of ABC transporters. Interestingly, FgABC1 is located towards one end of chromosome 2 in an area of high SNP density, and it has been suggested that these regions are enriched in genes involved in virulence and may be evolving more quickly compared with genes in other parts of the genome (Cuomo et al., 2007).
FgABC1 is important for disease of wheat but not barley
The homology of FgABC1 to transporters from other Fusaria that contribute to pathogen virulence suggested that FgABC1 may also be important in diseases caused by F. graminearum. To test for this role, the FgABC1 gene was deleted and replaced with a hygromycin selectable marker gene by homologous recombination (Fig. 1). Using PCR for verification, independent homologous recombinant mutants for FgABC1 were obtained (Fig. 1). No obvious defects in conidiation or perithecia formation were observed in any of the three independent FgABC1 mutants, and each of these mutants had similar radial growth rates on either defined synthetic nutrient poor (SNA) or complete (V8 juice) nutrient agar medium when compared with wild type (Fig. 1). To test the role of FgABC1 in plant disease, an assay using root inoculation was used. Shoot elongation after six days was used as a measure of disease severity. The assay showed a clear role for FgABC1 in disease on wheat, but this was not observed when barley was used as the host (Fig. 2). The wheat and barley assays presented in Fig. 2 are from plants inoculated at the same time from the same inoculum source, indicating that the mutation in FgABC1 does not affect general virulence of F. graminearum per se, but rather specifically affects wheat infection. Similarly, a specific role in wheat head infection, but not barley, was observed for the F. culmorum transporter FcABC1 (Skov et al., 2004), suggesting that FgABC1 and FcABC1 may be orthologous and transport an unidentified compound either uniquely produced by wheat or a fungal compound only required for infection of wheat. In addition, a role for FgABC1 in virulence towards wheat was demonstrated using an independent stem base infection assay (Fig. 3). Production of specific phytoalexins is typically restricted to individual species of plants which could explain the difference observed between the importance of FgABC1 during wheat and barley infection. Taken together, these results indicate that the importance of FgABC1 for virulence is not restricted to a specific plant tissue type and that the role of FgABC1 in virulence is likely to be similar to those of other transporters in this class in other fungi described in the introduction.
FgABC1 is likely to exert its function late in disease processes
ABC-G family transporters are typically involved in PDR. To determine the point(s) in infection at which FgABC1 acts, the expression of FgABC1 was analysed using publically available Affymetrix microarray data for FCR on wheat (Stephens et al., 2008), FHB on barley and wheat (Güldener et al., 2006b; Lysøe et al., 2011) and during infection of coleoptiles of wheat (Zhang et al., 2012). FgABC1 was highly expressed in all four interactions (Fig. 4). One of the technical challenges of analysing fungal gene expression during infection is the nonhomogeneous nature of the material being analysed. That is, some fungal cells will be at the leading edge of an infection, and these will most likely have a different gene expression profile compared with cells behind the invasive hyphae. The experiment conducted by Zhang et al. (2012), where gene expression during coleoptile invasion was analysed in developmentally synchronous material, provided a high resolution representation of the phasic nature of the infection process. FgABC1 expression was highest at 64 and 240 h postinoculation, coinciding with highly destructive colonization of the host (Fig. 4). Slightly higher expression was also observed later in the FCR on wheat and during the middle to later time points of the FHB time courses on wheat and barley (Fig. 4). These time points are concomitant with extensive visual symptom development, which in FHB is much shorter than FCR (Mudge et al., 2006). Taken together, these data indicate that FgABC1 most likely exerts its function late in the infection of all tissue types. Given ABC-G family transporters are involved in the transport of xenobiotics, the expression of all 19 ABC-G transporters in F. graminearum was analysed (Fig. 4). Most family members appeared to have similar expression dynamics in each of the four experiments despite the different host and tissue types (Fig. 4). This suggests that the F. graminearum programme of pathogenesis on different tissues, at least in part, shares many commonalities despite very different rates of disease symptom expression. A number of other ABC-G family transporters were highly expressed during infection (e.g. FGSG_08309 and FGSG_16825), and these may also have roles in F. graminearum virulence.
FgABC1 is important for resistance to xenobiotics but not known grass defence molecules
Transporters within the ABC family have been reported to transport a range of compounds, and PDR-type transporters often provide protection to cells via efflux of toxic compounds (Gulshan & Moye-Rowley, 2007; Kretschmer et al., 2009). As a primary screen for a role in resistance to toxic compounds, the phenotype array system (Biolog) was used to identify potential substrates. The compound benalaxyl had a strong inhibitory effect on a selected mutant (ΔFgABC1.4) when compared with wild type in this primary screen (data not shown). Salicylanilide also was identified in the primary screen as showing differences between the mutant and wild type, but subsequent assays revealed both the wild type and mutant were highly sensitive to this compound (data not shown). The inhibitory effect when the mutant was grown on benalaxyl was confirmed using a drop inoculation on half-strength potato dextrose agar (Fig. 5). Benalaxyl is a commercial systemic acylanilide fungicide primarily used against Oomycete pathogens in horticultural crops, but it also has some inhibitory activity against Ascomycete fungi (Meneau & Sanglard, 2005). Benalaxyl is not known to have a structural equivalent in plants, but these findings demonstrate that FgABC1 provides protection against antimicrobial compounds.
To test a role for FgABC1 in transporting putative grass defence compounds, spore droplet germination assays were also performed against the wheat compound 2-benzoxazolinone (BOA), the barley compound gramine and the more widely distributed tryptamine (Smith, 1977; Niemeyer, 2009). These compounds were not present in the phenotype arrays. While F. graminearum appears moderately sensitive to all three compounds, the FgABC1 mutant and parental strain showed no differential sensitivity (Fig. 5). While the virulence assays clearly show a role for FgABC1 during disease and the in vitro assays suggested BOA, gramine and tryptamine were not the targets of FgABC1, there remained the possibility that in the less complex in vitro environment, alternative detoxification mechanisms, such as other PDR transporters, may provide sufficient tolerance to these compounds. To rule this out, the F. pseudograminearum homologue of FgABC1 was expressed in yeast under the control of the galactose-inducible promoter GAL1. While FpABC1 was able to impart increased resistance to benalaxyl in a wild-type yeast background (Fig. 6), this gene could not provide increased tolerance to BOA, gramine or tryptamine in either a wild-type strain (data not shown), or the PDR mutant strain AD12345678 that lacks eight different PDR transporters (Decottignies et al., 1998; Fig. 6).
Currently, the function that FgABC1 plays in disease development is unknown. Fungal toxins can be exported from fungal plant pathogens by ABC transporters (e.g. Gardiner et al., 2005), and it has also been shown that the PDR transporter of yeast, Pdr5p, is able to export the Fusarium toxin deoxynivalenol, a known virulence factor (Mitterbauer & Adam, 2002). FgABC1 is unlikely to be involved in deoxynivalenol efflux as this is mediated by a major facilitator protein TRI12 (Alexander et al., 1999). Wheat can produce a large spectrum of molecules with potential antimicrobial activity (Moraes et al., 2008). The production of defence metabolites in barley roots has been shown to be induced by F. graminearum (Lanoue et al., 2010), and wheat genes that encode enzymes involved in secondary metabolism that can potentially produce defence metabolites are also transcriptionally induced during FCR (Desmond et al., 2008a). Likewise, the entire tryptophan pathway including a final step converting tryptophan to tryptamine is up regulated during the mid to late phases of barley FHB (Boddu et al., 2006). It is therefore probable that FgABC1 protects the fungus from specific host defence molecules. Future work should focus on assessing the relative sensitivity of the ΔFgABC1 deletion mutants to specific wheat metabolites produced during infection. However, at present, there appears to be a dearth of information surrounding phytoalexins produced by wheat.
AES acknowledges the support of a CSIRO/IMB postgraduate scholarship. DMG and ALM acknowledge support from the Australian Research Council (DP0985486 and DP110100389, respectively). DMG was also supported by the Australian Grains Research and Development Corporation. We thank Andrew Beacham, Rothamsted Research, UK for the kind gift of pHYG4.1 and Professor Frederick Roth, University of Toronto, for providing yeast strains AD12345678.