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α-Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of tomatinases that degrade α-tomatine into the less fungitoxic compounds β-tomatine and tomatidine. Although pathogenic on tomato, it has been reported that the biotrophic fungus Cladosporium fulvum is unable to detoxify α-tomatine.
Here, we present a functional analysis of the glycosyl hydrolase (GH10), CfTom1, which is orthologous to fungal tomatinases.
We show that C. fulvum hydrolyzes α-tomatine into tomatidine in vitro and during the infection of tomato, which is fully attributed to the activity of CfTom1, as shown by the heterologous expression of this enzyme in tomato. Accordingly, ∆cftom1 mutants of C. fulvum are more sensitive to α-tomatine and are less virulent than the wild-type fungus on tomato. Although α-tomatine is thought to be localized in the vacuole, we show that it is also present in the apoplast, where it is hydrolyzed by CfTom1 on infection. The accumulation of tomatidine during infection appears to be toxic to tomato cells and does not suppress defense responses, as suggested previously.
Altogether, our results show that CfTom1 is responsible for the detoxification of α-tomatine by C. fulvum, and is required for full virulence of this fungus on tomato.
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Plants have developed basal defense barriers that protect them from infection by potential pathogens. These include physical barriers, such as lignin or callose, and chemical barriers, such as antimicrobial compounds (Hammerschmidt et al., 1984; de Wit et al., 2009). A well-known class of antimicrobial compounds present in plants comprises the saponins, which are glycosylated steroids or steroidal alkaloids representing a constitutive chemical barrier against a wide range of fungal and bacterial pathogens (Bowyer et al., 1995; Osbourn, 1996). Saponins cause loss of membrane integrity in target organisms by forming complexes with sterols, resulting in pore formation and cell lysis (Keukens et al., 1995; Osbourn, 1996). Sensitivity to saponins is correlated with the type of sterols present in the membranes of the potential pathogens. Fungal membranes that contain sterols with free 3β-hydroxy groups are sensitive to saponins, whereas plant cell membranes are insensitive because of the presence of sterol glycosides (Steel & Drysdale, 1988). Similarly, oomycetes are insensitive to saponins because their membranes lack 3β-hydroxy sterols (Steel & Drysdale, 1988).
In response to the inherent resistance mechanism present in plants, bacteria and fungi that are pathogenic on saponin-producing plants have developed a detoxification mechanism by secreting saponin-detoxifying enzymes (Ford et al., 1977; Roldan-Arjona et al., 1999; Kaup et al., 2005). Saponin detoxification by pathogens has mainly been studied for avenacin and α-tomatine, which are present in oat and tomato, respectively. During infection of oat roots, the fungus Gaeumannomyces graminis var. avenae secretes the avenacinase enzyme that detoxifies avenacin, a triterpenoid saponin. Mutants deleted for the avenacinase gene are no longer able to infect oat, but are still virulent on wheat, a host that does not produce saponins (Bowyer et al., 1995). In tomato (Solanum lycopersicum L.), the major saponin is α-tomatine, a steroidal glycoalkaloid that is present in leaves and green fruits in concentrations as high as 1 mM (Roddick, 1977; Osbourn, 1996). α-Tomatine consists of the aglycon tomatidine and the tetrasaccharide lycotetraose (Fig. 1a). The toxicity of α-tomatine depends on the presence of lycotetraose, because removal of one or all four sugar residues renders α-tomatine less toxic (Osbourn, 1996). During tomato infection, bacterial and fungal pathogens secrete various types of tomatinase enzyme that can detoxify α-tomatine by removing one or more sugar residues (Martin-Hernandez et al., 2000; Kaup et al., 2005; Pareja-Jaime et al., 2008). Tomatinase enzymes secreted by Septoria lycopersici, Botrytis cinerea and Verticillium albo-atrum belong to the glycosyl hydrolase family 3 (GH3) of carbohydrate-degrading enzymes (CAZY; Martin-Hernandez et al., 2000). They remove the terminal β-1,2-d-glucose or the β-1,3-d-xylose residues from α-tomatine (Osbourn et al., 1995; Quidde et al., 1998). Other tomato pathogens, such as the fungus Fusarium oxysporum f. sp. lycopersici and the bacterium Clavibacter michiganensis ssp. michiganensis, secrete a tomatinase that belongs to the glycosyl hydrolase family 10 (GH10; Roldan-Arjona et al., 1999; Kaup et al., 2005). GH10 tomatinase enzymes remove lycotetraose from α-tomatine to form the aglycon tomatidine (Fig. 1a; Roldan-Arjona et al., 1999; Pareja-Jaime et al., 2008). Although several knock-out studies have been performed to assess the role of tomatinase enzymes in the virulence of bacterial and fungal tomato pathogens (Martin-Hernandez et al., 2000; Kaup et al., 2005), only the GH10 tomatinase FoTom1 from F. oxysporum f. sp. lycopersici has been shown to play a role in the virulence of this vascular pathogen (Pareja-Jaime et al., 2008). It has also been suggested that products resulting from tomatinase activity play an indirect role in the virulence of tomato pathogens by suppressing plant defense responses. The different breakdown products of α-tomatine (β-tomatine, tomatidine and lycotetraose) have been reported to suppress various types of defense response, including the oxidative burst and the hypersensitive response (Bouarab et al., 2002; Ito et al., 2004).
The nonobligate biotrophic fungus Cladosporium fulvum is a well-studied tomato pathogen that causes leaf mold. Cladosporium fulvum enters tomato leaves through stomata and colonizes the apoplastic space surrounding mesophyll cells (Stergiopoulos & de Wit, 2009). Although pathogenic on tomato, it has been reported previously that C. fulvum is not able to detoxify α-tomatine, and the vacuolar location of α-tomatine has been hypothesized to allow the fungus to infect tomato (Melton et al., 1998). However, heterologous expression of the GH3 tomatinase gene from S. lycopersici in C. fulvum resulted in a transformant showing increased sporulation relative to the wild-type during infection of tomato (Melton et al., 1998). These results suggest that C. fulvum may be exposed to α-tomatine during the colonization of the apoplastic space of tomato, and that it does not produce functional tomatinase enzymes. However, recent sequencing of the C. fulvum genome revealed the presence of 19 genes encoding GH3 and two genes encoding GH10 enzymes (de Wit et al., 2012). Here, we demonstrate that one of the two GH10 genes encodes a functional tomatinase, CfTom1, which degrades α-tomatine into nontoxic tomatidine both in vitro and during infection of tomato. Functional analysis of ∆cftom1 mutants of C. fulvum showed that the degradation of α-tomatine is required for full virulence of the fungus on tomato, which is probably a result of the increased sensitivity of these mutants to α-tomatine rather than to the suppression of basal defense responses by its breakdown products.
Materials and Methods
Fungal and plant materials
The Cladosporium fulvum race 0WU (CBS131901; de Wit et al., 2012) was grown on half-strength potato dextrose agar (19.5 g l−1 PDA and 15 g l−1 technical agar, Oxoid, Cambridge, UK) at 20°C for 2–3 wk for conidia production and DNA isolation. Stocks of conidia were maintained in 25% glycerol at −80°C. For tomatinase activity assays, the fungus was grown in liquid CA medium (10 g l−1 casamino acids, 10 mM ammonium sulfate and 0.5 g l−1 yeast nitrogen base, pH 6.5). To induce the expression of the CfTom1 gene, C. fulvum was pre-incubated in Gamborg B5 medium supplemented with 20 g l−1 sucrose at 22°C and incubated in an orbital shaker at 200 rpm for 6 d. Mycelium was then transferred to several B5 induction media at pH 4, pH 7, in the absence or presence of 50 μM α-tomatine, and cultured for 24 h.
Susceptible Money Maker (MM) Cf-0 tomato (Solanum lycopersicum L.) was used for inoculation experiments. Plants were grown in the glasshouse at 70% relative humidity, at 23–25°C during the daytime and at 19–21°C at night, with a light/dark regime of 16/8 h and 100 W m−2 supplemental light when the light intensity was < 150 W m−2.
Nucleic acid methods
DNA was isolated from freeze–dried mycelia of C. fulvum strains, which were scraped from PDA plates, or from ground C. fulvum-inoculated tomato leaves frozen in liquid nitrogen, using the DNeasy plant mini kit (Qiagen Benelux BV, Venlo, the Netherlands), according to the manufacturer's instructions. Total RNA was isolated from ground mycelia or C. fulvum-inoculated tomato leaves using the hybrid method, as described by van Esse et al. (2008).
cDNA was synthesized from 5 μg of total RNA using the SuperScript II reverse transcriptase kit (Invitrogen), as described previously (van Esse et al., 2008). Quantitative PCR was performed with the 7300 System (Applied Biosystems, Foster City, CA, USA): each reaction was performed in 25 μl containing 12.5 μl Sensimix (Bioline, London, UK), 1 μl of 10 μM deoxynucleoside triphosphate (dNTP), 1 μl of each forward and reverse oligonucleotide (5 μM), 100 ng of template cDNA and 9.5 μl of double-distilled H2O. The thermal profile included an initial 95°C denaturation step for 10 min, followed by denaturation for 15 s at 95°C and annealing/extension for 45 s at 60°C for 40 cycles. qCfActin_F and qCfActin_R and qCfTom1_F and qCfTom1_R oligonucleotide pairs were designed with Primer3 Plus (Supporting Information Table S1; Untergasser et al., 2007). The efficiency and specificity of the oligonucleotide pairs were determined with a dilution series of genomic DNA before use. The C. fulvum actin gene was used as a reference gene for normalization, and results were analyzed using the method (Livak & Schmittgen, 2001). The results are the average of three biological repeats.
All PCRs were performed in 25 μl using Pfu or GoTaq DNA polymerase (Promega), following the manufacturer's recommendations, and using 100 ng of genomic DNA as template. The PCR program was initiated by a denaturation step at 94°C for 5 min, followed by 32 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 45 s and extension at 72°C for 1 min, with a final extension step at 72°C for 7 min.
Southern blotting was performed with digoxigenin-labeled probes using a DIG DNA Labeling and Detection Kit (Roche, Basel, Switzerland), according to the manufacturer's instructions. The probe was generated using the pCfTom1_US_F and pCfTom1_US_R primer pair (Table S1).
Identification and cloning of the CfTom1 gene and its expression by the Potato virus X (PVX) expression system in tomato
Amino acid sequences of GH3 and GH10 tomatinases were aligned with ClustalW2 (Larkin et al., 2007) and edited in GeneDoc software (Nicholas et al., 1997). A consensus phylogenetic tree was constructed using the minimum-evolution algorithm with default parameters and 1000 bootstrap replications in MEGA5 software (Tamura et al., 2011).
The GH10 tomatinase gene (CfTom1; jgiID 188986) from C. fulvum was amplified by PCR using the following oligonucleotide set: the CfTom1_F oligonucleotide excludes the native signal peptide sequence of the tomatinase gene and includes a 15-nucleotide overhang sequence corresponding to the PR1A signal peptide sequence; the CfTom1_R oligonucleotide contains the NotI restriction site (Table S1). A second PCR was performed to amplify the PR1A signal peptide sequence using the PR1A_F oligonucleotide containing the ClaI restriction site and the PR1A_R oligonucleotide. Overlapping PCR was performed to fuse the PR1A signal peptide sequence to the CfTom1 gene using the PR1A_F and CfTom1_R oligonucleotides (Table S1). The amplified PR1A-CfTom1 fragment was purified from agarose gel using the Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Buckinghamshire, UK). The purified PR1A-CfTom1 fragment was cloned into the pGEM-T Easy vector (Promega) according to the manufacturer's instructions. The recombinant pGEM-T vector was introduced into chemically competent Escherichia coli cells (DH5α) by the standard heat shock transformation protocol. The plasmid was retrieved from a positive clone using the Miniprep plasmid isolation kit (Qiagen) and the insert was sequenced by Macrogen Inc. (Amsterdam, the Netherlands). The correct PR1A-CfTom1 insert was isolated using ClaI and NotI restriction enzymes (Promega), and was ligated (Promega) into the binary PVX-based vector pSfinx (Hammond-Kosack et al., 1995; Takken & Lu, 2001), which was digested with the same restriction enzymes. The ligation reaction was introduced into E. coli and the pSfinx::PR1A-CfTom1 plasmid obtained was finally introduced into Agrobacterium tumefaciens (GV3101) by electroporation. A positive A. tumefaciens clone containing the pSfinx::PR1A-CfTom1 construct was cultured on plates containing modified Luria–Bertani (LB) medium (10 g l−1 bacto-peptone; 5 g l−1 yeast extract; 5 g l−1 NaCl) supplemented with 100 μg ml−1 kanamycin and 25 μg ml−1 rifampicin for 48 h at 28°C. Heterologous expression of pSfinx::PR1A-CfTom1 in tomato seedlings was performed as described previously (Stergiopoulos et al., 2010).
Construction of the cftom1 targeted deletion plasmid
The gene replacement vector pR4R3∆cftom1 was constructed using the MultiSite Gateway® Three-Fragment Vector Construction Kit (Invitrogen), according to the manufacturer's instructions. The upstream (US, 1.8 kb) and downstream (DS, 1.55 kb) flanking regions of CfTom1 were amplified using oligonucleotides with overhang sequences homologous to the AttB4, AttB1r, AttB2r and AttB3 recombination sites (Table S1). Purified US and DS fragments were cloned into pDONR™ P4-P1R and pDONR™ P2R-P3, respectively, using 1 μl of BP clonase™ II enzyme mix (Invitrogen), 70 ng of insert DNA and 70 ng of pDNOR™ (Invitrogen, Carlsbad, CA, USA) in a reaction volume of 5 μl, and introduced into E. coli. The p221_GFP_HYG pENTRY vector was kindly provided by Rafael Arango Isaza (Plant Research International, Wageningen, the Netherlands). To construct the final replacement vector, an LR reaction was performed with 50 ng of pDONR™ P4-P1R_cftom1US, 50 ng of pDONR™ P2R-P_cftom1DS, 50 ng of p221_GFP_HYG and 70 ng of pDEST™R4-R3 destination vector in the presence of 1 μl of LR clonase™ II enzyme mix (Invitrogen) in a total volume of 5 μl. The LR reaction was introduced into E. coli to obtain the final pR4R3∆cftom1 plasmid. Insertion of all fragments was confirmed by PCR using insert-specific primers (Table S1).
Agrobacterium tumefaciens-mediated fungal transformation and mutant screening
The pR4R3∆cftom1 construct was transformed into A. tumefaciens AGL1 by electroporation. Agrobacterium tumefaciens-mediated transformation of C. fulvum was performed as described previously (Zwiers & De Waard, 2001) with the following modifications. Agrobacterium tumefaciens AGL1 cells containing the pR4R3∆cftom1 construct were grown in minimal medium (MM; 10 mM K2HPO4, 10 mM KH2PO4, 2.5 mM NaCl, 2 mM MgSO4, 0.7 mM CaCl2, 9 μM FeSO4, 4 mM NH4SO4 and 10 mM glucose; adjusted to pH 5.5) supplemented with 100 μg ml−1 spectinomycin and 10 μg ml−1 rifampicin for 2–3 d at 28°C. Agrobacterium tumefaciens AGL1 cells were collected by centrifugation at 3363 g for 8 min and resuspended in induction medium (IM) (MM; 40 mM 2-(N-morpholino)ethanesulfonic acid, pH 5.3; 0.5% glycerol (w/v); 200 μM acetosyringone) supplemented with appropriate antibiotics to the final optical density at 600 nm (OD600) of 0.15. Before co-cultivation, the A. tumefaciens AGL1 culture was incubated at 28°C for 4–6 h to an OD600 of 0.25. Cladosporium fulvum conidia were collected from half-strength PDA plates with sterile water and passed through two layers of miracloth to remove mycelium. The A. tumefaciens AGL1 culture (200 μl; OD600 = 0.25) was co-cultivated with 200 μl conidia suspension (1 × 107 conidia ml−1). The co-cultivation mixture was plated on H Bond-nitrocellulose membrane that had been placed previously on IM plates and incubated for 2 d at 20°C. After 2 d co-incubation, membranes were transferred to MM plates supplemented with 100 μg ml−1 hygromycin and 200 μM cefotaxime. Transformed fungal colonies (2–3-wk-old) were transferred onto PDA plates supplemented with 100 μg ml−1 hygromycin and 200 μM cefotaxime, and incubated at 20°C for 2–3 wk. Fungal transformants were subcultured twice on selective PDA plates and once on nonselective PDA plates. Subsequently, transformants were tested on PDA plates containing 100 μg ml−1 hygromycin.
α-Tomatine sensitivity assay
Conidia from wild-type C. fulvum, one ectopic transformant and two independent ∆cftom1 mutants were collected and diluted to a final concentration of 2 × 104 conidia ml−1. Agar plugs were prepared on sterile microscope slides by the addition of 250 μl CA medium (CA medium supplemented with 1% technical agar). Conidia (20 μl) were mixed with 10 mM α-tomatine (in methanol; Sigma-Aldrich) to obtain a final concentration of 100 μM, and incubated on the CA agar plugs at 20°C for 2 d. As a negative control, conidia were incubated in methanol (1% v/v), but without α-tomatine. For each sample, the germination of 100 conidia was analyzed. The results are the average of two biological repeats.
Monitoring of tomatinase activity by LC-MS
Extraction and liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analysis of semi-polar secondary metabolites were performed according to the protocols described previously (De Vos et al., 2007; Tikunov et al., 2010). Briefly, 0.1 g of ground frozen tomato leaves and 2 ml of freeze–dried apoplastic fluid (AF; 0.5 ml of AF is obtained from 1 g of leaves) were extracted with 300 μl (99.9% methanol with 0.1% formic acid (v/v)) and 200 μl (75% methanol with 0.1% formic acid (v/v)), respectively, and sonicated for 15 min. The mixture was centrifuged at 20 000 g for 10 min and the supernatant was filtered using a 0.45-μm inorganic membrane filter (Anotop 10; Whatman, NH, USA), fitted onto a disposable syringe, and transferred to a glass vial. Ionization was performed using electrospray ionization in positive mode. A collision energy of 10 eV was used for full-scan mass detection in the range of m/z 100–1500. Leucine enkephalin, [M + H]+ = 556.6305, was used for online mass calibration (lock mass) using a separate spray inlet. For the monitoring of rutin and α-tomatine in the AF and total extract (TE) of tomato leaflets, an LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific) was used, with Xcalibur software to control the instrument, for data acquisition and analysis (van der Hooft et al., 2011). The mass chromatograms that were generated in LTQ-Orbitrap were processed (peak picking and baseline correction) using the MetAlign software package (Lommen, 2009). From the processed data, information on the relative peak heights of the representative masses [M + H]+ for α-tomatine, dehydrotomatine and their degradation products, tomatidine and tomatidenol, was extracted at their recorded retention times for tomato fruit (Table S2), in the MOTO database (Moco et al., 2006) and the Komics database (http://webs2.kazusa.or.jp/komics/; Iijima et al., 2008). Absolute quantification of α-tomatine was performed using a standard curve of commercial α-tomatine (Sigma-Aldrich).
For in vitro α-tomatine degradation, 1 × 106 conidia of C. fulvum wild-type strain, ectopic transformant and ∆cftom1 mutants were cultured in 30 ml of liquid CA medium at 22°C with shaking at 200 rpm for 5–6 d, using three biological replicates. The fungal cultures were filtered to remove mycelia and the supernatants were used to determine tomatinase activity. The culture filtrate (4 ml) of each strain was incubated with 100 μg ml−1 α-tomatine (Sigma-Aldrich) for 24 h with shaking at 28°C. After 24 h of incubation, samples were prepared for LC-MS analysis to determine α-tomatine, and its degradation products tomatidine and β-tomatine, as described above. This experiment was performed with three biological replicates.
Agrobacterium tumefaciens strains containing the pSfinx::CfTom1 and the original pSfinx (empty virus) vectors were inoculated on 10-d-old MM-Cf-0 tomato seedlings for transient heterologous CfTom1 gene expression. This experiment was performed in three biological replicates. After 5 wk, pSfinx::CfTom1 and pSfinx::Empty construct-transformed tomato leaves were collected for AF isolation. AF isolation was performed by vacuum infiltration according to the method described by de Wit & Spikman (1982). Fifteen milliliters of AF for each sample were freeze–dried and re-suspended in 6 ml of methanol containing 0.1% formic acid. Before LC-MS analysis, 1 ml of each sample was filtered through 0.45-μm pore size filters and samples were analyzed by LC-MS in positive ionization mode to determine the depletion of α-tomatine and the accumulation of tomatidine. This experiment was also performed for wild-type, ectopic and ∆cftom1 mutant-inoculated tomato plants. Here, AF was isolated from inoculated tomato plants at 10 dpi with the different strains (this experiment was performed with two biological replicates).
Wild-type C. fulvum (race 0WU; CBS131901), one ectopic and two independent ∆cftom1 mutants (∆cftom1-2.6 and ∆cftom1-5.1) were grown on half-strength PDA plates for 2–3 wk at 20°C. Conidia were collected from the plates with water and subsequently passed through one layer of miracloth to remove fungal mycelium. Conidia were diluted to a final concentration of 5 × 105 conidia ml−1. Five-week-old MM-Cf-0 tomato plants were inoculated with conidia from the wild-type, ectopic and two ∆cftom1 mutants via spray inoculation on the abaxial side of the tomato leaves. Cladosporium fulvum-inoculated tomato plants were kept in plastic-covered cages for 2 d to ensure 100% relative humidity for conidial germination. After 2 d, the plastic covers were removed and disease development of the wild-type, ectopic transformant and ∆cftom1 mutants was assayed every 2 d. Inoculated leaves were collected at different days post-inoculation (dpi) (2, 4, 8, 10 and 12 dpi; the experiment was performed with three biological replicates). Fungal biomass quantification was performed using quantitative PCR analysis as described previously, but employing genomic DNA. Genomic DNAs were isolated from wild-type, ectopic transformant and ∆cftom1 mutant-infected leaves and were diluted to a final concentration of 100 ng μl−1. A standard curve was constructed using serial dilutions of C. fulvum genomic DNA (10, 1, 0.1, 0.01, 0.001 ng μl−1) employing actin as a reference gene. Logarithms (base 10) of DNA concentrations were plotted against the crossing point of Ct values.
The effect of tomatidine on H2O2 accumulation
Cell suspensions of MSK8 tomato (Lycopersicon esculentum Mill.) were grown in Murashige and Skoog plant salt base (4.3 g l−1) liquid medium supplemented with B5 vitamins (myo-inositol (100 g l−1), nicotinic acid (1 g l−1), pyridoxine.HCl (1 g l−1) and thiamine.HCl (10 g l−1)), sucrose (3%) and kinetin (0.1 mg ml−1). The MSK8 cells were continuously rotated at 125 rpm in the dark at 25°C. Five-day-old MSK8 cell suspensions were used for the H2O2 accumulation assay. Two milliliters of MSK8 cell suspensions were transferred to 12-well plates and stabilized for 2 h in a rotary shaker before further use. These cells were co-incubated with either α-tomatine (final concentration, 40 μM) or tomatidine (final concentration, 40 μM) in the presence of the histochemical stain 3,3′-diaminobenzidine (100 μg ml−1) and with or without 1 μM chitin hexamer. The MSK8 cell suspensions treated with the same dilution rate of methanol (0.8% v/v) were used as a control. Photographs were taken at 15 h post co-incubation. A reddish-brown precipitate indicates the accumulation of H2O2.
Assessment of the phytotoxic effects of α-tomatine and tomatidine towards tomato
α-Tomatine and tomatidine (100, 250 and 500 μM, diluted from a 5-mM stock of α-tomatine and tomatidine in methanol; Sigma-Aldrich) were infiltrated into 5- or 6-wk-old tomato leaves. The same dilution rates of methanol (10%, 5% and 2% v/v) were used as negative controls. Photographs were taken 3 d after treatment with α-tomatine or tomatidine. The experiment was performed with three biological replicates.
Cladosporium fulvum secretes a functional tomatinase enzyme that degrades α-tomatine in vitro and in planta
Unlike other tomato pathogens, it has been reported previously that C. fulvum is unable to degrade α-tomatine (Melton et al., 1998). During infection, C. fulvum colonizes the apoplastic space surrounding tomato mesophyll cells without entering them. It has been hypothesized that C. fulvum does not need any tomatinase enzyme as α-tomatine has been suggested to reside solely in the vacuole. However, AFs of tomato leaves have not been critically inspected for the presence of α-tomatine. In this study, we performed LC-MS analysis on both total leaf extracts and AFs to monitor the relative levels of α-tomatine and its degradation products. As a control, we also analyzed rutin, a glycoside of the flavonoid quercetin, which has been reported to be present in the cytoplasm and/or vacuole of plants (Marrs et al., 1995; Markham et al., 2001). As expected, rutin was abundantly present in the total leaf extracts, but was undetectable in AFs, whereas α-tomatine was detected in AFs at a concentration of 0.02 ± 0.005 μmol g−1 fresh leaf (Figs 1b, S1). Although this concentration is lower than that of total leaf extracts (1.0 ± 0.1 μmol g−1 fresh leaf), it indicates that α-tomatine is detected at a significant level in AFs. Similar distributions of rutin and α-tomatine were seen in both infected and mock-inoculated leaves (Fig. S1). In contrast with the observations reported in the literature, this result shows that C. fulvum encounters α-tomatine on colonization of the tomato apoplastic space. The ability of C. fulvum to degrade α-tomatine during the infection of tomato was subsequently addressed. AFs from both C. fulvum-infected and healthy tomato leaves were isolated at different time points and analyzed by LC-MS. Again in contrast with previous reports, we observed a clear depletion of α-tomatine with the concomitant accumulation of tomatidine in AFs isolated from C. fulvum-infected plants from 10 dpi onwards (Fig. 1c,d). At 14 dpi, the concentration of α-tomatine in AFs of C. fulvum-infected tomato leaves was 17 times lower than that in AFs obtained from mock-inoculated tomato leaves. In addition, LC-MS analysis revealed the depletion of dehydrotomatine, another glycoalkaloid, in C. fulvum-infected tomato leaves (Fig. S2a,b). Accordingly, the accumulation of tomatidenol, the degradation product of dehydrotomatine, was also detected in AFs of C. fulvum-infected tomato leaves (Fig. S2c). However, the observed depletion of α-tomatine could be caused by the induction of plant-derived α-tomatine-degrading enzymes in infected plants. In order to prove that the fungus was responsible for this degradation, we first incubated culture filtrate from C. fulvum grown in vitro with α-tomatine (100 μg ml−1) for 24 h. Subsequently, the relative levels of α-tomatine and its degradation product tomatidine were monitored by LC-MS. The concentration of α-tomatine after incubation with culture filtrate was reduced relative to that of the noninoculated control medium, and α-tomatine degradation coincided with the accumulation of tomatidine (Fig. S3), which was not detected in the noninoculated control medium. These results suggest that the α-tomatine degradation observed during tomato infection is probably a result of the secretion of a tomatinase enzyme by C. fulvum. It was noteworthy that, in all the experiments, no β-tomatine could be detected, suggesting that C. fulvum does not secrete any functional GH3 tomatinase enzymes. Thus, a secreted GH10 enzyme is expected to be responsible for the degradation of α-tomatine by C. fulvum.
Identification of C. fulvum CfTom1, a GH10 tomatinase that degrades α-tomatine into tomatidine in planta
The availability of the genome sequence of C. fulvum facilitated the identification of GH10 enzyme-encoding genes. Only two genes belonging to this family were identified, but no substrate specificity could be assigned (de Wit et al., 2012). However, one of the genes encodes a protein that shares 61% amino acid identity with the F. oxysporum f. sp. lycopersici GH10 tomatinase enzyme; this gene was named CfTom1 (JGI protein ID: 188986). A phylogenetic analysis of selected GH3 and GH10 family enzymes confirmed that CfTom1 belongs to the clade of characterized fungal and bacterial GH10 tomatinases (Fig. 2). Although no GH3 tomatinase activity could be detected, the C. fulvum genome was also found to contain genes related to the GH3 tomatinase of S. lycopersici and avenacinase of G. graminis. As enzymes in the GH3 clade have been shown to provide different substrate specificities, even when they share high similarity, the GH3 enzymes of C. fulvum are believed to target other tomato metabolites. Given that the toxicity of α-tomatine is pH dependent, the expression of CfTom1 was assessed by quantitative PCR in vitro at different pH values and also in the presence or absence of α-tomatine. Consistent with the higher toxicity of α-tomatine at neutral pH, CfTom1 is weakly expressed at low pH, whereas its expression is induced at pH 7 (Fig. 3; Dow & Callow, 1978; Roddick & Drysdale, 1984). Similar to tomatinase induction by α-tomatine in F. oxysporum (Pareja-Jaime et al., 2008), the presence of α-tomatine also induces the expression of CfTom1, even at pH 4 (Fig. 3). CfTom1 shows a very low expression level during the early stages of tomato infection, but its expression is induced significantly at 12 and 15 dpi (Fig. 3).
In order to verify that CfTom1 is responsible for the degradation of α-tomatine into tomatidine, the CfTom1 gene was constitutively expressed in tomato seedlings using the PVX expression system (Hammond-Kosack et al., 1995). Five weeks after agroinoculation, AFs were isolated from PVX::CfTom1- and empty PVX-expressing tomato plants. LC-MS analysis of AF from control plants showed detectable levels of α-tomatine, but also the presence of dehydrotomatine (Fig. 4). The control AF also contained limited, but detectable, levels of tomatidine, which points to a low level of α-tomatine degradation by plant enzymes. In contrast, clear accumulation of tomatidine was detected in AF obtained from CfTom1-expressing plants, demonstrating that CfTom1 has GH10 tomatinase activity. The LC-MS analysis also revealed the accumulation of tomatidenol in AF from CfTom1-expressing tomato plants, indicating that CfTom1 can also degrade another glycoalkaloid present in tomato (Figs 4, S2c).
∆cftom1 mutants of C. fulvum can no longer degrade α-tomatine in vitro
Functional analysis of CfTom1 was continued by targeted gene deletion in the sequenced C. fulvum strain. For this, the CfTom1 gene was replaced by the hygromycin resistance (HYG) and green fluorescent protein (GFP) genes. Two independent ∆cftom1 mutants (∆cftom1-2.6 and ∆cftom1-5.1), for which gene replacement was confirmed by Southern blot analysis, were selected (Fig. S4). An ectopic transformant and the wild-type strain were used as controls in all experiments. Both the ∆cftom1-2.6 and ∆cftom1-5.1 mutants did not show any visible phenotype with regard to morphology, in vitro growth and sporulation rates when compared with the wild-type strain and ectopic transformant. To check for altered sensitivity of the mutants to α-tomatine, the compound was tested in a conidium germination assay. Conidia of the wild-type strain, ectopic transformant and two ∆cftom1 mutants were allowed to germinate in the presence of control solution (1% methanol) or 100 μM α-tomatine. In the absence of α-tomatine, no difference in the germination of conidia was observed between all strains (c. 90% of conidia germinated; Fig. 5a,b). However, when incubated in 100 μM α-tomatine, the germination of conidia of both ∆cftom1 mutants was reduced to 70% (Fig. 5a,b). The higher sensitivity of the mutants is probably a result of their inability to degrade α-tomatine.
To verify this hypothesis, culture filtrates of the deletion mutants were incubated with α-tomatine (100 μg ml−1), together with culture filtrates of the wild-type strain and ectopic transformant as positive controls, and noninoculated culture medium as a negative control. After 24 h of incubation, both α-tomatine depletion and tomatidine accumulation were observed for the wild-type strain and ectopic transformant (Fig. 5c). In contrast, culture filtrates from both mutants were not able to degrade α-tomatine and no tomatidine accumulation was detected. These results show that the higher sensitivity of the ∆cftom1 mutants to α-tomatine is a result of their inability to degrade the compound in vitro.
In planta degradation of α-tomatine is required for full virulence of C. fulvum on tomato
The ability to degrade α-tomatine appears to be important for the successful infection of tomato by fungal pathogens (Pareja-Jaime et al., 2008). The role of CfTom1 in C. fulvum virulence was analyzed by the inoculation of susceptible tomato plants with the wild-type strain, ectopic transformant and two ∆cftom1 mutants. Virulence was measured by the quantification of the fungal biomass by quantitative PCR. No significant difference in fungal biomass was observed for the wild-type, ectopic transformant and two Δcftom1 mutants during the early stages of infection (until 8 dpi; Fig. 6a). However, from 10 dpi onwards, the biomass of the two ∆cftom1 mutants was reduced by c. 63% when compared with the wild-type strain and ectopic transformant (Fig. 6a). The reduction in fungal biomass was correlated with a delay in disease progression at 10 dpi, indicating that CfTom1 is required for full virulence of C. fulvum on tomato.
The depletion of α-tomatine and the accumulation of tomatidine were measured at 10 dpi in the wild-type, ectopic transformant and two Δcftom1 mutants. Similar to the in vitro results, no tomatidine could be detected in AFs obtained from plants inoculated with the ∆cftom1 mutants, whereas this compound was clearly accumulated in the AFs of plants inoculated with the wild-type and the ectopic transformant (Fig. 6b). The reduction in fungal biomass production by the deletion mutants might be explained by their inability to degrade α-tomatine.
It has been reported previously that the breakdown products of α-tomatine, such as tomatidine and β-tomatine, can suppress plant defense responses (Bouarab et al., 2002; Ito et al., 2004), suggesting that the accumulation of tomatidine can also contribute indirectly to virulence. Thus, the reduced virulence of the ∆cftom1 mutants may not only be a result of their increased sensitivity to α-tomatine, but also of their reduced ability to suppress plant defense responses, as tomatidine no longer accumulates. We assayed the presumed defense-suppressing activity of tomatidine on H2O2 production in MSK8 tomato cell suspensions, but such activity was not confirmed in our experiments. In contrast, MSK8 tomato cells incubated with α-tomatine or tomatidine showed higher H2O2 production when compared with the negative control (Fig. S5). Furthermore, α-tomatine or tomatidine even enhanced the production of H2O2 after treatment of the cells with chitin, showing that they were unable to suppress chitin-triggered basal defense responses. This unexpected effect of tomatidine on H2O2 production suggested that it may even be toxic to tomato cells. Indeed, Itkin et al. (2011) have shown recently that the down-regulation of the glycoalkaloid metabolism-1 (GAME1) gene, which is responsible for the glycosylation of the aglycone tomatidine to α-tomatine, results in growth retardation of this tomato mutant as a consequence of the accumulation of its precursor tomatidine. To further investigate this observation, different concentrations of α-tomatine and tomatidine (100, 250 and 500 μM) were injected in the intercellular space of tomato leaves. The results clearly showed that tomatidine induces cell death in tomato leaves at concentrations of 250 and 500 μM, whereas α-tomatine does not cause cell death even at the highest concentration (Fig. 7). These results do not point to a role in plant defense suppression for tomatidine, but rather indicate that the compound is toxic to tomato. Thus, the decrease in virulence of the ∆cftom1 mutants is caused by their inability to degrade α-tomatine.
Saponins are antimicrobial compounds present in plants which provide constitutive protection against a broad range of pathogens (Osbourn, 1996). In tomato, the major characterized saponin is α-tomatine, a compound that disrupts fungal membranes. Tomato pathogens, however, can overcome this chemical barrier by the production of tomatinase enzymes that detoxify α-tomatine into β-tomatine (by GH3 enzymes) or tomatidine and lycotetraose (by GH10 enzymes; Roldan-Arjona et al., 1999; Martin-Hernandez et al., 2000).
The concentration of α-tomatine in tomato leaves can reach levels as high as 1 mM, assuming a uniform distribution in cells (Arneson & Durbin, 1967). In previous reports, it was assumed that most of the α-tomatine is localized in the vacuoles (Roddick, 1977). For this reason, and the fact that C. fulvum grows biotrophically in the intercellular space (Stergiopoulos & de Wit, 2009), it was expected that this fungus would not need to detoxify α-tomatine during infection. However, the assumed vacuolar localization is questionable, as the original conclusion of Roddick (1977) was mainly based on the fact that α-tomatine was present in the supernatant after sequential centrifugation, including ultracentrifugation, without taking into account the possibility for possible localization in the apoplast. Our analysis reports, for the first time, the concentration of α-tomatine in AFs as 0.02 ± 0.005 μmol g−1 fresh leaf. This quantification seems to be reliable because the concentration of α-tomatine in total leaf extract (1.0 ± 0.1 μmol g−1 fresh leaf) is comparable with that reported by others (Melton et al., 1998). This higher concentration suggests that α-tomatine is indeed more abundant inside plant cells, but is also present significantly in AFs. It is likely that the concentration of α-tomatine encountered by C. fulvum around the mesophyll cells inside tomato leaves is higher than that measured here. Monitoring α-tomatine degradation and tomatidine accumulation during tomato infection by C. fulvum revealed that this fungus is able to detoxify this saponin. This result contrasts with previous reports, which could possibly be explained by a lower sensitivity of the methods used at the time. In their analysis, Melton et al. (1998) performed thin-layer chromatography (TLC), which is less sensitive than LC-MS, to detect the breakdown products of α-tomatine after incubation with proteinaceous extracts isolated from culture filtrates of C. fulvum (Melton et al., 1998). More importantly, the culture filtrates they used in the assay originate from C. fulvum grown on B5 medium, for which the pH is c. 4.5. Our expression data showed that CfTom1 is barely expressed in the same medium at pH 4, suggesting that the absence of tomatinase activity in this previous study may have been a result of the limited expression of CfTom1.
Analysis of the C. fulvum CAZY enzymes revealed two GH10 and 19 GH3 genes in its genome (de Wit et al., 2012). A phylogenetic analysis showed that only one of the GH10 enzymes belongs to the GH10 tomatinase clade (CfTom1), whereas three GH3 enzymes belong to the GH3 tomatinase/avenacinase clade. Our results showed that C. fulvum degrades α-tomatine into tomatidine both in vitro and during infection of tomato, suggesting that CfTom1 is responsible for the observed activity. A specific search for the presence of β-tomatine was unsuccessful both in vitro and during infection of tomato, suggesting that the enzymes encoded by the three putative GH3 genes cannot degrade α-tomatine into β-tomatine. Alternatively, CfTom1 could also degrade β-tomatine to tomatidine. However, this is unlikely because the presence of β-tomatine was not observed in vitro or in planta when α-tomatine degradation was determined for the ∆cftom1 mutants. These results indicate that CfTom1 is probably the only enzyme responsible for the degradation of α-tomatine by C. fulvum, and the three putative GH3 tomatinases cannot complement ∆cftom1 mutants for α-tomatine degradation. The predicted protein sequences suggest that the GH3 enzymes are functional, but might be involved in the degradation of other (secondary metabolite) compounds present in tomato. Indeed, although the homology between the GH3 enzymes is high, they could have different substrate specificities. For example, the amino acid identity between S. lycopersici GH3 tomatinase and G. graminis var. avenae GH3 avenacinase is only 53% (Osbourn et al., 1995). In addition, recent RNAseq analysis performed on C. fulvum-infected tomato leaves has revealed that these three GH3 genes are poorly expressed both in vitro and in planta (P. J. G. M. de Wit, unpublished).
Our results showed a good correlation between α-tomatine depletion (Figs 1c, 6a), CfTom1 gene expression (Fig. 3) and C. fulvum growth (Fig. 6a, wild-type). α-Tomatine depletion was measured at 10 dpi, which is the time point at which the CfTom1 gene starts to be induced significantly and the fungal biomass starts to increase significantly. At later time points, the fungal biomass is much higher (Fig. 6a, wild-type) and α-tomatine depletion is also higher (Fig. 1c). Therefore, we assume that the depletion of α-tomatine is related to fungal biomass and the expression level of CfTom1.
The ability of tomato pathogens to specifically degrade α-tomatine suggests that tomatinase enzymes play an important role in the infection process. Several tomatinase enzymes have been characterized from bacterial and fungal tomato pathogens (Martin-Hernandez et al., 2000; Sandrock & Vanetten, 2001; Kaup et al., 2005; Seipke & Loria, 2008). However, neither GH3 tomatinase knock-out mutants in S. lycopersici (Martin-Hernandez et al., 2000) nor GH10 tomatinase knock-out mutants in C. michiganensis ssp. michiganensis (Kaup et al., 2005) showed a significant decrease in virulence when compared with wild-type strains. These results could be caused by only subtle effects on virulence that were difficult to detect, or by the presence of additional tomatinase-encoding genes in the genomes of these pathogens (Sandrock & Vanetten, 2001; Pareja-Jaime et al., 2008; this study). So far, the only strong evidence for the involvement of a tomatinase enzyme in pathogenicity was found for the GH10 tom1 gene of F. oxysporum f. sp. lycopersici, although the genome of this fungus also contains putative GH3 tomatinase genes (Pareja-Jaime et al., 2008). Our study shows that the GH10 CfTom1 gene of C. fulvum is involved in virulence. Colonization of tomato leaves by ∆cftom1 mutants was reduced, as shown by the significant reduction in fungal biomass from 10 dpi onwards. No significant difference in biomass was observed between the mutants, wild-type strain and ectopic transformant at the early stages of infection (2–8 dpi), at which time points the CfTom1 gene was only weakly expressed. CfTom1 gene expression is induced after 9 dpi, which explains the difference in growth between the mutants and controls at later stages of infection. Accordingly, the degradation of α-tomatine to tomatidine during infection was found to occur from 10 dpi onwards. This study confirms the previous finding that the heterologous expression of the S. lycopersici GH3 tomatinase gene in C. fulvum causes increased sporulation in susceptible tomato plants, and supports a role for α-tomatine degradation in C. fulvum virulence (Melton et al., 1998).
As it has been reported that the breakdown products of α-tomatine, such as β-tomatine and tomatidine, suppress plant defense responses (Bouarab et al., 2002; Ito et al., 2004), the absence of this indirect activity may also contribute to the reduction in virulence of ∆cftom1 mutants, in addition to the increased sensitivity of the fungus to α-tomatine. However, the suppression of plant defense responses by tomatidine could not be confirmed in our experiments. Rather, we found that tomatidine was toxic to tomato cells. A similar effect was also found by Itkin et al. (2011) in mutant tomato plants accumulating tomatidine. These results suggest that the reduction in virulence of the ∆cftom1 mutants is only caused by the increased sensitivity to α-tomatine.
Altogether, our results clearly show that the intercellular tomato pathogen C. fulvum encounters α-tomatine during the colonization of the apoplastic space of tomato leaves, and that CfTom1 is the major, and possibly only, α-tomatine-detoxifying enzyme that contributes to the full virulence of this fungus on tomato. This activity certainly contributed to the adaptation of C. fulvum to tomato after divergence from its close relative Dothistroma septosporum which is pathogenic on pine and lacks the CfTom1 gene (de Wit et al., 2012). The present work shows the powerful role of genome mining and sensitive assays in solving discrepancies reported in previous studies.
We thank Rafael Arango Isaza for supplying the p221_GFP_HYG pENTRY construct. R.C.H.d.V. acknowledges the Centre for Biosystems Genomics and the Netherlands Metabolomics Centre, which are both part of the Netherlands Genomics Initiative, for additional funding. P.J.G.M.W. was supported by the Royal Netherlands Academy of Arts and Sciences and J.C. by the European Research Area Plant Genomics, project PRR-CROP.