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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).
Figure 1. Detoxification of α-tomatine by Cladosporium fulvum. (a) Tomatinase enzymes of the glycosyl hydrolase family 10 (GH10) hydrolyze α-tomatine into tomatidine and lycotetraose. (b) LC-MS detection of rutin, a compound that has similar polarity to α-tomatine and is thought to be localized in the cell and/or vacuole, and α-tomatine, in apoplastic fluid (AF) and total extract (TE) from tomato leaves inoculated with C. fulvum at 6 d post-inoculation (dpi). α-Tomatine is present in total leaf extracts and in AFs at the concentrations of 1.0 ± 0.1 μmol g−1 and 0.02 ± 0.005 μmol g−1 of fresh leaf, respectively. LC-MS detection of the relative peak height of (c) α-tomatine and (d) tomatidine in AFs isolated at 6, 10 and 14 dpi from healthy (mock) and C. fulvum-inoculated tomato plants. A two-way ANOVA was performed and followed by a Bonferroni test. Only significant differences are indicated (**, P < 0.01; ***, P < 0.001; n = 4). Error bars represent the standard deviation of at least three biological repeats.
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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.
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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.