Bitter and sweet make tomato hard to (b)eat.

The glycoalkaloid saponin α-tomatine is a tomato-specific secondary metabolite that accumulates to millimolar levels in vegetative tissues and has antimicrobial and antinutritional activity that kills microbial pathogens and deters herbivorous insects. We describe recent insights into the biosynthetic pathway of α-tomatine synthesis and its regulation. We discuss the mode of action of α-tomatine by physically interacting with sterols, thereby disrupting membranes, and how tomato protects itself from its toxic action. Tomato pathogenic microbes can enzymatically hydrolyze, and thereby inactivate, α-tomatine using either of three distinct types of glycosyl hydrolases. We also describe findings that extend well beyond the simple concept of plants producing toxins and pathogens inactivating them. There are reports that toxicity of α-tomatine is modulated by external pH, that α-tomatine can trigger programmed cell death in fungi, that cellular localization matters for the impact of α-tomatine on invading microbes, and that α-tomatine breakdown products generated by microbial hydrolytic enzymes can modulate plant immune responses. Finally, we address a number of outstanding questions that deserve attention in the future.

Introduction

The conquest of South and Central America in the 16th and 17th centuries resulted in the import into Europe of several food crops indigenous to the America’s, including tomato and potato. These Solanaceae were soon discovered to produce bitter‐tasting glycoalkaloids in vegetative organs, of which consumption could result in serious poisoning. It was later observed that, apart from their antinutritional effect, glycoalkaloids also possess antibiotic activity, are repellent or toxic to pest insects, and may have useful medicinal applications. In the past decades, the tomato glycoalkaloid saponin α‐tomatine has been extensively studied for its role in the interaction of plants with pest insects and pathogens, often with the aim of improving plant health. In this paper we present an overview of α‐tomatine as a broad‐spectrum toxic plant compound that protects tomato from herbivores and pathogens, and we discuss recent insights into its biosynthesis and regulation. Furthermore, we describe how microbial pathogens cope with the inhibitory activity of α‐tomatine and may even exploit the hydrolytic breakdown products of α‐tomatine to modulate plant immune responses.

α‐Tomatine: the major tomato saponin with antibiotic activity

The study of α‐tomatine started from the exploration of fungistatic agents in tomato tissues. Over 70 yr ago, Fontaine et al. (1948) named the first purified compound from tomato leaves possessing antifungal properties as tomatine. It was identified as a glycosidal alkaloid, also known as steroidal glycoalkaloids (SGAs), which are a subgroup of saponins. Later studies revealed that its chemical structure is composed of a steroidal aglycone (‘tomatidine’) and a tetrasaccharide side branch (β‐lycotetraose) containing two molecules of glucose and one molecule each of galactose and xylose. α‐Tomatine is the name of the form with the tetrasaccharide, whereas the other forms lacking a terminal xylose or terminal glucose, or both terminal sugars were designated as β1‐tomatine, β2‐tomatine, and γ‐tomatine, respectively (Fig. 1; Kuhn et al., 1956, 1957). α‐Tomatine is the major SGA, as well as the main saponin, in vegetative tissues and green fruits; its concentration can be up to several millimolar on an FW basis (Keukens et al., 1995; Friedman & Levin, 1998; Kozukue et al., 2004; Iijima et al., 2013). The high α‐tomatine levels in immature fruit decrease during ripening by the conversion to esculeoside A (Fig. 1; Mintz‐Oron et al., 2008; Iijima et al., 2009; Cárdenas et al., 2019; Nakayasu et al., 2020). The antimicrobial activity of α‐tomatine was first demonstrated on the fungal pathogen Fusarium oxysporum f. sp. lycopersici (Roddick, 1974); thereafter, antibiotic properties were reported against many tomato pathogens (including fungi, oomycetes, and bacteria), as well as pest insects (Campbell & Duffey, 1979; Sandrock & VanEtten, 1998; Kaup et al., 2005; Seipke & Loria, 2008; Altesor et al., 2014; Chowánski et al., 2016). Because of its high concentration and broad spectrum of in vitro antibiotic activity, α‐tomatine has long been studied as a defense compound that might confer resistance to tomato pathogens. Here, we will discuss old and recent knowledge about the relevance and mode of action of α‐tomatine and illustrate its versatile biological properties, which extend well beyond the perception of a ‘simple’ membrane‐perforating toxin.

Fig. 1

The tomato (Solanum lycopersicum) biosynthetic pathway of α‐tomatine from cholesterol. Chemical structures and names of several biosynthetic intermediates are provided. The arrows represent catalytic conversions, with the gene name (if characterized) provided above the solid blue arrows; dashed blue arrows represent catalytic conversions for which genes are currently unknown. Genes in colored boxes are in close physical proximity in a genomic cluster on tomato chromosomes 7 and 12. The red dashed boxes highlight three key components: the phytotoxic precursor tomatidine, the defense compound α‐tomatine, and the nonbitter breakdown product esculeoside A.

The biosynthetic pathway of α‐tomatine and its regulation

Although α‐tomatine was identified as the major SGA in tomato more than 70 yr ago, the metabolism and regulation of its synthesis are not fully understood. Initial studies on α‐tomatine metabolism were driven by an interest to improve fruit quality by removing the antinutritional trait caused by α‐tomatine. However, as a potent defense metabolite, insight into its metabolism can also help to increase α‐tomatine levels in vegetative tissue, and thereby contribute to resistance.

SGAs, including α‐tomatine, are synthesized from cholesterol, although cholesterol biosynthesis in plants itself is not fully understood. Conversion of cholesterol to α‐tomatine (Fig. 1) involves multiple reactions mediated by enzymes encoded by GLYCOALKALOID METABOLISM (GAME) genes (Itkin et al., 2013). The first part of the pathway requires four enzymes (GAME7, GAME8, GAME11 and GAME6) and results in the synthesis of a saponin aglycone that serves as precursor for both glycoalkaloids and steroidal saponins. The first dedicated step towards glycoalkaloid production is the oxidation of saponin aglycone by GAME4, followed by multiple additional conversions to form the aglycone alkaloid tomatidine (Itkin et al., 2013). Recent research revealed that the conversion from dehydrotomatidine to tomatidine is not mediated by a single reaction, as suggested by Friedman (2002) and Itkin et al. (2013), but rather involves multiple steps, including oxidation, isomerization, and reduction, and requires a short‐chain dehydrogenase/reductase (GAME25, also known as Sl3βHSD) and steroid 5α‐reductase (SlS5αR2) (Sonawane et al., 2018; Akiyama et al., 2019; Lee et al., 2019). As shown in Fig. 1, the synthesis of α‐tomatine from its aglycon tomatidine requires four consecutive glycosylations by distinct glycosyltransferases GAME1, GAME17, GAME18 and GAME2 (Itkin et al., 2013, 2011). During tomato fruit ripening, the decrease of α‐tomatine content results from conversion to esculeoside A, a nonbitter steroidal glycoalkaloid. This process involves GAME31, a 2‐oxoglutarate‐dependent dioxygenase (also known as Sl23DOX) that catalyzes the hydroxylation of α‐tomatine, and the recently identified glycosyltransferase GAME5, which produces esculeoside A (Fig. 1; Cárdenas et al., 2019; Nakayasu et al., 2020; Szymański et al., 2020).

Interestingly, six GAME genes are physically clustered on tomato chromosome 7 (Fig. 1), including GAME11 and GAME6, which are required in the production of the furostanol‐type saponin aglycon along with the four genes encoding the glycosyltransferase that add the lycotetraose moiety to tomatidine. The potato has a similar cluster in the syntenic region; however, potato lacks orthologues to the GAME18 and GAME2 genes of tomato, which mediate the two final steps of tomatine biosynthesis (Cárdenas et al., 2015). Also, the genes GAME4 and GAME12, involved in the first two dedicated steps towards glycoalkaloid synthesis, are physically clustered in tomato chromosome 12, and this cluster is conserved in potato (Cárdenas et al., 2015).

The regulation of α‐tomatine metabolism involves the GAME9 gene, a member of the APETALA2/Ethylene Response Factor family, also referred to as JRE4 (Cárdenas et al., 2016; Thagun et al., 2016). More recently, additional genes involved in the (positive or negative) regulation of α‐tomatine metabolism have been identified (Wang et al., 2018; Zhao et al., 2018; Chen et al., 2019; Swinnen et al., 2020; see Table 1).

Table 1

Tomato genes shown to be involved in regulation of glyoalkaloid biosynthesis.

Gene name	Locus name	Role in regulation of α‐tomatine metabolism	Target genes	References
GAME9/JRE4	Solyc01g090340	Positive regulator	C5‐SD, DWF5, GAME4, GAME7, GAME17	Cárdenas et al. (2016), Nakayasu et al. (2018), Thagun et al. (2016), Yu et al. (2020)
MYC2	Solyc08g076930	Positive regulator	C5‐SD, GAME4, GAME7	Cárdenas et al. (2016), Swinnen et al. (2020)
MYC1	Solyc08g005050	Positive regulator	C5‐SD	Swinnen et al. (2020)
TAGL1	Solyc07g055920	Negative regulator	Unknown	Zhao et al. (2018)
TDR4/FUL1	Solyc06g069430	Negative regulator	Unknown	Zhao et al. (2019)
HY5	Solyc08g061130	Positive regulator	GAME1, GAME4, GAME17	Wang et al. (2018)
PIF3	Solyc01g102300	Negative regulator	GAME1, GAME4, GAME17	Wang et al. (2018)
MYB12	Solyc06g009710	Positive regulator	Unknown	Chen et al. (2019)

Toxicity mechanisms: membrane disruption or more than that?

Numerous studies have shown that α‐tomatine is toxic to a spectrum of tomato pathogens and pests. Membrane disruption, followed by cytoplasmic leakage and cell death, was observed in cells exposed to α‐tomatine (Arneson & Durbin, 1968; Campbell & Duffey, 1979; Osbourn, 1996a; Hoagland, 2009). The molecular basis for membranolytic action has been studied in depth. Membrane leakage caused by α‐tomatine is dependent on the presence of sterols in the plasma membrane. A mutant of Fusarium solani that accumulated 20% less sterol in the membrane manifested lower sensitivity to α‐tomatine (Défago & Kern, 1983). Phytophthora species do not synthesize sterols and, hence, were tolerant to α‐tomatine; however, they gained sensitivity when grown in medium supplemented with free sterols (Steel & Drysdale, 1988). Old studies showed that α‐tomatine can bind in vitro to different types of sterols, such as cholesterol and ergosterol, which are the major mammalian and fungal sterols, respectively, and sitosterol and stigmasterol, which are predominantly found in plant cells (Roddick, 1979). The membrane disrupting effect caused by this interaction required the intact tetrasaccharide group of α‐tomatine and the presence of sterol 3β‐hydroxy groups (Nepal & Stine, 2019). By contrast, membranes containing sterols lacking 3β‐hydroxy groups were insensitive to disruption by α‐tomatine (Roddick & Drysdale, 1984; Steel & Drysdale, 1988; Keukens et al., 1995). Chemical hydrolysis products of α‐tomatine lacking a single monosaccharide (β1‐tomatine, β2‐tomatine) or multiple sugars (γ‐tomatine and the aglycon tomatidine) showed > 95% reduction in their ability to disrupt membranes (Keukens et al., 1995).

Despite its disrupting activity on artificial membranes, infiltration of α‐tomatine into the apoplast of tomato leaves did not cause visible damage (Ökmen et al., 2013). Considering the high concentration of α‐tomatine in tomato tissue, tomato plants must be able to avoid self‐intoxication. Indeed, tomato and potato leaves had a lower content of free sterols (c. 10%) and were more resistant to α‐tomatine as they manifested less electrolyte leakage than plants containing higher proportions of free sterols, such as tobacco (Nicotiana benthamiana) and Nicandra physalodes (c. 50%) (Steel & Drysdale, 1988). The fact that tomato cells can withstand high concentrations of α‐tomatine is likely associated with substitution at 3β‐hydroxyl groups, thereby forming sterol conjugates that prevent binding with α‐tomatine (Steel & Drysdale, 1988). In contrast to plants, fungi predominantly accumulate ergosterol, which occurs as free sterol and in multiple esterified forms (Hartmann, 1998; Weete et al, 2010). The ratio between these two forms varies among fungal species, but it is unknown whether this ratio affects the sensitivity to α‐tomatine (Yuan et al., 2007).

Apart from the membranolytic action by sterol binding, Ito et al. (2007) reported that α‐tomatine may induce programmed cell death (PCD) in the fungus F. oxysporum. Hallmarks of apoptosis, such as DNA fragmentation, depolarization of the transmembrane potential of mitochondria, and generation of reactive oxygen species (ROS), were detected in fungal cells treated with α‐tomatine (Ito et al., 2007). The cell death induction by α‐tomatine in F. oxysporum was markedly reduced by the application of a specific inhibitor of PCD (Ito et al., 2007), the only report thus far of PCD induction by α‐tomatine in tomato pathogens. However, the PCD‐inducing activity in fungi is not unexpected as α‐tomatine was also reported to stimulate caspase‐independent PCD in mouse colon cells and human leukemia cell lines (Chao et al., 2012; Kim et al., 2015). The induction of apoptosis in plant pathogens by plant defense molecules was reported in the interaction between Arabidopsis and the fungus Botrytis cinerea (Shlezinger et al., 2011). The toxic effect of α‐tomatine seems to be based both on membranolytic activity and the activation of the PCD machinery, although the mechanism of PCD induction remains elusive. More efforts should be made to increase our understanding of the modes of action of α‐tomatine.

How pathogens deal with α‐tomatine: the role of tomatinase

One way of dealing with the toxic action of α‐tomatine is the accumulation of low levels of sterols in membranes, as occurring in Pythium and Phytophthora species (Défago & Kern, 1983; Steel & Drysdale, 1988). Besides such passive tolerance, fungi can actively repair membrane damage inflicted by α‐tomatine. Exposure of Neurospora crassa to α‐tomatine triggered the recruitment of the membrane repair protein PEF1 to the lysing point at the membrane, and deletion of pef1 increased the sensitivity of N. crassa to α‐tomatine and other pore‐forming drugs (Schumann et al., 2019). Furthermore, fungi can actively export exogenous toxic compounds through ATP‐binding cassette (ABC) transporters. The roles of ABC transporters in the efflux of plant secondary metabolites and synthetic fungicides are well documented (Andrade et al., 2000; Schoonbeek et al., 2001; Stergiopoulos & de Waard, 2002; Kretschmer et al., 2009; Stefanato et al., 2009). In the insect herbivore Helicoverpa armigera, transcript levels for ABC transporters were induced when larvae were fed α‐tomatine (Bretschneider et al., 2016), indicating that the adaption to α‐tomatine in H. armigera might require ABC transporters. However, a direct role of ABC transporters in the tolerance to α‐tomatine in tomato pathogens and pests remains to be characterized.

Another active way of dealing with α‐tomatine is to secrete enzymes that degrade α‐tomatine to reduce its toxicity (Osbourn, 1996a; Sandrock & VanEtten, 1998). Hydrolysis of α‐tomatine was first reported in the fungus Septoria lycopersici (Arneson & Durbin, 1968). Since then, the identification and characterization of tomatinase activity has been extended to more pathogens. Although enzymes catalyzing the hydrolysis of α‐tomatine are collectively referred to as tomatinase, they differ in the glycosidic cleavage sites, catalytic mechanisms, and the classification of corresponding genes (Fig. 2; Table 2). The degradation process is categorized into three main actions, based on the hydrolysis products β2‐tomatine, β1‐tomatine and tomatidine.

Fig. 2

Different hydrolytic activities that detoxify α‐tomatine. Chemical structures of α‐tomatine are simplified. The glycosidic bonds that are cleaved are indicated by a red arrow. Carbohydrate‐active enzyme activities that catalyze the reaction are indicated below the arrows, and microorganisms (Cladosporium fulvum, Clavibacter michiganensis subsp. michiganensis, Fusarium graminearum, Fusarium oxysporum f. sp. lycopersici, Fusarium solani, Gibberella pulicaris, Streptomyces scabies 87‐22, Colletotrichum coccodes, Septoria lycopersici, Verticillium albo‐atrum, Botrytis cinerea) that were shown to possess these activities are specified above the arrows.

Table 2

Microbial glycosyl hydrolases capable of degrading α‐tomatine.

Pathogen	GH family	Accession (database)	Degradation product	Reference
Colletotrichum coccodes	Unknown	not applicable	β2‐Tomatine	Sandrock & VanEtten (2001)
Septoria lycopersici	GH3	U35462 (NCBI)	β2‐Tomatine	Sandrock et al. (1995)
Verticillium albo‐atrum	Unknown	Not applicable	β2‐Tomatine	Pegg & Woodward (1986)
Botrytis cinerea	GH3, GH39, GH43?	Not applicable	β1‐Tomatine	Quidde et al. (1998)
Cladosporium fulvum	GH10	188986 (JGI)	Tomatidine	Ökmen et al. (2013)
Clavibacter michiganensis subsp. michiganensis	GH10	AAP57293 (NCBI)	Tomatidine	Kaup et al. (2005)
Fusarium graminearum	GH10	EYB27127 (NCBI)	Tomatidine	Carere et al. (2017)
Fusarium oxysporum f. sp. lycopersici	GH10	AJ012668 (NCBI)	Tomatidine	Roldán‐Arjona et al. (1999)
Fusarium solani	Unknown	Not applicable	Tomatidine	Lairini & Ruiz‐Rubio (1998)
Gibberella pulicaris	Unknown	Not applicable	Tomatidine	Weltring et al. (1998)
Streptomyces scabies 87‐22	GH10	CBG74701 (NCBI)	Tomatidine	Seipke & Loria (2008)
Alternaria alternata	Unknown	Not applicable	Unknown, but not tomatidine	Oka et al. (2006)
Corynespora cassiicola	Unknown	Not applicable	Unknown, but not tomatidine	Oka et al. (2006)

Regulation of tomatinase expression

β2‐Tomatine as the main product

Generation of β2‐tomatine by cleaving off the terminal d‐glucose has been reported in the fungi S. lycopersici, Verticillium albo‐atrum, and Colletotrichum coccodes (Arneson & Durbin, 1968; Pegg & Woodward, 1986; Sandrock et al., 1995; Sandrock & VanEtten, 2001). The enzyme possesses β‐glucosidase activity and is named β2‐tomatinase. The cloning and sequencing of the S. lycopersici β2‐tomatinase gene indicated that it belongs to the Glycosyl Hydrolase (GH) Family 3 of carbohydrate‐active enzymes (CAZymes) (Lombard et al., 2014).

β1‐Tomatine as the main product

Besides the terminal d‐glucose, the other terminal sugar moiety, d‐xylose, can be the target of enzymatic hydrolysis. Removal of the terminal d‐xylose and release of β1‐tomatine was reported in B. cinerea (Quidde et al., 1998). Genes encoding β1‐tomatinase have not been cloned, so we cannot yet attribute the activity to a GH family; however, this enzyme must possess β‐xylosidase activity and is likely from the GH39 or GH43 family of CAZymes. Using the sequence of S. lycopersici β2‐tomatinase as a probe, Quidde et al. (1999) cloned a homologue (sap1) from B. cinerea. Characterization of a B. cinerea sap1 knockout mutant revealed that this gene is not responsible for the β1‐tomatinase activity as the mutant can still hydrolyze α‐tomatine (Quidde et al., 1999). The characterization of genes encoding β1‐tomatinase in B. cinerea and other fungi awaits their cloning.

Tomatidine as the main product

Besides hydrolytic removal of single terminal sugar moieties, several microorganisms can convert α‐tomatine to the aglycon tomatidine (Fig. 2; Table 2): the fungi Cladosporium fulvum (Ökmen et al., 2013), F. oxysporum (Roldán‐Arjona et al., 1999), F. solani (Lairini & Ruiz‐Rubio, 1998), Fusarium graminearum (Carere et al., 2017), and Gibberella pulicaris (Weltring et al., 1998), and the bacterial pathogens Clavibacter michiganensis (Kaup et al., 2005) and Streptomyces scabies (Seipke & Loria, 2008). The generation of tomatidine is through the removal of the tetrasaccharide chain (lycotetraose). Unlike the β2‐tomatinase, which belongs to the CAZy GH3 category, all tomatinase activities that detoxify α‐tomatine through cleaving off the lycotetraose belong to the GH10 family (Table 2). Finally, there is one example of an organism possessing distinct, functionally redundant enzymes capable of detoxifying α‐tomatine. Removing the lycotetraose group was first considered to be the mode of action of α‐tomatine degradation in F. oxysporum (Roldán‐Arjona et al., 1999). However, a knockout mutant in the GH10 CAZyme gene remained able to degrade α‐tomatine because it also possesses several GH3 enzymes that convert α‐tomatine into β2‐tomatine instead of tomatidine. The β2‐tomatinase GH3 activity was not identified in the first place as it was masked by the presence of the GH10 tomatinase activity, which cleaves off the entire lycotetraose branch (Pareja‐Jaime et al., 2008).

Several aspects of the regulation of expression of tomatinase genes have been described. First, the tomatinase activity and expression of tomatinase genes can often be induced by α‐tomatine (Quidde et al., 1998; Roldán‐Arjona et al., 1999; Ökmen et al., 2013). Second, induction seemed to be specific to α‐tomatine treatment, as there was no induction by other saponins, such as chaconine or solanine in B. cinerea (Quidde et al., 1998). Finally, the effect of carbon catabolite repression differed between fungi: β1‐tomatinase from B. cinerea was not subject to catabolite repression (Quidde et al., 1998), but expression of the F. oxysporum GH10 tomatinase gene was repressed when glucose is present (Roldán‐Arjona et al., 1999).

Degradation of α‐tomatine is more than detoxification

Pathogens achieve detoxification of α‐tomatine by enzymatically converting it to less‐toxic products. In addition to detoxification, tomatinase activity may have additional biological repercussions. A GH3 tomatinase‐deficient mutant of S. lycopersici caused more intense plant cell death than the wild‐type in early stages of infection and induced enhanced expression of defense‐related genes on tomato leaves (Martin‐Hernandez et al., 2000). In a different study, inoculation on N. benthamiana of the S. lycopersici GH3 tomatinase‐deficient mutant, but not the wild‐type, elicited intense cell death in mesophyll tissue; this resembled a hypersensitive response, and the infection was fully contained within 2 d post‐inoculation (Bouarab et al., 2002). These observations indicated that GH3 tomatinase not only detoxifies α‐tomatine, but also mediates the suppression of plant defense responses. Further experiments showed that pre‐infiltration of β2‐tomatine in N. benthamiana leaves enabled the S. lycopersici GH3 tomatinase mutant to cause expanding lesions and also compromised plant resistance to the bacterial pathogen Pseudomonas syringae pv tabaci. By contrast, infiltration of α‐tomatine did not have such effects (Bouarab et al., 2002). Moreover, silencing of the N. benthamiana SGT1 gene, required for disease resistance in plants, restored the pathogenicity of S. lycopersici GH3 tomatinase‐deficient mutant (Austin et al., 2002; Bouarab et al., 2002; Peart et al., 2002). These observations suggest that the capacity of tomatinase to suppress plant defense depends on the breakdown product(s) generated by tomatinase, rather than the protein itself. A dual function of tomatinase was also reported in F. oxysporum, which converts α‐tomatine by a GH10 hydrolase to the aglycon tomatidine (Roldán‐Arjona et al., 1999). The addition of either tomatidine or lycotetraose to suspension‐cultured tomato cells can suppress the oxidative burst and hypersensitive cell death triggered by fungal elicitor (Ito et al., 2004). The effect of tomatidine and lycotetraose on the production of ROS was studied in more detail. In vitro assays revealed that tomatidine could scavenge superoxide anions as effectively as ascorbic acid, whereas lycotetraose did not possess antioxidant activity. These observations suggest that the suppression of an oxidative burst by degradation products of α‐tomatine is based on different mechanisms: tomatidine can directly scavenge ROS, whereas lycotetraose might block the generation of ROS through an as yet unknown mechanism. Furthermore, treatment of tomato plant with tomatidine or lycotetraose promoted the colonization of hypocotyls by a nonpathogenic F. oxysporum strain lacking tomatinase activity (Ito et al., 2004). Besides modulating plant defense responses, tomatidine was reported to exhibit phytotoxic effects. Transgenic tomato plants in which the GAME1 gene (Fig. 1) was silenced accumulated excessive levels of tomatidine and exhibited severe developmental defects (Itkin et al., 2011). This observation was substantiated by the cell‐death‐inducing effect on tomato leaves of exogenously applied tomatidine (Fig. 3; Ökmen et al., 2013). Based on these studies, it is apparent that the hydrolysis of α‐tomatine during pathogen infection is not merely reducing its toxicity but is also affecting the physiology and defense responses of the plant through α‐tomatine breakdown products. In some situations, the latter role appeared to be important for virulence of tomato pathogens (Bouarab et al., 2002; Ito et al., 2004).

Fig. 3

Phytotoxic effects of α‐tomatine and tomatidine on tomato (Solanum lycopersicum) leaves. Taken from Ökmen et al. (2013) with permission.

Subcellular localization: arsenal or battlefield?

Like other saponins, α‐tomatine is thought to be localized within tomato cells and to be released upon cell damage resulting from pathogen invasion (Dow & Callow, 1978) or pest feeding. The subcellular localization can define the spatial and temporal contribution of α‐tomatine to the inhibition of pathogen infection. Theoretically, if a tomato pathogen can avoid the release of α‐tomatine from the host cells, it would circumvent the inhibition. Studies on the biotrophic tomato pathogen C. fulvum have shed light on the importance of the distribution of α‐tomatine because this fungus exclusively colonizes the apoplast and causes limited damage to host cells until the final stage of infection (Stergiopoulos & de Wit, 2009). Initially, it was proposed that C. fulvum was less likely to encounter inhibitory concentrations of α‐tomatine during infection if the glycoalkaloid predominantly localizes intracellularly. Based on this assumption, it was hypothesized that tomatinase activity might not be important for full virulence of C. fulvum despite high sensitivity of the fungus to α‐tomatine (Dow & Callow, 1978; Kohmoto & Yoder, 1998; Melton et al., 1998). In order to test whether tomatinase activity contributes to virulence, Melton et al. (1998) expressed a GH3 tomatinase gene from S. lycopersici in C. fulvum, as this was the only characterized tomatinase gene at that time. Expression of the heterologous tomatinase resulted in enhanced virulence of C. fulvum (as assessed by increased sporulation) and provided evidence for a positive role of α‐tomatine degradation to C. fulvum infection (Melton et al., 1998). A later study described the identification of the C. fulvum endogenous GH10 tomatinase gene CfTom1 and further substantiated the role of tomatinase activity. A knockout mutant in the CfTom1 gene displayed increased sensitivity to α‐tomatine and reduced virulence on tomato (Ökmen et al., 2013). This study also detected the presence of α‐tomatine in apoplastic fluid at 0.02 mM, which is low compared with the levels of c. 1 mM in total leaf extract (Ökmen et al., 2013). In light of these studies, there is no doubt that α‐tomatine predominantly accumulates inside plant cells; however, the amounts of α‐tomatine in intercellular spaces might be sufficient to exert some inhibition to invading microbes. It is unknown whether the apoplastic localization of α‐tomatine involves active secretion or merely results from the leakage from cells. The impact of the intercellular distribution of α‐tomatine in the defense against pathogens that employ different infection strategies, such as necrotrophic and hemibiotrophic pathogens, remains unclear.

Effect of pH on tolerance to α‐tomatine

α‐Tomatine is more toxic at higher pH (Arneson & Durbin, 1968; Dow & Callow, 1978). At pH 3.0, α‐tomatine concentrations almost 300 times higher were required to achieve the same inhibitory effect on fungi as at pH 8.0 (Arneson & Durbin, 1968). This effect might be partially caused by increased protonation of α‐tomatine in acidic conditions, as the unprotonated α‐tomatine can bind to cholesterol in vitro but the protonated form cannot (Arneson & Durbin, 1968). Besides influencing the toxicity of α‐tomatine, ambient pH may also affect the expression of tomatinase genes. The C. fulvum GH10 tomatinase gene Cftom1 was barely expressed in liquid medium containing α‐tomatine at pH 4.0, whereas abundant transcript levels were detected at pH 7 (Ökmen et al., 2013). This observation explained why a previous study could not detect α‐tomatine degradation, as the medium used to grow the mycelium was adjusted to pH 4.5 (Melton et al., 1998). Studies showing the impact of pH on in vitro assays raise questions about the role of ambient pH at infection sites in tomato–pathogen interactions, and highlight the possible impact of ambient pH manipulation by microbes during infection, such as the host tissue acidification reported for B. cinerea (Müller et al., 2018). Although the effect of pH manipulation might not occur with the specific purpose to decrease sensitivity to α‐tomatine, it likely affects its toxicity and thereby could have an impact on the outcome of tomato–microbe interactions.

Typical phytoanticipin or more than that?

The term phytoanticipin was first proposed and defined by VanEtten et al. (1994). Phytoanticipins are low molecular weight metabolites with antibiotic properties that are either preformed or generated from accumulated precursors when plants are challenged by pathogens. Phytoanticipins differ from the phytoalexins, which are induced upon pathogen infection. α‐Tomatine has long been considered as a potent phytoanticipin because of its high accumulation in healthy tomato tissues and its toxicity against different pathogens (Osbourn, 1996b; Piasecka et al., 2015). A recent study on resistance to early blight (Alternaria solani) described a difference in metabolic profiles between a resistant wild tomato (Solanum arcanum) and the susceptible cultivated tomato (Solanum lycopersicum). This study indicated that α‐tomatine can also serve as a phytoalexin in certain conditions (Shinde et al., 2017). A pronounced increase of α‐tomatine content was detected in S. arcanum after A. solani infection, to levels 10 times higher than before infection. By contrast, the susceptible cultivated tomato had more severe symptoms, and its α‐tomatine level increased by only 2.5‐fold. Counter‐intuitively, the expression of α‐tomatine biosynthetic genes GAME1, GAME17 and GAME18, as well as the regulator gene GAME9, were much higher in susceptible cultivated tomato despite the lower increase of α‐tomatine levels, compared with the wild tomato upon infection. By contrast, GAME2, which encodes the enzyme that performs the last step of α‐tomatine synthesis, was expressed at much higher levels in resistant wild tomato, highlighting an important (rate‐limiting) role of GAME2 expression in α‐tomatine stimulation in response to A. solani invasion. To date, this is the only report showing that α‐tomatine biosynthesis can be elicited by the challenge of microbes.

The balance between α‐tomatine accumulation and degradation defines the outcome on the battlefield

Although being referred to as a defense compound in many studies because of its high accumulation in tomato tissue and its toxicity against many pathogens, direct evidence of the contribution of α‐tomatine to plant immunity is lacking. The importance of α‐tomatine in basal defense is indirectly implied from various studies on tomato pathogens. First, tomato pathogens tend to be more resistant to α‐tomatine than organisms that are nonpathogenic on tomato (Arneson & Durbin, 1968; Steel & Drysdale, 1988). For instance, mycelia of the fungal tomato pathogens B. cinerea, V. albo‐atrum and F. solani exhibited less electrolyte leakage than nontomato pathogens, such as Alternaria tenuis, Ascochyta pisi and F. graminearum, when incubated with α‐tomatine (Steel & Drysdale, 1988). A comprehensive study among 23 fungal strains revealed a strong correlation between the tolerance to α‐tomatine, the ability to degrade α‐tomatine, and pathogenicity on tomato (Sandrock & VanEtten, 1998). A similar phenomenon was observed in pea pathogens: among 50 plant pathogenic microbes, only the taxa that were able to metabolize the pea phytoalexin pisatin could infect pea, and all isolates that were nonpathogenic on pea were unable to detoxify pisatin (Delserone et al., 1999). Moreover, the pea pathogen Nectria haematococca can infect mature tomato fruit (low in α‐tomatine) but not green fruit, which accumulates high concentrations of α‐tomatine, whereas expression of the S. lycopersici GH3 tomatinase gene in N. haematococca conferred the ability to colonize green tomato fruit (Sandrock & VanEtten, 2001). These observations implicate that degrading α‐tomatine is essential to achieving successful infection on tomato or determining the host range. A similar concept was described for the oat pathogen Gaeumannomyces graminis var. tritici, in which mutants that were unable to degrade the oat saponin avenacin A‐1 lost their ability to infect oat (Osbourn et al., 1995).

However, the mutagenesis of genes encoding tomatinase in several microbes thus far does not support the role of tomatinase as an essential determinant in pathogenicity on tomato but rather contributes quantitatively to virulence. For example, C. fulvum GH10 tomatinase‐deficient mutants remained pathogenic on tomato despite accumulating less fungal biomass, whereas the heterologous overexpression of S. lycopersici GH3 tomatinase in C. fulvum enhanced fungal sporulation during tomato infection (Melton et al., 1998). Also, in F. oxysporum, GH10 tomatinase‐deficient mutant caused delayed disease development compared with the wild‐type (Pareja‐Jaime et al., 2008). Moreover, the natural field isolate M3a of B. cinerea (from grape) was deficient in α‐tomatine degrading activity and accordingly was less virulent on tomato leaves, compared with the α‐tomatine‐degrading strain B05.10. When infecting plant tissues lacking α‐tomatine, such as bean leaves, similar lesion sizes were observed for M3a and B05.10 (Quidde et al., 1998). In addition, the infection on tomato was unaffected when tomatinase was disrupted in S. lycopersici (Martin‐Hernandez et al., 2000) or in the bacterium S. scabies (Seipke & Loria, 2008). In these cases, it suggested that these organisms possess additional mechanisms that confer tolerance to α‐tomatine. These observations are indicative of the importance of tomatinase in tomato–microbe interactions and consequently highlight the potential role of α‐tomatine in tomato basal defense.

Moreover, the contribution of ‘tomatinase’ to plant infection might not necessarily be (exclusively) related to α‐tomatine degradation. As already described, tomatinases are glycosyl hydrolases of distinct CAZyme families, which might also act on substrates other than α‐tomatine and thereby play a different role in the infection. For instance, the virulence of GH3 tomatinase‐deficient mutants of S. lycopersici was not reduced on tomato leaves; however, they failed to infect N. benthamiana leaves, which do not accumulate α‐tomatine (Bouarab et al., 2002). Similarly, a glycosyl hydrolase from F. graminearum possessing hydrolytic activity, an α‐tomatine, acted as a virulence factor on wheat (Carere et al., 2017).

In a recent study, tomato leaves overexpressing a gene encoding tomato strictosidine synthase (STR‐2) accumulated more α‐tomatine and exhibited enhanced resistance against B. cinerea and Phytophthora infestans (Chen et al., 2019). Taken together, there are strong indications that α‐tomatine participates in the basal defenses against pathogens. However, whether an increase in α‐tomatine levels may increase resistance to presently notorious pathogens and the absence of α‐tomatine will render tomato plants more susceptible to organisms not normally infecting tomato remain to be studied. Such studies would benefit from using α‐tomatine‐deficient transgenic tomato (using CRISPR) and lines accumulating a higher level of α‐tomatine to provide direct evidence of the role of α‐tomatine in plant immunity.

Conclusion and perspectives

The data discussed herein provide circumstantial evidence that α‐tomatine is a specialized metabolite that confers important levels of protection from herbivory and pathogen invasion. The final proof of its important function in plant defense remains to be provided by knocking out its biosynthesis or increasing its levels by selective overexpression of GAME genes and testing the impact of altered α‐tomatine levels on the susceptibility to herbivores or pathogens. The mode of action on plant, microbial, and insect membrane and the mechanisms by which α‐tomatine induces PCD in plants need to be resolved.

Enzymatic degradation of α‐tomatine by three distinct types of microbial secreted CAZymes – GH3, GH10 or GH43, which, respectively, remove the terminal glucose moiety, terminal xylose moiety, or the entire lycotetraose group – provides a beautiful example of independent, convergent evolution in several pathogenic bacteria and fungi towards the detoxification of a potent antimicrobial compound. These genes probably evolved from ancestral GH genes with the appropriate catalytic site, towards specialization on a substrate that was a major obstacle for pathogen development and reproduction in a toxic environment. For detoxification of other phytoanticipins, such as avenacin and pisatin, there is generally just a single enzymatic activity reported that can inactivate these compounds. The finding of three separate detoxification activities for the same antimicrobial compound is remarkable.

It is noteworthy that enzymatic degradation products of α‐tomatine, such as β2‐tomatine, tomatidine, and lycotetraose, can modulate the immune response of a plant, suggesting that the removal of sugar moieties benefits a pathogenic microbe in two ways: by reducing membrane permeating activity of α‐tomatine and by lowering the plant defense machinery. The impact of antimicrobial plant metabolites on the plant immune response through different mechanisms (other than being toxic to microbes) deserves further attention. The observation that sterol glycosylation in tomato confers tolerance to the toxicity of α‐tomatine raises the question of whether microbes, and especially tomato pathogens, could protect themselves from membrane damage by glycosylating their sterols, either constitutively or in the presence of α‐tomatine.

Author contributions

YY collected the literature and prepared the figures. YY and JALvK wrote the manuscript.