Introduction: Plant genomes harbor a great diversity of glycosyl hydrolases

Carbohydrates are abundant biomolecules on our planet and play a prominent role in plants. In plants, carbohydrates are found in two forms: simple and complex. Glucose is the simplest monosaccharide synthesized during photosynthesis. These sugars are used for energy and serve as substrates for building complex carbohydrates. Monosaccharide sugars such as glucose are linked via glycosidic bonds, resulting in the formation of complex polysaccharides such as starch and cellulose. Sometimes, monosaccharides are linked to noncarbohydrate species, such as metabolites or proteins via glycosidic bonds leading to the formation of glycoconjugates (Szeja et al. 2017). These complex carbohydrates do not remain static but are hydrolyzed during various physiological processes in the plant life cycle. Therefore, the hydrolysis of polysaccharides is an important biochemical process that is essential for plant survival.

Glycosidases or glycosyl hydrolases (GHs) are a family of carbohydrate-active enzymes (CAZymes) that hydrolyze glycosidic bonds in carbohydrates and glycoconjugates (Kötzler et al. 2014). Based on the mode of action of glycosidases on their target substrates they are divided into exo- and endoglycosidases. Exoglycosidases hydrolyze glycosidic bonds at the termini of substrates, whereas endoglycosidases hydrolyze internal glycosidic bonds in complex sugars or oligosaccharides. To hydrolyze glycosidic bonds, glycosidases typically carry two catalytic amino acids, such as glutamate or aspartate in their active site. The distance between two catalytic residues determines the mechanism of hydrolysis of the glycosidic bond, i.e., either retaining or inverting (Davies and Henrissat 1995). In retaining glycosidases, the two catalytic residues are approximately ∼5.5 Å apart, and their hydrolysis of glycosides results in the retention of the C1 anomeric configuration of the released sugar moiety (Fig. 1A). In contrast, in inverting glycosidases, the two catalytic residues are approximately ∼10 Å apart, and their hydrolysis of glycosides leads to the inversion of the C1 anomeric configuration of the released sugar moiety (Fig. 1B). Based on sequence similarities, these enzymes are further classified into different glycosyl hydrolase (GH) families (Henrissat and Davies 1997). In general, the members of a particular GH family follow one of two mechanisms of hydrolysis of glycosidic bonds. However, there are exceptions. For example, members of the GH97 family contain both retaining and inverting GHs (Gloster et al. 2008). In particular, it has been shown that members of GH families such as GH4 and GH109, also follow an unusual mechanism of glycosidic bond hydrolysis, and for certain GH families, such as GH152, the mechanism of glycosidic hydrolysis is not known (Yip et al. 2007; Drula et al. 2022). Compared with other sequenced genomes, plant genomes contain a large number of glycosidase genes (Coutinho et al. 2003). For example, in the genomes of four important plants, Arabidopsis, rice, soybean, and sorghum, there are many glycosidases that follow different mechanisms of hydrolysis of glycosidic bonds, among which the GHs that follow the retaining mechanism are abundant (Fig. 2A). The glycosidases from these GH families target various substrates derived from glucose, galactose, xylose, mannose, and other sugar moieties. The activities of these glycosidases on these substrates are important not only for various physiological processes but also during plant–pathogen interactions (Minic 2008).

Fig. 1
figure 1

Mechanisms of glycosidic bond hydrolysis. A Retaining mechanism of glycosidic bond hydrolysis. Hydrolysis of glycosides by retaining glycosidases leads to retention of the C1 anomeric configuration of the released sugar moiety. Retaining glycosidases involve a two-step mechanism, in which one of the active glutamate/aspartate residues acts as a nucleophile, and the other glutamate/aspartate residue acts as an acid/base catalyst. In the first step, the nucleophile attacks the C1 anomeric carbon of the glycoside, leading to the formation of a glycosyl-enzyme intermediate. In the second step, water acts as a nucleophile and hydrolyzes the glycosyl-enzyme intermediate. B Inverting mechanism of glycosidic bond hydrolysis. Hydrolysis of glycosides by inverting glycosidases leads to inversion of the C1 anomeric configuration of the released sugar moiety. Inverting glycosidases involves a one-step mechanism, in which one of the active glutamate/aspartate residues acts as a general acid, and the other glutamate/aspartate residue acts as a general base. Figures were created using ChemDraw and CorelDRAW software

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Fig. 2
figure 2

Plant genomes harbor a great diversity of glycosyl hydrolase (GH) families. A The abundance of retaining and inverting glycosidases in the four different plant species. Plant proteome sequences of Arabidopsis thaliana, Glycine max, Oryza sativa, and Sorghum bicolor were downloaded from the Phytozome v13 database and annotated for CAZy prediction using the standalone version of dbCAN. The GH family was parsed from the dbCAN output and represented. The mechanism of action for each annotated GH family has been obtained from the Carbohydrate-Active Enzyme (CAZy) Database. The circle size was adjusted based on the abundance of total glycosidases present in the respective analyzed plant species. B A survey of the number of GH families and glycosidases in four plant species as of August 2023. Distribution of various glycosyl hydrolase (GH) families in four plant species. The number of glycosidases present in each family of the respective plant species analyzed is represented. * Represents the inverting glycosidase families and # represents the families for which the exact mechanism of glycosidic bond hydrolysis is unknown or follow other new mechanisms. The GH families shown to play important roles during pathogen infections are highlighted in red

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In recent years, plant glycosidases from various GH families have emerged as key players in pathogen infections (Fig. 2B). The members of the respective GH families have varied substrate targets, which leads to further diversity in glycosidases. Plant glycosidases have been shown to act upon pathogen components, host secondary metabolites, host apoplastic sugars, host cell walls, and host N-glycans to mediate immunity against invading pathogens. Furthermore, we classified the activities of the plant defense GHs under eleven different mechanisms through which they operate during pathogen infections. We also provided “Box 1” information summarizing the various strategies that microbial pathogens use to circumvent the direct action of plant GH activities. We have provided detailed information on the GH family classification, catalytic activities, subcellular localization, domain structure, and functional roles of defense-related plant GHs. We have also outlined open questions that could be helpful for advancing this field of research.

GHs in targeting pathogen components

β-galactosidases (BGALs)

Β-galactosidases (BGALs) (EC 3.2.1.23) are exoglycosidases that act on nonreducing β-d-galactosyl residues present in β-d-galactosides. In plants, all BGALs belong to the GH35 family. In addition to the GH35 catalytic domain, BGALs also carry an additional GHD (β-sandwich-like domain) and gal_lectin domain in their C-terminus (Fig. 3) (Chandrasekar and van der Hoorn 2016). The relevance of these additional domains is yet to be studied, but they might assist BGALs in substrate specificity. Plant BGALs have been shown to play important cell wall-associated physiological roles, such as altering the hydration properties of seed coat mucilage, cell wall metabolism, and fruit ripening (Carey et al. 2001; Dean et al. 2007; Chandrasekar and van der Hoorn 2016). The importance of BGALs in plant–pathogen interactions has only recently been elucidated. NbBGAL1 is an active β-galactosidase in N. benthamiana, that is localized in the apoplast (Chandrasekar et al. 2014). N. benthamiana plants lacking NbBGAL1 showed increased susceptibility to the virulent P. syringae strain Pseudomonas syringae pv. tomato (Pto) DC3000(ΔhQ) (Buscaill et al. 2019). Similarly, heterologous expression of AtBGAL8, an orthologue of NbBGAL1, or NbBGAL1 in N. benthamiana increased resistance to P. syringae. pv. tabaci 6605 (Pta6605) (Buscaill et al. 2021). Functionally, NbBGAL1 mediates the release of flg22 elicitor peptides from the flagellin polymer. NbBGAL1 potentially targets the O-glycan protective layer in the flagella of P. syringae to facilitate the action of other glycosidases and proteases that process the flagellin polymer to release flg22 peptides (Buscaill et al. 2019) (Fig. 4). The released flg22 peptides are recognized by plants and trigger innate immune responses. Manipulation of BGAL activity is an important strategy employed by P. syringae to survive in the apoplast. P. syringae evades the activities of NbBGAL1 through two different strategies. First, P. syringae produces a BGAL inhibitor during infection (Chandrasekar et al. 2017) (Strategy 1, Fig. 5), and second, through glycan polymorphisms. P. syringae strains carry polymorphic O-glycan structures in their flagella that are insensitive to BGAL1 activity (Strategy 2, Fig. 5). By incorporating different O-glycoforms these bacterial pathogens avoid the release of flagellin elicitor peptides. It is not known which glycosidases unmask these polymorphic O-glycans. The identification of these glycosidases is an important topic to explore for revealing the processing events leading to elicitor release from these bacterial strains. Thus, in plants, only two BGALs (AtBGAL8 and NbBGAL1) have been characterized during bacterial infections. In several studies, biochemical analysis of cell walls isolated from plant fungal pathogens revealed the presence of galactose and β-d-galactose-containing polysaccharides, such as galactomannan (Cantu et al. 2009; Schoffelmeer et al. 1999; de Oliveira et al. 2019). Hence, exploring the biological role of BGALs during fungal infections and investigating whether they mediate the release of elicitors from fungal glycan components are interesting topics. Plant BGALs have also been shown to target β-d-galactosyl residues in hemicellulosic cell wall components, such as xyloglucan (Sampedro et al. 2012). The activities of these BGALs are important for regulating cell wall extension and remodeling. Therefore, it would be interesting to investigate whether BGALs also play a role in cell wall remodeling during plant pathogen infections.

Fig. 3
figure 3

The domain architecture of prominent defense-related GHs. The sequences were retrieved from either the UniPort database or the Phytozome database. The domains in the respective sequences were analyzed using Pfam software. The presence of the signal peptide in the respective sequence was predicted using the SignalP 6.0 prediction tool. GH, glycosyl hydrolase; Fn3-like, fibronectin type III-like; CBM, carbohydrate binding module; CBD, chitin binding domain; GHD, glycosyl hydrolase domain; Y, yes; N, no

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Fig. 4
figure 4

Prominent elicitor and bioactive glycan-releasing GHs. HvBGLUII is a GH17 family β-1,3-endoglucanase from barley that targets the extracellular polysaccharide matrix of fungi to release a ROS-scavenging decasaccharide fragment. Gmβ-1,3-endoglucanase (GmBGLU) is an endoglucanase from soybean that targets the cell walls of oomycete pathogens to release β-glucan elicitor fragments. AtLYS1, a member of the GH18 family of class III chitinases from Arabidopsis thaliana, hydrolyzes the peptidoglycan (PGN) layer in bacteria to release PGN elicitor fragments. NbBGAL1 is a GH35 family β-galactosidase from Nicotiana benthamiana that acts on O-glycans in flagella to initiate the release of flagellin peptides. Figures were created using CorelDRAW software

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Fig. 5
figure 5

Five different microbial strategies are employed by various microbial pathogens to circumvent direct action of plant GHs. Details of these strategies are provided in the “Box 1” section. The part highlighted in orange in the figure represents the component employed by the respective microbial pathogen. Figures were created using CorelDRAW software

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Endochitinases

Endochitinases (EC 3.2.1.14) are glycosidases that hydrolyze the internal β-1,4-glycosidic linkages between N-acetylglucosamine (GlcNAc) residues in the chitin polymer. Endochitinases are induced during pathogen infections and are classified as PR proteins of the PR-3, PR4, PR-8, and PR-11 families (van Loon et al. 2006). Based on their sequence similarities and structural features, plant chitinases are generally divided into five different classes, class I to class V. Chitinases of classes III and V belong to the GH18 family, and chitinases of other classes belong to the GH19 family (Passarinho and de Vries 2002). Chitin is an important component that provides structural support to the cell walls of fungi (Geoghegan et al. 2017). Plant chitinases are thought to disintegrate the cell wall of fungal pathogens through hydrolysis of the chitin polymer. Accordingly, under in vitro conditions, purified chitinases from several plant species have been shown to exhibit antifungal activities against several plant pathogens (Kirubakaran and Sakthivel 2007; Xayphakatsa et al. 2008; Rajninec et al. 2020). Furthermore, the overexpression of plant chitinases in crop plants enhances resistance, and the downregulation of plant chitinases results in increased susceptibility to plant fungal pathogens, indicating their contribution to plant immunity (Maximova et al. 2006; Iqbal et al. 2012; Prasad et al. 2013; Han et al. 2019). The hydrolytic activities of plant chitinases on fungal cell walls lead to the generation of chitin oligosaccharides with different degrees of polymerization (Boller et al. 1983). These chitin oligosaccharides are recognized by the lysine motif receptor kinase (LYK) receptor complex, which is localized in the plasma membrane where it activates the plant’s innate immune response (Kaku et al. 2006; Miya et al. 2007; Cao et al. 2014). Chitin oligosaccharides with a degree of polymerization less than four are not effective elicitors, whereas chitin oligosaccharides with a degree of polymerization greater than six have been shown to be effective at triggering plant immune responses (Roby et al. 1987; Ren and West 1992; Felix el al. 1993; Yamada et al. 1993). These larger chitin oligosaccharides have been shown to bind effectively to the receptor that triggers plant immunity (Cao et al. 2014). However, the exact length of the chitin oligosaccharide produced in the apoplast for plant immune responses remains to be investigated (Sánchez-Vallet et al. 2015; Pusztahelyi 2018). Furthermore, not all classes of chitinases hydrolyze chitin polymers efficiently. In tobacco, class I chitinases have been shown to effectively target chitin polymers, while class V chitinases target oligomers rather than chitin polymers (Brunner et al. 1998). Therefore, studying the action of multiple chitinases to produce biologically relevant chitin oligomers that act as elicitors would be useful. Members of class III chitinases from the GH18 family have been shown to behave as bifunctional enzymes. This class of enzymes exhibits chitinase- and lysozyme-like activities. Lysozymes (EC 3.2.1.17) are endoglycosidases that hydrolyze internal β-1–4 glycosidic linkages between N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues in the peptidoglycan (PGN) polymer (Phillips 1967). Notably, AtLYS1, a class III chitinase from A. thaliana has been shown to exhibit chitinase- and lysozyme-like activities (Liu et al. 2014). AtLYS1 is localized in the apoplast and targets the PGN layer in the cell walls of plant pathogenic bacteria (Fig. 4). The hydrolytic activities of AtLYS1 resulted in the release of PGN fragments, which are recognized by the PGN receptor complex LYM1-LYM3-CERK1 to activate plant immune responses (Fig. 4). Suppression of AtLYS1 expression resulted in increased susceptibility to PtoDC3000, demonstrating its role during bacterial infections. Plant chitinases have also been shown to be important in cell death-related processes during pathogen infections (Ali et al. 2020). The transcript levels of CaChitIV, a class IV chitinase gene from pepper plants of the GH19 family, were induced during infection with Xanthomonas campestris pv. vesicatoria (Xcv), an avirulent bacterial strain that triggers cell death (Kim et al. 2015). CaChitIV interacts with the receptor-like cytoplasmic protein kinase PIK1 to promote cell death and plant defense responses (Kim et al. 2015). Notably, certain plant chitinases have acquired new functions when their enzymatic activities have been lost. These ‘neo-functionalized’ chitinases play important roles in pathogen infections. Xylanase inhibitor protein I (XIP-1) is a member of the GH18 family from wheat (Triticum aestivum) and is annotated as a class III chitinase. Although XIP-1 carries the conserved catalytic residues in its active site, the protein has lost its activities due to natural substitution mutations in adjacent amino acid residues (glycine to tyrosine) in the active site (Juge et al. 2004). Although XIP-1 is catalytically inactive, it serves as an inhibitor of the GH11 family xylanases produced by Botrytis cinerea (B. cinerea) under in vitro conditions (Brutus et al. 2005). Xylanases of the GH11 family are important virulence factors in B. cinerea during infections (Brito et al. 2006). Furthermore, the transcript levels of XIP-1 were significantly increased in wheat leaves infected with the powdery mildew pathogen Erysiphe graminis, supporting its role in plant defense (Igawa et al. 2005). CacIXIP is another xylanase inhibitor protein from coffee (Coffea arabica) that belongs to the GH18 family. CacIXIP has lost its chitinase activity due to a natural mutation in its catalytic glutamate residue (glutamate to glutamine) (Vasconcelos et al. 2011). However, CacIXIP has antifungal effects on P. pachyrhizi (Vasconcelos et al. 2011). Recently, a ‘neo-functionalized’ chitinase, NbPR3, belonging to the GH19 family was identified in Nicotiana benthamiana (Sueldo et al. 2024). Like CacIXIP, NbPR3 has lost its chitinase activity due to a natural mutation in its catalytic glutamate residue (glutamate to glutamine). Interestingly, NbPR3 has antibacterial effects on P. syringae (Sueldo et al. 2024). The neo-functionalization of members of the GH18 and GH19 families is an exciting research topic. Identification of additional such neo-functionalized proteins would open opportunities for crop improvement against fungal pathogens. The importance of plant chitinases is well reflected by various strategies employed by pathogens (Bakhat et al. 2023). Cladosporium fulvum (C. fulvum), a leaf mold-causing fungal pathogen, secretes the chitin-binding effector protein Avr4, which binds to chitin in fungal cell walls, thereby preventing hydrolysis by plant chitinase enzymes (Strategy 3, Fig. 5) (van den Burg et al. 2006). The same pathogen secretes another effector, Ecp6, which binds chitin fragments that trigger the plant immune system (de Jonge et al. 2010). Moniliophthora perniciosa, a plant fungal pathogen, secretes MpChi, a ‘neo-functionalized’ chitinase of the GH18 family, to bind chitin elicitor fragments (Fiorin et al. 2018). Several fungal pathogens have been shown to secrete polysaccharide deacetylases (PDA) that convert chitin oligomers into inactive chitosan fragments (Gao et al. 2019; Xu et al. 2020). Verticillium dahliae (V. dahliae) is a fungal pathogen that secretes a serine protease (Ssep1) to degrade the cotton chitinase Chi28 of the GH19 family (Strategy 4, Fig. 5) (Han et al. 2019). Plant chitinases are likely targeted by inhibitors produced by plant pathogens. Activity-based protein profiling (ABPP) studies have shown that the activities of plant chitinases are suppressed during P. syringae infections (Sueldo et al. 2024). Furthermore, a recent computational study using the Alphafold multimer platform revealed several candidates for small secreted proteins (SSPs) that could act as plant chitinase inhibitors (Homma et al. 2023). However, additional experimental studies are needed to validate the inhibitory activities of these SSPs. To date, chitinase inhibitors secreted by plant pathogens have not been reported. Therefore, identifying chitinase inhibitors and investigating their biological significance would be interesting research topics.

Endo-β-1,3-glucanases

Endo-β-1,3-glucanases (EC 3.2.1.39) are endoglycosidases that hydrolyze internal β-1,3-glycosyl linkages in polysaccharides such as β-1,3 glucan. Endo-β-1,3-glucanases are classified as members of the PR-2 family of PR proteins and are induced during infections. In barley (Hordeum vulgare), GII, a specific isoform of endo-β-1,3-glucanase is well characterized. These enzymes are induced upon elicitor treatment or during fungal infections (Kaku et al. 1997; Roulin et al. 1997). The apoplastic GII endo-β-1,3-glucanase, HvBGLUII from barley (Hordeum vulgare) which belongs to the GH17 family, targets the extracellular polysaccharide matrix (EPS) of the plant fungal pathogen Bipolaris sorokiniana, and the beneficial fungal endophyte S. indica (Chandrasekar et al. 2022). The activities of HvBGLUII on the EPS matrix of these fungi resulted in the release of β-GD, a conserved decasaccharide fragment that consists of seven β-1,3-linked D-glucose backbone units substituted with three terminal-β-1,6-glucosyl residues (Fig. 4). The β-GD fragment released from the EPS matrix does not trigger plant immune responses but rather displays reactive oxygen species (ROS) scavenging activity. HvBGLUII is also capable of hydrolyzing laminariheptose, an unsubstituted β-1,3-glucan oligosaccharide into glucose and laminaribiose. These products triggered an increase in early ROS burst accumulation in barley roots. Therefore, HvBGLUII is a plant defense enzyme that releases potential Microbe-associated Molecular Patterns (MAMPs) from unsubstituted β-1,3-glucans. However, HvBGLUII is hijacked by fungi to release β-GD, a decasaccharide with antioxidant properties that subverts the plant immune system. Endo-β-1,3-glucanases have also been shown to release elicitors or MAMPs from the insoluble mycelial cell walls of oomycete plant pathogens. Endo-β-1,3-glucanases from soybean (Glycine max) cotyledons (GmBGLU) target the cell wall of Phytophthora sojae (P. sojae) and release a β-glucan elicitor (Fig. 4) (Keen and Yoshikawa 1983). β-glucan elicitors trigger the production of phytoalexin compounds such as glyceollins, which are important for resistance to the oomycete pathogen P. sojae (Yoshikawa et al. 1981; Keen et al. 1983; Takeuchi et al. 1990). Partial structural characterization of the released elicitor revealed that the β-glucan elicitor contains a varying length of the β-1,6-glucan backbone substituted with one or two β-1,3-glucosyl moieties (Fig. 4) (Okinaka et al. 1995). Acid/enzyme hydrolysis of the cell walls of the fungal pathogens M. oryzae and Septoria tritici results in the release of β-glucan elicitors, which consist of only the β-1,3-glucan backbone or β-1,3-glucan substituted with β-1,6-glucosyl units (Yamaguchi et al. 2000; Shetty et al. 2009). Therefore, these findings suggest that glucan MAMPs released from fungal cell walls by plant β-1,3-endoglucanase may have different structures than elicitors released from oomycete cell walls. The plant endo-β-1,3-glucanases responsible for the release of biologically relevant MAMPs from fungal cell walls remain to be characterized. The activities of plant endo-β-1,3-glucanases are circumvented by two strategies employed by microbial pathogens. P. sojae secretes a glucanase inhibitor protein (Gip) to target the isoforms of plant endo-β-1,3-glucanases that release elicitors from the pathogen cell wall (Strategy 1, Fig. 5) (Ham et al. 1997). Colletotrichum lindemuthianum, a fungal pathogen that causes anthracnose disease, has been shown to produce inhibitors against plant endo-β-1,3-glucanases (Albersheim and Valent 1974). In a recent study, an effector from Phakopsora pachyrhizi, a fungal pathogen that causes soybean rust was shown to suppress the activities of plant endo-β-1,3-glucanases (Strategy 1) (Bueno et al. 2022). B. sorokiniana produces an extracellular polysaccharide (EPS) matrix that is rich in β-1,3-glucans substituted with terminal-β-1,6-glucosyl residues. The β-1,6-glucosyl residues protect fungi from the activities of endo-β-1,3-glucanases and prevent the release of β-1,3-glucan elicitor fragments from the cell walls (Chandrasekar et al. 2022) (Strategy 5, Fig. 5).

Proteins that bind to β-glucan elicitors with high affinity have been identified in plants from the Fabaceae family and are thought to function as receptors to initiate defense signaling (Cheong et al. 1993; Mithöfer et al. 1996; Umemoto et al. 1997; Leclercq et al. 2008). These glucan elicitor binding proteins (GEBPs) carry an additional glycosyl hydrolase domain that belongs to the GH81 family (Fliegmann et al. 2004). GEBP with the GH81 domain exhibited endo-β-1,3-glucanase activity on the β-1,3-glucan substrates (Fliegmann et al. 2005). GEBPs not only are found in the Fabaceae family but are also widely distributed in other monocotyledonous plants, such as barley. Recently, GEBP from barley was characterized and found to play an important role in fungal colonization. GEBPs in barley act as broad compatibility factors during interactions with beneficial and pathogenic fungi. The endo-β-1,3-glucanase activities of GEBPs have been shown to be important for modulating ROS homeostasis in plants (Wanke et al. 2023). GEBPs hydrolyze the long-chain β-1,3-linked glucans of laminarin into shorter fragments. These shorter fragments are recognized differently by N. benthamiana and barley. Hence, GEBPs might act upon oligosaccharides released from fungal cell walls to modulate ROS production through pattern-triggered immune (PTI) responses. Mutation of two GEBPs, GEBP1 and GEBP2, in barley resulted in increased accumulation of carbohydrate-rich cell wall appositions (CWAs) and hyperresponse at the host cell wall during colonization by beneficial fungi such as S. indica, Serendipita vermifera, and Rhizophagus irregularis. Furthermore, the gebp1 and gebp2 barley mutants showed increased resistance to fungal pathogens such as B. sorokiniana and Blumeria graminis f. sp. hordei (Wanke et al. 2023). Elucidating the mechanism of hyperresponse in the host cell walls of gebp mutants, particularly by monitoring the compositional changes that occur in the cell walls of gebp mutants during fungal colonization, is an interesting topic to explore. Overall, the novel roles of endo-β-1,3-glucanases are emerging in various plant species during pathogen infections.

Fructan exohydrolases (FEHs)

Fructan exohydrolases (FEHs) are exoglycosidases that hydrolyze terminal nonreducing β-d-fructosyl residues in fructan polysaccharides (Ende 2018). In several plant species, fructans serve as storage carbohydrates and protective agents under low-temperature and abiotic stress conditions (Valluru and Ende 2008; Livingston et al. 2009). These enzymes belong to the GH32 family and are similar to plant cell wall invertases (Van den Ende 2022). It has been speculated that FEHs may target microbial fructan polysaccharides to release MAMPs that trigger plant immunity (Van den Ende et al. 2004). Zm-6-FEH (EC 3.2.1.154) is an apoplast-localized maize FEH that specifically hydrolyzes levan, a fructan polysaccharide containing fructosyl residues linked via β-2,6-glycosyl bonds (Huang et al. 2020). Levan is an abundant fructan polysaccharide found in the EPS of several bacterial plant pathogens (Gross et al. 1992; Yuan et al. 2022). Furthermore, the activities and transcript levels of Zm-6-FEH were induced by levan-producing bacterial plant pathogens such as Erwinia carotovora (Huang et al. 2020). Similarly, Ci6-FEH (EC 3.2.1.154) II is another apoplast-localized chicory FEH that hydrolyzes microbial levan polysaccharides (Versluys et al. 2022). The exact role of these FEHs in plant immunity is not yet clear, but FEHs are emerging as potential GHs that release elicitors from bacterial plant pathogens.

GHs in targeting host cell walls

β-xylosidases (BXLs)

Β-xylosidases (BXLs) (EC 3.2.1.37) are exoglycosidases that hydrolyze nonreducing β-1,4-xylosyl residues from β-xylosides, β-xylooligosaccharides, and xylan polysaccharides. Plant BXLs play an important role in secondary cell wall metabolism and plant development (Goujon et al. 2003). Of the seven BXL members in the Arabidopsis genome, BXL4, from GH3 family has been well characterized during pathogen infections. β-xylosidase 4 (BXL4) is a candidate protein that accumulates in the apoplast of Arabidopsis leaves expressing AvrRpm1 (Breitenbach et al. 2014). AvrRpm1 is an effector protein from P. syringae that induces systemic acquired resistance (SAR) when recognized by the CC-NLR protein, RPM1 (Resistance to Pseudomonas syringae pathovar maculicola 1). In a recent study, Arabidopsis bxl4 mutants were impaired in SAR activation, leading to increased growth of PtoDC3000 in systemic leaves of SAR-induced bxl4 mutants (Bauer et al. 2023). Furthermore, bxl4 mutants were also impaired in phloem-mediated and airborne long-distance signaling (Bauer et al. 2023). Therefore, BXL4 appears to be a central component of SAR signaling during bacterial infections. In another recent study, BXL4 was shown to contribute to resistance against B. cinerea (Guzha et al. 2022). The Arabidopsis bxl4 mutant showed increased susceptibility to B. cinerea, and Arabidopsis plants overexpressing the BXL4 gene showed increased resistance to the pathogen. Previously, BXL4 was characterized biochemically and shown to exhibit specific activity toward polysaccharides with β-1,4-linkages, such as xylan (Minic et al. 2004). The specific mechanism through which BXL4 contributes to resistance to B. cinerea is unknown. It has been speculated that BXL4 may act on xylan or arabinan side chains in the plant cell wall to contribute to resistance by remodeling these polysaccharides during infection. Our domain analysis revealed the presence of a fibronectin type III (Fn-3)-like domain in the C-terminus of the BXL4 enzyme (Fig. 3). The Fn-3-like domain was shown to be associated with several members of the GH family. However, the exact function of this domain is unclear (Sidar et al. 2020). Therefore, it would be interesting to investigate the functional relevance of these domains during pathogen infections.

Endo-β-1,4-glucanases (EGases)

Endo-β-1,4-glucanases (EGases) (EC 3.2.1.4) are endoglycosidases that hydrolyze internal β-1,4-glycosyl linkages in β-1,4 glucan polysaccharides such as cellulose. EGases are involved in various cell wall-associated physiological processes, such as fruit ripening, organ abscission, cell wall expansion, and remodeling (Minic and Jouanin 2006). EGases have also been implicated in plant–pathogen interactions. Downregulation of two EGase genes, CEL1 and CEL2, in tomato leaves results in increased susceptibility to P. syringae, which indicates their positive role in disease resistance (Flors et al. 2007). However, interestingly, downregulation of these EGases has also resulted in increased resistance to B. cinerea (Flors et al. 2007). Like in tomato plants, Arabidopsis EGase mutants also exhibit contrasting disease-associated phenotypes. cel2 or cel3 Arabidopsis mutants were susceptible to P. syringae, whereas cel2 Arabidopsis mutants were resistant to B. cinerea infection (Finiti et al. 2013). Interestingly, several other EGase Arabidopsis mutants, such as eg4, eg10, and kor-1, also exhibited increased resistance to B. cinerea. However, Arabidopsis kor-1 mutants are highly susceptible to P. syringae (López-Cruz et al. 2014). Korrigan1 (KOR1) is a plasma membrane-anchored EGase that is involved in remodeling cellulose microfibrils during biosynthesis (Nicol et al. 1998). The contrasting disease-associated phenotypes caused by mutations in plant EGase genes might be due to imbalances in the levels of defense hormones. For instance, Arabidopsis kor-1 plants exhibited increased levels of jasmonic acid (JA) and activation of jasmonic acid (JA)-biosynthetic pathways during P. syringae infections (López-Cruz et al. 2014). In general, during infections, activation of the JA signaling pathway is required for the resistance against necrotrophs, and the salicylic acid (SA) signaling pathway is required for the resistance against the biotrophs and hemibiotrophs (Glazebrook 2005). Furthermore, the JA and SA signaling pathways are antagonistic during pathogen infections (Takahashi et al. 2004). Since biochemical analysis revealed increased levels of JA in several Arabidopsis EGase mutants (Finiti et al. 2013), these mutants might be resistant to the necrotrophic pathogen, B. cinerea. Due to the antagonistic nature of the JA/SA signaling pathways, these mutants might be more susceptible to P. syringae, a hemibiotrophic pathogen. However, studies on the complex feedback mechanism that exists between EGases and plant defense hormones are yet to be unraveled. Elucidating these details would be helpful for introducing broad-spectrum resistance in plants to pathogens with different lifestyles.

Endo-β-1,4-xylanases

Endo-β-1,4-xylanases (EC 3.2.1.8) are endoglycosidases that hydrolyze internal β-1,4-glycosyl linkages in xylan polysaccharides. Plant endo-β-1,4-xylanases are involved in several cell wall-associated physiological processes, such as secondary cell wall synthesis, cell wall deposition, regulation of plant growth, and facilitation of pollen tube penetration (Suen and Huang 2007; Hu et al. 2020; Tu et al. 2020). In plants, the GH10 family predominantly includes glycosidases that exhibit only endo-β-1,4-xylanase activities. To date, only one member of the GH10 family has been shown to be important during pathogen infections. MORE1 (Magnaporthe oryzae resistance 1) is a member of the GH10 family in A. thaliana and its disruption in the A. thaliana ecotype Wassilewskija (Ws-0) resulted in increased susceptibility to fungal pathogens such as Magnaporthe oryzae (M. oryzae) and Alternaria brassicicola (Kim et al. 2022). Interestingly, in contrast to the observed phenotype in Arabidopsis, disruption of a MORE1 homolog, OsMORE1a, in rice resulted in increased resistance to pathogens such as M. oryzae and Xanthomonas oryzae pv. oryzae (Xoo). Interestingly, rice osmore1a mutants showed increased susceptibility to the fungal pathogen Cochliobolus miyabeanus. Like other EGases, contrasting disease-associated phenotypes can be observed due to mutations in the GH10 family gene. The reason might be due to the lifestyle of the investigated pathogens and the imbalance in hormone levels. M. oryzae and Xoo are hemibiotrophic pathogens, whereas C. miyabeanus is a necrotrophic pathogen. Notably, rice osmore1a showed an increase in the expression of genes controlled by SA and a decrease in the expression of genes regulated by JA (Kim et al. 2022). Hence, the antagonistic nature of SA and JA might be the reason for the contrasting disease-associated phenotypes. However, biochemical analysis is needed to quantify the levels of SA and JA in these mutants during pathogen infections. Since members of the GH10 family predominantly exhibit endo-β-1,4-xylanase activities, it would be interesting to investigate whether MORE1 is involved in cell wall remodeling during infections. In particular, our domain analysis revealed that OsMORE1a has an additional carbohydrate-binding module 4 (CBM4) in the N-terminus (Fig. 3). CBM4 has been shown to be specific to xylan cell wall polysaccharide, and therefore, these modules may aid in the recognition of plant cell wall substrates (Simpson et al. 2002). The precise functional roles of these additional modules remain to be investigated.

Polygalacturonases (PGs)

Polygalacturonases (PGs) are enzymes that hydrolyze α-1,4-galacturonosyl residues in pectin cell wall polysaccharides. Plant PGs are involved in cell wall-associated physiological processes such as fruit ripening, cell elongation, and pollen tube growth (Hadfield and Bennett 1998). In plants, pectin integrity is important for preventing the colonization of various pathogens, and pectin metabolism plays a major role in determining the outcome of plant–pathogen interactions. Studies have shown that the loss of function of individual PGs does not result in a distinct disease-associated phenotype. For example, downregulation of LePG or CRISPR-Cas9-based mutagenesis of the PG2a gene (Solyc10g080210) in tomato plants did not improve resistance to B. cinerea (Cantu et al. 2008; Silva et al. 2021). However, simultaneous suppression of LePG together with LeEXP1 reduced the susceptibility of tomato fruits to B. cinerea. LeEXP1 is an expansin involved in the loosening of plant cell walls for cell wall remodeling (Cosgrove 2015). PG and expansin are important proteins that act cooperatively to promote fruit ripening processes by modifying pectins. Hence, self-disassembly of fruit pectins mediated by PG and expansin contributes to susceptibility to fungal pathogens. Like in other cell wall-associated GHs, depending on the lifestyle of the invading pathogen, mutations in PGs have also led to contrasting disease-associated phenotypes. Tomato fruits with reduced activity of multiple PGs showed resistance to necrotrophic fungal pathogens such as Geotrichum candidum and Rhizopus stolonifer but were susceptible to Colletotrichum gleosporioides, a hemibiotrophic fungal pathogen (Kramer et al. 1992; Cooper et al. 1998). Hence, mutations in PGs might have led to an increase in JA levels in these plants resulting in resistance to these necrotrophs. In recent years, new roles for PGs have emerged. ZmPGH1, a PG from maize, suppressed the HR induced by RP1-D21. RP1-D21 is an autoactive nucleotide-binding domain leucine-rich repeat (NLR) protein that induces HR in maize. Transient expression of ZmPGH1 in N. benthamiana suppressed the HR induced by RP1-D21 and another active NLR RPM1(D505V) (He et al. 2019). In addition, ZmPGH1 suppressed cell death caused by SA and (9S,13S)-10-oxo-phytoenoic acid (10-OPEA). (He et al. 2019). However, the exact mechanism by which ZmPGH1 regulates the cell death process is interesting to explore. It is speculated that cell wall-mediated changes may be responsible for these effects. In another recent study, OsPG1 was shown to be associated with the leaf tip necrosis phenotype in rice (Cao et al. 2021). Mutation of the OsPG1 gene led to changes in the cell wall composition and increased resistance to various Xoo races. Therefore, it would be interesting to investigate the cell death mechanism mediated by PGs and the associated disease resistance phenotypes caused due to changes in the cell wall composition.

GHs in targeting host apoplastic sugars

Cell wall invertases (CWIs)

Invertases (EC 3.2.1.26) are exoglycosidases that hydrolyze nonreducing α,1-β,2-glycosidic linkages in sucrose, resulting in the formation of hexose sugars, glucose, and fructose. Sucrose is an important disaccharide sugar that is transported from source tissue to sink tissue via the phloem apoplast. During pathogen infections, plants modulate carbohydrate flux in infected tissues by increasing hexose levels in the apoplast (Berger et al. 2007; Tauzin and Giardina 2014). These sugars are further transported into plant cells by hexose transporters and activate the expression of defense genes through sugar signaling (Liu et al. 2022). Based on their localization in plant cells invertases are classified into vacuolar invertases, cytoplasmic invertases, and cell wall invertases. Of the three types of invertases, cell wall invertases play an important role in the interaction between plants and pathogens. During pathogen infections, the activities of cell wall invertases lead to an increase in the hexose concentration, which is used by infected cells to efficiently activate plant immune responses (Proels and Hückelhoven 2014). In several studies, the activities and expression of extracellular cell wall invertases increase during infection with plant pathogens ranging from biotrophic to necrotrophic lifestyles (Fotopoulos et al. 2003; Scharte et al. 2005; Swarbrick et al. 2006). Although the induction of plant cell wall invertases may suggest a positive role in disease resistance, studies have shown that these enzymes play contrasting roles, i.e., either positive or negative during plant–pathogen interactions. In general, cell wall invertases have been shown to play a positive role in disease resistance in hemibiotrophic pathogens and a negative role in disease resistance in biotrophic pathogens. RNAi-mediated suppression of the cell wall invertase gene in tobacco (Nicotiana tabacum) resulted in increased susceptibility to Phytophthora nicotianae, an oomycete plant pathogen (Essmann et al. 2008). GIF1 (GRAIN INCOMPLETE FILLING 1) is an invertase localized in rice cell walls (Wang et al. 2008). gif mutant rice plants were susceptible to postharvest fungal pathogens such as Alternaria sp. (Sun et al. 2014). Furthermore, overexpression of GIF1 results in increased resistance to rice pathogens such as X. oryzae and M. oryzae (Sun et al. 2014). Mutation of TaCWI-B1, a cell wall invertase gene in wheat results in increased susceptibility to fungal pathogens such as Fusarium pseudograminearum and Rhizoctonia cerealis which cause fusarium crown rot (FCR) and sharp eye spot (SE), respectively, in wheat (Lv et al. 2023). Furthermore, the overexpression of TaCWI-B1 in wild-type plants improved resistance against these pathogens. In addition, TaCWI-B1 also modulates cell wall components such as cellulose and pectin probably through TaGAL1, an α-galactosidase involved in mannan degradation (Lv et al. 2023). In contrast to studies on the positive role of CWIs in disease resistance, some studies have shown their negative role in disease resistance. By suppressing the expression of a cell wall invertase gene, LIN8 in tomato plants delayed the development of symptoms and tolerance to the biotrophic bacterial pathogen Xcv (Kocal et al. 2008). The reason for the opposing roles of cell wall invertases is not yet clear. Although the lifestyle of the invading pathogen can be a reason for the observed contrasting phenotypes, the influence of SA and JA due to mutation or overexpression of CWIs cannot be excluded. To date, no studies have evaluated the levels of SA/JA or their marker genes in cwi mutants in the context of plant–pathogen interactions. Hence, elucidating these missing links is an interesting topic for further investigation.

GHs in targeting host secondary metabolites

β-glucosidases (BGLUs)

Β-glucosidases (BGLUs) (EC 3.2.1.21) are exoglycosidases that hydrolyze nonreducing β-d-glucosyl residues from β-glucosides and β-glucooligosaccharides. β-glucosidases (BGLUs) in plants act as mediators of chemical defense during pathogen infections. The BGLUs characterized so far in pathogen infections belong to the GH1 family. These enzymes catalyze the hydrolysis of O-linked or S-linked glucosyl residues present in secondary metabolites such as glucosinolates which are associated with chemical defense (Ketudat Cairns et al. 2015). During the hydrolysis of the glucosidic bond, the aglucone part of the secondary metabolite associated with chemical defense is converted into toxic forms and limits the penetration of pests and pathogens. Normally, BGLUs and their substrates are localized in separate compartments, and their activities unfold only during pathogen invasion or tissue injury. Myrosinases (β-thioglucoside glucohydrolase (TGG)) are a special class of vacuolar and endoplasmic reticulum (ER)-localized BGLUs that carry only one catalytic glutamate residue in their active site and hydrolyze glucosinolates to achieve broad-spectrum antimicrobial activity during pathogen invasion. TGG1 and TGG2 are functional myrosinases in Arabidopsis leaf guard cells that regulate stomatal movement (Islam et al. 2009). These enzymes function upstream of the Ca2+ elevation step of the Ca2+ signaling pathway, which is mediated by the plant hormones abscisic acid (ABA) and methyl jasmonate (Me-JA). Overexpression of a homologous TGG1 gene from broccoli (BoTGG1) in Arabidopsis improved stomatal defense against the bacterial pathogen PtoDC3000 (Zhang et al. 2019). Overexpression of the BoTGG1 gene accelerated stomatal closure and inhibited stomatal reopening, thereby increasing resistance to the bacterial pathogen. In some studies, TGG1 and TGG2 have been shown to play important roles in the hypersensitive response (HR) and programmed cell death (PCD) during pathogen infections. In plants, HR and PCD are involved in effector-triggered immunity (ETI), which limits pathogen progression. The hydrolytic activities of TGG1 and TGG2 on aliphatic glucosinolates are important for the production of sulforaphane, a cell death-inducing compound generated during HR (Andersson et al. 2015). As a result, Arabidopsis tgg1/tgg2 double mutants exhibited reduced cell death and increased susceptibility to Hyaloperonospora arabidopsis, an oomycete plant pathogen (Andersson et al. 2015). In another study, the hydrolytic activities of TGG1 and TGG2 on indole glucosinolates were shown to be important for attenuating the PCD response caused by fumonisin, a mycotoxin (Zhao et al. 2015). Accordingly, Arabidopsis tgg1/tgg2 mutants exhibited severe cell death symptoms when treated with fumonisin. Myrosinases are not the only enzymes that hydrolyze glucosinolates. BGLUs with two glutamate residues at their catalytic site have also been shown to hydrolyze these compounds. These BGLUs are often referred to as atypical myrosinases (Sugiyama and Hirai 2019). PYK10 and PEN2 (Penetration 2) are well-known atypical myrosinases that act on glucosinolates, which are important for plant defense. PYK10 is a BGLU23 protein that is localized in the ER body of Arabidopsis and can hydrolyze the glucosylated secondary metabolites such as scopoline (O-linked glucoside) and indole glucosinolates, which are important for plant defense (Ahn et al. 2010; Nakano et al. 2017). The activities of PYK10 limit the rapid colonization of Serendipita indica (S. indica), a beneficial plant fungal endophyte, and are important for balanced mutualistic interactions (Sherameti et al. 2008). Therefore, investigating the role of PYK10 during infection by various microbial pathogens would be interesting. The hydrolytic activities of PEN2 are important for broad-spectrum disease resistance against various filamentous fungal pathogens (Bednarek et al. 2009). PEN2 is BGLU26 protein localized both in the mitochondria and peroxisomes of Arabidopsis thaliana (A. thaliana) and resists entry of fungal pathogens at the site of infection through hydrolysis of indole glucosinolates (Bednarek et al. 2009; Fuchs et al. 2016). PEN2 is also involved in the production of cell death-inducing compounds during HR through the hydrolysis of indole glucosinolates. As a result, Arabidopsis pen2 mutants exhibit reduced hypersensitive cell death caused by the recognition of effectors of various microbial pathogens (Johansson et al. 2014). PEN2 also plays an important role in the innate immune response. PEN2 is required for callose deposition upon recognition of flg22, a bacterial microbe-associated molecular pattern (MAMP) (Clay et al. 2009). In a recent study, G1, a BGLU from Medicago truncatula (Barrel clover), was shown to contribute to resistance against Rhizoctonia solani, a necrotrophic fungal pathogen. G1 is a nucleolus-localized BGLU that catalyzes the hydrolysis of glucosyl residues linked via an ester bond in saponin glucosides, and the hydrolysis products likely play a role in the defense against this pathogen (Lacchini et al. 2023). In addition to secondary metabolites, BGLUs also modulate the activities of phytohormones that are important for plant defense. SA is a plant hormone that plays an important role in the activation of several pathogenesis-related (PR) genes and in local and systemic acquired resistance (SAR). However, a higher concentration of active SA is toxic to plant cells and is therefore stored in an inactive form as SA-2-O-β-d-glucoside (SAG) (Kawano et al. 2004). SA: BGLUs hydrolyze the glucosidic linkage in SAG to activate SA (Hennig et al. 1993; Seo et al. 1995). In rice (Oryza sativa, Os), Os4BGLU12, Os4BGLU13, and Os1BGLU4 have been shown to hydrolyze SAG (Himeno et al. 2013; Hua et al. 2015; Xu et al. 2022). However, the phenotypic effects due to loss of function of any SA: BGLUs against plant pathogens have not been investigated so far. In a recent study, the rice mutants bglu19 and bglu23 showed increased susceptibility to Xanthomonas oryzae pv. oryzicola (Xoc) (Li et al. 2019). Since the expression of phytohormone marker genes is downregulated in these mutants, it was speculated that these BGLUs may play a role in the regulation of defense hormones. However, the exact functional roles of OsBGLU19 and OsBGLU23 in plant immunity remain to be investigated. ABA is an abiotic stress phytohormone that exists as an ABA glucose ester (ABA-GE, inactive form). BGLUs catalyze the hydrolysis of glucosyl residues linked via an ester bond in ABA-GE, resulting in the release of active ABA. Several studies have shown that ABA acts antagonistically to SA and JA, leading to increased susceptibility to plant pathogens (Anderson et al. 2004; Schmidt et al. 2008; Jiang et al. 2010). In one study, the transcript levels of the ABA-GE hydrolyzing BGLU (ABA: BGLU) were upregulated in grapes during B. cinerea infection (Jia et al. 2016). Therefore, certain ABA:BGLUs may act as susceptibility factors in plant–pathogen interactions. Similarly, the downregulation of an ABA-active BGLU gene in strawberry plants increased resistance to B. cinerea (Li et al. 2013). In addition to BGLUs that target glucosides or glucose-conjugated hormones, they have also been shown to hydrolyze cell wall-derived oligosaccharides. Os4BGLU12 hydrolyzed oligosaccharides such as cellobiose (β-1,4), laminaribiose (β-1,3), and glucotriose (β-1,3:1,4) (Opassiri et al. 2010). Os4BGLU6 hydrolyzed laminaribiose (β-1,3) (Seshadri et al. 2009) and Os4BGLU7 hydrolyzed cellotriose (β-1,4) (Chuenchor et al. 2008). A barley BGLU, BGQ60, hydrolyzed cellobiose (β-1,4) and laminaribiose (β-1,3) (Leah et al. 1995). Oligosaccharides such as cellobiose, cellotriose, and laminaribiose are well-characterized elicitors that trigger plant immune responses (Klarzynski et al. 2000; Johnson et al. 2018; He et al. 2023). Therefore, it would be interesting to investigate whether plant pathogens manipulate plant BGLU activity to dampen oligosaccharide-mediated immune responses during infections.

GHs in targeting host N-glycans

α-glucosidases

Α-glucosidases are exoglycosidases that hydrolyze nonreducing α-d-glucosyl residues from α-glucosides. Plant α-glucosidases are involved in the breakdown of starch and the processing of N-glycans attached to proteins (Beers et al. 1990; Burn et al. 2002). Glucosidase II (GII) is an ER-resident α-glucosidase that is involved in the trimming of α-1,3-linked glucosyl units from the N-glycans attached to proteins. GII is required for the biogenesis and stable accumulation of the EF-Tu receptor (EFR) in A. thaliana. These proteins also carry an additional Gal_Mutarotase domain in their N-terminus (Fig. 3). However, the functional significance of this domain is unknown. GII is a heterodimeric protein with a catalytic α-subunit (GIIα) and an ER-retention signal-containing β-subunit (GIIβ). Arabidopsis gIIα or gIIβ mutants exhibit impaired EFR-mediated immune signaling (Lu et al. 2009; von Numers et al. 2010). EFR is an important innate immune receptor that recognizes the bacterial elongation factor Tu (EF-Tu) as a MAMP and triggers plant defense responses. Furthermore, gIIα Arabidopsis mutants of GII were hypersusceptible to Pto DC3000 (Lu et al. 2009). Protein glycosylation is an important posttranslational modification required for the proper function of several proteins. Therefore, it would be interesting to investigate other glycosyl hydrolases in the endomembrane system that are important for the quality control of several immunity-related proteins and enzymes.

Conclusion and future perspectives

Plant genomes harbor a large diversity of glycosidases. In recent years, glycosidases from various GH families have emerged as important players in pathogen infections. To date, glycosidases from twelve different GH families have been shown to play functional roles during pathogen invasion, and we were able to categorize their roles into eleven different mechanisms (Table 1): First, the release of bioactive elicitors. Second, the release of susceptibility factors. Third, acting as antimicrobial agents. Fourth, as a compatibility factor for fungal colonization. Fifth, acting as an inhibitor of CAZymes produced by pathogens. Sixth, cell wall-mediated defense. Seventh, modulation of apoplastic sugar fluxes by hydrolysis of sucrose. Eighth, attenuating the PCD process. Ninth, mediate chemical defense through hydrolysis of inactive secondary metabolites. Tenth, producing cell death-inducing compounds for HR. Eleventh, quality control of immunity-related proteins. The members of the GH families involved in these defense-related processes are summarized with their subcellular localization (Table 1). Notably, several studies have shown that knocking out certain GHs that are involved in cell wall-associated processes can act as a double-edged sword during pathogen infections (Fig. 6). Knocking out these GHs results in an imbalance in defense hormone levels, resulting in contrasting disease-associated phenotypes depending on the lifestyle of the pathogen. Elucidating the mechanistic links between these GHs and the regulation of defense hormones could be helpful for introducing broad-spectrum resistance to plant pathogens. Cell wall remodeling mediated by GHs play an important role in plant defense. However, mechanistic insights into cell wall remodeling processes during infection are not known. Using a glycomics approach that includes complex cell wall analytics such as monosaccharide analysis, methylation, and ELISA-based glycome profiling, would be beneficial for determining the exact cell wall modifications caused by GHs during infections (Pattathil et al. 2017; Kim et al. 2020). This knowledge would ultimately be helpful for the generation of plants with smart cell walls to combat invading pathogens. The neo-functionalization of members of the GH18 and GH19 families is an interesting research topic. It would be interesting to explore the possibility of converting the active members of these families into ‘neo-functionalized’ ones through genome editing. The base editing approach can be used to precisely mutate the active site residue or the key residues in the active site for neo-functionalization. Such a strategy would be an efficient way to confer disease resistance to crops without incurring metabolic costs, as the members of these families are induced during infections. Taken together, these findings indicate that plant GHs play a crucial role in plant defense. The functional roles of these various plant glycosidases from unexplored GH families during pathogen infections remain to be discovered. Interdisciplinary technologies would be a value-added approach to accelerate the discovery and functional validation of multiple plant glycosidases. Activity-based protein profiling (ABPP) is a functional proteome approach in which chemical probes are used to monitor the activities of various enzymes. ABPP was instrumental in the identification of NbBGAL1, an important immune glycosidase of the GH35 family (Buscaill et al. 2019). In recent years, several chemical probes targeting glycosidases from different GH families have been synthesized (Wu et al. 2019). These probes can be applied during plant–pathogen interactions to monitor the activities of glycosidases from different GH families. Several open questions related to GHs in the context of plant defense are summarized below. We believe that answering these questions would not only help gain mechanistic insights into defense-related GHs but also provide opportunities for translating discoveries into field applications.

Table 1 Summary of functional roles of prominent GHs during pathogen infections
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Fig. 6
figure 6

Certain glycosidases act as double-edged swords during infections. A Disruption of KOR-1, a member of the GH9 family in Arabidopsis thaliana leads to increased JA levels of JA and decreased SA levels, resulting in resistance tp necrotrophs and susceptibility to hemibiotrophs. B Disruption of OsMORE1, a GH10 family member in rice, led to increased expression of SA marker genes and decreased expression of JA marker genes, resulting in resistance to hemibiotrophs and susceptibility to necrotrophs. Figures were created using CorelDRAW software

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Key open questions

  1. 1.

    Can the active members of the GH18 and GH19 families be converted to neo-functionalized proteins using the base editing approach? Do pathogens use any strategies to combat the ‘neo-functionalized proteins’? Do ‘neo-functionalized proteins’ exist in other plant GH families?

  2. 2.

    Is it possible to prevent the suppression of plant GHs by using genome editing technologies to mutate key amino acid residues important for interactions with the inhibitors produced by plant pathogens? Would such an approach improve plant disease resistance?

  3. 3.

    Why do certain plant GHs function as double-edged swords during infections? What are the mechanistic reasons for this phenomenon?

  4. 4.

    What is the relevance of additional domains in plant defense-related GHs?

  5. 5.

    What are the mechanistic insights into the contribution of plant GHs to hypersensitive response-mediated cell death and cell wall remodeling in disease resistance?

  6. 6.

    Which glycosidases unmask the polymorphic O-glycans in flagella for the elicitor release?

  7. 7.

    Do plant GHs mediate the release of bioactive elicitors from the extracellular polysaccharide matrix of pathogenic bacteria?

  8. 8.

    Are the activities of plant chitinases regulated by inhibitors produced by plant pathogens?

  9. 9.

    Which plant GHs mediate the release of elicitors from the cell walls of fungal pathogens?

  10. 10.

    Do the bioactive elicitors or glycans released by retaining and inverting plant GHs have any biological significance?