Lectin receptor kinases (LecRKs). Typically, LecRKs are two-domain proteins composed of an N-terminal extracellular lectin domain and a C-terminal cytosolic Ser/Thr kinase domain, separated by a transmembrane region (Figure 1C). Based on their lectin domain LecRKs are classified into 4 types; G-, C-, L-, and LysM-type (Singh and Zimmerli, 2013; Vaid et al., 2013). Although these LecRKs consist of at least one domain that shows striking sequence similarity with a lectin motif, very little information is available with respect to the ability of this domain to recognize and interact with specific carbohydratestructures.

G-type LecRKs contain an extracellular lectin domain which resembles the Galanthus nivalis agglutinin (GNA). However, it remains to be shown whether this sugar binding domain is indeed involved in ligand binding. Based on genome-wide analyses, 32 G-type LecRKs have been identified in Arabidopsis thaliana and 100 G-type LecRKs in rice (Vaid et al., 2012). G-type LecRKs function in self-incompatibility reactions in flowering plants (the so-called SRKs) and in plant defense to biotic stress as well as to abiotic stress (Sherman-Broyles et al., 2007; Kim et al., 2009; Sun et al., 2013).

C-type (calcium-dependent) LecRKs are mostly found in mammalian proteins that mediate immune responses and play a role in pathogen recognition. In plants, C-type RLKs are rather rare. At present only one C-type LecRK encoding gene has been identified in A. thaliana (At1g52310), though its function has not been elucidated yet (Cambi et al., 2005; Bouwmeester and Govers, 2009).

L-type (legume-like) LecRKs represent a more abundant group of LecRKs. Thus far, 45 L-type LecRKs have been identified in A. thaliana. Based on phylogenetic relationships the genes encoding Arabidopsis L-type LecRK can be classified into nine clusters and nine clades (designated with the Roman numerals I to IX). These genes showed variable expression patterns in different tissues and developmental stages in response to stimuli (Bouwmeester and Govers, 2009). Some LecRKs were indeed reported to be involved in plant resistance to pathogens, e.g., AtLecRK-I.9 is involved in sensing cell wall integrity and defense response to Phytophthora infestans (Bouwmeester et al., 2011). AtLecRK-VI.2 is critical for resistance against Pseudomonas syringae and Pectobacterium carotovorum (Singh et al., 2012; Huang et al., 2014) while AtLecRK-IV.3 induces resistance against Botrytis cinerea (Huang et al., 2013). Some AtLecRKs have also been reported to act in hormone signaling (ABA) and stomatal immunity (e.g., AtLecRK-VI.2 and AtLecRK-V.5; Singh et al., 2012). L-type LecRKs have also been identified in other plants. For instance, tobacco plants express L-type LecRKs with a role in plant immunity against pathogens and insects (Kanzaki et al., 2008; Gilardoni et al., 2011). In turn, Medicago plants contain L-type LecRKs that are involved in symbiosis (Navarro-Gochicoa et al., 2003).

At present, it is not yet clear whether the L-type LecRKs possess lectin activity since the amino acids important for interaction of the legume lectin domain with its specific carbohydrate ligand are poorly conserved. In contrast the hydrophobic site present in the legume-type lectin domain is preserved, suggesting that LecRKs may act in the recognition of small hydrophobic ligands (Huang et al., 2013; Choi et al., 2014). Recently, evidence was obtained that the plasma membrane localized DORN1, encoded by the AtLecRK-I.9 gene, plays an important role as a receptor for extracellular ATP (Figure 1C; Choi et al., 2014). DORN1 lacks the conserved Ca²⁺ and Mn²⁺ binding residues that are critical for carbohydrate binding activity of legume lectins. Early studies also suggested the ability of the legume lectin domain to bind adenine, a component of ATP (Roberts and Goldstein, 1983). However, since adenine was unable to compete with ATP for binding to DORN1, the exact ATP binding site in DORN1 remains to be determined. These data are in good agreement with the fact that extracellular ATP is now perceived as a central signaling molecule in plant stress responses (Cao et al., 2014; Choi et al., 2014).

LysM LecRKs are the most studied LecRKs (Figures 1C,D and Table 2). They contain ectopic lysin motifs, referred to as LysM domains, which are considered to mediate binding to various types of bacterial PGN and fungal chitin, upon recognition of the GlcNAc moieties (Buist et al., 2008; Gust et al., 2012). The lysine motif, approximately 40 amino acids in length, is a ubiquitous protein domain found in most living organisms except the Archaea. It can be used as a single domain, but is also present in the form of two or occasionally three repeats in a large number of proteins. In most cases LysM motifs are coupled to other protein domains exhibiting some enzymatic activity, such as GlcNAc modification in the case of microbial hydrolases or intracellular signaling for plant kinases.

The Arabidopsis chitin elicitor receptor kinase 1 (AtCERK1, also known as LYK1/RLK1) is the major chitin receptor found in A. thaliana (Miya et al., 2007; Petutschnig et al., 2010; Tanaka et al., 2013). It is a membrane-anchored protein with an extracellular domain containing three LysM motifs coupled to an intracellular kinase domain. This kinase domain contains a canonical RD (Arginine-Aspartate) motif in its catalytic loop and possesses autophosphorylation activity, unlike the non-RD kinase domain of typical PRRs. AtCERK1 was reported to be involved in fungal resistance. The protein directly binds to fungal chitooligosaccharides (GlcNAc_n with n > 2) through its LysM domains, but only longer oligomers (n > 4) trigger immune responses. Liu et al. (2012b) demonstrated that binding of chitin oligomers (n = 8) to AtCERK1 induces homodimerization of the receptor, which is essential for the activation of downstream intracellular signaling, most likely by phosphorylation of both CERK1 cytoplasmic kinase domains.

AtCERK1 can also mediate perception of PGN when part of a complex with AtLYM1 and AtLYM3, two other transmembrane LysM containing proteins lacking an intracellular kinase domain (the so-called LYP proteins; Willmann et al., 2011). Next to AtCERK1 A. thaliana contains four additional LysM RLKs, designated AtLYK2–5. Since AtLYK4 and AtLYK5 can also recognize and bind to chitin molecules but have a non-functional pseudokinase domain it has been suggested they should form a complex with AtCERK1 to compose a functional chitin receptor (Wan et al., 2012). AtLYK3 possesses a functional kinase domain, but was suggested be involved in ABA signaling rather than in chitin recognition (Paparella et al., 2014).

Unlike AtCERK1, the rice ortholog OsCERK1 cannot bind directly to chitooligosaccharides. OsCERK1, a LysM LecRK with a single extracellular LysM domain, requires heterodimerization with its co-receptor OsCEBiP (chitin elicitor binding protein) for chitin binding and subsequent activation of innate immunity (Figure 1D). OsCEBiP is a transmembrane LysM receptor protein containing three LysM domains and lacking a kinase domain, resembling the Arabidopsis proteins AtLYM1 and AtLYM3. However, whereas AtLYM1 and AtLYM3 are involved in PGN binding through receptor formation with AtCERK1, OsCEBiP seems to play a major role in fungal chitin perception. Upon binding to the fungal GlcNAc_6-8 oligomers, OsCEBiP homo-dimerizes at the plasma membrane of the plant cell. After this ligand-induced dimerization, the OsCEBiP sandwich-like structure forms a heteromeric complex with two OsCERK1 proteins, to activate intracellular defenses, including ROS signaling, callose deposition, and defense gene expression (Shimizu et al., 2010; Shinya et al., 2012; Hayafune et al., 2014; Kouzai et al., 2014). OsLYP4 and OsLYP6 are two additional rice LysM proteins lacking a kinase domain which presumably mediate perception of both PGN and chitin, but their action and transmembrane signal transfer remain unclear (Liu et al., 2012a).

Plants not only use LysM LecRKs to recognize pathogenic organisms, they also use them to perceive beneficial organisms such as mycorrhizal fungi and rhizobacteria implicating a dual role of LysM in both innate immunity and symbiosis (Table 2; Gust et al., 2012). Examples include the Lotus japonicus NRF1 and NRF5 (Radutoiu et al., 2003) and the Medicago truncatula LYK3 and LYK4 (Knogge and Scheel, 2006) which can recognize rhizobial lipochitin-oligosaccharide signals or Nod factors.

An overview of soluble proteins with a lectin domain involved in plant defense signaling is given in Table 3.

Amaranthins. The Amaranthin family groups all proteins related to amaranthin, a lectin present in the seeds of Amaranthus caudatus. Native amaranthin is a homodimeric protein built of two 33 kDa subunits, each comprising two tandem-arrayed homologous amaranthin domains. Amaranthin specifically recognizes the T-antigen disaccharide Galβ(1,3)GalNAc but also interacts with GalNAc. Interestingly, the amaranthin domain itself possesses no sugar binding site(s), but the specific head-to-tail arrangement of two amaranthin subunits is necessary to establish the T-antigen disaccharide binding site (Van Damme et al., 2008). Up till now, only amaranthins originating from Amaranthus species have been purified and biochemically characterized. This nucleocytoplasmic lectin was reported to enhance the plant’s resistance against aphids when ectopically expressed in transgenic tobacco, potato, and cotton by affecting growth and development of the invading aphids (Wu et al., 2006; Xin et al., 2011).

Screening of the publicly available genome databases revealed that the amaranthin domain is widespread throughout the plant kingdom. Several chimeric proteins containing N-terminal amaranthin domain(s) coupled to unrelated protein domains have been identified (Van Damme et al., 2011). Columbine plants (Aquilegia formosa× Aquilegia pubescens) encode a protein in which two amaranthin domains are coupled to a kinase domain. Since this protein does not have a transmembrane domain, it is suggested to reside inside the cell. Cucumber, maize, and wheat plants were found to contain genes that encode proteins with amaranthin domain(s) coupled to an aerolysin domain. Aerolysins are cytolytic toxins that are mostly produced by the bacterium Aeromonas and can kill host cells upon pore formation in the plasma membrane (Degiacomi et al., 2013). In wheat, a chimeric protein called Hfr-2 (Hessian fly responsive-2) is up-regulated in the leaf sheaths after feeding of virulent Hessian fly larvae and armyworms, and enhances wheat tolerance against Hessian fly larvae (Puthoff et al., 2005).

Calreticulin/calnexin. Calreticulin (CRT) and calnexin (CNX) are glucose binding lectins residing in the endoplasmic reticulum (ER) of eukaryotic cells. Both CRT and CNX act as molecular chaperones and are essential ER components ensuring proper folding and quality control of newly synthesized secretory and membrane-bound glycoproteins before ER release (Ellgaard and Frickel, 2003; Kapoor et al., 2004; Williams, 2006). While CNX is a type-I integral membrane protein, CRT is a soluble protein. Both CRT and CNX act together with their co-chaperones ERp57 and PDI, two soluble thiol-disulfide oxidoreductases. Whereas the classical chaperones associate with the peptide moiety of their substrates, CNX and CRT bind to their glycoprotein substrates primarily through specific recognition and binding to the oligosaccharide intermediates Glc₁Man_7-9GlcNAc₂ present on nascent glycoproteins.

In the ER quality control system, a growing polypeptide initially gets N-glycosylated with the core glycan Glc₃Man₉GlcNAc₂. By successive action of glucosidase I and II, the outer glucoses are trimmed resulting in a monoglucosylated glycoprotein which then serves as the substrate of CNX/CRT for proper folding. Once the glycoprotein is correctly folded, the terminal glucose of its oligosaccharide is cleaved by glucosidase II and the glycoprotein is released from the CNX/CRT/ERp57 complex for further processing. If the glycoprotein is not correctly folded, it is recognized by the UDP-glucose:glycoprotein glucosyltransferase enzyme, which acts as a folding sensor, and gets re-glucosylated to promote its renewed association with CNX/CRT. As such, de- and re-glucosylation of glycoproteins facilitates their correct folding. When folding ultimately fails, the misfolded glycoproteins are sorted out of the ER and are degraded by the proteasome, a system known as ER associated degradation or ERAD. Under adverse environmental conditions, the demand for protein folding exceeds the capacity of the system resulting in the accumulation of misfolded proteins in the ER, giving rise to so-called ER stress (Howell, 2013; Liu and Li, 2014).

The correct folding of membrane-bound PRRs is a critical step in plant immunity. The ER quality control system not only regulates the abundance and quality of transmembrane receptors, it also affects downstream signaling of the receptor (Tintor and Saijo, 2014). The LRR-RLKs AtEFR and AtFLS2 from A. thaliana as well as the LysM LecRK NFP from M. truncatula require proper N-glycosylation for accurate functioning. Nevertheless, AtEFR and AtFLS2 production are coordinated by different ER components (Li et al., 2009; Häweker et al., 2010). Arabidopsis plants contain two types of CRTs: CRT1/2 and CRT3 isoforms (Thelin et al., 2011). Whereas CRT1 is a key chaperone in plant ER stress, CRT3 is typically involved in the quality control of AtEFR and the brassinosteroid receptor BRI1, but is not essential for AtFLS2 biogenesis (Jin et al., 2009). CRT2 appears to have a dual regulatory role in plant defense against the biotrophic pathogen P. syringae pv tomato DC3000 (Qiu et al., 2012). Upon pathogen invasion, CRT2 is involved in the up-regulation of SA-dependent immune signaling through its Ca²⁺ buffering capacity. However, CRT2 negatively influences these SA-dependent responses through its chaperone activity, resulting in the overall suppression of plant resistance toward P. syringae pv tomato DC3000.

EUL-related lectins. The family of EUL-related lectins groups all nucleocytoplasmic proteins that comprise at least one Euonymus lectin (EUL) domain. The prototype of this family is the so-called Euonymus europaeus agglutinin (EEA) which is present at very high concentrations in the arillus tissue of the spindle tree (E. europaeus). EEA is a non-glycosylated homodimeric protein composed of 17 kDa subunits, and recognizes two structurally different classes of glycans. Glycans with carbohydrate epitopes containing galactose, such as Galα1–3Gal and Galα1–3Galβ1–4GlcNAc, blood group B [Galα1–3(Fucα1-2)Gal-], and O (Fucα1–2Gal-) epitopes are bound with a higher affinity compared to high-mannose N-glycans. Based on inhibition studies, it was suggested that the EUL domain might contain two different binding sites (Fouquaert et al., 2008).

Sequences with an EUL domain are present in almost all sequenced plant genomes from Embryophyta, ranging from liverworts to flowering plants. The sequence of the EUL domain is well conserved suggesting that the corresponding EUL proteins fulfill an essential role in plants. Based on the overall protein domain architecture, the EUL family can be divided into two groups containing proteins either composed of a single EUL domain (S-type EUL proteins) or of two tandemly arrayed EUL domains separated by a spacer sequence (D-type EUL proteins; Fouquaert and Van Damme, 2012). The majority of the EUL sequences encode chimeric proteins, in which the EUL domain is linked to other unknown domains. Whereas most dicot species encode one or two EUL S-type proteins, monocot, and lower plant species contain a whole set of S- and D-type EUL sequences.

In contrast to EEA, which is expressed at high concentrations in the arilli of spindle tree seeds, the EUL proteins from Oryza sativa and A. thaliana are very low abundant proteins. In both plants the amount of EUL transcripts is enhanced after the plant was subjected to different abiotic (such as dehydration, salinity, osmotic stress, and ABA treatment) and biotic (such as bacterial and fungal infection) stresses (Fouquaert and Van Damme, 2012; Al Atalah et al., 2014a).

Next to the differential regulation of gene expression, the EUL proteins show different carbohydrate binding properties. ArathEULS3 from A. thaliana preferentially interacts with N-glycans containing galactosylated structures such as Lewis X [Galβ1–4(Fucα1–3)GlcNAc], Lewis Y [Fucα1–2Galβ1–4(Fucα1–3)GlcNAc] and lactosamine (Galβ1–4GlcNAc) motifs (Van Hove et al., 2011). Similarly, both EUL domains composing the two-domain EUL protein from rice, OrysaEULD1A, show carbohydrate specificity toward galactose containing glycans (Al Atalah et al., 2014b). In contrast, the rice protein OrysaEULS2 preferably binds mannosylated N-glycan structures (Al Atalah et al., 2012). All these EUL proteins are located in the nucleus and the cytoplasm of the plant cell. In search for interacting proteins, Li et al. (2014) recently reported that ArathEULS3 interacts with the nuclear/cytosolic ABA receptor RCAR1. Furthermore, Berendzen et al. (2012) demonstrated interaction of ArathEULS3 with CPK3, a Ca²⁺ dependent kinase involved in the ABA response in guard cells, supporting a role for ArathEULS3 in ABA signaling and stomatal closure.

Jacalin-related lectins (JRL). The family of jacalin-related lectins is named after jacalin, a 18 kDa T-antigen disaccharide-binding lectin domain first isolated from the seeds of jackfruit (Artocarpus integrifolia). Based on differences in molecular structure, subcellular localization, and carbohydrate binding properties, the large group of jacalins can be subdivided into two subgroups, further referred to as the galactose binding and mannose binding JRLs, residing in the vacuolar and nucleocytoplasmic compartment of the plant cell, respectively.

Galactose-specific JRLs have been reported mainly within the family Moraceae, whereas the mannose-specific JRLs are widespread in Viridiplantae. Furthermore, recent studies have shown that the jacalin domain is not restricted to plant proteins, since a similar domain has been reported in eukaryotes outside the plant kingdom as well as in some prokaryotes (Van Damme et al., 2008; Kanagawa et al., 2014). In plants, chimeric proteins comprising one or more jacalin domains linked to an unrelated domain are widely distributed. For instance, in A. thaliana, sequences composed of one or two jacalin domains C-terminally linked to five in tandem arranged Kelch domains are present. In addition, multiple Arabidopsis genes encode a putative F-box protein with a C-terminal jacalin domain (Nagano et al., 2008). Several Poaceae species (wheat, rice, maize) express proteins consisting of an N-terminal dirigent domain (also called disease-response domain) C-terminally fused to a jacalin domain. In rice, additional types of chimerolectins were identified, either composed of an N-terminal tyrosine kinase domain coupled to two or three jacalin domains or an N-terminal NB-ARC motif coupled to a LRR and a C-terminal jacalin domain (Van Damme et al., 2008).

Many jacalin-related lectin genes have been shown to be associated with disease resistance, abiotic stress signaling, wounding, insect damage or multiple stresses (Song et al., 2014). Especially the jacalin proteins with a dirigent domain are functionally involved in plant defense. To our knowledge, these chimeric proteins have only been reported in Poaceae species. In wheat, nearly half of the jacalin-related lectin genes encode dirigent domain-containing jacalin-related proteins. Several of these proteins have been studied in some detail, amongst them TaVER2, TaHfr-1, and TaJA-1 (Song et al., 2014). Interestingly all these mannose binding lectins are expressed as a response toward plant stress. TaVER2 is specifically expressed during vernalization in wheat (Yong et al., 2003) but is also up-regulated upon jasmonate and ABA treatment (Feng et al., 2009). TaHfr-1 is up-regulated after herbivory of Hessian fly larvae (Williams et al., 2002) and TaJA-1 is specifically accumulating after jasmonate treatment (Ma et al., 2013). The mannose specific TaHfr-1 was shown to effectively inhibit Hessian fly larval feeding resulting in the delay of larval development and premature death of the pest insects (Subramanyam et al., 2008). Transgenic tobacco plants overexpressing Ta-JA1 revealed increased resistance to bacterial, fungal, and viral pathogens (Ma et al., 2010). TaVER2 homologs have been found in maize and sorghum (β-glucosidase aggregating factor) and in rice (OsJAC1; Esen and Blanchard, 2000; Jiang et al., 2006; Kittur et al., 2009). Transgenic rice plants overexpressing OsJAC1 indicated the importance of OsJAC1 for rice growth and development (Jiang et al., 2007).

Similar to the chimerolectins also jacalin-related proteins composed only of jacalin domains are up-regulated in plant tissues subjected to certain stress treatments. For instance, Orysata was first reported as a salt inducible mannose binding lectin in the leaves of O. sativa (Zhang et al., 2000). Later it was shown that Orysata is also expressed upon JA and ABA treatment, after infection with an incompatible Magnaporthe grisea strain as well as during senescence (Lee et al., 2001; de Souza Filho et al., 2003; Qin et al., 2003). Glycan array analyses revealed that Orysata preferentially interacts with high-mannose and some more complex N-glycans (Al Atalah et al., 2011). In recent years, several orthologs of Orysata have been identified in Gramineae species but also in other plants, such as Helianthus tuberosus and Ipomoea batatas. The mannose specific wheat protein TaJRLL1 consisting of two jacalin-like domains is considered a component of SA and JA dependent plant defense signaling mechanisms and is activated upon fungal infection (Fusarium graminearum and Blumeria graminis; Xiang et al., 2011). However, not all jacalin-related defense proteins depend on hormone signaling. For example, the Arabidopsis jacalin-related JAX1 confers broad but specific resistance to potex viruses by inhibition of viral RNA accumulation, independent of hormone signaling (Yamaji et al., 2012). Other Arabidopsis jacalin-related proteins interact with proteins of ER bodies, i.e., ER-derived organelles presumably involved in defense against herbivores and/or pathogens (Nagano et al., 2008). Recently, the jacalin-related protein from sunflower seedlings named Helja was reported as a lectin with antifungal properties toward some pathogenic fungi of the genus Candida. Helja induces morphological changes as well as ROS production in yeast cells. Furthermore, lectin treatment also altered the membrane permeability of the cells (Regente et al., 2014).

Nictaba-related lectins. The family of Nictaba-related lectins was named after the Nicotiana tabacum agglutinin, abbreviated as Nictaba, a 19 kDa lectin domain originally discovered in tobacco leaves (Chen et al., 2002a) after jasmonate treatment. Though the lectin was first reported as a chito-oligosaccharide binding protein, glycan array analyses revealed that Nictaba also reacts with the inner core structure (Man)₃β1–4GlcNAcβ1–4GlcNAcβ-N-Asn of high-mannose and complex N-glycans. Biochemical assays confirmed that Nictaba can interact in a sugar-inhibitable way with many N-glycosylated proteins (Lannoo et al., 2006, 2007). A nuclear proteomics approach revealed the interaction of Nictaba with the core histone proteins H2A, H2B, and H4 through their O-GlcNAc modification (Schouppe et al., 2011), which was later confirmed at the microscopical level (Delporte et al., 2014a).

An extensive survey of the genome/transcriptome databases indicated that Nictaba-like domains are widespread among the Embryophyta but are absent from other eukaryotes and prokaryotes. Few proteins belonging to the Nictaba-like family consist of a single Nictaba domain. Furthermore, numerous sequences were identified encoding chimeric proteins comprising the Nictaba domain fused to unrelated N-terminal domains (e.g., F-box domain) or a Nictaba domain C-terminally fused to an N-terminal TIR (toll/interleukin-1 receptor) domain (found in Arabidopsis and tomato), an AIG1 (avrRpt2-induced gene) domain (identified in Arabidopsis) or a kinase domain (found in rice; Delporte et al., 2014b).

At present, only few Nictaba-related proteins have been studied for their biological properties and physiological role. A comparative analysis of the carbohydrate binding properties of Nictaba from N. tabacum, the Cucurbitaceae phloem lectin PPL, the A. thaliana homolog PP2-A1 and the A. thaliana F-box-Nictaba protein encoded by the gene At2g02360 revealed that despite the sequence similarity and the presence of conserved amino acids in the carbohydrate binding site, different Nictaba domains can interact with different glycan motifs (Delporte et al., 2014b), suggesting different biological roles.

Since insect herbivory by Lepidopteran insects also triggers the JA pathway the tobacco lectin also accumulates after caterpillar attack. Furthermore, feeding assays with (transgenic) tobacco lines demonstrated the entomotoxic activity of Nictaba. It was suggested that the entomotoxic effect of Nictaba is caused by interaction of the lectin with glycoconjugates present in the digestive tract of the insect (Vandenborre et al., 2010, 2011). Within the plant cell, insect herbivory results in enhanced Nictaba expression in the cytoplasm, followed by partial translocation of the lectin to the nucleus, where it can interact with core histone proteins through their O-GlcNAc modification. It is hypothesized that Nictaba binding to chromatin results in enhanced transcription of defense related genes (Lannoo and Van Damme, 2010).

The Cucurbitaceae phloem lectins are a group of Nictaba-related lectins that are found in phloem exudates of different Cucurbitaceae species. Unlike Nictaba, the PP2 lectins are exclusively and continuously expressed in the companion cells of the phloem and then translocated into the phloem sap. The pumpkin lectin PPL and the PP2-like protein from A. thaliana, PP2-A1, show high binding affinity for chitin oligomers (GlcNAc_3-6). Similar to Nictaba, PP2-A1 also binds with the Man₃GlcNAc₂ core of high-mannose N-glycans (Beneteau et al., 2010). Interestingly, the expression of PP2-A1 was enhanced by ethylene treatment and Pseudomonas infection. Furthermore, PP2-A1 represses phloem feeding of the green peach aphid Myzus persicae (Zhang et al., 2011), displayed antifungal activity against various fungal strains (Lee et al., 2014), and negatively affects Cucurbit aphid borne yellow virus transmission (Bencharki et al., 2010), strongly supporting a role in plant defense.

Several F-box Nictaba proteins are encoded in the A. thaliana genome. Glycan array analyses revealed the F-box protein encoded by At2g02360 exhibits carbohydrate binding activity toward N- and O-glycans with N-acetyllactosamine (LacNAc; Galβ1–3GlcNAc and Galβ1–4GlcNAc) and poly-LacNAc structures as well as with Lewis A (Galβ1–3(Fucα1–4)GlcNAc), Lewis X (Galβ1–4(Fuca1–3)GlcNAc), Lewis Y (Fucα1–2Galβ1–4(Fucα1–3)GlcNAc), and type-1 B motifs (Galα1–3(Fucα1–2)Galβ1–3GlcNAc. Since these glycan structures have been reported in bacteria, viruses, and animals rather than in plants the physiological importance of this glycan interaction remains enigmatic (Lannoo et al., 2008; Stefanowicz et al., 2012). Furthermore, the same Arabidopsis F-box Nictaba protein was shown to interact with core members of an E3-type ubiquitin ligase complex which resulted in the hypothesis assuming a role of the Nictaba domain in glycoprotein degradation (Takahashi et al., 2004; Arabidopsis Interactome Mapping Consortium, 2011).

Ricin-B lectins. The ricin-B lectin family is one of the most widespread families of carbohydrate binding proteins in nature. The most famous member of this family is ricin, a toxic protein from castor bean (Ricinus communis L.) seeds, which was the very first lectin discovered in plants by Peter Hermann Stillmark in 1888. Ricin is a chimeric protein consisting of an A chain with enzymatic activity linked through a disulfide bridge with a B chain with lectin activity. This B chain consists of a duplicated ricin-B domain, responsible for the carbohydrate binding activity of the protein toward galactosylated structures. The enzymatic activity of ricin involves RNA N-glycosidase activity and is responsible for the removal of a highly conserved adenine residue from the sarcin/ricin loop of the 28S ribosomal RNA. As a result, the ribosomes are no longer able to bind elongation factor 2 and protein synthesis is blocked. Because of their catalytic activity these chimeric proteins are also referred to as type 2 ribosome-inactivating proteins (RIPs), and are considered as toxic proteins if they succeed in entering the cell. The uptake of the protein by the host cell is aided by their lectinic B-chain which can specifically interact with glycoconjugate structures on the cell surface. Except for ricin and abrin (from the jequirity bean Abrus precatorius), most type 2 RIPs are only moderately or even weakly toxic (Van Damme et al., 2001; Stirpe and Battelli, 2006).

The family of ricin-B related lectins is widespread in the plant kingdom and has been characterized in detail for what concerns its biological activity and toxicity in several plant species, especially R. communis (castor bean), Abrus precatorius (jequirity bean), Viscum album (mistletoe), and Sambucus nigra (elderberry). Unlike the other soluble lectins described above, most ricin-B related proteins accumulate in the plant vacuole or are secreted to the extracellular space (Van Damme et al., 2008). Over the years the ricin-B domain was identified in numerous plants, animals, fungi, and bacteria. All these proteins with ricin-B domains are also classified as the R-type lectins (Cummings and Etzler, 2009).

Within the genus Sambucus (elderberry) an extended family of ricin-B related proteins, including several chimerolectins and hololectins has been identified (Shang and Van Damme, 2014). Detailed hapten inhibition assays and glycan array studies revealed that all these S. nigra proteins exhibit different carbohydrate binding properties, and allowed to classify the Sambucus lectins into three groups. A first group covers the lectins SNA-II and SNA-IV as well as the type 2 RIP SNA-V with specificity toward Gal/GalNAc and Gal/GalNAc-containing glycan structures. The second group comprises only the type 2 RIP SNA-I, which specifically interacts with terminal sialic acid residues (Neu5Ac; α2–6) linked to Gal/GalNAc. Finally, the type 2 RIP SNLRP represents a third specificity group with strong interaction with GlcNAc oligomers (Shang and Van Damme, 2014).

Several lines of evidence support the idea that ricin-B related lectins play a role in plant defense against pathogens (Chen et al., 2002b; Vandenbussche et al., 2004a,b) and insects (Wei et al., 2004; Shahidi-Noghabi et al., 2009). Over-expressing SNA-I′ or SNA-V from S. nigra in transgenic tobacco (Samsun NN) plants enhances the tobacco plant’s resistance against infection with tobacco mosaic virus. Though the antiviral effect is clearly related to the amount of protein expressed it cannot be related to an increased expression of pathogenesis-related proteins (Chen et al., 2002b; Vandenbussche et al., 2004a,b). Furthermore, no clear correlation was observed between in planta antiviral activity of the transgenic tobacco lines and the in vitro N-glycosidase activity of the proteins toward genomic RNA of the tobacco mosaic virus, suggesting that the in planta antiviral activity of these RIPs may rely on a direct interaction with the virus (Vandenbussche et al., 2004a). At present the importance of the lectin activity in the antiviral activity of the proteins remains unclear.

The first evidence for the insecticidal activity of ricin-B related proteins came from feeding assays with ricin and cinnamomin (from Cinnamomum camphora tree). Ricin showed strong toxicity to several insects such as cowpea weevil (Callosobruchus macultatus), cotton boll weevil (Anthonomus grandis), house fly (Musca domestica), and larvae of the silkworm Bombyx mori (Wei et al., 2004). The differences in activity between ricin and cinnamomin could not be related to the enzymatic activity but rather were attributed to differences in the activity of the lectin chain of the proteins (Wei et al., 2004). Shahidi-Noghabi et al. (2009) reported the enhanced resistance of transgenic tobacco plants overexpressing SNA-I or its isoform SNA-I′ toward different pest insect species including aphids and caterpillars. Since mutation of the carbohydrate binding site can abolish or reduce the toxic effect, the entomotoxic properties of the proteins can be linked to their carbohydrate binding activity (Shahidi-Noghabi et al., 2008). In addition, the cytotoxic effects of S. nigra RIPs toward insect cells was accompanied by caspase 3-like protease-induced apoptosis (Shahidi-Noghabi et al., 2008, 2011). More research is needed to identify the interacting proteins for the Sambucus lectins on the cell surface.