Review
Research review Lipochitooligosaccharide recognition: an ancient story Author for correspondence: Gary Stacey Tel: +1 5738844752 Email: [email protected]
Yan Liang, Katalin T oth, Yangrong Cao, Kiwamu Tanaka, Catherine Espinoza and Gary Stacey
Received: 31 March 2014 Accepted: 18 May 2014
University of Missouri, Columbia, MO 65211, USA
Divisions of Plant Science and Biochemistry, National Center for Soybean Biotechnology, Christopher S. Bond Life Sciences Center,
Summary New Phytologist (2014) doi: 10.1111/nph.12898
Key words: arbuscular mycorrhizal (AM) symbiosis, innate immunity, lipochitooligosaccharides (LCOs), Myc factors, Nod factors, nodulation, peptidoglycan.
Chitin is the second most abundant polysaccharide in nature, found in crustacean shells, insect exoskeletons and fungal cell walls. The action of chitin and chitin derivatives on plants has become a very interesting story of late. Chitin is a b1-4-linked polymer of N-acetyl-Dglucosamine (GlcNAc). In this unmodified form, chitooligosaccharides (degree of polymerization (dp) = 6–8)) are strong inducers of plant innate immunity. By contrast, when these chitooligosaccharides are acylated (so-called lipochitooligosaccharides, LCOs) and further modified, they can act as Nod factors, the key signaling molecules that play an important role in the initiation of the legume–rhizobium symbiosis. In a similar form, these molecules can also act as Myc factors, the key signaling molecules involved in the arbuscular mycorrhizal (AM) symbiosis. It has been proposed that Nod factor perception might have evolved from the more ancient AM symbiosis. Increasing evidence now suggests that LCO perception might have evolved from plant innate immunity signaling. In this review, we will discuss the evolutionary origin of symbiotic LCO recognition.
Introduction The legume–rhizobium symbiosis is a major contributor to biological nitrogen fixation and, therefore, to the global nitrogen cycle (Bohlool et al., 1992). During the symbiosis, legume roots form a novel organ, the nodule, in which the bacteria fix atmospheric dinitrogen and convert it into ammonia that can be utilized by the host plants. Over the past few decades, the legume–rhizobium symbiosis has been of intense interest to researchers, as this nodulation ability offers the potential to reduce the use of fossil fuel-derived chemical fertilizer, improve soil health and mitigate negative environmental effects resulting from agricultural practices. However, nodulation ability is mostly restricted to the family of leguminous plants (Fabaceae) and it would be a huge challenge to transfer this ability to nonlegumes (Charpentier & Oldroyd, 2010; Oldroyd & Dixon, 2014; Rogers & Oldroyd, 2014). Formation of functional legume root nodules requires two separate but tightly coordinated developmental processes: nodule organogenesis and bacterial infection. In most legumes, nodule formation is initiated by rhizobial attachment to root Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
hair tips, stimulating subsequent cortical cell redifferentiation and root hair curling. Cortical cell redifferentiation gives rise to a nodule primordium, leading to the eventual development of a mature nodule. Curling of the root hair entraps bacteria, leading to the subsequent formation of a plant cell wall-derived infection thread. The infection thread delivers the bacteria from the root hair into the newly formed nodule primordium, where the infection thread branches and bacteria are released by exocytosis. Inside the nodule, the bacteria differentiate into their endosymbiotic form, bacteroids, which fix dinitrogen into ammonium (Geurts et al., 2005; Oldroyd & Downie, 2008; Ferguson et al., 2010). The key step in establishing the legume–rhizobium symbiosis is the plant recognition of bacterial signal molecules, Nod factors (Oldroyd et al., 2011). The longstanding belief is that nonlegumes cannot form nodules in response to rhizobia because of their inability to recognize Nod factors (Beatty & Good, 2011). In this review, we focus on new information regarding the recognition of Nod factors by legumes and nonlegumes, as well as structurally similar molecules, such as Myc factors, chitin and peptidoglycan (PGN). New Phytologist (2014) 1 www.newphytologist.com
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polymerization (dp) = 3–5) with various substitutions on the reducing and nonreducing ends, defined as lipochitooligosaccharides (LCOs). Fig. 1(a) shows the unmodified chitooligosaccharides, and Fig. 1(b) shows the representative Nod factor (Nod Bj V C18:1Δ11, MeFuc) produced by Bradyrhizobium
Nod factor recognition Nod factor structures have been extensively reviewed (Stacey et al., 1992; Denarie et al., 1996; D’Haeze & Holsters, 2002). Briefly, Nod factors are short chitooligosaccharides (degree of OH
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Fig. 1 Structure of chitin, representative Nod factor, Myc factor and peptidoglycan. (a) Chitin, a polymer of N-acetylglucosamine. The acetyl moiety (red) is important for chitinreceptor binding. (b) Nod factor (Nod Bj V C18:1Δ11, MeFuc) produced by Bradyrhizobium japonicum. The substitutions of chitin backbone are in red. (c) Myc factor (LCO-IV C18:1 Δ9) produced by Glomus intraradices. (d) Monomer of peptidoglycan produced by Escherichia coli. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist japonicum (Sanjuan et al., 1992). The Nod factor nonreducing end is N-acylated by a fatty acid, and the reducing end is modified by 2-O-methylfucose (Sanjuan et al., 1992). Nod factors are structurally diverse and it is generally thought that plant host specificity is determined by the specific chemistry of the signal produced by the compatible rhizobial symbiont. For example, different rhizobia produce Nod factors that differ in chitin chain length, specific fatty acylation, sulfation, acetylation, etc. (Lerouge et al., 1990; Carlson et al., 1993; Staehelin et al., 2000). Knowledge of the chemistry of the Nod factors immediately raised the question as to how legumes recognize this molecule. This led to studies of plant mutants in both Medicago truncatula and (a)
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Lotus japonicus, which were found to be insensitive to Nod factor addition. The cloning of the underlying genes in these mutants identified two plasma membrane Nod factor receptors, termed Nod factor receptor 1 (LjNFR1) and LjNFR5, in Lotus japonicus (Madsen et al., 2003; Radutoiu et al., 2003) and MtLYK3 and MtNFP in Medicago truncatula (Fig. 2a,b; Limpens et al., 2003; Arrighi et al., 2006). These Nod factor receptors belong to the lysin motif (LysM)-containing receptor-like kinase (LYKs) family, consisting of an extracellular domain with one to three LysM motifs (ancient and conserved 40 amino acid motif), a transmembrane domain and an intracellular serine/threonine kinase domain. However, the lack of critical amino acids within the LjNFR5/ MtNFP clade indicates that these proteins lack kinase activity (b)
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Fig. 2 Model for Nod factor, Myc factor, chitin and peptidoglycan recognition. (a) Nod factors are recognized by LjNFR1/MtLYK3 and LjNFR5/MtNFP in nodule organogenesis in both Lotus japonicus and Medicago truncatula. (b) LjNFR5/MtNFP is essential for Nod factor recognition in bacterial infection in both L. japonicus and M. truncatula. (c) PaNFP is required for both rhizobial and arbuscular mycorrhizal (AM) symbiosis in Parasponia andersonii. (d) MtNFP is involved in Myc factor stimulation of lateral root growth in M. truncatula. (e) Long-chain chitooligosaccharides (degree of polymerization (dp) = 6–8) are recognized by OsCERK1 and OsCEBiP, leading to the immune response in rice. (f) Long-chain chitooligosaccharides (dp = 6–8) are recognized by AtCERK1, leading to the immune response in Arabidopsis. (g) AtLYK4 is involved in chitooligosaccharide (dp = 6–8)mediated immune response in Arabidopsis. (h) AtLYM2 is involved in chitooligosaccharide (dp = 6–8)-induced suppression of molecular flux in plasmodesmata. (i) Peptidoglycan is recognized by AtLYM1/AtLYM3 and AtCERK1, leading to the immune response in Arabidopsis. (j) Peptidoglycan is recognized by OsLYP4/OsLYP6, leading to the immune response in rice. (k) MtNFP is involved in plant immune response in M. truncatula, but the ligand is still unknown. (l) AtLYK3 is required for immunosuppression of Nod factors and short-chain chitooligosaccharides (dp = 4–5) in Arabidopsis. The inactive kinase activity is labeled as X. Conserved LysM domains are shown in color, while those less conserved (perhaps nonfunctional) are shown in white. MAMP, microbe-associated molecular pattern.
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(Zhang et al., 2007; Lohmann et al., 2010). In plants, LYKs and another group of LysM-containing receptor proteins (LYM, lacking the intracellular kinase domain) are important membrane proteins for recognition of GlcNAc-containing molecules, such as PGN, chitin, and LCOs (Buist et al., 2008; Antolin-Llovera et al., 2012). Transformation of LjNFR1 and LjNFR5 into nonhost M. truncatula and L. filicaulis was shown to confer the ability to recognize the Nod factors produced by Mesorhizobium loti, the compatible symbiont of L. japonicus (Radutoiu et al., 2007). Point mutations in LjNFR5 and experiments that swapped the extracellular domains between MtNFP and the ortholog of NFP in pea suggest that the second LysM domain of LjNFR5/MtNFP is the critical binding region for Nod factor recognition (Radutoiu et al., 2007; Bensmihen et al., 2011). Measurements of binding affinity by surface plasmon resonance or thermophoresis indicates that LjNFR1 and LjNFR5 bind Nod factor with very high affinity (Kd values in the nanomolar range), which is comparable to the Nod factor concentrations found to elicit physiological responses in legumes (Miwa et al., 2006; Broghammer et al., 2012). These studies suggest that the Nod factor receptors have evolved to possess high binding affinity and chemical specificity for the Nod factors produced by the compatible rhizobia. Although nodule organogenesis (taking place in cortical cells) and bacterial infection (occurring in epidermal cells) are tightly coupled, they can be genetically separated and Nod factor receptors are necessary for both processes (Oldroyd & Downie, 2008; Madsen et al., 2010). However, epidermal expression of LjNFR1 using tissue-specific promoters could not complement Ljnfr1 nodule organogenesis (Hayashi et al., 2014), whereas epidermal expression of MtNFP/LjNFR5 could rescue Mtnfp/Ljnfr5 mutants, including nodule organogenesis in cortical cells (Rival et al., 2012), suggesting that LjNFR1 expression is required in both epidermal and cortical cells, but LjNFR5 is only necessary in the epidermis. In addition, these two processes can be distinguished via the activation of intracellular calcium spiking and calcium influx. Calcium spiking is essential for nodule organogenesis, whereas calcium influx is more associated with bacterial infection and requires 100-fold higher concentrations of Nod factors compared with the activation of calcium spiking (Shaw & Long, 2003; Miwa et al., 2006). Analysis of Nod factor-triggered calcium influx in nodulation mutants suggested that MtNFP is indispensable to this response, whereas MtLYK3 is not (Morieri et al., 2013). Given that MtNFP, within the LjNFR5 subclade, has an inactive kinase and LysM-mediated signaling typically needs two receptors, it is likely that another, as yet unidentified, kinase-active LysM receptor is required for Nod factor-triggered calcium influx (Fig. 2b).
Nod factor recognition might have evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis Compared with legume–rhizobium symbioses, AM symbioses are more widespread. The fossil record indicates that AM symbioses are > 400 million yr old and occur in almost all land plants (Redecker et al., 2000). In this symbiosis, AM fungi benefit plants through promoting the acquisition of phosphate, nitrogen, sulfur and water from soil (Schmitz & Harrison, 2014). Studies of plant mutants New Phytologist (2014) www.newphytologist.com
New Phytologist established that AM and rhizobial symbioses share some common symbiotic signaling components, including MtDMI2/LjSYMRK (plasma membrane receptor kinase), MtDMI1/LjPOLLUX (a cation ion channel), and MtDMI3/LjCCaMK (calcium- and calmodulin-dependent protein kinase; reviewed in Kouchi et al., 2010; Oldroyd, 2013). The discovery of this common symbiotic pathway led to the hypothesis that rhizobia coopted signaling and cellular pathways from AM symbiosis, and Nod factor recognition evolved from the receptors for AM signaling molecules. Indeed, Nod factor-like LCOs, so-called Myc factors (Fig. 1c), were found to be signaling molecules in AM symbioses (Maillet et al., 2011). Analysis of Myc factor-induced lateral root formation in nodulation mutants indicated that Myc factors also shared most common signaling components with Nod factors, such as DMI1 and DMI2 (Maillet et al., 2011). As might be expected from these results, the Myc factor receptors also belong to the LYK family. This is shown nicely in the report on Parasponia andersonii NFP. Parasponia is a nonlegume able to establish symbiosis with both rhizobia and AM fungi. When a bacterial artificial chromosome library from P. andersonii was screened with an MtNFP probe, one ortholog of MtNFP was found. RNAi-induced silencing of PaNFP strongly inhibited both AM and rhizobial symbiosis (Fig. 2c) (Op den Camp et al., 2011). The Parasponia–rhizobium symbiosis is thought to be relatively young compared with legumes, suggesting that the ancestry of receptors for AM signaling molecules might be the same as Nod factor receptors in legumes. Phylogenetic analysis of MtNFP and MtLYK3 family genes suggests that these genes experienced duplication events during their evolution (Zhang et al., 2007). A likely hypothesis is that one copy maintained the presumed ancestral function as a Myc factor receptor, while in legumes the paralogous gene underwent neofunctionalization to become a Nod factor receptor (De Mita et al., 2014). However, Myc factor might not be the only signaling molecule mediating AM symbiosis. Mtnfp mutant plants are defective in Myc factor-induced lateral root formation (Fig. 2d), but they show normal AM colonization (Amor et al., 2003; Maillet et al., 2011). Recently, Genre et al. (2013) proposed an answer to this apparent conundrum by showing that short-chain chitooligosaccharides (chitotetraose and chitopentaose), devoid of the acyl substitution, support AM colonization by a mechanism independent of MtNFP. These chitooligosaccharides were found in large abundance (nM) in AM exudates, in contrast to Myc factors, which appear to be present in very low concentrations (Maillet et al., 2011). The addition of these short-chain chitooligosaccharides could trigger calcium spiking identical to that induced by inoculation with the AM fungus. Taken at face value, the findings of Maillet et al. (2011) and Genre et al. (2013) suggest that the Myc factor acts through MtNFP to activate lateral root branching, but it is the action of the chitooligosaccharide signals that actually mediates AM fungal colonization. This is a rather confusing picture that requires further research for clarification. However, this situation is very similar to various studies on the response of legumes to both Nod factor and chitooligosaccharides, with the latter shown to induce intracellular calcium flux (Walker et al., 2000) and to induce expression of nodulation-associated genes (Minami et al., 1996) dependent on chemistry and concentration. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist LCO recognition might have evolved from plant– fungal pathogen interaction Both Nod factors and Myc factors are recognized by plants in support of a beneficial symbiosis. In sharp contrast, unsubstituted chitooligosaccharides (Fig. 1a, dp = 6–8) are recognized as a microbe-associated molecular pattern (MAMP) by plants, leading to the induction of innate immunity (so-called MAMP-triggered immunity, MTI). The structural similarity between Nod/Myc factors and chitin MAMPs raises the question of how these similar molecules could induce apparently opposite responses in plants; that is, one supporting symbiotic infection while the other defends against pathogen infection. MAMPs are usually conserved structures among different classes of microbes but are distinct from host molecules. Examples include fungal chitin, bacterial flagellin, elongation factor Tu, PGN and lipopolysaccharides (Macho & Zipfel, 2014). MTI is the first line of immunity and plays an important role in the plant resistance against various pathogens (Boller & Felix, 2009). A typical MTI response includes calcium influx, elevation of reactive oxygen species, induction of defenserelated gene expression and some longer-term responses, such as callose deposition and pathogen growth restriction (Boller & Felix, 2009). MAMPs are recognized by plant pattern recognition receptors (PRRs). For example, PGN and chitooligosaccharides are recognized by LysM receptors (Antolin-Llovera et al., 2012). In order to avoid MTI, pathogens evolved effector proteins that are directly delivered into the plant cell, such as through the action of a pathogen type III protein secretion system (Jones & Dangl, 2006; Feng & Zhou, 2012). Plant resistance (R) genes recognize these pathogen effector proteins, leading to increased pathogen resistance. This form of resistance is termed effector-triggered immunity (ETI). The plant ETI response is usually stronger than MTI, but as it is based on a single R gene it can be easily overcome by genetic adaptation of the pathogen. Therefore, in an agricultural setting, MTI may be of greater importance because it is broader and multigenic, making it more difficult for the pathogen to overcome (Zhang & Zhou, 2010). Plants do not produce chitin but have chitinases that can hydrolyze chitin found in fungal hyphal tips (Punja & Zhang, 1993). Presumably these chitinases release long-chain chitooligosaccharides (dp = 6–8 GlcNAc) that are necessary to elicit plant innate immunity (Stacey & Shibuya, 1997; Shibuya & Minami, 2001). Some fungal pathogens produce effectors, such as LysM Protein1 (Slp1) from rice blast fungus Magnaporthe oryzae (Mentlak et al., 2012) and ECP6 from tomato leaf mold fungus Cladosporium fulvum (de Jonge et al., 2010), that compete for chitin binding with the plant chitin receptor. Slp1 and ECP6 show very high affinity for chitin binding and presumably sequester the chitooligosaccharides at the fungal–plant interface so that they are not recognized by the plant (de Jonge et al., 2010; Mentlak et al., 2012; Sanchez-Vallet et al., 2013). Recognition of long-chain chitooligosaccharides (dp = 6–8) has been well studied in Arabidopsis and rice. Given the previous discussion, it should not come as a surprise that plant chitin receptors are also found within the LysM protein family. The first chitin receptor was identified by cloning the rice chitin Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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elicitor-binding protein (OsCEBiP) isolated as a result of its ability to bind directly to chitin (Kaku et al., 2006). OsCEBiP encodes a LysM receptor protein without an intracellular kinase domain. It now seems clear that OsCEBiP functions by forming a heterodimer with OsCERK1 (chitin elicitor receptor kinase 1) in the presence of chitin, leading to activation of the OsCERK1 intracellular kinase domain (Shimizu et al., 2010). Recent data suggest that OsCEBiP binds chitin as a dimer in which one long-chain chitooligosaccharide is sandwiched between two monomers (Fig. 2e) (Hayafune et al., 2014). These data suggest that the functional, chitin-bound OsCEBiP–OsCERK1 complex functions as a heterotetramer, although this awaits biochemical confirmation (Fig. 2e). Chitin recognition in Arabidopsis appears to be somewhat different from the case in rice. The prevalent model is that AtCERK1, the ortholog of OsCERK1, is essential for chitininduced plant innate immunity (Miya et al., 2007; Wan et al., 2008). However, the crystal structure of AtCERK1 revealed that the chitin binding site is located in the central domain of the extracellular region, with long-chain chitooligosaccharides acting as a bivalent ligand to promote receptor homodimerization (Liu et al., 2012b), leading to activation of the intracellular kinase domain (Fig. 2f). In addition to AtCERK1, other LysM proteins, including AtLYK4, AtLYK5, and AtLYM2, are able to bind chitin, as demonstrated by their precipitation using chitin beads (Petutschnig et al., 2010; Wan et al., 2012). Indeed, Atlyk4 mutants showed a reduced response to chitin but not to the extent shown by Atcerk1 mutants (Fig. 2g) (Wan et al., 2012). AtLYM2, the Arabidopsis homolog of the rice OsCEBiP protein, is not required for chitin-triggered innate immunity (Shinya et al., 2012; Wan et al., 2012). However, LYM2 was recently shown to be necessary for chitin-induced suppression of intracellular flux through plasmodesmata (Fig. 2h), a response that does not require AtCERK1 (Faulkner et al., 2013; Narusaka et al., 2013). Therefore, both rice and Arabidopsis employ CERK1 but differ in the identity of the coreceptor, with OsCEBiP filling this role in rice and perhaps AtLYK4 filling the role in Arabidopsis (Fig. 2g). However, in both rice and Arabidopsis, the functional receptor appears to require a functional kinase (CERK1) with a coreceptor that lacks intracellular kinase function, either because of the lack of an intracellular kinase domain (OsCEBiP) or because of mutations that inactivate the kinase (AtLYK4). In addition to Nod factors, Myc factors and chitin, LysM receptors also recognize another GlcNAc-containing ligand, PGN (Fig. 1d), the major chemical component of the bacterial cell wall. In Arabidopsis, AtLYM1 and AtLYM3 bind directly to PGN. AtCERK1 is required for PGN-triggered innate immunity but does not bind PGN (Fig. 2I; Willmann et al., 2011). In rice, orthologs of AtLYM1 and AtLYM3, OsLYP4 and OsLYP6, can recognize both PGN and chitin, and play a dual role in PGN- and chitin-triggered innate immunity (Fig. 2j) (Liu et al., 2012a). To date, there have been no reports implicating a role for OsCERK1 in PGN signaling in rice. Phylogenetic analysis indicates that CERK1 is within the LjNFR1 family, indicating that LCO, PGN and chitin recognition clearly have a single evolutionary origin. Indeed, transgenic expression of a chimeric protein with the LjNFR1 extracellular domain fused to the AtCERK1 kinase domain, slightly modified to New Phytologist (2014) www.newphytologist.com
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match the LjNFR1 kinase domain more closely, was shown to rescue the Ljnfr1 mutant nodulation phenotype (Nakagawa et al., 2011). Therefore, conservation of structure between the various LYKs appears to extend to the degree of kinase specificity. Consistent with this notion, there are a number of reports that implicate Nod factors and Nod factor receptors in plant innate immunity. For example, in M. truncatula, Mtnfp mutants were shown to be more susceptible to the fungi Verticillium albo-atrum (Ben et al., 2012) and Colletotrichum trifolii, and also to the oomycete Aphanomyces euteiches (Rey et al., 2013). Therefore, these authors proposed that MtNFP has dual roles in both symbiosis and innate immunity (Fig. 2k). Coexpression of LjNFR1 and LjNFR5, or MtNFP and MtLYK3, in Nicotiana benthamiana caused a celldeath response in the absence of Nod factors, resembling the effects of AtCERK1 overexpression (Nakagawa et al., 2011; Pietraszewska-Bogiel et al., 2013). In addition, the application of purified Nod factors (5 lM) was shown to induce culture acidification (a typical MTI response) in suspension-cultured soybean cell (Day et al., 2001) and induce the production of reactive oxygen species in Arabidopsis with very high concentrations of Nod factors (100 lg ml 1, or 60 lM) (Wang et al., 2014). However, such high concentrations are unlikely to be found at the in planta site of rhizobial infection. Understandably, previous research on Nod factors focused exclusively on their role as symbiotic signals necessary for rhizobial infection. However, Mitra & Long (2004) suggested that Nod factors could also function to suppress the expression of pathogen defense-related genes in legumes. It should be noted that current dogma supports the notion that only legumes recognize Nod factors and this may be one of the important reasons why nonlegumes cannot be nodulated by rhizobia (Beatty & Good, 2011). However, given our discussion earlier concerning the similarity between chitin and Nod factor recognition, it seems surprising that nonlegumes cannot recognize Nod factors, at least when presented in sufficient concentration. Recently, Liang et al. (2013) found that the ability of Nod factors to suppress plant innate immunity is conserved in both legumes and nonlegumes. Indeed, this response was found in soybean, Arabidopsis, corn and tomato upon the addition of nM concentrations of Nod factors, a concentration equivalent to that needed for symbiotic responses in legumes (Lerouge et al., 1990). Short-chain chitooligosaccharides (dp = 4–5) were also capable of suppressing innate immunity responses but required a concentration 100-fold higher than the active Nod factor concentration. Nod factor suppression of innate immunity was independent of the Nod factor receptor genes (NFR1 and NFR5) when tested in soybean. However, an examination of mutants defective in each of the five LYK genes in Arabidopsis showed that Atlyk3 mutant plants did not respond to Nod factor, suggesting that this protein is the Nod factor receptor. However, this suggestion requires further biochemical testing (Liang et al., 2013). Similar to AtCERK1, AtLYK3 has an active, intracellular kinase domain but only has a single conserved LysM domain in the extracellular region. Previous analysis demonstrated that a single LysM domain was capable of binding to chitotetraose (Ohnuma et al., 2008). Therefore, the presence of a single LysM domain in AtLYK3 may explain the specificity for short-chain Nod factors and chitooligosacharides. Liang et al. New Phytologist (2014) www.newphytologist.com
(2013) suggested a model by which Nod factors are recognized by AtLYK3, resulting in activation of the intracellular kinase domain (Fig. 2l), ultimately leading to a suppression of innate immunity. Consistent with the negative role of AtLYK3 in plant innate immunity, Atlyk3 mutant plants are more resistant to the fungal pathogens Botrytis cinerea and Pectobacterium carotovorum (Paparella et al., 2014). Although not directly tested, it is interesting to speculate that fungal pathogens may seek to counter chitin MTI by further hydrolyzing chitooligosacharides to produce short-chain molecules that can suppress innate immunity.
LCO recognition: an ancient story Phylogenetic analysis of the LYK family clearly indicates that Myc factor, Nod factor and chitooligosaccharide recognition share a common origin (Zhang et al., 2007). The fossil record suggests that AM symbioses originated > 400 million yr ago (Redecker et al., 2000). Therefore, specialists in symbiosis have tended to view this symbiosis as predating that of the rhizobium–legume interaction. The discovery of the common symbiotic pathway, essential for both symbioses, underscores their evolutionary connection. Although the origin of plant–pathogen interaction has not been documented, the fossil record suggests that some parasitic fungi, chytridiomycetes, were found within cells of algae c. 400 million yr ago (Taylor & Taylor, 1997). Since algae do not form symbiosis with AM fungi, it seems reasonable to assume that plants interacted with pathogenic fungi before the complex mycorrhizal symbiosis evolved. In addition, the LysM domain for recognizing the GlcNAc-containing molecules is an ancient motif that is present in the algal lineages that predate emergence of higher plants (Zhang et al., 2007). LysM domains are involved in both bacterial PGN and fungal chitooligosaccharide signaling (Buist et al., 2008). Therefore, we hypothesize that it is this LysM domain function that predates that of Myc factor recognition. An intriguing suggestion is that this system first evolved to function in MTI. Later microbes adapted to use these signaling systems to recognize molecules that could suppress MTI. Based on the work of Liang et al. (2013), this function is broadly conserved among both dicots and monocots. It is quite possible that the ability to suppress innate immunity was the first function for Nod factors and Myc factors, with further coevolution leading to a more profound developmental role for these molecules. This was probably driven by selection for greater and greater efficiency and specificity (Madsen et al., 2010). Indeed, it is now clear that in specific cases, rhizobia can nodulate legumes without Nod factors (Giraud et al., 2007; Okazaki et al., 2013). However, in the case of soybean at least, mutants that lack the ability to recognize the Nod factor symbiotic signal can still suppress innate immunity upon Nod factor application (Liang et al., 2013).
Acknowledgements The work in the authors’ laboratory was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy (grant no. DEFG02-08ER15309) and the Next-Generation BioGreen 21 Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist Program Systems and Synthetic Agrobiotech Center, Rural Development Administration, Republic of Korea (grant no. PJ009068).
References Amor BB, Shaw SL, Oldroyd GE, Maillet F, Penmetsa RV, Cook D, Long SR, Denarie J, Gough C. 2003. The NFP locus of Medicago truncatula controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation. Plant Journal 34: 495–506. Antolin-Llovera M, Ried MK, Binder A, Parniske M. 2012. Receptor kinase signaling pathways in plant-microbe interactions. Annual Review Phytopathology 50: 451–473. Arrighi JF, Barre A, Ben Amor B, Bersoult A, Soriano LC, Mirabella R, de Carvalho-Niebel F, Journet EP, Gherardi M, Huguet T et al. 2006. The Medicago truncatula lysin motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes. Plant Physiology 142: 265–279. Beatty PH, Good AG. 2011. Plant science. Future prospects for cereals that fix nitrogen. Science 333: 416–417. Ben C, Toueni M, Montanari S, Tardin MC, Fervel M, Negahi A, Saint-Pierre L, Mathieu G, Gras MC, Noel D et al. 2012. Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt. Journal of Experimental Botany 64: 317–332. Bensmihen S, de Billy F, Gough C. 2011. Contribution of NFP LysM domains to the recognition of Nod factors during the Medicago truncatula/Sinorhizobium meliloti symbiosis. PLoS ONE 6: e26114. Bohlool BB, Ladha JK, Garrity DP, Geogre T. 1992. Biological nitrogen fixation for sustainable agriculture: a perspective. Plant and Soil 141: 1–11. Boller T, Felix G. 2009. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual Review of Plant Biology 60: 379–406. Broghammer A, Krusell L, Blaise M, Sauer J, Sullivan JT, Maolanon N, Vinther M, Lorentzen A, Madsen EB, Jensen KJ et al. 2012. Legume receptors perceive the rhizobial lipochitin-oligosaccharide signal molecules by direct binding. Proceedings of the National Academy of Sciences, USA 109: 13859–13864. Buist G, Steen A, Kok J, Kuipers OP. 2008. LysM, a widely distributed protein motif for binding to (peptido)glycans. Molecular Microbiology 68: 838–847. Carlson RW, Sanjuan J, Bhat UR, Glushka J, Spaink HP, Wijfjes AH, van Brussel AA, Stokkermans TJ, Peters NK, Stacey G. 1993. The structures and biological activities of the lipo-oligosaccharide nodulation signals produced by type I and II strains of Bradyrhizobium japonicum. Journal of Biology Chemistry 268: 18372– 18381. Charpentier M, Oldroyd G. 2010. How close are we to nitrogen-fixing cereals? Current Opinion in Plant Biology 13: 556–564. Day RB, Okada M, Ito Y, Tsukada K, Zaghouani H, Shibuya N, Stacey G. 2001. Binding site for chitin oligosaccharides in the soybean plasma membrane. Plant Physiology 126: 1162–1173. De Mita S, Streng A, Bisseling T, Geurts R. 2014. Evolution of a symbiotic receptor through gene duplications in the legume–rhizobium mutualism. New Phytologist 201: 961–972. Denarie J, Debelle F, Prome JC. 1996. Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Annual Review of Biochemistry 65: 503–535. D’Haeze W, Holsters M. 2002. Nod factor structures, responses, and perception during initiation of nodule development. Glycobiology 12: 79R–105R. Faulkner C, Petutschnig E, Benitez-Alfonso Y, Beck M, Robatzek S, Lipka V, Maule AJ. 2013. LYM2-dependent chitin perception limits molecular flux via plasmodesmata. Proceedings of the National Academy of Sciences, USA 110: 9166– 9170. Feng F, Zhou JM. 2012. Plant-bacterial pathogen interactions mediated by type III effectors. Current Opinion in Plant Biology 15: 469–476. Ferguson BJ, Indrasumunar A, Hayashi S, Lin MH, Lin YH, Reid DE, Gresshoff PM. 2010. Molecular analysis of legume nodule development and autoregulation. Journal of Integrative Plant Biology 52: 61–76. Genre A, Chabaud M, Balzergue C, Puech-Pages V, Novero M, Rey T, Fournier J, Rochange S, Becard G, Bonfante P et al. 2013. Short-chain chitin oligomers Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
Research review Lipochitooligosaccharide recognition: an ancient story Author for correspondence: Gary Stacey Tel: +1 5738844752 Email: [email protected]
Yan Liang, Katalin T oth, Yangrong Cao, Kiwamu Tanaka, Catherine Espinoza and Gary Stacey
Received: 31 March 2014 Accepted: 18 May 2014
University of Missouri, Columbia, MO 65211, USA
Divisions of Plant Science and Biochemistry, National Center for Soybean Biotechnology, Christopher S. Bond Life Sciences Center,
Summary New Phytologist (2014) doi: 10.1111/nph.12898
Key words: arbuscular mycorrhizal (AM) symbiosis, innate immunity, lipochitooligosaccharides (LCOs), Myc factors, Nod factors, nodulation, peptidoglycan.
Chitin is the second most abundant polysaccharide in nature, found in crustacean shells, insect exoskeletons and fungal cell walls. The action of chitin and chitin derivatives on plants has become a very interesting story of late. Chitin is a b1-4-linked polymer of N-acetyl-Dglucosamine (GlcNAc). In this unmodified form, chitooligosaccharides (degree of polymerization (dp) = 6–8)) are strong inducers of plant innate immunity. By contrast, when these chitooligosaccharides are acylated (so-called lipochitooligosaccharides, LCOs) and further modified, they can act as Nod factors, the key signaling molecules that play an important role in the initiation of the legume–rhizobium symbiosis. In a similar form, these molecules can also act as Myc factors, the key signaling molecules involved in the arbuscular mycorrhizal (AM) symbiosis. It has been proposed that Nod factor perception might have evolved from the more ancient AM symbiosis. Increasing evidence now suggests that LCO perception might have evolved from plant innate immunity signaling. In this review, we will discuss the evolutionary origin of symbiotic LCO recognition.
Introduction The legume–rhizobium symbiosis is a major contributor to biological nitrogen fixation and, therefore, to the global nitrogen cycle (Bohlool et al., 1992). During the symbiosis, legume roots form a novel organ, the nodule, in which the bacteria fix atmospheric dinitrogen and convert it into ammonia that can be utilized by the host plants. Over the past few decades, the legume–rhizobium symbiosis has been of intense interest to researchers, as this nodulation ability offers the potential to reduce the use of fossil fuel-derived chemical fertilizer, improve soil health and mitigate negative environmental effects resulting from agricultural practices. However, nodulation ability is mostly restricted to the family of leguminous plants (Fabaceae) and it would be a huge challenge to transfer this ability to nonlegumes (Charpentier & Oldroyd, 2010; Oldroyd & Dixon, 2014; Rogers & Oldroyd, 2014). Formation of functional legume root nodules requires two separate but tightly coordinated developmental processes: nodule organogenesis and bacterial infection. In most legumes, nodule formation is initiated by rhizobial attachment to root Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
hair tips, stimulating subsequent cortical cell redifferentiation and root hair curling. Cortical cell redifferentiation gives rise to a nodule primordium, leading to the eventual development of a mature nodule. Curling of the root hair entraps bacteria, leading to the subsequent formation of a plant cell wall-derived infection thread. The infection thread delivers the bacteria from the root hair into the newly formed nodule primordium, where the infection thread branches and bacteria are released by exocytosis. Inside the nodule, the bacteria differentiate into their endosymbiotic form, bacteroids, which fix dinitrogen into ammonium (Geurts et al., 2005; Oldroyd & Downie, 2008; Ferguson et al., 2010). The key step in establishing the legume–rhizobium symbiosis is the plant recognition of bacterial signal molecules, Nod factors (Oldroyd et al., 2011). The longstanding belief is that nonlegumes cannot form nodules in response to rhizobia because of their inability to recognize Nod factors (Beatty & Good, 2011). In this review, we focus on new information regarding the recognition of Nod factors by legumes and nonlegumes, as well as structurally similar molecules, such as Myc factors, chitin and peptidoglycan (PGN). New Phytologist (2014) 1 www.newphytologist.com
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polymerization (dp) = 3–5) with various substitutions on the reducing and nonreducing ends, defined as lipochitooligosaccharides (LCOs). Fig. 1(a) shows the unmodified chitooligosaccharides, and Fig. 1(b) shows the representative Nod factor (Nod Bj V C18:1Δ11, MeFuc) produced by Bradyrhizobium
Nod factor recognition Nod factor structures have been extensively reviewed (Stacey et al., 1992; Denarie et al., 1996; D’Haeze & Holsters, 2002). Briefly, Nod factors are short chitooligosaccharides (degree of OH
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Fig. 1 Structure of chitin, representative Nod factor, Myc factor and peptidoglycan. (a) Chitin, a polymer of N-acetylglucosamine. The acetyl moiety (red) is important for chitinreceptor binding. (b) Nod factor (Nod Bj V C18:1Δ11, MeFuc) produced by Bradyrhizobium japonicum. The substitutions of chitin backbone are in red. (c) Myc factor (LCO-IV C18:1 Δ9) produced by Glomus intraradices. (d) Monomer of peptidoglycan produced by Escherichia coli. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist japonicum (Sanjuan et al., 1992). The Nod factor nonreducing end is N-acylated by a fatty acid, and the reducing end is modified by 2-O-methylfucose (Sanjuan et al., 1992). Nod factors are structurally diverse and it is generally thought that plant host specificity is determined by the specific chemistry of the signal produced by the compatible rhizobial symbiont. For example, different rhizobia produce Nod factors that differ in chitin chain length, specific fatty acylation, sulfation, acetylation, etc. (Lerouge et al., 1990; Carlson et al., 1993; Staehelin et al., 2000). Knowledge of the chemistry of the Nod factors immediately raised the question as to how legumes recognize this molecule. This led to studies of plant mutants in both Medicago truncatula and (a)
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Lotus japonicus, which were found to be insensitive to Nod factor addition. The cloning of the underlying genes in these mutants identified two plasma membrane Nod factor receptors, termed Nod factor receptor 1 (LjNFR1) and LjNFR5, in Lotus japonicus (Madsen et al., 2003; Radutoiu et al., 2003) and MtLYK3 and MtNFP in Medicago truncatula (Fig. 2a,b; Limpens et al., 2003; Arrighi et al., 2006). These Nod factor receptors belong to the lysin motif (LysM)-containing receptor-like kinase (LYKs) family, consisting of an extracellular domain with one to three LysM motifs (ancient and conserved 40 amino acid motif), a transmembrane domain and an intracellular serine/threonine kinase domain. However, the lack of critical amino acids within the LjNFR5/ MtNFP clade indicates that these proteins lack kinase activity (b)
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Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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Fig. 2 Model for Nod factor, Myc factor, chitin and peptidoglycan recognition. (a) Nod factors are recognized by LjNFR1/MtLYK3 and LjNFR5/MtNFP in nodule organogenesis in both Lotus japonicus and Medicago truncatula. (b) LjNFR5/MtNFP is essential for Nod factor recognition in bacterial infection in both L. japonicus and M. truncatula. (c) PaNFP is required for both rhizobial and arbuscular mycorrhizal (AM) symbiosis in Parasponia andersonii. (d) MtNFP is involved in Myc factor stimulation of lateral root growth in M. truncatula. (e) Long-chain chitooligosaccharides (degree of polymerization (dp) = 6–8) are recognized by OsCERK1 and OsCEBiP, leading to the immune response in rice. (f) Long-chain chitooligosaccharides (dp = 6–8) are recognized by AtCERK1, leading to the immune response in Arabidopsis. (g) AtLYK4 is involved in chitooligosaccharide (dp = 6–8)mediated immune response in Arabidopsis. (h) AtLYM2 is involved in chitooligosaccharide (dp = 6–8)-induced suppression of molecular flux in plasmodesmata. (i) Peptidoglycan is recognized by AtLYM1/AtLYM3 and AtCERK1, leading to the immune response in Arabidopsis. (j) Peptidoglycan is recognized by OsLYP4/OsLYP6, leading to the immune response in rice. (k) MtNFP is involved in plant immune response in M. truncatula, but the ligand is still unknown. (l) AtLYK3 is required for immunosuppression of Nod factors and short-chain chitooligosaccharides (dp = 4–5) in Arabidopsis. The inactive kinase activity is labeled as X. Conserved LysM domains are shown in color, while those less conserved (perhaps nonfunctional) are shown in white. MAMP, microbe-associated molecular pattern.
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(Zhang et al., 2007; Lohmann et al., 2010). In plants, LYKs and another group of LysM-containing receptor proteins (LYM, lacking the intracellular kinase domain) are important membrane proteins for recognition of GlcNAc-containing molecules, such as PGN, chitin, and LCOs (Buist et al., 2008; Antolin-Llovera et al., 2012). Transformation of LjNFR1 and LjNFR5 into nonhost M. truncatula and L. filicaulis was shown to confer the ability to recognize the Nod factors produced by Mesorhizobium loti, the compatible symbiont of L. japonicus (Radutoiu et al., 2007). Point mutations in LjNFR5 and experiments that swapped the extracellular domains between MtNFP and the ortholog of NFP in pea suggest that the second LysM domain of LjNFR5/MtNFP is the critical binding region for Nod factor recognition (Radutoiu et al., 2007; Bensmihen et al., 2011). Measurements of binding affinity by surface plasmon resonance or thermophoresis indicates that LjNFR1 and LjNFR5 bind Nod factor with very high affinity (Kd values in the nanomolar range), which is comparable to the Nod factor concentrations found to elicit physiological responses in legumes (Miwa et al., 2006; Broghammer et al., 2012). These studies suggest that the Nod factor receptors have evolved to possess high binding affinity and chemical specificity for the Nod factors produced by the compatible rhizobia. Although nodule organogenesis (taking place in cortical cells) and bacterial infection (occurring in epidermal cells) are tightly coupled, they can be genetically separated and Nod factor receptors are necessary for both processes (Oldroyd & Downie, 2008; Madsen et al., 2010). However, epidermal expression of LjNFR1 using tissue-specific promoters could not complement Ljnfr1 nodule organogenesis (Hayashi et al., 2014), whereas epidermal expression of MtNFP/LjNFR5 could rescue Mtnfp/Ljnfr5 mutants, including nodule organogenesis in cortical cells (Rival et al., 2012), suggesting that LjNFR1 expression is required in both epidermal and cortical cells, but LjNFR5 is only necessary in the epidermis. In addition, these two processes can be distinguished via the activation of intracellular calcium spiking and calcium influx. Calcium spiking is essential for nodule organogenesis, whereas calcium influx is more associated with bacterial infection and requires 100-fold higher concentrations of Nod factors compared with the activation of calcium spiking (Shaw & Long, 2003; Miwa et al., 2006). Analysis of Nod factor-triggered calcium influx in nodulation mutants suggested that MtNFP is indispensable to this response, whereas MtLYK3 is not (Morieri et al., 2013). Given that MtNFP, within the LjNFR5 subclade, has an inactive kinase and LysM-mediated signaling typically needs two receptors, it is likely that another, as yet unidentified, kinase-active LysM receptor is required for Nod factor-triggered calcium influx (Fig. 2b).
Nod factor recognition might have evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis Compared with legume–rhizobium symbioses, AM symbioses are more widespread. The fossil record indicates that AM symbioses are > 400 million yr old and occur in almost all land plants (Redecker et al., 2000). In this symbiosis, AM fungi benefit plants through promoting the acquisition of phosphate, nitrogen, sulfur and water from soil (Schmitz & Harrison, 2014). Studies of plant mutants New Phytologist (2014) www.newphytologist.com
New Phytologist established that AM and rhizobial symbioses share some common symbiotic signaling components, including MtDMI2/LjSYMRK (plasma membrane receptor kinase), MtDMI1/LjPOLLUX (a cation ion channel), and MtDMI3/LjCCaMK (calcium- and calmodulin-dependent protein kinase; reviewed in Kouchi et al., 2010; Oldroyd, 2013). The discovery of this common symbiotic pathway led to the hypothesis that rhizobia coopted signaling and cellular pathways from AM symbiosis, and Nod factor recognition evolved from the receptors for AM signaling molecules. Indeed, Nod factor-like LCOs, so-called Myc factors (Fig. 1c), were found to be signaling molecules in AM symbioses (Maillet et al., 2011). Analysis of Myc factor-induced lateral root formation in nodulation mutants indicated that Myc factors also shared most common signaling components with Nod factors, such as DMI1 and DMI2 (Maillet et al., 2011). As might be expected from these results, the Myc factor receptors also belong to the LYK family. This is shown nicely in the report on Parasponia andersonii NFP. Parasponia is a nonlegume able to establish symbiosis with both rhizobia and AM fungi. When a bacterial artificial chromosome library from P. andersonii was screened with an MtNFP probe, one ortholog of MtNFP was found. RNAi-induced silencing of PaNFP strongly inhibited both AM and rhizobial symbiosis (Fig. 2c) (Op den Camp et al., 2011). The Parasponia–rhizobium symbiosis is thought to be relatively young compared with legumes, suggesting that the ancestry of receptors for AM signaling molecules might be the same as Nod factor receptors in legumes. Phylogenetic analysis of MtNFP and MtLYK3 family genes suggests that these genes experienced duplication events during their evolution (Zhang et al., 2007). A likely hypothesis is that one copy maintained the presumed ancestral function as a Myc factor receptor, while in legumes the paralogous gene underwent neofunctionalization to become a Nod factor receptor (De Mita et al., 2014). However, Myc factor might not be the only signaling molecule mediating AM symbiosis. Mtnfp mutant plants are defective in Myc factor-induced lateral root formation (Fig. 2d), but they show normal AM colonization (Amor et al., 2003; Maillet et al., 2011). Recently, Genre et al. (2013) proposed an answer to this apparent conundrum by showing that short-chain chitooligosaccharides (chitotetraose and chitopentaose), devoid of the acyl substitution, support AM colonization by a mechanism independent of MtNFP. These chitooligosaccharides were found in large abundance (nM) in AM exudates, in contrast to Myc factors, which appear to be present in very low concentrations (Maillet et al., 2011). The addition of these short-chain chitooligosaccharides could trigger calcium spiking identical to that induced by inoculation with the AM fungus. Taken at face value, the findings of Maillet et al. (2011) and Genre et al. (2013) suggest that the Myc factor acts through MtNFP to activate lateral root branching, but it is the action of the chitooligosaccharide signals that actually mediates AM fungal colonization. This is a rather confusing picture that requires further research for clarification. However, this situation is very similar to various studies on the response of legumes to both Nod factor and chitooligosaccharides, with the latter shown to induce intracellular calcium flux (Walker et al., 2000) and to induce expression of nodulation-associated genes (Minami et al., 1996) dependent on chemistry and concentration. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist LCO recognition might have evolved from plant– fungal pathogen interaction Both Nod factors and Myc factors are recognized by plants in support of a beneficial symbiosis. In sharp contrast, unsubstituted chitooligosaccharides (Fig. 1a, dp = 6–8) are recognized as a microbe-associated molecular pattern (MAMP) by plants, leading to the induction of innate immunity (so-called MAMP-triggered immunity, MTI). The structural similarity between Nod/Myc factors and chitin MAMPs raises the question of how these similar molecules could induce apparently opposite responses in plants; that is, one supporting symbiotic infection while the other defends against pathogen infection. MAMPs are usually conserved structures among different classes of microbes but are distinct from host molecules. Examples include fungal chitin, bacterial flagellin, elongation factor Tu, PGN and lipopolysaccharides (Macho & Zipfel, 2014). MTI is the first line of immunity and plays an important role in the plant resistance against various pathogens (Boller & Felix, 2009). A typical MTI response includes calcium influx, elevation of reactive oxygen species, induction of defenserelated gene expression and some longer-term responses, such as callose deposition and pathogen growth restriction (Boller & Felix, 2009). MAMPs are recognized by plant pattern recognition receptors (PRRs). For example, PGN and chitooligosaccharides are recognized by LysM receptors (Antolin-Llovera et al., 2012). In order to avoid MTI, pathogens evolved effector proteins that are directly delivered into the plant cell, such as through the action of a pathogen type III protein secretion system (Jones & Dangl, 2006; Feng & Zhou, 2012). Plant resistance (R) genes recognize these pathogen effector proteins, leading to increased pathogen resistance. This form of resistance is termed effector-triggered immunity (ETI). The plant ETI response is usually stronger than MTI, but as it is based on a single R gene it can be easily overcome by genetic adaptation of the pathogen. Therefore, in an agricultural setting, MTI may be of greater importance because it is broader and multigenic, making it more difficult for the pathogen to overcome (Zhang & Zhou, 2010). Plants do not produce chitin but have chitinases that can hydrolyze chitin found in fungal hyphal tips (Punja & Zhang, 1993). Presumably these chitinases release long-chain chitooligosaccharides (dp = 6–8 GlcNAc) that are necessary to elicit plant innate immunity (Stacey & Shibuya, 1997; Shibuya & Minami, 2001). Some fungal pathogens produce effectors, such as LysM Protein1 (Slp1) from rice blast fungus Magnaporthe oryzae (Mentlak et al., 2012) and ECP6 from tomato leaf mold fungus Cladosporium fulvum (de Jonge et al., 2010), that compete for chitin binding with the plant chitin receptor. Slp1 and ECP6 show very high affinity for chitin binding and presumably sequester the chitooligosaccharides at the fungal–plant interface so that they are not recognized by the plant (de Jonge et al., 2010; Mentlak et al., 2012; Sanchez-Vallet et al., 2013). Recognition of long-chain chitooligosaccharides (dp = 6–8) has been well studied in Arabidopsis and rice. Given the previous discussion, it should not come as a surprise that plant chitin receptors are also found within the LysM protein family. The first chitin receptor was identified by cloning the rice chitin Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
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elicitor-binding protein (OsCEBiP) isolated as a result of its ability to bind directly to chitin (Kaku et al., 2006). OsCEBiP encodes a LysM receptor protein without an intracellular kinase domain. It now seems clear that OsCEBiP functions by forming a heterodimer with OsCERK1 (chitin elicitor receptor kinase 1) in the presence of chitin, leading to activation of the OsCERK1 intracellular kinase domain (Shimizu et al., 2010). Recent data suggest that OsCEBiP binds chitin as a dimer in which one long-chain chitooligosaccharide is sandwiched between two monomers (Fig. 2e) (Hayafune et al., 2014). These data suggest that the functional, chitin-bound OsCEBiP–OsCERK1 complex functions as a heterotetramer, although this awaits biochemical confirmation (Fig. 2e). Chitin recognition in Arabidopsis appears to be somewhat different from the case in rice. The prevalent model is that AtCERK1, the ortholog of OsCERK1, is essential for chitininduced plant innate immunity (Miya et al., 2007; Wan et al., 2008). However, the crystal structure of AtCERK1 revealed that the chitin binding site is located in the central domain of the extracellular region, with long-chain chitooligosaccharides acting as a bivalent ligand to promote receptor homodimerization (Liu et al., 2012b), leading to activation of the intracellular kinase domain (Fig. 2f). In addition to AtCERK1, other LysM proteins, including AtLYK4, AtLYK5, and AtLYM2, are able to bind chitin, as demonstrated by their precipitation using chitin beads (Petutschnig et al., 2010; Wan et al., 2012). Indeed, Atlyk4 mutants showed a reduced response to chitin but not to the extent shown by Atcerk1 mutants (Fig. 2g) (Wan et al., 2012). AtLYM2, the Arabidopsis homolog of the rice OsCEBiP protein, is not required for chitin-triggered innate immunity (Shinya et al., 2012; Wan et al., 2012). However, LYM2 was recently shown to be necessary for chitin-induced suppression of intracellular flux through plasmodesmata (Fig. 2h), a response that does not require AtCERK1 (Faulkner et al., 2013; Narusaka et al., 2013). Therefore, both rice and Arabidopsis employ CERK1 but differ in the identity of the coreceptor, with OsCEBiP filling this role in rice and perhaps AtLYK4 filling the role in Arabidopsis (Fig. 2g). However, in both rice and Arabidopsis, the functional receptor appears to require a functional kinase (CERK1) with a coreceptor that lacks intracellular kinase function, either because of the lack of an intracellular kinase domain (OsCEBiP) or because of mutations that inactivate the kinase (AtLYK4). In addition to Nod factors, Myc factors and chitin, LysM receptors also recognize another GlcNAc-containing ligand, PGN (Fig. 1d), the major chemical component of the bacterial cell wall. In Arabidopsis, AtLYM1 and AtLYM3 bind directly to PGN. AtCERK1 is required for PGN-triggered innate immunity but does not bind PGN (Fig. 2I; Willmann et al., 2011). In rice, orthologs of AtLYM1 and AtLYM3, OsLYP4 and OsLYP6, can recognize both PGN and chitin, and play a dual role in PGN- and chitin-triggered innate immunity (Fig. 2j) (Liu et al., 2012a). To date, there have been no reports implicating a role for OsCERK1 in PGN signaling in rice. Phylogenetic analysis indicates that CERK1 is within the LjNFR1 family, indicating that LCO, PGN and chitin recognition clearly have a single evolutionary origin. Indeed, transgenic expression of a chimeric protein with the LjNFR1 extracellular domain fused to the AtCERK1 kinase domain, slightly modified to New Phytologist (2014) www.newphytologist.com
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match the LjNFR1 kinase domain more closely, was shown to rescue the Ljnfr1 mutant nodulation phenotype (Nakagawa et al., 2011). Therefore, conservation of structure between the various LYKs appears to extend to the degree of kinase specificity. Consistent with this notion, there are a number of reports that implicate Nod factors and Nod factor receptors in plant innate immunity. For example, in M. truncatula, Mtnfp mutants were shown to be more susceptible to the fungi Verticillium albo-atrum (Ben et al., 2012) and Colletotrichum trifolii, and also to the oomycete Aphanomyces euteiches (Rey et al., 2013). Therefore, these authors proposed that MtNFP has dual roles in both symbiosis and innate immunity (Fig. 2k). Coexpression of LjNFR1 and LjNFR5, or MtNFP and MtLYK3, in Nicotiana benthamiana caused a celldeath response in the absence of Nod factors, resembling the effects of AtCERK1 overexpression (Nakagawa et al., 2011; Pietraszewska-Bogiel et al., 2013). In addition, the application of purified Nod factors (5 lM) was shown to induce culture acidification (a typical MTI response) in suspension-cultured soybean cell (Day et al., 2001) and induce the production of reactive oxygen species in Arabidopsis with very high concentrations of Nod factors (100 lg ml 1, or 60 lM) (Wang et al., 2014). However, such high concentrations are unlikely to be found at the in planta site of rhizobial infection. Understandably, previous research on Nod factors focused exclusively on their role as symbiotic signals necessary for rhizobial infection. However, Mitra & Long (2004) suggested that Nod factors could also function to suppress the expression of pathogen defense-related genes in legumes. It should be noted that current dogma supports the notion that only legumes recognize Nod factors and this may be one of the important reasons why nonlegumes cannot be nodulated by rhizobia (Beatty & Good, 2011). However, given our discussion earlier concerning the similarity between chitin and Nod factor recognition, it seems surprising that nonlegumes cannot recognize Nod factors, at least when presented in sufficient concentration. Recently, Liang et al. (2013) found that the ability of Nod factors to suppress plant innate immunity is conserved in both legumes and nonlegumes. Indeed, this response was found in soybean, Arabidopsis, corn and tomato upon the addition of nM concentrations of Nod factors, a concentration equivalent to that needed for symbiotic responses in legumes (Lerouge et al., 1990). Short-chain chitooligosaccharides (dp = 4–5) were also capable of suppressing innate immunity responses but required a concentration 100-fold higher than the active Nod factor concentration. Nod factor suppression of innate immunity was independent of the Nod factor receptor genes (NFR1 and NFR5) when tested in soybean. However, an examination of mutants defective in each of the five LYK genes in Arabidopsis showed that Atlyk3 mutant plants did not respond to Nod factor, suggesting that this protein is the Nod factor receptor. However, this suggestion requires further biochemical testing (Liang et al., 2013). Similar to AtCERK1, AtLYK3 has an active, intracellular kinase domain but only has a single conserved LysM domain in the extracellular region. Previous analysis demonstrated that a single LysM domain was capable of binding to chitotetraose (Ohnuma et al., 2008). Therefore, the presence of a single LysM domain in AtLYK3 may explain the specificity for short-chain Nod factors and chitooligosacharides. Liang et al. New Phytologist (2014) www.newphytologist.com
(2013) suggested a model by which Nod factors are recognized by AtLYK3, resulting in activation of the intracellular kinase domain (Fig. 2l), ultimately leading to a suppression of innate immunity. Consistent with the negative role of AtLYK3 in plant innate immunity, Atlyk3 mutant plants are more resistant to the fungal pathogens Botrytis cinerea and Pectobacterium carotovorum (Paparella et al., 2014). Although not directly tested, it is interesting to speculate that fungal pathogens may seek to counter chitin MTI by further hydrolyzing chitooligosacharides to produce short-chain molecules that can suppress innate immunity.
LCO recognition: an ancient story Phylogenetic analysis of the LYK family clearly indicates that Myc factor, Nod factor and chitooligosaccharide recognition share a common origin (Zhang et al., 2007). The fossil record suggests that AM symbioses originated > 400 million yr ago (Redecker et al., 2000). Therefore, specialists in symbiosis have tended to view this symbiosis as predating that of the rhizobium–legume interaction. The discovery of the common symbiotic pathway, essential for both symbioses, underscores their evolutionary connection. Although the origin of plant–pathogen interaction has not been documented, the fossil record suggests that some parasitic fungi, chytridiomycetes, were found within cells of algae c. 400 million yr ago (Taylor & Taylor, 1997). Since algae do not form symbiosis with AM fungi, it seems reasonable to assume that plants interacted with pathogenic fungi before the complex mycorrhizal symbiosis evolved. In addition, the LysM domain for recognizing the GlcNAc-containing molecules is an ancient motif that is present in the algal lineages that predate emergence of higher plants (Zhang et al., 2007). LysM domains are involved in both bacterial PGN and fungal chitooligosaccharide signaling (Buist et al., 2008). Therefore, we hypothesize that it is this LysM domain function that predates that of Myc factor recognition. An intriguing suggestion is that this system first evolved to function in MTI. Later microbes adapted to use these signaling systems to recognize molecules that could suppress MTI. Based on the work of Liang et al. (2013), this function is broadly conserved among both dicots and monocots. It is quite possible that the ability to suppress innate immunity was the first function for Nod factors and Myc factors, with further coevolution leading to a more profound developmental role for these molecules. This was probably driven by selection for greater and greater efficiency and specificity (Madsen et al., 2010). Indeed, it is now clear that in specific cases, rhizobia can nodulate legumes without Nod factors (Giraud et al., 2007; Okazaki et al., 2013). However, in the case of soybean at least, mutants that lack the ability to recognize the Nod factor symbiotic signal can still suppress innate immunity upon Nod factor application (Liang et al., 2013).
Acknowledgements The work in the authors’ laboratory was supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy (grant no. DEFG02-08ER15309) and the Next-Generation BioGreen 21 Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust
New Phytologist Program Systems and Synthetic Agrobiotech Center, Rural Development Administration, Republic of Korea (grant no. PJ009068).
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8 Review
Research review
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New Phytologist (2014) www.newphytologist.com
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