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Arbuscular mycorrhizal (AM) fungi establish mutualistic symbiosis with a wide range of terrestrial plants, including rice. However, the mechanisms underlying the initiation of AM symbiosis are yet to be elucidated, particularly in nonleguminous plants. We previously demonstrated that chitin elicitor receptor kinase 1 (OsCERK1), a lysin motif receptor–like kinase essential for chitin-triggered immunity, also plays a key role in AM symbiosis in rice. However, the mechanisms underlying the regulation of switching between immunity and symbiosis by OsCERK1 are yet to be fully elucidated. SYMBIOSIS RECEPTOR–LIKE KINASE (SYMRK)/DOES NOT MAKE INFECTIONS 2 (DMI2) is a leucine-rich repeat receptor–like kinase associated with both root nodule symbiosis and AM symbiosis in legumes. The homolog of SYMRK in rice, OsSYMRK, has a shorter form than that in legumes because OsSYMRK lacks a malectin-like domain (MLD). The MLD reportedly contributes to symbiosis in Lotus japonicus; however, the contribution of OsSYMRK to AM symbiosis in rice remains unclear. Phylogenetic analyses indicated that the MLD of SYMRK/DMI2 is widely conserved even in mosses and ferns but absent in commelinids, including rice. To understand the function of OsSYMRK, we produced an Ossymrk knockout mutant using genome editing technology. AM colonization was mostly abolished in Ossymrk with a more severe phenotype than Oscerk1. Ca2+ spiking against chitin tetramer was also diminished in Ossymrk. In contrast, comparable defense responses against chitin heptamer to the wild type were observed in Ossymrk. Bimolecular fluorescence complementation studies demonstrating an interaction between OsSYMRK and OsCERK1 indicate that OsSYMRK may play an important role in switching from immunity to symbiosis through the interaction with OsCERK1 in rice.

In rice, the lysin motif (LysM) receptor-like kinase OsCERK1, originally identified as the essential molecule for chitin-triggered immunity, plays a key role in arbuscular mycorrhizal (AM) symbiosis. As we previously reported, although AM colonization was largely repressed at 2 weeks after inoculation (WAI), arbuscules were observed at 5 WAI in oscerk1 mutant. Conversely, most mutant plants that defect the common symbiosis signaling pathway exhibited no arbuscule formation. Concerning the reason for this characteristic phenotype of oscerk1, we speculated that OsRLK10, which is a putative paralog of OsCERK1, may have a redundant function in AM symbiosis. The protein sequences of these two genes are highly conserved and it is estimated that the gene duplication occurred 150 million years ago. Here we demonstrated that OsCERK2/OsRLK10 induced AM colonization and chitin-triggered reactive oxygen species production in oscerk1 knockout mutant as similar to OsCERK1. The oscerk2 mutant showed a slight but significant reduction of AM colonization at 5 WAI, indicating the contribution of OsCERK2 for AM symbiosis. However, the oscerk2;oscerk1 double-knockout mutant produced arbuscules at 5 WAI as similar to the oscerk1 mutant, indicating that the redundancy of OsCERK1 and OsCERK2 did not explain the mycorrhizal colonization in oscerk1 at 5 WAI. These results indicated that OsCERK2 has a potential to regulate both chitin-triggered immunity and AM symbiosis and at least partially contributes to AM symbiosis in rice though the contribution of OsCERK2 appears to be weaker than that of OsCERK1.

Receptor complex formation at the cell surface is a key step to initiate downstream signaling but the contribution of this process for the regulation of the direction of downstream responses is not well understood. In the plant-microbe interactions, while CERK1, an Arabidopsis LysM-RLK, mediates chitin-induced immune responses, NFR1, a Lotus homolog of CERK1, regulates the symbiotic process with rhizobial bacteria through the recognition of Nod factors. Concerning the mechanistic insight of the regulation of such apparently opposite biological responses by the structurally related RLKs, Nakagawa et al. previously showed that the addition of YAQ sequence, conserved in NFR1 and other symbiotic LysM-RLKs, to the kinase domain of CERK1 switched downstream responses from defense to symbiosis using a set of chimeric receptors, NFR1-CERK1s. These results indicated that such a subtle difference in the cytoplasmic domain of LysM-RLKs could determine the direction of host responses from defense to symbiosis. On the other hand, it is still not understood how such structural differences in the cytoplasmic domains determine the direction of host responses. We here analyzed the interaction between chimeric NFR1s and NFR5, a partner receptor of NFR1, by co-immunoprecipitation (Co-IP) of these proteins transiently expressed in Nicotiana benthamiana. These results indicated that the cytoplasmic interaction between the LysM-RLKs is important for the symbiotic receptor complex formation and the YAQ containing region of NFR1 contributes to trigger symbiotic signaling through the successful formation of NFR1/NFR5 complex.

Significance Fungal disease is a major threat to agriculture and consequently human nutrition. Many pathogenic fungi penetrate through stomata. This study reveals that chitin oligosaccharide (CTOS) from fungal cell wall induces stomatal closure through its receptor CERK1, Ca2+, and S-type anion channel SLAC1, which would prevent fungal penetration. It is also shown that conversion of CTOS to chitosan oligosaccharide (CSOS) is a possible fungal strategy to circumvent stomatal immunity. At higher concentration, guard cells perceive CSOS, resulting in guard cell death, which potentially contributes to plant defense halting fungal infection through stomata. The finding of active guard cell death represents a conceptual advance in understanding of stomatal immunity and would guide future research on guard cell–microbe interaction and crop protection., Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca2+, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca2+ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.

Reactive oxygen species generation is one of the most popular index of plant immune responses. Leaf disk assay has been commonly used for MAMP/elicitor-induced ROS analysis by many groups. However, the reproducibility of the leaf disk assay relies on the skills of the people engaged in the experiments and the experiment itself seems not suitable for some plant species, which had a tough leaf structure and lower penetration efficiency of MAMPs/elicitors. In this study, we prepared a handmade leaf cutter to cut out the leaf fragments with uniform size and slits. The use of such fragments obtained by the new leaf cutter as well as the increase of the number of leaf fragments for each experiment improved the reliability and reproducibility of the leaf disk assay. This cutter was also successfully applied to rice leaf disk assay, indicating the applicability to other plant spices.

Lysin motif (LysM) is a carbohydrate-binding modules found in all kingdoms. LysM binds to N-acetylglucosamine-containing molecules such as peptidoglycan, chitin, Nod factor, and Myc factor and is found in peptidoglycan hydrolases, chitinases, and plant pathogen effectors and plant receptor/co-receptor for defense and symbiosis signaling. This chapter describes the synthesis of a nonradioactive chitin ligand, biotinylated chitin octasaccharide, (GlcNAc)

Lysin motif (LysM) is a carbohydrate-binding modules found in all kingdoms. LysM binds to N-acetylglucosamine-containing molecules such as peptidoglycan, chitin, Nod factor, and Myc factor and is found in peptidoglycan hydrolases, chitinases, and plant pathogen effectors and plant receptor/co-receptor for defense and symbiosis signaling. This chapter describes the synthesis of a nonradioactive chitin ligand, biotinylated chitin octasaccharide, (GlcNAc)8-Bio, and its application for the detection and characterization of chitin-binding LysM receptor CEBiP in the microsomal membrane fraction of rice suspension-cultured cells by affinity labeling. We also describe the purification of CEBiP from the plasma membrane of the rice cells by affinity chromatography with the synthesized (GlcNAc)8-APEA-CH-Sepharose as an affinity matrix.

Lysin motif (LysM) receptor-like proteins/kinases (LysM-RLPs/RLKs) are well known to play an important role in the induction of defense or symbiosis signaling through the recognition of carbohydrate ligands in plants. Chitin elicitor receptor kinase 1 (CERK1) is the receptor-like kinase (RLK) essential for chitin-induced defense signaling in Arabidopsis and rice. At/OsCERK1 is also known to be important for peptidoglycan (PGN)-induced defense signaling. To induce chitin and PGN responses, CERK1 forms a receptor complex with corresponding LysM-type receptors such as CEBiP, LYM1/3 or LYP4/6. Furthermore, OsCERK1 in rice also plays a key role in lipopolysaccharide (LPS) signaling, and AtCERK1 in Arabidopsis is involved in unbranched β-glucan signaling. In any case, CERK1 plays a crucial role in the activation of defense signaling by these ligands and is an essential hub-RLK/co-receptor in these systems. After chitin perception, autophosphorylation of the CERK1 kinase domain is essential for inducing immune signaling, and some functionally important phosphorylation sites have been identified. In addition, several CERK1-interacting proteins and their contribution to the downstream signaling have been reported. In particular, phosphorylation of receptor-like cytoplasmic kinases (RLCKs) by CERK1 is important for the activation of chitin signaling. These findings have contributed to our understanding of the early steps of chitin signaling. This review focuses on current knowledge about CERK1-mediated receptor complex formation and subsequent intracellular signaling.

While ligand-induced autophosphorylation of receptor-like kinases (RLKs) is known to be critical for triggering the downstream responses, biochemical mechanism by which each phosphorylation site contributes to the initiation of corresponding signaling cascades is only poorly understood, except the involvement of some phosphorylation sites in the regulation of catalytic activity of these RLKs. In this article, we first confirmed that the phosphorylation of S493 of AtCERK1 is involved in the regulation of chitin-induced defense responses by the complementation of an atcerk1 mutant with AtCERK1(S493A) cDNA. In vitro kinase assay with the heterologously expressed kinase domain of AtCERK1, GST-AtCERK1cyt, showed that the S493A mutation did not affect the autophosphorylation of AtCERK1 itself but diminished the transphosphorylation of downstream signaling components, PBL27 and PUB4. On the other hand, a phosphomimetic mutant, GST-AtCERK1(S493D)cyt, transphosphorylated these substrates as similar to the wild type AtCERK1. These results suggested that the phosphorylation of S493 does not contribute to the regulation of catalytic activity but plays an important role for the transphosphorylation of the downstream signaling components, thus contributing to the initiation of chitin signaling. To our knowledge, it is a novel finding that a specific phosphorylation site contributes to the regulation of transphosphorylation activity of RLKs. Further studies on the structural basis by which S493 phosphorylation contributes to the regulation of transphosphorylation would contribute to the understanding how the ligand-induced autophosphorylation of RLKs properly regulates the downstream signaling.

Perception of microbe‐associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen‐activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho‐signaling transduction pathway from PRR‐mediated pathogen recognition to MAPK activation in plants. We found that the receptor‐like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1‐LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin‐induced MAPK activation and disease resistance to Alternaria brassicicola . PBL27 phosphorylates MAPKKK5 in vitro , which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo . Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin‐induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants. ![][1] Chitin receptor CERK1 transmits immune signals to the intracellular MAPK cascade in plants. This occurs via phosphorylation of MAPKKK5 by the CERK1‐associated kinase PBL27, providing a missing link between pathogen perception and signaling output. [1]: /embed/graphic-1.gif

In legume-specific rhizobial symbiosis, host plants perceive rhizobial signal molecules, Nod factors, by a pair of LysM receptor-like kinases, NFR1/LYK3 and NFR5/NFP, and activate symbiotic responses through the downstream signaling components also required for arbuscular mycorrhizal (AM) symbiosis. Recently, the rice NFR1/LYK3 ortholog, OsCERK1, was shown to play crucial roles for AM symbiosis. On the other hand, the roles of the NFR5/NFP ortholog in rice have not been elucidated, while it has been shown that NFR5/NFP orthologs, Parasponia PaNFR5 and tomato SlRLK10, engage in AM symbiosis. OsCERK1 also triggers immune responses in combination with a receptor partner, OsCEBiP, against fungal or bacterial infection, thus regulating opposite responses against symbiotic and pathogenic microbes. However, it has not been elucidated how OsCERK1 switches these opposite functions. Here, we analyzed the function of the rice NFR5/NFP ortholog, OsNFR5/OsRLK2, as a possible candidate of the OsCERK1 partner for symbiotic signaling. Inoculation of AM fungi induced the expression of OsNFR5 in the rice root, and the chimeric receptor consisting of the extracellular domain of LjNFR5 and the intracellular domain of OsNFR5 complemented the Ljnfr5 mutant for rhizobial symbiosis, indicating that the intracellular kinase domain of OsNFR5 could activate symbiotic signaling in Lotus japonicus. Although these data suggested the possible involvement of OsNFR5 in AM symbiosis, osnfr5 knockout mutants were colonized by AM fungi similar to the wild-type rice. These observations suggested several possibilities including the presence of functionally redundant genes other than OsNFR5 or involvement of novel ligands, which do not require OsNFR5 for recognition.

Plants have the ability to recognize microbe-associated molecular patterns (MAMPs) and initiate various defense responses. Chitin is a representative fungal MAMP that triggers defense signaling in a wide range of plant species. In rice, OsCEBiP and OsCERK1 form a receptor complex and play critical roles in chitin-triggered defense signaling. Recently, we found the formation of a unique sandwich-type dimer of OsCEBiP plays an important role for activation of chitin signaling. We now understand why N -acetyl groups and the longer chitin-oligosaccharides are required for receptor binding. We also found OsCERK1 is a bifunctional molecule acting in defense and also in AM symbiosis.

Cell cultures established from various plant species have been used for a range of physiological and biochemical studies. Homogeneity of cell types and size of clusters in the cell culture often gave a clearer and simpler results compared to those obtained with the whole plant. On the other hand, possible variability of physiological conditions and responsiveness to external stimuli between the cell lines could be problematic for comparative studies. Aiming at combining the usefulness of plant cell culture with the rich information and genetic resources of Arabidopsis, we systemically examined the methods/conditions to establish cell lines for comparative studies, which could be applicable to a variety of genetic resources. Arabidopsis cell lines thus established from the meristem of mature seeds showed reproducible and comparable MAMP responses such as ROS generation and defense-related gene expression. MAMP responses of the cultured cells showed the specificity depending on the presence/absence of the corresponding MAMP receptor. Pharmacological study with a protein kinase inhibitor, K252a, also showed the usefulness of the cell culture for such studies. These results indicated the usefulness of the method to establish Arabidopsis cell lines, which are useful for comparative studies between genetic resources.

Plant cell surface receptor-like kinases (RLKs) mediate the signals from microbe-associated molecular patterns (MAMPs) that induce immune responses. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, is a common MAMP perceived by animals and plants; however, the plant receptors/co-receptors are unknown except for LORE, a bulb-type lectin S-domain RLK (B-lectin SD1-RLK) in Arabidopsis. OsCERK1 is a multifunctional RLK in rice that contains lysin motifs (LysMs) and is essential for the perception of chitin, a fungal MAMP, and peptidoglycan, a bacterial MAMP. Here, we analyzed the relevance of OsCERK1 to LPS perception in rice. Using OsCERK1-knockout mutants (oscerk1), we evaluated hydrogen peroxide (H2 O2 ) production and gene expression after LPS treatment. We also examined the LPS response in knockout mutants for the B-lectin SD1-RLK genes in rice and for all LysM-protein genes in Arabidopsis. Compared with wild-type rice cells, LPS responses in oscerk1 cells were mostly diminished. By contrast, rice lines mutated in either of three B-lectin SD1-RLK genes and Arabidopsis lines mutated in the LysM-protein genes responded normally to LPS. From these results, we conclude that OsCERK1 is an LPS receptor/co-receptor and that the LPS perception systems of rice and Arabidopsis are significantly different.

Plants can detect infecting fungi through the perception of chitin oligosaccharides by lysin motif receptors such as CEBiP and CERK1. A major function of CERK1 seems to be as a signaling molecule in the receptor complex formed with ligand-binding molecules and to activate downstream defense signaling. Fungal pathogens, however, have developed counter strategies to escape from the chitin-mediated detection by using effectors and/or changing their cell walls. Common structural features between chitin and Nod-/Myc-factors and corresponding receptors have suggested the close relationships between the chitin-mediated immunity and rhizobial/arbuscular mycorrhizal symbiosis. The recent discovery of the dual function of OsCERK1 in both plant immunity and mycorrhizal symbiosis sheds new light on the evolutionary relationships between defense and symbiotic systems in plants.

Pattern recognition receptors on the plant cell surface mediate the recognition of microbe-associated molecular patterns, in a process which activates downstream immune signaling. These receptors are plasma membrane-localized kinases which need to be autophosphorylated to activate downstream responses. Perception of attacks from fungi occurs through recognition of chitin, a polymer of an N-acetylglucosamine which is a characteristic component of the cell walls of fungi. This process is regulated in Arabidopsis by chitin elicitor receptor kinase CERK1. A more complex process characterizes rice, in which regulation of chitin perception is operated by a complex composed of OsCERK1, a homolog of CERK1, and the chitin elicitor binding protein OsCEBiP. Recent literature has provided a mechanistic description of the complex regulation of activation of innate immunity in rice and an advance in the structural description of molecular players involved in this process. This review describes the current status of the understanding of molecular events involved in innate immunity activation in rice.

Plants possess the ability to recognize microbe-associated molecular patterns (MAMPs) and PAMPs through the PRRs, and initiate pattern-triggered immunity. MAMPs are derived from cell-envelope components, secreted materials and cytosolic proteins from bacteria, oomycetes or fungi, and some MAMPs play a similar function in the innate immunity in mammals. Chitin is a representative fungal MAMP and triggers defense signaling in a wide range of plant species. The chitin receptors CEBiP and CERK1 on the plasma membrane have LysM (lysin motif) in their ectodomains. These molecules play an important role for the defense responses in rice and Arabidopsis, strictly recognizing the size and acetylated form of chitin oligosaccharides. However, related LysM receptors also play major roles for the signaling in root nodule and arbuscular mycorrhizal symbiosis. This review summarizes current knowledge on the molecular mechanisms of the defense and symbiosis signaling mediated by LysM receptors, including the activation steps of chitin-induced defense signaling downstream of LysM receptors.

The rice lysin-motif (LysM) receptor-like kinase OsCERK1 is now known to have a dual role in both pathogenic and symbiotic interactions. Following the recent discovery that the Oscerk1 mutant is unable to host arbuscular mycorrhizal (AM) fungi, we have examined whether OsCERK1 is directly involved in the perception of the short-chain chitin oligomers (Myc-COs) identified in AM fungal exudates and shown to activate nuclear calcium (Ca(2+) ) spiking in the rice root epidermis. An Oscerk1 knockout mutant expressing the cameleon NLS-YC2.60 was used to monitor nuclear Ca(2+) signaling following root treatment with either crude fungal exudates or purified Myc-COs. Compared with wild-type rice, Ca(2+) spiking responses to AM fungal elicitation were absent in root atrichoblasts of the Oscerk1 mutant. By contrast, rice lines mutated in OsCEBiP, encoding the LysM receptor-like protein which associates with OsCERK1 to perceive chitin elicitors of the host immune defense pathway, responded positively to Myc-COs. These findings provide direct evidence that the bi-functional OsCERK1 plays a central role in perceiving short-chain Myc-CO signals and activating the downstream conserved symbiotic signal transduction pathway.

Plants are constantly exposed to threats from pathogenic microbes and thus developed an innate immune system to protect themselves. On the other hand, many plants also have the ability to establish endosymbiosis with beneficial microbes such as arbuscular mycorrhizal (AM) fungi or rhizobial bacteria, which improves the growth of host plants. How plants evolved these systems managing such opposite plant-microbe interactions is unclear. We show here that knockout (KO) mutants of OsCERK1, a rice receptor kinase essential for chitin signaling, were impaired not only for chitin-triggered defense responses but also for AM symbiosis, indicating the bifunctionality of OsCERK1 in defense and symbiosis. On the other hand, a KO mutant of OsCEBiP, which forms a receptor complex with OsCERK1 and is essential for chitin-triggered immunity, established mycorrhizal symbiosis normally. Therefore, OsCERK1 but not chitin-triggered immunity is required for AM symbiosis. Furthermore, experiments with chimeric receptors showed that the kinase domains of OsCERK1 and homologs from non-leguminous, mycorrhizal plants could trigger nodulation signaling in legume-rhizobium interactions as the kinase domain of Nod factor receptor1 (NFR1), which is essential for triggering the nodulation program in leguminous plants, did. Because leguminous plants are believed to have developed the rhizobial symbiosis on the basis of AM symbiosis, our results suggest that the symbiotic function of ancestral CERK1 in AM symbiosis enabled the molecular evolution to leguminous NFR1 and resulted in the establishment of legume-rhizobia symbiosis. These results also suggest that OsCERK1 and homologs serve as a molecular switch that activates defense or symbiotic responses depending on the infecting microbes.

Recognition of microbe-associated molecular patterns (MAMPs) initiates pattern-triggered immunity in host plants. Pattern recognition receptors (PRRs) and receptor-like cytoplasmic kinases (RLCKs) are the major components required for sensing and transduction of these molecular patterns. However, the regulation of RLCKs by PRRs and their specificity remain obscure. In this study we show that PBL27, an Arabidopsis ortholog of OsRLCK185, is an immediate downstream component of the chitin receptor CERK1 and contributes to the regulation of chitin-induced immunity in Arabidopsis. Knockout of PBL27 resulted in the suppression of several chitin-induced defense responses, including the activation of MPK3/6 and callose deposition as well as in disease resistance against fungal and bacterial infections. On the other hand, the contribution of PBL27 to flg22 signaling appears to be very limited, suggesting that PBL27 selectively regulates defense signaling downstream of specific PRR complexes. In vitro phosphorylation experiments showed that CERK1 preferentially phosphorylated PBL27 in comparison to BIK1, whereas phosphorylation of PBL27 by BAK1 was very low compared with that of BIK1. Thus, the substrate specificity of the signaling receptor-like kinases, CERK1 and BAK1, may determine the preference of downstream RLCKs.

WRKY transcription factors form a large family of plant-specific transcription factors and participate in plant defense responses either as positive or negative regulators. In this study, we comprehensively analyzed the role of one of the group IIa WRKY transcription factors in rice, OsWRKY28, in the regulation of basal defense responses to a compatible race of the rice blast fungus Magnaporthe oryzae, strain Ina86-137. The expression analyses of the group IIa WRKY transcription factors in rice revealed that OsWRKY28, together with OsWRKY71, exhibit an early-induced expression prior to the late-induced expressions of OsWRKY62 and OsWRKY76. The GFP-OsWRKY28 fusion protein localized mainly in the nuclei of onion epidermal cells, and the maltose-binding protein-fused OsWRKY28 recombinant protein specifically bound to W-box elements. A transient reporter gene assay clearly showed that OsWRKY28 functions as a transcriptional repressor. Overexpression of OsWRKY28 in rice plants resulted in enhanced susceptibility to Ina86-137. Finally, transcriptome analysis revealed that the induction of several defense-related genes in the wild type after Ina86-137 infection was counteracted in OsWRKY28-overexpressing rice plants. These results strongly suggest that OsWRKY28 is a negative regulator of basal defense responses against Ina86-137 and acts as a modulator to maintain the responses at an appropriate level by attenuating the activation of defense-related gene expression levels.

Highlight Xyloglucan oligosaccharide metabolism by α-xylosidase impacts xyloglucan remodelling, the mechanical integrity of the primary cell wall of growing tissues, cell expansion, and seed germination., Regulation and maintenance of cell wall physical properties are crucial for plant growth and environmental response. In the germination process, hypocotyl cell expansion and endosperm weakening are prerequisites for dicot seeds to complete germination. We have identified the Arabidopsis mutant thermoinhibition-resistant germination 1 (trg1), which has reduced seed dormancy and insensitivity to unfavourable conditions for germination owing to a loss-of-function mutation of TRG1/XYL1, which encodes an α-xylosidase. Compared to those of wild type, the elongating stem of trg1 showed significantly lower viscoelasticity, and the fruit epidermal cells were longitudinally shorter and horizontally enlarged. Actively growing tissues of trg1 over-accumulated free xyloglucan oligosaccharides (XGOs), and the seed cell wall had xyloglucan with a greatly reduced molecular weight. These observations suggest that XGOs reduce xyloglucan size by serving as an acceptor in transglycosylation and eventually enhancing cell wall loosening. TRG1/XYL1 gene expression was abundant in growing wild-type organs and tissues but relatively low in cells at most actively elongating part of the tissues, suggesting that α-xylosidase contributes to maintaining the mechanical integrity of the primary cell wall in the growing and pre-growing tissues. In germinating seeds of trg1, expression of genes encoding specific abscisic acid and gibberellin metabolism enzymes was altered in accordance with the aberrant germination phenotype. Thus, cell wall integrity could affect seed germination not only directly through the physical properties of the cell wall but also indirectly through the regulation of hormone gene expression.

Induced plant defense responses against insect herbivores are triggered by wounding and/or perception of herbivore elicitors from their oral secretions (OS) and/or saliva. In this study, we analyzed OS isolated from two rice chewing herbivores, Mythimna loreyi and Parnara guttata. Both types of crude OS had substantial elicitor activity in rice cell system that allowed rapid detection of early and late defense responses, i.e. accumulation of reactive oxygen species (ROS) and defense secondary metabolites, respectively. While the OS from M. loreyi contained large amounts of previously reported insect elicitors, fatty acid-amino acid conjugates (FACs), the elicitor-active P. guttata’s OS contained no detectable FACs. Subsequently, elicitor activity associated with the high molecular mass fraction in OS of both herbivores was identified and shown to promote ROS and metabolite accumulations in rice cells. Notably, the application of N-linolenoyl-Gln (FAC) alone had only negligible elicitor activity in rice cells; however, the activity of isolated elicitor fraction was substantially promoted by this FAC. Our results reveal that plants integrate various independent signals associated with their insect attackers to modulate their defense responses and reach maximal fitness in nature.

Historically viewed as primarily functioning in the regulation of gas and water vapor exchange, it is now evident that stomata serve an important role in plant immunity. Indeed, in addition to classically defined functions related to cell architecture and movement, the actin cytoskeleton has emerged as a central component of the plant immune system, underpinning not only processes related to cell shape and movement, but also receptor activation and signaling. Using high resolution quantitative imaging techniques, the temporal and spatial changes in the actin microfilament array during diurnal cycling of stomatal guard cells has revealed a highly orchestrated transition from random arrays to ordered bundled filaments. While recent studies have demonstrated that plant stomata close in response to pathogen infection, an evaluation of stimulus-induced changes in actin cytoskeletal dynamics during immune activation in the guard cell, as well as the relationship of these changes to the function of the actin cytoskeleton and stomatal aperture, remains undefined. In the current study, we employed quantitative cell imaging and hierarchical clustering analyses to define the response of the guard cell actin cytoskeleton to pathogen infection and the elicitation of immune signaling. Using this approach, we demonstrate that stomatal-localized actin filaments respond rapidly, and specifically, to both bacterial phytopathogens and purified pathogen elicitors. Notably, we demonstrate that higher order temporal and spatial changes in the filament array show distinct patterns of organization during immune activation, and that changes in the naïve diurnal oscillations of guard cell actin filaments are perturbed by pathogens, and that these changes parallel pathogen-induced stomatal gating. The data presented herein demonstrate the application of a highly tractable and quantifiable method to assign transitions in actin filament organization to the activation of immune signaling in plants.

Perception of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRRs) triggers various defense responses in plants. This MAMP-triggered immunity plays a major role in the plant resistance against various pathogens. To clarify the molecular basis of the specific recognition of chitin oligosaccharides by the rice PRR, CEBiP (chitin-elicitor binding protein), as well as the formation and activation of the receptor complex, biochemical, NMR spectroscopic, and computational studies were performed. Deletion and domain-swapping experiments showed that the central lysine motif in the ectodomain of CEBiP is essential for the binding of chitin oligosaccharides. Epitope mapping by NMR spectroscopy indicated the preferential binding of longer-chain chitin oligosaccharides, such as heptamer-octamer, to CEBiP, and also the importance of N-acetyl groups for the binding. Molecular modeling/docking studies clarified the molecular interaction between CEBiP and chitin oligosaccharides and indicated the importance of Ile(122) in the central lysine motif region for ligand binding, a notion supported by site-directed mutagenesis. Based on these results, it was indicated that two CEBiP molecules simultaneously bind to one chitin oligosaccharide from the opposite side, resulting in the dimerization of CEBiP. The model was further supported by the observations that the addition of (GlcNAc)(8) induced dimerization of the ectodomain of CEBiP in vitro, and the dimerization and (GlcNAc)(8)-induced reactive oxygen generation were also inhibited by a unique oligosaccharide, (GlcN beta 1,4GlcNAc)(4), which is supposed to have N-acetyl groups only on one side of the molecule. Based on these observations, we proposed a hypothetical model for the ligand-induced activation of a receptor complex, involving both CEBiP and Oryza sativa chitin-elicitor receptor kinase-1.

Pattern recognition receptors on the plant cell surface mediate the recognition of microbe/damage-associated molecular patterns (MAMPs/DAMPs) and activate downstream immune signaling. Autophosphorylation of signaling receptor-like kinases is a critical event for the activation of downstream responses but the function of each phosphorylation site in the regulation of immune signaling is not well understood. In this study, 41 Ser/Thr/Tyr and 15 Ser/Thr residues were identified as in vitro and in vivo autophosphorylation sites of Arabidopsis CERK1, which is essential for chitin signaling. Comprehensive analysis of transgenic plants expressing mutated CERK1 genes for each phosphorylation site in the cerk1-2 background indicated that the phosphorylation of T479 in the activation segment and Y428 located upstream of the catalytic loop is important for the activation of chitin-triggered defense responses. Contribution of the phosphorylation of T573 to the chitin responses was also suggested. In vitro evaluation of kinase activities of mutated kinase domains indicated that the phosphorylation of T479 and T573 is directly involved in the regulation of kinase activity of CERK1 but the phosphorylation of Y428 regulates chitin signaling independently of the regulation of kinase activity. These results indicated that the phosphorylation of specific residues in the kinase domain contributes to the regulation of downstream signaling either through the regulation of kinase activity or the different mechanisms, e.g. regulation of protein-protein interactions.

The structure of the lipooligosaccharide (LOS) from the rice pathogen Xanthomonas oryzae pv. oryzae has been elucidated. The characterization of the core oligosaccharide structure was obtained by the employment of two chemical degradation protocols and by analysis of the products via NMR spectroscopy. The structure of the lipid A portion was achieved by MALDI mass spectrometry analysis on purified lipid A. The LOS from Xanthomonas oryzae pv. oryzae revealed to possess the same core structure of Xanthomonas campestris pv. campestris and interesting novel features on its lipid A domain. The evaluation of the biological activity of both LOS and isolated lipid A was also executed. (C) 2016 Elsevier Ltd. All rights reserved.

It has been well known that oligosaccharides generated from fungal, bacterial, or plant cell walls can induce various defense responses in plant cells. Traditionally they have been called as “elicitors” or “general elicitors” that induce defense responses in a wide range of plant species. These molecules are now classified as representative MAMPs/PAMPs (microbe/pathogen-associated molecular patterns) or DAMPs (damage/danger-associated molecular patterns) and believed to play important roles in plant immunity, as a trigger of the so-called pattern-triggered immunity (PTI). Biological activities of these oligosaccharide elicitors, including structure/function relationships, corresponding receptors, and downstream signaling, have been the subject of intense studies, by which these oligosaccharides became most well-characterized oligosaccharins. In this chapter, present knowledge on the plant receptors of carbohydrate elicitors generated from the cell walls of pathogenic microbes and also host plants is summarized. Mechanism of the activation of downstream signaling is discussed for chitin receptor, as this system has become the most well-characterized oligosaccharin receptor in plant immunity. It is also discussed how pathogenic microbes try to escape from the MAMP-mediated detection of their invasion by host plants.

International audience; The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.

We previously reported that rice plants expressing the chimeric receptor consisting of rice chitin oligosaccharides binding protein (CEBiP) and the intracellular protein kinase region of Xa21, which confers resistance to rice bacterial blight, showed enhanced cellular responses to a chitin elicitor N-acetylchitoheptaose and increased resistance to the rice blast fungus Magnaporthe oryzae. Here, we investigated whether CEBiP fused with another type of receptor-like protein kinase (RLK) also functions as a chimeric receptor. Fusion proteins CRPis consist of CEBiP and the intracellular protein kinase region of a true resistance gene Pi-d2. Transgenic rice expressing a CRPi showed enhanced cellular responses specifically to N-acetylchitoheptaose in cultured cells and increased levels of disease resistance against M. oryzae in plants. These responses depended on the amino acid sequences predicted to be essential for the protein kinase activity of CRPi. The structure of the transmembrane domain in CRPi affected the protein accumulation, cellular responses, and disease resistance in transgenic rice. These results suggest that the chimeric receptor consisting of CEBiP and Pi-d2 functions as a receptor for chitin oligosaccharides and CEBiP-based chimeric receptors fused with other RLKs may also act as functional receptors.

Nitrogen-fixing symbiosis between legumes and rhizobia is initiated by the recognition of rhizobial Nod factors (NFs) by host plants. NFs are diversely modified derivatives of chitin oligosaccharide, a fungal elicitor that induces defense responses in plants. Recent evidence has shown that both NFs and chitin elicitors are recognized by structurally related LysM receptor kinases. Transcriptome analyses of Lotus japonicus roots indicated that NFs not only activate symbiosis genes but also transiently activate defense-related genes through NF receptors. Conversely, chitin oligosaccharides were able to activate symbiosis genes independently of NF receptors. Analyses using chimeric genes consisting of the LysM receptor domain of a Lotus japonicus NF receptor, NFR1, and the kinase domain of an Arabidopsis chitin receptor, CERK1, demonstrated that substitution of a portion of the αEF helix in CERK1 with the amino acid sequence YAQ from the corresponding region of NFR1 enables L. japonicus nfr1 mutants to establish symbiosis with Mesorhizobium loti. We also showed that the kinase domains of two Lotus japonicus LysM receptor kinases, Lys6 and Lys7, which also possess the YAQ sequence, suppress the symbiotic defect of nfr1. These results strongly suggest that, in addition to adaptation of extracellular LysM domains to NFs, limited alterations in the kinase domain of chitin receptors have played a crucial role in shifting the intracellular signaling to symbiosis from defense responses, thus constituting one of the key genetic events in the evolution of root nodule symbiosis in legume plants.

Chitin is a major molecular pattern for various fungi, and its fragments, chitin oligosaccharides, are known to induce various defense responses in plant cells. A plasma membrane glycoprotein, CEBiP (chitin elicitor binding protein) and a receptor kinase, CERK1 (chitin elicitor receptor kinase) (also known as LysM-RLK1), were identified as critical components for chitin signaling in rice and Arabidopsis, respectively. However, it is not known whether each plant species requires both of these two types of molecules for chitin signaling, nor the relationships between these molecules in membrane signaling. We report here that rice cells require a LysM receptor-like kinase, OsCERK1, in addition to CEBiP, for chitin signaling. Knockdown of OsCERK1 resulted in marked suppression of the defense responses induced by chitin oligosaccharides, indicating that OsCERK1 is essential for chitin signaling in rice. The results of a yeast two-hybrid assay indicated that both CEBiP and OsCERK1 have the potential to form hetero- or homo-oligomers. Immunoprecipitation using a membrane preparation from rice cells treated with chitin oligosaccharides suggested the ligand-induced formation of a receptor complex containing both CEBiP and OsCERK1. Blue native PAGE and chemical cross-linking experiments also suggested that a major portion of CEBiP exists as homo-oligomers even in the absence of chitin oligosaccharides.

Chitin is a component of fungal cell walls, and its fragments act as elicitors in many plants. The plasma membrane glycoprotein CEBiP, which possesses LysM domains, is a receptor for the chitin elicitor (CE) in rice. Here, we report that the perception of CE by CEBiP contributes to disease resistance against the rice blast fungus, Magnaporthe oryzae, and that enhanced responses to CE by engineering CEBiP increase disease tolerance. Knockdown of CEBiP expression allowed increased spread of the infection hyphae. To enhance defense responses to CE, we constructed chimeric genes composed of CEBiP and Xa21, which mediate resistance to rice bacterial leaf blight. The expression of either CRXa1 or CRXa3, each of which contains the whole extracellular portion of CEBiP, the whole intracellular domain of XA21, and the transmembrane domain from either CEBiP or XA21, induced cell death accompanied by an increased production of reactive oxygen and nitrogen species after treatment with CE. Rice plants expressing the chimeric receptor exhibited necrotic lesions in response to CE and became more resistant to M. oryzae. Deletion of the first LysM domain in CRXA1 abolished these cellular responses. These results suggest that CEs are produced and recognized through the LysM domain of CEBiP during the interaction between rice and M. oryzae and imply that engineering pattern recognition receptors represents a new strategy for crop protection against fungal diseases.

OsWRKY53, a chitin oligosaccharide elicitor-responsive rice WRKY gene, has been found to be involved in defense responses in rice. We identified three tandem W-box elements, putative recognition sites for WRKY transcription factors, as cis elements that are essential to the elicitor-responsiveness of OsWRKY53 by deletion and mutation analysis of the promoter by dual luciferase assay.

The dasABC genes encode an ATP-binding cassette (ABC) transporter, which is one of the uptake systems for N,N'-diacetylchitobiose [(GlcNAc)(2)] in Streptomyces coelicolor A3(2), although the gene encoding the ABC subunit that provides ATP hydrolysis for DasABC has not been identified. In this study, we disrupted the sequence that is highly homologous to the msiK gene, the product of which is an ABC subunit assisting several ABC permeases in other Streptomyces species. Disruption of msiK severely affected the ability of S. coelicolor A3(2) to utilize maltose, cellobiose, starch, cellulose, chitin and chitosan, but not glucose. The msiK null mutant lacked (GlcNAc)(2)-uptake activity, but GlcNAc transport activity was unaffected. The data indicated that msiK is essential for (GlcNAc)(2) uptake, which in S. coelicolor A3(2) is governed by ABC transporters including the DasABC-MsiK system, in contrast to Escherichia coli and Serratia marcescens, in which (GlcNAc)(2) uptake is mediated by the phosphotransferase system. Interestingly, the induction of chitinase production by (GlcNAc)(2) or chitin was absent in the msiK null mutant, unlike in the parent strain M145. The defect in chitinase gene induction was rescued by expressing the His-tagged MsiK protein under the control of the putative native promoter on a multicopy plasmid. The data suggest that uptake of (GlcNAc)(2) is necessary for induction of chitinase production. The msiK gene was constitutively transcribed, whereas the transcription of dasA [(GlcNAc)(2)-binding protein gene], malE (putative maltose-binding protein gene), cebE1 (putative cellobiose-binding protein gene) and bxlE1 (putative xylobiose-binding protein gene) was induced by their corresponding sugar ligands. This is believed to be the first report to indicate that (GlcNAc)(2) uptake mediated by ABC transporters is essential for chitinase production in streptomycetes, which are known to be the main degraders of chitin in soil.

A conidia suspension of Magnaporthe grisea carried elicitor activity that induced the expression of defense-related genes and the production of H(2)O(2) in suspension-cultured rice cells. The levels of H(2)O(2) produced were dependent on fungal isolates and were correlated with the catalase activity in the supernatant fraction of each conidia suspension, not with gene-for-gene interactions.

An elicitor of rice defense responses was recently isolated from human feces and was identified as cholic acid (CA). Pathogen infection in rice leaves induces phytocassanes and momilactones, both of which are major diterpenoid phytoalexins in rice, whereas CA mainly induces phytocassanes. We established a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry protocol for the rapid and accurate quantification of phytocassanes and momilactones. Using this method, we showed that CA preferentially induced the formation of phytocassanes in suspension-cultured rice cells, while a fungal chitin oligosaccharide elicitor induced that of both phytocassanes and momilactones. We further investigated the effects of CA on the expression of diterpene cyclase genes involved in phytoalexin biosynthesis. CA induced the transcription of the genes OsCPS2 (OsCyc2) and OsKSL7 (OsDTC1), which are involved in phytocassane biosynthesis, to a greater extent than the genes OsCPS4 (OsCyc1) and OsKSL4, which are involved in momilactone biosynthesis. OsCPS2 was particularly strongly induced, suggesting that it is one of the main mechanisms by which CA induces high levels of phytocassanes.

Chitin is a major component of fungal cell walls and serves as a microbe-associated molecular pattern (MAMP) for the detection of various potential pathogens in innate immune systems of both plants and animals. We recently showed that chitin elicitor-binding protein (CEBiP), plasma membrane glycoprotein with LysM motifs, functions as a cell surface receptor for chitin elicitor in rice. The predicted structure of CEBiP does not contain any intracellular domains, suggesting that an additional component(s) is required for signaling through the plasma membrane into the cytoplasm. Here, we identified a receptor-like kinase, designated CERK1, which is essential for chitin elicitor signaling in Arabidopsis . The KO mutants for CERK1 completely lost the ability to respond to the chitin elicitor, including MAPK activation, reactive oxygen species generation, and gene expression. Disease resistance of the KO mutant against an incompatible fungus, Alternaria brassicicola , was partly impaired. Complementation with the WT CERK1 gene showed cerk1 mutations were responsible for the mutant phenotypes. CERK1 is a plasma membrane protein containing three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain with autophosphorylation/myelin basic protein kinase activity, suggesting that CERK1 plays a critical role in fungal MAMP perception in plants.

A rice diterpene cyclase, OsDTC2, functions as a stemar-13-ene synthase that converts syn-copalyl diphosphate into stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The transcriptional expression of OsDTC2 is induced by treatment of suspension-cultured rice cells with a chitin oligosaccharide elicitor. To investigate the molecular mechanisms of the elicitor signaling pathway that leads to OsDTC2 expression, we carried out deletion and mutation analysis of the region -1939 bp upstream of the transcription start site of OsDTC2 in rice cells using dual luciferase assays. The region between -1709 and -1450 bp was found to contain six W-box motifs, which are putative recognition sites for WRKY transcription factors, as cis elements involved in elicitor-responsiveness and/or basic promoter activity of OsDTC2.

Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.