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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.

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.

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 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.

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.

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.

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.

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.

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.

Diterpenoid phytoalexins such as momilactones and phytocassanes are produced via geranylgeranyl diphosphate in suspension-cultured rice cells after treatment with a chitin elicitor. We have previously shown that the production of diterpene hydrocarbons leading to phytoalexins and the expression of related biosynthetic genes are activated in suspension-cultured rice cells upon elicitor treatment. To better understand the elicitor-induced activation of phytoalexin biosynthesis, we conducted microarray analysis using suspension-cultured rice cells collected at various times after treatment with chitin elicitor. Hierarchical cluster analysis revealed two types of early-induced expression (EIE-1, EIE-2) nodes and a late-induced expression (LIE) node that includes genes involved in phytoalexins biosynthesis. The LIE node contains genes that may be responsible for the methylerythritol phosphate (MEP) pathway, a plastidic biosynthetic pathway for isopentenyl diphosphate, an early precursor of phytoalexins. The elicitor-induced expression of these putative MEP pathway genes was confirmed by quantitative reverse-transcription PCR. 1-Deoxy-D: -xylulose 5-phosphate synthase (DXS), 1-deoxy-D: -xylulose 5-phosphate reductoisomerase (DXR), and 4-(cytidine 5'-diphospho)-2-C-methyl-D: -erythritol synthase (CMS), which catalyze the first three committed steps in the MEP pathway, were further shown to have enzymatic activities that complement the growth of E. coli mutants disrupted in the corresponding genes. Application of ketoclomazone and fosmidomycin, inhibitors of DXS and DXR, respectively, repressed the accumulation of diterpene-type phytoalexins in suspension cells treated with chitin elicitor. These results suggest that activation of the MEP pathway is required to supply sufficient terpenoid precursors for the production of phytoalexins in infected rice plants.

PAMP (pathogen-associated molecular pattern) recognition plays an important role during the innate immune response in both plants and animals. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are representative of typical PAMP molecules and have been reported to induce defense-related responses, including the suppression of the hypersensitive response, the expression of defense genes and systemic resistance in plants. However, the details regarding the precise molecular mechanisms underlying these cellular responses, such as the molecular machinery involved in the perception and transduction of LPS molecules, remain largely unknown. Furthermore, the biological activities of LPS on plants have so far been reported only in dicots and no information is thus available regarding their functions in monocots. In our current study, we report that LPS preparations for various becteria, including plant pathogens and non-pathogens, can induce defense responses in rice cells, including reactive oxygen generation and defense gene expression. In addition, global analysis of gene expression induced by two PAMPs, LPS and chitin oligosaccharide, also reveals a close correlation between the gene responses induced by these factors. This indicates that there is a convergence of signaling cascades downstream of their corresponding receptors. Furthermore, we show that the defense responses induced by LPS in the rice cells are associated with programmed cell death (PCD), which is a finding that has not been previously reported for the functional role of these molecules in plant cells. Interestingly, PCD induction by the LPS was not detected in cultured Arabidopsis thaliana cells.

The Arabidopsis mutant cad1 (constitutively activated cell death 1) shows a phenotype that mimics hypersensitive response (HR)-like cell death. The CAD1 gene, which encodes a protein containing a domain with significant homology to the MACPF (membrane attach complex and perforin) domain of complement components and perforin, is likely to control plant immunity negatively and has a W-box cis-element in its promoter region. We found that expression of the CAD1 gene and other W-box containing genes, such as NPR1 and PR2, was promoted by salicylic acid (SA) and benzothiadiazole (BTH) as a SA agonist. The CAD1 gene was also stimulated by a purified chitin oligosaccharide elicitor (degree of polymerization = 8). This latter control was not under SA, because CAD1 expression was not suppressed in 35SnahG transgenic plants, which are unable to accumulate SA. These expression profiles were confirmed by promoter analysis using pCAD1::GUS transgenic plants. The CAD1 expression promoted by BTH and the chitin elicitor was not suppressed in the npr1 mutant, which is insensitive to SA signaling. These results indicate that the CAD1 gene is regulated by two distinct pathways involving SA and a chitin elicitor: viz., SA signaling mediated through an NPR1-independent pathway, and chitin elicitor signaling, through an SA-independent pathway. Three CAD1 homologs that have multiple W-box elements in their promoters were also found to be under the control of SA.

The mode of action of a potent elicitor, N-acetylchitooligosaccharide, in rice plants was examined. In intact seedlings, no significant uptake of the elicitor via the roots was observed within 3 h, whereas rapid uptake was observed in excised leaves. Rapid and transient expression of an elicitor-responsive gene, EL2, was induced in the leaves of intact seedlings sprayed with the elicitor or in the roots and leaves of intact seedlings by immersing roots in the elicitor solution. Histochemical analysis indicated that EL2 was expressed in cells exposed to the elicitor of root and leaves. In seedlings treated with the elicitor for 1 d or longer, hyphal growth of rice blast fungus was significantly delayed, and an accumulation of auto-fluorescence around the infection site was observed. Two defense-related genes, PR-1 and PR-10 (PBZ1), were induced in a systemic and local manner by elicitor treatment, in correlation with the induction of resistance against rice blast fungus. N-Acetylchitoheptaose did not inhibit the hyphal growth of the fungi. These results indicate the occurrence of systemic signal transmission from N-acetylchitooligosaccharide in rice plants.

Chitin is a major component of fungal cell walls and serves as a molecular pattern for the recognition of potential pathogens in the innate immune systems of both plants and animals. In plants, chitin oligosaccharides have been known to induce various defense responses in a wide range of plant cells including both monocots and dicots. To clarify the molecular machinery involved in the perception and transduction of chitin oligosaccharide elicitor, a high-affinity binding protein for this elicitor was isolated from the plasma membrane of suspension-cultured rice cells. Characterization of the purified protein, CEBiP, as well as the cloning of the corresponding gene revealed that CEBiP is actually a glycoprotein consisting of 328 amino acid residues and glycan chains. CEBiP was predicted to have a short membrane spanning domain at the C terminus. Knockdown of CEBiP gene by RNA interference resulted in the suppression of the elicitor-induced oxidative burst as well as the gene responses, showing that CEBiP plays a key role in the perception and transduction of chitin oligosaccharide elicitor in the rice cells. Structural analysis of CEBiP also indicated the presence of two LysM motifs in the extracellular portion of CEBiP. As the LysM motif has been known to exist in the putative Nod-factor receptor kinases involved in the symbiotic signaling between leguminous plants and rhizobial bacteria, the result indicates the involvement of partially homologous plasma membrane proteins both in defense and symbiotic signaling in plant cells.

A novel elicitor that induces chitinases in tobacco BY-2 cells was isolated from Alternaria alternata 102. Six other fungi, including A. alternata IFO 6587, could not induce, or weakly induce chitinase activity. The purified elicitor was soluble in 75% methanol and showed the chitinase-inducing activity when applied at concentrations of as low as 25 ng·mL−1. Structural determination by methylation analysis, reducing-end analysis, MALDI-TOF/MS, and NMR spectroscopy indicated that the elicitor was a mixture of β-1,3-, 1,6-oligoglucans mostly with a degree of polymerization of between 8 and 17. Periodate oxidation of the elicitor suggested that the 1,6-linked and nonreducing terminal residues are essential for the elicitor activity. Further analysis of the elicitor responses in BY-2 cells indicated that the activity of this β-1,3-, 1,6-glucan elicitor was about 1000 times more potent than that of laminarin, which is a known elicitor of defense responses in tobacco. Analyzing the expression of defense-related genes indicated that a phenylalanine ammonia-lyase gene and a coumaroyl-CoA O-methyltransferase gene were transiently expressed by this β-1,3-, 1,6-glucan elicitor. The elicitor induced a weak oxidative burst but did not induce cell death in the BY-2 cells. In the tissue of tobacco plants, this β-1,3-, 1,6-glucan elicitor induced the expression of basic PR-3 genes, the phenylpropanoid pathway genes, and the sesquiterpenoid pathway genes. In comparison with laminarin and laminarin sulfate, which are reported to be potent elicitors of defense responses in tobacco, the expression pattern of genes induced by the purified β-1,3-, 1,6-glucan elicitor was more similar to that induced by laminarin than to that induced by laminarin sulfate.

Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with jasmonic acid (JA) for 1/2h yielded a cDNA of a gene tentatively named RERJ1 that is upregulated in response to exogenous JA. Northern blot analysis indicated that the RERJ1 mRNA levels peaked at 1/2-1h after the addition of jasmonic acid and then decreased gradually. RERJ1 encodes a transcriptional regulator with a basic helix-loop-helix motif. The phenotypes of transgenic rice plants overexpressing sense or antisense RERJ1 mRNA demonstrated that RERJ1 is involved in the growth inhibition of rice shoots caused by JA. Other biological functions of RERJ1 are discussed from an evolutionary standpoint.

The generation of reactive oxygen species (ROS) is a central component of the elicitor-induced defence reactions in cultured cells as well as the resistance responses of plants to pathogen challenge. We show that N-acetylchitooligosaccharide elicitor induces rapid and transient activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and that phosphatidylcholine-specific phospholipase D (PC-PLD) in suspension-cultured rice cells and their products, phosphatidic acid (PA) and diacylglycerol (DG), especially the former, play an important role in the elicitor-induced ROS generation based on the following observations: (1) the amount of PA and DG in rice cells was rapidly increased by the elicitor treatment. (2) Elicitor-induced activation of PI-PLC and PC-PLD in the membrane fraction was confirmed by the analysis of enzymatic products from radio-labelled phospholipids as well as by 1-butanol (1-ButOH)-specific formation of phosphatidylbutanol (PtdBut) (for PC-PLD). Inhibitors of these phospholipases at least partly inhibited the elicitor-induced ROS generation. (3) Exogenously applied PA and DG could induce ROS generation in the rice cells in the absence of the elicitor. (4) PA phosphohydrolase (PAPH) and diacylglycerol kinase (DGK) activities, which catalyse the conversion of PA and DG with each other, are present in the rice cells and the inhibitors of these enzymes inhibited/stimulated the elicitor-induced ROS generation depending on the direction of the PA accumulation. These results indicate the important role of PI-PLC/PC-PLD and their products, especially PA, in the signal transduction cascade downstream of the N-acetylchitooligosaccharide receptor.

We identified two new members of the GRAS gene family from rice, CIGR1 and CIGR2, which are rapidly induced upon N-acetylchitooligosaccharide elicitor perception. The predicated proteins encoded by CIGR1 and CIGR2 possess significant sequence similarity with previously identified members of the GRAS family, such as Arabidopsis SCARECROW, GAI, RGA, tomato Lateral suppressor, and rice SLR1, all of which have VHIID regions, likely to play a role in cellular signaling. Fusions of CIGR1 and CIGR2 with Green Fluorescent Protein were detected exclusively in the nuclei of onion epidermal cells. The expression of CIGR1 and CIGR2 was dependent on the structure of N-acetylchitooligosaccharides, which parallels the structural specificity for chitin binding to the plasma membrane-localized chitin-binding protein, and independent of de novo protein synthesis. Co-cultivation of rice cells with rice blast fungus strongly induced the expression of CIGR1 and CIGR2, whereas inoculation of suspension cells with phytopathogenic bacteria did not. We hypothesize that CIGR1 and CIGR2 act as transcriptional regulators in the early events of the elicitor-induced defense response in rice.

We present evidence that the rice receptor for N-acetylchitooligosaccharide elicitor does not couple to heterotrimeric G-protein (G-protein), one of the most important signal transducers from the cell surface to down-stream effectors in various cellular responses of many organisms. Using mutant rice cells lacking functional G-protein α-subunit, cellular responses of suspension-cultured rice cells derived from Daikoku dwarf (d1) mutants that were shown to contain mutations in the coding region of the G-protein α-subunit (Fujisawa et al. 1999) to N-acetylchitooligosaccharide (oligochitin) elicitor were compared with those of the corresponding parent cell lines. All the elicitor-induced cellular responses, such as medium alkalinization, generation of reactive oxygen species, expression of early responsive genes, PAL and PR genes, and production of phytoalexin, were basically identical in the mutant and wild type cell lines. Considering the reported presence of a single copy gene for the G-protein α-subunit in many plant species including rice, these results strongly support the above conclusion.

Summary Presence of a high-affinity binding protein for N-acetylchitooligosaccharide (fragments of chitin) elicitor in the plasma membrane from rice leaf and root cells was shown by affinity labeling experiments with an 125I-labeled N-acetylchitooligosaccharide derivative. Binding studies also showed that binding site in the leaf cells has a high affinity to highly elicitor-active, larger chitin fragments but much lower or no affinity to less elicitor-active or elicitor-inactive oligosaccharides. The amount of the binding protein in the leaf cells was slightly smaller than that in the suspension-cultured cells but much larger compared to that in the root cells. These results indicate the possible- involvement of the elicitor binding protein in the perception of the elicitor signal in intact rice plant.

Partial acid/enzymatic hydrolysis of the beta-(1--3, 1--6)-glucan from the cell walls of the rice blast disease fungus Pyricularia oryzae (Magnaporthe grisea) released elicitor-active fragments that induced phytoalexin biosynthesis in suspension-cultured rice cells. From the digestion of the glucan by an endo-beta-(1--3)-glucanase, one highly elicitor-active glucopentaose was purified as a reduced compound, tetraglucosyl glucitol. The structure of this tetraglucosyl glucitol as well as two other related tetraglucosyl glucitols was elucidated as follows: (1) Glcbeta(1--3)Glcbeta(1--3)(Glcbeta(1--6)) Glcbeta(1--3)Glucitol (most active fragment); (2) Glcbeta(1--3)(Glcbeta(1--6))Glcbeta(1--3)Glcbeta (1--3)Glucitol; and (3) Glcbeta(1--6) Glcbeta(1--3)Glcbeta(1--3)Glcbeta(1--3)Glucitol. However, a synthetic hexa-beta-glucoside, known as a minimal structural element for the phytoalexin elicitor for soybean cotyledon cells, did not induce phytoalexin biosynthesis in the rice cells. Conversely, the beta-glucan fragment from P. oryzae did not induce phytoalexin biosynthesis in the soybean cotyledon cells, indicating differences in the recognition of glucooligosaccharide elicitor signals in these two plants. Because rice cells have been shown to recognize chitin fragments larger than pentamers as potent elicitors, these results also indicate that the rice cells can recognize at least two types of oligosaccharides from fungal cell walls as signal molecules to initiate defense response.

Expression patterns of chitinase transcripts induced by N-acetylchitooligosaccharide elicitor were analyzed by northern blot hybridization in order to reveal a signal transduction pathway leading to the activation of class I chitinase genes (Cht-1 and Cht-3), which may play an important role in producing N-acetylchitooligosaccharide elicitor. The transcription level of both genes was enhanced in response to N-acetylchitooligosaccharides larger than pentaose at subnanomolar concentrations. These structure and dose dependencies were consistent not only with those for a 75 kDa high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane, but also with other series of cellular responses including phytoalexin production and the expression of elicitor-responsive genes (EL2, EL3). Therefore, the elicitor signal to evoke these cellular responses including the activation of the chitinase genes could be common and transmitted into cells through the 75 kDa protein. However, the signal transduction pathway for the activation of the chitinase gene appeared to diverge from those for the other elicitor-responsive genes shortly after the signal perception. It was shown that the induction of chitinase expression by N-acetylchitooligosaccharide would require protein phosphorylation, but not de novo protein synthesis. The oxidative burst was demonstrated not to be necessary for transcriptional induction of the all four elicitor-responsive genes (Cht, PAL, EL2, EL3) by N-acetylchitooligosaccharide.

Pseudomonas avenae is a Gram-negative phytopathogenic bacterium that causes the symptom of a brown stripe in infected susceptible plants. The host range of P. avenae is wide among the monocotyledonous plants, however, individual strains can infect only one or a few host species. A rice-incompatible strain, N1141, caused rapid cell death in sheath sections and in cultured rice cells. A rice-compatible strain, H8301, also induced cell death, however, this cell death in a compatible interaction was delayed compared to the cell death induced by the N1141 incompatible strain. Inoculation of N1141 strain induced expression of EL2 gene which is thought to be one of the defense-related gene. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) of cultured rice cells showed that DNA cleavage occurred only in N1141-inoculated rice cells. N1141 strain caused cytoplasmic condensation, shrinkage, and plasma membrane blebbing, all of which are important morphological characteristics of programmed cell death (PCD). In contrast, H8301 strain inoculated rice cells appeared to show weakening of the cell wall instead of cytoplasm condensation, shrinkage and membrane blebbing. These results suggest that the rapid cell death of rice induced by the incompatible strain is characterized as PCD.

A (1→3,1→4)- β -glucanase was purified from rice bran to electrophoretic homogeneity by fractionation with ammonium sulfate followed by ion-exchange chromatography’s on DEAE- and CM-Sepharose, chromatofocusing on Polybuffer exchanger 94 and hydrophobic interaction chromatography on Phenyl-Sepharose. The purified enzyme had a molecular mass of 31 kDa as judged by SDS-PAGE and a basic isoelectric point of 7.9. The enzyme specifically hydrolyzed (1→3,1→4)- β -glucans such as β -glucans of barley and oat, and lichenins, but not CM-cellulose and laminarin. The purified enzyme was most active at pH 4.5 and stable for 10 min at 40°C. When barley (1→3,1→4)- β -glucan was used as the substrate, two oligosaccharides were formed as the major products of hydrolysis after prolonged incubation with the enzyme. No glucose was detected however, indicating that the enzyme may be classified as an endo-(1→3,1→4)- β -glucanase (EC 3.2.1.73). NH 2 -terminal amino acid sequencing (40 residues) of rice endo-(1→3,1→4)- β -glucanase revealed that it had a high similarity (83–85%) to that of endo-(1→3,1→4)- β -glucanase from barley and wheat.

CEBiP, a plasma membrane-localized glycoprotein of rice, directly binds with chitin elicitors (CE), and has been identified as a receptor for CE by using CEBiP-RNAi rice cells. To further clarify the function of CEBiP, we produced CEBiP-disrupted rice plants by applying an efficient Agrobacterium-mediated gene-targeting system based on homologous recombination, which has recently been developed for rice. Homologous recombination occurred at the CEBiP locus in ~0.5 % of the positive/negative selected calli. In the self-pollinated next generation, it was confirmed that the first exon of CEBiP was replaced with the hygromycin selection cassette as designed, and that the expression of CEBiP was completely deficient in homozygous cebip lines. Affinity-labeling analysis using biotinylated N-acetylchitooctaose demonstrated that CEBiP is the major CE-binding protein in rice cultured cells and leaves, which was consistent with the result that the response to CE in cebip cells was greatly diminished. Nevertheless, we observed a significant decrease in disease resistance against Magnaporthe oryzae, the causal agent of rice blast disease, only when the cebip leaf sheaths were inoculated with a weakly virulent strain, suggesting that CE perception during the infection process of M. oryzae is limited. The response to peptidoglycan and lipopolysaccharides in cebip cells was not affected, strongly suggesting that CEBiP is a CE-specific receptor.

Analysis of free sugars in developing rice anthers by high-performance anion-exchange chromatography (HPAEC) showed that a very high concentration of a novel oligosaccharide accumulated specifically during microsporogenesis. Structural analysis of the purified oligosaccharide by methylation analysis, mass spectrometry/mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy revealed its structure to be beta-L- Ara(f)-(1-->3)-alpha-L-Ara(f)-(1-->3)-beta-D-Gal(p)-(1-->6)-D-Gal, which is closely related to a tetrasaccharide unit found in the glycan chain of a plant cell surface proteoglycan, the arabinogalactan-protein (AGP). Chilling treatment (12 degrees C, 4 days), which injures rice anthers during microsporogenesis, decreased the concentration of the tetrasaccharide, but the sucrose level increased. This effect was especially evident in a chilling-sensitive mutant line, YM56-1. These results suggest that this unique tetrasaccharide may play an important role in both the development of the rice anther and its response to chilling.

By using subtracted probes, two cDNA clones of rice, EL2 and EL3, were isolated as genes responsive within 6 min to jV-acetylchit oheptaose, a potent biotic elicitor for phytoalexin biosynthesis. Analyses of the sequence of the cDNAs showed that both of EL2 and EL3 encoded basic proteins with no significant similarities to those of known genes.

Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue.

To isolate genes responsive to N-acetylchitooligosaccharide elicitor in suspension-cultured rice cells, we screened rice ESTs by DNA microarray analysis, and were able to identify novel elicitor-responsive genes. Sequencing analysis of three ESTs revealed that the up-regulated genes include a transcription factor, Myb, small G-protein, Rac, and calmodulin. The results indicated the genome-wide change of gene expression in response to N-acetylchitooligosaccharide elicitor.

Chitinase treatment of a commercial laminarioligosaccharide preparation from a mushroom resulted in a loss of previously reported elicitor activity in rice cells, indicating that the activity was attributable not to the laminarioligosaccharide but rather to the contaminating chitin fragments. This suggests that the elicitor activity of laminarioligosacchraides from such sources containing chitinaceous substances should be carefully interpreted.

International audience; The chitin elicitor-binding protein (CEBiP) from rice was the first plant lysin motif (LysM) protein for which the biological and biochemical function had been established. It belongs to a plant-specific family of extracellular LysM proteins (LYMs) for which we analyzed the phylogeny. LYMs are present in vascular plants only, where an early gene duplication event might have resulted in two types which were retained in present day genomes. LYMs consist of a signal peptide, three consecutive LysMs, separated by cysteine pairs, and a C-terminal region without any known signature, whose length allows the distinction between the two types, and which may be followed by a glycosylphosphatidylinositol (GPI) anchor motif. We analyzed a representative of each type, MtLYM1 and MtLYM2, from Medicago truncatula at the biochemical level and with respect to their expression patterns and observed some similarities but also marked differences. MtLYM1 and MtLYM2 proved to be very different with regard to abundance and apparent molecular mass on SDS-PAGE. Both undergo several post-translational modifications, including N-glycosylation and the addition of a GPI anchor, which would position the proteins at the outer face of the plasma membrane. Only MtLYM2, but not MtLYM1, showed specific binding to biotinylated N-acetylchitooctaose in a manner similar to CEBiP, which belongs to the same type. We postulate that LYM2-type proteins likely function in the perception of chitin-related molecules, whereas possible functions of LYM1-type proteins remain to be elucidated.

Induction of phytoalexin formation in suspension-cultured rice cells by a series of N-acetylchitooligosaccharides and chitooligosaccharides was studied. N-acetylchitooligosaccharides larger than hexaose induced the formation of momilactones A and B as well as oryzalexins A, B, and D at very low concentrations like 10− 9–10− 6 M (N-acetylchitoheptaose). GlcNAc oligomers smaller than trimers had almost no activity and a series of deacetylated chitooligosaccharides were also inactive. Strict requirement for the size and structure of GlcNAc oligomers as well as the sensitivity to them strongly indicates the presence of recognition systems specific for these compounds in rice cells. The level of momilactone A produced reached 100–500 μg/g of cultured cells, which appeared to be enough to prevent the growth of pathogenic fungi such as Pyricularia oryzae, thus indicating the importance of this phenomenon in the defense systems of rice plants. Suspension-cultured cells obtained only from a suitable period of cult...

The biological role of a secretory catalase of the rice blast fungus Magnaporthe oryzae was studied. The internal amino acid sequences of the partially purified catalase in the culture filtrate enabled us to identify its encoding gene as a catalase-peroxidase gene, CPXB, among four putative genes for catalase or catalase-peroxidase in M. oryzae. Knockout of the gene drastically reduced the level of catalase activity in the culture filtrate and supernatant of conidial suspension (SCS), and increased the sensitivity to exogenously added H2O2 compared with control strains, suggesting that CPXB is the major gene encoding the secretory catalase and confers resistance to H2O2 in hyphae. In the mutant, the rate of appressoria that induced accumulation of H2O2 in epidermal cells of the leaf sheath increased and infection at early stages was delayed; however, the formation of lesions in the leaf blade was not affected compared with the control strain. These phenotypes were complimented by reintroducing the putative coding regions of CPXB driven by a constitutive promoter. These results suggest that CPXB plays a role in fungal defense against H2O2 accumulated in epidermal cells of rice at the early stage of infection but not in pathogenicity of M. oryzae.

Fungal Defenses One of the major driving forces of evolution is the constant arms race between plants and animals and the microbial pathogens that infect them. The fungus Cladosporium fulvum causes leaf mold on tomato plants. One of the ways tomato plants sense infections by C. fulvum is by detecting chitin, a component of fungal cell walls. In response, the fungus has evolved strategies to evade detection. De Jonge et al. (p. 953 ) have now identified one such mechanism in C. fulvum , mediated by the effector protein Ecp6. Secreted Ecp6 is able to bind to chitin oligosaccharides that are released upon degradation of the fungal cell wall and sequester them so that they are not detected by tomato chitin receptors. Proteins with domain structure similar to Ecp6 are conserved throughout the fungal kingdom, which suggests that chitin sequestration may represent a general mechanism used by fungi to evade immune detection.

Plants recognize potential microbial pathogens through microbial-associated molecular patterns (MAMPs) and activate a series of defense responses, including cell death and the production of reactive oxygen species (ROS) and diverse anti-microbial secondary metabolites. Mitogen-activated protein kinase (MAPK) cascades are known to play a pivotal role in mediating MAMP signals; however, the signaling pathway from a MAPK cascade to the activation of defense responses is poorly understood. Here, we found in rice that the chitin elicitor, a fungal MAMP, activates two rice MAPKs (OsMPK3 and OsMPK6) and one MAPK kinase (OsMKK4). OsMPK6 was essential for the chitin elicitor-induced biosynthesis of diterpenoid phytoalexins. Conditional expression of the active form of OsMKK4 (OsMKK4(DD) ) induced extensive alterations in gene expression, which implied dynamic changes of metabolic flow from glycolysis to secondary metabolite biosynthesis while suppressing basic cellular activities such as translation and cell division. OsMKK4(DD) also induced various defense responses, such as cell death, biosynthesis of diterpenoid phytoalexins and lignin but not generation of extracellular ROS. OsMKK4(DD) -induced cell death and expression of diterpenoid phytoalexin pathway genes, but not that of phenylpropanoid pathway genes, were dependent on OsMPK6. Collectively, the OsMKK4-OsMPK6 cascade plays a crucial role in reprogramming plant metabolism during MAMP-triggered defense responses.

The plant genome encodes a wide range of receptor-like proteins but the function of most of these proteins is unknown. We propose the use of affinity cross-linking of biotinylated ligands for a ligand-based survey of the corresponding receptor molecules. Biotinylated ligands not only enable the analysis of receptor-ligand interactions without the use of radioactive compounds but also the isolation and identification of receptor molecules by a simple affinity trapping method. We successfully applied this method for the characterization, isolation and identification of the chitin elicitor binding protein (CEBiP). A biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the detection of CEBiP in the plasma or microsomal membrane preparations from rice and carrot cells. Binding characteristics of CEBiP analyzed by inhibition studies were in good agreement with the previous results obtained with the use of a radiolabeled ligand. The biotin-tagged CEBiP could be purified by avidin affinity chromatography and identified by LC-MALDI-MS/MS after tryptic digestion. We also used this method to detect OsFLS2, a rice receptor-like kinase for the perception of the peptide elicitor flg22, in membrane preparations from rice cells overexpressing OsFLS2. This work demonstrates the applicability of this method to the purification and identification of plant receptor proteins.

Phospholipase D (PLD) plays an important role in plants, including responses to abiotic as well as biotic stresses. A survey of the rice (Oryza sativa) genome database indicated the presence of 17 PLD genes in the genome, among which OsPLDα1, OsPLDα5, and OsPLDβ1 were highly expressed in most tissues studied. To examine the physiological function of PLD in rice, we made knockdown plants for each PLD isoform by introducing gene-specific RNA interference constructs. One of them, OsPLDβ1-knockdown plants, showed the accumulation of reactive oxygen species in the absence of pathogen infection. Reverse transcription-polymerase chain reaction and DNA microarray analyses revealed that the knockdown of OsPLDβ1 resulted in the up-/down-regulation of more than 1,400 genes, including the induction of defense-related genes such as pathogenesis-related protein genes and WRKY/ERF family transcription factor genes. Hypersensitive response-like cell death and phytoalexin production were also observed at a later phase of growth in the OsPLDβ1-knockdown plants. These results indicated that the OsPLDβ1-knockdown plants spontaneously activated the defense responses in the absence of pathogen infection. Furthermore, the OsPLDβ1-knockdown plants exhibited increased resistance to the infection of major pathogens of rice, Pyricularia grisea and Xanthomonas oryzae pv oryzae. These results suggested that OsPLDβ1 functions as a negative regulator of defense responses and disease resistance in rice.

Expression of OsWRKY71, a rice WRKY gene, was induced by biotic elicitors and pathogen infection. It was also found that OsWRKY71 has features characteristic of a transcriptional repressor. Microarray analysis revealed that several elicitor-induced defense-related genes were upregulated in rice cells overexpressing OsWRKY71. These results indicate that the activation of defense-related genes by OsWRKY71 was probably indirect.

We present a detailed characterization of the chitin oligosaccharide elicitor-induced gene OsWRKY53. OsWRKY53 was also induced in suspension-cultured rice cells by a fungal cerebroside elicitor and in rice plants by infection with the blast fungus Magnaporthe grisea. A fusion of OsWRKY53 with green fluorescent protein was detected exclusively in the nuclei of onion epidermal cells, and OsWRKY53 protein specifically bound to W-box elements. A transient assay using the particle bombardment method showed that OsWRKY53 is a transcriptional activator. A microarray analysis revealed that several defense-related genes, including pathogenesis-related protein genes such as PBZ1, were upregulated in rice cells overexpressing OsWRKY53. Finally, overexpression of OsWRKY53 in rice plants resulted in enhanced resistance to M. grisea. These results strongly suggest that OsWRKY53 is a transcription factor that plays important roles in elicitor-induced defense signaling pathways in rice.