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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.

Streptomycetes are known as saprophytic soil bacteria and the main decomposers of chitin, which is a polymer of N-acetylglucosamine (GlcNAc) linked by β-1,4 bonds. Streptomycetes degrade chitin by producing a variety of chitinase (EC 3.2.1.14) (for a review, see reference 27) that hydrolyzes chitin into N,N′-diacetylchitobiose [(GlcNAc)2] as the final product (3, 21). Streptomyces coelicolor A3(2) strain M145 possesses 13 chitinase (chi) genes in its genome, although 6 of them are putative ones (2, 6, 12, 29). The expression of five chi genes (chiA, chiB, chiC, chiD, and chiF) is induced by chitin in the bacterium (30), and each of the chitinases (ChiA, ChiC, ChiD, and ChiF) has its own enzymatic properties (12). It is assumed that these chitinases act synergistically with each other in chitin degradation, as has been reported in the chitinase system of a marine bacterium, Alteromonas sp. strain O-7 (22). It is generally known that chitinase genes in streptomycetes are induced in the presence of the chitin substrate and repressed when both glucose (Glc) and chitin are present (for a review, see reference 27). The regulation mechanisms are, however, still unclear. The 12-bp direct repeat (DR) sequences overlapping the promoters are required for regulation of the chi63 gene in Streptomyces plicatus and the chiA gene in Streptomyces lividans (8, 16, 20). The DR-like sequences are also present upstream of other chitinase genes, the expression of which is regulated as described above. It is therefore believed that the DR-like sequences are common cis-acting elements involved in the regulation of chitinase genes in streptomycetes. The transcription of chi genes in S. coelicolor A3(2) and S. lividans is induced by (GlcNAc)2 as well as by chitin (17, 30). Because of the more immediate induction activity of (GlcNAc)2, it has been suggested that (GlcNAc)2 production, which occurs by hydrolysis of chitin, is necessary for the induction of chi gene expression (30). Although chitinase systems have been studied in streptomycetes for decades, the nature of the systems of transport of chitin degradation products was unclear until two transporters for GlcNAc uptake were identified in Streptomyces olivaceoviridis. One is Ngc, an ABC (ATP-binding cassette) transport system for GlcNAc and probably for (GlcNAc)2 (40). The corresponding solute-binding protein NgcE has high affinities for GlcNAc and (GlcNAc)2. The other transporter protein is PtsC2 of the phosphotransferase system which is specific for GlcNAc (38). During the course of our studies, we obtained data suggesting that (GlcNAc)2 uptake activity was induced by the amino sugar itself. In light of this implication, we explored transporter genes for (GlcNAc)2 in the genome of S. coelicolor A3(2) by using the DR sequence as a probe. We successfully identified a novel ABC transporter gene for (GlcNAc)2, the deduced products of which exhibit properties distinct from those of Ngc of S. olicaceoviridis. Interestingly, the gene cluster for the new transporter is located adjacent to the dasR gene, which is involved in the regulation of morphological differentiation in Streptomyces griseus (35) as well as in S. coelicolor A3(2) (23).

1997年12月にFCCAおよび生化学工業の共同制作で開始されたグライコワード (GlycoWord) の編集作業は、2006年末をもってひとまず終了した。グライコワードは、ネット上で糖質科学のキーワードを解説する世界初の用語解説集で、コンパクトかつ的確な解説が英語と日本語の両方で読むことができるという特徴をもっており、日本だけでなく世界において大勢の方々に親しみをもって利用されてきた。グライコワードは、掲載開始から今日までの糖質科学隆盛期における役割を終え、今、来るべき新しい発展期における役割を模索する転機を迎えている。

Most plant pattern recognition receptors induce immune responses by detecting molecular patterns typical to one group of microbes. A newly identified complex, on the other hand, monitors effector proteins widely distributed among bacteria, fungi and oomycetes, casting a new light on the evolution of pattern recognition in plants.

We show that a rice GRAS family protein, CIGR2, is a bonafide transcriptional activator, and through this function, targets the B-type heat shock protein-encoding gene OsHsf23 (Os09g0456800). CIGR2 (Os07g0583600) is an N-acetylchitooligosaccharide elicitor-responsive gene whose activity, through the direct transcriptional control of OsHsf23, is required for mediating hypersensitive cell death activation during pathogen infection. RNAi lines of CIGR2 and OsHsf23 similarly exhibited the higher level of granulation in the epidermal cells of leaf sheath inoculated with an avirulent isolate of rice blast fungus. Interestingly, we did not observe altered levels of resistance, suggesting that CIGR2 suppresses excessive cell death in the incompatible interaction with blast fungus via activation of OsHsf23.

The csnR gene, localized at the beginning of an operon, csnR-K, which organization is conserved through many actinomycete genomes, was previously shown to repress the transcription of the chitosanase gene csnA in Streptomyces lividans. However, knowledge on the function of the whole csnR-K operon in the metabolism of chitosan (an N-deacetylated derivative of chitin) remained limited. Mutants of S. coelicolor A3(2) harboring partial or total deletions of the csnR-K operon were analyzed for their capacity to uptake glucosamine oligosaccharides (GlcN)n. The csnR-K operon was autoregulated by CsnR repressor and its transcription was inducible by GlcN oligosaccharides. The operon controlled the uptake of GlcN oligosaccharides in S. coelicolor A3(2), with a minor contribution to the consumption of monomeric GlcN but not chitin-related N-acetylated derivatives. The deletion of the whole operon abolished the uptake of GlcN oligosaccharides. The CsnEFG transporter encoded by this operon is the front door for the assimilation of chitosan-derived hydrolysis products in S. coelicolor A3(2). The ATP-binding component MsiK was essential for CsnEFG transport function. Also, deletion of msiK abolished the induction of csnA transcription by GlcN oligosaccharides.

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.

This paper describes an efficient evaluation system of Arabidopsis mutants for defense responses. Young A. thaliana seedlings were analyzed for the induction of reactive oxygen species (ROS) as well as defense gene expression by chitin oligosaccharide elicitor in microtiter plates. Combined with the analysis of ROS in microtiter plates, the system enabled to evaluate thousands of mutant plants within a couple of weeks. Pharmacological studies could be applied and provided information about the molecular machinery involved in the defense responses. Experiments with the dissected organs showed that the roots were responsible for most ROS generation while the gene activation was observed in all the organs. An example of the successful application of this system for the screening of Arabidopsis muntats is also presented. Thus, the system provides a promising approach to screen, or evaluate, novel mutants for a confined defense signaling cascade downstream of a specific elicitor.

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.

Two monomeric lectins, SSA-b-3 and SSA-b-4, were purified from the bark tissue of Japanese elderberry, Sambucus sieboldiana. SDS-PAGE of the purified lectins showed the presence of single bands of 35 and 33 kDa for SSA-b-3 and SSA-b-4, respectively, irrespective of the presence of reducing agent. MS analysis as well as gel filtration of these lectins indicated that they exist mostly as monomeric lectins. Analysis of the N-terminal amino acid sequences of SSA-b-3 and SSA-b-4 yielded an identical sequence, indicating their close structural relationship. Four cDNA clones with extensive homology were obtained from the bark cDNA library and indicated to encode SSA-b-3 or SSA-b-4 from the comparison with the N-terminal sequences of these lectins. These clones were classified into two groups, three for SSA-b-3 and one for SSA-b-4, based on the predicted isoelectric points. The amino acid sequences of the encoded polypeptides were almost identical with the B-chain of a type 2 ribosome-inactivating protein from the same bark tissue, sieboldin-b, except for the absence of a small peptide containing a cystein residue, which is critical for the heteromeric dimerization with an A-subunit. Carbohydrate binding specificity and biological activity of these lectins are also reported.

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.

N-acetylchitooligosaccharides are potent elicitors to suspension-cultured rice cells, inducing a set of defense reactions. Expression of defense-related genes is considered to play an important role in defense reactions, and we employed microarray analysis of 8987 randomly selected expressed sequence tags to analyze the changes in gene expression caused by N-acetylchitooctaose. In this experiment, 166 genes were significantly induced and 93 genes were repressed. RNA gel blot analysis of 16 of these genes confirmed the microarray results. Of the 259 ESTs identified as responsive to N-acetylchytooctaose, 18 genes are related to signal transduction, including five calcium-dependent protein kinases (CDPKs). Among these, three novel CDPKs responsive to N-acetylchitooctaose were isolated.

Summary The role of myrosinase (β-thioglucoside glucohydrolase, EC 3.2.3.1) in the phototropic response in radish hypocotyls was investigated. Unilateral illumination with blue light abruptly up-regulated the activity of myrosinase, which releases bioactive 4-methylthio-3-butenyl isothiocyanate (MTBI) from inactive 4-methylthio-3-butenyl glucosinolate (MTBG), in the illuminated halves of radish hypocotyls 10 min after onset of phototropic stimulation, peaking after 30 min and decreasing thereafter. The myrosinase activity in the shaded halves also increased, but was significantly lower than that in the illuminated halves. Furthermore, whether blue light illumination induces myrosinase gene expression was studied. Northern blotting analysis indicated that myrosinase mRNA levels were increased markedly in unilaterally illuminated hypocotyls, reaching maximum signal intensity within 10 min after onset of blue illumination, declining nearly to the control level thereafter. These results suggested that phototropic stimulation promotes myrosinase gene expression and myrosinase activity in the illuminated side, resulting in the conversion of inactive MTBG to active MTBI and simultaneously producing more active raphanusanins, causing a phototropic response.

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.

OsCERK1 is a rice receptor-like kinase that mediates the signal of a fungal cell wall component, chitin, by coordinating with a lysin motif (LysM)-containing protein CEBiP. To further elucidate the function of OsCERK1 in the defense response, we disrupted OsCERK1 using an Agrobacterium-mediated gene targeting system based on homologous recombination. In OsCERK1-disrupted lines, the generation of hydrogen peroxide and the alteration of gene expression in response to a chitin oligomer were completely abolished. The OsCERK1-disrupted lines also showed lowered responsiveness to a bacterial cell wall component, peptidoglycan. Yeast two-hybrid analysis indicated that OsCERK1 interacts with the LysM-containing proteins LYP4 and LYP6, which are known to participate in the peptidoglycan response in rice. Observation of the infection behavior of rice blast fungus (Magnaporthe oryzae) revealed that disruption of OsCERK1 led to increased hyphal growth in leaf sheath cells. Green fluorescent protein-tagged OsCERK1 was localized around the primary infection hyphae. These results demonstrate that OsCERK1 is indispensable for chitin perception and participates in innate immunity in rice, and also mediates the peptidoglycan response. It is also suggested that OsCERK1 mediates the signaling pathways of both fungal and bacterial molecular patterns by interacting with different LysM-containing receptor-like proteins.

The legume family includes important grain, forage, and agroforestry species. One of the major constraints for cultivation of these legumes is obviously production loss by disease (Graham and Vance in Plant Physiol 131(3):872–877, 2003). Thus, Lotus japonicus is utilized not only as a symbiotic model but also as a research platform for studying serious diseases in legumes. However, most of our knowledge about the defense mechanism in L. japonicus comes from the study of legume–rhizobia symbiosis. In this section, we mainly focus on the regulation of defense responses in host symbiotic process, the pathogenic aspect of symbiotic microbial partners, and then illustrate the Lotus pathogens.

We describe a highly convergent approach for the synthesis of the tetraglucosyl glucitol 1 and its derivatives 4 , 5 and 6 which exhibit phytoalexin-elicitor activity in rice cells based on sequential glycosylation.

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.

A CH3OH-utilizing bacterium that has the ability to produce extracellular polysaccharide (EPS) was isolated from a soil sample, and was identified as the obligate methylotroph Methylobacillus sp. strain 12S on the basis of its 16S rDNA sequence and growth-substrate specificity. The EPS produced by strain 12S was purified and the sugar composition was analysed by GC-MS and HPLC to reveal that the EPS was a heteropolymer composed of glucosyl, galactosyl, and mannosyl residues in the molar ratio 3:1:1. In order to produce mono- and/or oligosaccharides by single-step fermentation from CH3OH, stain 12S was mutagenized by transposon 5. Among eleven EPS-deficient mutants, three strains were found to accumulate significant amounts of reducing sugars in the media. The amounts of the reducing sugars produced by the mutants ( > ca. 700 mg glucose equivalent/l) were > 11-22 times higher than those produced by the wild-type strain (

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.

糸状菌細胞壁の構成成分であるキチンのオリゴ糖の特定サイズのものはイネの培養細胞に対して強いエリシター活性を示すだけではなく、単子葉植物を中心に広い範囲の植物でエリシターとして認識されることが示唆されている。筆者らは最近、イモチ病菌細胞壁グルカンの酵素分解物からイネ培養細胞に対して強いエリシター活性を示す1種のグルコペンタオースを精製し、その構造を明らかにした。その結果、イネとダイズは異なる構造のグルカン断片をエリシターとして認識していることが示唆された。植物病原菌の感染に対する防御反応の多くが、エリシター処理によっても再現できることから、防御関連遺伝子発現に至るシグナル伝達過程を解析する優れたモデル系として、多くの研究が行われている。エリシターシグナルの受容と伝達に関与する受容体分子を同定し、その性質を調べることはシグナル伝達機構を解明する上できわめて重要である。筆者らはこれまでに、キチンオリゴ糖エリシターの受容体候補として、イネ培養細胞原形質膜上に存在する75kDaのエリシター結合タンパク質を同定しこれを精製した。このタンパク質およびβ-グルカンエリシター結合タンパク質の構造と性質を調べることにより、これらのオリゴ糖エリシターがイネやその他の植物でどのように認識・伝達されていくか、また、こうした認識・応答系がどのように進化してきたかなどについての知見が得られるものと期待される。

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.

N-Acetylchitoheptaose strongly induces a set of defense reactions in suspension-cultured rice cells including cytoplasmic acidification (K. Kuchitsu, Y. Yazaki, K. Sakano, and N. Shibuya, Plant Cell Physiol. 38:1012-1018, 1997) and the accumulation of mRNAs for two rapidly activated genes, EL2 and EL3 (E. Minami, K. Kuchitsu, D.-Y. He, H. Kouchi, N. Midoh, Y. Ohtsuki, and N. Shibuya, Plant Cell Physiol. 37:563-567, 1996), as well as phenylalanine ammonia-lyase (PAL), chitinase and β-1,3-glucanase. Treatment of cells with propionic acid resulted in the accumulation of the mRNAs for EL2, EL3, and PAL in a manner similar to the accumulation induced by N-acetylchitoheptaose. Concomitantly, there was a rapid decrease in the cytoplasmic pH as detected with in vivo 31P-nuclear magnetic resonance (NMR) spectroscopy. Interestingly, K-252a, a potent inhibitor of Ser/Thr protein kinases, strongly inhibited gene induction by N-acetylchitoheptaose, but showed much less inhibition of gene induction caused by propionic acid. Calyculin A, a protein phosphatase inhibitor, induced mRNA accumulations for EL2, EL3 and PAL, with concomitant acidification of the cytoplasm. On the other hand, chitinase and β-glucanase mRNA levels did not change after addition of propionic acid or calyculin A. Treatment of the cells with propionic acid did not induce the production of reactive oxygen species. These results strongly suggest that cytoplasmic acidification at the early stage of elicitor action could be a key step in the signal transduction events leading to the expression of elicitor-responsive genes. A hypothetical model of elicitor signal pathway is proposed based on these results.

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.

A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [125I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([125I]-GN8-AzPEA) and a [125I]-labeled 2-(4-aminophenyl)ethylamino conjugate ([125]-GN8-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligosaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)8 showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)8, cellopentaose nor alpha-1,4 linked N-acetylgalactosamine octamer (GalNAc)8 at concentrations as high as 25 microM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the 'high-affinity binding site' previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.

N-acetylchitooligosaccharides of degree of polymerization 6 to 8 elicit the synthesis of (1→3)-β-glucan endohydrolase (EC 3.2.1.39) activity in suspension-cultured cells derived from immature barley (Hordeum vulgare) embryos. Corresponding de-acetylated chitooligosaccharides have no effect. Concentrations of N-acetylchitoheptaose in the 200 nM range are sufficient to elicit the response. A 2- to 3-fold increase in (1→3)-β-glucanase activity is detected 24 h after the addition of the oligosaccharide. Non-denaturing gel electrophoresis, coupled with the in situ detection of (1→3)-β-glucanase activity in the gels, has enabled the separation of specific isoforms of the enzyme. The increase in (1→3)-β-glucanase activity following N-acetylchitoheptaose induction is attributable predominantly to enhanced levels of isoenzyme GII, although the barley (1→3)-β-glucanase gene family encodes at least seven different isoforms of the enzyme. Northern hybridization analyses with gene-specific probes confirmed the presence of mRNA encoding isoenzyme GII as the major mRNA in cells treated with the oligosaccharide elicitor. The results therefore demonstrate a specific induction of the (1→3)-β-glucanase isoenzyme GII gene following stimulation of barley cells with oligosaccharides of fungal cell wall origin, and further suggest that a plant's response to microbial attack involves transcription of quite specific members of the gene families that encode pathogenesis-related proteins.

A type II ribosome-inactivating protein (RIP) was isolated from the bark tissue of Japanese elderberry (Sambucus sieboldiana) and named sieboldin-b. Sieboldin-b is a heterodimeric protein consisting of 27- and 33-kDa subunits and showed strong ribosome-inactivating activity in vitro but did not show in vivo toxicity. The amino acid sequence of sieboldin-b deduced from the structure of the cDNA showed that both subunits of sieboldin-b are encoded on a single precursor polypeptide. Sieboldin-b has a structure homologous with the Neu5Ac(alpha 2-6)Gal/GalNAc-specific bark lectin from S. sieboldiana (SSA) and also typical type II RIPs such as ricin and abrin. Detailed analyses of carbohydrate binding properties of sieboldin-b revealed that sieboldin-b binds to Gal/GalNAc, similar to ricin/abrin, in spite of its highly homologous structure with SSA. The biological properties of these toxins/lectins are compared, and the possible explanation for such diversity is discussed.

TV-acetylchitooIigosaccharides (fragments of chitin) elicit defense responses, including phytoalexin production, in suspension-cultured rice cells. They induced rapid and transient membrane depolarization accompanied by a transient increase in net CP-efflux. The membrane depolarization was not affected by anaerobiosis or azide, suggesting that the major part of the depolarization was mediated by ion channels, not by energy-dependent ion pumps. Depolarization was partly inhibited in the presence of Caor Cl~-channel blockers and highly inhibited by depletion of Ca in the extracellular medium. A calcium ionophore, A23187, caused a transient depolarization but not an increase in Cl~ efflux, while it did not inhibit the elicitor-induced transient depolarization and Cl~ efflux. These suggest that the influx of Ca from the extracellular space to the cytoplasm is necessary as an initial trigger but not sufficient for membrane depolarization, Cl~ efflux, and the following signaling processes.

/V-Acetylchitooligosaccharides, fragments of a main backbone polymer of fungal ceil wall, elicit defense responses including phytoalexin production in suspensioncultured rice cells. The purified oligosaccharide triggers rapid, transient membrane depolarization. Ion fluxes induced by the oligosaccharides were analyzed by using ionselective electrodes. Treatment of the cells with the oligosaccharides induced transient efflux of K + and influx of H + immediately after the elicitation. To monitor the pH values of the cytoplasm and the vacuoles noninvasively under a physiological condition, in vivo 31 P-nuclear magnetic resonance spectroscopy was applied to the cells to which oxygenated growth medium was perfused continuously. The cytoplasmic pH showed significant transient decrease, correspondingly. Only the N-acetylchitooligosaccharides with a degree of polymerization higher than 5 were active, whereas deacetylated chitosan oligomers caused no effect. Less than 1 nM of iV-acetylchitoheptaose was sufficient to induce rapid flux of ions. Such strict structural requirements for the induction of ion fluxes were similar to those of specific binding to the putative plasma membrane receptor as well as a series of signaling events specifically induced by the oligosaccharides, suggesting the involvement of transient changes in cytoplasmic ion concentration in oligosaccharide signaling for defense responses.

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.

Bark lectins from the elderberry species belonging to the genus Sambucus have a unique carbohydrate binding specificity for sialylated glycoconjugates containing NeuAc(alpha 2-6)Gal/GalNAc sequence. To elucidate the structure of the elderberry lectin, a cDNA library was constructed from the mRNA isolated from the bark tissue of Japanese elderberry (Sambucus sieboldiana) with lambda gt11 phage and screened with anti-S. sieboldiana agglutinin (SSA) antibody. The nucleotide sequence of a cDNA clone encoding full-length SSA (LecSSA1) showed the presence of an open reading frame with 1902 base pairs, which corresponded to 570 amino acid residues. This open reading frame encoded a signal peptide and a linker region (19 amino acid residues) between the two subunits of SSA, the hydrophobic (A-chain) and hydrophilic (B-chain) subunits. This indicates that SSA is synthesized as a preproprotein and post-translationally cleaved into two mature subunits. Homology searching as well as molecular modeling studies unexpectedly revealed that each subunit of SSA has a highly homologous structure to the galactose-specific lectin subunit and ribosome-inactivating subunit of plant toxic proteins such as ricin and abrin, indicating a close evolutionary relationship between these carbohydrate-binding proteins.

N-acetylchitooligosaccharides, fragments of the backbone of fungal cell wall, trigger rapid membrane responses such as transient depolarization, and elicit defense reactions including phytoalexin production in suspension-cultured rice cells. The generation of reactive oxygen species triggered by the oligosaccharide signal was analyzed with EPR spectroscopy using a spin trapping system, 4-pyridyl 1-oxideN-tert-butyl nitrone (4-POBN) and ethanol. OH generation was detected as the α-hydroxyethyl adduct of 4-POBN after elicitation. Superoxide dismutase, catalase or diethylenetriamine pentaacetic acid, a metal chelator, inhibited $${}^ \cdot O$$ generation, proposing the following reaction sequence: generation of $${}^ \cdot O_{2^ - } $$ in response to the oligosaccharide elicitor, followed by dismutation to H2O2, then generation of $${}^ \cdot OH$$ by the reaction of H2O2 with Fe2+ that is generated by the reduction of Fe3+ by $${}^ \cdot O_{2^ - } $$ . Generation of the same reactive oxygen species was also triggered by calyculin A, a protein phosphatase inhibitor, alone, suggesting the involvement of protein phosphorylation in its regulation during the oligosaccharide signal transduction.

The chain conformation of an arabinoxylan isolated from the rice endosperm cell wall, which was composed of the linear backbone of a 1,4-linked beta-xylose residue highly substituted at the C3 position with alpha-L-arabinofuranose, was studied by X-ray fiber diffraction and a conformational analysis. From the X-ray results the polysaccharide was suggested to take an extended left-handed, three-fold helical structure which is not a desirable conformation to make a complex firmly associated with cellulose or xyloglucan that was elucidated to be present in the cell wall. A conformational analysis, using the PFOS force field for the O-alpha-L-arabinofuranose-(1--3)-beta-D-xylose residue and O-alpha-L-arabinofuranose-(1--3)-[(1--4)-beta-D-xylose residue]3, in which the arabinose residue was linked to the C3 position of the middle residue of the xylotriose residue, and using the PS79 computer program for the arabinoxylan indicated that the backbone xylan chain could not take a two-fold helix similar to that of cellulose because of significant steric interaction between the arabinose side chain and the backbone. Thus, the left-handed, three-fold helix was found to be reasonable.

Chitin is a representative microbe-associated molecular pattern (MAMP) molecule for various fungi and induces immune responses in many plant species. It has been clarified that the chitin signaling in rice requires a receptor kinase OsCERK1 and a receptor-like protein (Os)CEBiP, which specifically binds chitin oligosaccharides. On the other hand, Arabidopsis requires a receptor kinase (At)CERK1 for chitin signaling but it is not clear whether the plant also requires a CEBiP-like molecule for chitin perception/signaling. To clarify the similarity/difference of the chitin receptor in these two model plants, we first characterized CEBiP homologs in Arabidopsis. Only one of three CEBiP homologs, AtCEBiP (LYM2), showed a high-affinity binding for chitin oligosaccharides similar to rice CEBiP. AtCEBiP also represented the major chitin-binding protein in the Arabidopsis membrane. However, the single/triple knockout (KO) mutants of Arabidopsis CEBiP homologs and the overexpressor of AtCEBiP showed chitin-induced defense responses similar to wild-type Arabidopsis, indicating that AtCEBiP is biochemically functional as a chitin-binding protein but does not contribute to signaling. Studies of the chitin binding properties of the ectodomains of At/OsCERK1 and the chimeric receptors consisting of ecto/cytosolic domains of these molecules indicated that AtCERK1 is sufficient for chitin perception by itself.

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.