Physiological and Molecular Plant Pathology (2001) 59, 223±233 doi:10.1006/pmpp.2001.0364, available online at http://www.idealibrary.com on

MINI-REVIEW

Oligosaccharide signalling for defence responses in plant

N. SHIBUYA and E. MINAMI Biochemistry Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan

(Accepted for publication October 2001)

Keywords: elicitor; receptor; OGA; chitin; chitosan; beta-glucan; signal transduction; ion ¯ux; protein phosphorylation; defence response.

INTRODUCTION transduction cascades and elicitor-responsive genes. Several reviews [5, 25, 34, 40] have already been Higher plants have the ability to initiate various defence published on these subjects and in this review the authors reactions such as the production of phytoalexins, try to focus mostly on recent progress. antimicrobial proteins, reactive oxygen species, and reinforcement of cell walls when they are infected by pathogens such as fungi, bacteria and viruses. If these reactions occur in a timely manner, the infection will not CHARACTERISTICS OF OLIGOSACCHARIDE proceed further. However, if the defence reactions occur ELICITORS too late or are suppressed, the infection process will proceed successfully [89]. Thus, it is critically important b-glucan oligosaccharides for plants to detect infecting pathogens e€ectively and Oligosaccharide elicitors derived from the b-glucans of deliver such information intracellularly/intercellularly to pathogenic Oomycetes, such as Phytophthora sojae,have activate their defence machinery. been very well characterized. A doubly-branched hepta- It is believed that the detection of pathogens is b-glucoside that was generated from P. sojae glucan by mediated by chemical substances secreted/generated by partial acid hydrolysis was shown to be a very active the pathogens. Various types of such compounds (elicitor elicitor for glyceollin biosynthesis in soybean cotyledon molecules) including oligosaccharides, (glyco)proteins, cells [86]. From detailed studies with a set of naturally (glyco)peptides and lipids, have been shown to induce obtained as well as synthetic oligosaccharides, it has been defence responses in plant cells and their involvement in shown that soybean cells recognize speci®c features of the the detection of (potential) pathogens in plant has been hepta-b-glucoside structure, including all three non- discussed [5, 24, 34]. Oligosaccharides derived from reducing terminal glucosyl residues and their spacing fungal and plant cell wall polysaccharides are one class of along the backbone of the molecule [10]. A partial well characterized elicitors that, in some cases, can induce hydrolysate of the P. sojae b-glucan also acts as an active defence responses at a very low concentration e.g. nM.In elicitor on various plant cells belonging to the family view of their well-de®ned chemical nature and the Fabaceae, indicating the presence of similar perception presence of highly sensitive perception systems in plants, systems in these plants [14]. In the case of tobacco cells, some of these elicitors have provided good model systems however, the hepta-b-glucoside did not act as an elicitor, to study how plant cells recognize such chemical signals but linear b-1,3-linked glucooligosaccharides (laminar- and transduce them for activation of the defence ioligosaccharides) were active elicitors [47]. Interestingly, machinery. These studies include structure±activity a cyclic 1,3- 1,6-linked b-glucan secreted by a symbiotic relationships of the elicitor molecules, characterization bacterium, Bradyrhizobioum japonicum, was reported to act of the corresponding receptors and analysis of signal as a suppressor for the induction of phytoalexin biosynthesis in soybean by b-glucan elicitor [62]. Abbreviations used in text: A9-C, anthracene 9-carboxylic acid; MBP, mannan binding protein; OGAs, oligogalacturo- There has been little information on the b-glucan nides; TLR, toll-like receptors; 2-D PAGE, two-dimensional gel fragment elicitor that acts on monocots. Inui et al.[38] electrophoresis. reported the induction of chitinase and PAL activity in 0885-5765/01/110223+11 $35.00/00 224 N. Shibuya and E. Minami cultured rice cells by laminarihexaose. Recently Yama- The requirements for the size and structure of active guchi et al.[104] reported that a pentasaccharide (in the chitin fragments are di€erent depending on the experi- reduced form) puri®ed from an enzymatic digest of the b- mental systems. In the case of rice and wheat, larger glucan from the rice blast fungus, Magnaporthe grisea oligosaccharides such as heptamer or octamer are (Pyricularia oryzae), shows potent elicitor activity to induce preferentially recognized and showed detectable activity phytoalexin biosynthesis in suspension-cultured rice cells. even at nM concentrations [3, 103]. On the other hand, in The elicitor-active glucopentaose induced phytoalexin the case of tomato cells, the elicitor activity was almost the biosynthesis in rice cells at 10 nM concentrations. Struc- same for oligosaccharides larger than tetramer [27]. Such tural elucidation of the elicitor-active glucopentaose di€erences in the speci®city probably re¯ect the charac- showed a quite contrasting structure compared to the teristics of the corresponding receptors and will be hepta-b-glucoside that is active on soybean. In the case of explained in future studies of the interaction of these the hepta-b-glucoside elicitor from P. sojae, the backbone oligosaccharides with these receptor molecules. In these is a 1,6-linked b-glucooligosaccharide with branches at plant cells, deacetylated oligosaccharides, chitosan frag- the 3-position of two 6-linked glucosyl residues. In the case ments, did not show elicitor activity. of the elicitor-active b-glucopentaose from M. grisea, the Chitosan or its fragments can induce defence responses backbone oligosaccharide is 1,3-linked and branched at in several plant systems, mostly dicots. Chitosan induces the 6-position of a 3-linked residue. Two related callose formation in soybean and parsley cells [13, 49], glucopentaoses that di€er only in the position of the proteinase inhibitors in tomato leaves [102] and phyto- glucosyl stub showed little elicitor activity, indicating a alexin biosynthesis in pea [31]. However, the concentra- strict recognition of the active oligosaccharide in rice cells. tions required for these responses are usually much higher Comparison of the elicitor activity of the b-glucan than those necessary for activity of chitin oligosaccharides fragments from M. grisea and synthetic hexa-b-glucoside, in other plant systems. The e€ects of the degree of the minimum structural unit for activity of the hepta-b- acetylation as well as the molecular size of chitosan and its glucoside in soybean, using both suspension-cultured rice oligosaccharides were examined with respect to callose cells and soybean cotyledons, indicated that each glucan formation, phytoalexin induction and ligni®cation [2, 43, fragment can only be recognized by the corresponding 99]. The elicitor activity of chitosan seems to be mediated host plants [104]. Although the structural requirement for through the interaction of this polycationic molecule with the recognition by the rice cells must be examined in more negatively charged phospholipids, rather than a speci®c detail, these results indicate clear di€erences in the speci- interaction with a receptor-like molecule [43]. ®city of the corresponding receptors in rice and soybean. It will be interesting from an evolutionary viewpoint to see Oligogalacturonides whether various plants can be grouped depending on their abilities to recognize di€erent b-glucan elicitors. Oligogalacturonides (OGAs) derived from pectic poly- saccharides of plant cell walls have also been known to act as elicitor molecules. OGAs have been shown to induce biosynthesis of phytoalexins in the cotyledons of soybean Chitin and chitosan oligosaccharides [70] and kidney bean [23], proteinase inhibitors in Chitin (b-1,4-linked polymer of N-acetylglucosamine) is tomato leaves [26], and ligni®cation of cucumber a common component of fungal cell walls and its cotyledons [78] and cultured castor bean cells [7]. So fragments, N-acetylchitooligosaccharides, have been far, there has been no report of elicitor activity of OGAs shown to act as potent elicitor signals in several plant in monocot plants, which might be reasonable in the light systems. These include the induction of ligni®cation in of the much lower content of pectic polysaccharides in wheat [3], ion ¯ux and protein phosphorylation in monocot cell walls. OGAs can be generated from pectic cultured tomato cells [27], chitinase activity in melon polysaccharides by partial acid hydrolysis or by the [79], and gene expression of glucanase in cultured barley action of pectinase or pectate lyase. Regardless of the cells [42]. In suspension-cultured rice cells, the action of method of generation, OGAs of DP ˆ 10±12 appear to be chitin fragments has been extensively studied. These the most active component. However, Reymond et al. studies showed the induction by chitin fragments of [77] showed that the ability of OGAs to induce protein enzyme activities involved in the biosynthesis of terpenoid phosphorylation in vitro in tomato increased with the size phytoalexins [75, 103], membrane depolarization [45, of the OGAs even to a DP more than 20. This result 50], Cl and K‡ e‚ux, cytoplasmic acidi®cation [52], indicates that the optimum size of OGAs for elicitor generation of reactive oxygen species [51], biosynthesis of activity observed with plant cells or tissues might re¯ect jasmonic acid [69] and expression of unique early the penetratability of the OGAs, rather than the responsive genes and typical defence related genes speci®city of the recognition. OGAs usually require [60, 68, 92] by chitin fragments. higher concentrations to show elicitor activity compared Oligosaccharide signalling for defence responses 225 to chitin or glucan fragment elicitor. Clear synergism, by enzymatic digestion of the P. sojae glucan (average which might be important to amplify the information on molecular mass was 10 kDa) for preparing the anity site, was observed for OGAs and the hepta-b-glucoside matrix. Using partial amino acid sequence information elicitor for the induction of phytoalexin biosynthesis in from this polypeptide, they obtained a cDNA encoding a soybean cotyledons [21]. polypeptide of 667 amino acids and a calculated mass of 74 424 Da. The predicted sequence had no homology with any known proteins, except some homology with Receptors for oligosaccharide elicitors three gene products from yeast, whose function is Receptor for b-glucan oligosaccharide elicitor. The search for unknown. An 125I-labelled b-glucan oligosaccharide receptors for glucan oligosaccharide elicitors was initiated mixture from P. sojae bound to the crude extracts from by Yoshikawa et al.[106] with the use of a radio-labelled Escherichia coli and suspension-cultured tobacco cells, both elicitor-active polysaccharide, mycolaminaran. Binding of which expressed the cDNA. An antibody to the assays with 125I-labelled, elicitor-active hepta-b-glucoside recombinant protein inhibited half of the binding of the or a mixture of elicitor-active b-glucan fragments has labelled ligand to the membrane preparation from provided a wealth of information on the high-anity soybean cotyledon cells and also inhibited half of the binding site present in membrane preparations from phytoalexin production elicited by the b-glucan frag- soybean roots. The binding speci®city of this site was most ments. From these results, the cDNA was concluded to extensively studied by Cheong and Hahn [11] by using encode a receptor for b-glucan elicitor. Closely related various synthetic oligosaccharides that are structurally cDNAs for glucan elicitor binding protein were recently related to the hepta-b-glucoside. These studies demon- cloned from French bean and soybean by another group strated the presence in membrane preparations from root and reported to contain a single transmembrane domain and other parts of soybean of a single class of high-anity [64, 65]. These recent studies showed that the soybean binding sites for this elicitor, whose speci®city correlated protein expressed in trans in tomato cells, bound the . very well with the elicitor activity of various b-glucan hepta-b-glucoside elicitor with a Kd of 4 5nM and showed oligosaccharides in the soybean cotyledon assay [11, 17, binding characteristics roughly similar to those observed 18]. Density gradient centrifugation indicated the enrich- for the microsomal fraction from soybean. However, the ment of the high-anity binding sites mostly in the soybean protein was inactive when expressed in trans in plasma membrane fraction [9, 84]. Similar high-anity insect cells [65]. An antibody generated against the insect binding sites for b-glucan elicitor were also detected in cell-expressed protein could eliminate over 80 % of hepta- the membrane preparation from the plant cells belonging b-glucoside binding activity from the solubilized mem- to the family Fabaceae that responded to the elicitor [14]. brane protein preparation. From these results the authors Coà te et al.[20] recently showed that two model legumes, suggested that the cDNA encodes the hepta-b-glucoside Medicago truncatula and Lotus japonicus, both possess hepta- binding protein identi®ed previously. b-glucoside binding sites in their membranes, but the The progress achieved to date provides an important speci®city of these sites seems to be di€erent from the clue to clarify the function of the 75 kDa elicitor-binding soybean site. protein in the signal transduction pathway activated by Photoanity labelling experiments using the solubil- the b-glucan oligosaccharide elicitor. To assess the ized microsomal proteins from soybean roots detected a biological function of this protein further, it will be major band of approx. 70 kDa as a candidate poly- necessary to answer the following questions. Firstly, the peptide for the binding protein [16]. biological function of the gene/protein should be more Various attempts have been made to solubilize and clearly demonstrated, hopefully with the use of transgenic purify the binding protein. These studies resulted in the plants or mutants in this gene. Secondly, it would also be identi®cation of e€ective detergents such as ZW 3±12 very interesting to know how the 75 kDa protein can and n-dodecanoylsucrose for the solubilization of the function as a receptor molecule, because this protein elicitor binding activity [9, 15]. From the analysis of the seems to have no domains/motifs typical for known behavior of the solubilized fraction on gel ®ltration, it was receptor families. Further, it would be desirable to assumed that the binding protein is present as a kind of examine the binding speci®city of the transgenically multimeric complex [9]. By using the anity chroma- expressed protein in more detail to assure that its glucan tography with the immobilized elicitor-active b-glucan elicitor binding characteristics coincide with those deter- fragments, MithoÈ fer et al.[66] succeeded in purifying the mined previously for the site in soybean membranes. 75 kDa protein that behaved as 240 kDa protein under No information is available for the glucan elicitor- native conditions. binding protein in rice, although the inability of the Umemoto et al.[97] also puri®ed a glucan elicitor- elicitor-active glucan fragments to inhibit binding com- binding protein from soybean roots using a similar petitively of the radio-labelled chitin oligosaccharide to approach, but used a mixture of oligosaccharides released plasma membranes of rice cells indicates the presence of 226 N. Shibuya and E. Minami di€erent binding sites/proteins for these two oligosacchar- supports a functional role of these binding proteins in the ide elicitors (Yamaguchi et al., unpublished work). perception of the oligochitin elicitor signal. Detection of Comparison of the characteristics of the glucan elicitor- the oligochitin binding protein in soybean is especially binding proteins of soybean and rice should be of great interesting because oligochitin perception, in addition to interest because of the di€erences in the responses of these the perception of lipochitin oligosaccharides (Nod plants to di€erent glucan fragments from corresponding factors), has been suggested to play a role in the initiation pathogenic fungi. of nodulation in legume plants [22, 59]. Cloning of the gene encoding the oligochitin elicitor- binding protein will not only reveal the structure of this Receptor for chitin oligosaccharide elicitor protein but also make it possible to analyse the biological/ A single class of high-anity binding site for chitin biochemical function of this protein by the use of a oligosaccharide elicitor was detected in the microsomal/ suitable expression system. It will also be interesting to plasma membrane preparation from suspension-cultured compare the characteristics of the glucan and chitin rice cells [87, 88], tomato cells [4] and soybean root and elicitor-binding proteins. cultured cells [22] by using radio-labelled, elicitor-active derivatives of the oligosaccharide. The binding speci®city Signal transduction of these sites corresponded well with the elicitor activity of chitin oligosaccharides in these systems, re¯ecting the Most of the defence reactions induced by oligosaccharide di€erent preferences of these plants for the size of the elicitors are also observed when plant cells are treated with oligosaccharides, as discussed already. The binding sites non-saccharide elicitors or infected with pathogens. One of in rice and soybean cells were shown to be mostly the major di€erences is that oligosaccharide elicitors, in concentrated in the plasma membrane [22, 87]. contrast to at least some other elicitors, do not induce the Photoanity labelling as well as anity cross-linking hypersensitive reaction (HR) leading to cell death. For experiments with 125I-labelled derivatives of N-acetyl- example, fungal xylanase from Tricoderma viride [105]ora chitooctaose revealed the presence of a high-anity secreted proteinaceous elicitor, INF1 elicitin from P. infes- binding protein with the size of 75 kDa in the plasma tans [82], induces the HR in suspension-cultured tobacco membrane from suspension-cultured rice cells and also cells, whereas OGAs do not. In suspension-cultured rice from rice leaves and roots [39, 72]. The speci®city of the cells, ¯agellin from Pseudomonas avena induces the HR [8], labelling of this protein again coincided with the binding but N-acetylchitooligosaccharides do not (Tanabe et al., speci®city of the membrane preparation and also with the unpublished work). In plant±pathogen interactions, the elicitor activity of the chitin oligosaccharides. The HR requires a correct combination of an avirulence gene coincidence of the binding speci®city with the biological of the pathogen and a corresponding resistance gene of the activity of the oligosaccharides as well as its localization in host plant. Until now, a number of pathogen-derived the plasma membrane strongly suggest that the 75 kDa elicitors causing HR have been characterized but no protein is the receptor molecule, or a part of the receptor oligosaccharide elicitors are included in this group. complex, for this elicitor. The earliest events after the recognition of elicitor Binding experiments as well as anity cross-linking molecules are membrane depolarization and ion ¯uxes. In with these radio-labelled oligochitin ligands detected the tobacco suspension cells, a rapid change in the membrane presence of high-anity binding sites as well as the potential, in¯ux of Ca2‡ and H‡ e‚ux of K‡, which corresponding binding proteins in plasma membrane resulted in cytoplasmic acidi®cation and extracellular preparations from several plants including soybean [22], alkalinization, were induced by OGAs [57]. The relation- cultured barley and carrot cells and wheat leaves (Okada ships between these events are unclear, except that the K‡ et al., unpublished work). Interestingly, these plant cells e‚ux was dependent on the extracellular Ca2‡, which can were also shown to respond to the chitin oligosaccharide be replaced with Mg2‡, indicating that the K‡ e‚ux is elicitor through the generation of reactive oxygen species the downstream event of Ca2‡ in¯ux. OGA-induced or the initiation of ligni®cation. In contrast, binding sites cytoplasmic acidi®cation was prevented by pre-treatment or binding proteins for oligochitins were not detected in with the protein kinase inhibitors, staurosporine and the membrane preparations from a cell line of tobacco dimethylaminopurine, while a protein phosphatase BY-2, which also did not respond to this elicitor in the inhibitor, calyculin A, induced cytoplasmic acidi®cation assay system. These results, along with the results for rice by itself. These results suggest that protein kinase(s)/ and tomato, indicate the distribution of similar oligo- phosphatase(s) sensitive to these inhibitors regulate the chitin perception systems among various plants belonging H‡ ¯ux across the plasma membrane [58]. to both monocots and dicots. The parallelism between N-acetylchitooligosaccharides of DP 4 or higher the presence of the oligochitin, binding protein and the induced extracellular alkalinization and the rapid phos- responsiveness of these plant cells to this elicitor again phorylation of proteins in suspension-cultured tomato Oligosaccharide signalling for defence responses 227 cells [27]. The extracellular alkalinization was transient Ca2‡ and abolished in the presence of La3‡, an inhibitor and ®nished within 10 min. It was signi®cantly inhibited of Ca2‡ channel. by the addition of the protein kinase inhibitor, K-252a, What are the roles of these early events in the elicitor- and induced by calyculin A, suggesting again that the activated signal-transduction pathway? Or how is the H‡ in¯ux is dependent on protein phosphorylation/ signal transmitted to later defence reactions? It has been dephosphorylation events. Interestingly, tomato cells well described that Ca2‡ plays important roles as a treated with N-acetylchitooligosaccharides did not second messenger in a variety of biochemical processes in respond to a second treatment with the same elicitor for plants [81] including defence reactions [83]. An inhibitor up to 8 h, i.e. stayed in a refractory stage, although the of anion channels, A9-C (anthracene 9-carboxylic acid), cells were still able to respond to another elicitor, T. viride has also been shown to inhibit many defence reactions xylanase, and alkalinized the medium. Another interest- [36, 41, 68]. It is likely that rapid changes in the ionic ing observation is that the addition of xylanase induced environment of the cytoplasm of the plant cells could later cellular responses such as the activation of PAL and modify enzyme activities, leading to the metabolic biosynthesis of ethylene in tomato cells [29], while N- changes. In this context, it is interesting that production acetylchitooligosaccharides seem to induce only early of secondary metabolites is enhanced in response to cellular responses, e.g. alkalinization of the medium and changes in cytoplasmic pH induced by the addition of protein phosphorylation, in tomato cells [27]. orthovanadate [33, 35, 90], an inhibitor of H‡-ATPase, In contrast, N-acetylchitooligosaccharides of DP 7 and H‡-speci®c ionophores [32], or elicitor [80]. 8 induced a variety of defence responses in suspension- In tobacco cells, it was shown that the level of mRNAs cultured rice cells as already described. N-acetylchito- for two key enzymes of secondary metabolism, PAL and heptaose induced a transient depolarization of the plasma 3-hydroxy-3-methylglutaryl CoA reductase, increased by membrane with a lag time of 20±60 s [45]. The treatment with propionic acid, inorganic phosphate, or a depolarization required the presence of extracellular H‡-ATPase inhibitor, erythrosine B, all of which induced Ca2‡ that could not be replaced with Mg2‡. The cytoplasmic acidi®cation [53]. In rice cells, activation of a depolarization was not a€ected by anaerobic treatment speci®c set of elicitor-responsive genes was observed in or the addition of azide, indicating that ion channels, but response to the addition of propionic acid or butyric acid, not energy-dependent ion pumps, have a central role in both of which signi®cantly acidi®ed the cytoplasm [36]. this process. However, none of the tested inhibitors of These results indicate that cytoplasmic acidi®cation or Ca2‡ channels and anion channels completely cancelled modi®cation of the electrochemical potential, such as the the depolarization. Thus, it was dicult to specify which H‡ gradient across the plasma membrane, could be a ion channels are responsible for the depolarization, signal for the induction of defence reactions. However, though the involvement of ion channel(s) that have rather some reports do not support this hypothesis. In soybean broad speci®city, as demonstrated in cultured parsley cells cells, cytoplasmic acidi®cation was not observed for at [108], was suggested. Recently, pore-forming activity least the ®rst 30 min when the pH of the OGAs was within plasma membranes was shown for yeast elicitor adjusted to that of the media, although whether or not fractions in parsley cells [48]. It is tempting to speculate later defence reactions occurred in the same experimental that other elicitors including oligosaccharide elicitors system were not examined [37]. In tomato cells carrying might have similar activities. In conclusion, Ca2‡ appears the host resistance gene, CF5, which confers resistance to essential for the elicitor-induced depolarization in rice Cladosporium fulvum carrying an avirulence gene, avr5, the cells. However, its mode of action is not clear at present. In plasma membrane H‡-ATPase was activated by addition this experimental system, N-acetylchitoheptaose also of a crude elicitor fraction prepared from the fungus induced transient e‚ux of K‡ and Cl, and in¯ux of including the avr5 product and the pH of the culture H‡, leading to the acidi®cation of the cytoplasm and medium decreased, implying the occurence of the concomitant alkalinization of the culture medium [45, cytoplasmic alkalinization, although this was not con- 52]. The H‡ in¯ux was inhibited by K-252a and induced ®rmed [100]. The di€erences in the various reports on by calyculin A in the absence of the elicitor, analogous to elicitor-induced cytoplasmic acidi®cation could be the response of tobacco cells to treatment with OGAs [58], ascribed to di€erences in the experimental conditions as indicating the involvement of protein phosphorylation/ well as to the plant species used for these experiments. dephosphorylation in the regulation of this response [36]. In tobacco cells, cytoplasmic acidi®cation was transi- Similar ion ¯uxes were observed in soybean cells ently induced when cells were treated with puri®ed treated with the b-glucan elicitor [24]. The dynamics of OGAs, whereas it was sustained for hours when cells were the cytosolic Ca2‡ levels were analysed using soybean treated with a crude preparation of the culture media of cells transformed with a Ca2‡ monitoring protein, P. megasperma. Regardless of the di€erent time pro®les of aequorin [63]. The change of Ca2‡ level induced by cytoplasmic acidi®cation, both OGA and the crude the elicitor was biphasic, dependent on the extracellular elicitor fraction induced PAL and o-diphenolmethyl 228 N. Shibuya and E. Minami transferase [30]. In general, the cytoplasmic acidi®cation class I chitinase gene activation by N-acetylchitoheptaose induced by addition of acid or inhibitors of H‡-ATPase is was inhibited by K-252a [68]. In cultured parsley cells persistent, whereas it is transient when induced by OGA and protoplasts, addition of K-252a activated the in tobacco cells [57]orbyN-acetylchitooligosacharides in synthesis of a phenylpropanoid phytoalexin, ¯ano- rice cells [52]. These observations lead us to speculate coumarin, and okadaic acid inhibited the synthesis that the rapid decrease in pH is the signal for the induced by a proteinaceous elicitor [76]. Irrespective of induction of defence reactions. the direction of the regulation, positive or negative, At present, the molecular basis of how the pH signal is protein phosphorylation/dephosphorylation appear to transmitted further within the cell has not been revealed. play critical roles in elicitor signalling. In tobacco cells, activation of a MAP kinase was observed In order to understand the molecular mechanisms of following cytoplasmic acidi®cation induced by acetic acid the elicitor-signal transduction, it is essential to identify or butyric acid [94]. MAP kinases play essential roles in a which proteins are phosphorylated or dephosphorylated wide range of biological processes including stress in the cell. In tobacco cells, a MAP kinase was phos- responses in plant cells [55, 85, 91, 98, 107]. In tobacco phorylated at its tyrosin residue after treatment with a cells treated with an elicitor fraction from the cell wall of crude elicitor-fraction from the fungal cell wall [91]. P. infestans, a rapid and transient activation of a MAP Similar results were shown in soybean cells treated with kinase was shown [91]. In cultured parsley cells treated OGA7±12 [93]. These phosphorylations were essential to with an oligopeptide elicitor, Pep-13, similar activation of the kinase activities. A proteinaceous elicitor, cryptogein, a MAP kinase and its re-location to the nucleus were was shown to induce phosphorylations of at least observed [55]. In the signalling pathway(s) activated by 19 proteins by in vivo 32P-labelling of tobacco cells followed oligosaccharide elicitors, similar activation of MAP kinase by two-dimensional gel electrophoresis (2-D PAGE) [54]. is likely but has not been demonstrated yet. By combination of 2-D PAGE and mass spectrometoric Many reports using di€erent elicitors and cells support analysis, a 43 kDa polypeptide, AtPhos43, which was the hypothesis that elicitor-signalling pathways involve phosphorylated in Arabidopsis cells immediately after protein phosphorylation/dephosphorylation steps [5]. In addition of a ¯agellin-derived peptide elicitor, ¯g22, was these works, the e€ects of protein kinase inhibitors, such shown to contain ankyrin-repeat. Two orthologous as K-252a or staurosporin, or protein phosphatase proteins were found to be rapidly phosphorylated in rice inhibitors, such as calyculin A or okadaic acid, have cells treated with N-acetylchitohexaose [73]. These been examined, and, in most plant systems with some proteomic approaches can be a powerful tool for both exceptions, protein kinase inhibitors blocked and protein the detection and identi®cation of the phosphorylated (or phosphatase inhibitors stimulated the defence reactions. dephosphorylated) proteins in response to environmental Inhibitors of protein phosphatase 1 and 2A have been stimuli including elicitors. reported to be the most e€ective in mimicing some part of plant defence reactions [28, 56]. The most e€ective inhibitors often vary depending on the plant species. For Potential role of oligosaccharide elicitor signalling the defence example, calyculin A, okadaic acid (both speci®c to 1 and machinery in plants 2A type) and cantharidin (speci®c to 2A type) are active in soybean cells [56], whereas only calyculin A is active in What is the function of oligosaccharide signalling in rice cells (Minami and Shibuya, unpublished work). In plants? It is dicult to answer this question thoroughly at soybean cotyledons, many kinds of phosphatase 1 and 2A this stage. However, it has been shown at a minimum that inhibitors potentially induced the biosynthesis of a many plants are equipped with sophisticated systems by ¯avonoid compound, daidzein [56], which is a precursor which they can respond to certain oligosaccharide of the soybean phytoalexin, glyceollin. Since the hepta-b- elicitors and activate a set of defence reactions. Concern- glucoside elicitor also induces the biosynthesis of glyceol- ing the possibility of the generation of such oligosacchar- lin in soybean cotyledons, it could imply that these ides at the site of infection, many plants have been shown inhibitors act on signalling steps that are involved in the to produce chitinases and glucanases constitutively or signal transduction pathway activated by the hepta-b- inducibly as a part of their defence machinery [19]. These glucoside. enzymes have been indicated to act on the (potential) It should be noted that some results are not consistent pathogens and inhibit their growth, but they can also with the e€ects of protein kinase/phosphatase inhibitors. generate various fragments that in turn can act as elicitors In rice cells, activation of a class II chitinase gene by an in the host plants, which then results in the induction/ elicitor fraction from the rice blast fungus, Magnaporthe ampli®cation of defence reactions. In addition, invading grisea, was inhibited by the protein phosphatase inhibi- microbes themselves may generate such cell wall tors, odakaic acid or calyculin A, but not by the protein fragments, as during spore germination of P. sojae [101]. kinase inhibitors, K-252a or staurosporin [46], whereas For OGA, many pathogenic microbes are known to Oligosaccharide signalling for defence responses 229 produce polygalacturonase or pectate lyase that can old, broader detection system in combination with a more release OGA from the cell walls of host plants [19]. developed, speci®c system in a whole defence array can be These observations indicate that oligosaccharide signal- seen in vertebrates. Mannan-binding protein (MBP) is a ling has the potential to function in plant defence. circulating component of mammalian serum and can Evolutionary conservation of oligosaccharide signalling bind to potential pathogens such as yeast. When MBP pathways in various types of plants also supports the binds to such microbes or cell surface macromolecules, biological importance of these systems. Interestingly, the complex can activate the complement system in the the use of di€erences in cell surface carbohydrates for absence of an antigen±antibody complex that is required the detection of self and non-self, and for further defence for the activation through the ``classical pathway'' [95]. reactions, are a common strategy for various living Thus, MBP seems to function as a complement to the systems. For example, chitin or fungal b-glucans, and immune system, which takes time for full activation. their fragments, have been known to induce defence Another example is the close connection between innate responses in vertebrates [61, 96] as well as non-vertebrates and acquired immunity through toll-like receptors [67]. Chitinase and glucanase, which degrade poly- (TLRs). Rapidly growing information on this ®eld saccharides not existing on the cell surfaces of plants and indicates that activation of the innate immune system vertebrates, have been considered to be important by various molecules from (potential) pathogenic components for defence systems. Recent identi®cation of microbes, such as LPS, ¯agellin, zymosan etc. plays an a chitinase in human serum has also been interpreted in a important role for the activation of acquired immune similar context [6]. Various types of lectin-like molecules system [1]. Somewhat analogous relationships between which bind to chitin or its fragments are also distributed race cultivar speci®c resistance and basic resistance in among higher plants [12, 74], non-vertebrates [44], and plant may be imagined. even bacteria [71] and have also been discussed for their Apart from their biological function in vivo, oligo- involvement in defence machinery. saccharide elicitors provide an excellent model system to Plant resistance mechanisms against (potential) patho- study the perception and transduction of external signals gens are often classi®ed into two categories. That is, a to induce defence responses in plant cells. The knowledge very speci®c resistance response between a speci®c race gained by such studies also provides a base for the carrying an avirulence gene and a cultivar carrying the development of novel agrochemicals for disease control corresponding resistance gene, and a more general one and also for the development of disease-resistant crops by that gives resistance to the majority of potential regulating the defence system in plant through genetic pathogens [5, 25, 34, 40, 89]. Potential coverage of manipulation. wide varieties of pathogens by oligosaccharide elicitors *c 2001 Government of Japan indicates the involvement of this system, as well as other ``non-speci®c elicitors'', in the latter type of mechanism, which is often called general resistance, basic incompat- ibility, or non-host resistance. The phrase ``non-speci®c REFERENCES elicitors'' may lead to misunderstandings about the features of this group of elicitors. These elicitors seem to 1. 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