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Plant growth-promoting rhizobacterial (PGPR) strains R62 and R81 have previously been isolated and characterized as part of the Indo-Swiss Collaboration in Biotechnology. Here we present the draft genome sequences of these two PGPR strains, with the aim of unraveling the mechanisms behind their ability to promote wheat growth.

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Recent studies have suggested that bacterial volatiles play an important role in bacterial-plant interactions. However, few reports of bacterial species that produce plant growth modulating volatiles have been published, raising the question whether this is just an anecdotal phenomenon. To address this question, we performed a large screen of strains originating from the soil for volatile-mediated effects on Arabidopsis thaliana. All of the 42 strains tested showed significant volatile-mediated plant growth modulation, with effects ranging from plant death to a sixfold increase in plant biomass. The effects of bacterial volatiles were highly dependent on the cultivation medium and the inoculum quantity. GC-MS analysis of the tested strains revealed over 130 bacterial volatile compounds. Indole, 1-hexanol and pentadecane were selected for further studies because they appeared to promote plant growth. None of these compounds triggered a typical defence response, using production of ethylene and of reactive oxygen species (ROS) as read-outs. However, when plants were challenged with the flg-22 epitope of bacterial flagellin, a prototypical elicitor of defence responses, additional exposure to the volatiles reduced the flg-22-induced production of ethylene and ROS in a dose-dependent manner, suggesting that bacterial volatiles may act as effectors to inhibit the plant's defence response.

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Since the 1995 discovery of the broad iron K-line emission from the Seyfert galaxy MCG-6-30-15 (ref. 1), broad iron K lines have been found in emission from several other Seyfert galaxies, from accreting stellar-mass black holes and even from accreting neutron stars. The iron K line is prominent in the reflection spectrum created by the hard-X-ray continuum irradiating dense accreting matter. Relativistic distortion of the line makes it sensitive to the strong gravity and spin of the black hole. The accompanying iron L-line emission should be detectable when the iron abundance is high. Here we report the presence of both iron K and iron L emission in the spectrum of the narrow-line Seyfert 1 galaxy 1H 0707-495. The bright iron L emission has enabled us to detect a reverberation lag of about 30 s between the direct X-ray continuum and its reflection from matter falling into the black hole. The observed reverberation timescale is comparable to the light-crossing time of the innermost radii around a supermassive black hole. The combination of spectral and timing data on 1H 0707-495 provides strong evidence that we are witnessing emission from matter within a gravitational radius, or a fraction of a light minute, from the event horizon of a rapidly spinning, massive black hole.

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The mutualistic symbiosis between flowering plants and arbuscular mycorrhizal fungi is extremely abundant in terrestrial ecosystems. In this symbiosis, obligately biotrophic fungi colonize the root of the host plants, which can benefit from these fungi by enhanced access to mineral nutrients in the soil, especially phosphorus. One of the main goals of research on this symbiosis is to find plant genes that control fungal development in the host plant. In this work, we show that mycorrhizal colonization is regulated by enod40, an early nodulin gene known to be involved in the nodule symbiosis of legumes with nitrogen-fixing bacteria. Medicago truncatula plants overexpressing enod40 exhibited stimulated mycorrhizal colonization in comparison with control plants. Overexpression of enod40 promoted fungal growth in the root cortex and increased the frequency of arbuscule formation. Transgenic lines with suppressed levels of enod40 transcripts, likely via a cosuppression phenomenon induced by the transgene, exhibited reduced mycorrhizal colonization. Hence, enod40 might be a plant regulatory gene involved in the control of the mycorrhizal symbiosis.

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In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properly addressed. At present, there is no "model system" to study these aspects in detail. Such a system with Lotus japonicus and the broad host range Rhizobium sp. NGR234 has been developed. L. japonicus L. cv. Gifu was inoculated with Rhizobium sp. NGR234 and grown over a 12 week time period. The first nodules could be harvested after 3 weeks. Nodulation reached a plateau after 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant. Nodules were harvested and homogenized at different stages of plant development. Microscopic inspection of the extracts revealed that, typically, nodules contained c. 15x10(9) bacteroids g(-1) FW, and that about 60% of the bacteroids were viable as judged by vital staining. When aliquots of the extracts were plated on selective media, a substantial number of "colony-forming units" was observed in all cases, indicating that a considerable fraction of the bacteroids had the potential to redifferentiate into growing bacteria. In nodules from the early developmental stages, the fraction of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slightly when the plants started to flower. In order to see how nodule senescence affected the survival and redifferentiation potential of bacteroids, some plants were placed in the dark for 1 week. This led to typical symptoms of senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins. However, surprisingly, the number of total and viable bacteroids g(-1) nodule FW remained virtually constant, and the fraction of total bacteroids yielding CFUs did not decrease but significantly increased up to 75% of the bacteroids judged to be viable after extraction. This result indicates that during nodule senescence bacteroids might be induced to redifferentiate into the state of free-living, growing bacteria.

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Flagellin, the main building block of the bacterial flagellum, acts as potent elicitor of defense responses in different plant species. Genetic analysis in Arabidopsis thaliana identified two distinct loci, termed FLS1 and FLS2, that are essential for perception of flagellin-derived elicitors. FLS2 was found to encode a leucine-rich repeat transmembrane receptor-like kinase with similarities to Toll-like receptors involved in the innate immune system of mammals and insects. Here we used a radiolabeled derivative of flg22, a synthetic peptide representing the elicitor-active domain of flagellin, to probe the interaction of flagellin with its receptor in A. thaliana. The high affinity binding site detected in intact cells and membrane preparations exhibited specificity for flagellin-derived peptides with biological activity as agonists or antagonists of the elicitor responses. Specific binding activity was measurable in all ecotypes of A. thaliana that show sensitivity to flagellin but was barely detectable in the flagellin-insensitive ecotype Ws-0 affected in FLS1. A strongly impaired binding of flagellin was observed also in several independent flagellin-insensitive mutants isolated from the flagellin-sensitive ecotype La-er. In particular, no binding was found in plants carrying a mutation in the LRR domain of FLS2. These data indicate that the formation of functional receptor-binding sites depends on genes encoded by both loci, FLS1 and FLS2. The tight correlation between the presence of the binding site and elicitor response provides strong evidence that this binding site acts as the physiological receptor of flagellin.

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Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.

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To find out more about the interaction between potato and Phytophthora infestans at the molecular level, we screened for genes induced early after infection using mRNA differential display. Among the twenty cDNA clones recovered in the screen, two were found to represent plant genes whose transcript levels increased during infection of intact plants. These two genes differed strikingly in their response to wounding. Stprx2, a putative peroxidase, responded slowly and transiently to wounding, and its expression pattern was similar to that of gst1, a well-described pathogen-induced gene of potato. The second gene, StNAC, was induced rapidly and strongly after wounding but not systemically. Transcript levels reached a maximum after around 1 h and returned to basal levels after ca. 24 h. StNAC has strong similarity to the ATAF subfamily of NAC domain proteins, a large family of putative transcriptional activators. Arabidopsis ATAF1 and ATAF2 were also shown to be induced by wounding. This implies that the ATAF genes are not merely structurally similar but also share a conserved role in stress responses.

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Carbohydrate metabolism and symbiont survival were studied in nodules of soybean (G. max [L.] Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101), induced to senesce simultaneously by application of the photosynthesis inhibitor dichloromethyl urea (DCMU). The plant-borne carbohydrates sucrose and starch started to decline after 2 d and reached background levels after 8 d, in parallel with the decline of nitrogenase. However, the microsymbiont-borne disaccharide trehalose declined only by about 40% and subsequently remained at a constant level of c. 6 mg x g(-1) dry weight up to 14 d, when nodules softened and decayed. The number of re-isolated viable bacteria was not significantly decreased in senescent nodules as compared to control nodules. These results indicate that during terminal senescence of nodules an appreciable part of the bacteria conserve their trehalose pools and survive.

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The perception of microbial signal molecules is part of the strategy evolved by plants to survive attacks by potential pathogens. To gain a more complete understanding of the early signaling events involved in these responses, we used radioactive orthophosphate to pulse-label suspension-cultured cells of Arabidopsis in conjunction with two-dimensional gel electrophoresis and mass spectrometry to identify proteins that are phosphorylated rapidly in response to bacterial and fungal elicitors. One of these proteins, AtPhos43, and related proteins in tomato and rice, are phosphorylated within minutes after treatment with flagellin or chitin fragments. By measuring (32)P incorporation into AtPhos43 immunoprecipitated from extracts of elicitor-treated hormone and defense-response mutants, we found that phosphorylation of AtPhos43 after flagellin treatment but not chitin treatment is dependent on FLS2, a receptor-like kinase involved in flagellin perception. Induction by both elicitors is not dependent on salicylic acid or EDS1, a putative lipase involved in defense signaling.

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In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.

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Transgenic soybean (Glycine max) culture cells expressing apoaequorin, a Ca2+ indicator, were exposed to glucan fragments derived from Phytophthora sojae or to chitin oligomers. The effects of these elicitors on cytosolic Ca2+ concentrations and on mRNA levels of two beta-tubulin isoforms, tubB1 and tubB2, were investigated. The glucan elicitors, to which the cells are known to react with a biphasic cytosolic Ca2+ increase, induced a down-regulation of the tubB1 mRNA levels while the tubB2 mRNA level remained constant. The decrease of tubB1 mRNA level was observed after 1 hour of glucan treatment. In contrast, chitin oligomers, known to provoke a monophasic Ca2+ increase of short duration, did not affect the tubB1 mRNA level. Pre-incubation with 10 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, an extracellular Ca2+ chelator, blocked the cytosolic Ca2+ increase as well as the decrease of tubB1 mRNA levels induced by glucan elicitors. Likewise, pre-incubation with 1 mM neomycin, which reduced only the second glucan-induced Ca2+ peak, blocked the decrease of tubB1 mRNA level. Experiments with cordycepin, a transcription inhibitor, indicated that glucan fragments induced the degradation of tubB1 mRNA. In conclusion, the glucan-induced cytosolic Ca2+ changes are correlated with a strong increase in tubB1 mRNA degradation.

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Trehalase is ubiquitous in higher plants. So far, indications concerning its function are scarce, although it has been implicated in the detoxification of exogenous trehalose. A putative trehalase gene, T19F6.15, has been identified in the genome sequencing effort in Arabidopsis. Here we show that this gene encodes a functional trehalase when its cDNA is expressed in yeast, and that it is expressed in various plant organs. Furthermore, we present results on the distribution and activity of trehalase in Arabidopsis and we describe how inhibition of trehalase by validamycin A affects the plants response to exogenous trehalose (alpha-D-glucopyranosyl-[1, 1]-alpha-D-glucopyranoside). Trehalase activity was highest in floral organs, particularly in the anthers (approximately 700 nkat g(-1) protein) and maturing siliques (approximately 250 nkat g(-1) protein) and much lower in leaves, stems, and roots (less than 50 nkat g(-1) protein). Inhibition of trehalase in vivo by validamycin A led to the accumulation of an endogenous substance that had all the properties of trehalose, and to a strong reduction in sucrose and starch contents in flowers, leaves, and stems. Thus, trehalose appears to be an endogenous substance in Arabidopsis, and trehalose and trehalase may play a role in regulating the carbohydrate allocation in plants.

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Summary Suppression Subtractive Hybridization (SSH) was applied in a search for genes induced during the compatible interaction between Phytophthora infestans and potato. Using potato leaves that had been treated with benzo(1,2,3)thiadiazole-7-carbothioic acid S-methylester (BTH) as the control tissue, a low redundancy library with a relatively low frequency of the classic plant Pathogenesis-Related (PR) genes was generated. 288 of the clones were screened for induced sequences using Inverse Northern analysis (hybridizing the arrayed clones with radiolabelled cDNA populations). Of the 75 clones that were detectable by this method, 43 appeared to be induced. Eleven of these clones were then analysed by total RNA blot analysis, and elevation of transcript levels during P. infestans infection was confirmed for 10 of them. Some of the cDNAs analysed by RNA blot analysis have homology to genes already known to be induced during infection, e.g. to beta-1,3-glucanase. Another group of cDNAs have homology to enzymes involved in detoxification: gamma-glutamylcysteine synthetase, cytochrome P450, glutathione S-transferase and an MRP-type ABC transporter. Other infection induced cDNAs encode putative proteins that have not previously been reported to be induced by infection: e.g. the ER-located chaperone BiP, and a homologue of Aspergillus nidulans SudD, which was isolated as a suppressor of a mutation in chromosome disjunction. The differential library therefore presents the opportunity to analyse the metabolic changes occurring during infection, and the disease process itself in more detail.

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Purified basic chitinase or ß-1,3-glucanase or a combination of the two enzymes were applied to hyphae of the arbuscular mycorrhizal fungus Glomus mosseae grown in vitro. Chitinase applied to the hyphal tip produced an inhibition of hyphal extension, lysis of the apex and alterations of the growth pattern of the fungus. No effect was observed, however, when chitinase was applied to subapical parts of the hyphae or when glucanase was applied to any part of the hyphae. Application of a combination of the two enzymes to the hyphal tip produced an effect similar to that of chitinase alone.

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To study the responses of forests to global change, model ecosystems consisting of beech and spruce trees were maintained in open top chambers for 4 years under four conditions, namely with normal and elevated CO2 and with low and high nitrogen input, each replicated four times. Each open top chamber (height 3 m, diameter 3 m, soil depth 1.5 m) contained two separate soil compartments containing nutrient-poor siliceous and nutrient-rich calcareous soil. Here, we focus on the fine roots and the soil microbial community in these model ecosystems. At the time of planting, the fine roots were cut back according to forestry practice, and the newly formed roots were colonized by the indigenous soil microflora. After 4 years, the total biomass of fine roots, when averaged over all treatments, was almost the same in each of the two soil types; it was highest in the top 100 mm of soil (60%) and decreased sharply in deeper soil layers. Fungal biomass associated with the fine roots, consisting mainly of ectomycorrhizal fungi, was estimated using the ergosterol content as a marker. It was much higher in fine roots in the siliceous than in the calcareous soil, indicating considerably enhanced ectomycorrhiza formation in the nutrient-poor siliceous soil. Elevated atmospheric CO2 stimulated fine root production by ca. 85% and 43% in the top 100 mm of calcareous and siliceous soils respectively. Increased nitrogen input caused a slightly reduced production of fine root biomass in the calcareous soil but increased it by 33% in the siliceous soil. Marker substances for microorganisms were analyzed in the root-free soil. The amounts of carbon released by fumigation/extraction (a general marker for microbial biomass) and chitin (a marker for fungal biomass) were significantly higher in the top layer of the siliceous than of the calcareous soil, but they did not respond significantly to the treatments with elevated CO2 or the nitrogen fertilizer. The total concentration of the phospholipid fatty acids (PLFAs) and the number of the PLFAs did not differ between the two soil types. However, four of the eight most abundant PLFAs differed significantly between the two soil types. Principal component analysis revealed clearly separated clusters for the two soil types. Although analysis did not reveal any significant changes in response to the treatments, the concentration of the PLFA typical for ectomycorrhizal fungi was significantly higher under conditions of elevated CO2 in the nutrient-rich calcareous soil.

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Cells of tomato (Lycopersicon esculentum) growing in suspension gradually depleted their culture medium and caused a steady decrease in its osmolality. When confronted with a sudden change in medium osmolality (a hypo-osmotic or hyperosmotic shock), respectively, these cells responded with volume changes and stress symptoms such as rapid extracellular alkalinization, efflux of K(+)-ions, and induction of 1-aminocyclopropane-1-carboxylate synthase acid, the key enzyme of ethylene biosynthesis. This array of stress symptoms is well known from cultured plant cells treated with microbial elicitors. Compared with elicitor treatment, induction of responses by hyperosmotic shock was slow and occurred only after increases of approximately 200,000 Pa in osmotic pressure. In contrast, hypo-osmotic shock induced responses without measurable lag and faster than elicitor treatments. Measurable medium alkalinization was induced when medium osmolality was reduced by as little as approximately 10 mosmol, a change corresponding to only approximately 0.2 bar in osmotic pressure. Like treatment with elicitors, hypo-osmotic shock induced specific changes in protein phosphorylations as demonstrated by in vivo labeling with [(33)P]orthophosphate. Exposure of cells to consecutive up- and down-shifts in medium osmolality showed that sensing of osmotic changes occurred within seconds, whereas adaptation to new osmotic conditions proceeded over hours. In conclusion, suspension-cultured plant cells display rapid, easily measurable macroscopic responses to osmotic shock and provide an interesting model system to study osmoregulation, a key process in plant growth and development.

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Enzymes of grasses involved in fructan synthesis are of interest since they play a major role in assimilate partitioning and allocation, for instance in the leaf growth zone. Several fructosyltransferases from tall fescue (Festuca arundinacea) have previously been purified (Lüscher and Nelson, 1995). It is surprising that all of these enzyme preparations appeared to act both as sucrose (Suc):Suc 1-fructosyl transferases (1-SST) and as fructan:fructan 6(G)-fructosyl transferases. Here we report the cloning of a cDNA corresponding to the predominant protein in one of the fructosyl transferase preparations, its transient expression in tobacco protoplasts, and its functional analysis in the methylotrophic yeast, Pichia pastoris. When the cDNA was transiently expressed in tobacco protoplasts, the corresponding enzyme preparations produced 1-kestose from Suc, showing that the cDNA encodes a 1-SST. When the cDNA was expressed in P. pastoris, the recombinant protein had all the properties of known 1-SSTs, namely 1-kestose production, moderate nystose production, lack of 6-kestose production, and fructan exohydrolase activity with 1-kestose as the substrate. The physical properties were similar to those of the previously purified enzyme, except for its apparent lack of fructan:fructan 6(G)-fructosyl transferase activity. The expression pattern of the corresponding mRNA was studied in different zones of the growing leaves, and it was shown that transcript levels matched the 1-SST activity and fructan content.

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Rhizobial Nod factors (NFs) function as nodulation signals that trigger symbiotic responses of leguminous host plants. NFs consist of a chitin oligomer backbone carrying a fatty acid at the non-reducing end. Depending on the rhizobial strain, NFs carry additional substituents, which may determine host specificity. Transgenic suspension-cultured soybean (Glycine max [L.] Merr.) cells expressing aequorin have been used to record cytosolic [Ca(2+)] changes upon treatment with purified NFs and chitin fragments. Both compounds elicited an increase of cytosolic [Ca(2+)] at nanomolar concentrations. The shape and amplitude of cytosolic [Ca(2+)] changes was similar to the response elicited by un-derivatized chitin oligomers. Cells challenged first with NFs did not respond to a subsequent treatment with chitin oligomers and vice versa. Dose-response experiments showed that un-derivatized chitin oligomers were more active compared with NFs. The capacity of NFs to elicit the calcium response depended on their structure. The presence of reducing end substituents in methylfucosylated NFs from Rhizobium sp. NGR234 and the O-acetyl group at the non-reducing end in NFs from Sinorhizobium meliloti attenuated the activity to cause the calcium changes. The sulfate group in NFs from Rhizobium tropici did not affect the elicitor activity. Pentameric S. meliloti NFs were more active than tetrameric molecules, whereas trimeric or dimeric degradation products were inactive. Substituents in NFs may have the function to avoid stimulation of defense reactions mediated by the perception system for chitin oligomers.

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flg22, a peptide corresponding to the most conserved domain of bacterial flagellin, acts as a potent elicitor in plants. Here, we have used an iodinated derivative of flg22 ((125)I-labeled Tyr-flg22) as a molecular probe for the flagellin receptor in tomato cells. This radioligand showed rapid binding to a single class of specific, saturable, high-affinity receptor sites in intact cells and membrane preparations. Binding, although essentially nonreversible under physiological conditions, was not covalent, and chemical cross-linking was required to specifically label a single polypeptide of 115 kD. Intact flagellin and elicitor-active flagellin peptides but not biologically inactive analogs efficiently competed for binding of radioligand. Peptides lacking the C terminus of the conserved domain, previously found to act as competitive antagonists of elicitor action in tomato cells, also competed for binding of radioligand. Thus, this novel, high-affinity binding site exhibited all the characteristics expected of a functional receptor of bacterial flagellin. For a model of receptor activation, we propose a two-step mechanism according to the address-message concept, in which binding of the N terminus (address) is the first step and activation of responses with the C terminus (message) is the second step.

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