Characterization of cyclic β-glucans of Bradyrhizobium by MALDI-TOF mass spectrometry

Periplasmic, cyclic β-glucans isolated from Bradyrhizobium elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium yuanmingense strains have been investigated by means of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), 1D and 2D nuclear magnetic resonance (NMR), as well as standard chemical methods. These compounds are built of 10–13 d-glucose residues. The main fractions contain molecules assembled of 12 hexose units (M_w = 1945.363 Da). Glucose monomers are linked by β-(1→3) or β-(1→6) glycosidic bonds. The ratio of β-(1→3) to β-(1→6) linked glucose is approximately 1:2. Moreover, methylation analysis demonstrated the presence of terminal, non-reducing, as well as branched (i.e., 3- and 6-substituted) glucoses. Thus, the basic structure of the investigated compounds is similar to that of periplasmic oligosaccharides from Bradyrhizobium japonicum and Azorhizobium caulinodans strains. The analyzed cyclic β-glucans are substituted by phosphocholine (PC) (one or two residues per ring) and highly decorated with acetate and succinate. The substituents are arranged diversely in the population of cyclic β-glucan molecules. The concentrations of cyclic β-glucans in Bradyrhizobium periplasmic space are osmotically regulated and increase in response to a decrease of medium osmolarity.     

Effect of Nitrate on Bradyrhizobium — Mungbean (Vigna radiata) Symbiosis in presence of Syringaldehyde

Bradyrhizoblum — mungbean (Vigna radiata) symbiosis was influenced by the application of nitrate in combination with.yrlngaldehyde. Application of nitrate alone at lower concentrations (2, 5 mM) caused a reduction at initial stage (10 DAS) while at later stage (35 DAS–45 DAS) enhanced the nodulation status (nodule number, nodule weight) and nodule efficiency (acetylene reduction activity). Higher concentration of nitrate (10 mM) caused a reduction in nodule number, nodule weight and acetylene reduction activity. Syringaldehyde alone improved the nodulation status and nodule efficiency while it acted synergistically when applied in combination with nitrate. These effects were also reflected in the biomass of plants.     

New Bradyrhizobium japonicum Strains That Possess High Copy Numbers of the Repeated Sequence RSα

In a survey of DNA fingerprints of indigenous Bradyrhizobium japonicum with the species-specific repeated sequences RSα and RSβ, 21 isolates from three field sites showed numerous RS-specific hybridization bands. The isolates were designated highly reiterated sequence-possessing (HRS) isolates, and their DNA hybridization profiles were easily distinguished from the normal patterns. Some HRS isolates from two field sites possessed extremely high numbers of RSα copies, ranging from 86 to 175 (average, 128), and showed shifts and duplications of nif- and hup-specific hybridization bands. The HRS isolates exhibited slower growth than normal isolates, although no difference in symbiotic properties was detected between the HRS and normal isolates. Nucleotide sequence analysis of 16S rRNA genes showed that HRS isolates were strains of B. japonicum. There was no difference in the spectra of serological and hydrogenase groupings of normal and HRS isolates. Some HRS isolates possessed a tandem repeat RSα dimer that is similar to the structure of (IS30)₂, which was shown to cause a burst of transpositional rearrangements in Escherichia coli. The results suggest that HRS isolates are derived from normal isolates in individual fields by genome rearrangements that may be mediated by insertion sequences such as RSα.Insertion sequence (IS) elements are discrete segments of DNA that are able to transpose to numerous sites on bacterial plasmids and chromosomes, usually with an increase in their copy number (7). IS elements can also promote rearrangement of genomes and other replicons (7). Many IS elements and uncharacterized repeated DNA sequences among plant-associated gram-negative bacteria, including Agrobacterium (5), Bradyrhizobium (10, 11, 17), Rhizobium (3, 5, 35), and Xanthomonas (2) spp., have been described. These repeated elements often cause genomic instability affecting genes responsible for plant associations (15, 23), and they have been postulated to play a role in evolution and genomic instability (1, 7, 17, 24, 29). Indeed, complete sequencing of a symbiotic plasmid pNGR234a from a Rhizobium sp. demonstrated that almost one-fifth of the total plasmid sequence is made up of IS elements and a mosaic sequence structure including nodulation loci (6).Members of the genus Bradyrhizobium are slow-growing, gram-negative, nitrogen-fixing heterotrophic bacteria which can form root nodules on several leguminous plants. In Bradyrhizobium japonicum, several repeated DNA sequences (RSα, RSβ, RSγ, RSδ, RSɛ, and RSζ) have been identified (10, 11, 17). At least one of these sequences, RSα, has structural properties similar to that of a prokaryotic IS element. Interestingly, the RS copies are often clustered around the regions of nitrogen-fixation and nodulation genes on the chromosome of B. japonicum USDA110 (17). An insertion sequence, HRS1, also was found to be closely linked to common and genotype-specific nodulation genes in B. japonicum serocluster USDA123 and USDA127 strains (15, 27).DNA fingerprints with RSα, RSβ, and HRS1 as probes revealed genetic diversity within natural populations of B. japonicum that nodulated soybeans (13, 15, 22, 27), indicating that RS fingerprinting is useful for isolate or strain identification and is a valuable tool for evaluating the genetic structure of indigenous B. japonicum populations.In a previous paper (22) two B. japonicum isolates, NC32a and NC3a, obtained from a Nakazawa field site showed numerous bands of RS-specific hybridization. Of 213 isolates of soybean bradyrhizobia indigenous to six field sites in Japan (reference 22 and unpublished data), 19 isolates have been found to exhibit numerous bands of RS-specific hybridization, suggesting that the distribution of such isolates is ubiquitous. In this study, we have genetically and phenotypically characterized field isolates of soybean bradyrhizobia showing numerous bands of RS-specific hybridization as first steps toward gaining some understanding of their ecological role.     

Sol–gel immobilization of haloalkane dehalogenase from Bradyrhizobium japonicum for the remediation 1,2-dibromoethane

Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon–halogen bonds in a broad range of environmental pollutants such as aliphatic mono-, di-, and polyhalogenated alkanes. From the biotechnology point of view haloalkane dehalogenases attract attention because of many potential uses for the bioremendation of soil, water and air. In the present study, different Rhizobium strains (Sinorhizobium meliloti 1021, Rhizobium leguminosarum bv. trifolii, Mesorhizobium loti MAFF, Bradyrhizobium japonicum usda 110) were screened for their ability to produce stable and active 1,2-dibromoethane-degrading dehalogenase. The results showed that B. japonicum produces the most potent dehalogenase. This enzyme was cloned, expressed in Escherichia coli BL21(DE3), purified and was entrapped in tetraethylorthosilicate derived sol–gel. The tetraethylorthosilicate sol–gel entrapped haloalkane dehalogenases exhibited higher storage and operational stability at 4 °C and 25 °C, compared to the free enzyme. Kinetic analysis of the entrapped enzyme using 1,2-dibromoethane showed that substrate turnover was limited by partitioning effects or diffusion through the sol–gel matrix. The biocatalyst was used in a packed bed bioreactor for the biodegradation of 1,2-DBE. Under selected conditions the sol–gel entrapped dehalogenase was able to hydrolyze 91.8% of the loaded 1,2-DBE, within 16.7 h. The results of the present study suggest that the use of HLD biocatalysis may provide a ‘green chemistry’ tool for sustainable remediation of 1,2-DBE.     

Autoregulation of soybean–Bradyrhizobium nodule symbiosis is controlled by shoot or/and root factors

Two strains of Bradyrhizobium japonicum were evaluated with five commercial cultivars of soybean (Clark, Crauford, Davis, Centaur, and Nessen) and one hypernodulating mutant NOD1-3. The hypernodulating NOD1-3 produced 30–50 times the number of nodules of commercial cultivars either inoculated with B. japonicum strain USDA 123 or RCR 3409. Grafting of NOD1-3 shoots to Clark and Davis roots induced hypernodulation on roots of Clark and Davis but did not enhance nodulation when grafted onto the roots of Crauford, Centaur, and Nessen. In contrast, the shoots of Clark, Davis, Centaur and Nessen significantly inhibited nodule formation on the root of NOD1-3. However, Crauford shoots did not alter nodule formation on the roots of NOD1-3 as compared with self-grafts of NOD1-3. It appears that the shoot of NOD1-3 has the ability to alter autoregulatory control of nodulation of Clark and Davis cultivars, but not of Crauford, Centaur and Nessen. The results suggest that the regulation of nodulation in soybean cultivars Clark and Davis is controlled by the shoot factors, while the Crauford was root controlled. Reciprocal grafts between NOD1-3 and Centaur or Nessen indicate that both shoot and root factors are involved in regulation of nodulation. The results suggested that the regulation of nodulation did not depend on bradyrhizobial strains. The shoot control of hypernodulation may be causally related to differential root isoflavonoid levels, which are also controlled by shoot. Application of daidzein significantly enhanced the nodulation and nitrogenase activity of soybean cv. Clark. Root control of restricted nodulation of soybean cv. Centaur did not respond to the addition of daidzein in nutrient solution indicating that this character is not related to isoflavonoids. Therefore, autoregulation in Clark and Centaur plants may be separate events in legume–rhizobia symbiosis and regulated by different kinds of signals.     

Phenotypic and phylogenetic characterization of native peanut Bradyrhizobium isolates obtained from Córdoba, Argentina

Peanut is an economically important legume nodulated by slow-growing bacteria of the genus Bradyrhizobium. In this study, a collection of native slow-growing peanut rhizobial isolates from Argentina was obtained and characterized. The phenotypical characterization included the determination of the symbiotic properties, whereas the genetic and phylogenetic diversity was assessed through ERIC-PCR and sequencing of the internal transcribed spacer (ITS) region, as well as the dnaK and nodA genes. The results obtained indicated that peanut nodulating bradyrhizobia were phenotypically and genotypically diverse, and included locally adapted variants of B. yuanmingense and B. iriomotense carrying novel nodA alleles.     

Genome-informed Bradyrhizobium taxonomy: where to from here?

Bradyrhizobium is thought to be the largest and most diverse rhizobial genus, but this is not reflected in the number of described species. Although it was one of the first rhizobial genera recognised, its taxonomy remains complex. Various contemporary studies are showing that genome sequence information may simplify taxonomic decisions. Therefore, the growing availability of genomes for Bradyrhizobium will likely aid in the delineation and characterization of new species. In this study, we addressed two aims: first, we reviewed the availability and quality of available genomic resources for Bradyrhizobium. This was achieved by comparing genome sequences in terms of sequencing technologies used and estimated level of completeness for inclusion in genome-based phylogenetic analyses. Secondly, we utilized these genomes to investigate the taxonomic standing of Bradyrhizobium in light of its diverse lifestyles. Although genome sequences differed in terms of their quality and completeness, our data indicate that the use of these genome sequences is adequate for taxonomic purposes. By using these resources, we inferred a fully resolved, well-supported phylogeny. It separated Bradyrhizobium into seven lineages, three of which corresponded to the so-called supergroups known for the genus. Wide distribution of key lifestyle traits such as nodulation, nitrogen fixation and photosynthesis revealed that these traits have complicated evolutionary histories. We present the first robust Bradyrhizobium species phylogeny based on genome sequence information for investigating the evolution of this important assemblage of bacteria. Furthermore, this study provides the basis for using genome sequence information as a resource to make important taxonomic decisions, particularly at the species and genus levels.     

Discovery of a Novel (+)-γ-Lactamase from Bradyrhizobium japonicum USDA 6 by Rational Genome Mining

A novel (+)-γ-lactamase used for the resolution of racemic γ-lactam from Bradyrhizobium japonicum USDA 6 was found as a result of sequence-structure guided genome mining. It consists of 409 amino acids, only 49% of which are identical to the amino acid sequences of the known (+)-γ-lactamase from Sulfolobus solfataricus. This is only the third (+)-γ-lactamase gene to be reported.     

Are Bradyrhizobium japonicum stable during a long stay in soil?

Two strains of Bradyrhizobium japonicum, recognizable by their intrinsic resistance to high levels of antibiotics and their serological features were introduced into three calcareous soils under field conditions. These strains were re-isolated 16 or 20 years later and compared with the parental strains kept lyophilized. In the Dijon location, the survival was high although soybean was never grown in the field. But the B. japonicum completely disappeared in the Montpellier field after 10 years under vineyard. In the Toulouse field after the two initially introduced strains, inoculation of subsequent soybean crops with a new strain enabled this strain to occupy 70–80% of the nodules; these results suggest that under such conditions the problem of competition can be solved by repeated inoculation. In this field, the number of introduced B. japonicum remained high during 4 years without soybeans, but a new inoculation would be necessary after 5 years. In the two fields where the survival was high, the two strains remained at about the same relative level as at introduction, there was no detectable exchange of characters between them. With regards to agronomic characteristics, there were no important changes in the competitivity of the strains. Among the eight field isolates tested in a greenhouse for efficiency by comparison with eight lyophilized isolates, seven showed no significant difference for the total weight of soybean or seed yield but one field isolate showed a loss of efficiency corresponding to 27% less seed weight. This long-term experiment allowed us to conclude that the B. japonicumstrains used were stable for many characters, but variations in efficiency may rarely occur.     

Straight and branched (ω-1)-hydroxylated very long chain fatty acids are components of Bradyrhizobium lipid A

Lipopolysaccharides of seven Bradyrhizobium strains and three whole-cell fatty acid preparations from bacteria isolated from nodules of Sarothamnus scoparius (common broom) were studied for the presence of very long chain (ω-1)-hydroxy fatty acids. Several such fatty acids were identified. Among them, straight-chain as well as mono- and dimethyl branched acids with chains in the range from 26 to 34 carbon atoms were found. Pyrrolidides and 4,4-dimethyloxazoline derivatives were used to determine the branching position. Carbons at the (ω-10) and/or (ω-11) positions in alkyl chains were points of attachment of methyl groups. These data complete the structure of bradyrhizobial lipid A with important details. The obtained results can be applied in the chemotaxonomy of Bradyrhizobium.     

Exopolysaccharide (EPS) synthesis in Bradyrhizobium japonicum : sequence, operon structure and mutational analysis of an exo gene cluster

The nucleotide sequence of a 8330-bp DNA fragment from Bradyrhizobium japonicum 110spc4 was determined. Sequence analysis revealed that six ORFs were present and the deduced amino acid sequences were homologous to enzymes involved in exopolysaccharide (EPS) biosynthesis. The genes appear to be organized into at least four different operons. One gene was found to be homologous to exoB, which encodes a UDP-galactose 4′-epimerase. Other ORFs were homologous to UDP-hexose transferases and one ORF showed similarity to Sinorhizobium (Rhizobium) meliloti ExoP, which has been suggested to be involved in EPS chain-length determination. A set of deletion and insertion mutants was constructed and the resulting B. japonicum strains were tested for their symbiotic traits. Deletion mutant ΔP22, which lacks the C-terminal part of ExoP, the UDP-hexose transferase ExoT and the N-terminal part of ExoB, shows a delayed nodulation phenotype and induces symptoms of plant defense reactions; its EPS does not contain galactose and no high molecular weight fraction is synthesized. In contrast, insertion mutant EH3, which expresses an exoP gene product that is truncated in its putative periplasmic domain, produced an EPS containing both HMW and LMW fractions. However, the interaction of EH3 with soybeans was severely perturbed. As a rule, only the initial steps of nodule formation were observed.     

Biosynthesis of cyclic β-(1–3),β-(1–6) glucan in Bradyrhizobium spp.

Inner membranes of Bradyrhizobium japonicum strain USDA 110 produced in vitro soluble and insoluble -(1–3),-(1–6) glucans. The reaction proceeded through a 90 kDa inner membrane intermediate protein; used UDP-glucose as sugar donor and required Mg²⁺. Gel chromatography of soluble glucans resolved a cyclic -(1–3) glucan with a degree of polymerization of eleven from a family of -(1–3),-(1–6) glucans with variable degree of polymerization higher than eleven. Bradyrhizobium strains BR4406 and BR8404 isolated from tree legume nodules in Southeast Brazil produce -(1–3),-(1–6) glucans very similar to that of B. japonicum. A 100 kDa protein was identified in these strains as intermediates in the synthesis of these glucans. Inner membranes of B. japonicum USDA110, B. japonicum I17, and Bradyrhizobium strains BR4406 and BR8404 incubated with UDP-glucose were unable to synthesize -(1–2) glucan and lacked the 235 kDa intermediate protein known to be involved in the synthesis of -(1–2) glucan in Agrobacterium tumefaciens, Rhizobium meliloti and Rhizobium loti.Abbreviations EPS= exopolysaccharides - CPS= capsular polysaccharides - LPS= lipopolysaccharides - AMA= Yeast extract-mannitol medium - TY= tryptone-yeast extract - PMSF= phenyl methyl sulfonil fluoride

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花生根瘤菌Bradyrhizobium sp.MM6 Ⅲ型分泌系统的结构和功能

【目的】探究花生根瘤菌Bradyrhizobium sp. MM6的Ⅲ型分泌系统(T3SS)的结构及其在根瘤菌与不同宿主建立共生关系中的作用。【方法】同源比对分析菌株MM6的T3SS基因簇的结构特征,并采用三亲本接合转移的方法构建T3SS调节基因ttsI突变菌株;通过蛭石结瘤和石蜡切片实验,比较突变体与野生型的共生固氮表型差异。【结果】经预测,MM6的T3SS基因簇编码区长约34.1 kb,可分为3个区域,包含10个保守结构基因和8个效应蛋白基因,与B. diazoefficiens USDA110相应基因的序列相似性为83%–93%;成功构建了MM6的ttsI突变株;ttsI突变株与野生型分别与花生(S523和Y45)、野大豆和大豆中黄57结瘤,ttsI突变体在花生中的总瘤数显著增加(P0.05),根瘤中含菌细胞更多;ttsI突变体在野大豆中平均每株植物增加4个根瘤,根瘤中含菌细胞更多,地上部干重相比野生型MM6显著增加(P0.05);在大豆中黄57中,野生型MM6能形成红色的有效根瘤,ttsI突变体不结瘤,且植株叶片发黄,地上部干重相比野生型MM6显著降低(P0.05)。【结论】MM6的T3SS在花生和野大豆共生体系中起着有害的作用,而在大豆中黄57的共生体系中起着有利的作用。     

花生根瘤菌Bradyrhizobium sp.MM6 Ⅲ型分泌系统的结构和功能

【目的】探究花生根瘤菌Bradyrhizobium sp.MM6的Ⅲ型分泌系统(T3SS)的结构及其在根瘤菌与不同宿主建立共生关系中的作用。【方法】同源比对分析菌株MM6的T3SS基因簇的结构特征,并采用三亲本接合转移的方法构建T3SS调节基因ttsI突变菌株;通过蛭石结瘤和石蜡切片实验,比较突变体与野生型的共生固氮表型差异。【结果】经预测,MM6的T3SS基因簇编码区长约34.1 kb,可分为3个区域,包含10个保守结构基因和8个效应蛋白基因,与B.diazoefficiens USDA110相应基因的序列相似性为83%–93%;成功构建了MM6的ttsI突变株;ttsI突变株与野生型分别与花生(S523和Y45)、野大豆和大豆中黄57结瘤,ttsI突变体在花生中的总瘤数显著增加(P<0.05),根瘤中含菌细胞更多;ttsI突变体在野大豆中平均每株植物增加4个根瘤,根瘤中含菌细胞更多,地上部干重相比野生型MM6显著增加(P<0.05);在大豆中黄57中,野生型MM6能形成红色的有效根瘤,ttsI突变体不结瘤,且植株叶片发黄,地上部干重相比野生型MM6显著降低(P<0.05)。【结论】MM6的T3SS在花生和野大豆共生体系中起着有害的作用,而在大豆中黄57的共生体系中起着有利的作用。     

Nitrate-dependent N₂O emission from intact soybean nodules via denitrification by Bradyrhizobium japonicum bacteroids

In the presence of nitrate, N₂O emission increased markedly from soybean roots inoculated with nosZ mutant of Bradyrhizobium japonicum, but not from soybean roots inoculated with a napA nosZ double mutant, indicating that B. japonicum bacteroids in soybean nodules are able to convert the exogenously supplied nitrate into N₂O via a denitrification pathway.     

花生根瘤菌Bradyrhizobium sp.MZ5 Ⅲ型分泌系统调节基因ttsI突变体的功能分析

【目的】探究慢生型花生根瘤菌Ⅲ型分泌系统在花生-根瘤菌互作的功能。【方法】本研究采用同源重组和三亲本接合转移的方法,构建Bradyrhizobium sp. MZ5的Ⅲ型分泌系统调节基因ttsI突变体;荧光定量PCR检测添加大豆苷元(Daidzein)和染料木黄酮(Genistein)诱导物后野生型和突变株转录水平上ttsI的表达量变化及其差异;蛭石结瘤实验分析ttsI基因突变对花生结瘤能力的影响。【结果】在转录水平上,大豆苷元和染料木黄酮对MZ5的Ⅲ型分泌系统调节基因ttsI的表达具有显著的抑制作用(P0.05)。在MZ5△ttsI突变体中ttsI基因的表达量都明显下调,与野生型菌株的相比都达到极显著水平(P0.001)。蛭石结瘤实验表明,与野生型菌株相比,MZ5△ttsI突变体在不同花生品种的结瘤数和地上部干重都显著性降低。根瘤石蜡切片表明,MZ5△ttsI突变体在根瘤内的含菌量少于野生型菌株。【结论】Bradyrhizobium sp. MZ5菌株中的Ⅲ型分泌系统在花生-根瘤菌互作中对结瘤有积极的促进作用。     

The lack of plastidal transit sequence cannot override the targeting capacity of <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> δ-aminolevulinic acid synthase in transgenic rice

The δ-aminolevulinic acid synthase (ALA-S) is an enzyme which catalyzes the synthesis of δ-aminolevulinic acid (ALA). The Bradyrhizobium japonicum ALA-S coding sequence lacking plastidal transit sequence was introduced into the rice genome (C line). The transgenic lines, C4 and C5, were compared with the transgenic lines expressing TALA-S gene with plastidal transit sequence (P line) to investigate whether the plastidal sequence affects the targeting capacity of B. japonicum ALA-S gene and the ALA-synthesizing capacity in rice plants. The B. japonicum ALA-S mRNA was expressed efficiently in C lines and the protein was localized in the stroma of chloroplasts regardless of the transit sequence as in P lines. The resulting transgenic plants, C line, had similar levels of ALA-S activity, ALA, protoporphyrin IX and chlorophylls, compared to those of P lines. In response to irradiance of 350 μmol m⁻² s⁻¹, transgenic lines C4 and C5 displayed the characteristic phenotypes of photodynamic damage, i.e., decreases in photosynthetic parameter F_v/F_m, as in P5 and P14 lines, whereas wild type did not. These results indicate that the lack of the plastidal transit sequence influences neither chloroplast translocation of B. japonicum ALA-S nor ALA-synthesizing capacity in the transgenic rice.     

Enzymatic preparation of optically pure (+)-2-azabicyclo[2.2.1]hept-5-en-3-one by (−)-γ-lactamase from Bradyrhizobium japonicum USDA 6

Whole cells of Bradyrhizobium japonicum USDA 6 showed both (+)-γ-lactamase activity and (−)-γ-lactamase activity. Insight into the genome of B. japonicum USDA 6 revealed two potential γ-lactamases: a type I (+)-γ-lactamase and a (−)-γ-lactamase, making it the first strain to contain two totally different enantioselective lactamases. Both recombinant enzymes could easily be used to prepare either optically pure (+)-γ-lactam ((+)-2-azabicyclo[2.2.1]hept-5-en-3-one) or optically pure (−)-γ-lactam ((−)-2-azabicyclo[2.2.1]hept-5-en-3-one), which are versatile synthetic building blocks for the synthesis of various carbocyclic nucleosides and carbocyclic sugar analogues. Bioinformatic analysis showed that the type I (+)-γ-lactamase belongs to the amidase signature family, with 504 amino acids; the (−)-γ-lactamase, which consists of 274 amino acids, belongs to the hydrolase family. Here, we report that B. japonicum USDA contains a (−)-γ-lactamase in addition to a (+)-γ-lactamase, and it is the (−)-γ-lactamase from this strain that is examined in detail in this Letter. Enzymatic synthesis of optically pure (+)-γ-lactam with nearly 50% isolated yield and >99% ee was achieved.     

Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3-1,6-β-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6--glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6--glucans. Here we demonstrate that both the fungal and the bacterial -glucans are ligands of -glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal -glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6--glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6--glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6--glucans. Another type of cyclic -glucan, a 1,2--glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the -glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plant's defence response.Abbreviations HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-l-[hexaglucosyl]deoxyglucitol - HG-AzPEA l-[2-(4-azidophenyl)-ethyl]amino-l-[hexaglucosyl]deoxyglucitol - IC₅₀ concentration for half-maximal displacement We thank Ines Arlt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Fonds der Chemischen Industrie (J.E.), and USDA CSRS NRI Competitive Research grant 93373059233 (A.A.B.).