Lipo-chitooligosaccharides Research Articles

Stability and Activity of the Major Nod Factor Produced by Bradyrhizobium japonicum following Purification, Sterilization, and Storage

Nod factors, also known as lipo-chitooligosaccharides (LCOs), are signal molecules produced by rhizobia during rhizobia-legume symbiosis. These Nod factors often must be purified, sterilized, and stored before use in research experiments. Therefore, we evaluated the influence of purification, sterilization, and storage methods on degradation and biological activity of Nod factor comprised primarily of Nod BjV(C18:1 MeFuc). During filter sterilization, various types of filters affected the amount of LCO recovered: polyestersulfone (67.7%), cellulose acetate (54.9%), nylon (48.1%), polytetrafluoroethylene (38.0%), and mixed cellulose ester (31.8%). During autoclaving (durations of 15 to 30 min) ∼30% of the LCO was lost LCO degraded faster when stored at 23 ± 2°C (room temperature) than at 4 ± 1°C (refrigerator); after 16 mo, 74% of the LCO was recovered following room temperature storage and 84% following refrigerator storage. The biological activity (root hair deformation and stimulation of soybean seed germination) of LCO (10 -7 M) obtained from autoclaved and stored samples were not different from that of freshly prepared LCO; no reduction in biological activity was found.

Methyl jasmonate, alone or in combination with genistein, and Bradyrhizobium japonicum increases soybean ( Glycine max L.) plant dry matter production and grain yield under short season conditions

Jasmonic acid (JA) is a plant hormone produced via the octadecanoid pathway from its precursor, linolenic acid. Jasmonates are involved in plant wound responses and defense against insects and fungal elicitors. They can also act as signal molecules in the Bradyrhizobium-soybean symbiosis. Pre-incubation of Bradyrhizobium japonicum inocula with gensitein (Ge), an effective inducer of nodulation genes in this species enhances soybean nodulation, nitrogen fixation and yield under low spring soil temperature field conditions. Since jasmonates are also able to induce nodulation genes and cause the production of lipo-chitooligosaccharides (LCOs) by B. japonicum, we conducted two field experiments, in southwestern Quebec, Canada, to determine whether pre-incubation of B. japonicum with methyl jasmonate (MeJA) alone or in combination with genistein (Ge), prior to inoculation, increased soybean plant dry matter production and grain yield. Experiments at each site used a two factor randomized complete block design (RCBD) with four replicates. Two B. japonicum strains (USDA3 and 532C) and four inducer molecule treatments [control, Ge (20 μM), MeJA (50 μM), and Ge + MeJA (20 μM + 50 μM)] were used in the study. The bacterial cultures were induced for 24 h with the inducer molecules and then applied into the furrows at the time of planting. Both Ge and MeJA, alone or in combination, increased plant growth, dry matter accumulation, and grain yield. This study showed that MeJA, alone or in combination with Ge, can be used to promote soybean plant growth and grain yield under short season field conditions.

Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators.

Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor-induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor-inducible gene expression.

Propagation of Norway spruce via somatic embryogenesis

Somatic embryogenesis combined with cryopreservation is an attractive method to propagate Norway spruce (Picea abies) vegetatively both as a tool in the breeding programme and for large-scale clonal propagation of elite material. Somatic embryos are also a valuable tool for studying regulation of embryo development. Embryogenic cell lines of Norway spruce are established from zygotic embryos. The cell lines proliferate as proembryogenic masses (PEMs). Somatic embryos develop from PEMs. PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos. Withdrawal of plant growth regulators stimulates PEM-to-somatic embryo transition accompanied by programmed cell death (PCD) in PEMs. This PCD is mediated by a marked decrease in extracellular pH. If the acidification is abolished by buffering the culture medium, PEM-to-somatic embryo transition together with PCD is inhibited. Cell death, induced by withdrawal of PGRs, can be suppressed by extra supply of lipo-chitooligosaccharides (LCOs). Extracellular chitinases are probably involved in production and degradation of LCOs. During early embryogeny, the embryos form an embryonal mass surrounded by a surface layer. The formation of a surface layer is accompanied by a switch in the expression pattern of an Ltp-like gene (Pa18) and a homeobox gene (PaHB1), from ubiquitous expression in PEMs to surface layer-specific in somatic embryos. Ectopic expression of Pa18 and PaHB1 leads to an early developmental block. Transgenic embryos and plants of Norway spruce are routinely produced by using a biolistic approach. The transgenic material is used for studying the importance of specific genes for regulating plant development, but transgenic plants can also be used for identification of candidate genes for use in the breeding programme.

Biotic elicitors as a means of increasing isoflavone concentration of soybean seeds

SummarySoybean (Glycine max) seeds contain isoflavones that have positive impacts on human health. Four greenhouse experiments were conducted to determine if isoflavone concentration of mature soybean seeds could be increased using elicitor compounds. The effects on soybean seed isoflavone concentrations following foliar applications of two lipo‐chitooligosaccharides (LCO) [Bj V (C18:1 MeFuc) and Bj V (Ac, C16, MeFuc)], chitosan, actinomycetes spores (Streptomyces melanosporofaciens strain EF‐76) and yeast extract at different concentrations and growth stages were evaluated. Combined chitosan seed treatment and foliar applications were also evaluated. Concentrations of daidzein, genistein, glycitein, and total isoflavones were determined by HPLC. Foliar applications of LCOs, chitosan, and actinomycetes caused a marked increase in individual and total isoflavone concentration (ranging between 21% and 84%) of mature seeds when compared to untreated control plants. There were limited differences between the different concentrations and stages of application tested for chitosan and actinomycetes; however, response to LCOs was greatest at higher concentrations (i.e. 10‐6 M) when applied at the early podding stage. Compared to untreated plants, combined seed treatment and foliar applications of chitosan increased individual and total isoflavone concentration of mature soybean seeds by 16% to 93%. Trends were similar for different cultivars, however, the magnitude of the response varied. Finally, response to foliar applications of yeast extract was highly concentration dependent with increases of up to 56% in total isoflavone observed with 2 mg mL‐1. Results indicate that elicitors hold promise as a way of increasing isoflavone concentration of mature soybean seeds.

The relative orientation of the lipid and carbohydrate moieties of lipochitooligosaccharides related to nodulation factors depends on lipid chain saturation

Lipochitooligosaccharides (LCOs) signal the symbiosis of rhizobia with legumes and the formation of nitrogen-fixing root nodules. LCOs 1 and 2 share identical tetrasaccharide scaffolds but different lipid moieties (1, LCO-IV(C16:1[9Z], SNa) and , LCO-IV(C16:2[2E,9Z], SNa)). The conformational behaviors of both LCOs were studied by molecular modeling and NMR. Modeling predicts that a small lipid modification would result in a different relative orientation of the lipid and tetrasaccharide moieties. Diffusion ordered spectroscopy reports that both LCOs form small aggregates above 1 mM. Nuclear Overhauser spectroscopy (NOESY) data, collected under monomeric conditions, reveals lipid-carbohydrate contacts only for 1, in agreement with the modeling data. The distinct molecular structures of 1 and 2 have the potential to contribute to their selective binding by legume proteins.

Nod factor-treated Medicago truncatula roots and seeds show an increased number of nodules when inoculated with a limiting population of Sinorhizobium meliloti.

Medicago truncatula is a model legume plant that interacts symbiotically with Sinorhizobium meliloti, the alfalfa symbiont. This process involves a molecular dialogue between the bacterium and the plant. Legume roots exude flavonoids that induce the expression of a set of rhizobial genes, the nod genes, which are essential for nodulation and determination of the host range. In turn, nod genes control the synthesis of lipo-chito-oligosaccharides (LCOs), Nod factors, which are bacteria-to-plant signal molecules mediating recognition and nodule organogenesis. M. truncatula roots or seeds have been treated with Nod factors and hydroponically growing seedlings have been inoculated with a limiting population of S. meliloti. It has been shown that submicromolar concentrations of Nod factors increase the number of nodules per plant on M. truncatula. Compared with roots, this increase is more noticeable when seeds are treated. M. truncatula seeds are receptive to submicromolar concentrations of Nod factors, suggesting the possibility of a high affinity LCO perception system in seeds or embryos as well.

Identical accumulation and immobilization of sulfated and nonsulfated Nod factors in host and nonhost root hair cell walls.

Nod factors are signaling molecules secreted by Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can elicit specific responses at subnanomolar concentrations on a compatible host. How plants perceive LCOs is unclear. In this study, using fluorescent Nod factor analogs, we investigated whether sulfated and nonsulfated Nod factors were bound and perceived differently by Medicago truncatula and Vicia sativa root hairs. The bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair deformation assay on M. truncatula, showing bioactivity down to 0.1 to 1 nM. Fluorescence microscopy of plasmolyzed M. truncatula root hairs shows that sulfated fluorescent Nod factors accumulate in the cell wall of root hairs, whereas they are absent from the plasma membrane when applied at 10 nM. When the fluorescent Nod factor distribution in medium surrounding a root was studied, a sharp decrease in fluorescence close to the root hairs was observed, visualizing the remarkable capacity of root hairs to absorb Nod factors from the medium. Fluorescence correlation microscopy was used to study in detail the mobilities of sulfated and nonsulfated fluorescent Nod factors which are biologically active on M. truncatula and V. sativa, respectively. Remarkably, no difference between sulfated and nonsulfated Nod factors was observed: both hardly diffuse and strongly accumulate in root hair cell walls of both M. truncatula and V. sativa. The implications for the mode of Nod factor perception are discussed.

Signals exchanged between legumes and Rhizobium: agricultural uses and perspectives

Legumes and rhizobia exchange at least three different, but sometimes complementary sets of signals. Amongst the variety of substances normally and continuously secreted into the rhizosphere by plants are phenolic compounds. Flavonoid components of these mixtures are especially active in inducing rhizobial nodulation genes. Many nod-genes exist. Some (e.g., nodD) serve as regulators of transcription, but most code for enzymes involved in the synthesis of a family of lipo-chito-oligosaccharides (LCOs) called Nod-factors. Nod-factors possess hormone-like properties, are key determinants in nodulation, and allow rhizobia to enter the plant. As Nod-factors also stimulate the synthesis and release of flavonoids from legume roots, the response to inoculation is amplified. Once the bacteria enter the plant, other sets of signals are exchanged between the symbionts. These include extra-cellular polysaccharides (EPSs) as well as proteins externalised via type-three secretion systems. These carbohydrates/proteins may be active in invasion of the root. At the time of writing, only flavonoids and Nod-factors have been chemically synthesised and of these only the former are available in large quantities. Field trials in North America show that seed application of flavonoids stimulates nodulation and nitrogen fixation in soybeans grown at low soil temperatures. The biological basis to these responses is discussed.

Structural determination of the lipo-chitin oligosaccharide nodulation signals produced by Rhizobium giardinii bv. giardinii H152

Rhizobium giardinii bv. giardinii is a microsymbiont of plants of the genus Phaseolus and produces extracellular signal molecules that are able to induce deformation of root hairs and nodule organogenesis. We report here the structures of seven lipochitooligosaccharide (LCO) signal molecules secreted by R. giardinii bv. giardinii H152. Six of them are pentamers of GlcNAc carrying C 16:0, C 18:0, C 20:0 and C 18:1 fatty acyl chains on the non-reducing terminal residue. Four are sulfated at C-6 of the reducing terminal residue and one is acetylated in the same position. Six of them are N-methylated on the non-reducing GlcN residue and all the nodulation factors are carbamoylated on C-6 of the non-reducing terminal residue. The structures were determined using monosaccharide composition and methylation analyses, 1D- and 2D-NMR experiments and a range of mass spectrometric techniques. The position of the carbamoyl substituent on the non-reducing glucosamine residue was determined using a CID-MSMS experiment and an HMBC experiment.

A host-specific bacteria-to-plant signal molecule (Nod factor) enhances germination and early growth of diverse crop plants.

Lipo-chitooligosaccharides (LCOs), or Nod factors, are host-specific bacteria-to-plant signal molecules essential for the establishment of a successful N(2)-fixing legume-rhizobia symbiosis. At submicromolar concentrations Nod factors induce physiological changes in host and non-host plants. Here we show that the Nod factor Nod Bj V(C18:1,MeFuc) of Bradyrhizobium japonicum 532C enhances germination of a variety of economically important plants belonging to diverse botanical families: Zea mays, Oryza sativa (Poaceae), Beta vulgaris (Chenopodaceae), Glycine max, Phaseolus vulgaris (Fabaceae), and Gossypium hirsutum (Malvaceae), under laboratory, greenhouse and field conditions. Similar increases in germination were observed for filtrates of genistein-induced cultures of B. japonicum 532C, while non-induced B. japonicum, induced Bj 168 (a nodC mutant of B. japonicum deficient in Nod factor synthesis) or the pentamer of chitin did not invoke such responses, demonstrating the role of Nod factor in the observed effects. In addition, three out of four synthetic LCOs evaluated also promoted germination of corn, soybean and Arabidopsis thaliana seeds. LCO also enhanced the early growth of corn seedlings under greenhouse conditions. These findings suggest the possible use of LCOs for improved crop production.

Rapid colorimetric quantification of lipo-chitooligosaccharides from Mesorhizobium loti and Sinorhizobium meliloti.

Nod factors are lipids with a chitinlike headgroup produced by gram-negative Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are essential signaling molecules for accomplishing symbiosis between the bacteria and roots of legume plants. Despite their important role in the Rhizobium-legume interaction, no fast and sensitive Nod factor quantification methods exist. Here, we report two different quantification methods. The first is based on the enzymatic hydrolysis of Nod factors to release N-acetylglucosamine (GlcNAc), which can subsequently be quantified. It is shown that the degrading enzyme, glusulase, releases exactly two GlcNAc units per pentameric nodulation factor from Mesorhizobium loti factor, allowing quantification of LCOs from Mesorhizobium loti. The second method is based on a specific type of Nod factors that are sulfated on the reducing GlcNAc, allowing quantification analogous to the quantification of sulfolipids. Here, a two-phase extraction method is used in the presence of methylene blue, which specifically forms an ion pair with sulfated lipids. The blue ion pair partitions into the organic phase, after which the methylene blue signal can be quantified. To enable Nod factor quantification with this method, the organic phase was modified and the partitioning was evaluated using fluorescent and radiolabeled sulfated Nod factors. It is shown that sulfated LCOs can be quantified with this method, using sodium dodecyl sulfate for calibration. Both methods allow Nod factor quantification in parallel enabling a fast and easy detection of nanomole quantities of Nod factors. Accurate Nod factor quantification will be crucial for characterization and cross-comparison of the affinity for Nod factors of newly identified Nod factor binding proteins or putative Nod factor receptors.

An inducible activator produced by a Serratia proteamaculans strain and its soybean growth-promoting activity under greenhouse conditions

Serratia proteamaculans 1-102 (1-102) promotes soybean-bradyrhizobia nodulation and growth, but the mechanism is unknown. After adding isoflavonoid inducers to 1-102 culture, an active peak with a retention time of about 105 min in the HPLC fractionation was isolated using a bioassay based on the stimulation of soybean seed germination. The plant growth-promoting activity of this material was compared with 1-102 culture (cells) and supernatant under greenhouse conditions. The activator was applied to roots in 83, 830 and 8300 HPLC microvolts (microV) per seedling when plants were inoculated with bradyrhizobia or sprayed onto the leaves in same concentrations at 20 d after inoculation. The root-applied activator, especially at 1 ml of 830 microV per seedling, enhanced soybean nodulation and growth at the same level as 1-102 culture under both optimal and sub-optimal root zone temperatures. Thus, this activator stimulating soybean seed germination is also responsible for the plant growth-promoting activity of 1-102 culture. However, when sprayed onto the leaves, the activator did not increase growth and in higher concentrations decreased average single leaf area. The results suggest that this inducible activator might be a lipo-chitooligosaccharide (LCO) analogue. LCOs act as rhizobia-to-legume signals stimulating root nodule formation. The activator could provide additional 'signal', increasing in the signal quality (the signal-to-noise ratio, SNR) of the plant-rhizobia signal exchange process.

An inducible activator produced by a Serratia proteamaculans strain and its soybean growth‐promoting activity under greenhouse conditions

Serratia proteamaculans 1‐102 (1‐102) promotes soybean–bradyrhizobia nodulation and growth, but the mechanism is unknown. After adding isoflavonoid inducers to 1‐102 culture, an active peak with a retention time of about 105 min in the HPLC fractionation was isolated using a bioassay based on the stimulation of soybean seed germination. The plant growth‐promoting activity of this material was compared with 1‐102 culture (cells) and supernatant under greenhouse conditions. The activator was applied to roots in 83, 830 and 8300 HPLC microvolts (μV) per seedling when plants were inoculated with bradyrhizobia or sprayed onto the leaves in same concentrations at 20 d after inoculation. The root‐applied activator, especially at 1 ml of 830 μV per seedling, enhanced soybean nodulation and growth at the same level as 1‐102 culture under both optimal and sub‐optimal root zone temperatures. Thus, this activator stimulating soybean seed germination is also responsible for the plant growth‐promoting activity of 1‐102 culture. However, when sprayed onto the leaves, the activator did not increase growth and in higher concentrations decreased average single leaf area. The results suggest that this inducible activator might be a lipo‐chitooligosaccharide (LCO) analogue. LCOs act as rhizobia‐to‐legume signals stimulating root nodule formation. The activator could provide additional ‘signal’, increasing in the signal quality (the signal‐to‐noise ratio, SNR) of the plant–rhizobia signal exchange process.

The effect of temperature and genistein concentration on lipo-chitooligosaccharide (LCO) production by wild-type and mutant strains of Bradyrhizobium japonicum

Lipo-chitooligosaccharides (LCOs), also known as nod factors, are the bacteria-to-plant signal molecules synthesized in response to the plant-to-bacteria signals, usually flavonoids. In Canada, low soil temperature is potentially a major factor limiting soybean growth and symbiotic nitrogen fixation. Low temperatures cause reductions in the amount of flavonoids, especially genistein, in soybean roots and also inhibit the expression of nod genes resulting in a delay in the onset of nodulation. The addition of genistein can overcome the earlier negative effects of low temperature. However, genistein is expensive and more cost-effective approaches to this problem may exist. We used UV mutagenesis to generate 10 mutants from Bradyrhizobium japonicum strain USDA 110, that expressed nod genes in the absence of plant-to-bacteria signal molecules (e.g. genistein) and they were further screened for their ability to synthesize LCO at three different temperatures (15, 17 and 25 °C). No detectable LCO was produced by these strains and mutants at 0 and 0.01 μM genistein. However, at higher genistein levels (0.1 and 1 μM) all mutants produced more LCO, at the three temperatures tested, than the wild type USDA 110 and 532C. Mutants Bj30054, Bj30056 and Bj30057 were most promising, producing the maximum concentration of LCO at low incubation temperatures (15 and 17 °C). Therefore, we envisage that under short season conditions that are characterized by low spring soil-temperatures these mutants (Bj30054, Bj30056 and Bj30057) would produce more LCO than USDA 110 or 532C, possibly leading to better nodulation and N 2 fixation and eventually soybean yield.

In vitro induction of lipo-chitooligosaccharide production in Bradyrhizobium japonicum cultures by root extracts from non-leguminous plants

Bradyrhizobium japonicum can form a N2-fixing symbiosis with compatible leguminous plants. It can also act as a plant-growth promoting rhizobacterium (PGPR) for non-legume plants, possibly through production of lipo-chitooligosaccharides (LCOs), which should have the ability to induce disease resistance responses in plants. The objective of this work was to determine whether non-leguminous crop plants can induce LCO formation by B. japonicum cultures. Cultures treated with root extracts of soybean, corn, cotton or winter wheat were assayed for presence and level of LCO. Root extracts of soybean, corn and winter wheat all induced LCO production, with extracts of corn inducing the greatest amounts. Root washings of corn also induced LCO production, but less than the root extract. These results indicated that the stimulation of non-legume plant growth by B. japonicum could be through the production of LCOs, induced by materials excreted by the roots of non-legume plants.

Isolation and characterization of the major nod factor of Bradyrhizobium japonicum strain 532C

Bradyrhizobium japonicum 532C nodulates soybean effectively under cool Canadian spring conditions and is used in Canadian commercial inoculants. The major lipo-chitooligosaccharide (LCO), bacteria-to-plant signal was characterized by HPLC, FAB-mass spectroscopy MALDI-TOF mass spectroscopy and revealed to be LCO Nod Bj-V (C18:1, MeFuc). This LCO is produced by type I strains of B. japonicum and is therefore unlikely to account for this strains superior ability to nodulate soybean under Canadian conditions. We also found that use of yeast extract mannitol medium gave similar results to that of Bergerson minimal medium.

Signalling and Development of Rhizobium: Legume Symbioses

SIGNALLING AND DEVELOPMENT OF R?IIZOBIUM ]LEGUM4E SYMB LOSES Kristina Lindstrom, Zewdu Terefework, Leena Suominen and Gilles Lortet Kristina Lindstrbm (corresponding author; e-mail: Kristina. Lindstrom@Helsinki.fi), Zewdu Terefework, Leena Suominenand Gilles Lortet, Department ofApplied Chemistry and Microbiology, University ofHelsinki, Viikki Biocenter, P.O.Box56, FIN-00014, Finland. INTRODUCTION The legume root nodule is a specialised organ on the plant root and is formed as a result of specific signal exchanges between the rhizobial microsym biont and the host plant. The signal exchange between rhizobia and legumes and the processes leading to a nitrogen-fixng symbiosis involve sev eral steps: (i) growth of the rhizobia in the rhizo sphere of the host; (ii) induction of rhizobial nodulation genes by plant exudate; (iii) production of the bacterial signal molecule, the Nod factor; (iv) attachment of rhizobia to the roots of the host; (v) induction of cell division in the plant; (vi) penetration by rhizobia into the plant via the infection thread; and (vii) engulfnent of rhizobia into intracellular symbiosomes (Hadri et al. 1998). In the root nodule, the bacterial nitrogenase en zyme system can reduce atmospheric dinitrogen to ammonia, which is then transported to the plant. SIGNAL EXCHANGE AND HOST SPECIFICITY Legumes excrete a number of secondary metab olites into their rhizosphere. Flavonoids and chal chones are the most important of these for the interaction with rhizobia. Depending on the host and the bacterium, specific compounds will serve as signals, or inducers, for the bacterial nodulation genes via the activator, NodD. This is the first level of specificity in the interaction. The bacterial nodulation genes will act together to produce a return signal, a lipochitooligosaccharide (LCO) commonly called the Nod factor (NF). This is the second level of host specificity (Fig. 1). The NF has an N-acetylglucosamiine backbone, consisting of three to five sugar units, wlth a fatty-acid moiety (C16-C20) attached to the non-reducing end. In addition, there are other substituents, such as acetyl, carbamoyl, methyl, sulphate and sugar groups, on the reducing or non-reducing end. The nature of the fatty acid and the substituents seems to determine host specificity at this level (Perret et al. 2000). Rhizobium galegae, which is very host specific for nodulation, has a multi-unsaturated fatty-acid chain, a carbamoyl group on the non reducing end and an acetyl group on the non-terminal sugar moiety next to the non-reduc ing end (Yang et al. 1999). According to our hypothesis, it is the acetyl group that confers the narrow host range. Several models have been pro posed for the perception of the NF by the plant, but no conclusive results have so farbeen reported. INFECTION, NODULE FORMATION AND NITROGEN FIXATION The mode of infection by rhizobia varies, depend ing mainly on the host plant (Hadri et al. 1998). In many legume genera, e.g. Pisum, Tnfolium, Medicago and Galega, the NF causes root-hair curling or deformation, followed by the formation of an infection thread in the root hair. The rhizo bia pass through the infection thread into the root, to a nodule primordium formed by the plant in the inner cortex. The rhizobia change to their bac teroid form and become surrounded by the plant-derived symbiosome membrane. The inde terminate nodule type formed in thisway is elong ated and has an apical meristem devoid of infected cells. The organisation of the symbiotic plant cells within the indetenninate nodule follows a charac teristic pattern: plant cells are infected near the apex, fix nitrogen in the centre and contain senesc ing bacteroids in the oldest part. The determinate nodule type, characteristic of genera such as Lotus, Glycine and Phaseolus, is formed after rhizobial penetration at the deformed root-hair base. The first Rhizobium-induced cell divisions occur in the outer cortex, and meristematic cells become in fected with symbiosomes, leading to colonisation of the central tissue, subsequent abortion of the menstems and a spherical nodule. A third form of infection is the 'crack entry', during which rhizo bia enter intercellularly at the point of emergence of the lateral roots. This infection type occurs in genera such as Arachis, Stylosanthes, Andira and Neptunia. In effective symbioses, the rhizobia within the...

Perception and Signal Transduction of Rhizobial NOD Factors

Referee: Dr. Gary Stacey, Director, Center for Legume Research, Department of Microbiology, M409 Walters Life Science Bldg., University of Tennessee, Knoxville, TN 37966-0845Soil bacteria belonging to genera Rhizobium, Bradyrhizobium, Allorhizobium, Azorhizobium, Mesorhizobium, and Sinorhizobium are able to induce nodule formation on the roots of leguminous plants. In the differentiated root nodules bacteria fix as bacteroids atmospheric nitrogen and deliver it to the host plant. The interaction between bacteria and host plant starts with a complex signal exchange. After induction by plant flavonoids, rhizobia synthesize and secrete lipo-chitooligosaccharides (LCOs), known as Nod factors, which induce morphological changes and expression of early nodulin genes in the roots of host plants. Specific recognition of Nod factors by host plants and early stages of signal transduction are discussed.

Rhizobium sp. BR816 produces a complex mixture of known and novel lipochitooligosaccharide molecules.

Rhizobial lipochitooligosaccharide (LCO) signal molecules induce various plant responses, leading to nodule development. We report here the LCO structures of the broadhost range strain Rhizobium sp. BR816. The LCOs produced are all pentamers, carrying common C18:1 or C18:0 fatty acyl chains, N-methylated and C-6 carbamoylated on the nonreducing terminal N-acetylglucosamine and sulfated on the reducing/terminal residue. A second acetyl group can be present on the penultimate N-acetylglucosamine from the nonreducing terminus. Two novel characteristics were observed: the reducing/terminal residue can be a glucosaminitol (open structure) and the degree of acetylation of this glucosaminitol or of the reducing residue can vary.