Lipo-chitin Oligosaccharides Research Articles

Shoot control of root development and nodulation is mediated by a receptor-like kinase.

In legumes, root nodule organogenesis is activated in response to morphogenic lipochitin oligosaccharides that are synthesized by bacteria, commonly known as rhizobia. Successful symbiotic interaction results in the formation of highly specialized organs called root nodules, which provide a unique environment for symbiotic nitrogen fixation. In wild-type plants the number of nodules is regulated by a signalling mechanism integrating environmental and developmental cues to arrest most rhizobial infections within the susceptible zone of the root. Furthermore, a feedback mechanism controls the temporal and spatial susceptibility to infection of the root system. This mechanism is referred to as autoregulation of nodulation, as earlier nodulation events inhibit nodulation of younger root tissues. Lotus japonicus plants homozygous for a mutation in the hypernodulation aberrant root (har1) locus escape this regulation and form an excessive number of nodules. Here we report the molecular cloning and expression analysis of the HAR1 gene and the pea orthologue, Pisum sativum, SYM29. HAR1 encodes a putative serine/threonine receptor kinase, which is required for shoot-controlled regulation of root growth, nodule number, and for nitrate sensitivity of symbiotic development.

Novel lipochitin oligosaccharide structures produced by Rhizobium etli KIM5s

The novel lipochitin oligosaccharide (LCOs) structures produced by Rhizobium etli KIM5s were characterized using a nanoHPLC reverse-phase system coupled to an ion-trap mass spectrometer. This technique was shown to be more sensitive for structural elucidation of LCOs than previously used mass spectrometric methods. The structures of the LCOs of R. etli KIM5s, the majority containing six monosaccharide residues, differed from those synthesized by all other rhizobia analyzed to date. In addition, novel structures in which the chitin backbone was deacetylated at one or more GlcNAc moieties were found as minor compounds. The difference in host range of this strain compared to that of other known bean microsymbionts is discussed.

Bradyrhizobium japonicum mutants with enhanced sensitivity to genistein resulting in altered nod gene regulation.

Bradyrhizobium japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. Plasmid pZB32, carrying a nodY::lacZ transcriptional gene fusion, was introduced into B. japonicum cells that had been subjected to UV mutagenesis. Ten independent transformants producing a blue color on plates containing 5bromo-4chloro-3indolyl-beta-D-galactopyranoside but lacking genistein, indicative of constitutive expression of the nodY::lacZ reporter gene, were isolated. Beta-galactosidase activity assays revealed that while all of the 10 strains were sensitive to low concentrations of genistein, none exhibited truly constitutive nodY::lacZ expression in liquid culture. Soybean plants inoculated with three of the mutants were chlorotic and stunted, with shoot dry weights close to those of the uninoculated plants, indicating the absence of nitrogen fixation. Differences in the kinetics of nodY::lacZ expression and lipochitin oligosaccharide Nod signal production suggested that the strains carried different mutations. Some of these strains may be useful in mitigating the low root zone temperature-associated delay in soybean nodulation at the northern extent of soybean cultivation.

Responses of a model legume Lotus japonicus to lipochitin oligosaccharide nodulation factors purified from Mesorhizobium loti JRL501.

Lotus japonicus has been proposed as a model legume for molecular genetic studies of symbiotic plant-microbe interactions leading to the fixation of atmospheric nitrogen. Lipochitin oligosaccharides (LCOs), or Nod factors, were isolated from the culture of Mesorhizobium loti strain JRL501 (MAFF303099), an efficient microsymbiont of L. japonicus B-129 cv. Gifu. High-performance liquid chromatography and mass spectrometric analyses allowed us to identify at least five different structures of LCOs that were produced by JRL501. The major component was NodMl-V(C18:1, Me, Cb, AcFuc), an N-acetyl-glucosamine pentamer in which the nonreducing residue is N-acylated with a C18:1 acyl moiety, N-methylated, and carries a carbamoyl group and the reducing N-acetylglucosamine residue is substituted with 4-O-acetyl-fucose. Additional novel LCO structures bearing fucose instead of acetyl-fucose at the reducing end were identified. Mixtures of these LCOs could elicit abundant root hair deformation on L. japonicus roots at a concentration of 10(-7) to 10(-9) M. Spot inoculation of a few nanograms of LCOs on L. japonicus roots induced the formation of nodule primordia in which the early nodulin genes, ENOD40 and ENOD2, were expressed in a tissue-specific manner. We also observed the formation of a cytoplasmic bridge (preinfection thread) in the swollen outermost cortical cells. This is the first description of cytoplasmic bridge formation by purified LCOs alone in a legume-forming determinate nodules.

Binding site for chitin oligosaccharides in the soybean plasma membrane.

Affinity cross-linking of the plasma membrane fraction to an (125)I-labeled chitin oligosaccharide led to the identification and characterization of an 85-kD, chitin binding protein in plasma membrane-enriched fractions from both suspension-cultured soybean cells and root tissue. Inhibition analysis indicated a binding preference for larger (i.e. degrees of polymerization = 8) N-acetylated chitin molecules with a 50% inhibition of initial activity value of approximately 50 nM. N-Acetyl-glucosamine and chitobiose showed no inhibitory effects at concentrations as high as 250 microM. It is noteworthy that the major lipo-chitin oligosaccharide Nod signal produced by Bradyrhizobium japonicum was also shown to be a competitive inhibitor of ligand binding. However, the binding site appeared to recognize the chitin portion of the Nod signal, and it is unlikely that this binding activity represents a specific Nod signal receptor. Chitooligosaccharide specificity for induction of medium alkalinization and the generation of reactive oxygen in suspension-cultured cells paralleled the binding activity. Taken together, the presence of the chitin binding protein in the plasma membrane fraction and the specificity and induction of a biological response upon ligand binding suggest a role for the protein as an initial response mechanism for chitin perception in soybean (Glycine max).

Rhizobial NodL O -Acetyl Transferase and NodS N -Methyl Transferase Functionally Interfere in Production of Modified Nod Factors

The products of the rhizobial nodulation genes are involved in the biosynthesis of lipochitin oligosaccharides (LCOs), which are host-specific signal molecules required for nodule formation. The presence of an O-acetyl group on C-6 of the nonreducing N-acetylglucosamine residue of LCOs is due to the enzymatic activity of NodL. Here we show that transfer of the nodL gene into four rhizobial species that all normally produce LCOs that are not modified on C-6 of the nonreducing terminal residue results in production of LCOs, the majority of which have an acetyl residue substituted on C-6. Surprisingly, in transconjugant strains of Mesorhizobium loti, Rhizobium etli, and Rhizobium tropici carrying nodL, such acetylation of LCOs prevents the endogenous nodS-dependent transfer of the N-methyl group that is found as a substituent of the acylated nitrogen atom. To study this interference between nodL and nodS, we have cloned the nodS gene of M. loti and used its product in in vitro experiments in combination with purified NodL protein. It has previously been shown that a chitooligosaccharide N deacetylated on the nonreducing terminus (the so-called NodBC metabolite) is the preferred substrate for NodS as well as for NodL. Here we show that the NodBC metabolite, acetylated by NodL, is not used by the NodS protein as a substrate while the NodL protein can acetylate the NodBC metabolite that has been methylated by NodS.

The Nodulation Protein NodG Shows the Enzymatic Activity of an 3-Oxoacyl-Acyl Carrier Protein Reductase

The acyl carrier protein NodF is required for the synthesis of unusual polyunsaturated fatty acids that confer specificity to lipochitin oligosaccharide nodulation (Nod) factors of Rhizobium leguminosarum. In this study, homogeneous NodF protein was used as a ligand to identify proteins of R. leguminosarum that specifically interact with NodF and presumably are involved in the biosynthesis or transfer of the unusual fatty acids. The N-terminal amino acid sequence of a 29-kDa protein that interacts strongly with NodF revealed high similarity to NodG of Rhizobium sp. N33 and to NodG of Sinorhizobium meliloti We cloned and sequenced the gene coding for the NodG-like protein of R. leguminosarum and found it to be the product of the constitutively expressed gene fabG. FabG is the 3-oxoacyl-acyl carrier protein reductase that catalyzes the first reduction step in each cycle of fatty acid elongation. FabG of R. leguminosarum and NodG of Rhizobium sp. N33 were expressed in Escherichia coli. In both cases, the purified protein showed 3-oxoacyl-acyl carrier protein reductase activity in vitro. Therefore, NodG has the same biochemical function as FabG, and the high degree of similarity at the protein and DNA level suggest that nodG is a duplication of the housekeeping genefabG.

The Rhizobium etli argC gene is essential for Arginine biosynthesis and nodulation of Phaseolus vulgaris.

A Tn5-induced mutant strain (CTNUX5) of Rhizobium etli unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into an argC-homologous gene. Unlike its wild-type parent (strain CE3), the mutant strain CTNUX5 had an absolute dependency on arginine to grow. The argC gene was cloned from the wild-type strain CE3, and the resulting plasmid, pAR207, after transformation was shown to relieve the arginine auxotrophy of strain CTNUX5. Unlike strain CE3 or CTNUX5-pAR207, strain CTNUX5 showed undetectable levels of N-acetyl-gamma-glutamylphosphate reductase activity. Unless arginine was added to the growth medium, strain CTNUX5 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (nodulation factors) and to induce nodules or nodulelike structures on the roots of Phaseolus vulgaris.

Growth temperature regulation of host-specific modifications of rhizobial lipo-chitin oligosaccharides: the function of nodX is temperature regulated.

Lipo-chitin oligosaccharides (LCOs) are usually produced and isolated for structural analysis from bacteria cultured under laboratory rather than field conditions. We have studied the influence of bacterial growth temperature on the LCO structures produced by different Rhizobium leguminosarum strains, using thin-layer chromatographic, high-performance liquid chromatographic, and mass spectrometric analyses. Wild-type R. leguminosarum bv. viciae A1 was shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12 degrees C than at 28 degrees C, indicating that the activity of nodX (a gene encoding an LCO O-acetyl transferase) is temperature dependent. Interestingly, symbiotic resistance genes sym1 and sym2 found in primitive pea cultivars are also temperature sensitive, only being active at low temperatures, at which they block nodulation by R. leguminosarum bv. viciae strains lacking nodX. We therefore propose that the gene-for-gene relationship between plant and bacterium has a temperature-sensitive mechanism as an adaptation to environmental conditions. An R. leguminosarum bv. trifolii strain was also shown to produce larger relative amounts of nodX-mediated, acetylated LCOs at 12 degrees C than at 28 degrees C. The major components synthesized by the two strains are produced at both temperatures but in different relative amounts, while some minor components are only produced at one of the two temperatures.

Functional analysis of chimeras derived from the Sinorhizobium meliloti and Mesorhizobium loti nodC genes identifies regions controlling chitin oligosaccharide chain length.

The rhizobial nodulation gene nodC encodes an N-acetylglucosaminyltransferase that is responsible for the synthesis of chitin oligosaccharides. These oligosaccharides are precursors for the synthesis of the lipo-chitin oligosaccharides that induce cell division and differentiation during the development of nitrogen-fixing root nodules in leguminous plants. The NodC proteins of Mesorhizobium loti and Sinorizobium meliloti yield chitinpentaose and chitintetraose as their main products, respectively. In order to localize regions in these enzymes that are responsible for this difference in product chain length, a set of six chimeric enzymes, comprising different combinations of regions of the NodC proteins from these two bacteria, was expressed in Escherichia coli. The oligosaccharides produced were analyzed using thin-layer chromatography. The major conclusion from this work is that a central 91-amino acid segment does not play any obvious role in determining the difference in the chain length of the major product. Furthermore, the characteristically predominant synthesis of chitintetraose by S. meliloti NodC is mainly dependent on a C-terminal region of maximally 164 amino acids; exchange of only this C-terminal region is sufficient to completely convert the M. loti chitinpentaose synthase into an S. meliloti-like chitintetraose synthase. The N-terminal region of 170 amino acids also plays a role in restricting the length of the major product to a tetramer. However, the role of the C-terminal region is clearly dominant, since exchanging the N-terminal region has no effect on the relative amounts of chitintetraose and -pentaose produced when the C-terminal region of S. meliloti NodC is present. The length of a predicted beta-strand around residue 300 in the C-terminal region of various NodC proteins is the only structural element that seems to be related to the length of the chitin oligosaccharides produced by these enzymes; the higher the amount of chitintetraose relative to chitinpentaose, the shorter the predicted beta-strand. This element may therefore be important for the effect of the C-terminal 164 amino acids on chitin oligosaccharide chain length.

A Lotus japonicus nodulation system based on heterologous expression of the fucosyl transferase NodZ and the acetyl transferase NoIL in Rhizobium leguminosarum.

Heterologous expression of NodZ and NolL proteins in Rhizobium leguminosarum bv. viciae led to the production of acetyl fucosylated lipo-chitin oligosaccharides (LCOs), indicating that the NolL protein obtained from Mesorhizobium loti functions as an acetyl transferase. We show that the NolL-dependent acetylation is specific for the fucosyl penta-N-acetylglucosamine species. In addition, the NolL protein caused elevated production of LCOs. Efficient nodulation of Lotus japonicus by the NodZ/NolL-producing strain was demonstrated. Nodulation efficiency was further improved by the addition of the ethylene inhibitor L-alpha-(2-aminoethoxyvinyl) glycine (AVG).

Heterologous rhizobial lipochitin oligosaccharides and chitin oligomers induce cortical cell divisions in red clover roots, transformed with the pea lectin gene.

Division of cortical cells in roots of leguminous plants is triggered by lipochitin oligosaccharides (LCOs) secreted by the rhizobial microsymbiont. Previously, we have shown that presence of pea lectin in transgenic white clover hairy roots renders these roots susceptible to induction of root nodule formation by pea-specific rhizobia (C. L. Díaz, L. S. Melchers, P. J. J. Hooykaas, B. J. J. Lugtenberg, and J. W. Kijne, Nature 338:579-581, 1989). Here, we report that pea lectin-transformed red clover hairy roots form nodule primordium-like structures after inoculation with pea-, alfalfa-, and Lotus-specific rhizobia, which normally do not nodulate red clover. External application of a broad range of purified LCOs showed all of them to be active in induction of cortical cell divisions and cell expansion in a radial direction, resulting in formation of structures that resemble nodule primordia induced by clover-specific rhizobia. This activity was obvious in about 50% of the red clover plants carrying hairy roots transformed with the pea lectin gene. Also, chitopentaose, chitotetraose, chitotriose, and chitobiose were able to induce cortical cell divisions and cell expansion in a radial direction in transgenic roots, but not in control roots. Sugar-binding activity of pea lectin was essential for its effect. These results show that transformation of red clover roots with pea lectin results in a broadened response of legume root cortical cells to externally applied potentially mitogenic oligochitin signals.

A plant regulator controlling development of symbiotic root nodules.

Symbiotic nitrogen-fixing root nodules on legumes are founded by root cortical cells that de-differentiate and restart cell division to establish nodule primordia. Bacterial microsymbionts invade these primordia through infection threads laid down by the plant and, after endocytosis, membrane-enclosed bacteroids occupy cells in the nitrogen-fixing tissue of functional nodules. The bacteria excrete lipochitin oligosaccharides, triggering a developmental process that is controlled by the plant and can be suppressed. Nodule inception initially relies on cell competence in a narrow infection zone located just behind the growing root tip. Older nodules then regulate the number of nodules on a root system by suppressing the development of nodule primordia. To identify the regulatory components that act early in nodule induction, we characterized a transposon-tagged Lotus japonicus mutant, nin (for nodule inception), arrested at the stage of bacterial recognition. We show that nin is required for the formation of infection threads and the initiation of primordia. NIN protein has regional similarity to transcription factors, and the predicted DNA-binding/dimerization domain identifies and typifies a consensus motif conserved in plant proteins with a function in nitrogen-controlled development.

Lipochitin Oligosaccharides from Rhizobium leguminosarum bv. viciae Reduce Auxin Transport Capacity in Vicia sativa subsp. nigra Roots

Induction of the formation of root nodule primordia in legume roots by symbiotic rhizobia is probably preceded by a change in plant hormone physiology. We used a Vicia sativa (vetch) split root system to study the effect of inoculation with rhizobia or purified Nod factors (lipochitin oligosaccharides, LCOs) on polar auxin transport in roots. Addition of R. leguminosarum bv. viciae, the infective symbiote of vetch, to roots of its host plant reduced polar auxin transport capacity of these roots within 24 h, in contrast to addition of non-nodulating, Sym plasmid-cured rhizobia. Addition of purified vetch-specific LCOs (NodRlv-IV/V[18:4,Ac]) caused a transient reduction in as little as 4 h after application, while after 16 h a second, stronger, and prolonged inhibition was observed that lasted at least 48 h. This reduction of auxin transport capacity was in the same order of magnitude as inhibition by N-(1-naphthyl)phthalamic acid (NPA). Purified LCOs (NodRm-IV[16:2,Ac,S]) from Sinorhizobium meliloti, the symbiote of alfalfa, and chitopentaose were inactive, which indicates a specific effect of LCOs produced by R. leguminosarum bv. viciae. Auxin transport inhibition was restricted to the apical nodulation-susceptible part of the roots, whereas the upper parts of the roots showed no difference in auxin transport after treatment. The effect could be observed with as low as 10-9 M NodRlv-IV/V[18:4,Ac] LCOs. Reduction of auxin transport by LCOs could not be inhibited by nitrate. Since inhibition of auxin transport capacity preceded the first root cortical cell divisions that result in root primordium formation, our results suggest a direct relationship between LCOs, polar auxin transport, and root nodule initiation, consistent with the hypothesis of U. Mathesius, H. R. M. Schlaman, H. P. Spaink, C. Sautter, B. G. Rolfe, and M. A. Djordjevic (Plant J. 14:23–34, 1998). However, nonmitogenic NodRlv-IV/V[18:1,Ac] showed a similar effect, which suggests that mitogenicity results from additional effects, in concert with auxin transport inhibition.

Chemical synthesis of N-acetylglucosamine derivatives and their use as glycosyl acceptors by the Mesorhizobium loti chitin oligosaccharide synthase NodC

Rhizobial bacteria synthesize lipo-chitin oligosaccharide signal molecules (Nod factors) that are essential for the formation of symbiotic organs on the roots of host plants, a process known as nodulation. Biosynthesis of the chitin oligosaccharide moiety in Nod factors is carried out by the rhizobial N-acetylglucosaminyltransferase NodC. The initial acceptor or primer used for the synthesis of chitin oligosaccharides in vivo is unknown. To investigate the acceptor specificity of NodC, we have synthesized derivatives of N-acetylglucosamine (GlcNAc) with different aglycones and tested whether they are acceptors for NodC in vitro using a membrane preparation of an Escherichia coli strain expressing the Mesorhizobium loti chitin oligosaccharide synthase NodC. Analysis of reaction products using thin-layer chromatography shows that GlcNAc derivatives containing simple alkyl chains or other hydrophobic groups linked to C-1 are acceptors for NodC. The enzyme appears to be specific for acceptors in which the aglycone is β-linked. GlcNAc derivatives in which the methyl group of the N-acetyl moiety of GlcNAc is replaced by an allyloxy or benzyloxy group are still used as acceptors by NodC. The original methyl group at this position therefore does not appear to be essential for the interaction between NodC and GlcNAc. A NodC-dependent reaction product that is more hydrophobic than GlcNAc was detected in reaction mixtures containing 5% methanol but lacking an exogenously added acceptor. This may be due to the presence of a natural hydrophobic glycosyl acceptor for NodC in the membranes of E. coli, but the structure of this reaction product remains to be investigated.

The Rhizobium etli trpB gene is essential for an effective symbiotic interaction with Phaseolus vulgaris.

A mutant strain (CTNUX4) of Rhizobium etli carrying Tn5 unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Sequence analysis showed that Tn5 is inserted into a trpB (tryptophan synthase)-homologous gene. When tested on the roots of Phaseolus vulgaris, strain CTNUX4 was able to induce only small, slightly pink, ineffective (Fix-) nodules. However, under free-living conditions, strain CTNUX4 was unable to produce flavonoid-inducible lipo-chitin oligosaccharides (Nod factors) unless tryptophan was added to the growth medium. These data and histological observations indicate that the lack of tryptophan biosynthesis affects the symbiotic behavior of R. etli.

Aberrant nodulation response of Vigna umbellata to a Bradyrhizobium japonicum NodZ mutant and nodulation signals.

The (Brady)rhizobium nodulation gene products synthesize lipo-chitin oligosaccharide (LCO) signal molecules that induce nodule primordia on legume roots. In spot inoculation assays with roots of Vigna umbellata, Bradyrhizobium elkanii LCO and chemically synthesized LCO induced aberrant nodule structures, similar to the activity of these LCOs on Glycine soja (soybean). LCOs containing a pentameric chitin backbone and a reducing-end 2-O-methyl fucosyl moiety were active on V. umbellata. In contrast, the synthetic LCO-IV(C16:0), which has previously been shown to be active on G. soja, was inactive on V. umbellata. A B. japonicum NodZ mutant, which produces LCO without 2-O-methyl fucose at the reducing end, was able to induce nodule structures on both plants. Surprisingly, the individual, purified, LCO molecules produced by this mutant were incapable of inducing nodule formation on V. umbellata roots. However, when applied in combination, the LCOs produced by the NodZ mutant acted cooperatively to produce nodulelike structures on V. umbellata roots.

Structural characterisation of lipo-chitin oligosaccharides isolated from Bradyrhizobium aspalati, microsymbionts of commercially important South African legumes

The shoots of the South African legume Aspalathus linearis spp. linearis ( A. linearis) are used in the manufacture of an increasingly popular beverage that has acclaimed beneficial effects on health; this important export product is known as Rooibos (or Redbush) tea. Three strains of Bradyrhizobium aspalati, which are the nitrogen-fixing symbionts of Aspalathus carnosa, A. hispida and A. linearis, were tested for the production of lipo-chitin oligosaccharide signal molecules using thin-layer chromatographic analysis after induction with different inducers, including Rooibos tea extract, and radioactive labelling. Large-scale separation, using high-performance liquid chromatography, of lipo-chitin oligosaccharides from B. aspalati isolated from A. carnosa was performed for structural characterisation using fast-atom bombardment mass spectrometry and chemical modifications followed by gas chromatography–mass spectrometric analysis. The strain was shown to secrete a family of unusual lipo-chitin oligosaccharides that are highly substituted on the nonreducing-terminal residue but unsubstituted on the reducing-terminal residue. They have a backbone of three to five β-(1→4)-linked N-acetyl- d-glucosamine residues substituted on the nonreducing terminus with a C16:0, C16:1, C18:0, C18:1, C19:1cy, or C20:1 fatty acyl chain, and are both N-methylated and 4,6-dicarbamoylated.

Chitin oligosaccharide synthesis by rhizobia and zebrafish embryos starts by glycosyl transfer to O4 of the reducing-terminal residue.

Lipochitin oligosaccharides are organogenesis-inducing signal molecules produced by rhizobia to establish the formation of nitrogen-fixing root nodules in leguminous plants. Chitin oligosaccharide biosynthesis by the Mesorhizobium loti nodulation protein NodC was studied in vitro using membrane fractions of an Escherichia coli strain expressing the cloned M. loti nodC gene. The results indicate that prenylpyrophosphate-linked intermediates are not involved in the chitin oligosaccharide synthesis pathway. We observed that, in addition to N-acetylglucosamine (GlcNAc) from UDP-GlcNAc, NodC also directly incorporates free GlcNAc into chitin oligosaccharides. Further analysis showed that free GlcNAc is used as a primer that is elongated at the nonreducing terminus. The synthetic glycoside p-nitrophenyl-beta-N-acetylglucosaminide (pNPGlcNAc) has a free hydroxyl group at C4 but not at C1 and could also be used as an acceptor by NodC, confirming that chain elongation by NodC takes place at the nonreducing-terminal residue. The use of artificial glycosyl acceptors such as pNPGlcNAc has not previously been described for a processive glycosyltransferase. Using this method, we show that also the DG42-directed chitin oligosaccharide synthase activity, present in extracts of zebrafish embryos, is able to initiate chitin oligosaccharide synthesis on pNPGlcNAc. Consequently, chain elongation in chitin oligosaccharide synthesis by M. loti NodC and zebrafish DG42 occurs by the transfer of GlcNAc residues from UDP-GlcNAc to O4 of the nonreducing-terminal residue, in contrast to earlier models on the mechanism of processive beta-glycosyltransferase reactions.

Function of chitin oligosaccharides in plant and animal development.

In plant development chitin oligosaccharides have been studied intensively as part of the communication between leguminous plants and Rhizobium bacteria. The Rhizobium bacteria synthesize and secrete lipochitin oligosaccharides (LCOs) to induce the development of a root nodule, in which the bacteria will infiltrate to start a symbiotic relation with the plant. Here we will give an overview of the biosynthetic route used by the bacteria to synthesize these LCOs. Perception by the plant will also be discussed as well as early responses to the LCOs. By working with the genes from the biosynthetic route, other genes were identified that share homology with the chitin synthase genes from Rhizobium. These genes are now isolated from human, mouse, chick, Xenopus and zebrafish and can be divided into three classes. They are mainly expressed during early development at the same stage as chitin oligosaccharide synthase activity can be detected. A controversy has been risen about their biochemical activity and will be further discussed here.