Lipo-chitin Oligosaccharides Research Articles

Regulation of Plant Morphogenesis by Lipo-Chitin Oligosaccharides

Regulation of Plant Morphogenesis by Lipo-Chitin Oligosaccharides

Induction of nodule primordia on Phaseolus and Acacia by lipo-chitin oligosaccharide nodulation signals from broad-host-range Rhizobium strain GRH2.

Rhizobium wild-type strain GRH2 was originally isolated from the tree, Acacia cyanophylla, and has a broad host-range which includes herbaceous legumes, such as Phaseolus and Trifolium species. Here we show that strains of Rhizobium sp. GRH2, into which heterologous nodD alleles have been introduced, produce a large diversity of both sulphated and non-sulphated lipo-chitin oligosaccharides (LCOs). Most of the molecular species contain an N-methyl group on the reducing-terminal N-acetyl-glucosamine. The LCOs vary in the nature of the fatty acyl chain and in the length of the chitin backbone. The majority of the LCOs have an oligosaccharide chain length of five GlcNAc residues, but a few are oligomers having six GlcNAc units. LCOs purified from GRH2 are able to induce root hair formation and deformation on Acacia cyanophylla and A. melanoxylon plants. We show that an N-vaccenoyl-chitopentaose bearing an N-methyl group is able to induce nodule primordia on Phaseolus vulgaris, A. cyanophylla, and A. melanoxylon, indicating that for these plants an N-methyl modification is sufficient for nodule primordia induction.

Isolation, chemical structures and biological activity of the lipo-chitin oligosaccharide nodulation signals from Rhizobium etli

Rhizobium etli is a microsymbiont of plants of the genus Phaseolus. Using mass spectrometry we have identified the lipo-chitin oligosaccharides (LCOs) that are produced by R. etli strain CE3. They are N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with cis-vaccenic acid (C18:1) or stearic acid (C18:0) and carries a carbamoyl group at C4. The reducing residue is substituted at the C6 position with O-acetylfucose. Analysis of their biological activity on the host plant Phaseolus vulgaris shows that these LCOs can elicit the formation of nodule primordia which develop to the stage where vascular bundles are formed. The formation of complete nodule structures, including an organized vascular tissue, is never observed. Considering the very close resemblance of the R. etli LCO structures to those of R. loti (I. M. López-Lara, J. D. J. van den Berg, J. E. Thomas Oates, J. Glushka, B. J. J. Lugtenberg, H. P. Spaink, Mol Microbiol 15: 627-638, 1995) we tested the ability of R. etli strains to nodulate various Lotus species and of R. loti to nodulate P. vulgaris. The results show that R. etli is indeed able to nodulate Lotus plants. However, several Lotus species are only nodulated when an additional flavonoid independent transcription activator (FITA) nodD gene is provided. Phaseolus plants can also be nodulated by R. loti bacteria, but only when the bacteria contain a FITA nodD gene. Apparently, the type of nod gene inducers secreted by the plants is the major basis for the separation of Phaseolus and Lotus into different cross inoculation groups.

Substrate Specificity and Kinetic Studies of Nodulation Protein NodL of Rhizobium leguminosarum

All lipo-chitin oligosaccharides identified from Rhizobium leguminosarum carry an O-acetyl moiety on C6 of the nonreducing terminal N-acetylglucosamine residue. Previously, we have shown that purified NodL protein, using acetyl-CoA as acetyl donor, in vitro acetylates N-acetylglucosamine, chitin oligosaccharides, and lipo-chitin oligosaccharides. In this paper, the enzymatic properties and substrate specificity of NodL protein were analyzed, using a spectrophotometric assay to quantify NodL transacetylating activity. NodL functions optimally under alkaline conditions. Transacetylating activity has a broad temperature optimum between 28 and 42 degrees C. NodL protein is stable for at least 15 min up to 48 degrees C. Glucosamine, chitosan oligosaccharides, terminally de-N-acetylated chitin derivatives, and cellopentaose were identified as acetyl-accepting substrates for NodL protein. Quantitative substrate specificity studies show that chitin derivatives with a free amino group on the nonreducing terminal residue are the preferred substrates of the NodL protein. Our results strongly indicate that the nonreducing terminally de-N-acetylated chitin oligosaccharides produced by the NodC and NodB enzymes are the in vivo acetyl-accepting substrates for NodL protein.

Structural Requirements of Synthetic and Natural Product Lipo-Chitin Oligosaccharides for Induction of Nodule Primordia on Glycine soja

Rhizobia synthesize a class of lipo-chitin oligosaccharides that induce root hair deformation and induce the initiation of nodule structures on legume roots. These lipo-chitin oligosaccharides are tetra- and penta-lipo-oligosaccharides of N-acetylglucosamine with an acyl substitution on the nonreducing end and are commonly known as Nod factors. In this study, we demonstrate that synthetic analogs of natural product Nod factors have the same biological activities. To determine structure-activity relationships, a collection of synthetic and natural product lipo-chitin oligosaccharides was assayed on Glycine soja. All biologically active lipo-chitin oligosaccharides induced both root hair deformation and nodule initiations on G. soja. The most active lipo-chitin oligosaccharides deformed root hairs at 10(-15) M and induced nodules at 1 ng of lipo-chitin oligosaccharide per spot inoculation. Plant responses demonstrate an interdependence of backbone length and the presence of substitutions on the reducing end. Lipo-chitin oligosaccharides containing four N-acetylglucosamine residues were active only without a reducing end modification, whereas lipo-chitin oligosaccharides containing five N-acetylglucosamine residues were active only with reducing end modification. The plant thus recognizes lipo-chitin oligosaccharides without reducing end substitutions despite the importance of these modifications for host range.

A central domain of Rhizobium NodE protein mediates host specificity by determining the hydrophobicity of fatty acyl moieties of nodulation factors

Previously, we have shown that the nodE gene is a major determinant of the difference in host range between Rhizobium leguminosarum biovars viciae and trifolii. A new genetic test system for stringent functional analysis of nodE genes was constructed. By testing chimeric nodE genes constructed by the exchange of polymerase chain reaction (PCR)-generated restriction cassettes, we show that a central domain, containing only 44 non-conserved amino acid residues, determines the host specificity of the NodE protein (401 amino acid residues). Mass spectrometric analysis of the lipo-chitin oligosaccharides (LCOs) produced by the new test strain containing the biovar viciae nodE gene shows that molecules containing a polyunsaturated C18:4 (trans-2, trans-4, trans-6, cis-11-octadecatetraenoic) fatty acyl moiety are produced, as is the case for wild-type R. leguminosarum bv. viciae. The LCOs determined by the biovar trifolii nodE gene, which was overproduced in our test strain, carry C18:2 and C18:3 fatty acyl chains containing two or three conjugated trans double bonds, respectively. Therefore, the main difference between the nodE-determined LCOs of biovar viciae and trifolii in this system is the presence or absence of one cis double bond, resulting in the very different hydrophobicity of the LCOs. Using a newly developed spot application assay, we show that the C18:2- and C18:3-containing LCOs are able to induce the formation of nodule primordia on roots of Trifolium pratense. On the basis of these and other recent results, we propose that the host range of nodulation of the R. leguminosarum biovars viciae and trifolii is determined by the degree of hydrophobicity of the polyunsaturated fatty acyl moieties of their LCOs, which is mediated by the host-specific central domain of the NodE protein.

Structural identification of the lipo-chitin oligosaccharide nodulation signals of Rhizobium loti.

Rhizobium loti is a fast-growing Rhizobium species that has been described as a microsymbiont of plants of the genus Lotus. Nodulation studies show that Lotus plants are nodulated by R. loti, but not by most other Rhizobium strains, indicating that R. loti produces specific lipo-chitin oligosaccharides (LCOs) which are necessary for the nodulation of Lotus plants. The LCOs produced by five different Rhizobium loti strains have been purified and were shown to be N-acetylglucosamine pentasaccharides of which the non-reducing residue is N-methylated and N-acylated with cis-vaccenic acid (C18:1) or stearic acid (C18:O) and carries a carbamoyl group. In one R. loti strain, NZP2037, an additional carbamoyl group is present on the non-reducing terminal residue. The major class of LCO molecules is substituted on the reducing terminal residue with 4-O-acetylfucose. Addition of LCOs to the roots of Lotus plants results in abundant distortion, swelling and branching of the root hairs, whereas spot inoculation leads to the formation of nodule primordia.

Role of rhizobial lipo-chitin oligosaccharide signal molecules in root nodule organogenesis

The role of oligosaccharide molecules in plant development is discussed. In particular the role of the rhizobial lipo-chitin oligosaccharide (LCO) signal molecules in the development of the root nodule indicates that oligosaccharides play an important role in organogenesis in plants. Recent results of the analyses of structures and of the biosynthesis of the LCO molecules are summarized in this paper. The knowledge and technologies that resulted from these studies will be important tools for further studying the function of LCO signals in the plant and in the search for analogous signal molecules produced by plants.

Serine residue 45 of nodulation protein NodF from Rhizobium leguminosarum bv. viciae is essential for its biological function

A system for testing the role of the Rhizobium nodF gene in the production of host-specific lipochitin oligosaccharides and in nodulation was developed. We show that a mutant nodF gene, in which the codon for serine residue 45 was changed to that for threonine, still expresses NodF, which, however, is no longer functional.

Structural identification of metabolites produced by the NodB and NodC proteins of Rhizobium leguminosarum.

The Rhizobium nodulation genes nodABC are involved in the synthesis of lipo-chitin oligosaccharides. We have analysed the metabolites which are produced in vivo and in vitro by Rhizobium strains which express the single nodA, nodB and nodC genes or combinations of the three. In vivo radioactive labelling experiments, in which D-[1-14C]-glucosamine was used as a precursor, followed by mass spectrometric analysis of the purified radiolabelled metabolic products, showed that Rhizobium strains that only express the combination of the nodB and nodC genes do not produce lipo-chitin oligosaccharides but instead produce chitin oligomers (mainly pentamers) which are devoid of the N-acetyl group on the non-reducing terminal sugar residue (designated NodBC metabolites). Using the same procedure we have shown that when the nodL gene is expressed in addition to the nodBC genes the majority of metabolites contain an additional O-acetyl substituent on the non-reducing terminal sugar residue (designated NodBCL metabolites). The NodBC and NodBCL metabolites purified after in vivo labelling were compared with the radiolabelled metabolites produced in vitro by Rhizobium bacterial cell lysates to which UDP-N-acetyl-D-[U-14C]-glucosamine was added using thin-layer chromatography. The results show that the lysates of strains which expressed the nodBC or nodBCL genes can also produce NodBC and NodBCL metabolites. The same results were obtained when the NodB and NodC proteins were produced separately in two different strains. On the basis of these and other recent results, we propose that NodB is a chitin oligosaccharide deacetylase, NodC an N-acetylglucosaminyltransferase and, by default, NodA is involved in lipid attachment.

The A.R.M. Decision

Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor receptor 1 (NFR1) bind Nod factor directly at high-affinity binding sites. Both receptor proteins were posttranslationally processed when expressed as fusion proteins and extracted from purified membrane fractions of Nicotiana benthamiana or Arabidopsis thaliana. The N-terminal signal peptides were cleaved, and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N-glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man3XylFucGlcNAc4, were identified by mass spectrometry and located at amino acid positions N68 and N198. Receptor–ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The Kd values obtained for Nod factor binding were in the nanomolar range and comparable to the concentration range sufficient for biological activity. Structure-dependent ligand specificity was shown by using chitin oligosaccharides. Taken together, our results suggest that ligand recognition through direct ligand binding is a key step in the receptor-mediated activation mechanism leading to root nodule development in legumes.