A. A. N. van Brussel

Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs.

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.

The ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar, viciae on Vicia sativa subsp, nigra by suppressing the 'Thick and short roots' phenotype

Nodulation of Vicia sativa subsp. nigra L. by Rhizobium bacteria is coupled to the development of thick and short roots (Tsr). This root phenotype as well as root-hair induction (Hai) and root-hair deformation (Had) are caused by a factor(s) produced by the bacteria in response to plant flavonoids. When very low inoculum concentrations (0.5–5 bacteria·ml-1) were used, V. sativa plants did not develop the Tsr phenotype and became nodulated earlier than plants with Tsr roots. Furthermore, the nodules of these plants were located on the primary root in contrast to nodules on Tsr roots, which were all located at sites of lateral-root emergence. The average numbers of nodules per plant were not significantly different for these two types of nodulation. Root-growth inhibition and Hai, but not Had, could be mimicked by ethephon, and inhibited by aminoethoxyvinylglycine (AVG). Addition of AVG to co-cultures of Vicia sativa and the standard inoculum concentration of 5·105 bacteria·ml-1 suppressed the development of the Tsr phenotype and restored nodulation to the pattern that was observed with very low concentrations of bacteria (0.5–5 bacteria·ml-1). The delay in nodulation on Tsr roots appeared to be caused by the fact that nodule meristems did not develop on the primary root, but only on the emerging laterals. The relationship between Tsr, Hai, Had, and nodulation is discussed.

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 olgosaccharide 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.

Repression of Small bacteriocin excretion in Rhizobium leguminosarum and Rhizobium trifolii by transmissible plasmids

SummaryAll of the fast growing Rhizobium leguminosarum and R. trifolii strains studied, except four, excrete a small bacteriocin (small) into the culture medium. Only the four non-excreting strains harbour highly transmissible plasmids among which are the Sym plasmids pRL1JI (pea cross-inoculation group) and pRtr5a (clover cross-inoculation group). Small production genes were demonstrated in three of these strains and a plasmid function preventing excretion of small was present in all four strains (Rps). Two of the plasmids rendered the cells sensitive to small (Sbs). The plasmid functions Rps, Sbs, and medium bacteriocin production (Mep) on pRL1JI were expressed in R. leguminosarum, R. trifolii, R. phaseoli and A. tumefaciens. The formation of thick and short roots (Tsr) on Vicia sativa and nodulation (Nod) were also expressed in these hosts. No expression of above mentioned functions was found in R. meliloti.

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.

Expression of a Rhizobium phaseoli Sym plasmid in R. trifolii and Agrobacterium tumefaciens: incompatibility with a R. trifolii Sym plasmid.

Identification of the Sym plasmid in Rhizobium phaseoli strain RCC3622 is described. Introduction of this plasmid into R. trifolii or Agrobacterium tumefaciens strains resulted in bacteria capable of forming characteristic spherical root nodules on beans. This Sym plasmid, designated pSym9, was characterized as 275 MDa and nonconjugative. pSym9 was incompatible with the R. trifolii Sym plasmid pSym5, and carries genes determining a melanin-like black pigment. A second plasmid of 135 MDa, pRph3622a, was also transferred from R. phaseoli to R. trifolii and A. tumefaciens. Transconjugants carrying this plasmid did not form root nodules on beans. In contrast to other Rhizobium plasmids, pRph3622a was unstable in A. tumefaciens.

High NaCl Concentrations Induce the nod Genes of Rhizobium tropici CIAT899 in the Absence of Flavonoid Inducers

The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors.

The Biochemical Function of the Rhizobium Leguminosarum Proteins Involved in the Production of Host Specific Signal Molecules

In R.leguminosarum the NodF and NodE proteins play an important role in the determination of host specificity of nodulation [1]. We have shown that the NodF protein contains a 4’-phosphopantetheine prosthetic group. This result suggests that NodF protein is involved in the synthesis of polyketide derivatives. After labeling with 14C-acetate we have isolated the compounds produced by this nodulation protein. We have also investigated the role of the other nodulation proteins. In addition to the regulatory NodD protein, the NodABC and NodFEL proteins appear to be sufficient to produce the five detected wild-type Nod metabolites. Physical and chemical studies indicate that these R. leguminosarum compounds differ significantly from the reported R. meliloti signal compound [2]. The biological activity of several Nod metabolites was studied in three different bioassays on Vicia sativa plants showing a biological functionality of the 0-acetyl modification produced by NodL protein.

An improved large-scale isolation procedure for bacteroids of Rhizobium leguminosarum Frank. from Pisum savitum L.

SummaryBy manipulation of the growth medium level we have been able to concentrate the bulk of root nodules on a small proximal part of the main root. Measurements of several plant parameters on plants grown in a specially developed cultivation bin, show how changes in growth medium level effect nodulation.An isolation procedure is described for aerobic and anaerobic isolation of bacteroids of up to 1200 plants within 1 hour. Specific activities of nitrogenase preparations in these were found to be upto 21 n moles C2H2 red. per min per mg protein. re]19750724

Signals Involved in Nodulation and Nitrogen Fixation

One of the most fundamental aspects in biology is the regulation of biochemical pathways, which is brought about by an interplay between signals, genetic elements and transacting factors. Our knowledge of the signals involved in nodulation and nitrogen fixation is progressing rapidly but our understanding of their mechanisms of action is rather poor. In this overview we will deal with signals known or assumed to be involved in the various steps which lead to nodulation and nitrogen fixation. Rhizobium will be the leading microsymbiont but, when appropriate, other microbes will be dealt with. Because of severe space limitation many valuable references had to be omitted.