The Influence of Lipopolysaccharides and Glucans from Two Rhizobium leguminosarum bv. viciae Strains on the Formation and Efficiency of Their Symbioses with Pea Plants

The influence of lipopolysaccharides (LPS), glucans, and their unseparated complexes on nodulation activity of rhizobia and efficiency of their symbioses with pea plants was studied in vegetation tests. Two Rhizobium leguminosarum bv. viciae strains which differed in their symbiotic properties were used: strain 31 (fix⁺, efficient, moderately virulent, and moderately competitive) and strain 248b (fix^–, inefficient, highly virulent, and highly competitive). Preparations of LPS–glucan complex and the respective LPS from the highly virulent strain 248b increased the nodulation activity of both strains by 10–26%. Analogous preparations from a less virulent strain 31 did not have this ability. Unseparated LPS–glucan complexes from these strains increased the productivity of plants infected with the efficient strain by 18–23% but did not change it in plants inoculated with the other, inefficient strain. No significant influence of LPS preparations on the symbiosis productivity was observed. Glucans from both strains enhanced the nodulation ability of the highly virulent strain by 36–56%. In addition, treatment of pea plants with glucan from strain 248b increased nitrogen fixation by root nodules by 27% in plants inoculated with strain 31. In general, the formation and efficiency of the symbiosis of R. leguminosarum bv. viciae with pea plants was more influenced by preparations from strain 248b, highly virulent but deficient in nitrogen fixation, than by preparations from the nitrogen fixation–proficient but less virulent strain 31.     

Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.     

Distribution of O-acetyl groups in the exopolysaccharide synthesized by Rhizobium leguminosarum strains is not determined by the Sym plasmid

The patterns of O-acetylation of the exopolysaccharide (EPS) from the Sym plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii strain LPR5, R. leguminosarum bv. trifolii strain ANU843 and R. leguminosarum bv. viciae strain 248 were determined by 1H and 13C NMR spectroscopy. Beside a site indicative of the chromosomal background, these strains have one site of O-acetylation in common, namely residue b of the repeating unit. The O-acetyl esterification pattern of EPS of the Sym plasmid-cured derivatives of strains LPR5, ANU843, and 248 was not altered by the introduction of a R. leguminosarum bv. viciae Sym plasmid or a R. leguminosarum bv. trifolii Sym plasmid. The induction of nod gene expression by growth of the bacteria in the presence of Vicia sativa plants or by the presence of the flavonoid naringenin, produced no significant changes in either amount or sites of O-acetyl substitution. Furthermore, no such changes were found in the EPS from a Rhizobium strain in which the nod genes are constitutively expressed. The substitution pattern of the exopolysaccharide from R. leguminosarum is, therefore, determined by the bacterial genome and is not influenced by genes present on the Sym plasmid. This conclusion is inconsistent with the suggestion of Philip-Hollingsworth et al. (Philip-Hollingsworth, S., Hollingsworth, R. I., Dazzo, F. B., Djordjevic, M. A., and Rolfe, B. G. (1989) J. Biol. Chem. 264, 5710-5714) that nod genes of R. leguminosarum bv. trifolii, by influencing the acetylation pattern of EPS, determine the host specificity of nodulation.     

A cultivar-specific interaction between Rhizobium leguminosarum bv. trifolii and subterranean clover is controlled by nodM, other bacterial cultivar specificity genes, and a single recessive host gene.

Insertion mutagenesis identified two negatively acting gene loci which restrict the ability of Rhizobium leguminosarum bv. trifolii TA1 to infect the homologous host Trifolium subterraneum cv. Woogenellup. One locus was confirmed by DNA sequence analysis as the nodM gene, while the other locus, designated csn-1 (cultivar-specific nodulation), is not located on the symbiosis plasmid. The presence of these cultivar specificity loci could be suppressed by the introduction of the nodT gene from ANU843, a related R. leguminosarum bv. trifolii strain. Other nod genes, present in R. leguminosarum bv. viciae (including nodX) and R. meliloti, were capable of complementing R. leguminosarum bv. trifolii TA1 for nodulation on cultivar Woogenellup. Nodulation studies conducted with F2 seedlings from a cross between cultivar Geraldton and cultivar Woogenellup indicated that a single recessive gene, designated rwt1, is responsible for the Nod- association between strain TA1 and cultivar Woogenellup. Parallels can be drawn between this association and gene-for-gene systems common in interactions between plants and biotrophic pathogens.     

The major chemotaxis gene cluster of Rhizobium leguminosarum bv. viciae is essential for competitive nodulation

Rhizobium leguminosarum biovar viciae strain 3841 is a motile alpha-proteobacterium that can establish a nitrogen-fixing symbiosis within the roots of pea plants. In order to determine the contribution of chemotaxis to the lifestyle of R. leguminosarum, we have characterized the function of two chemotaxis gene clusters (che1 and che2) in controlling motility behaviour. We have found that both chemotaxis gene clusters modulate the motility swimming bias of R. leguminosarum cells and that the che1 cluster is the major pathway controlling swimming bias and chemotaxis. The che2 cluster also contributes to swimming bias, but has a minor effect on chemotaxis. Using competitive nodulation assays, we have demonstrated that a functional che1 cluster, but not the che2 cluster, promotes competitive nodulation of the peas. This finding implies that the environmental cue(s) triggering chemotaxis of R. leguminosarum bv. viciae cells towards the roots of pea and facilitating colonization are likely to be processed through the che1 cluster despite the contribution of both che clusters to swimming behaviour. A phylogenetic analysis of the distribution of che1 and che2 orthologues in the alpha-proteobacteria together with our results allow us to propose that che1 homologues are major controllers of chemotaxis and host association in the Rhizobiaceae.     

Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.     

Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells.

Reporter strains of soil-borne bacteria were used to test the hypothesis that chemicals released by root border cells can influence the expression of bacterial genes required for the establishment of plant-microbe associations. Promoters from genes known to be activated by plant factors included virE, required for Agrobacterium tumefaciens pathogenesis, and common nod genes from Rhizobium leguminosarum bv viciae and Rhizobium meliloti, required for nodulation of pea (Pisum sativum) and alfalfa (Medicago sativum), respectively. Also included was phzB, an autoinducible gene encoding the biosynthesis of antibiotics by Pseudomonas aureofaciens. The virE and nod genes were activated to different degrees, depending on the source of border cells, whereas phzB activity remained unaffected. The homologous interaction between R. leguminosarum bv viciae and its host, pea, was examined in detail. Nod gene induction by border cells was dosage dependent and responsive to environmental signals. The highest levels of gene induction by pea (but not alfalfa) border cells occurred at low temperatures, when little or no bacterial growth was detected. Detached border cells cultured in distilled water exhibited increased nod gene induction (ini) in response to signals from R. leguminosarum bv viciae.     

Natural variation in host-specific nodulation of pea is associated with a haplotype of the SYM37 LysM-type receptor-like kinase

Rhizobium leguminosarum bv. viciae, which nodulates pea and vetch, makes a mixture of secreted nodulation signals (Nod factors) carrying either a C18:4 or a C18:1 N-linked acyl chain. Mutation of nodE blocks the formation of the C18:4 acyl chain, and nodE mutants, which produce only C18:1-containing Nod factors, are less efficient at nodulating pea. However, there is significant natural variation in the levels of nodulation of different pea cultivars by a nodE mutant of R. leguminosarum bv. viciae. Using recombinant inbred lines from two pea cultivars, one which nodulated relatively well and one very poorly by the nodE mutant, we mapped the nodE-dependent nodulation phenotype to a locus on pea linkage group I. This was close to Sym37 and PsK1, predicted to encode LysM-domain Nod-factor receptor-like proteins; the Sym2 locus that confers Nod-factor-specific nodulation is also in this region. We confirmed the map location using an introgression line carrying this region. Our data indicate that the nodE-dependent nodulation is not determined by the Sym2 locus. We identified several pea lines that are nodulated very poorly by the R. leguminosarum bv. viciae nodE mutant, sequenced the DNA of the predicted LysM-receptor domains of Sym37 and PsK1, and compared the sequences with those derived from pea cultivars that were relatively well nodulated by the nodE mutant. This revealed that one haplotype (encoding six conserved polymorphisms) of Sym37 is associated with very poor nodulation by the nodE mutant. There was no such correlation with polymorphisms at the PsK1 locus. We conclude that the natural variation in nodE-dependent nodulation in pea is most probably determined by the Sym37 haplotype.     

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. leguminosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutant, or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.     

Competitive nodulation blocking of cv. Afghanistan pea is related to high levels of nodulation factors made by some strains of Rhizobium leguminosarum bv. viciae

Cultivar Afghanistan peas are resistant to nodulation by many strains of Rhizobium leguminosarum bv. viciae but are nodulated by strain TOM, which carries the host specificity gene nodX. Some strains that lack nodX can inhibit nodulation of cv. Afghanistan by strain TOM. We present evidence that this "competitive nodulation-blocking" (Cnb) phenotype may result from high levels of Nod factors inhibiting nodulation of cv. Afghanistan peas. The TOM nod gene region (including nodX) is cloned on pIJ1095, and strains (including TOM itself) carrying pIJ1095 nodulate cv. Afghanistan peas very poorly but can nodulate other varieties normally. The presence of pIJ1095, which causes increased levels of Nod factor production, correlates with Cnb. Nodulation of cv. Afghanistan by TOM is also inhibited by a cloned nodD gene that increases nod gene expression and Nod factor production. Nodulation of cv. Afghanistan can be stimulated if nodD on pIJ1095 is mutated, thus severely reducing the level of Nod factor produced. Repression of nod gene expression by nolR eliminates the Cnb phenotype and can stimulate nodulation of cv. Afghanistan. Addition of Nod factors to cv. Afghanistan roots strongly inhibits nodulation. The Cnb+ strains and added Nod factors inhibit infection thread initiation by strain TOM. The sym2A allele determines resistance of cv. Afghanistan to nodulation by strains of R. leguminosarum bv. viciae lacking nodX. We tested whether sym2A is involved in Cnb by using a pea line carrying the sym2A region introgressed from cv. Afghanistan; nodulation in the introgressed line was inhibited by Cnb+ strains. Therefore, the sym2A region has an effect on Cnb, although another locus (or loci) may contribute to the stronger Cnb seen in cv. Afghanistan.     

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment

Abstract An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II ( npt II) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.     

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.     

A plant arabinogalactan-like glycoprotein promotes a novel type of polar surface attachment by Rhizobium leguminosarum

Rhizobium leguminosarum bv. viciae can attach to the roots of legume and non-legume plants. We wanted to determine whether root exudates could affect in vitro surface attachment in a confocal microscopy assay. Root exudate from pea, other legumes, wheat, and Arabidopsis induced R. leguminosarum bv. viciae to attach end-on (in a polar manner) to glass in hexagonal close-packed arrays, rather than attaching along their long axis. This did not involve a reorientation but was probably due to altered growth. The polar attachment involves a novel bacterial component because it occurred in mutants lacking a symbiosis plasmid (and hence nodulation genes) and polar glucomannan. The major surface (acidic) exopolysaccharide was required, and mutations affecting exported proteins and flagella delayed but did not block polar attachment. The polar attachment activity was purified as a high molecular weight fraction from pea root exudate and is an arabinogalactan protein (AGP) based on its carbohydrate content, reactivity with AGP-specific monoclonal antibodies and Yariv reagent, and sensitivity to enzymes that degrade proteins and carbohydrates. We propose that this novel mode of AGP-induced attachment may be important for growth of these bacteria on the roots of both legumes and non-legumes.     

Nod factors stimulate seed germination and promote growth and nodulation of pea and vetch under competitive conditions

Nod factors are lipochitooligosaccharide (LCO) produced by soil bacteria commonly known as rhizobia acting as signals for the legume plants to initiate symbiosis. Nod factors trigger early symbiotic responses in plant roots and initiate the development of specialized plant organs called nodules, where biological nitrogen fixation takes place. Here, the effect of specific LCO originating from flavonoid induced Rhizobium leguminosarum bv. viciae GR09 culture was studied on germination, plant growth and nodulation of pea and vetch. A crude preparation of GR09 LCO significantly enhanced symbiotic performance of pea and vetch grown under laboratory conditions and in the soil. Moreover, the effect of GR09 LCOs seed treatments on the genetic diversity of rhizobia recovered from vetch and pea nodules was presented.     

Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.     

Entry of Rhizobium leguminosarum bv.viciae into root hairs requires minimal Nod factor specificity, but subsequent infection thread growth requires nodO or nodE

Using various mutant strains of Rhizobium leguminosarum bv. viciae, we have investigated the role of nodO in stimulating infection thread development in vetch and pea. Analysis of R. leguminosarum bv. viciae nodE and nodO mutants revealed no significant difference from the wild-type infection phenotype. Conversely, an R. leguminosarum bv. viciae nodE nodO double mutant was severely impaired in its ability to form normal infection threads. This strain displayed a number of novel infection-related events, including intracellular accumulations of bacteria at the base of root hairs, distended and enlarged infection threads, and reversed threads growing up root hairs. Since normal infection was seen in a nodE mutant, nodO must suppress these abnormal infection phenomena A deletion mutant, retaining only the nodD and nodABCIJ genes, also formed intracellular accumulations at the base of root hairs. Addition of R. leguminosarum bv. viciae nodO could alleviate this phenotype and restore some infection thread formation, although these threads appeared to be abnormal. Exogenous application of R. leguminosarum bv. viciae Nod factors could not alleviate the aberrant infection phenotype. Our results show that the most basic Nod factor structure can allow bacterial entry into the root hair, and that nodO can promote subsequent infection thread development.     

Allelic relationships of pea nodulation mutants

Thirteen stable nonnodulating mutant lines of pea (Pisum sativum L.) originating from cv. Finale were tested for allelism in pairwise crosses. The F(1) plants were evaluated for the symbiotic phenotype under controlled growth conditions against the nodule bacterium Rhizobium leguminosarum bv. viciae strain 248. All mutations were found to be recessive and the lines were classified into eight complementation groups comprising Risnod1-Risnod23, Risnod8, Risnod9-Risnod22, Risnod14, Risnod19-Risnod25, Risnod20, Risnod24-Risnod26, and Risnod32. Position of Risnod21 was not firmly established, leaving the possibility of allelism both with Risnod19-Risnod25 and Risnod20. The results were partially consistent with the previous reports on the allelism of these lines. Additional crosses confirmed the correspondence of Risnod14 with the locus sym7 and of Risnod19-Risnod25 with sym8. The high number of eight complementation groups formed by 13 mutants provides an indication of additional nodulation loci in pea to those already reported and confirms the complexity of the genetic control of the early stages of nodulation.