Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Evidence for two uptake systems in Rhizobium leguminosarum for hydroxy-aromatic compounds metabolized by the 3-oxoadipate pathway

Rhizobium leguminosarum biovar viciae and Rhizobium leguminosarum biovar trifolii have separate uptake systems for 4-hydroxybenzoate and protocatechuate. The 4-hydroxybenzoate uptake system (pobP) is inhibited by a range of compounds with substitution at the 4-position on the aromatic ring whereas the uptake system for protocatechuate (pcaP) is markedly inhibited only by other dihydroxybenzoic acids. The rate of 4-hydroxybenzoate uptake is very low in Rhizobium leguminosarum and Rhizobium trifolii grown on protocatechuate but mutants defective in 4-hydroxybenzoate uptake transport protocatechuate at rates similar to the wild-type grown under similar conditions.     

Sugar-binding activity of pea (Pisum sativum) lectin is essential for heterologous infection of transgenic white clover hairy roots by Rhizobium leguminosarum biovar viciae

Legume lectin stimulates infection of roots in the symbiosis between leguminous plants and bacteria of the genus Rhizobium. Introduction of the Pisum sativum lectin gene (psl) into white clover hairy roots enables heterologous infection and nodulation by the pea symbiont R. leguminosarum biovar viciae (R.l. viciae). Legume lectins contain a specific sugar-binding site. Here, we show that inoculation of white clover hairy roots co-transformed with a psl mutant encoding a non-sugar-binding lectin (PSL N125D) with R.l. viciae yielded only background pseudo-nodule formation, in contrast to the situation after transformation with wild type psl or with a psl mutant encoding sugar-binding PSL (PSL A126V). For every construct tested, nodulation by the homologous symbiont R.l. trifolii was normal. These results strongly suggest that (1) sugar-binding activity of PSL is necessary for infection of white clover hairy roots by R.l. viciae, and (2) the rhizobial ligand of host lectin is a sugar residue rather than a lipid.     

Biosynthesis of cyclic β-(1,2)-glucans inRhizobium leguminosarum biovarsviciae,phaseoli andtrifolii

Inner membranes of Rhizobium leguminosarum biovars viciae, phaseoli, and trifolii synthesized a heterogenous family of neutral cyclic -(1,2)-glucans in a reaction system that used oligosaccharide intermediates covalently bound to a large protein. This glucoprotein showed a slightly lower mobility on SDS-polyacrylamide gels (apparent mol. mass of 320 kDa) than the -(1,2)-glucan intermediate protein of Rhizobium meliloti. In vivo pulse-label experiments with growing cells of R. leguminosarum biovar trifolii RS800 using radioactive glucose showed that few species of cyclic -(1,2)-glucans were synthesized and up to 30% were substituted with charged non-glycosidic residues, probably sn-1-phosphoglycerol.     

Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis

In the biosynthesis of lipochitin oligosaccharides (LCOs) theRhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence ofnodA in thenodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which thenodA, nodB andnodC genes are separately present on different plasmids. Efficient nodulation was only obtained ifnodC was present on a low-copy-number vector. Our results confirm the notion thatnodA ofRhizobium leguminosarum biovarviciae is essential for nodulation onVicia. Surprisingly, replacement ofR. l. bv.viciae nodA by that ofBradyrhizobium sp. ANU289 results in a nodulation-minus phenotype onVicia. Further analysis revealed that theBradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of theR. l. bv.viciae nodF E-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion thatnodA is a commonnod gene should be revised.     

Hair Curling Induced in Heterologous Legumes and Monocots by Flavonoid-Treated Rhizobia

The curling of root hairs and the deformation response wereobserved when white clover was infected with homologous strainsof Rhizobium leguminosarum biovar trifolii 4S and 0403. In thecase of Rhizobium meliloti NZ and Rhizobium leguminosarum biovarviciae 128C53, however, curling was only induced when thesebacteria were pretreated with flavonoids: luteolin in the caseof R. meliloti and naringenin for R.I. viciae. The same resultswere obtained with oat, a monocotyledonous non-leguminous plant.The two flavonoids mentioned are secreted from the host plantsand induce the expression of genes for root hair curling (Hac)on Sym plasmid in homologous rhizobia, therefore, the curlingresponse in both white clover and oat appears to be correlatedwith the activation of the Hac genes. These results suggestthat a factor(s) that activates the Hac genes, such as 7,4'-dihydroxyflavonewhich is known as the factor required by R. I. trifolii, issecreted from the oat roots. (Received June 12, 1989; Accepted November 9, 1989)     

Application of physical and genetic map of Rhizobium leguminosarum bv. trifolii TA1 to comparison of three closely related rhizobial genomes

A combined physical and genetic map of Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) genome was constructed and used in comparison of chromosomal organization with the closely related R. leguminosarum bv. viciae 3841 (Rlv) and Rhizobium etli CNF42 (Rhe). This approach allowed evaluation of chromosome and genome plasticity and provided important insights into R. leguminosarum lineage diversity. MssI, SmiI, PacI, and I-CeuI restriction endonucleases were chosen for the analysis, generating fragments with suitable size distributions for RtTA1 genome mapping. The fragments were assembled into a physical map using a combination of complementary methods, including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization. About 100 genetic markers were located on the RtTA1 restriction map. Comparison of genetic maps of RtTA1, Rlv, and Rhe revealed extensive chromosomal colinearity despite differences in the physical maps. The comparison provides bases for comprehensive analysis of the evolution of R. leguminosarum genome, indicating that, at least on the chromosomal level, no major rearrangements had occurred after the evolutionary divergence of R. leguminosarum biovars. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rhizobium nodulation protein NodA is a host-specific determinant of the transfer of fatty acids in Nod factor biosynthesis

In the biosynthesis of lipochitin oligosaccharides (LCOs) theRhizobium nodulation protein NodA plays an essential role in the transfer of an acyl chain to the chitin oligosaccharide acceptor molecule. The presence ofnodA in thenodABCIJ operon makes genetic studies difficult to interpret. In order to be able to investigate the biological and biochemical functions of NodA, we have constructed a test system in which thenodA, nodB andnodC genes are separately present on different plasmids. Efficient nodulation was only obtained ifnodC was present on a low-copy-number vector. Our results confirm the notion thatnodA ofRhizobium leguminosarum biovarviciae is essential for nodulation onVicia. Surprisingly, replacement ofR. l. bv.viciae nodA by that ofBradyrhizobium sp. ANU289 results in a nodulation-minus phenotype onVicia. Further analysis revealed that theBradyrhizobium sp. ANU289 NodA is active in the biosynthesis of LCOs, but is unable to direct the transfer of theR. l. bv.viciae nodF E-dependent multi-unsaturated fatty acid to the chitin oligosaccharide acceptor. These results lead to the conclusion that the original notion thatnodA is a commonnod gene should be revised.     

Inoculation of Vicia sativa subsp. nigra roots with Rhizobium leguminosarum biovar viciae results in release of nod gene activating flavanones and chalcones

Flavonoids released by roots of Vicia sativa subsp. nigra (V. sativa) activate nodulation genes of the homologous bacterium Rhizobium leguminosarum biovar viciae (R. l. viciae). Inoculation of V. sativa roots with infective R. l. viciae bacteria largely increases the nod gene-inducing ability of V. sativa root exudate (A.A.N. van Brussel et al., J Bact 172: 5394–5401). The present study showed that, in contrast to sterile roots and roots inoculated with R. l. viciae cured of its Sym plasmid, roots inoculated with R. l. viciae harboring its Sym plasmid released additional nod gene-inducing flavonoids. Using ¹H-NMR, the structures of the major inducers released by inoculated roots, 6 flavanones and 2 chalcones, were elucidated. Roots extracts of (un)inoculated V. sativa contain 4 major non-inducing, most likely glycosylated, flavonoids. Therefore, the released flavonoids may either derive from the root flavonoids or inoculation with R. l. viciae activates de novo flavonoid biosynthesis.     

Nodulation of Trifolium repens with modified Bradyrhizobium and the nodulation of Parasponia with Rhizobium leguminosarum biovar trifolii

Thirty one strains of Rhizobium leguminosarum biovar trifolii isolated from the North and South American continents, New Guinea, USSR, Turkey and Australia, nodulated P. andersonii ineffectively when grown in plant growth tubes and in Leonard jars. Nodules were slow to form, sometimes taking over 100 days. Reisolates of R. leguminosarum biovar trifolii from P. andersonii nodulated Trifolium repens and their identity was confirmed using serological techniques. Dual occupation of nodules by Rhizobium and Bradyrhizobium in P. andersonii is reported. The reduced effectiveness of the Bradyrhizobium symbiosis depended on the relative numbers of Rhizobium occupants in this dual system. R. leguminosarum biovar trifolii and Bradyrhizobium strains from Parasponia were able to co-exist in nodules on P. andersonii and maintain similar populations in the rhizosphere and on culture media. Bradyrhizobium strains, separated from R. leguminosarum biovar trifolii, were able to initiate and form nodule-like structures on T. repens. Bradyrhizobium bacteria were identified as the sole occupants of the cells of the nodule-like structures on Trifolium repens using an immunogold labelling technique applied to ultrathin sectins. The re-isolates of Bradyrhizobium obtained from these nodule-like structures on T. repens were able to effectively nodulate P. andersonii.     

Melanin Production by Rhizobium Strains

Different Rhizobium and Bradyrhizobium strains were screened for their ability to produce melanin. Pigment producers (Mel⁺) were found among strains of R. leguminosarum biovars viceae, trifolii, and phaseoli, R. meliloti, and R. fredii; none of 19 Bradyrhizobium strains examined gave a positive response. Melanin production and nod genes were plasmid borne in R. leguminosarum biovar trifolii RS24. In R. leguminosarum biovar phaseoli CFN42 and R. meliloti GR015, mel genes were located in the respective symbiotic plasmids. In R. fredii USDA 205, melanin production correlated with the presence of its smallest indigenous plasmid.     

Major flavonoids in uninoculated and inoculated roots of Vicia sativa subsp. nigra are four conjugates of the nodulation gene-inhibitor kaempferol

Inoculation of Vicia sativa subsp. nigra (V. sativa) roots with Rhizobium leguminosarum biovar. viciae (R.l. viciae) bacteria substantially increases the ability of V. sativa to induce rhizobial nodulation (nod) genes. This increase is caused by the additional release of flavanones and chalcones which all induce the nod genes of R.l. viciae (K. Recourt et al., Plant Mol Biol 16: 841–852). In this paper, we describe the analyses of the flavonoids present in roots of V. sativa. Independent of inoculation with R.l. viciae, these roots contain four 3-O-glycosides of the flavonol kaempferol. These flavonoids appeared not capable of inducing the nod genes of R.l. viciae but instead are moderately active in inhibiting the activated state of those nod genes. Roots of 7-day-old V. sativa seedlings did not show any kaempferol-glycosidase activity consistent with the observation that kaempferol is not released upon inoculation with R.l. viciae. It is therefore most likely that inoculation with infective (nodulating) R.l. viciae bacteria results in de novo flavonoid biosynthesis and not in liberation of flavonoids from a pre-existing pool.     

Genetic Diversity among Rhizobium leguminosarum bv. Trifolii Strains Revealed by Allozyme and Restriction Fragment Length Polymorphism Analyses

Allozyme electrophoresis and restriction fragment length polymorphism (RFLP) analyses were used to examine the genetic diversity of a collection of 18 Rhizobium leguminosarum bv. trifolii, 1 R. leguminosarum bv. viciae, and 2 R. meliloti strains. Allozyme analysis at 28 loci revealed 16 electrophoretic types. The mean genetic distance between electrophoretic types of R. leguminosarum and R. meliloti was 0.83. Within R. leguminosarum, the single strain of bv. viciae differed at an average of 0.65 from strains of bv. trifolii, while electrophoretic types of bv. trifolii differed at a range of 0.23 to 0.62. Analysis of RFLPs around two chromosomal DNA probes also delineated 16 unique RFLP patterns and yielded genetic diversity similar to that revealed by the allozyme data. Analysis of RFLPs around three Sym (symbiotic) plasmid-derived probes demonstrated that the Sym plasmids reflect genetic divergence similar to that of their bacterial hosts. The large genetic distances between many strains precluded reliable estimates of their genetic relationships.     

Resistance to nodulation of cv. Afghanistan peas is overcome by nodX, which mediates an O-acetylation of the Rhizobium leguminosarum lipo-oligosaccharide nodulation factor

Only some strains of Rhizobium leguminosarum biovar viciae can efficiently nodulate varieties of peas such as cv. Afghanistan, which carry a recessive allele that blocks efficient nodulation by most western isolates of R. I. viciae. One strain (TOM) which can nodulate cv. Afghanistan peas has a gene (nodX) that is required to overcome the nodulation resistance. Strain TOM makes significantly lower amounts of lipo-oligosaccharide nodulation factors than other strains of R. I. viciae. and this effect appears to be due to lower levels of nod gene induction. These nodulation factors are similar to those from other R. I. viciae. strains in that they consist of an oligomer of four or five β1-4-linked N-acetylglucosamine residues in which the terminal non-reducing glucosamine carries an O-acetyl group and a C_18:4 or C_18:1N-acyl group. However, one of the nodulation factors made by strain TOM differs from the factors made by other strains of R. I. viciae. in that it carries an O-acetyl group on the C-6 of the reducing N-acetylglucosamine residue. This acetylation is NodX-dependent and the pentameric nodulation factor is acetylated on the reducing N-acetylglucosamine residue whereas the tetrameric nodulation factor is not. Although the nodL gene product is also an O-acetyl transferase (it O-acetylates the C-6 of the terminal non-reducing glucosamine), there is very little similarity between the amino acid sequences of these two acetyl transferases.     

A Rhizobium tropici DNA region carrying the amino-terminal half of a nodD gene and a nod-box-like sequence confers host-range extension

Rhizobium tropici CIAT899 is a broad-host-range strain that, in addition to Phaseolus, nodulates other plant legumes such as Leucaena and Macroptilium. The narrow-host-range of Rhizobium leguminosarum biovars phaseoli (strain CE3) and trifolii (strain RS1051) can be extended to Leucaena esculents and Phaseolus vulgaris plants, respectively, by the introduction of a DNA fragment 521 bp long, which carries 128 amino acids of the amino-terminal region of a nodD gene from R. tropici, as well as a putative nod-box-like sequence, divergently oriented. The 521 bp fragment, in the presence of L. esculenta or P. vulgaris root exudates, induced a R. leguminosarum bv. viciae nodA-lacZ fusion in either a CE3 or RS1051 background, respectively.     

Competition among Strains of Rhizobium leguminosarum biovar trifolii and Use of a Diallel Analysis in Assessing Competition

Competition between indigenous Rhizobium leguminosarum biovar trifolii strains and inoculant strains or between mixtures of inoculant strains was assessed in field and growth-room studies. Strain effectiveness under competition was compared with strain performance in the absence of competition. Field inoculation trials were conducted at Elora, Ontario, Canada, with soil containing indigenous R. leguminosarum biovar trifolii. The indirect fluorescent-antibody technique was used for the identification of nodule occupants. Treatments consisted of 10 pure strains, a commercial peat inoculant containing a mixture of strains, and an uninoculated control. Inoculant strains occupied 17.5 to 85% of nodules and resulted in increased dry weight and nitrogen content, as compared with the uninoculated control. None of the strains was capable of completely overcoming resident rhizobia, which occupied, on average, 50% of the total nodules tested. In growth-room studies single commercial strains were mixed in all possible two-way combinations and assessed in a diallel mating design. Significant differences in plant dry weight of red clover were observed among strain combinations. Specific combining ability effects were significant at the 10% level, suggesting that the effectiveness of strain mixtures depended on the specific strain combinations. Strains possessing superior effectiveness and competitive abilities were identified by field and growth-room studies. No relationship was detected between strain effectiveness and competitive ability or between strain recovery and host cultivar. The concentration of indigenous populations was not considered to be a limiting factor in the recovery of introduced strains at this site.     

Rhizobium Attachment and Curling in Asparagus, Rice and Oat Plants

Observations by scanning electron microscopy revealed that rhizobiaattach to the surface of rice protoplasts with regenerated cellwalls, isolated mesophyll cells of asparagus, and root hairsof rice and oat seedlings. Those strains of rhizobia, namelyRhizobium leguminosarum biovar trifolii, Bradyrhizobium japonicumand Bradyrhizobium sp., attach to the cells of these monocotsin the same manner as they attach to the host dicots tested.Escherichia coli did not attach. These results suggest thatthe attachment of rhizobia is not a host-specific process. Whenoat seedlings were infected by R. l. trifolii, hair curlingwas observed. The interactions between monocot plants and rhizobiais discussed in this paper. (Received June 12, 1989; Accepted November 9, 1989)     

Genetic Structure of Rhizobium leguminosarum biovar trifolii and viciae Populations Found in Two Oregon Soils under Different Plant Communities

An investigation was carried out to determine the genetic structure in soil populations of Rhizobium leguminosarum bv. trifolii and viciae at each of two Oregon sites (A and C) that were 1 km apart. Although the soils were similar, the plant communities were quite different because grazing by domestic animals had been allowed (site A) or prevented (site C). Analysis of allelic variation at 13 enzyme-encoding loci by multilocus enzyme electrophoresis delineated 202 chromosomal types (ETs) among a total of 456 isolates representing two populations of R. leguminosarum bv. trifolii (AT and CT) and two populations of R. leguminosarum bv. viciae (AV and CV). Regardless of their site of origin or biovar affiliation, isolates of the same ET were confirmed to be more closely related to each other than to isolates of other ETs by repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus sequences and the PCR technique. Despite the wide range in densities of the Rhizobium populations (<10² to >10⁵/g of soil), their overall genetic diversities were similar (mean genetic diversity, 0.45 to 0.51), indicating that low-density populations of soil-borne bacterial species are not necessarily of little genetic diversity. Linkage disequilibrium analysis revealed significant multilocus structure (nonrandom associations of alleles) within each of the four populations. From a combination of cluster and linkage disequilibrium analyses, a total of eight distinct groups of ETs were defined in the four populations. Two groups (I and III) contributed significant numbers of ETs and isolates to each population. The two populations of R. leguminosarum bv. viciae (AV and CV) exhibited similar genetic structures despite existing at different densities, in different plant communities, and in the presence (CV) or absence (AV) of their local Vicia hosts. In contrast, total linkage disequilibrium was partitioned differently in two biovar populations occupying the same soil (AV and AT), with disequilibrium in the latter being due entirely to the presence of group V.     

Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.