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.     

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.     

Sym2 of Pea Is Involved in a Nodulation Factor-Perception Mechanism That Controls the Infection Process in the Epidermis

In pea (Pisum sativum) up to 50 nodulation mutants are known, several of which are affected in the early steps of the symbiotic interaction with Rhizobium sp. bacteria. Here we describe the role of the sym2 gene in nodulation (Nod) factor perception. Our experiments show that the sym2A allele from the wild pea variety Afghanistan confers an arrest in infection-thread growth if the Rhizobium leguminosarum bv viciae strain does not produce Nod factors with a NodX-mediated acetylation at their reducing end. Since the induction of the early nodulin gene ENOD12 in the epidermis and the formation of a nodule primordium in the inner cortex were not affected, we conclude that more than one Nod factor-perception mechanism is active. Furthermore, we show that sym2A-mediated control of infection-thread growth was affected by the bacterial nodulation gene nodO.     

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.     

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.     

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.     

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum.

Mutants of Rhizobium leguminosarum bv. viciae unable to respire via the cytochrome aa3 pathway were identified by the inability to oxidize N,N'-dimethyl-p-phenylenediamine. Two mutants which were complemented by cosmid pIJ1942 from an R. leguminosarum clone bank were identified. Although pea nodules induced by these mutants contained many bacteroids, no symbiotic nitrogen fixation was detected. Heme staining of cellular proteins revealed that all cytochrome c-type heme proteins were absent. These mutants lacked spectroscopically detectable cytochrome c, but cytochromes aa3 and d were present, the latter at a higher-than-normal level. DNA sequence analysis of complementing plasmids revealed four apparently cotranscribed open reading frames (cycH, cycJ, cycK, and cycL). CycH, CycJ, CycK, and CycL are homologous to Bradyrhizobium japonicum and Rhizobium meliloti proteins thought to be involved in the attachment of heme to cytochrome c apoproteins; CycK and CycL are also homologous to the Rhodobacter capsulatus ccl1 and ccl2 gene products and the Escherichia coli nrfE and nrfF gene products involved in the assembly of c-type cytochromes. The absence of cytochrome c heme proteins in these R. leguminosarum mutants is consistent with the view that the cycHJKL operon could be involved in the attachment of heme to apocytochrome c.     

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.     

Rhizobium leguminosarum bv. viciae contains a second fnr/fixK-like gene and an unusual fixL homologue

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK–fixL region is located upstream of the fixNOQP operon on the non-nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N-terminal domain of response regulators) fused to its transmitter domain. An oxygen-sensing haem-binding domain, found in other FixL proteins, is conserved in R. leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr -like gene, designated fixK , whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix⁺ phenotype. A reduced nitrogen-fixation activity was found for a R. leguminosarum bv. viciae fnrN -deletion mutant, whereas no nitrogen-fixation activity was detectable for a fixK / fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified downstream of fixK–fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.     

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.     

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.     

Mass Spectrometric Analysis of Acidic Exo-Oligosaccharides of Root Nodule Bacterium Rhizobium leguminosarum bv. viciae VF39

Microbiology - The structures of exo-oligosaccharides produced by a symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum bv. viciae VF39 were determined using the methods of mass...     

The nifA gene product from Rhizobium leguminosarum biovar trifolii lacks the N-terminal domain found in other NifA proteins

The nifA gene has been identified between the fixX and nifB genes in the clover microsymbiont Rhizobium leguminosarum biovar trifolii (R.I. bv. trifolii) strain ANU843. Expression of the nifA gene is induced in the symbiotic state and site-directed mutagenesis experiments indicate that nifA expression is essential for symbiotic nitrogen fixation. Interestingly, the predicted R.I. bv. trifolii NifA protein lacks an N-terminal domain that is present in the homologous proteins from R.I. bv. viciae, Rhizobium meliloti, Bradyrhizobium japonicum, Klebsiella pneumoniae and all other documented NifA proteins. This indicates that this N-terminal domain is not essential for NifA function in R.I. bv. trifolii.     

N-acyl-homoserine lactone inhibition of rhizobial growth is mediated by two quorum-sensing genes that regulate plasmid transfer

The growth of some strains of Rhizobium leguminosarum bv. viciae is inhibited by N-(3-hydroxy-7-cis tetradecenoyl)-L-homoserine lactone (3OH-C(14:1)-HSL), which was previously known as the small bacteriocin before its characterization as an N-acyl homoserine lactone (AHL). Tn5-induced mutants of R. leguminosarum bv. viciae resistant to 3OH-C(14:1)-HSL were isolated, and mutations in two genes were identified. These genes, bisR and triR, which both encode LuxR-type regulators required for plasmid transfer, were found downstream of an operon containing trb genes involved in the transfer of the symbiotic plasmid pRL1JI. The first gene in this operon is traI, which encodes an AHL synthase, and the trbBCDEJKLFGHI genes were found between traI and bisR. Mutations in bisR, triR, traI, or trbL blocked plasmid transfer. Using gene fusions, it was demonstrated that bisR regulates triR in response to the presence of 3OH-C(14:1)-HSL. In turn, triR is then required for the induction of the traI-trb operon required for plasmid transfer. bisR also represses expression of cinI, which is chromosomally located and determines the level of production of 3OH-C(14:1)-HSL. The cloned bisR and triR genes conferred 3OH-C(14:1)-HSL sensitivity to strains of R. leguminosarum bv. viciae normally resistant to this AHL. Furthermore, bisR and triR made Agrobacterium tumefaciens sensitive to R. leguminosarum bv. viciae strains producing 3OH-C(14:1)-HSL. Analysis of patterns of growth inhibition using mutant strains and synthetic AHLs revealed that maximal growth inhibition required, in addition to 3OH-C(14:1)-HSL, the presence of other AHLs such as N-octanoyl-L-homoserine lactone and/or N-(3-oxo-octanoyl)-L-homoserine lactone. In an attempt to identify the causes of growth inhibition, a strain of R. leguminosarum bv. viciae carrying cloned bisR and triR was treated with an AHL extract containing 3OH-C(14:1)-HSL. N-terminal sequencing of induced proteins revealed one with significant similarity to the protein translation factor Ef-Ts.     

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.     

Use of nodulation pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA analysis to discriminate genotypes of Rhizobium leguminosarum biovar viciae

Twenty-seven new Rhizobium isolates were obtained from root nodules of wild and crop legumes belonging to the genera Vicia, Lathyrus and Pisum from different agroecological areas in central and southern Italy. A polyphasic approach including phenotypic and genotypic techniques was used to study their diversity and their relationships with other biovars and species of rhizobia. Analysis of symbiotic properties and stress tolerance tests revealed that wild isolates showed a wide spectrum of nodulation and a marked variation in stress tolerance compared with reference strains tested in this study. All rhizobial isolates (except for the isolate CG4 from Galega officinalis) were presumptively identified as Rhizobium leguminosarum biovar viciae both by their symbiotic properties and the specific amplification of the nodC gene. In particular, we found that the nodC gene could be used as a diagnostic molecular marker for strains belonging to the bv. viciae. RFLP-PCR 16S rDNA analysis confirms these results, with the exception of two strains that showed different RFLP-genotypes from those of the reference strains of R. leguminosarum bv. viciae. Analysis of intraspecies relationship among strains by using the RAPD-PCR technique showed a high level of genetic polymorphism, grouping our isolates and reference strains into six different major clusters with a similarity level of 20%. Data from seven parameters of phenotypic and genotypic analyses were evaluated by using principal component analysis which indicated the differences among strains and allowed them to be divided into seven different groups.     

固氮相关的两个植物基因转化烟草及其表达

豆科植物凝集和血红蛋白分别在植物识别其相应的根瘤菌和在根瘤内降低氧分压保护固氮酶的共生固氮作用中起重要作用。将豌豆（Ｐｉｓｕｍ ｓａｔｉｖａ Ｌ．）凝集素基因（ｐｌ）和Ｐａｒａｑｓｐｏｎｉａ ａｎｄｅｒｓｏｎｉｉ血红蛋白基因（ｐｈｂ）构建到同一植物表达载体上,通过根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导法转化烟草（Ｎｉｃｓ     

The expression of the nodD and nodABC genes of Rhizobium leguminosarum is not regulated in response to combined nitrogen

Abstract Using a Rhizobium leguminosarum bv. viciae strain harboring nodD :: lacZ or nodC :: lacZ translational fusions, grown in minimal media containing different concentrations of nitrate and/or ammonium salts, lacZ expression was monitored. Based on these experiments it is shown that the induction of Rhizobium leguminosarum bv. viciae nodD and nodABC operons by the flavanone naringenin is not regulated in response to nitrate and/or ammonium salts.