Biosynthesis and degradation of nodule-specific Rhizobium loti compounds in Lotus nodules.

Two nodule-specific Rhizobium loti compounds were identified in Lotus tenuis and Lotus pedunculatus nodules induced by strain NZP2037. One, a silver nitrate-positive cation called rhizolotine, has been characterized as the riboside of a novel alpha-hydroxyimino acid containing a 1,4,5,6-tetrahydropyrimidine ring (G. J. Shaw, R. D. Wilson, G. A. Lane, L. D. Kennedy, D. B. Scott, and G. J. Gainsford, J. Chem. Soc. Chem. Commun., p. 180-181, 1986), and the other, yellow-1, stains yellow with ninhydrin. Both compounds were degraded by R. loti NZP2037 but not by strains of Rhizobium meliloti, Rhizobium trifolii, or Agrobacterium tumefaciens. Under the conditions tested neither compound was able to serve as a sole source of C or N for growth of R. loti NZP2037. Rhizolotine and yellow-1 were found in nodules from a range of different legumes inoculated with NZP2037, suggesting that the Rhizobium and not the host plant determines their synthesis. Neither compound was found in nodulelike structures of L. pedunculatus induced by transposon Tn5-induced noninfectious (Inf-) mutants of NZP2037 or in similar structures induced by a transconjugant of NZP2037 containing the symbiotic (Sym) cointegrate plasmid pPN1 of R. trifolii. Both compounds were also absent in the ineffective nodules induced by the bacterial-release-negative (Bar-) mutant, strain PN239. However, both compounds were present in nodules induced by the fixation-negative (Fix-) mutant PN235 and in Fix+ nodules formed by a plasmid-cured derivative of NZP2037. These results would suggest that infection and bacterial release from the infection thread are necessary for nodule (symbiotic) synthesis of these compounds.     

Isolation and characterization of transposon Tn5-induced symbiotic mutants of Rhizobium loti

Rhizobium loti NZP2037 and NZP2213, each cured of its single large indigenous plasmid, formed effective nodules on Lotus spp., suggesting that the symbiotic genes are carried on the chromosome of these strains. By using pSUP1011 as a vector for introducing transposon Tn5 into R. loti NZP2037, symbiotic mutants blocked in hair curling (Hac), nodule initiation (Noi), bacterial release (Bar), and nitrogen fixation (Nif/Cof) on Lotus pedunculatus were isolated. Cosmids complementing the Hac, Noi, and Bar mutants were isolated from a pLAFR1 gene library of NZP2037 DNA by in planta complementation and found to contain EcoRI fragments of identical sizes to those into which Tn5 had inserted in the mutants. The cosmids that complemented the mutants of these phenotypic classes did not share common fragments, nor did cosmids that complemented four mutants within the Noi class, suggesting that these symbiotically important regions are not tightly linked on the R. loti chromosome.     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

The effect of bacterial strain and temperature changes on the nitrogenase activity of Lotus pedunculatus root nodules

After 40 days of growth at 25°C, Lotus pedunculatus cav., cv. Maku plants infected with Rhizobium loti strain NZP2037 displayed similar relative growth rates but had twice the nodule mass and only one third the whole plant dry weight of plants infected with Bradyrhizobium sp. (Lotus) strain CC814s. In the NZP2037 symbiosis, the rate of CO₂ evolution (per g dry weight of nodulated root) was 1.6 times as high as that in the CC814s symbiosis while the rate of C₂H₂ reduction (per g dry weight of nodule) was only 48% of that in the CC814s symbiosis. Studies of the effect of short term temperature changes on the gas exchange characteristics (CO₂ and H₂ evolution, C₂H₂ reduction) of these symbioses revealed wide differences in the optima for C₂H₂ reduction. Nodules infected with NZP2037 displayed maximal C₂H₂ reduction rates [157 μmol (g dry weight nodule)^?1 h^?1] at 12°C, whereas nodules infected with CC814s were optimal at 30°C [208 μmol (g dry weight nodule)^?1 h^?1]. These short term studies suggested that differences in temperature optima for N₂ may have partially accounted for the poorer effectivity, at 25°C, of strain NZP2037 when compared with strain CC-814s. The relative efficiency [RE = 1 – (H₂ evolution/C₂H₂ reduction)] of N₂ fixation varied widely with temperature in the two symbioses, but there was a general trend toward higher RE with lower temperatures. The ratio of CO₂ evolution: C₂H₂ reduction (mol/mol) in nodulated roots infected with CC814s was constant (ca 10 CO₂/C₂H₂) between 5°C and 30°C, whereas in plants infected with NZP2037 it reached a minimal value of 3.3 CO₂/C₂H₂ at 10°C and was 19 CO₂/C₂H₂ at the growing temperature (25°C).     

Symbiotic effectiveness of phage-resistant mutants of two strains of Lotus rhizobia

Summary Phage-resistant mutants were obtained from a fast-growing (NZP2037) and a slow-growing (CC814s) strain of Rhizobium nodulating Lotus. All the mutants were stable and did not differ from the original parent strain in their cultural characteristics. OnLotus pedunculatus the mutants of NZP2037 were as effective in N-fixation as the parent strain but most mutants of CC814s were less effective. These mutants of CC814s were also less effective than the parent strain on several other host plants.     

Transfer of an indigenous plasmid of Rhizobium loti to other rhizobia and Agrobacterium tumefaciens

Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.     

Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host.

By Tn5 mutagenesis of Rhizobium loti PN184 (NZP2037 str-1) and selection for nonfluorescence of colonies on Calcofluor agar, eight independently generated expolysaccharide (EPS) mutants (three smooth and five rough) were isolated. The parent strain, PN184, was found to produce an acidic EPS. This EPS was produced. with reduced O acetylation, by the smooth EPS mutants but not by the rough EPS mutants. Lipopolysaccharide was isolated from all mutants and was identical to that of PN184 as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All mutants were resistant to lysis by R. loti bacteriophage phi 2037/1. Cosmids that complemented the mutations in the rough EPS mutants were isolated from a pLAFR1 gene library of NZP2037 by complementation of the nonfluorescent phenotype. The genes identified were shown to be unlinked and located on the chromosome. All mutants were fully effective when inoculated onto Lotus pedunculatus, a determinate nodulating host, but were ineffective, inducing the formation of very small nodules or tumorlike growths, when inoculated onto Leucaena leucocephala, an indeterminate nodulating host. These results, obtained in an isogenic Rhizobium background, support suggestions that acidic EPS is required for effective nodulation of indeterminate nodulating legumes but is not required for effective nodulation of determinate nodulating legumes.     

Isolation and characterization of a Rhizobium loti gene required for effective nodulation of Lotus pedunculatus

A Rhizobium loti gene required for effective invasion of the host Lotus pedunculatus has been identified by transposon Tn5 mutagenesis. Cosmids that complemented a previously isolated mutation (239) at this invasion (inv) locus were identified by in planta complementation and used to construct a physical map of the gene region. The insertion site of Tn5 in PN239 was mapped to a 7.5-kb EcoRI fragment, which complemented the mutation when subcloned into pLAFR1. Further Tn5 mutagenesis of the 7.5-kb fragment was carried out in Escherichia coli using bacteriophage lambda 467, and the mutations homogenotized into R. loti NZP2037. Three additional Fix- mutations were isolated, and these were found to map adjacent to the position of the original mutation in strain PN239. All the other Tn5 insertions isolated in the 7.5-kb fragment gave a Fix+ phenotype on L. pedunculatus. Electron microscopic examination of the L. pedunculatus nodules induced by the isolated Fix- mutants showed that bacteria were either blocked in release from the infection threads or were unable to undergo normal bacteroid development. The inv locus as defined by the Tn5 insertions was sequenced, and a single open-reading frame (ORF) of 576 bp, corresponding to a polypeptide of 21.3 kDa, was identified. The position and orientation of this ORF were consistent with those of the isolated Tn5 Fix- insertions.     

Evaluation and improvement of the energy efficiency of nitrogen fixation in Lotus corniculatus nodules induced by Rhizobium loti strains indigenous to Uruguay

In order to evaluate energy efficiency of nitrogen fixation by the Lotus corniculatus/Rhizobium loti symbiosis, Uruguayan R. loti strains were tested for hydrogen-uptake (Hup) status. Nodules induced in L. corniculatus by all eight R. loti strains tested evolved high amounts of hydrogen (2.0–8.7 mol H2/h.g nodule fresh weight). This production of hydrogen corresponds to 38–69% of total nitrogenase activity estimated as acetylene reduction, suggesting that hydrogen is not recycled within these nodules. This was confirmed by the lack of hydrogenase activity in bacteroid suspensions. Additionally, no hybridization signals were observed in total DNA restriction digests from these strains when a DNA fragment containing part of hydrogenase structural genes from Rhizobium leguminosarum bv. viciae was used as probe. Cosmid pHU52, containing the complete gene cluster required for hydrogen oxidation in Bradyrhizobium japonicum, was introduced into two R. loti strains. Transconjugants from only one of the strains were able to express hydrogenase activity in vegetative cells incubated under the derepression conditions described for B. japonicum. Bacteroids induced by both transconjugant strains in L. corniculatus and Lotus tenuis expressed hydrogenase activity in nodules. The level of hydrogenase activity induced in L. tenuis nodules was two-fold higher than those induced in L. corniculatus. This implies the existence of a strong host effect on hydrogenase expression in this symbiotic system.     

Effectiveness of Lotus Root Nodules: I. MORPHOLOGY AND FLAVOLAN CONTENT OF NODULES FORMED ON LOTUS PEDUNCULATUS BY FAST-GROWING LOTUS RHIZOBIA

The morphology of root nodules formed on Lotus pedunculatusby two fast-growing strains of Rhizobium, NZP2037 which formseffective (nitrogen-fixing) nodules and NZP2213 which formsineffective (non-nitrogen-fixing) nodules, has been studied.The nodules formed by NZP2037 contained a central zone of bacteroid-filledplant cells surrounded by a cortex. In contrast the nodulesformed by NZP2213 contained no Rhizoblum-infected plant cells,but rhizobia were found in localized areas on the nodule surfaceand between the outer two or three cell layers of the nodule.Electron-dense osmiophilic deposits identified as flavolans(condensed tannins) were present in the vacuoles of many uninfectedplant cells in the nodules formed by both Rhizobium strains.This is the first time that flavolans have been positively identifiedin legume root nodules. In the NZP2037 nodule flavolans werepresent in the outer cortical and epidermal cells. In the ineffecitveNZP2213 nodule fiavolans were present in many of the centralnodule cells. The concentration of flavolan in the NZP2213 nodulewas 12 times higher than in the NZP2037 nodule.     

Rhizobium strain effects on pea: The relation between nitrogen accumulation, phosphoenolpyruvate carboxylase activity in nodules and asparagine in root bleeding sap

Pisum sativum L. cv. Bodil was infected with various strains of Rhizobium leguminosarum (R501, 128c53, B155, 18a or 1044). The Rhizobium genotype influenced the activity of the plant enzyme phosphoenoipyruvate (PEP) carboxylase (EC 4.1.1.31), and the assimilation of fixed N in the root nodules. The specific activity of nodule PEP carboxylase was lowest in the symbioses, which accumulated the least total N (R501 and 128c53). The root bleeding sap of the less effective symbioses contained a lower proportion of asparagine and a higher proportion of glutamine than the more effective symbioses (B155,18a and 1044). The N yield of the symbioses was related neither to the net respiratory CO₂ evolution of the root system nor to the nitrogenase linked nodule respiration. The lower yielding symbioses accumulated a larger proportion of the fixed N in the nodules due to a higher proportion of total dry weight contained in the nodule tissue. However, the concentration of soluble protein in the nodules of the lower-yielding symbioses was lower than that recorded for the higher yileding symbioses. The effect of the Rhizobium strains on N yield was maintained at maturity, and reflected in seed yields.     

Mutations conferring azide resistance enhance symbiotic nitrogen fixation in Rhizobium loti

Azide-resistant (AzR) mutants of Rhizobium loti strain NZP2037 were isolated. Mutations conferring azide resistance (azi) appeared at a frequency of 0.5 × 10-7. Nine AzR mutants of R. loti were characterised for their symbiotic behaviour with Lotus pedunculatus plants. In comparison to the wild type parent strain, AzR mutants exhibited either similar or higher symbiotic effectiveness. The azi mutations which enhanced nitrogen fixation as well as improving shoot dry weight of the inoculated plants also increased nodulation. Unlike several azi mutations in Escherichia coli, these azi mutations did not alter sensitivity of R. loti to phenethyl alcohol. One of the AzR mutants exhibited higher micro-aerobic, N, N, N, N-tetramethyl-p-phenylenediamine (TMPD) oxidase activity.     

Carbon and nitrogen partitioning in young nodulated pea (wild type and nitrate reductase-deficient mutant) plants exposed to NH4NO3

Carbon and nitrogen partitioning was examined in a wild-type and a nitrate reductase-deficient mutant (A317) of Pisum sativum L. (ev. Juneau), effectively inoculated with two strains of Rhizobium leguminosarum (128C23 and 128C54) and grown hydroponically in medium without nitrogen for 21 days, followed by a further 7 days in medium without and with 5 mM NH₄NO₃. In wild-type symbioses the application of NH₄NO₃ significantly reduced nodule growth, nitrogenase (EC 1.7.99.2) activity, nodule carbohydrates (soluble sugars and starch) and allocation of [¹⁴C]-labelled (NO₃⁻, NH₄⁺, amino acids) in roots. In nodules, there was a decline in amino acids together with an increase in inorganic nitrogen concentration. In contrast, symbioses involving A317 exhibited no change in nitrogenase activity or nodule carbohydrates, and the concentrations of all nitrogenous solutes measured (including asparagine) in roots and nodules were enhanced. Photosynthate allocation to the nodule was reduced in the 128C23 symbiosis. Nitrite accumulation was not detected in any case. These data cannot be wholly explained by either the carbohydrate deprivation hypothesis or the nitrite hypothesis for the inhibition of symbiotic nitrogen fixation by combined nitrogen. Our result with A317 also provided evidence against the hypothesis that NO₃⁻ and NH₄⁺ or its assimilation products exert a direct effect on nitrogenase activity. It is concluded that more than one legume host and Rhizobium strain must be studied before generalizations about Rhizobium /legume interactions are made.     

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

Differential effects of combined N sources on early steps of the Nod factor-dependent transduction pathway in Lotus japonicus

The development of nitrogen-fixing nodules in legumes is induced by perception of lipochitin-oligosaccharide signals secreted by a bacterial symbiont. Nitrogen (N) starvation is a prerequisite for the formation, development, and function of root nodules, and high levels of combined N in the form of nitrate or ammonium can completely abolish nodule formation. We distinguished between nitrate and ammonium inhibitory effects by identifying when and where these combined N sources interfere with the Nod-factor-induced pathway. Furthermore, we present a small-scale analysis of the expression profile, under different N conditions, of recently identified genes involved in the Nod-factor-induced pathway. In the presence of high levels of nitrate or ammonium, the NIN gene fails to be induced 24 h after the addition of Nod factor compared with plants grown under N-free conditions. This induction is restored in the hypernodulating nitrate-tolerant har1-3 mutant only in the presence of 10 and 20 mM KNO3. These results were confirmed in Lotus plants inoculated with Mesorhizobium loti. NIN plays a key role in the nodule organogenesis program and its downregulation may represent a crucial event in the nitrate-dependent pathway leading to the inhibition of nodule organogenesis.     

Molecular cloning of a nodulation gene from fast- and slow-growing strains of Lotus rhizobia

Summary Cosmids containing a nodulation gene from Rhizobium loti NZP2037 were isolated using a 12.8 kb nod:: Tn5 EcoRI fragment from the Nod^- mutant strain PN233, as a hybridisation probe. A physical map of the nod region was established using the enzymes EcoRI and HindIII and the site of insertion of Tn5 in PN233 determined. Site-specific exchange of the cloned nod:: Tn5 fragment demonstrated that Tn5, and not an indigenous insertion sequence, was responsible for the nod mutation in PN233. The nod cosmids isolated complemented the Nod^- phenotype of strain PN233 but restoration of the Fix phenotype was variable suggesting a need for marker rescue to occur before nitrogen fixation occurred.Corresponding nod cosmids were isolated from a R. loti strain, NZP2213, that forms ineffective tumour-like structures on Lotus pedunculatus and from the slow-growing strain (Bradyrhizobium sp), CC814s, by in planta complementation of PN233. Hybridisation experiments suggested that the nod gene region of R. loti NZP2037 was more homologous to Bradyrhizobium strain CC814s than with a nod gene region of R. trifolii strain PN100. However, transfer of the R. trifolii nod cosmid into the R. loti Nod mutant PN233, restored the ability of this strain to initiate nodules on Lotus pedunculatus.     

Interplay of flg22-induced defence responses and nodulation in Lotus japonicus

In this study the interplay between the symbiotic and defence signalling pathways in Lotus japonicus was investigated by comparing the responses to Mesorhizobium loti, the symbiotic partner of L. japonicus, and the elicitor flg22, a conserved peptide motif present in flagellar protein of a wide range of bacteria. It was found that defence and symbiotic pathways overlap in the interaction between L. japonicus and M. loti since similar responses were induced by the mutualistic bacteria and flg22. However, purified flagellin from M. loti did not induce any response in L. japonicus, which suggests the production of other elicitors by the symbiotic bacteria. Defence responses induced by flg22 caused inhibition of rhizobial infection and delay in nodule organogenesis, as demonstrated by the negative effect of flg22 in the formation of spontaneous nodules in the snf1 L. japonicus mutant, and the inhibition of NSP1 and NSP2 genes. This indicates the antagonistic effect of the defence pathway on the nodule formation in the initial rhizobium-legume interaction. However, the fact that flg22 did not affect the formation of new nodules once the symbiosis was established indicates that after the colonization of the host plant by the symbiotic partner, the symbiotic pathway has prevalence over the defensive response. This result is also supported by the down-regulation of the expression levels of the flg22 receptor FLS2 in the nodular tissue.     

Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus.

Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J. T. Sullivan, H. N. Patrick, W. L. Lowther, D. B. Scott, and C. W. Ronson, Proc. Natl. Acad. Sci. USA 92:8985-8989, 1995). In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L. corniculatus. These included two from plants at the field site sampled by Sullivan et al. and five from plants at a new field plot adjacent to that site. The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti. Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R. loti NZP2213T were determined by three methods. Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence. The nonsymbiotic isolates represented four genomic species that were related to R. loti; the diverse symbiotic isolates from the site belonged to one of these species. The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii. These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes.