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A non-nodulating alfalfa mutant displays neither root hair curling nor early cell division in response to Rhizobium meliloti.

The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex. These early steps have been studied previously by analysis of R. meliloti mutants. Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R. meliloti stimulate host cell division but the resulting nodules are uninfected. As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating plants were inoculated with wild-type R. meliloti and scored for root hair curling and cell divisions. MN-1008 was found to be defective in both responses. Mutant plants inoculated with Exo- bacteria also showed no cell division response. Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R. meliloti nodABC genes. These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.

Cell Division↗

Characterization of a symbiotically defective serine auxotroph of Mesorhizobium ciceri.

A Tn5-induced mutant strain (TL68) of Mesorhizobium ciceri unable to grow with ammonium as the sole nitrogen source was isolated and characterized. Unlike its wild-type parent (strain TAL620), the mutant had an absolute dependence on serine to grow. Cloning of the DNA region containing Tn5 and sequence analysis showed that Tn5 was inserted into the gene coding for 3-phosphoglycerate dehydrogenase, which catalyses the first step in the serine biosynthetic pathway. The role of serine biosynthesis of M. ciceri in the establishment of nitrogen-fixing symbiosis with chickpea (Cicer arietinum L) was investigated using the mutant TL68. The serA(-) mutant (TL68) was unable to elicit the development of efficient nodules on the roots of Cicer arietinum L. The addition of serine to the plant-growth medium restored the ability of the mutant to nodulate Cicer arietinum, and the nodules were able to fix nitrogen.

Alphaproteobacteria↗

Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes.

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasmid includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine biosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf-) and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix- nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.

DNA Transposable Elements↗

Heme Synthesis in Soybean Root Nodules: I. On the Role of Bacteroid delta-Aminolevulinic Acid Synthase and delta-Aminolevulinic Acid Dehydrase in the Synthesis of the Heme of Leghemoglobin.

During nodulation of soybean (Glycine max) by Rhizobium japonicum, variations in the activities of two enzymes of heme biosynthesis, delta-aminolevulinic acid synthase (ALAS) and delta-aminolevulinic acid dehydrase (ALAD) are described. delta-Aminolevulinic acid synthase activity is found in the bacteroid fraction of nodules, but is not detected in the plant fraction. Bacteroid ALAS activity parallels heme accumulation during nodule development. delta-Aminolevulinic acid dehydrase activity is found in both bacteroid and plant cytosol fractions. Bacteroid ALAD activity is constant or increases during nodulation while plant ALAD activity falls.Bacteroid ALAD activity is found in effective, not in inefficient nodules. Plant ALAD activity is found in both effective and inefficient nodules. Plant ALAD activity falls during development of both types of root nodules.These results support the contention that it is the bacteroid ALAS and ALAD activities, not those of the plant, that are directly involved in formation of leghemoglobin heme, suggesting that the bacteroid may be solely responsible for formation of leghemoglobin heme in the nodule symbiosis.

Journal Article↗

Nodule infection by bean yellow mosaic virus in Phaseolus vulgaris.

Infection of root nodules of beans, Phaseolus vulgaris L., by bean yellow mosaic virus (BYMV) and the effect of the disease on the specific activity of the nodule are reported. Infectivity and serological microprecipitin assays with two sources of BYMV antiserum demonstrated that nodules from bean plants whose leaves had been inoculated with BYMV contain BYMV antigen. The disease reduced the fresh weights of tops, roots, and root nodules and induced premature nodule decay and/or nodule drop. The disease also reduced leghemoglobin content, on a plant weight basis, and N2 fixation rate, on an individual plant basis, as measured by the acetylene reduction assay. The increased leghemoglobin content per gram-nodule in BYMV-infected nodules relative to healthy nodules might be associated with multiplication of the virus in the nodule and/or unknown cellular effects derived from the BYMV-Rhizobium interaction.

Antigens, Viral↗

Symbiotic host specificity between leguminous plants and rhizobia is determined by substituted and acylated glucosamine oligosaccharide signals.

Rhizobia are nitrogen-fixing bacteria which invade root hairs of leguminous plants and induce, in a specific manner, the formation of root nodules in which they fix nitrogen. The early steps of the symbiosis can be considered as a reciprocal molecular communication between the two partners. Initially, the plant excretes a gene inducer which stimulates the expression of bacterial nodulation genes. These nodulation genes are responsible for the synthesis of extracellular host-specific signals, called nodulation factors. The bacterial nodulation factors were isolated and structurally identified as substituted and N-acylated chitin oligosaccharides. These prokaryotic lipo-oligosaccharide signals play a key role in the symbiosis by controlling the host specificity of the bacteria. They constitute a new class of signalling molecules able to elicit nodule organogenesis in leguminous plants in the absence of bacteria.

Carbohydrate Conformation↗

An inducible activator produced by a Serratia proteamaculans strain and its soybean growth-promoting activity under greenhouse conditions.

Serratia proteamaculans 1-102 (1-102) promotes soybean-bradyrhizobia nodulation and growth, but the mechanism is unknown. After adding isoflavonoid inducers to 1-102 culture, an active peak with a retention time of about 105 min in the HPLC fractionation was isolated using a bioassay based on the stimulation of soybean seed germination. The plant growth-promoting activity of this material was compared with 1-102 culture (cells) and supernatant under greenhouse conditions. The activator was applied to roots in 83, 830 and 8300 HPLC microvolts (microV) per seedling when plants were inoculated with bradyrhizobia or sprayed onto the leaves in same concentrations at 20 d after inoculation. The root-applied activator, especially at 1 ml of 830 microV per seedling, enhanced soybean nodulation and growth at the same level as 1-102 culture under both optimal and sub-optimal root zone temperatures. Thus, this activator stimulating soybean seed germination is also responsible for the plant growth-promoting activity of 1-102 culture. However, when sprayed onto the leaves, the activator did not increase growth and in higher concentrations decreased average single leaf area. The results suggest that this inducible activator might be a lipo-chitooligosaccharide (LCO) analogue. LCOs act as rhizobia-to-legume signals stimulating root nodule formation. The activator could provide additional 'signal', increasing in the signal quality (the signal-to-noise ratio, SNR) of the plant-rhizobia signal exchange process.

Biological Assay↗

Medicago truncatula transformation using root explants.

Medicago truncatula is a forage crop that has been developed into a model legume. As a model plant, M. truncatula is particularly useful for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. The development of different transformation methods is an important aspect for functional genomic studies in the species. This protocol describes an efficient system for generating transgenic plants from M. truncatula roots based on Agrobacterium tumefaciens-mediated transformation. Furthermore, the protocol can be easily adapted for recovering fertile transgenic plants from transformed hairy roots.

Agrobacterium tumefaciens↗

Nodulation of Glycine max by Six Bradyrhizobium japonicum Strains with Different Competitive Abilities.

The root nodule locations of six Bradyrhizobium japonicum strains were examined to determine if there were any differences which might explain their varying competitiveness for nodule occupancy on Glycine max. When five strains were added to soybeans in plastic growth pouches in equal proportions with a reference strain (U.S. Department of Agriculture, strain 110), North Carolina strain 1028 and strain 110 were the most competitive for nodule occupancy, followed by U.S. Department of Agriculture strains 122, 76, and 31 and Brazil strain 587. Among all strains, nodule double occupancy was 17% at a high inoculum level (10 CFU pouch) and 2% at a low inoculum level (10 CFU pouch). The less competitive strains increased their nodule representation by an increase in the doubly occupied nodules at the high inoculum level. Among all strains, the number of taproot and lateral root nodules was inversely related at both the high and low inoculum levels (r = -0.62 and -0.69, respectively; P = 0.0001). This inverse relationship appeared to be a result of the plant host control of bacterial infection. Among each of the six strains, greater than 95% of the taproot nodules formed at the high inoculum density were located on 25% of the taproot length, the nodules centering on the position of the root tip at the time of inoculation. No differences among the six strains were observed in nodule initiation rates as measured by taproot nodule position. Taproot nodules were formed in the symbiosis before lateral root nodules. One of the poorly competitive strains (strain 76) occupied three times as many taproot nodules as lateral root nodules when competing with strain 110 (nodules were harvested from 4-week-old plants). Among these six wild-type strains of B. japonicum, competitive ability evidently is not related to nodule initiation rates.

Journal Article↗

Effects of phosphorus and nitrogen on nodulation are seen already at the stage of early cortical cell divisions in Alnus incana.

BACKGROUND AND AIMS: The present work aimed to study early stages of nodulation in a chronological sequence and to study phosphorus and nitrogen effects on early stages of nodulation in Alnus incana infected by Frankia. A method was developed to quantify early nodulation stages in intact root systems in the root hair-infected actinorhizal plant A. incana. Plant tissue responses were followed every 2 d until 14 d after inoculation. Cortical cell divisions were already seen 2 d after inoculation with Frankia. Cortical cell division areas, prenodules, nodule primordia and emerging nodules were quantified as host responses to infection. METHODS: Seedlings were grown in pouches and received different levels of phosphorus and nitrogen. Four levels of phosphorus (from 0.03 to 1 mM P) and two levels of nitrogen (0.71 and 6.45 mM N) were used to study P and N effects on these early stages of nodule development. KEY RESULTS: P at a medium concentration (0.1 mM) stimulated cell divisions in the cortex and a number of prenodules, nodule primordia and emerging nodules as compared with higher or lower P levels. A high N level inhibited early cell divisions in the cortex, and this was particularly evident when the length of cell division areas and presence of the nodulation stages were related to root length. CONCLUSIONS: Extended cortical cell division areas were found that have not been previously shown in A. incana. The results show that effects of P and N are already expressed at the stage when the first cortical cell divisions are induced by Frankia.

Alnus↗

Increase in Internode Length of Phaseolus lunatus L. Caused by Inoculation with a Nitrate Reductase-deficient Strain of Rhizobium sp.

Dramatic differences in the height of lima beans (Phaseolus lunatus L.) treated with two different Rhizobium strains were studied. Lima beans were grown in Perlite in the greenhouse or in a minus-N culture solution in the growth chamber. The plants were inoculated with either Rhizobium sp. (lima bean) strain 127E15, which contains the constitutive nitrate reductase activity, or strain 127E14, which lacks that activity. For up to 3 weeks, no growth differences were observed in the plants inoculated with either strain. Five weeks after inoculation, however, those plants inoculated with strain 127E14 were significantly taller and had a larger number of leaves than those inoculated with strain 127E15. The difference in plant height was the result of increased internode elongation caused by inoculation with Rhizobium sp. 127E14. This response was observed with all lima bean cultivars tested, including Henderson, Fordhook, Allgreen, and Early Thorogreen. The growth difference occurred in plants cultured in the greenhouse or in the growth chamber.No differences were observed in fresh weights of nodules, roots, axes, leaves, or flowers. The number of nodules, pods, or flowers also did not differ. Acetylene reduction rates and total N content were equal in plants inoculated with strains 127E14 or 127E15.Inasmuch as only internode length and leaf number differed between Rhizobium treatments, the response appeared to be hormone-mediated. Application of gibberellic acid to the apex of plants inoculated with strain 127E15 caused an increase in plant height similar to that observed in untreated plants inoculated with strain 127E14. Conversely, the height of plants infected with strain 127E14 was decreased by application of gibberellin synthesis inhibitors to the root system. These data suggest that the increased growth caused by inoculation with strain 127E14 could be the result of increased gibberellin synthesis in lima bean nodules infected with that strain. Whether this response was related to the absence of nitrate reductase activity in strain 127E14 is unknown.

Journal Article↗

[Characteristics of certain symbiotic pea mutants by traits related to hormonal status].

Association between traits for hormonal status and nodulation in the mutants of pea Pisum sativum L. with abnormal nodulation and original forms was analyzed. The sensitivity of plant tissues to exogenous phytohormones and changes in the concentration of the major auxin, indolyl-3-acetic acid, in plant roots during interaction with rhizobia were examined. Association between Nod(++)-phenotype and auxin balance was revealed: the supernodulating mutants were more sensitive to auxin treatment than the parental cultivars. Mutations in the sym8 gene, in contrast to those in the sym5 gene, had no effect on plant hormonal status. The level of indolyl-3-acetic acid during interaction with rhizobia depended on the time after inoculation and plant genotype. The mutations affecting nodulation were suggested to change auxin level in roots.

Genotype↗

Induction and spatial organization of polyamine biosynthesis during nodule development in Lotus japonicus.

Putrescine and other polyamines are produced by two alternative pathways in plants. One pathway starts with the enzyme arginine decarboxylase (ADC; EC 4.1.1.19), the other with ornithine decarboxylase (ODC; EC 4.1.1.17). Metabolite profiling of nitrogen-fixing Lotus japonicus nodules, using gas chromatography coupled to mass spectrometry, revealed a two- to sixfold increase in putrescine levels in mature nodules compared with other organs. Genes involved in polyamine biosynthesis in L japonicus nodules were identified by isolating cDNA clones encoding ADC (LjADC1) and ODC (LjODC) from a nodule library. Searches of the public expressed sequence tag databases revealed the presence of a second gene encoding ADC (LjADC2). Real-time reverse-transcription-polymerase chain reaction analysis showed that LjADC1 and LjADC2 were expressed throughout the plant, while LjODC transcripts were detected only in nodules and roots. Induction of LjODC and LjADC gene expression during nodule development preceded symbiotic nitrogen fixation. Transcripts accumulation was maximal at 10 days postinfection, when a 6.5-fold increase in the transcript levels of LjODC was observed in comparison with the uninfected roots, while a twofold increase in the transcript levels of LjADC1 and LjADC2 was detected. At later stages of nodule development, transcripts for ADC drastically declined, while in the case of ODC, transcript accumulation was higher than that in roots until after 21 days postinfection. The expression profile of genes involved in putrescine biosynthesis correlated well with the expression patterns of genes involved in cell division and expansion, including a L. japonicus Cyclin D3 and an alpha-expansin gene. Spatial localization of LjODC and LjADC1 gene transcripts in developing nodules revealed that both transcripts were expressed in nodule inner cortical cells and in the central tissue. High levels of LjADC1 transcripts were also observed in both nodule and connecting root vascular tissue, suggesting that putrescine and other polyamines may be subject to long-distance transport. Our results indicate that polyamines are primarily involved in physiological and cellular processes involved in nodule development, rather than in processes that support directly symbiotic nitrogen fixation and assimilation.

Amino Acid Sequence↗

Evolution of signal transduction in intracellular symbiosis.

Plant roots form intracellular symbioses with fungi and bacteria resulting in arbuscular mycorrhiza and nitrogen-fixing root nodules, respectively. A novel receptor like-kinase has been discovered that is required for the transduction of both bacterial and fungal symbiotic signals. This kinase defines an ancient signalling pathway that probably evolved in the context of arbuscular mycorrhiza and has been recruited subsequently for endosymbiosis with bacteria. An ancestral symbiotic interaction of roots with intracellular bacteria might have emerged from such a recruitment, in the progenitor of the nodulating clade of plants. Analysis of symbiotic mutants of host plants and bacterial microsymbionts has revealed that present-day endosymbioses require the coordinated induction of more than one signalling pathway for development.

Bacteria↗

Invasion of Lotus japonicus root hairless 1 by Mesorhizobium loti involves the nodulation factor-dependent induction of root hairs.

In many legumes, including Lotus japonicus and Medicago truncatula, susceptible root hairs are the primary sites for the initial signal perception and physical contact between the host plant and the compatible nitrogen-fixing bacteria that leads to the initiation of root invasion and nodule organogenesis. However, diverse mechanisms of nodulation have been described in a variety of legume species that do not rely on root hairs. To clarify the significance of root hairs during the L. japonicus-Mesorhizobium loti symbiosis, we have isolated and performed a detailed analysis of four independent L. japonicus root hair developmental mutants. We show that although important for the efficient colonization of roots, the presence of wild-type root hairs is not required for the initiation of nodule primordia (NP) organogenesis and the colonization of the nodule structures. In the genetic background of the L. japonicus root hairless 1 mutant, the nodulation factor-dependent formation of NP provides the structural basis for alternative modes of invasion by M. loti. Surprisingly, one mode of root colonization involves nodulation factor-dependent induction of NP-associated cortical root hairs and epidermal root hairs, which, in turn, support bacterial invasion. In addition, entry of M. loti through cracks at the cortical surface of the NP is described. These novel mechanisms of nodule colonization by M. loti explain the fully functional, albeit significantly delayed, nodulation phenotype of the L. japonicus ROOT HAIRLESS mutant.

Base Sequence↗