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Rearrangement of actin microfilaments in plant root hairs responding to rhizobium etli nodulation signals

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.

Journal Article↗

Nod factor-treated Medicago truncatula roots and seeds show an increased number of nodules when inoculated with a limiting population of Sinorhizobium meliloti.

Medicago truncatula is a model legume plant that interacts symbiotically with Sinorhizobium meliloti, the alfalfa symbiont. This process involves a molecular dialogue between the bacterium and the plant. Legume roots exude flavonoids that induce the expression of a set of rhizobial genes, the nod genes, which are essential for nodulation and determination of the host range. In turn, nod genes control the synthesis of lipo-chito-oligosaccharides (LCOs), Nod factors, which are bacteria-to-plant signal molecules mediating recognition and nodule organogenesis. M. truncatula roots or seeds have been treated with Nod factors and hydroponically growing seedlings have been inoculated with a limiting population of S. meliloti. It has been shown that submicromolar concentrations of Nod factors increase the number of nodules per plant on M. truncatula. Compared with roots, this increase is more noticeable when seeds are treated. M. truncatula seeds are receptive to submicromolar concentrations of Nod factors, suggesting the possibility of a high affinity LCO perception system in seeds or embryos as well.

Medicago truncatula↗

Purification and characterization of a specific nucleoside diphosphatase from soybean root nodules.

A specific nucleoside diphosphatase was purified from the plant portion of soybean (Glycine max L.) root nodules. This enzyme is highly specific for nucleotide diphosphates; it is unable to hydrolyze nucleotide tri- and monophosphates or a variety of other phosphorylated compounds. It will, however, hydrolyze any nucleotide disphosphate tested. The pH optimum of the enzyme is about 7.5; it requires a divalent cation for activity; and it is neither inhibited nor activated by any of the metabolites tested. It appears that in vivo this enzyme would be very active, but its function is not clear.

Journal Article↗

Isolation and characterization of hydrogen-oxidizing bacteria induced following exposure of soil to hydrogen gas and their impact on plant growth.

In many legumes, the nitrogen fixing root nodules produce H2 gas that diffuses into soil. It has been demonstrated that such exposure of soil to H2 can promote plant growth. To assess whether this may be due to H2-oxidizing microorganisms, bacteria were isolated from soil treated with H2 under laboratory conditions and from soils collected adjacent to H2 producing soybean nodules. Nineteen isolates of H2-oxidizing bacteria were obtained and all exhibited a half-saturation coefficient (Ks) for H2 of about 1 ml l(-1). The isolates were identified as Variovorax paradoxus, Flavobacterium johnsoniae and Burkholderia spp. using conventional microbiological tests and 16S rRNA gene sequence analysis. Seventeen of the isolates enhanced (57-254%) root elongation of spring wheat seedlings. Using an Arabidopsis thaliana bioassay, plant biomass was increased by 11-27% when inoculated by one of four isolates of V. paradoxus or one isolate of Burkholderia that were selected for evaluation. The isolates of V. paradoxus found in both H2-treated soil and in soil adjacent to soybean nodules had the greatest impact on plant growth. The results are consistent with the hypothesis that H2-oxidizing bacteria in soils have plant growth promoting properties.

Arabidopsis↗

[A Sinorhizoboium meliloti strain that can nodulate soybean plants].

Sinorhizobium meliloti XJ96077 was isolated from root nodules of alfalfa (Medicago sativa) in Xinjiang Region of China. Nodulation experiments showed that both soybean and alfalfa were effectively nodulated by XJ96077. The DNA (G+ C) mol% of strain XJ96077 was 61.9%. The DNA homologies of strain XJ96077 were 93% and 80% with S. meliloti USDA1002T and 042BM, respectively. These results showed that XJ96077 belongs to Sinorhizobium meliloti. To prove the capability of XJ96077 to nodulate both soybean and alfalfa, constitutively expressed green fluorescence protein gene gfp was introduced to XJ96077, and the recombinant strain XJ96077(G) was obtained. Root nodules of the soybean and alfalfa inoculated with XJ96077(G) and the expression of gfp were observed using the confocal laser scanning microscope. XJ96077 showed various nodulation capacities with different soybean cultivars.

Medicago sativa↗

[The influence of lipopolysaccharides and glucans from two Rhizobium leguminosarum bv. viciae strains on the formation and efficiency of their symbioses with pea plants].

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

Glucans↗

Root Isoflavonoid Response to Grafting between Wild-Type and Nodulation-Mutant Soybean Plants.

It was previously reported that the hypernodulating soybean (Glycine max [L.] Merr.) mutants, derived from the cultivar Williams, had higher root concentration of isoflavonoid compounds (daidzein, genistein, and coumestrol) than did Williams at 9 to 12 days after inoculation with Bradyrhizobium japonicum. These compounds are known inducers of nod genes in B. japonicum and may be involved in subsequent nodule development. The current study involving reciprocal grafts between NOD1-3 (hypernodulating mutant) and Williams showed that root isoflavonoid concentration and content was more than twofold greater when the shoot genotype was NOD1-3. When grafted, NOD1-3 shoots also induced hypernodulation on roots of both Williams and NOD1-3, while Williams shoots induced normal nodulation on both root genotypes. This shoot control of hypernodulation may be causally related to differential root isoflavonoid levels, which are also controlled by the shoot. In contrast, the nonnodulating characteristic of the NN5 mutant was strictly root controlled, based on reciprocal grafts. Delayed inoculation (7 days after planting) resulted in greater nodule numbers on both NOD1-3 and Williams, compared with a seed inoculation treatment. The nodulation pattern of grafted plants was independent of whether the shoot portion was derived from inoculated seed or uninoculated seed, when grafted at day 7 onto seedling roots derived from inoculated seed. This observation, coupled with the fact that no difference existed in nodule number of NOD1-3 and Williams until after 9 days from seed inoculation, indicated that if isoflavonoids play a role in differential nodulation of the hypernodulating mutant and the wild type, the effect is on advanced stages of nodule ontogeny, possibly related to autoregulation, rather than on initial infection stages.

Journal Article↗

The PIN and LAX families of auxin transport genes in Medicago truncatula.

Auxin transport proteins may be involved in nodule development. As a prelude to investigating the roles of these proteins in nodule development, we took advantage of the genetic and molecular resources available in the legume Medicago truncatula to characterize the gene families encoding auxin efflux and influx carriers. We identified ten auxin efflux carrier sequences (MtPINs) and five auxin influx/permease sequences (MtLAXs). The genomic sequence of each of these fifteen genes was determined, the genes were mapped on the publicly available map of M. truncatula, and their expression was examined in shoot and root tissue of nodulating plants. With one exception, transcripts of all MtPIN genes were detected. The expression of MtPIN2 was limited to nodulating roots, while transcripts of all other expressed genes were detected in both shoots and roots. Both the PIN and LAX gene families contain more members in M. truncatula than in Arabidopsis, but the gene families are not significantly expanded. Sequence comparison of the M. truncatula PIN and LAX genes with PIN and LAX genes from other dicots and monocots indicates that both gene families share a common overall structure, with areas of high homology both within M. truncatula and across species boundaries. Molecular phylogenies of both the PIN and LAX gene families were constructed. Combined with intron position and expression data, the phylogenies were used to assign relationships between MtPIN and MtLAX genes and the orthologous Arabidopsis PIN and LAX genes. MtPIN2 and MtPIN7 appear to be the result of a recent gene duplication with subsequent divergence of expression patterns. These results set the stage for the use of these genes in research on the role of auxin in nodulation.

Base Sequence↗

Carbohydrates in Soybean Nodules: II. DISTRIBUTION OF COMPOUNDS IN SEEDLINGS DURING THE ONSET OF NITROGEN FIXATION.

During the first few days of nitrogen fixation activity by soybean (Glycine max (L.) Merr) root nodules, d-chiro-inositol, myo-inositol, sucrose, alpha,alpha-trehalose, and maltose accumulate rapidly and reach concentrations several fold greater than concentrations in other plant organs. Concentrations of d-pinitol in nodules (>/=1.0 milligrams per gram fresh weight) were similar to concentrations in leaf blades. The concentration of fructose in nodules was lower than concentrations in other plant organs.Comparison of nonnodulated roots, nodulated roots (after removal of nodules), and nodules indicated that nodules may compete successfully with roots for carbohydrates, especially the disaccharides sucrose, alpha,alpha-trehalose, and maltose. Based on the isolation of protoplasts and bacteroids, it was tentatively concluded that the highest concentrations of cyclitols in soybean nodules are located in the infected region and that, inside infected cells, the highest concentrations of d-pinitol and myo-inositol are outside of bacteroids.Evidence for the identification of d-chiro-inositol and maltose in soybean nodules is presented.

Journal Article↗

Split-Root Assays Using Trifolium subterraneum Show that Rhizobium Infection Induces a Systemic Response That Can Inhibit Nodulation of Another Invasive Rhizobium Strain.

Subterranean clover plants possessing two equally infectible and robust lateral root systems ("split roots") were used in conjunction with several specific mutant strains (derived from Rhizobium trifolii ANU843) to investigate a systemic plant response induced by infective Rhizobium strains. This plant response controls and inhibits subsequent nodulation on the plant. When strain ANU843 was inoculated onto both root systems simultaneously or 24, 48, 72, or 96 h apart, an inhibitory response occurred which retarded nodulation on the root exposed to the delayed inoculum but only when the delay period between inocula was greater than 24 h. Equal numbers of nodules were generated on both roots when ANU843 was inoculated simultaneously or 24 h apart. The ability to infect subterranean clover plants was required to initiate the plant inhibitory response since preexposure of one root system to non-nodulating strains did not retard the ability of the wild-type strain to nodulate the opposing root system (even when the delay period was 96 h). Moreover, the use of specific Tn5-induced mutants subtly impaired in their ability to nodulate demonstrated that the plant could effectively and rapidly discriminate between infections initiated by either the parent or the mutant strains. When inoculated alone onto clover plants, these mutant strains were able to infect the most susceptible plant cells at the time of inoculation and induce nitrogen-fixing nodules. However, the separate but simultaneous inoculation on opposing root systems of the parent and the mutant strains resulted in the almost complete inhibition of the nodulation ability of the mutant strains. We concluded that the mutants were affected in their competitive ability, and this finding was reflected by poor nodule occupancy when the mutants were coinoculated with the parent strain onto a single root system. Thus the split-root system may form the basis of a simple screening method for the ranking of competitiveness of various rhizobia on small seeded legumes.

Journal Article↗

Rhizobium meliloti exopolysaccharides: synthesis and symbiotic function.

Bacterial exopolysaccharide (EPS) is required for establishment of the nitrogen-fixing symbiosis between Rhizobium meliloti and its host plant, Medicago sativa (alfalfa), but the precise role of EPS in this interaction is not well defined. Bacterial mutants which fail to produce EPS induce nodules on the roots of the host plant, but fail to invade these root nodules. Research conducted in our lab and others suggests that EPS plays a specific role in the R. meliloti-M. sativa symbiosis. A common theme emerging from these studies is that small quantities of low-molecular-weight (LMW) EPS are sufficient to mediate successful invasion by R. meliloti mutants which fail to produce EPS, implying that LMW EPS may act as a signaling molecule during this process.

Carbohydrate Sequence↗

Low genetic diversity among Frankia spp. strains nodulating sympatric populations of actinorhizal species of Rosaceae, Ceanothus (Rhamnaceae) and Datisca glomerata (Datiscaceae) west of the Sierra Nevada (California).

Frankia spp. strains typically induce N2-fixing root nodules on actinorhizal plants. The majority of host plant taxa associated with the uncultured Group 1 Frankia strains, i.e., Ceanothus of the Rhamnaceae, Datisca glomerata (Datiscaceae), and all actinorhizal members of the Rosaceae except Dryas, are found in California. A study was conducted to determine the distribution of Frankia strains among root nodules collected from both sympatric and solitary stands of hosts. Three DNA regions were examined, the 5' end of the 16S rRNA gene, the internal transcribed spacer region between the 16S and 23S rRNA genes, and a portion of the glutamine synthetase gene (glnA). The results suggest that a narrow range of Group 1 Frankia spp. strains dominate in root nodules collected over a large area of California west of the Sierra Nevada crest with no apparent host-specificity. Comparisons with Group 2 Frankia strain diversity from Alnus and Myrica within the study range suggest that the observed low diversity is peculiar to Group 1 Frankia strains only. Factors that may account for the observed lack of genetic variability and host specificity include strain dominance over a large geographical area, current environmental selection, and (or) a past evolutionary bottleneck.

California↗

Differential gene expression in an actinorhizal symbiosis: evidence for a nodule-specific cysteine proteinase.

Nodules formed on the roots of actinorhizal plants as a consequence of nitrogen-fixing symbioses with the actinomycete Frankia appear to result from modification of the developmental pathway that leads to lateral root formation. Presently no information exists about factors that control this developmental switch or, until now, about genes that are differentially expressed as a result of an altered developmental pathway. Differential screening of an Alnus glutinosa nodule cDNA library revealed altered levels of gene expression in nodules as compared with roots and allowed isolation of host plant nodule-specific cDNA sequences. The deduced amino acid sequence of one full-length cDNA, AgNOD-CP1, represents a nodule-specific cysteine proteinase similar to cysteine proteinases of the papain superfamily. Residues critical to catalysis, active site, and disulfide bridges are conserved. Suggested roles for this enzyme are as a defense response to Frankia invasion, as a component of tissue remodeling in root and nodule tissues, as a cell cycle component, or as an element of protein turnover. Complexity of hybridization patterns revealed by Southern blot analysis suggests that the gene for AgNOD-CP1 is a member of a multigene family. Northern hybridization results indicate that this gene may have been recruited for a role specific to this symbiosis, a phenomenon observed in the Rhizobium-legume symbioses, perhaps common to many microbe-plant interactions.

Actinomycetales↗

Nodulation genes in the Rhizobium--plant signal exchange.

The process of the host-plant recognition by rhizobia is complex and multi-step. The interaction between legumes and microorganisms results in the induction of the root nodule. This symbiotic interaction is highly host-specific. Bacteria within nodules fix atmospheric nitrogen. This process is of immense ecological and economic significance. The subject of this presentation is the molecular mechanism by which the bacterium determines its host-specific characteristics. First flavonoids secreted by the plant roots induce the transcription of bacterial genes involved in nodulation, the so-called nod genes. This leads to the next step of the signalling system, i.e. the production and secretion of lipo-oligosaccharide molecules by rhizobia. These signal molecules have various discernible effects on the roots of the host leguminous plants. The bacterial nodulation factors were isolated and structurally identified as substituted and N-acylated chitin oligosaccharides. These prokaryotic 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. More recent studies implicate involvement of root cell membrane depolarization and ion selective channels in the communication processes that initiate nodule formation.

Gene Expression Regulation, Bacterial↗

Effects of storage time and temperature on the infectivity and effectiveness of Frankia entrapped in polyacrylamide gel.

Four Frankia-Casuarina endosymbiont strains were tested for their infectivity and effectiveness on C. equisetifolia plants after 1 d, 3 and 6 months of Frankia storage at 7, 28 and 40 degrees C as liquid-cultures and polyacrylamide gel (PAG)-immobilized inocula. At lower temperature the number of nodules was the same or higher than control for liquid inocula except after 6 months of storage. For the PAG-entrapped Frankia lower numbers of nodules were recorded due to the use of high Frankia titers. In general, the results showed comparable plant dry mass, total nitrogen, root to shoot and nodules to plant ratios at lower temperature treatments. Increasing time and temperature was accompanied with reduced infectivity and effectiveness on inoculated plants. No nodulation was scored on plants inoculated with liquid and PAG-entrapped inocula stored at 40 degrees C for 6 months; subsequently, plant growth was inhibited. The variations in results obtained for different strains and treatments lead to variations in plant nitrogen-fixing ability. The use of PAG as a carrier for different Frankia strains is suggested; the recommended storage temperature for PAG-immobilized Frankia in 7-28 degrees C for up to 3 months.

Acrylic Resins↗

Cell biological changes of outer cortical root cells in early determinate nodulation.

In the symbiosis of leguminous plants and Rhizobium bacteria, nodule primordia develop in the root cortex. This can be either in the inner cortex (indeterminate-type of nodulation) or outer cortex (determinate-type of nodulation), depending upon the host plant. We studied and compared early nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus, both known as determinate-type nodulation plants. Special attention was paid to the occurrence of cytoplasmic bridges, the influence of rhizobial Nod factors (lipochitin oligosaccharides [LCOs]) on this phenomenon, and sensitivity of the nodulation process to ethylene. Our results show that i) both plant species form initially broad, matrix-rich infection threads; ii) cytoplasmic bridges occur in L. japonicus but not in bean; iii) formation of these bridges is induced by rhizobial LCOs; iv) formation of primordia starts in L. japonicus in the middle root cortex and in bean in the outer root cortex; and v) in the presence of the ethylene-biosynthesis inhibitor aminoethoxyvinylglycine (AVG), nodulation of L. japonicus is stimulated when the roots are grown in the light, which is consistent with the role of cytoplasmic bridges during nodulation of L. japonicus.

Ethylenes↗

Infection-related activation of the cg12 promoter is conserved between actinorhizal and legume-rhizobia root nodule symbiosis.

Two nitrogen-fixing root nodule symbioses between soil bacteria and higher plants have been described: the symbiosis between legume and rhizobia and actinorhizal symbioses between plants belonging to eight angiosperm families and the actinomycete Frankia. We have recently shown that the subtilisin-like Ser protease gene cg12 (isolated from the actinorhizal plant Casuarina glauca) is specifically expressed during plant cell infection by Frankia. Here we report on the study of C. glauca cg12 promoter activity in the transgenic legume Medicago truncatula. We found that cg12 promoter activation is associated with plant cell infection by Sinorhizobium meliloti. Furthermore, applications of purified Nod factors and mycorrhizal inoculation failed to trigger expression of the cg12-reporter gene construct. This indicates that at least part of the transcriptional environment in plant cells infected by endosymbiotic nitrogen-fixing bacteria is conserved between legume and actinorhizal plants. These results are discussed in view of recent data concerning molecular phylogeny that suggest a common evolutionary origin of all plants entering nitrogen-fixing root nodule symbioses.

Gene Expression Regulation, Plant↗

Respiratory Elicitors from Rhizobium meliloti Affect Intact Alfalfa Roots

Molecules produced by Rhizobium meliloti increase respiration of alfalfa (Medicago sativa L.) roots. Maximum respiratory increases, measured either as CO2 evolution or as O2 uptake, were elicited in roots of 3-d-old seedlings by 16 h of exposure to living or dead R. meliloti cells at densities of 10(7) bacteria/mL. Excising roots after exposure to bacteria and separating them into root-tip- and root-hair-containing segments showed that respiratory increases occurred only in the root-hair region. In such assays, CO2 production by segments with root hairs increased by as much as 100% in the presence of bacteria. Two partially purified compounds from R. meliloti 1021 increased root respiration at very low, possibly picomolar, concentrations. One factor, peak B, resembled known pathogenic elicitors because it produced a rapid (15-min), transitory increase in respiration. A second factor, peak D, was quite different because root respiration increased slowly for 8 h and was maintained at the higher level. These molecules differ from lipo-chitin oligosaccharides active in root nodulation for the following reasons: (a) they do not curl alfalfa root hairs, (b) they are synthesized by bacteria in the absence of known plant inducer molecules, and (c) they are produced by a mutant R. meliloti that does not synthesize known lipo-chitin oligosaccharides. The peak-D compound(s) may benefit both symbionts by increasing CO2, which is required for growth of R. meliloti, and possibly by increasing the energy that is available in the plant to form root nodules.

Journal Article↗