Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 ⁻⁷ M and 10⁻⁶ M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non-host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.     

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.     

Rhizobial lipo-oligosaccharide nodulation factors: multidimensional chromatographic analysis of symbiotic signals involved in the development of legume root nodules

Nod factors are a group of biologically active oligosaccharidesignals that are secreted by symbiotically competent bacteriaof the family Rhizobiaceae. Their biosynthesis is determinedby rhizobial nodulation (nod) genes, and is specifically inducedin response to flavonoids secreted from the roots of host leguminousplants. The biological activity of Nod factors on these hostlegumes dramatically mimics the early developmental symptomsof the Rhizobium-legame symbiosis including, amongst other effects,root hair deformations and nodule initiation. Structurally,all Nod factors are short oligomers of ß-1,4-linkedN-acetylglucos-amine residues [usually degree of polymerization(dp) 4 or 5] that are N-acylated on the distal glucosarnine.This common ‘core’ structure may be modified bya number of species-specific substituents on the distal or reducingsugars. These modifications are governed by rhizobial host specificitynod genes. The biological activity of purified Nod factors mirrorsthis host specificity, indicating that the symbiotic host rangeof individual Rhizobium species is, at least partially, determinedby the variety of Nod factors they are able to produce. Herewe describe techniques that are universally applicable to theextraction, chromatographic separation and identification ofNod factors. We have applied these techniques to Nod factorsfrom the broad-host-range species Rhizobium fredii USDA257 andRhizobium spp. NGR234, and the more narrow-host-range Bradyrhizobiumjaponicum USDA110, and have identified a group of novel, relativelyhydrophilic Nod factors from the NGR234 species that may haveimplications for Nod factor biosynthesis. lipo-oligosaccharide Nod factor rhibozobia singals TLC     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

Symbiotic conditions induce structural modifications of Sinorhizobium sp. NGR234 surface polysaccharides

When the rhizosphere is starved of nitrogen, the soil bacteria Rhizobium are able to infect legume roots and invade root nodules, where they can fix atmospheric nitrogen. Nod boxes, the nod gene promoters located on the rhizobial symbiotic plasmid, are activated by means of flavonoids present in the legume root exudates, leading to the synthesis of lipochitooligomers: the Nod factors. Several recent works pointed out the importance of rhizobial surface polysaccharides in establishing the highly specific symbiosis between rhizobia and legumes. Lipopolysaccharides (LPSs) exhibit specific active roles in the later stages of the nodulation processes, such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen-fixing phenotype. The study reported here concerns the structural modifications affecting surface (lipo)polysaccharides when Sinorhizobium sp. NGR234 strains are grown with nod gene induction under nitrogen starvation. In the absence of induction, NGR234 only produces fast-migrating LPSs. When cultured in the presence of flavonoids, the same strain produces large quantities of a high-molecular-weight rhamnose-rich lipopolysaccharide (RLPS). Because the synthesis of this compound seems to be coded by the symbiotic plasmid under direct or indirect gene induction by flavonoids, this RLPS is thought to be biologically relevant.     

Rhizobium sp. Strain NGR234 Possesses a Remarkable Number of Secretion Systems

Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.Diverse soil bacteria interact with plants in ways that range from symbiotic to pathogenic. Symbiotic Eubacteria (both alpha- and betaproteobacteria, collectively called rhizobia) form nitrogen-fixing associations of tremendous environmental importance (41, 66). Although some rhizobia are able to reduce atmospheric nitrogen to ammonia under saprophytic, free-living conditions, the reduced oxygen tensions found within the intracellular environment of specialized organs called nodules, maximizes this process (16). As legume roots penetrate the soil, they come in contact with rhizobia. Symbiotic interactions are initiated by the exchange of diverse molecules between the partners. Among them, plants liberate flavonoids into the rhizosphere that upregulate rhizobial genes. As a result, lipo-chito-oligo-saccharidic Nod factors are produced that trigger the nodulation pathway in susceptible legumes. Then, in many hosts, rhizobia enter the roots through root hairs, make their way to the cortex, multiply and fill the intracellular spaces of mature nodules. Centripetal progression of rhizobia into the plant and their maturation into nitrogen-fixing symbiosomes depends on the continued exchange of diverse signals. Many, but not all of these signals have been identified; one sure way to take stock of what is necessary for effective symbiosis is to sequence the partners. We began this work by assembling overlapping sets of cosmids (contigs) of the microsymbiont Rhizobium sp. strain NGR234 (hereafter NGR234) (63), which enabled us to elucidate the nucleotide sequence of the symbiotic (pNGR243a) plasmid (29). Similar techniques permitted the assembly of sections of the extremely large megaplasmid pNGR234b (86), and some snapshot genome information was made available earlier (91); however, the use of pyrosequencing methods greatly facilitated this process. We report here the genome sequence of NGR234 that is able to nodulate more than 120 genera of legumes and the nonlegume Parasponia andersonii (69). It seems likely that the vast richness of secretory systems might be a major key to the broad host range.     

Rhizobial Nod factors stimulate somatic embryo development in Picea abies

 Nod factors are lipochitooligosaccharides (LCOs) secreted by rhizobia. Nod factors trigger the nodulation programme in a compatible host. A bioassay was set up to test how crude (NGR234) and purified (NodS) Nod factors influence cell division and somatic embryogenesis in a conifer, Norway spruce (Picea abies). The Nod factors promoted cell division in the absence of auxin and cytokinin. More detailed studies showed that NodS stimulates development of proembryogenic masses from small cell aggregates and further embryo development. However, stimulation was only observed in low-density cell cultures. Our data suggest that rhizobial Nod factors substitute for conditioning factors in embryogenic cultures of Norway spruce. Received: 20 January 1999 / Revision received: 26 March 1999 / Accepted: 27 April 1999     

Rhizobial communication with rice roots: Induction of phenotypic changes,mode of invasion and extent of colonization

Legume-rhizobial interactions culminate in the formation of structures known as nodules. In this specialized niche, rhizobia are insulated from microbial competition and fix nitrogen which becomes directly available to the legume plant. It has been a long-standing goal in the field of biological nitrogen fixation to extend the nitrogen-fixing symbiosis to non-nodulated cereal plants, such as rice. To achieve this goal, extensive knowledge of the legume-rhizobia symbioses should help in formulating strategies for developing potential rice-rhizobia symbioses or endophytic interactions. As a first step to assess opportunities for developing a rice-rhizobia symbiosis, we evaluated certain aspects of rice-rhizobia associations to determine the extent of predisposition of rice roots for forming an intimate association with rhizobia. Our studies indicate that: a. Rice root exudates do not activate the expression of nodulation genes such as nodY of Bradyrhizobium japonicum USDA110, nodA of R. leguminosarum bv. trifolii, or nodSU of Rhizobium. sp. NGR234; b. Neither viable wild-type rhizobia, nor purified chitolipooligosaccharide (CLOS) Nod factors elicit root hair deformation or true nodule formation in rice; c. Rhizobia-produced indole-3-acetic acid, but neither trans-zeatin nor CLOS Nod factors, seem to promote the formation of thick, short lateral roots in rice; d. Rhizobia develop neither the symbiont-specific pattern of root hair attachment nor extensive cellulose microfibril production on the rice root epidermis; e. A primary mode of rhizobial invasion of rice roots is through cracks in the epidermis and fissures created during emergence of lateral roots; f. This infection process is nod-gene independent, nonspecific, and does not involve the formation of infection threads; g. Endophytic colonization observed so far is restricted to intercellular spaces or within host cells undergoing lysis. h. The cortical sclerenchymatous layer containing tightly packed, thick walled fibers appears to be a significant barrier that restricts rhizobial invasion into deeper layers of the root cortex. Therefore, we conclude that the molecular and cell biology of the Rhizobium-rice association differs in many respects from the biology underlying the development of root nodules in the Rhizobium-legume symbiosis.     

Effect of plant root symbionts on performance of native woody species in competition with an invasive grass in multispecies microcosms

The majority of terrestrial plants form mutualistic associations with arbuscular mycorrhizal fungi (AMF) and rhizobia (i.e., nitrogen‐fixing bacteria). Understanding these associations has important implications for ecological theory and for restoration practice. Here, we tested whether the presence of AMF and rhizobia influences the performance of native woody plants invaded by a non‐native grass in experimental microcosms. We planted eight plant species (i.e., Acacia acuminata, A. microbotrya, Eucalyptus loxophleba subsp. loxophleba, E. astringens, Calothamnus quadrifidus, Callistemon phoeniceus, Hakea lissocarpha and H. prostrata) in microcosms of field‐conditioned soil with and without addition of AMF and rhizobia in a fully factorial experimental design. After seedling establishment, we seeded half the microcosms with an invasive grass Bromus diandrus. We measured shoot and root biomass of native plants and Bromus, and on roots, the percentage colonization by AMF, number of rhizobia‐forming nodules and number of proteaceous root clusters. We found no effect of plant root symbionts or Bromus addition on performance of myrtaceous, and as predicted, proteaceous species as they rely little or not at all on AMF and rhizobia. Soil treatments with AMF and rhizobia had a strong positive effect (i.e., larger biomass) on native legumes (A. microbotrya and A. acuminata). However, the beneficial effect of root symbionts on legumes became negative (i.e., lower biomass and less nodules) if Bromus was present, especially for one legume, i.e., A. acuminata, suggesting a disruptive effect of the invader on the mutualism. We also found a stimulating effect of Bromus on root nodule production in A. microbotrya and AMF colonization in A. acuminata which could be indicative of legumes’ increased resource acquisition requirement, i.e., for nitrogen and phosphorus, respectively, in response to the Bromus addition. We have demonstrated the importance of measuring belowground effects because the aboveground effects gave limited indication of the effects occurring belowground.     

Symbiosis-stimulated chitinase isoenzymes of soybean (Glycine mas (L.) Merr.)

Isoforms of endochitinase in soybean were studied in relation to root symbiosis. Five selected cultivars differing in their nodulation potential were inoculated with two strains of Bradyrhizobium japonicum, the broad host-range Rhizobium sp. NGR234, and with the mycorrhizal fungus Glomus mosseae. Total chitinase activity in nodules was up to 7-fold higher than in uninoculated roots and in mycorrhizal roots. The chitinase activity in nodules varied depending on the strain-cultivar combination. On semi-native polyacrylamide gels, four acidic isoforms were identified. Two isoforms (CH 2 and CH 4) were constitutively present in al analysed tissues. The other two isoforms (CH 1 and CH 3) were strongly induced in nodules and were simulated in mycorrhizal roots as compared to uninoculated roots. The induction of CH 1 varied in nodules depending on the soybean cultivar. This isoform was also stimulated in uninfected roots when they were treated with tri-iodobenzoic acid, rhizobial lipochitooloigosaccharides (Nod factors) and chitotetraose. CH 3 was not affected by these stimuli indicating that this isoform could represent a marker for enzymes induced in later stages of the symbiotic interactions.Key words: (Brady)rhizobium, chitinase isoenzymes, mycorrhiza, (restricted) nodulation, Nod factors      

Studies on fungi in the root region

Summary Tabulated information on the colonization of roots of barley, cabbage and dwarf bean by fungi during the first 10 days of root development is given. These data, obtained by isolation and direct observation studies, are discussed in relation to previous observations on the association of fungi with the roots of healthy crop plants.The results indicate that initial root colonization may be by any of a wide range of soil fungi, but that this mixed population rapidly gives way to a stable and typical root-surface mycoflora (dominated by such fungi asFusarium spp.,Cylindrocarpon radicicola, Gliocladium spp., andPenicillium spp.     

Rhizobial lipochitooligosaccharide nodulation factors activate expression of the legume early nodulin gene ENOD12 in rice

Lipochitooligosaccharide nodulation factors (Nod factors) produced by rhizobia are a major host range determinant. These factors play a pivotal role in the molecular signal exchange, infection and induction of symbiotic developmental responses in legumes leading to the formation of a nodule in which rhizobia carry out N₂ fixation. Determining whether rice ( Oryza sativa ) can respond to Nod factors could lead to strategies that would make rice amenable to develop a nitrogen-fixing endosymbiotic association with rhizobia. We introduced into rice the promoter of the infection-related gene MtENOD12 (from Medicago truncatula ) fused to the β-glucuronidase (GUS) reporter gene to serve as a molecular marker to aid in the detection of Nod factor signal perception by rice cells. Treatment of the transgenic rice roots with Nod factors (10^–6–10^–9 m ) under nitrogen-limiting conditions induced MtENOD12 -GUS expression in cortical parenchyma, endodermis and pericycle. In contrast, chitooligosaccharide backbone alone failed to elicit such a response in the root tissues. These findings demonstrate that rice roots perceive Nod factors and that these lipochitooligosaccharides, but not simple chitin oligomers, act as signal molecules in activating MtENOD12 in cortical parenchyma as in legumes. Exogenous application of N -naphthaleneacetic acid mimicked the Nod factor-elicited tissue-specific expression of MtENOD12 in roots while cytokinins inhibited it, thus evidencing that Nod factors, auxin and cytokinins probably act on similar signaling elements responsible for the regulation of MtENOD12 activation in rice. Taken together, these results suggest that at least a portion of the signal transduction machinery important for legume nodulation is likely to exist in rice.      

Fungal root colonization responses in natural grasslands after long-term exposure to elevated atmospheric CO2

Arbuscular mycorrhizae, ubiquitous mutualistic symbioses between plant roots and fungi in the order Glomales, are believed to be important controllers of plant responses to global change, in particular to elevated atmospheric CO₂. In order to test if any effects on the symbiosis can persist after long-term treatment, we examined root colonization by arbuscular mycorrhizal (AM) and other fungi of several plant species from two grassland communities after continuous exposure to elevated atmospheric CO₂ for six growing seasons in the field. For plant species from both a sandstone and a serpentine annual grassland there was evidence for changes in fungal root colonization, with changes occurring as a function of plant host species. We documented decreases in percentage nonmycorrhizal fungal root colonization in elevated CO₂ for several plant species. Total AM root colonization (%) only increased significantly for one out of the five plant species in each grassland. However, when dividing AM fungal hyphae into two groups of hyphae (fine endophyte and coarse endophyte), we could document significant responses of AM fungi that were hidden when only total percentage colonization was measured. We also documented changes in elevated CO₂ in the percentage of root colonized by both AM hyphal types simultaneously. Our results demonstrate that changes in fungal root colonization can occur after long-term CO₂ enrichment, and that the level of resolution of the study of AM fungal responses may have to be increased to uncover significant changes to the CO₂ treatment. This study is also one of the first to document compositional changes in the AM fungi colonizing roots of plants grown in elevated CO₂. Although it is difficult to relate the structural data directly to functional changes, possible implications of the observed changes for plant communities are discussed.     

NPR1 is required for root colonization and the establishment of a mutualistic symbiosis between the beneficial bacterium Rhizobium radiobacter and barley

Non-expressor of pathogenesis-related genes 1 (NPR1) is a key regulator of plant innate immunity and systemic disease resistance. The model for NPR1 function is based on experimental evidence obtained largely from dicots; however, this model does not fit all aspects of Poaceae family, which includes major crops such as wheat, rice and barley. In addition, there is little scientific data on NPR1's role in mutualistic symbioses. We assessed barley (Hordeum vulgare) HvNPR1 requirement during the establishment of mutualistic symbiosis between barley and beneficial Alphaproteobacterium Rhizobium radiobacter F4 (RrF4). Upon RrF4 root-inoculation, barley NPR1-knockdown (KD-hvnpr1) plants lost the typical spatiotemporal colonization pattern and supported less bacterial multiplication. Following RrF4 colonization, expression of salicylic acid marker genes were strongly enhanced in wild-type roots; whereas in comparison, KD-hvnpr1 roots exhibited little to no induction. Both basal and RrF4-induced root-initiated systemic resistance against virulent Blumeria graminis were impaired in leaves of KD-hvnpr1. Besides these immune-related differences, KD-hvnpr1 plants displayed higher root and shoot biomass than WT. However, RrF4-mediated growth promotion was largely compromised in KD-hvnpr1. Our results demonstrate a critical role for HvNPR1 in establishing a mutualistic symbiosis between a beneficial bacterium and a cereal crop.     

Flavonoids repress the production of antifungal 2,4-DAPG but potentially facilitate root colonization of the rhizobacterium Pseudomonas fluorescens

In the well-known legume–rhizobia symbiosis, flavonoids released by legume roots induce expression of the Nod factors and trigger early plant responses involved in root nodulation. However, it remains largely unknown how the plant-derived flavonoids influence the physiology of non-symbiotic beneficial rhizobacteria. In this work, we demonstrated that the flavonoids apigenin and/or phloretin enhanced the swarming motility and production of cellulose and curli in Pseudomonas fluorescens 2P24, both traits of which are essential for root colonization. Using a label-free quantitative proteomics approach, we showed that apigenin and phloretin significantly reduced the biosynthesis of the antifungal metabolite 2,4-DAPG and further identified a novel flavonoid-sensing TetR regulator PhlH, which was shown to modulate 2,4-DAPG production by regulating the expression of 2,4-DAPG hydrolase PhlG. Although having similar structures, apigenin and phloretin could also influence different physiological characteristics of P. fluorescens 2P24, with apigenin decreasing the biofilm formation and phloretin inducing expression of proteins involved in the denitrification and arginine fermentation processes. Taken together, our results suggest that plant-derived flavonoids could be sensed by the TetR regulator PhlH in P. fluorescens 2P24 and acts as important signalling molecules that strengthen mutually beneficial interactions between plants and non-symbiotic beneficial rhizobacteria.     

Use of ELISA and GUS‐transformed strains to study competition between pathogenic and non‐pathogenic Fusarium oxysporum for root colonization

To characterize the ability of different strains of Fusarium oxysporum to colonize roots, and to analyze competition for root colonization between pathogenic and non‐pathogenic strains of F. oxysporum, it was necessary to develop specific labelling techniques for quantification of root colonization. Two methods were selected: the production of polyclonal antibodies, and the use of GUS‐transformed strains of F. oxysporum. The polyclonal antibodies recognized infected plants, and gave a minimum reaction with healthy plants, but were not specific for individual strains of F. oxysporum. These antibodies enabled total density of F. oxysporum to be assessed on roots, by ELISA. Metabolic activity of the root population of GUS‐marked strains was assessed by measuring the glucuronidase activity. Strains showed a diversity in their ability to colonize roots: patterns of root colonization were similar, but the intensity and the speed of colonization differed according to the plant—fungus combination used. Results demonstrated competition between the pathogenic and the non‐pathogenic strains for root colonization. In the presence of the non‐pathogenic strain Fo 47, the competition seems to be reciprocal, affecting both the pathogen and non‐pathogenic strain. Other non‐pathogenic strains reduced root colonization by the pathogenic strain, but some strains did not reduce the metabolic activity of the pathogen, suggesting that different mechanisms are involved in the interaction between pathogenic and non‐pathogenic F. oxysporum.