Autoregulation of root nodule formation: signals of both symbiotic partners studied in a split-root system of Vicia sativa subsp. nigra

Inhibition of root nodule formation on leguminous plants by already induced or existing root nodules is called autoregulation of root nodule formation (AUT). Optimal conditions for AUT were determined using a split-root technique newly developed for Vicia sativa subsp. nigra. Infection of a root A with nodulating Rhizobium leguminosarum bv. viciae bacteria systemically inhibited nodulation of a spatially separated root B inoculated 2 days later with the same bacteria. This treatment gives complete AUT (total absence of nodules on root B). Only partial AUT of root B was obtained by incubation of root A with mitogenic nodulation (Nod) factors or with a noninfective strain producing normal mitogenic Nod factors. Nonmitogenic Nod factors did not evoke AUT. We identified two systemic plant signals induced by Rhizobium bacteria. Signal 1 (at weak buffering) was correlated with sink formation in root A and induced acidification of B-root medium. This signal is induced by treatment of root A with (i) nodulating rhizobia, (ii) mitogenic Nod factors, (iii) nonmitogenic Nod factors, or (iv) the cytokinin zeatin. Signal 2 (at strong buffering) could only be evoked by treatment with nodulating rhizobia or with mitogenic Nod factors. Most probably, this signal represents the specific AUT signal. Induction of complete AUT appears to require actively dividing nodule cells in nodule primordia, nodule meristems, or both of root A.     

Salicylic acid inhibits indeterminate-type nodulation but not determinate-type nodulation

LCOs (lipochitin oligosaccharides, Nod factors) produced by the rhizobial symbiote of Vicia sativa subsp. nigra (vetch, an indeterminate-type nodulating plant) are mitogenic when carrying an 18:4 acyl chain but not when carrying an 18:1 acyl chain. This suggests that the 18:4 acyl chain specifically contributes to signaling in indeterminate-type nodulation. In a working hypothesis, we speculated that the 18:4 acyl chain is involved in oxylipin signaling comparable to, for example, signaling by derivatives of the 18:3 fatty acid linolenic acid (the octadecanoid pathway). Because salicylic acid (SA) is known to interfere with oxylipin signaling, we tested whether nodulation of vetch could be affected by addition of 10(-4) M SA. This concentration completely blocked nodulation of vetch by Rhizobium leguminosarum bv. viciae and inhibited the mitogenic effect of 18:4 LCOs but did not affect LCO-induced root-hair deformation. SA did not act systemically, and only biologically active SA derivatives were capable of inhibiting nodule formation. SA also inhibited R. leguminosarum bv. viciae association with vetch roots. In contrast, addition of SA to Lotus japonicus (a determinate-type nodulating plant responding to 18:1 LCOs) did not inhibit nodulation by Mesorhizobium loti. Other indeterminate-type nodulating plants showed the same inhibiting response toward SA, whereas SA did not inhibit the nodulation of other determinate-type nodulating plants. SA may be a useful tool for studying fundamental differences between signal transduction pathways of indeterminate- and determinate-type nodulating plants.     

Differential response of soybean (Glycine max (L.) Merr.) genotypes to lipo-chito-oligosaccharide Nod Bj V (C(18:1) MeFuc)

Lipo-chito-oligosaccharides (LCOs) are bacteria-to-plant signal molecules essential for the establishment of rhizobia-legume symbioses. LCOs invoke a number of physiological changes in the host plants, such as root hair deformation, cortical cell division and ontogeny of complete nodule structures. The responses of five soybean cultivars to Nod BJ: V (C(18:1) MeFuc) isolated from Bradyrhizobium japonicum strain 532C were studied with a new technique. Two distinct types of root hair deformation were evident (i) bulging, in which root hairs were swollen at the tip or at the base depending on the cultivars and (ii) curling. The nodulating capacity of B. japonicum 532C varied among cultivars. Cultivars that produced a bulging reaction when treated with LCO had fewer nodules and the roots had low phenol contents. Cultivars that produced curling had higher numbers of nodules and the roots had higher amounts of phenol. Further, the roots of cultivars that showed root hair bulging were able to degrade LCO much faster than cultivars that manifested curling. The results of the present study establish relationships among the type of LCO-induced root hair deformation, root system LCO-degrading ability and nodulation capacity of soybean cultivars.     

Regulation of plant morphogenesis by Lipo‐Chitin oligosaccharides

Lipo‐chitin oligosaccharides (LCOs), produced by rhizobia, are causative agents of the formation of root nodules in leguminous plants. As outlined in this review, the root nodulation process presents a valuable model system to study plant morphogenesis. The knowledge that resulted from the studies of the biological function and biosynthesis of the rhizobial LCOs is summarized. It has been postulated that LCOs are representatives of a general class of signal molecules involved in plant and animal morphogenesis. Discussed is how the present knowledge can be used for future studies on the function of LCOs in morphogenesis and in the search for analogue signal molecules produced by plants and animals.     

Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum

In a previous work, we showed that non-nodulating agrobacteria strains were able to colonize root nodules of common bean. Both rhizobia and agrobacteria co-existed in the infected nodules. No impact on symbiosis was found in laboratory conditions when using sterile gravel as a support for growth. In this study, soil samples originating from different geographic and agronomic regions in Tunisia were inoculated with a mixture of agrobacteria strains isolated previously from root nodules of common bean. A significant effect on nodulation and vegetal growth of common bean was observed. Characterization of nodulating rhizobia and comparison with non-inoculated controls showed a biased genetic structure. It seemed that Rhizobium gallicum was highly inhibited, whereas nodulation by Sinorhizobium medicae was favored. Co-inoculation of non-sterile soils with R. gallicum and agrobacteria confirmed these findings. In vitro antibiosis assays indicated that agrobacteria exercised a significant antagonism against R. gallicum.     

Nod factor-induced root hair curling: continuous polar growth towards the point of nod factor application

A critical step in establishing a successful nitrogen-fixing symbiosis between rhizobia and legume plants is the entrapment of the bacteria between root hair cell walls, usually in characteristic 180 degrees to 360 degrees curls, shepherd's crooks, which are formed by the host's root hairs. Purified bacterial signal molecules, the nodulation factors (NFs), which are lipochitooligosaccharides, induce root hair deformation in the appropriate host legume and have been proposed to be a key player in eliciting root hair curling. However, for curling to occur, the presence of intact bacteria is thought to be essential. Here, we show that, when spot applied to one side of the growing Medicago truncatula root hair tip, purified NF alone is sufficient to induce reorientation of the root hair growth direction, or a full curl. Using wild-type M. truncatula containing the pMtENOD11::GUS construct, we demonstrate that MtENOD11::GUS is expressed after spot application. The data have been incorporated into a cell biological model, which explains the formation of shepherd's crook curls around NF-secreting rhizobia by continuous tip growth reorientation.     

Pre-infection events in non-nodulating species of African Acacia

Non-nodulation occurs sporadically throughout the family Leguminosae, the genus Acacia is unusual as there are a range of species which are consistently non-nodulating, but which are closely related to species able to form nodules. A comparative study of these and coexisting related nodulating species has been made to establish whether the non-nodulating species exhibited any pre-infection characteristics which are typical of their nodulating counterparts. The non-nodulating species did not form any nodule-like structures, but exhibited pre-infection characteristics such as rhizobial attachment similar to that of their nodulating counterparts. The root exudate of nodulating A. polyacantha contained stimulatory compounds which aided binding of rhizobia to the roots of the non-nodulating species and nodulation was completely inhibited when A. polyacantha was co-cultured with a non-nodulating species.     

Signaling and Gene Expression for Water-Tolerant Legume Nodulation

In the symbiotic interaction with rhizobia, legumes develop nodules in which nitrogen fixation takes place. Upon submersion, most temperate legumes are incapable of nodulation, but tropical legumes that grow in waterlogged soils have acquired water stress tolerance for growth and nodulation. One well-studied model plant, the tropical, semi-aquatic Sesbania rostrata, develops stem-located adventitious root primordia that grow out into adventitious roots upon submergence and develop into stem nodules after inoculation with the microsymbiont, Azorhizobium caulinodans. Sesbania rostrata also has a nodulated underground root system. On well-aerated roots, nodules form via root hair curling infection in the zone, just above the root tip, where root hairs develop; on hydroponic roots, an alternative process is used, recruiting a cortical intercellular invasion program at the lateral root bases that skips the epidermal responses. This intercellular cortical invasion entails infection pocket formation, a process that involves cell death features and reactive oxygen species. The plant hormones ethylene and gibberellin are the major signals that act downstream from the bacterial nodulation factors in the nodulation and invasion program. Both hormones block root hair curling infection, but cooperate to stimulate lateral root base invasion and play a role in infection thread formation, meristem establishment, and differentiation of meristem descendants.     

Acetylation of a fucosyl residue at the reducing end of Mesorhizobium loti nod factors is not essential for nodulation of Lotus japonicus

NodMl-V(C(18:1), Me, Cb, AcFuc) is a major component of lipo-chitin oligosaccharides (LCOs), or Nod factors, produced by Mesorhizobium loti. The presence of a 4-O-acetylated fucosyl residue (AcFuc) at the reducing end has been thought to be essential for symbiotic interactions with the compatible host plant, Lotus japonicus. We generated an M. loti mutant in which the nolL gene is disrupted; nolL has been shown to encode acetyltransferase that is responsible for acetylation of the fucosyl residue. The nolL disruptant Ml107 produced LCOs that lacked acetylation of fucosyl residues as expected, but exhibited nodulation performance on L. japonicus as efficiently as the wild-type M. loti strain MAFF303099. We show that LCOs without acetylation of a fucosyl residue purified from Ml107 are also able to induce abundant root hair deformation and nodule primordium formation. These results indicate that NolL-dependent acetylation of a fucosyl residue at the reducing end of M. loti LCOs is not essential for nodulation of L. japonicus.     

Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi

The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod(-) and Myc(-) co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod(-) mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod(-) and Myc(-). In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.     

SPIKE1 Activates the GTPase ROP6 to Guide the Polarized Growth of Infection Threads in Lotus japonicus

In legumes, rhizobia attach to root hair tips and secrete nodulation factor to activate rhizobial infection and nodule organogenesis. Endosymbiotic rhizobia enter nodule primordia via a specialized transcellular compartment known as the infection thread (IT). The IT elongates by polar tip growth, following the path of the migrating nucleus along and within the root hair cell. Rho-family ROP GTPases are known to regulate the polarized growth of cells, but their role in regulating polarized IT growth is poorly understood. Here, we show that LjSPK1, a DOCK family guanine nucleotide exchange factor (GEF), interacts with three type I ROP GTPases. Genetic analyses showed that these three ROP GTPases are involved in root hair development, but only LjROP6 is required for IT formation after rhizobia inoculation. Misdirected ITs formed in the root hairs of Ljspk1 and Ljrop6 mutants. We show that LjSPK1 functions as a GEF that activates LjROP6. LjROP6 enhanced the plasma membrane localization LjSPK1 in Nicotiana benthamiana leaf cells and Lotus japonicus root hairs, and LjSPK1 and LjROP6 interact at the plasma membrane. Taken together, these results shed light on how the LjROP6-LjSPK1 module mediates the polarized growth of ITs in L. japonicus.     

Transient susceptibility of root cells in four common legumes to nodulation by rhizobia

Root cells of four common legumes were found to remain susceptible to nodulation by rhizobia for only a short period of time. Delayed inoculation experiments conducted with these legume hosts indicated that the initially susceptible region of the root became progressively less susceptible if inoculations were delayed by a few hours. Profiles of the frequency of nodule formation relative to marks indicating the regions of root and root hair development at the time of inoculation indicated that nodulation of Vigna sinensis (L.) Endl. cv California Black Eye and Medicago sativa L. cvs Moapa and Vernal roots was inhibited just below the region that was most susceptible at the time of inoculation. This result suggests the existence of a fast-acting regulatory mechanism in these hosts that prevents overnodulation. Nodulation in white clover may occur in two distinct phases. In addition to the transient susceptibility of preemergent and developing root hair cells, there appeared to be an induced susceptibility of mature clover root hair cells. A cell-free bacterial exudate preparation from Rhizobium trifolii cells was found to render mature root hair cells of white clover more rapidly susceptible to nodulation.     

Formation of Tumor-Like Structures on Legume Roots by Rhizobium

Tumor-like structures appeared on the roots of Medicago sativa, Alysicarpus vaginalis, and Trifolium pratense inoculated with a non-nodulating strain of Rhizobium trifolii or with irradiated cultures of either of two nodulating Rhizobium strains. The structures were composed of disorganized plant tissues which, on the basis of microscopic examination, were devoid of bacterial cells. Rhizobia which could nodulate legumes of one cross-inoculation group and which were able to induce formation of such tumor-like structures on plants of a second cross-inoculation group were isolated from extracts of these root growths. The apparent tumorogenic activity of some of the rhizobia, but not their nodulating capacity, was lost when the bacteria were transferred in laboratory media.     

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.     

Identical accumulation and immobilization of sulfated and nonsulfated Nod factors in host and nonhost root hair cell walls

Nod factors are signaling molecules secreted by Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can elicit specific responses at subnanomolar concentrations on a compatible host. How plants perceive LCOs is unclear. In this study, using fluorescent Nod factor analogs, we investigated whether sulfated and nonsulfated Nod factors were bound and perceived differently by Medicago truncatula and Vicia sativa root hairs. The bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair deformation assay on M. truncatula, showing bioactivity down to 0.1 to 1 nM. Fluorescence microscopy of plasmolyzed M. truncatula root hairs shows that sulfated fluorescent Nod factors accumulate in the cell wall of root hairs, whereas they are absent from the plasma membrane when applied at 10 nM. When the fluorescent Nod factor distribution in medium surrounding a root was studied, a sharp decrease in fluorescence close to the root hairs was observed, visualizing the remarkable capacity of root hairs to absorb Nod factors from the medium. Fluorescence correlation microscopy was used to study in detail the mobilities of sulfated and nonsulfated fluorescent Nod factors which are biologically active on M. truncatula and V. sativa, respectively. Remarkably, no difference between sulfated and nonsulfated Nod factors was observed: both hardly diffuse and strongly accumulate in root hair cell walls of both M. truncatula and V. sativa. The implications for the mode of Nod factor perception are discussed.     

Molecular mechanisms of Nod factor diversity

The rhizobia–legume symbiosis is highly specific. Major host specificity determinants are the bacterial Nod factor signals that trigger the nodulation programme in a compatible host. Nod factors are lipo-chitooligosaccharides (LCOs) varying in the oligosaccharide chain length, the nature of the fatty acids and substitutions on the oligosaccharide. The nod genotype of rhizobia, which forms the genetic basis for this structural variety, includes a set of nodulation genes encoding the enzymes that synthesize LCOs. Allelic and non-allelic variation in these genes ensures the synthesis of different LCO structures by the different rhizobia. The nod genotypes co-evolved with host plant divergence in contrast to the rhizobia, which followed a different evolution. Horizontal gene transfer probably played an important role during evolution of symbiosis. The nod genotypes are particularly well equipped for horizontal gene transfer because of their location on transmissible plasmids and/or on 'symbiosis islands', which are symbiotic regions associated with movable elements.     

Correlation between extracellular fibrils and attachment of Rhizobium leguminosarum to pea root hair tips.

As part of a project meant to characterize molecules involved in nodulation, a semiquantitative microscopic assay was developed for measuring attachment of Rhizobium leguminosarum cells to pea root hair tips, i.e., the site at which R. leguminosarum initiates nodulation. This form of attachment, designated as cap formation, was dependent on the incubation pH and growth phase, with optimal attachment at pH 7.5 and with bacteria in the early stationary phase of growth. Addition of glucose to the growth medium delayed the initiation of the stationary phase and cap formation, suggesting a correlation between cap formation and carbon limitation. Attachment of R. leguminosarum was not inhibited by pea lectin haptens which makes it unlikely that lectins are involved under the tested conditions. Moreover, heterologous fast-growing rhizobia adhered equally well to pea root hair tips. Since the attachment characteristics of a Sym plasmid-cured derivative were indistinguishable from those of the wild-type strain, the Sym plasmidborne nodulation genes are not necessary for attachment. Sodium chloride and various other salts abolished attachment when present during the attachment assay in final concentrations of 100 mM. R. leguminosarum produced extracellular fibrils. A positive correlation between the percentage of fibrillated cells and the ability of the bacteria to form caps and to adhere to glass and erythrocytes was observed under various conditions, suggesting that these fibrils play a role in attachment of the bacteria to pea root hair tips, to glass, and to erythrocytes.     

Microscopic analysis of the effect ofRhizobium leguminosarum biovartrifolii host specific nodulation genes in the infection of white clovers

Summary A microscopic assessment is presented of the comparative infection capacity of wild-type and hybrid strains ofRhizobium leguminosarum bv.viciae withR. l. bv.trifolii strain ANU 843 on white clover seedlings. TheR. l. bv.viciae hybrid strains contained defined DNA segments coding for different combinations ofR. l. bv.trifolii host-specific nodulation genes. White clover plants were examined over a 72 h period to assessRhizobium infectivity, the morphological changes in root hair growth; colonisation ability of rhizobia; infection thread initiation and the ability to induce cortical cell division.R. l. bv.viciae strain 300 induced root hair curling more slowly than strain ANU 843 or any of the hybrid strain 300 bacteria, and when curling had taken place, there was poorer colonization by strain 300 within the folded hair cell, no evidence of infection thread formation and only limited cortical cell division 72 h after inoculation. The addition of the host-specific nodulation genes ofR. l. bv.trifolii to strain 300 was necessary to induce infection threads and establish a normal pattern of nodulation of the roots of white clovers.     

Lipochitooligosaccharides and legume Rhizobium symbiosis--a new concept

Legume-Rhizobium symbiosis is a multistep process characterized by the formation of root nodules on the host plant. A number of genes from both symbiotic partners share information during the interaction process. Nodulation genes (nod, nol and noe) have been classified as common nodulation genes and host specific (hsn) nodulation genes. Though common nodulation genes are enough to form root nodules, host specific nodulation genes are needed for specific interaction leading to formation of functional nodules. Core lipochitooligosaccharides (LCOs), the products of common nodulation genes are modified by the action of host specific nodulation genes. LCOs seem to be present in legumes as well as nonlegume and are known to act as a morphogen by acting as auxin-transport inhibitor. The understanding of Nod factor may contribute to reveal complex biological functions such as developmental regulation, signal transduction and plant morphogenesis.     

Manipulation of Ethylene Synthesis in Roots Through Bacterial ACC Deaminase for Improving Nodulation in Legumes

Symbiotic association between rhizobia and legumes results in the development of unique structures on roots, called nodules. Nodulation is a very complex process involving a variety of genes that control NOD factors (bacterial signaling molecules), which are essential for the establishment, maintenance and regulation of this process and development of root nodules. Ethylene is an established potent plant hormone that is also known for its negative role in nodulation. Ethylene is produced endogenously in all plant tissues, particularly in response to both biotic and abiotic stresses. Exogenous application of ethylene and ethylene-releasing compounds are known to inhibit the formation and functioning of nodules. While inhibitors of ethylene synthesis or its physiological action enhance nodulation in legumes, some rhizobial strains also nodulate the host plant intensively, most likely by lowering endogenous ethylene levels in roots through their 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Co-inoculation with ACC deaminase containing plant growth promoting rhizobacteria plus rhizobia has been shown to further promote nodulation compared to rhizobia alone. Transgenic rhizobia or legume plants with expression of bacterial ACC deaminase could be another viable option to alleviate the negative effects of ethylene on nodulation. Several studies have well documented the role of ethylene and bacterial ACC deaminase in development of nodules on legume roots and will be the primary focus of this critical review.