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.     

Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation.

Rhizobia induce the formation of root nodules on the roots of leguminous plants. In temperate legumes, nodule organogenesis starts with the induction of cell divisions in regions of the root inner cortex opposite protoxylem poles, resulting in the formation of nodule primordia. It has been postulated that the susceptibility of these inner cortical cells to Rhizobium nodulation (Nod) factors is conferred by an arrest at a specific stage of the cell cycle. Concomitantly with the formation of nodule primordia, cytoplasmic rearrangement occurs in the outer cortex. Radially aligned cytoplasmic strands form bridges, and these have been called preinfection threads. It has been proposed that the cytoplasmic bridges are related to phragmosomes. By studying the in situ expression of the cell cycle genes cyc2, H4, and cdc2 in pea and alfalfa root cortical cells after inoculation with Rhizobium or purified Nod factors, we show that the susceptibility of inner cortical cells to Rhizobium is not conferred by an arrest at the G2 phase and that the majority of the dividing cells are arrested at the G0/G1 phase. Furthermore, the outer cortical cells forming a preinfection thread enter the cell cycle although they do not divide.     

Effects of endogenous salicylic acid on nodulation in the model legumes Lotus japonicus and Medicago truncatula

The exogenous addition of salicylic acid (SA) was previously shown to inhibit indeterminate but not determinate-type nodulation. We sought to extend these results by modulating endogenous levels of SA through the transgenic expression of salicylate hydroxylase (NahG) in both stably transformed Lotus japonicus and composite Medicago truncatula plants. NahG expression in L. japonicus resulted in a marked reduction of SA levels. This reduction correlated with an increase in the number of infections and mean nodule number when compared to controls. However, a complicating factor was that NahG-expressing plants had greater root growth. Spot inoculations of NahG-expressing L. japonicus plants confirmed increased nodulation in these plants. Consistent with the reported inhibitory effects of exogenous SA on indeterminate-type nodulation, NahG expression in M. truncatula plants led to enhanced nodulation and infection. These data point to an important role for SA-mediated plant defense pathways in controlling nodule formation on both determinate and indeterminate nodule-forming hosts.     

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.     

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.     

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.     

Symbiotic performance of common bean and soybean co-inoculated with rhizobia and <Emphasis Type="Italic">Chryseobacterium balustinum</Emphasis> Aur9 under moderate saline conditions

The effect of co-inoculating beans and soybeans with rhizobia and Chryseobacterium, a plant growth promoting bacteria (PGPR), was studied under conditions of mild saline stress. Chryseobacterium balustinum Aur9 was used with Rhizobium tropici CIAT899 or R. etli ISP42 to inoculate common bean (Phaseolus vulgaris L.), or jointly with Ensifer (Sinorhizobium) fredii SMH12 and HH103 to inoculate soybean (Glycine max (L.) Merrill). The effect of co-inoculation was studied by following nodule primordia initiation, nodulation kinetics and symbiotic performance in plants grown under moderate saline conditions (25 mM NaCl). In common bean, co-inoculation improved nodule primordia formation when compared with single inoculation (R. tropici CIAT899). However, co-inoculation did not provide benefits in the development of nodule primordia in soybean with E. fredii SMH12. The kinetic of nodulation in bean was also favored by double inocula resulting in a higher number of nodules. Long-term effects of co-inoculation on beans and soybeans depended on the rhizobial species used. In both, control and saline conditions, co-inoculation of R. tropici CIAT899 and C. balustinum Aur9 improved bean growth when compared with the single inoculation (CIAT899). However, the positive effect of double inocula on plant growth did not occur when using R. etli ISP42. Soybean plants receiving double inoculation (E. fredii SMH12 and C. balustinum Aur9) showed better symbiotic performance, mostly under saline stress, than with a single inoculation. The results indicate that co-inoculation with C. balustinum and rhizobia under mild saline conditions partially relieves the salt-stress effects, although do not always result advantageous for symbiotic N₂ fixation in legume plants.     

Effects of ethylene and inhibitors of ethylene synthesis and action on nodulation in common bean (Phaseolus vulgaris L.)

The ethylene releasing compound, 2-chloroethylphosphonic acid (ethephon) inhibited nodule development in common bean (Phaseolus vulgaris L.) plants. In contrast, inhibitors of ethylene synthesis or its physiological activity enhanced nodulation. In a co-culture of bean seeds and rhizobia, ethephon inhibited rhizobial growth while inhibitors of ethylene synthesis or action did not influence the growth and proliferation of rhizobia. These data emphasize the role of ethylene as a regulator of nodulation in determinate nodulators and indicate that the ethylene signaling pathway involved in the nodulation process is not limited to the plant host but also involves the bacterial symbiont.     

Grafting analysis indicates that malfunction of TRICOT in the root causes a nodulation-deficient phenotype in Lotus japonicus

Leguminous plants develop root nodules in symbiosis with soil rhizobia. Nodule formation occurs following rhizobial infection of the host root that induces dedifferentiation of some cortical cells and the initiation of a new developmental program to form nodule primordia. In a recent study, we identified a novel gene, TRICOT (TCO), that acts as a positive regulator of nodulation in Lotus japonicus. In addition to its role in nodulation, tco mutant plants display pleiotropic defects including abnormal shoot apical meristem formation. Here, we investigated the effect of the tco mutation on nodulation using a grafting approach. The results strongly indicate that the nodulation-deficient phenotype of the mutant results from malfunction of the TCO gene in the root.     

Two effects of combined nitrogen on the adhesion of Rhizobium etli to bean roots

Combined forms of nitrogen negatively influence rhizobia-legume symbiosis. The effects of combined nitrogen are known for nodulation and dinitrogen (N₂) fixation, but little is known about the effect on preinfection events. Here, we studied the effects of combined nitrogen on the adhesion of Rhizobium etli to common bean (Phaseolus vulgaris L.) roots. When potassium nitrate (KNO₃) or sodium glutamate was added to an incubation mixture of rhizobia and plants that were previously grown in nitrogen-free solution, rhizobial adhesion to roots was stimulated. However, the rhizobial adhesion to bean roots that were previously grown with 10 mM KNO₃ was reduced by half. A fraction of the bean root exudates, which is thermolabile and has molecular mass larger than 12 kDa stimulated rhizobial adhesion, but this stimulatory activity was lost in root exudates obtained with 10 mM KNO₃. Thus, the inhibition of symbiosis in response to combined nitrogen may be controlled by the plant at the preinfection stage as well.     

Expression of LjENOD40 genes in response to symbiotic and non-symbiotic signals: LjENOD40-1 and LjENOD40-2 are differentially regulated in Lotus japonicus

Nitrogen fixation in nodules provides leguminous plants with an ability to grow in nitrogen-starved soil. Infection of the host plants by microsymbionts triggers various physiological and morphological changes during nodule formation. In Lotus japonicus, expression of early nodulin (ENOD) genes is triggered by perception of bacterial signal molecules, nodulation factors (Nod factors). We examined the expression patterns of ENOD40 genes during the nodule formation process. Two ENOD40 genes of L. japonicus were specifically expressed in the nodule formation process, but they showed different expression patterns upon infection. Each ENOD40 gene demonstrates an individual specificity and regulation with regard to rhizobial infection.     

A Small GTPase of the Rab Family Is Required for Root Hair Formation and Preinfection Stages of the Common Bean–Rhizobium Symbiotic Association

Legume plants are able to establish a symbiotic relationship with soil bacteria from the genus Rhizobium, leading to the formation of nitrogen-fixing root nodules. Successful nodulation requires both the formation of infection threads (ITs) in the root epidermis and the activation of cell division in the cortex to form the nodule primordium. This study describes the characterization of RabA2, a common bean (Phaseolus vulgaris) cDNA previously isolated as differentially expressed in root hairs infected with Rhizobium etli, which encodes a protein highly similar to small GTPases of the RabA2 subfamily. This gene is expressed in roots, particularly in root hairs, where the protein was found to be associated with vesicles that move along the cell. The role of this gene during nodulation has been studied in common bean transgenic roots using a reverse genetic approach. Examination of root morphology in RabA2 RNA interference (RNAi) plants revealed that the number and length of the root hairs were severely reduced in these plants. Upon inoculation with R. etli, nodulation was completely impaired and no induction of early nodulation genes (ENODs), such as ERN1, ENOD40, and Hap5, was detected in silenced hairy roots. Moreover, RabA2 RNAi plants failed to induce root hair deformation and to initiate ITs, indicating that morphological changes that precede bacterial infection are compromised in these plants. We propose that RabA2 acts in polar growth of root hairs and is required for reorientation of the root hair growth axis during bacterial infection.     

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.     

Heterologous rhizobial lipochitin oligosaccharides and chitin oligomers induce cortical cell divisions in red clover roots, transformed with the pea lectin gene

Division of cortical cells in roots of leguminous plants is triggered by lipochitin oligosaccharides (LCOs) secreted by the rhizobial microsymbiont. Previously, we have shown that presence of pea lectin in transgenic white clover hairy roots renders these roots susceptible to induction of root nodule formation by pea-specific rhizobia (C. L. Díaz, L. S. Melchers, P. J. J. Hooykaas, B. J. J. Lugtenberg, and J. W. Kijne, Nature 338:579-581, 1989). Here, we report that pea lectin-transformed red clover hairy roots form nodule primordium-like structures after inoculation with pea-, alfalfa-, and Lotus-specific rhizobia, which normally do not nodulate red clover. External application of a broad range of purified LCOs showed all of them to be active in induction of cortical cell divisions and cell expansion in a radial direction, resulting in formation of structures that resemble nodule primordia induced by clover-specific rhizobia. This activity was obvious in about 50% of the red clover plants carrying hairy roots transformed with the pea lectin gene. Also, chitopentaose, chitotetraose, chitotriose, and chitobiose were able to induce cortical cell divisions and cell expansion in a radial direction in transgenic roots, but not in control roots. Sugar-binding activity of pea lectin was essential for its effect. These results show that transformation of red clover roots with pea lectin results in a broadened response of legume root cortical cells to externally applied potentially mitogenic oligochitin signals.     

Reactions of Lotus japonicus ecotypes and mutants to root parasitic plants

Witchweeds (Striga spp.) and broomrapes (Orobanche spp.) are obligate root parasitic plants on economically important field and horticultural crops. The parasites' seeds are induced to germinate by root-derived chemical signals. The radicular end is transformed into a haustorium which attaches, penetrates the host root and establishes connection with the vascular system of the host. Reactions of Lotus japonicus, a model legume for functional genomics, were studied for furthering the understanding of host-parasite interactions. Lotus japonicus was compatible with Orobanche aegyptiaca, but not with Orobanche minor, Striga hermonthica and Striga gesnerioides. Orobanche minor successfully penetrated Lotus japonicus roots, but failed to establish connections with the vascular system. Haustoria in Striga hermonthica attached to the roots, but penetration and subsequent growth of the endophyte in the cortex were restricted. Striga gesnerioides did not parasitize Lotus japonicus. Among seven mutants of Lotus japonicus (castor-5, har1-5, alb1-1, ccamk-3, nup85-3, nfr1-3 and nsp2-1) with altered characteristics in relation to rhizobial nodulation and mycorrhizal colonization, castor-5 and har1-5 were parasitized by Orobanche aegyptiaca with higher frequency than the wild type. In contrast, Orobanche aegyptiaca tubercle development was delayed on the mutants nup85-3, nfr1-3 and nsp2-1. These results suggest that nodulation, mycorrhizal colonization and infection by root parasitic plants in Lotus japonicus may be modulated by similar mechanisms and that Lotus japonicus is a potential model legume for studying plant-plant parasitism.     

Stem Nodulation in Legumes: Diversity,Mechanisms, and Unusual Characteristics

Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the roots. A few aquatic legumes, however, can form nodules on their stem at dormant root primordia. The stem-nodulating legumes described so far are all members of the genera Aeschynomene, Sesbania, Neptunia, and Discolobium. Their rhizobial symbionts belong to four genera already described: Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium. This review summarizes our current knowledge on most aspects of stem nodulation in legumes, the infection process and nodule development, the characterization and unusual features of the associated bacteria, and the molecular genetics of nodulation. Potential use as green manure in lowland rice of these stem-nodulating legumes, giving them agronomical importance, is also discussed.