Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

Identification and characterization of KpsS, a novel polysaccharide sulphotransferase in Mesorhizobium loti

Plants enter into symbiotic relationships with bacteria that allow survival in nutrient-limiting environments. The bacterium Mesorhizobium loti enters into a symbiosis with the legume host, Lotus japonicus, which results in the formation of novel plant structures called root nodules. The bacteria colonize the nodules, and are internalized into the cytoplasm of the plant cells, where they reduce molecular dinitrogen for the plant. Symbiosis between M. loti and L. japonicus requires bacterial synthesis of secreted and cell-surface polysaccharides. We previously reported the identification of an unusual sulphate-modified form of capsular polysaccharide (KPS) in M. loti. To better understand the physiological function of sulphated KPS, we isolated the sulphotransferase responsible for KPS sulphation from M. loti extracts, determined its amino acid sequence and identified the corresponding M. loti open reading frame, mll7563 (which we have named kpsS). We demonstrated that partially purified KpsS functions as a fucosyl sulphotransferase in vitro. Furthermore, mutants deficient for this gene exhibit a lack of KPS sulphation and a decreased rate of nodule formation on L. japonicus. Interestingly, the kpsS gene product shares no significant amino acid similarity with previously identified sulphotransferases, but exhibited sequence identity to open reading frames of unknown function in diverse bacteria that interact with eukaryotes.     

A novel fix- symbiotic mutant of Lotus japonicus, Ljsym105, shows impaired development and premature deterioration of nodule infected cells and symbiosomes

Nitrogen-fixing symbiosis between legume plants and rhizobia is established through complex interactions between two symbiotic partners. To identify the host legume genes that play crucial roles in such interactions, we isolated a novel Fix- mutant, Ljsym105, from a model legume Lotus japonicus MG-20. The Ljsym105 plants displayed nitrogen-deficiency symptoms after inoculation with Mesorhizobium loti under nitrogen-free conditions, but their growth recovered when supplied with nitrogen-rich nutrients. Ljsym105 was recessive and monogenic and mapped on the upper portion of chromosome 4. The mutant Ljsym105 formed an increased number of small and pale-pink nodules. Nitrogenase (acetylene reduction) activity per nodule fresh weight was low but retained more than 50% of that of the wild-type nodules. Light and electron microscopic observations revealed that the Ljsym105 nodule infected cells were significantly smaller than those of wild-type plants, contained enlarged symbiosomes with multiple bacteroids, and underwent deterioration of the symbiosomes prematurely as well as disintegration of the whole infected cell cytoplasm. These results indicate that the ineffectiveness of the Ljsym105 nodules is primarily due to impaired growth of infected cells accompanied with the premature senescence induced at relatively early stages of nodule development. These symbiotic phenotypes are discussed in respect to possible functions of the LjSym105 locus in the symbiotic interactions required for establishment of the nitrogen-fixing symbiosis.     

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.     

Interplay of flg22-induced defence responses and nodulation in Lotus japonicus

In this study the interplay between the symbiotic and defence signalling pathways in Lotus japonicus was investigated by comparing the responses to Mesorhizobium loti, the symbiotic partner of L. japonicus, and the elicitor flg22, a conserved peptide motif present in flagellar protein of a wide range of bacteria. It was found that defence and symbiotic pathways overlap in the interaction between L. japonicus and M. loti since similar responses were induced by the mutualistic bacteria and flg22. However, purified flagellin from M. loti did not induce any response in L. japonicus, which suggests the production of other elicitors by the symbiotic bacteria. Defence responses induced by flg22 caused inhibition of rhizobial infection and delay in nodule organogenesis, as demonstrated by the negative effect of flg22 in the formation of spontaneous nodules in the snf1 L. japonicus mutant, and the inhibition of NSP1 and NSP2 genes. This indicates the antagonistic effect of the defence pathway on the nodule formation in the initial rhizobium-legume interaction. However, the fact that flg22 did not affect the formation of new nodules once the symbiosis was established indicates that after the colonization of the host plant by the symbiotic partner, the symbiotic pathway has prevalence over the defensive response. This result is also supported by the down-regulation of the expression levels of the flg22 receptor FLS2 in the nodular tissue.     

crinkle,a novel symbiotic mutant that affects the infection thread growth and alters the root hair,trichome, and seed development in Lotus japonicus

To elucidate the mechanisms involved in Rhizobium-legume symbiosis, we examined a novel symbiotic mutant, crinkle (Ljsym79), from the model legume Lotus japonicus. On nitrogen-starved medium, crinkle mutants inoculated with the symbiont bacterium Mesorhizobium loti MAFF 303099 showed severe nitrogen deficiency symptoms. This mutant was characterized by the production of many bumps and small, white, uninfected nodule-like structures. Few nodules were pale-pink and irregularly shaped with nitrogen-fixing bacteroids and expressing leghemoglobin mRNA. Morphological analysis of infected roots showed that nodulation in crinkle mutants is blocked at the stage of the infection process. Confocal microscopy and histological examination of crinkle nodules revealed that infection threads were arrested upon penetrating the epidermal cells. Starch accumulation in uninfected cells and undeveloped vascular bundles were also noted in crinkle nodules. Results suggest that the Crinkle gene controls the infection process that is crucial during the early stage of nodule organogenesis. Aside from the symbiotic phenotypes, crinkle mutants also developed morphological alterations, such as crinkly or wavy trichomes, short seedpods with aborted embryos, and swollen root hairs. crinkle is therefore required for symbiotic nodule development and for other aspects of plant development.     

Genetic suppressors of the Lotus japonicus har1-1 hypernodulation phenotype

Lotus japonicus har1 mutants respond to inoculation with Mesorhizobium loti by forming an excessive number of nodules due to genetic lesions in the HAR1 autoregulatory receptor kinase gene. In order to expand the repertoire of mutants available for the genetic dissection of the root nodule symbiosis (RNS), a screen for suppressors of the L. japonicus har1-1 hypernodulation phenotype was performed. Of 150,000 M2 plants analyzed, 61 stable L. japonicus double-mutant lines were isolated. In the context of the har1-1 mutation, 26 mutant lines were unable to form RNS, whereas the remaining 35 mutant lines carried more subtle symbiotic phenotypes, either forming white ineffective nodules or showing reduced nodulation capacity. When challenged with Glomus intraradices, 18 of the 61 suppressor lines were unable to establish a symbiosis with this arbuscular mycorrhiza fungus. Using a combined approach of genetic mapping, targeting induced local lesions in genomics, and sequencing, all non-nodulating mutant lines were characterized and shown to represent new alleles of at least nine independent symbiotic loci. The class of mutants with reduced nodulation capacity was of particular interest because some of them may specify novel plant functions that regulate nodule development in L. japonicus. To facilitate mapping of the latter class of mutants, an introgression line, in which the har1-1 allele was introduced into a polymorphic background of L. japonicus ecotype MG20, was constructed.     

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.     

A Sinorhizobium meliloti lipopolysaccharide mutant altered in cell surface sulfation

The Rhizobium-legume symbiosis involves the formation of a novel plant organ, the nodule, in which intracellular bacteria reduce molecular dinitrogen in exchange for plant photosynthates. Nodule development requires a bacterial signal referred to as Nod factor, which in Sinorhizobium meliloti is a beta-(1,4)-linked tetramer of N-acetylglucosamine containing N-acyl and O-acetyl modifications at the nonreducing end and a critical 6-O-sulfate at the reducing end. This sulfate modification requires the action of three gene products: nodH, which catalyzes the sulfonyl transfer, and nodPQ, which produce the activated form of sulfate, 3'-phosphoadenosine-5'-phosphosulfate. It was previously reported that S. meliloti cell surface polysaccharides are also covalently modified by sulfate in a reaction dependent on NodPQ. We have further characterized this unique form of bacterial carbohydrate modification. Our studies have determined that one of the nodPQ mutant strains used in the initial study of sulfation of cell surface harbored a second unlinked mutation. We cloned the gene affected by this mutation (referred to as lps-212) and found it to be an allele of lpsL, a gene previously predicted to encode a UDP-glucuronic acid epimerase. We demonstrated that lpsL encoded a UDP-glucuronic acid epimerase activity that was reduced in the lps-212 mutant. The lps-212 mutation resulted in an altered lipopolysaccharide structure that was reduced in sulfate modification in vitro and in vivo. Finally, we determined that the lps-212 mutation resulted in a reduced ability to elicit the formation of plant nodules and by altered infection thread structures that aborted prematurely.     

Alteration of O-specific polysaccharide structure of symbiotically defective Mesorhizobium loti mutant 2213.1 derived from strain NZP2213

Mesorhizobium loti mutant 2213.1 derived from the wild-type strain NZP2213 by Tn5 mutagenesis showed impaired effectiveness of symbiosis with the host plant Lotus corniculatus (Turska-Szewczuk et al., 2007 Microbiol Res, in press). The inability of lipopolysaccharide (LPS) isolated from the mutant 2213.1 strain or de-O-acetylated LPS of the parental cells to inactivate phage A1 particles implicated alterations in the LPS structure. The O-specific polysaccharide of the mutant was studied by chemical analyses along with (1)H and (13)C NMR spectroscopy, which clearly confirmed alterations in the O-chain structure. 2D NMR data showed that the mutant O-polysaccharide consists of a tetrasaccharide repeating unit containing non-substituted as well as O-acetylated or O-methylated 6-deoxytalopyranose residues. Additionally, an immunogold assay revealed a reduced number of gold particles on the mutant bacteroid cell surface, which could result from both a diminished amount of an O-antigenic determinant in mutant LPS and modifications of structural epitopes caused by alterations in O-acetylation or O-methylation of sugar residues. Western immunoblot assay of alkaline de-O-acetylated lipophilic M. loti NZP2213 LPS showed no reactivity with homologous serum indicating a role of O-acetyl groups in its O-specificity.     

Nitric oxide production induced in roots of Lotus japonicus by lipopolysaccharide from Mesorhizobium loti

Lipopolysaccharide (LPS) is a bacterial molecule that induces nitric oxide (NO) production and triggers defense systems in plant-pathogen interactions. NO production is induced in the roots of Lotus japonicus after inoculation of the roots with its microsymbiont Mesorhizobium loti. However, the rhizobial molecule that induces NO production has not yet been identified. We investigated NO production in the roots of L. japonicus by treatment with LPS of M. loti. LPS was prepared by phenol-hot water extraction and separated into several fractions: polysaccharide, lipooligosaccharide, oligosaccharide and lipid A. In the roots of L. japonicus, NO production was observed with an NO-specific fluorescent dye 4, 10 and 24 h after treatment with each fraction of LPS. NO production was detected 4 h after treatment with all fractions. NO production was also detectable 24 h after treatment, except after treatment with the polysaccharide and oligosaccharide fractions. Expression of a class 1 hemoglobin gene and application of an NO scavenger showed that the treatment with LPS and LOS induced a similar response to inoculation with M. loti. These data suggest that LPS of M. loti induces NO production after inoculation with M. loti.     

Molecular basis of symbiotic host specificity in Rhizobium meliloti: nodH and nodPQ genes encode the sulfation of lipo-oligosaccharide signals.

The symbiosis between Rhizobium and legumes is highly specific. For example, R. meliloti elicits the formation of root nodules on alfalfa and not on vetch. We recently reported that R. meliloti nodulation (nod) genes determine the production of acylated and sulfated glucosamine oligosaccharide signals. We now show that the biochemical function of the major host-range genes, nodH and nodPQ, is to specify the 6-O-sulfation of the reducing terminal glucosamine. Purified Nod factors (sulfated or not) from nodH+ or nodH- strains exhibited the same plant specificity in a variety of bioassays (root hair deformations, nodulation, changes in root morphology) as the bacterial cells from which they were purified. These results provide strong evidence that the molecular mechanism by which the nodH and nodPQ genes mediate host specificity is by determining the sulfation of the extracellular Nod signals.     

A novel ankyrin-repeat membrane protein, IGN1, is required for persistence of nitrogen-fixing symbiosis in root nodules of Lotus japonicus

Nitrogen-fixing symbiosis of legume plants with Rhizobium bacteria is established through complex interactions between two symbiotic partners. Similar to the mutual recognition and interactions at the initial stages of symbiosis, nitrogen fixation activity of rhizobia inside root nodules of the host legume is also controlled by specific interactions during later stages of nodule development. We isolated a novel Fix(-) mutant, ineffective greenish nodules 1 (ign1), of Lotus japonicus, which forms apparently normal nodules containing endosymbiotic bacteria, but does not develop nitrogen fixation activity. Map-based cloning of the mutated gene allowed us to identify the IGN1 gene, which encodes a novel ankyrin-repeat protein with transmembrane regions. IGN1 expression was detected in all organs of L. japonicus and not enhanced in the nodulation process. Immunoanalysis, together with expression analysis of a green fluorescent protein-IGN1 fusion construct, demonstrated localization of the IGN1 protein in the plasma membrane. The ign1 nodules showed extremely rapid premature senescence. Irregularly enlarged symbiosomes with multiple bacteroids were observed at early stages (8-9 d post inoculation) of nodule formation, followed by disruption of the symbiosomes and disintegration of nodule infected cell cytoplasm with aggregation of the bacteroids. Although the exact biochemical functions of the IGN1 gene are still to be elucidated, these results indicate that IGN1 is required for differentiation and/or persistence of bacteroids and symbiosomes, thus being essential for functional symbiosis.     

New nodulation mutants responsible for infection thread development in Lotus japonicus

Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.     

LysM domains mediate lipochitin-oligosaccharide recognition and Nfr genes extend the symbiotic host range

Legume-Rhizobium symbiosis is an example of selective cell recognition controlled by host/non-host determinants. Individual bacterial strains have a distinct host range enabling nodulation of a limited set of legume species and vice versa. We show here that expression of Lotus japonicus Nfr1 and Nfr5 Nod-factor receptor genes in Medicago truncatula and L. filicaulis, extends their host range to include bacterial strains, Mesorhizobium loti or DZL, normally infecting L. japonicus. As a result, the symbiotic program is induced, nodules develop and infection threads are formed. Using L. japonicus mutants and domain swaps between L. japonicus and L. filicaulis NFR1 and NFR5, we further demonstrate that LysM domains of the NFR1 and NFR5 receptors mediate perception of the bacterial Nod-factor signal and that recognition depends on the structure of the lipochitin-oligosaccharide Nod-factor. We show that a single amino-acid variation in the LysM2 domain of NFR5 changes recognition of the Nod-factor synthesized by the DZL strain and suggests a possible binding site for bacterial lipochitin-oligosaccharide signal molecules.     

Identification of a third sulfate activation system in Sinorhizobium sp. strain BR816: the CysDN sulfate activation complex

Sinorhizobium sp. strain BR816 possesses two nodPQ copies, providing activated sulfate (3'-phosphoadenosine-5'-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors. It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain. In this study, we describe the characterization of a third sulfate activation locus. Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes. The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined. FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent. On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors. Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources. In this way, it could be demonstrated that the "household" CysDN sulfate activation complex of Sinorhizobium sp. strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids. Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution.     

Nodule development induced by Mesorhizobium loti mutant strains affected in polysaccharide synthesis

The role of Mesorhizobium loti surface polysaccharides on the nodulation process is not yet fully understood. In this article, we describe the nodulation phenotype of mutants affected in the synthesis of lipopolysaccharide (LPS) and beta(1,2) cyclic glucan. M. loti lpsbeta2 mutant produces LPS with reduced amount of O-antigen, whereas M. loti lpsbeta1 mutant produces LPS totally devoid of O-antigen. Both genes are clustered in the chromosome. Based on amino acid sequence homology, LPS sugar composition, and enzymatic activity, we concluded that lpsbeta2 codes for an enzyme involved in the transformation of dTDP-glucose into dTDP-rhamnose, the sugar donor of rhamnose for the synthesis of O-antigen. On the other hand, lpsbeta1 codes for a glucosyltransferase involved in the biosynthesis of the O-antigen. Although LPS mutants elicited normal nodules, both show reduced competitiveness compared with the wild type. M. loti beta(1-2) cyclic glucan synthase (cgs) mutant induces white, empty, ineffective pseudonodules in Lotus tenuis. Cgs mutant induces normal root hair curling but is unable to induce the formation of infection threads. M. loti cgs mutant was more sensitive to deoxycholate and displayed motility impairment compared with the wild-type strain. This pleiotropic effect depends on calcium concentration and temperature.     

Dual effect of Mesorhizobium loti T3SS functionality on the symbiotic process

Mesorhizobium loti MAFF303099 has a functional type III secretory system (T3SS) involved in the nodulation process on Lotus tenuis and Lotus japonicus. Four putative M.?loti T3SS effectors (Mlr6358, Mlr6331, Mlr6361, and Mlr6316) have been previously described, and it has been demonstrated that the N-terminal regions of Mlr6361 and Mlr6358 mediate the secretion via a T3SS. Here, we demonstrate the capacity of Mlr6316 and Mlr6331 N-terminal regions to direct the secretion of a translational fusion to a reporter peptide through T3SS. By using single, double, and triple mutants, we demonstrated the positive and negative participation of some of these proteins in the determination of competitiveness on Lotus spp. Low competitiveness values correlated with low nodulation efficiency for a mutant deficient in three of the putative M.?loti effectors. Our data suggest that the net effect of M.?loti T3SS function on symbiotic process with Lotus results from a balance between positive and negative effects.