Contribution of NFP LysM domains to the recognition of Nod factors during the Medicago truncatula/Sinorhizobium meliloti symbiosis

The root nodule nitrogen fixing symbiosis between legume plants and soil bacteria called rhizobia is of great agronomical and ecological interest since it provides the plant with fixed atmospheric nitrogen. The establishment of this symbiosis is mediated by the recognition by the host plant of lipo-chitooligosaccharides called Nod Factors (NFs), produced by the rhizobia. This recognition is highly specific, as precise NF structures are required depending on the host plant. Here, we study the importance of different LysM domains of a LysM-Receptor Like Kinase (LysM-RLK) from Medicago truncatula called Nod factor perception (NFP) in the recognition of different substitutions of NFs produced by its symbiont Sinorhizobium meliloti. These substitutions are a sulphate group at the reducing end, which is essential for host specificity, and a specific acyl chain at the non-reducing end, that is critical for the infection process. The NFP extracellular domain (ECD) contains 3 LysM domains that are predicted to bind NFs. By swapping the whole ECD or individual LysM domains of NFP for those of its orthologous gene from pea, SYM10 (a legume plant that interacts with another strain of rhizobium producing NFs with different substitutions), we showed that NFP is not directly responsible for specific recognition of the sulphate substitution of S. meliloti NFs, but probably interacts with the acyl substitution. Moreover, we have demonstrated the importance of the NFP LysM2 domain for rhizobial infection and we have pinpointed the importance of a single leucine residue of LysM2 in that step of the symbiosis. Together, our data put into new perspective the recognition of NFs in the different steps of symbiosis in M. truncatula, emphasising the probable existence of a missing component for early NF recognition and reinforcing the important role of NFP for NF recognition during rhizobial infection.     

Expression of the apyrase-like APY1 genes in roots of Medicago truncatula is induced rapidly and transiently by stress and not by Sinorhizobium meliloti or Nod factors

The model legume Medicago truncatula contains at least six apyrase-like genes, five of which (MtAPY1;1, MtAPY1;2, MtAPY1;3, MtAPY1;4, and MtAPY1;5) are members of a legume-specific family, whereas a single gene (MtAPY2) has closer homologs in Arabidopsis. Phylogenetic analysis has revealed that the proteins encoded by these two plant gene families are more similar to yeast (Saccharomyces cerevisiae) GDA1 and to two proteins encoded by newly described mammalian genes (ENP5 and 6) than they are to mammalian CD39- and CD39-like proteins. Northern analyses and analyses of the frequencies of expressed sequence tags (ESTs) in different cDNA libraries suggest that in roots, leaves, and flowers, the more highly expressed genes are MtAPY1;3/MtAPY2, MtAPY1;3/MtAPY1;5 and MtAPY1;2/MtAPY1;3 respectively. In roots, at least four of the MtAPY1 genes are induced transiently within 3 to 6 h by a stress response that seems to be ethylene independent because it occurs after treatment with an ethylene synthesis inhibitor and also in the skl ethylene-insensitive mutant. This response also occurs in roots of the following symbiotic mutants: dmi1, dmi2, dmi3, nsp, hcl, pdl, lin, and skl. No evidence was obtained for a rapid, transient, and specific induction of the MtAPY genes in roots in response to rhizobia or rhizobial lipochitooligosaccharidic Nod factors. Thus, our data suggest that the apyrase-like genes, which in several legumes have been implicated to play a role in the legume-rhizobia symbiosis (with some members being described as early nodulin genes), are not regulated symbiotically by rhizobia in M. truncatula.     

Architecture of infection thread networks in developing root nodules induced by the symbiotic bacterium Sinorhizobium meliloti on Medicago truncatula

During the course of the development of nitrogen-fixing root nodules induced by Sinorhizobium meliloti on the model plant Medicago truncatula, tubules called infection threads are cooperatively constructed to deliver the bacterial symbiont from the root surface to cells in the interior of the root and developing nodule. Three-dimensional reconstructions of infection threads inside M. truncatula nodules showed that the threads formed relatively simple, tree-like networks. Some characteristics of thread networks, such as branch length, branch density, and branch surface-to-volume ratios, were remarkably constant across nodules in different stages of development. The overall direction of growth of the networks changed as nodules developed. In 5-d-old nodules, the overall growth of the network was directed inward toward the root. However, well-defined regions of these young networks displayed an outward growth bias, indicating that they were likely in the process of repolarizing their direction of development in response to the formation of the outward-growing nodule meristem. In 10- and 30-d-old nodules, the branches of the network grew outward toward the meristem and away from the roots on which the nodules developed.     

Overlap of proteome changes in Medicago truncatula in response to auxin and Sinorhizobium meliloti

We used proteome analysis to identify proteins induced during nodule initiation and in response to auxin in Medicago truncatula. From previous experiments, which found a positive correlation between auxin levels and nodule numbers in the M. truncatula supernodulation mutant sunn (supernumerary nodules), we hypothesized (1) that auxin mediates protein changes during nodulation and (2) that auxin responses might differ between the wild type and the supernodulating sunn mutant during nodule initiation. Increased expression of the auxin response gene GH3:beta-glucuronidase was found during nodule initiation in M. truncatula, similar to treatment of roots with auxin. We then used difference gel electrophoresis and tandem mass spectrometry to compare proteomes of wild-type and sunn mutant roots after 24 h of treatment with Sinorhizobium meliloti, auxin, or a control. We identified 131 of 270 proteins responding to treatment with S. meliloti and/or auxin, and 39 of 89 proteins differentially displayed between the wild type and sunn. The majority of proteins changed similarly in response to auxin and S. meliloti after 24 h in both genotypes, supporting hypothesis 1. Proteins differentially accumulated between untreated wild-type and sunn roots also showed changes in auxin response, consistent with altered auxin levels in sunn. However, differences between the genotypes after S. meliloti inoculation were largely not due to differential auxin responses. The role of the identified candidate proteins in nodule initiation and the requirement for their induction by auxin could be tested in future functional studies.     

Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti

Flavonoids play critical roles in legume–rhizobium symbiosis. However, the role of individual flavonoid compounds in this process has not yet been clearly established. We silenced different flavonoid-biosynthesis enzymes to generate transgenic Medicago truncatula roots with different flavonoid profiles. Silencing of chalcone synthase, the key entry-point enzyme for flavonoid biosynthesis led to flavonoid-deficient roots. Silencing of isoflavone synthase and flavone synthase led to roots deficient for a subset of flavonoids, isoflavonoids (formononetin and biochanin A) and flavones (7,4'-dihydroxyflavone), respectively. When tested for nodulation by Sinorhizobium meliloti , flavonoid-deficient roots had a near complete loss of nodulation, whereas flavone-deficient roots had reduced nodulation. Isoflavone-deficient roots nodulated normally, suggesting that isoflavones might not play a critical role in M. truncatula nodulation, even though they are the most abundant root flavonoids. Supplementation of flavone-deficient roots with 7, 4'-dihydroxyflavone, a major inducer of S. meliloti nod genes, completely restored nodulation. However, the same treatment did not restore nodulation in flavonoid-deficient roots, suggesting that other non- nod gene-inducing flavonoid compounds are also critical to nodulation. Supplementation of roots with the flavonol kaempferol (an inhibitor of auxin transport), in combination with the use of flavone pre-treated S. meliloti cells, completely restored nodulation in flavonoid-deficient roots. In addition, S. meliloti cells constitutively producing Nod factors were able to nodulate flavone-deficient roots, but not flavonoid-deficient roots. These observations indicated that flavones might act as internal inducers of rhizobial nod genes, and that flavonols might act as auxin transport regulators during nodulation. Both these roles of flavonoids appear critical for symbiosis in M. truncatula .     

Increased production of the exopolysaccharide succinoglycan enhances Sinorhizobium meliloti 1021 symbiosis with the host plant Medicago truncatula

The nitrogen-fixing rhizobial symbiont Sinorhizobium meliloti 1021 produces acidic symbiotic exopolysaccharides that enable it to initiate and maintain infection thread formation on host legume plants. The exopolysaccharide that is most efficient in mediating this process is succinoglycan (exopolysaccharide I [EPSI]), a polysaccharide composed of octasaccharide repeating units of 1 galactose and 7 glucose residues, modified with succinyl, acetyl, and pyruvyl substituents. Previous studies had shown that S. meliloti 1021 mutants that produce increased levels of succinoglycan, such as exoR mutants, are defective in symbiosis with host plants, leading to the hypothesis that high levels of succinoglycan production might be detrimental to symbiotic development. This study demonstrates that increased succinoglycan production itself is not detrimental to symbiotic development and, in fact, enhances the symbiotic productivity of S. meliloti 1021 with the host plant Medicago truncatula cv. Jemalong A17. Increased succinoglycan production was engineered by overexpression of the exoY gene, which encodes the enzyme responsible for the first step in succinoglycan biosynthesis. These results suggest that the level of symbiotic exopolysaccharide produced by a rhizobial species is one of the factors involved in optimizing the interaction with plant hosts.     

The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti

Legumes develop different types of lateral organs from their primary root, lateral roots and nodules, the latter depending on a symbiotic interaction with Sinorhizobium meliloti. Phytohormones have been shown to function in the control of these organogeneses. However, related signaling pathways have not been identified in legumes. We cloned and characterized the expression of Medicago truncatula genes encoding members of cytokinin signaling pathways. RNA interference of the cytokinin receptor homolog Cytokinin Response1 (Mt CRE1) led to cytokinin-insensitive roots, which showed an increased number of lateral roots and a strong reduction in nodulation. Both the progression of S. meliloti infection and nodule primordia formation were affected. We also identified two cytokinin signaling response regulator genes, Mt RR1 and Mt RR4, which are induced early during the symbiotic interaction. Induction of these genes by S. meliloti infection is altered in mutants affected in the Nod factor signaling pathway; conversely, cytokinin regulation of the early nodulin Nodule Inception1 (Mt NIN) depends on Mt CRE1. Hence, cytokinin signaling mediated by a single receptor, Mt CRE1, leads to an opposite control of symbiotic nodule and lateral root organogenesis. Mt NIN, Mt RR1, and Mt RR4 define a common pathway activated during early S. meliloti interaction, allowing crosstalk between plant cytokinins and bacterial Nod factors signals.     

Temporal effects on the composition of a population of Sinorhizobium meliloti associated with Medicago sativa and Melilotus alba

An assessment was made of the impact of temporal separation on the composition of a population of Sinorhizobium meliloti associated with Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a single site that had no known history of alfalfa cultivation. Root nodules were sampled on six occasions over two seasons, and a total of 1620 isolates of S. meliloti were characterized on the basis of phage sensitivity using 16 typing phages. Plant infection tests indicated that symbiotic S. meliloti were deficient in the soil at the time of planting and that these bacteria were present at low density during the first season (<10(2)/g of soil); in the second season numbers increased markedly to about 10(5)/g of soil. Overall, 37 and 51 phage types, respectively, were encountered among the nodule isolates from M. sativa and M. alba. The data indicate significant temporal shifts in the frequency and diversity of types associated with the two legume species. Apparent temporal variation with respect to the frequency of types appeared largely unpredictable and was not attributable to any one sampling time. The results indicate an apparent reduction in phenotypic diversity over the course of the experiment. Differential host plant selection of specific types with respect to nodule occupancy was indicated by significant interactions between legume species and either the frequency or diversity of phage types. Isolates from M. sativa that were resistant to lysis by all typing phages (type 14) were unusual in that they were predominant on this host at all sampling times (between 53% and 82% nodule occupancy) and were relatively homogeneous on the basis of DNA hybridization with 98% of the isolates analysed sharing the same nod EFG hybridization profile. In contrast, those isolates from M. alba comprising type 14 were encountered at low total frequency (2%) and were genetically heterogeneous on the basis of Southern hybridization. The implications of the observed temporal and host plant variation for ecological studies are discussed.     

Single-plant,Sterile Microcosms for Nodulation and Growth of the Legume Plant Medicago truncatula with the Rhizobial Symbiont Sinorhizobium meliloti

Rhizobial bacteria form symbiotic, nitrogen-fixing nodules on the roots of compatible host legume plants. One of the most well-developed model systems for studying these interactions is the plant Medicago truncatula cv. Jemalong A17 and the rhizobial bacterium Sinorhizobium meliloti 1021. Repeated imaging of plant roots and scoring of symbiotic phenotypes requires methods that are non-destructive to either plants or bacteria. The symbiotic phenotypes of some plant and bacterial mutants become apparent after relatively short periods of growth, and do not require long-term observation of the host/symbiont interaction. However, subtle differences in symbiotic efficiency and nodule senescence phenotypes that are not apparent in the early stages of the nodulation process require relatively long growth periods before they can be scored. Several methods have been developed for long-term growth and observation of this host/symbiont pair. However, many of these methods require repeated watering, which increases the possibility of contamination by other microbes. Other methods require a relatively large space for growth of large numbers of plants. The method described here, symbiotic growth of M. truncatula/S. meliloti in sterile, single-plant microcosms, has several advantages. Plants in these microcosms have sufficient moisture and nutrients to ensure that watering is not required for up to 9 weeks, preventing cross-contamination during watering. This allows phenotypes to be quantified that might be missed in short-term growth systems, such as subtle delays in nodule development and early nodule senescence. Also, the roots and nodules in the microcosm are easily viewed through the plate lid, so up-rooting of the plants for observation is not required.     

N-deacetylation of Sinorhizobium meliloti Nod factors increases their stability in the Medicago sativa rhizosphere and decreases their biological activity

Nod factors excreted by rhizobia are signal molecules that consist of a chitin oligomer backbone linked with a fatty acid at the nonreducing end. Modifications of the Nod factor structures influence their stability in the rhizosphere and their biological activity. To test the function of N-acetyl groups in Nod factors, NodSm-IV(C16:2,S) from Sinorhizobium meliloti was enzymatically N-deacetylated in vitro with purified chitin deacetylase from Colletotrichum lindemuthianum. A family of partially and completely deacetylated derivatives was produced and purified. The most abundant chemical structures identified by mass spectrometry were GlcN(C16:2)-GlcNAc-GlcNH2-GlcNAc(OH)(S), GlcN(C16,2)-GlcNAc-GlcNH2-GlcNH2(OH)(S), and GlcN(C16:2)-GlcNH2-GlcNH2-GlcNH2(OH)(S). In contrast to NodSm-IV(C16:2,S), the purified N-deacetylated derivatives were stable in the rhizosphere of Medicago sativa, indicating that the N-acetyl groups make the carbohydrate moiety of Nod factors accessible for glycosyl hydrolases of the host plant. The N-deacetylated derivatives displayed only a low level of activity in inducing root hair deformation. Furthermore, the N-deacetylated molecules were not able to stimulate Nod factor degradation by M. sativa roots, a response elicited by active Nod factors. These data show that N-acetyl groups of Nod factors are required for biological activity.     

Characterization of four lectin-like receptor kinases expressed in roots of Medicago truncatula. Structure, location, regulation of expression, and potential role in the symbiosis with Sinorhizobium meliloti

To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.     

Evidence that the exoH gene of Sinorhizobium meliloti does not appear to influence symbiotic effectiveness with Medicago truncatula 'Jemalong A17'

The purpose of this study was to identify strains of Sinorhizobium meliloti that formed either an effective or completely ineffective symbiosis with Medicago truncatula L. 'Jemalong A17' and, subsequently, to determine whether differences existed between their exoH genes. Sinorhizobium meliloti TII7 and A5 formed an effective and ineffective symbiosis with M. truncatula 'Jemalong A17', respectively. Using a multilocus sequence typing method, both strains were shown to have chromosomes identical with S. meliloti Rm1021 and RCR2011. The 2260-bp segments of DNA stretching from the 3' end of exoI through open reading frames of hypothetical proteins SM_b20952 and SM_b20953 through exoH into the 5' end of exoK in strains TII7 and Rm1021 differed by a single nucleotide at base 127 of the hypothetical protein SM_b20953. However, the derived amino acid sequences of the exoH genes of effective TII7, ineffective A5, and strain Rm1021 were shown to be identical with each other. Therefore, it would seem unlikely that the gene product of exoH is directly involved with the low efficiency of a symbiosis of strain Rm1021 with M. truncatula 'Jemalong A17'. Complementation or complete genome sequence analyses involving strains TII7 and A5 might be useful approaches to investigate the molecular bases for the differential symbiotic response with M. truncatula 'Jemalong A17'.     

The typA gene is required for stress adaptation as well as for symbiosis of Sinorhizobium meliloti 1021 with certain Medicago truncatula lines

In this article, we describe the typA gene of Sinorhizobium meliloti, the orthologue of typA/bipA genes found in a wide range of bacteria. We found that typA was required for survival of S. meliloti under certain stress conditions, such as growth at low temperature or low pH and in the presence of sodium dodecyl sulfate (SDS). The cold-sensitive phenotype of both Escherichia coli bipA and S. meliloti typA mutants were cross-complemented, indicating that the two genes are functionally equivalent. typA was indispensable for symbiosis on Medicago truncatula Jemalong and F83005.5 and contributes to the full efficiency of symbiosis on other host plant lines such as DZA315.16 or several cultivars of M. sativa. Hence, the symbiotic requirement for typA is host dependent. Interestingly, the symbiotic defect was different on Jemalong and F83005.5 plants, thus indicating that typA is required at a different stage of the symbiotic interaction.     

Population genetic structure of Sinorhizobium meliloti and S. medicae isolated from nodules of Medicago spp. in Mexico

We studied the genetic structure of 176 bacterial isolates from nodules of Medicago sativa, M. lupulina and M. polymorpha in fifteen sites distributed in three localities in Mexico. The strains were characterized by multilocus enzyme electrophoresis, plasmid profiles, PCR restriction fragment length polymorphism of 16S rRNA genes and of the intergenic spacer between 16S and 23S rRNA genes, and partial sequences of glnII, recA and nodB. Most of the strains were classified as Sinorhizobium meliloti, and a high genetic diversity was recorded. Six strains were classified as Sinorhizobium medicae, with no genetic variation. Phylogenetic and population genetic analyses revealed evidence of frequent recombination and migration within species.     

Microdiversity of Burkholderiales associated with mycorrhizal and nonmycorrhizal roots of Medicago truncatula

The genetic diversity of bacterial communities associated with mycorrhizal and nonmycorrhizal roots of Medicago truncatula was characterized by two approaches. Firstly, phylogenetic analysis was performed on 164 partial 16S rRNA gene-intergenic spacer (IGS) sequences from operational taxonomic units previously shown to be preferentially associated with mycorrhizal roots. These sequences were distributed into three branches corresponding to Comamonadaceae, Oxalobacteraceae and Rubrivivax subgroups. Most sequences were obtained from mycorrhizal roots, indicating the preferential association of the corresponding families with mycorrhizal roots. A second phylogenetic analysis was performed on the partial 16S rRNA gene-IGS sequences of 173 isolates among a large collection of isolates, from mycorrhizal and nonmycorrhizal roots, belonging to Comamonadaceae and Oxalobacteraceae on the basis of their positive hybridization with a partial 16S rRNA gene-IGS probe obtained in this study. Sequence analysis confirmed the affiliation of 166 isolates to Comamonadaceae and seven to Oxalobacteraceae. Oxalobacteraceae isolates were more abundant in mycorrhizal (five) than in nonmycorrhizal (two) roots, whereas Comamonadaceae isolates were more abundant in nonmycorrhizal (109) than mycorrhizal roots (57). Further analysis of Comamonadaceae isolates by BOX-PCR showed that the genetic structure of culturable populations belonging to this family differed significantly in mycorrhizal and nonmycorrhizal roots, as indicated by distributions in different BOX types, differences being significantly explained by BOX types only including isolates from mycorrhizal roots. These data are discussed in an ecological context.