Rhizobium nodule diversity and composition are influenced by clover host selection and local growth conditions.

While shaping of plant microbiome composition through 'host filtering' is well documented in legume-rhizobium symbioses, it is less clear to what extent different varieties and genotypes of the same plant species differentially influence symbiont community diversity and composition. Here, we compared how clover host varieties and genotypes affect the structure of Rhizobium populations in root nodules under conventional field and controlled greenhouse conditions. We first grew four Trifolium repens (white clover) F2 crosses and one variety in a conventional field trial and compared differences in root nodule Rhizobium leguminosarum symbiovar trifolii (Rlt) genotype diversity using high-throughput amplicon sequencing of chromosomal housekeeping (rpoB and recA) genes and auxiliary plasmid-borne symbiosis genes (nodA and nodD). We found that Rlt nodule diversities significantly differed between clover crosses, potentially due to host filtering. However, variance in Rlt diversity largely overlapped between crosses and was also explained by the spatial distribution of plants in the field, indicative of the role of local environmental conditions for nodule diversity. To test the effect of host filtering, we conducted a controlled greenhouse trial with a diverse Rlt inoculum and several host genotypes. We found that different clover varieties and genotypes of the same variety selected for significantly different Rlt nodule communities and that the strength of host filtering (deviation from the initial Rhizobium inoculant composition) was positively correlated with the efficiency of symbiosis (rate of plant greenness colouration). Together, our results suggest that selection by host genotype and local growth conditions jointly influence white clover Rlt nodule diversity and community composition.

Increasing morphological disparity and decreasing optimality for jaw speed and strength during the radiation of jawed vertebrates.

The Siluro-Devonian adaptive radiation of jawed vertebrates, which underpins almost all living vertebrate biodiversity, is characterized by the evolutionary innovation of the lower jaw. Multiple lines of evidence have suggested that the jaw evolved from a rostral gill arch, but when the jaw took on a feeding function remains unclear. We quantified the variety of form in the earliest jaws in the fossil record from which we generated a theoretical morphospace that we then tested for functional optimality. By drawing comparisons with the real jaw data and reconstructed jaw morphologies from phylogenetically inferred ancestors, our results show that the earliest jaw shapes were optimized for fast closure and stress resistance, inferring a predatory feeding function. Jaw shapes became less optimal for these functions during the later radiation of jawed vertebrates. Thus, the evolution of jaw morphology has continually explored previously unoccupied morphospace and accumulated disparity through time, laying the foundation for diverse feeding strategies and the success of jawed vertebrates.

Cyclic-ß-glucans of Rhizobium (Sinorhizobium) sp. strain NGR234 are required for hypo-osmotic adaptation, motility, and efficient symbiosis with host plants.

Cyclic-ß-glucans (CßG) consist of cyclic homo-polymers of glucose that are present in the periplasmic space of many Gram-negative bacteria. A number of studies have demonstrated their importance for bacterial infection of plant and animal cells. In this study, a mutant of Rhizobium (Sinorhizobium) sp. strain NGR234 (NGR234) was generated in the cyclic glucan synthase (ndvB)-encoding gene. The great majority of CßG produced by wild-type NGR234 are negatively charged and substituted. The ndvB mutation abolished CßG biosynthesis. We found that, in NGR234, a functional ndvB gene is essential for hypo-osmotic adaptation and swimming, attachment to the roots, and efficient infection of Vigna unguiculata and Leucaena leucocephala.

Synthesis of the flavonoid-induced lipopolysaccharide of Rhizobium Sp. strain NGR234 requires rhamnosyl transferases encoded by genes rgpF and wbgA.

In the presence of flavonoids, Rhizobium sp. strain NGR234 synthesizes a new lipopolysaccharide (LPS), characterized by a rhamnan O-antigen. The presence of this rhamnose-rich LPS is important for the establishment of competent symbiotic interactions between NGR234 and many species of leguminous plants. Two putative rhamnosyl transferases are encoded in a cluster of genes previously shown to be necessary for the synthesis of the rhamnose-rich LPS. These two genes, wbgA and rgpF, were mutated. The resulting mutant strains synthesized truncated rough LPS species rather than the wild-type rhamnose-rich LPS when grown with flavonoids. Based on the compositions of these purified mutant LPS species, we inferred that RgpF is responsible for adding the first one to three rhamnose residues to the flavonoid-induced LPS, whereas WbgA is necessary for the synthesis of the rest of the rhamnan O-antigen. The NGR234 homologue of lpsB, which, in other bacteria, encodes a glycosyl transferase acting early in synthesis of the core portion of LPS, was identified and also mutated. LpsB was required for all the LPS species produced by NGR234, in the presence or absence of flavonoids. Mutants (i.e., of lpsB and rgpF) that lacked any portion of the rhamnan O-antigen of the induced LPS were severely affected in their symbiotic interaction with Vigna unguiculata, whereas the NGR?wbgA mutant, although having very few rhamnose residues in its LPS, was able to elicit functional nodules.

Role of BacA in lipopolysaccharide synthesis, peptide transport, and nodulation by Rhizobium sp. strain NGR234.

BacA of Sinorhizobium meliloti plays an essential role in the establishment of nitrogen-fixing symbioses with Medicago plants, where it is involved in peptide import and in the addition of very-long-chain fatty acids (VLCFA) to lipid A of lipopolysaccharide (LPS). We investigated the role of BacA in Rhizobium species strain NGR234 by mutating the bacA gene. In the NGR234 bacA mutant, peptide import was impaired, but no effect on VLCFA addition was observed. More importantly, the symbiotic ability of the mutant was comparable to that of the wild type for a variety of legume species. Concurrently, an acpXL mutant of NGR234 was created and assayed. In rhizobia, AcpXL is a dedicated acyl carrier protein necessary for the addition of VLCFA to lipid A. LPS extracted from the NGR234 mutant lacked VLCFA, and this mutant was severely impaired in the ability to form functional nodules with the majority of legumes tested. Our work demonstrates the importance of VLCFA in the NGR234-legume symbiosis and also shows that the necessity of BacA for bacteroid differentiation is restricted to specific legume-Rhizobium interactions.

Roles of flavonoids and the transcriptional regulator TtsI in the activation of the type III secretion system of Bradyrhizobium elkanii SEMIA587.

Bradyrhizobium elkanii SEMIA587 is a symbiotic nitrogen-fixing bacterium of the group commonly called rhizobia, which induce nodule formation in legumes, and is widely used in Brazilian commercial inoculants of soybean. In response to flavonoid compounds released by plant roots, besides Nod factors, other molecular signals are secreted by rhizobia, such as proteins secreted by type III secretion systems (T3SSs). Rhizobial T3SSs are activated by the transcription regulator TtsI, which binds to sequences present in the promoter regions of T3SS genes via a conserved sequence called the tts box. To study the role of the T3SS of B. elkanii SEMIA587, ttsI was mutated. Protein secretion and flavonoid induction analysis, as well as nodulation tests, were performed with the wild-type and mutant strains. The results obtained showed that B. elkanii SEMIA587 secretes at least two proteins (NopA and NopL, known rhizobial T3SS substrates) after genistein induction, whilst supernatants of the ttsI mutant did not contain these Nops. Unusually for rhizobia, the promoter region of the B. elkanii SEMIA587 ttsI gene contains a tts box, which is responsive to flavonoid induction and to which TtsI can bind. Nodulation tests performed with three different leguminous plants showed that the B. elkanii SEMIA587 ttsI mutant displays host-dependent characteristics; in particular, nodulation of two soybean cultivars, Peking and EMBRAPA 48, was more efficient when TtsI of B. elkanii was functional.

How inefficient rhizobia prolong their existence within nodules.

Legumes are important food sources and therefore, the nitrogen fixing ability of legume-rhizobia symbioses have great potential to improve crop yields and/or reduce the use of nitrogenous fertilisers. Unfortunately the nitrogen fixing efficiency of many legume-rhizobial combinations is low. What restricts nodule efficiency? We believe that one answer lies in the neglected field of signal exchange within mature nodules. Indeed molecular determinants that permit rhizobia to persist for months within plants cells are still unknown. Here, we dissect acute infection that triggers nodulation from chronic infection in which the bacteria persist within nitrogen-fixing nodules. We suggest that defence responses are disabled in mature nodules and superseded by specialised mechanisms of bacterial population control.

Characterization of the Mesorhizobium loti MAFF303099 type-three protein secretion system.

Type III secretion systems (T3SS) have been found in several species of rhizobia. Proteins (termed effectors) secreted by this system are involved in host-range determination and influence nodulation efficiency. Mesorhizobium loti MAFF303099 possesses a functional T3SS in its symbiotic island whose expression is induced by flavonoids. As in other rhizobia, conserved cis-elements (tts box) were found in the promoter regions of genes or operons encoding T3SS components. Using a bioinformatics approach, we searched for other tts-box-controlled genes, and confirmed this transcriptional regulation for some of them using lacZ fusions to the predicted promoter regions. Translational fusions to a reporter peptide were created to demonstrate T3SS-mediated secretion of two new MAFF303099 effectors. Finally, we showed that mutation of the M. loti MAFF303099 T3SS affects its competitiveness on Lotus glaber and investigated, at the molecular level, responses of the model legume L. japonicus to the T3SS.

Symbiotic use of pathogenic strategies: rhizobial protein secretion systems.

Rhizobia - a diverse group of soil bacteria - induce the formation of nitrogen-fixing nodules on the roots of legumes. Nodulation begins when the roots initiate a molecular dialogue with compatible rhizobia in the soil. Most rhizobia reply by secreting lipochitooligosaccharidic nodulation factors that enable entry into the legume. A molecular exchange continues, which, in compatible interactions, permits rhizobia to invade root cortical cells, differentiate into bacteroids and fix nitrogen. Rhizobia also use additional molecular signals, such as secreted proteins or surface polysaccharides. One group of proteins secreted by rhizobia have homologues in bacterial pathogens and may have been co-opted by rhizobia for symbiotic purposes.

Rhizobia utilize pathogen-like effector proteins during symbiosis.

A type III protein secretion system (T3SS) is an important host range determinant for the infection of legumes by Rhizobium sp. NGR234. Although a functional T3SS can have either beneficial or detrimental effects on nodule formation, only the rhizobial-specific positively acting effector proteins, NopL and NopP, have been characterized. NGR234 possesses three open reading frames potentially encoding homologues of effector proteins from pathogenic bacteria. NopJ, NopM and NopT are secreted by the T3SS of NGR234. All three can have negative effects on the interaction with legumes, but NopM and NopT also stimulate nodulation on certain plants. NopT belongs to a family of pathogenic effector proteases, typified by the avirulence protein, AvrPphB. The protease domain of NopT is required for its recognition and a subsequent strong inhibition in infection of Crotalaria juncea. In contrast, the negative effects of NopJ are relatively minor when compared with those induced by its Avr homologues. Thus NGR234 uses a mixture of rhizobial-specific and pathogen-derived effector proteins. Whereas some legumes recognize an effector as potentially pathogen-derived, leading to a block in the infection process, others perceive both the negative- and positive-acting effectors concomitantly. It is this equilibrium of effector action that leads to modulation of symbiotic development.

Delayed maturation of nodules reduces symbiotic effectiveness of the Lotus japonicus-Rhizobium sp. NGR234 interaction.

Lotus japonicus, a model legume, develops an efficient, nitrogen-fixing symbiosis with Mesorhizobium loti that promotes plant growth. Lotus japonicus also forms functional nodules with Rhizobium sp. NGR234 and R. etli. Yet, in a plant defence-like reaction, nodules induced by R. etli quickly degenerate, thus limiting plant growth. In contrast, nodules containing NGR234 are long-lasting. It was found that NGR234 initiates nodule formation in a similar way to M. loti MAFF303099, but that the nodules which develop on eleven L. japonicus ecotypes are less efficient in fixing nitrogen. Detailed examination of nodulation of L. japonicus cultivar MG-20 revealed that symbiosomes formed four weeks after inoculation by NGR234 are enlarged in comparison with MAFF303099 and contain multiple bacteroids. Nevertheless, nodules formed by NGR234 fix sufficient nitrogen to avoid rejection by the plant. With time, these nodules develop into fully efficient organs containing bacteroids tightly enclosed in symbiosome membranes, just like those formed by M. loti MAFF303099. This work demonstrates the usefulness of using the well-characterized micro-symbiont NGR234 to study symbiotic signal exchange in the later stages of rhizobia-legume symbioses, especially given the large range of bacterial (NGR234) and plant (L. japonicus) mutants that are available.

TtsI regulates symbiotic genes in Rhizobium species NGR234 by binding to tts boxes.

Infection of legumes by Rhizobium sp. NGR234 and subsequent development of nitrogen-fixing nodules are dependent on the coordinated actions of Nod factors, proteins secreted by a type III secretion system (T3SS) and modifications to surface polysaccharides. The production of these signal molecules is dependent on plant flavonoids which trigger a regulatory cascade controlled by the transcriptional activators NodD1, NodD2, SyrM2 and TtsI. TtsI is known to control the genes responsible for T3SS function and synthesis of a symbiotically important rhamnose-rich lipo-polysaccharide, most probably by binding to cis elements termed tts boxes. Eleven tts boxes were identified in the promoter regions of target genes on the symbiotic plasmid of NGR234. Expression profiles of lacZ fusions to these tts boxes showed that they are part of a TtsI-dependent regulon induced by plant-derived flavonoids. TtsI was purified and demonstrated to bind directly to two of these tts boxes. DNase I footprinting revealed that TtsI occupied not only the tts box consensus sequence, but also upstream and downstream regions in a concentration-dependent manner. Highly conserved bases of the consensus tts box were mutated and, although TtsI binding was still observed in vitro, gfp fusions were no longer transcribed in vivo. Random mutagenesis of a tts box-containing promoter revealed more nucleotides critical for transcriptional activity outside of the consensus.

Protein-protein interactions within type III secretion system-dependent pili of Rhizobium sp. strain NGR234.

Pili synthesized by the type III secretion system of Rhizobium species strain NGR234 are essential for protein secretion and thus for efficient symbiosis with many legumes. Isolation and partial purification of these pili showed that they are composed of at least three proteins, NopA, NopB, and NopX. Using biochemical assays, we show here that these proteins interact directly with one another.

Structural characterization of a K-antigen capsular polysaccharide essential for normal symbiotic infection in Rhizobium sp. NGR234: deletion of the rkpMNO locus prevents synthesis of 5,7-diacetamido-3,5,7,9-tetradeoxy-non-2-ulosonic acid.

Many early molecular events in symbiotic infection have been documented, although factors enabling Rhizobium to progress within the plant-derived infection thread and ultimately survive within the intracellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity. Relatively few studies have examined the role of K-antigens in these events. However, we constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid, in the broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure -->4)-beta-d-glucuronic acid-(1-->4)-beta-5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid-(2-->. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN, and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected. The NGRDeltarkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. K-antigen production was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.

NopP, a phosphorylated effector of Rhizobium sp. strain NGR234, is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii.

Rhizobium sp. NGR234 nodulates many plants, some of which react to proteins secreted via a type three secretion system (T3SS) in a positive- (Flemingia congesta, Tephrosia vogelii) or negative- (Crotalaria juncea, Pachyrhizus tuberosus) manner. T3SSs are devices that Gram-negative bacteria use to inject effector proteins into the cytoplasm of eukaryotic cells. The only two rhizobial T3SS effector proteins characterized to date are NopL and NopP of NGR234. NopL can be phosphorylated by plant kinases and we show this to be true for NopP as well. Mutation of nopP leads to a dramatic reduction in nodule numbers on F. congesta and T. vogelii. Concomitant mutation of nopL and nopP further diminishes nodulation capacity to levels that, on T. vogelii, are lower than those produced by the T3SS null mutant NGR(Omega)rhcN. We also show that the T3SS of NGR234 secretes at least one additional effector, which remains to be identified. In other words, NGR234 secretes a cocktail of effectors, some of which have positive effects on nodulation of certain plants while others are perceived negatively and block nodulation. NopL and NopP are two components of this mix that extend the ability of NGR234 to nodulate certain legumes.

NopA is associated with cell surface appendages produced by the type III secretion system of Rhizobium sp. strain NGR234.

Rhizobium sp. strain NGR234, which is capable of interacting with a large number of legumes, utilizes a variety of signaling molecules to establish nitrogen-fixing symbioses. Among these are nodulation outer proteins (Nops) that transit through a type III secretion system (TTSS). Abolition of Nop secretion affects nodulation of certain legumes. Under free-living conditions, the secretion of Nops can be induced by the addition of flavonoids. Here, we show that an in-frame deletion of nopA abolishes secretion of all other Nops and has the same impact on nodule formation as mutations that lead to a nonfunctional TTSS. This secretion-minus phenotype of the nopA mutant, as well as bioinformatics analysis of NopA itself, suggests that NopA could be an external component of the TTSS. Electron microscopy showed that NGR234 synthesizes fibrillar structures on the cell surface in a flavonoid-inducible and NopA-dependent manner. Purification of the macromolecular surface appendages revealed that NopA is a major component of these structures.

NopB, a type III secreted protein of Rhizobium sp. strain NGR234, is associated with pilus-like surface appendages.

Rhizobium sp. strain NGR234 possesses a functional type three secretion system (TTSS), through which a number of proteins, called nodulation outer proteins (Nops), are delivered to the outside of the cell. A major constraint to the identification of Nops is their low abundance in the supernatants of NGR234 strains grown in culture. To overcome this limitation, a more sensitive proteomics-based strategy was developed. Secreted proteins from wild-type NGR234 were separated by two-dimensional gel electrophoresis, and the gel was compared to similar gels containing the proteins from a TTSS mutant (NGROmegarhcN). To identify the proteins, spots unique to the NGR234 gels were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and the data were compared to the sequence of the symbiotic plasmid of NGR234. A nonpolar mutant of one of these proteins was generated called NopB. NopB is required for Nop secretion but inhibits the interaction with Pachyrhizus tuberosus and augments nodulation of Tephrosia vogelii. Flavonoids and a functional TTSS are required for the formation of some surface appendages on NGR234. In situ immunogold labeling and isolation of these pili showed that they contain NopB.

TtsI, a key regulator of Rhizobium species NGR234 is required for type III-dependent protein secretion and synthesis of rhamnose-rich polysaccharides.

Formation of nitrogen-fixing nodules on legume roots by Rhizobium sp. NGR234 requires an array of bacterial factors, including nodulation outer proteins (Nops) secreted through a type III secretion system (TTSS). Secretion of Nops is abolished upon inactivation of ttsI (formerly y4xI), a protein with characteristics of two-component response regulators that was predicted to activate transcription of TTSS-related genes. During the symbiotic interaction, the phenotype of NGR omega ttsI differs from that of a mutant with a nonfunctional secretion machine, however. This indicated that TtsI regulates the synthesis of other symbiotic factors as well. Conserved sequences, called tts boxes, proposed to act as binding sites for TtsI, were identified not only within the TTSS cluster but also in the promoter regions of i) genes predicted to encode homologs of virulence factors secreted by pathogenic bacteria, ii) loci involved in the synthesis of a rhamnose-rich component (rhamnan) of the lipopolysaccharides (LPS), and iii) open reading frames that play roles in plasmid partitioning. Transcription studies showed that TtsI and tts boxes are required for the activation of TTSS-related genes and those involved in rhamnose synthesis. Furthermore, extraction of polysaccharides revealed that inactivation of ttsI abolishes the synthesis of the rhamnan component of the LPS. The phenotypes of mutants impaired in TTSS-dependent protein secretion, rhamnan synthesis, or in both functions were compared to assess the roles of some of the TtsI-controlled factors during symbiosis.

Characterization of NopP, a type III secreted effector of Rhizobium sp. strain NGR234.

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.

NopL, an effector protein of Rhizobium sp. NGR234, thwarts activation of plant defense reactions.

Bacterial effector proteins delivered into eukaryotic cells via bacterial type III secretion systems are important virulence factors in plant-pathogen interactions. Type III secretion systems have been found in Rhizobium species that form symbiotic, nitrogen-fixing associations with legumes. One such bacterium, Rhizobium sp. NGR234, secretes a number of type III effectors, including nodulation outer protein L (NopL, formerly y4xL). Here, we show that expression of nopL in tobacco (Nicotiana tabacum) prevents full induction of pathogenesis-related (PR) defense proteins. Transgenic tobacco plants that express nopL and were infected with potato virus Y (necrotic strain 605) exhibited only very low levels of chitinase (class I) and beta-1,3-glucanase (classes I and III) proteins. Northern-blot analysis indicated that expression of nopL in plant cells suppresses transcription of PR genes. Treatment with ethylene counteracted the effect of NopL on chitinase (class I). Transgenic Lotus japonicus plants that expressed nopL exhibited delayed development and low chitinase levels. In vitro experiments showed that NopL is a substrate for plant protein kinases. Together, these data suggest that NopL, when delivered into the plant cell, modulates the activity of signal transduction pathways that culminate in activation of PR proteins.