Arthrobacter agilis UMCV2 induces iron acquisition in Medicago truncatula (strategy I plant) in vitro via dimethylhexadecylamine emission

Background and aims

Iron is an essential nutrient for plant growth. Although abundant in soil, iron is poorly available. Therefore, plants have evolved mechanisms for iron mobilization and uptake from the rhizospheric environment. In this study, we examined the physiological responses to iron deficiency in Medicago truncatula plants exposed to volatile organic compounds (VOCs) produced by Arthrobacter agilis UMCV2.

Methods

The VOC profiles of the plant and bacterium were determined separately and during interaction assays using gas chromatography. M. truncatula plants exposed to A. agilis VOCs and pure dimethylhexadecylamine were transferred to conditions of iron deficiency, and parameters associated with iron nutritional status were measured.

Results

The relative abundance of the bacterial VOC dimethylhexadecylamine increased 12-fold when in co-cultures of A. agilis and M. truncatula, compared to axenic cultures. Plants exposed to bacterial VOCs or dimethylhexadecylamine exhibited a higher rhizosphere acidification capacity, enhanced ferric reductase activity, higher biomass generation, and elevated chlorophyll and iron content relative to controls.

Conclusions

The VOCs emitted by A. agilis UMCV2 induce iron acquisition mechanisms in vitro in the Strategy I plant M. truncatula. Dimethylhexadecylamine is the signal molecule responsible for producing the beneficial effects.     

Phosphate concentration alters the effective bacterial quorum in the symbiosis of Medicago truncatula-Sinorhizobium meliloti

The symbiosis of Medicago truncatula-Sinorhizobium meliloti is affected by phosphate (P) deficiency in the environment. Quorum sensing (QS) is a regulatory pathway in S. meliloti that controls various functions of free-living and symbiotic bacteria in response to phosphate availability and regulation is mediated by a periplasmic protein PstS, and also bacterial density. The quorum sensing pathway of S. meliloti, involves three genes named sinI, sinR and expR and also some bacterial auto-inducers such as N-acyl homoserine lactones (AHLs). In the current study, the expression of the different genes of quorum sensing and pstS were evaluated under 0.1, 0.5 and 2 mM P. The qRT-PCR results showed an increased expression of pstS and also the quorum sensing genes sinI and sinR but not expR, following phosphate starvation. Indeed, the enhanced level of sinR induces the expression of sinI that is responsible for the N-acyl homoserine lactones (AHL) production in S. meliloti. The different response of expR may be due to its negative control on sinR expression. In the symbiosis of M. truncatula-S. meliloti, it was shown that the concentration of phosphate in the medium alters the effective inoculating bacterial quorum (density). By increasing the phosphate concentration in the medium from 0.1 to 0.5 and 2 mM, considering the optimal plant growth and pink nodule (nitrogen-fixing) formation, the effective inoculating bacterial densities were 10⁵, 10⁷ and 10⁹ CFU ml^?1, respectively. Therefore, low phosphate concentrations can compensate for a low bacterial density by inducing the quorum sensing pathway and establishing a symbiosis. Conversely, bacterial density plays the main role in the formation of symbiosis at high phosphate concentrations.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Influence of salt stress on the genetically polymorphic system of Sinorhizobium meliloti-Medicago truncatula

The impacts of salt stress (75 mM NaCl) on the ecological efficiency of the genetically polymorphic Sinorhizobium meliloti-Medicago truncatula system were studied. Its impact on a symbiotic system results in an increase of the partners’ variability for symbiotic traits and of the symbiosis integrity as indicated by: (a) the specificity of the partners’ interactions-the nonadditive inputs of their genotypes into the variation of symbiotic parameters and (b) the correlative links between these parameters. The structure of the nodD1 locus and the plasmid content correlates to the efficiency of the symbiosis between S. meliloti and M. truncatula genotypes under stress conditions more sufficiently than in the absence of stress. Correlations between the symbiotic efficiency of rhizobia strains and their growth rate outside symbiosis are expressed under stress conditions, not in the absence of stress. Under salt stress symbiotic effectiveness was decreased for M. truncatula line F83005.5, which was salt sensitive for mineral nutrition. The inhibition of symbiotic activity for this line is linked with decreased nodule formation, whereas for Jemalong 6 and DZA315.16 lines it is associated with repressed N₂-fixation. It was demonstrated for the first time that salt stress impairs the M. truncatula habitus (the mass: height ratio increased 2- to 6-fold), which in the salt-resistant cultivar Jemalong 6 is normalized as the result of rhizobia inoculation.     

Biodiversity within Medicago truncatula genotypes toward response to iron deficiency: Investigation of main tolerance mechanisms

Iron (Fe) deficiency is one of the major environmental stresses affecting plant production in the world. The selection of tolerant genotypes is considered an effective remediation strategy for this stress. The present study was carried out in order to investigate the biodiversity within Medicago truncatula plants in response to Fe deficiency, to identify tolerant genotypes and to assess the main tolerance mechanisms. To do this, a screening test was performed on 20 M. truncatula genotypes cultivated in minimal medium. Biometric and physiological markers were analyzed, including plant biomass, chlorophyll and root architecture. Results showed a biodiversity among the 20 genotypes. Interestingly, Fe deficiency tolerance was highest in TN8.20 and A17 genotypes. However, the lowest tolerance behavior was observed in TN1.11 and TN6.18. In order to investigate the main tolerance mechanisms, an experiment was conducted in the hydroponic system on already selected genotypes. Assessment of Fe deficiency tolerance was performed mainly on plant growth parameters, Fe (III)-chelate-reductase activity, rhizosphere acidification and antioxidant system defense. The relative better tolerance of A17 and TN8.20 to Fe deficiency was positively correlated with their capacity to maintain higher Fe-acquisition efficiency in roots via rhizosphere acidification and the stimulation of Fe (III)-chelate-reductase activity. Moreover, tolerant genotypes showed the lowest decreases in chlorophyll content and photosynthetic activity (CO₂ assimilation) compared to the sensitive ones. The efficiency of antioxidant capacity of the tolerant genotypes was revealed in stimulation of catalase (CAT) and peroxidase (POX) activities as well as accumulation of polyphenols, leading to the maintenance of cell integrity under Fe deficiency.     

Kinetics and Strain Specificity of Rhizosphere and Endophytic Colonization by Enteric Bacteria on Seedlings of Medicago sativa and Medicago truncatula

The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (Medicago sativa) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of K. pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 10² CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was colonized in higher numbers on the rhizosphere and in the interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appears to occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi.     

Control of NO level in rhizobium-legume root nodules: Not only a plant globin story

Nitric oxide (NO) is a gaseous signaling molecule which plays both regulatory and defense roles in animals and plants. In the symbiosis between legumes and rhizobia, NO has been shown to be involved in bacterial infection and nodule development steps as well as in mature nodule functioning. We recently showed that an increase in NO level inside Medicago truncatula root nodules also could trigger premature nodule senescence. Here we discuss the importance of the bacterial Sinorhizobium meliloti flavohemoglobin to finely tune the NO level inside nodules and further, we demonstrate that S. meliloti possesses at least two non redundant ways to control NO and that both systems are necessary to maintain efficient nitrogen fixing activity.     

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.     

Genome-Wide Identification and Expression Profiling Analysis of the Aux/IAA Gene Family in Medicago truncatula during the Early Phase of Sinorhizobium meliloti Infection

Background

Auxin/indoleacetic acid (Aux/IAA) genes, coding a family of short-lived nuclear proteins, play key roles in wide variety of plant developmental processes, including root system regulation and responses to environmental stimulus. However, how they function in auxin signaling pathway and symbiosis with rhizobial in Medicago truncatula are largely unknown. The present study aims at gaining deeper insight on distinctive expression and function features of Aux/IAA family genes in Medicago truncatula during nodule formation.

Principal Findings

Using the latest updated draft of the full Medicago truncatula genome, a comprehensive identification and analysis of IAA genes were performed. The data indicated that MtIAA family genes are distributed in all the M. truncatula chromosomes except chromosome 6. Most of MtIAA genes are responsive to exogenous auxin and express in tissues-specific manner. To understand the biological functions of MtIAA genes involved in nodule formation, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expression profiling of MtIAA genes during the early phase of Sinorhizobium meliloti (S. meliloti) infection. The expression patterns of most MtIAA genes were down-regulated in roots and up-regulated in shoots by S. meliloti infection. The differences in expression responses between roots and shoots caused by S. meliloti infection were alleviated by 1-NOA application.

Conclusion

The genome-wide identification, evolution and expression pattern analysis of MtIAA genes were performed in this study. The data helps us to understand the roles of MtIAA-mediated auxin signaling in nodule formation during the early phase of S. meliloti infection.     

Effects of Medicago truncatula Genetic Diversity, Rhizobial Competition, and Strain Effectiveness on the Diversity of a Natural Sinorhizobium Species Community

We investigated the genetic diversity and symbiotic efficiency of 223 Sinorhizobium sp. isolates sampled from a single Mediterranean soil and trapped with four Medicago truncatula lines. DNA molecular polymorphism was estimated by capillary electrophoresis-single-stranded conformation polymorphism and restriction fragment length polymorphism on five loci (IGS_NOD, typA, virB11, avhB11, and the 16S rRNA gene). More than 90% of the rhizobia isolated belonged to the Sinorhizobium medicae species (others belonged to Sinorhizobium meliloti), with different proportions of the two species among the four M. truncatula lines. The S. meliloti population was more diverse than that of S. medicae, and significant genetic differentiation among bacterial populations was detected. Single inoculations performed in tubes with each bacterial genotype and each plant line showed significant bacterium-plant line interactions for nodulation and N₂ fixation levels. Competition experiments within each species highlighted either strong or weak competition among genotypes within S. medicae and S. meliloti, respectively. Interspecies competition experiments showed S. meliloti to be more competitive than S. medicae for nodulation. Although not highly divergent at a nucleotide level, isolates collected from this single soil sample displayed wide polymorphism for both nodulation and N₂ fixation. Each M. truncatula line might influence Sinorhizobium soil population diversity differently via its symbiotic preferences. Our data suggested that the two species did not evolve similarly, with S. meliloti showing polymorphism and variable selective pressures and S. medicae showing traces of a recent demographic expansion. Strain effectiveness might have played a role in the species and genotype proportions, but in conjunction with strain adaptation to environmental factors.     

Identification of Sinorhizobium meliloti Early Symbiotic Genes by Use of a Positive Functional Screen

The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis with its leguminous host plant, alfalfa, following a series of continuous signal exchanges. The complexity of the changes of alfalfa root structures during symbiosis and the amount of S. meliloti genes with unknown functions raised the possibility that more S. meliloti genes may be required for early stages of the symbiosis. A positive functional screen of the entire S. meliloti genome for symbiotic genes was carried out using a modified in vivo expression technology. A group of genes and putative genes were found to be expressed in early stages of the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes were further separated into two groups based on their responses to apigenin, a known nodulation (nod) gene inducer. The group of six genes not inducible by apigenin included the lsrA gene, which is essential for the symbiosis, and the dgkA gene, which is involved in the synthesis of cyclic β-1,2-glucan required for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible genes, most have not been previously characterized in S. meliloti, and none of them belongs to the nod gene family. The identification of this large group of alfalfa root exudate-inducible S. meliloti genes suggests that the interactions in the early stages of the S. meliloti and alfalfa symbiosis could be complex and that further characterization of these genes will lead to a better understanding of the symbiosis.     

Partner choice in Medicago Truncatula–Sinorhizobium symbiosis

In nitrogen-fixing symbiosis, plant sanctions against ineffective bacteria have been demonstrated in previous studies performed on soybean and yellow bush lupin, both developing determinate nodules with Bradyrhizobium sp. strains. In this study, we focused on the widely studied symbiotic association Medicago truncatula–Sinorhizobium meliloti, which forms indeterminate nodules. Using two strains isolated from the same soil and displaying different nitrogen fixation phenotypes on the same fixed plant line, we analysed the existence of both partner choice and plant sanctions by performing split-root experiments. By measuring different parameters such as the nodule number, the nodule biomass per nodule and the number of viable rhizobia per nodule, we showed that M. truncatula is able to select rhizobia based on recognition signals, both before and after the nitrogen fixation step. However, no sanction mechanism, described as a decrease in rhizobia fitness inside the nodules, was detected. Consequently, even if partner choice seems to be widespread among legumes, sanction of non-effective rhizobia might not be universal.     

Determination of nitrogen fixation effectiveness in selected <Emphasis Type="Italic">Medicago truncatula</Emphasis> isolates by measuring nitrogen isotope incorporation into pheophytin

Effectiveness is a term used to describe the input that a bacterial nitrogen-fixing symbiosis makes to plant nitrogen metabolism. In legumes, effectiveness is considered a polymorphic trait where specific interactions between the plant and symbiotic rhizobia contribute to the success of the interaction. Evaluation of effectiveness using model legumes like Medicago truncatula may open new avenues for genetic studies. In previous work, an isotope dilution mass spectrometry method, which uses the effect of nitrogen fixation on the nitrogen isotope composition of chlorophyll in plants grown on ¹⁵N fertilizer as a measure of effectiveness, was developed for estimating the contribution of symbiotic nitrogen fixation to plant nitrogen content. This ¹⁵N-dilution assay was used to evaluate the level of nitrogen fixation effectiveness in three Medicago truncatula lines that have been used as parents in generating recombinant inbred lines. Three Sinorhizobium meliloti strains, USDA 1600, 102F51 and MK506, differ in this measure of effectiveness on three lines of M. truncatula: Jemalong A17, DZA315.16 and F83005.5. Plant–rhizobia combinations grown in two different conditions showed comparable differences in effectiveness.     

Sinorhizobium meliloti succinylated high‐molecular‐weight succinoglycan and the Medicago truncatula LysM receptor‐like kinase MtLYK10 participate independently in symbiotic infection

The formation of nitrogen‐fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen‐fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin‐motif receptor‐like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild‐type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild‐type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan‐defective strains was achieved by in trans rescue with a Nod factor‐deficient S. meliloti mutant. While the Nod factor‐deficient strain was always more abundant inside nodules, the succinoglycan‐deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.     

Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium,but not nitrogen‐fixing rhizobial symbiosis

Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad‐spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen‐associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae‐specific PRR ELONGATION FACTOR‐THERMO UNSTABLE RECEPTOR (EFR), conferring recognition of the bacterial EF‐Tu protein, from Arabidopsis thaliana to the legume Medicago truncatula. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF‐Tu‐derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR‐Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.