Paired Medicago receptors mediate broad-spectrum resistance to nodulation by Sinorhizobium meliloti carrying a species-specific gene.

Plants have evolved the ability to distinguish between symbiotic and pathogenic microbial signals. However, potentially cooperative plant-microbe interactions often abort due to incompatible signaling. The Nodulation Specificity 1 (NS1) locus in the legume Medicago truncatula blocks tissue invasion and root nodule induction by many strains of the nitrogen-fixing symbiont Sinorhizobium meliloti. Controlling this strain-specific nodulation blockade are two genes at the NS1 locus, designated NS1a and NS1b, which encode malectin-like leucine-rich repeat receptor kinases. Expression of NS1a and NS1b is induced upon inoculation by both compatible and incompatible Sinorhizobium strains and is dependent on host perception of bacterial nodulation (Nod) factors. Both presence/absence and sequence polymorphisms of the paired receptors contribute to the evolution and functional diversification of the NS1 locus. A bacterial gene, designated rns1, is required for activation of NS1-mediated nodulation restriction. rns1 encodes a type I-secreted protein and is present in approximately 50% of the nearly 250 sequenced S. meliloti strains but not found in over 60 sequenced strains from the closely related species Sinorhizobium medicae. S. meliloti strains lacking functional rns1 are able to evade NS1-mediated nodulation blockade.

Gene Expression in Nitrogen-Fixing Symbiotic Nodule Cells in Medicago truncatula and Other Nodulating Plants.

Root nodules formed by plants of the nitrogen-fixing clade (NFC) are symbiotic organs that function in the maintenance and metabolic integration of large populations of nitrogen-fixing bacteria. These organs feature unique characteristics and processes, including their tissue organization, the presence of specific infection structures called infection threads, endocytotic uptake of bacteria, symbiotic cells carrying thousands of intracellular bacteria without signs of immune responses, and the integration of symbiont and host metabolism. The early stages of nodulation are governed by a few well-defined functions, which together constitute the common symbiosis-signaling pathway (CSSP). The CSSP activates a set of transcription factors (TFs) that orchestrate nodule organogenesis and infection. The later stages of nodule development require the activation of hundreds to thousands of genes, mostly expressed in symbiotic cells. Many of these genes are only active in symbiotic cells, reflecting the unique nature of nodules as plant structures. Although how the nodule-specific transcriptome is activated and connected to early CSSP-signaling is poorly understood, candidate TFs have been identified using transcriptomic approaches, and the importance of epigenetic and chromatin-based regulation has been demonstrated. We discuss how gene regulation analyses have advanced our understanding of nodule organogenesis, the functioning of symbiotic cells, and the evolution of symbiosis in the NFC.

The Medicago truncatula IEF Gene Is Crucial for the Progression of Bacterial Infection During Symbiosis.

Legumes are able to meet their nitrogen need by establishing nitrogen-fixing symbiosis with rhizobia. Nitrogen fixation is performed by rhizobia, which has been converted to bacteroids, in newly formed organs, the root nodules. In the model legume Medicago truncatula, nodule cells are invaded by rhizobia through transcellular tubular structures called infection threads (ITs) that are initiated at the root hairs. Here, we describe a novel M. truncatula early symbiotic mutant identified as infection-related epidermal factor (ief), in which the formation of ITs is blocked in the root hair cells and only nodule primordia are formed. We show that the function of MtIEF is crucial for the bacterial infection in the root epidermis but not required for the nodule organogenesis. The IEF gene that appears to have been recruited for a symbiotic function after the duplication of a flower-specific gene is activated by the ERN1-branch of the Nod factor signal transduction pathway and independent of the NIN activity. The expression of MtIEF is induced transiently in the root epidermal cells by the rhizobium partner or Nod factors. Although its expression was not detectable at later stages of symbiosis, complementation experiments indicate that MtIEF is also required for the proper invasion of the nodule cells by rhizobia. The gene encodes an intracellular protein of unknown function possessing a coiled-coil motif and a plant-specific DUF761 domain. The IEF protein interacts with RPG, another symbiotic protein essential for normal IT development, suggesting that combined action of these proteins plays a role in nodule infection.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ploidy-dependent changes in the epigenome of symbiotic cells correlate with specific patterns of gene expression.

The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed only in nodules. We show here that the transcriptional waves correlate with growing ploidy levels and have investigated how the epigenome changes during endoreduplication cycles. Differential DNA methylation was found in only a small subset of symbiotic nodule-specific genes, including more than half of the NCR genes, whereas in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes' active or repressed state. On the other hand, expression of nodule-specific genes correlated with ploidy-dependent opening of the chromatin as well as, in a subset of tested genes, with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in the differentiation of symbiotic cells.

Morphotype of bacteroids in different legumes correlates with the number and type of symbiotic NCR peptides.

In legume nodules, rhizobia differentiate into nitrogen-fixing forms called bacteroids, which are enclosed by a plant membrane in an organelle-like structure called the symbiosome. In the Inverted Repeat-Lacking Clade (IRLC) of legumes, this differentiation is terminal due to irreversible loss of cell division ability and is associated with genome amplification and different morphologies of the bacteroids that can be swollen, elongated, spherical, and elongated-branched, depending on the host plant. In Medicago truncatula, this process is orchestrated by nodule-specific cysteine-rich peptides (NCRs) delivered into developing bacteroids. Here, we identified the predicted NCR proteins in 10 legumes representing different subclades of the IRLC with distinct bacteroid morphotypes. Analysis of their expression and predicted sequences establishes correlations between the composition of the NCR family and the morphotypes of bacteroids. Although NCRs have a single origin, their evolution has followed different routes in individual lineages, and enrichment and diversification of cationic peptides has resulted in the ability to impose major morphological changes on the endosymbionts. The wide range of effects provoked by NCRs such as cell enlargement, membrane alterations and permeabilization, and biofilm and vesicle formation is dependent on the amino acid composition and charge of the peptides. These effects are strongly influenced by the rhizobial surface polysaccharides that affect NCR-induced differentiation and survival of rhizobia in nodule cells.

Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula.

Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula-Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix-). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.

From Intracellular Bacteria to Differentiated Bacteroids: Transcriptome and Metabolome Analysis in Aeschynomene Nodules Using the Bradyrhizobium sp. Strain ORS285 bclA Mutant.

Soil bacteria called rhizobia trigger the formation of root nodules on legume plants. The rhizobia infect these symbiotic organs and adopt an intracellular lifestyle within the nodule cells, where they differentiate into nitrogen-fixing bacteroids. Several legume lineages force their symbionts into an extreme cellular differentiation, comprising cell enlargement and genome endoreduplication. The antimicrobial peptide transporter BclA is a major determinant of this process in Bradyrhizobium sp. strain ORS285, a symbiont of Aeschynomene spp. In the absence of BclA, the bacteria proceed until the intracellular infection of nodule cells, but they cannot differentiate into enlarged polyploid and functional bacteroids. Thus, the bclA nodule bacteria constitute an intermediate stage between the free-living soil bacteria and the nitrogen-fixing bacteroids. Metabolomics on whole nodules of Aeschynomene afraspera and Aeschynomene indica infected with the wild type or the bclA mutant revealed 47 metabolites that differentially accumulated concomitantly with bacteroid differentiation. Bacterial transcriptome analysis of these nodules demonstrated that the intracellular settling of the rhizobia in the symbiotic nodule cells is accompanied by a first transcriptome switch involving several hundred upregulated and downregulated genes and a second switch accompanying the bacteroid differentiation, involving fewer genes but ones that are expressed to extremely elevated levels. The transcriptomes further suggested a dynamic role for oxygen and redox regulation of gene expression during nodule formation and a nonsymbiotic function of BclA. Together, our data uncover the metabolic and gene expression changes that accompany the transition from intracellular bacteria into differentiated nitrogen-fixing bacteroids.IMPORTANCE Legume-rhizobium symbiosis is a major ecological process, fueling the biogeochemical nitrogen cycle with reduced nitrogen. It also represents a promising strategy to reduce the use of chemical nitrogen fertilizers in agriculture, thereby improving its sustainability. This interaction leads to the intracellular accommodation of rhizobia within plant cells of symbiotic organs, where they differentiate into nitrogen-fixing bacteroids. In specific legume clades, this differentiation process requires the bacterial transporter BclA to counteract antimicrobial peptides produced by the host. Transcriptome analysis of Bradyrhizobium wild-type and bclA mutant bacteria in culture and in symbiosis with Aeschynomene host plants dissected the bacterial transcriptional response in distinct phases and highlighted functions of the transporter in the free-living stage of the bacterial life cycle.

An anthocyanin marker for direct visualization of plant transformation and its use to study nitrogen-fixing nodule development.

The development and functioning of the nitrogen fixing symbiosis between legume plants and soil bacteria collectively called rhizobia requires continuous chemical dialogue between the partners using different molecules such as flavonoids, lipo-chitooligosaccharides, polysaccharides and peptides. Agrobacterium rhizogenes mediated hairy root transformation of legumes is widely used to study the function of plant genes involved in the process. The identification of transgenic plant tissues is based on antibiotics/herbicide selection and/or the detection of different reporter genes that usually require special equipment such as fluorescent microscopes or destructive techniques and chemicals to visualize enzymatic activity. Here, we developed and efficiently used in hairy root experiments binary vectors containing the MtLAP1 gene driven by constitutive and tissue-specific promoters that facilitate the production of purple colored anthocyanins in transgenic tissues and thus allowing the identification of transformed roots by naked eye. Anthocyanin producing roots were able to establish effective symbiosis with rhizobia. Moreover, it was shown that species-specific allelic variations and a mutation preventing posttranslational acetyl modification of an essential nodule-specific cysteine-rich peptide, NCR169, do not affect the symbiotic interaction of Medicago truncatula cv. Jemalong with Sinorhizobium medicae strain WSM419. Based on the experiments, it could be concluded that it is preferable to use the vectors with tissue-specific promoters that restrict anthocyanin production to the root vasculature for studying biotic interactions of the roots such as symbiotic nitrogen fixation or mycorrhizal symbiosis.

Nodule-Specific Cysteine-Rich Peptides Negatively Regulate Nitrogen-Fixing Symbiosis in a Strain-Specific Manner in Medicago truncatula.

Medicago truncatula shows a high level of specificity when interacting with its symbiotic partner Sinorhizobium meliloti. This specificity is mainly manifested at the nitrogen-fixing stage of nodule development, such that a particular bacterial strain forms nitrogen-fixing nodules (Nod+/Fix+) on one plant genotype but ineffective nodules (Nod+/Fix-) on another. Recent studies have just begun to reveal the underlying molecular mechanisms that control this specificity. The S. meliloti strain A145 induces the formation of Fix+ nodules on the accession DZA315.16 but Fix- nodules on Jemalong A17. A previous study reported that the formation of Fix- nodules on Jemalong A17 by S. meliloti A145 was conditioned by a single recessive allele named Mtsym6. Here we demonstrate that the specificity associated with S. meliloti A145 is controlled by multiple genes in M. truncatula, including NFS1 and NFS2 that encode nodule-specific cysteine-rich (NCR) peptides. The two NCR peptides acted dominantly to block rather than promote nitrogen fixation by S. meliloti A145. These two NCR peptides are the same ones that negatively regulate nitrogen-fixing symbiosis associated with S. meliloti Rm41.

Transcriptomic dissection of Bradyrhizobium sp. strain ORS285 in symbiosis with Aeschynomene spp. inducing different bacteroid morphotypes with contrasted symbiotic efficiency.

To circumvent the paucity of nitrogen sources in the soil legume plants establish a symbiotic interaction with nitrogen-fixing soil bacteria called rhizobia. During symbiosis, the plants form root organs called nodules, where bacteria are housed intracellularly and become active nitrogen fixers known as bacteroids. Depending on their host plant, bacteroids can adopt different morphotypes, being either unmodified (U), elongated (E) or spherical (S). E- and S-type bacteroids undergo a terminal differentiation leading to irreversible morphological changes and DNA endoreduplication. Previous studies suggest that differentiated bacteroids display an increased symbiotic efficiency (E > U and S > U). In this study, we used a combination of Aeschynomene species inducing E- or S-type bacteroids in symbiosis with Bradyrhizobium sp. ORS285 to show that S-type bacteroids present a better symbiotic efficiency than E-type bacteroids. We performed a transcriptomic analysis on E- and S-type bacteroids formed by Aeschynomene afraspera and Aeschynomene indica nodules and identified the bacterial functions activated in bacteroids and specific to each bacteroid type. Extending the expression analysis in E- and S-type bacteroids in other Aeschynomene species by qRT-PCR on selected genes from the transcriptome analysis narrowed down the set of bacteroid morphotype-specific genes. Functional analysis of a selected subset of 31 bacteroid-induced or morphotype-specific genes revealed no symbiotic phenotypes in the mutants. This highlights the robustness of the symbiotic program but could also indicate that the bacterial response to the plant environment is partially anticipatory or even maladaptive. Our analysis confirms the correlation between differentiation and efficiency of the bacteroids and provides a framework for the identification of bacterial functions that affect the efficiency of bacteroids.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.