MtESN2 is a subgroup II sulphate transporter required for symbiotic nitrogen fixation and prevention of nodule early senescence in Medicago truncatula.

Adequate distribution of mineral sulphur (S) nutrition to nodules mediated by sulphate transporters is crucial for nitrogen fixation in symbiosis establishment process. However, the molecular mechanisms underlying this process remain largely unknown. In this study, we characterized the function of Early Senescent Nodule 2 (MtESN2), a gene crucial to nitrogen fixation in Medicago truncatula. Mutations in MtESN2 resulted in severe developmental and functional defects including dwarf shoots, early senescent nodules, and lower nitrogenase activity under symbiotic conditions compared to wild-type plants. MtESN2 encodes an M. truncatula sulphate transporter that is expressed only in roots and nodules, with the highest expression levels in the transition zone and nitrogen-fixing zone of nodules. MtESN2 exhibited sulphate transport activity when expressed in yeast. Immunolocalization analysis showed that MtESN2-yellow fluorescent protein fusion protein was localized to the plasma membranes of both uninfected and infected cells of nodules, where it might transport sulphate into both rhizobia-infected and uninfected cells within the nodules. Our results reveal an unreported sulphate transporter that contributes to effective symbiosis and prevents nodule early senescence in M. truncatula.

Nodule-specific cysteine-rich peptide 343 is required for symbiotic nitrogen fixation in Medicago truncatula.

Symbiotic interactions between legumes and rhizobia lead to the development of root nodules and nitrogen fixation by differentiated bacteroids within nodules. Differentiation of the endosymbionts is reversible or terminal, determined by plant effectors. In inverted repeat lacking clade legumes, nodule-specific cysteine-rich (NCR) peptides control the terminal differentiation of bacteroids. Medicago truncatula contains ∼700 NCR-coding genes. However, the role of few NCR peptides has been demonstrated. Here, we report characterization of fast neutron 2106 (FN2106), a symbiotic nitrogen fixation defective (fix-) mutant of M. truncatula. Using a transcript-based approach, together with linkage and complementation tests, we showed that loss-of-function of NCR343 results in impaired bacteroid differentiation and/or maintenance and premature nodule senescence of the FN2106 mutant. NCR343 was specifically expressed in nodules. Subcellular localization studies showed that the functional NCR343-YFP fusion protein colocalizes with bacteroids in symbiosomes in infected nodule cells. Transcriptomic analyses identified senescence-, but not defense-related genes, as being significantly upregulated in ncr343 (FN2106) nodules. Taken together, results from our phenotypic and transcriptomic analyses of a loss-of-function ncr343 mutant demonstrate an essential role of NCR343 in bacteroid differentiation and/or maintenance required for symbiotic nitrogen fixation.

NCR343 is required to maintain the viability of differentiated bacteroids in nodule cells in Medicago truncatula.

Bacteroid (name for rhizobia inside nodule cells) differentiation is a prerequisite for successful nitrogen-fixing symbiosis. In certain legumes, under the regulation of host proteins, for example, a large group of NCR (nodule cysteine rich) peptides, bacteroids undergo irreversible terminal differentiation. This process causes them to lose the ability to propagate inside nodule cells while boosting their competency for nitrogen fixation. How host cells maintain the viability of differentiated bacteroids while maximizing their nitrogen-reducing activities remains elusive. Here, through mutant screen, map-based cloning, and genetic complementation, we find that NCR343 is required for the viability of differentiated bacteroids. In Medicago truncatula debino1 mutant, differentiated bacteroids decay prematurely, and NCR343 is proved to be the casual gene for debino1. NCR343 is mainly expressed in the nodule fixation zone, where bacteroids are differentiated. In nodule cells, mature NCR343 peptide is secreted into the symbiosomes. RNA-Seq assay shows that many stress-responsive genes are significantly induced in debino1 bacteroids. Additionally, a group of stress response-related rhizobium proteins are identified as putative interacting partners of NCR343. In summary, our findings demonstrate that beyond promoting bacteroid differentiation, NCR peptides are also required in maintaining the viability of differentiated bacteroids.

A signal peptide peptidase is required for ER-symbiosome proximal association and protein secretion.

During legume-rhizobia symbiosis, differentiation of the symbiosome (engulfed intracellular rhizobia) is necessary for successful nitrogen fixation. To control symbiosome differentiation, host cell subcellular components, e.g., ER (endoplasmic reticulum), must adapt robustly to ensure large-scale host protein secretion to the new organelle. However, the key components controlling the adaption of ER in nodule cells remain elusive. We report that Medicago BID1, a nodule-specific signal peptide peptidase (SPP), is central to ER structural dynamics and host protein secretion. In bid1, symbiosome differentiation is blocked. BID1 localizes specifically to the ER membrane and expresses exclusively in nodule cells with symbiosomes. In the wild type ER forms proximal association structures with symbiosomes, but not in bid1. Consequently, in bid1 excessive ER stress responses are induced and ER-to-symbiosome protein secretion is impaired. In summary, a nodule-specific SPP is necessary for ER-symbiosome proximal association, host protein secretion, and symbiosome differentiation.

The Medicago truncatula nodule-specific cysteine-rich peptides, NCR343 and NCR-new35 are required for the maintenance of rhizobia in nitrogen-fixing nodules.

In the nodules of IRLC legumes, including Medicago truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen-fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence-related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray- or transcriptome-based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR-new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. We found that the expression of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localized to the symbiotic compartment. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula.

Nitrogen and Phosphorus Signaling and Transport During Legume-Rhizobium Symbiosis.

Nitrogen (N) and phosphorus (P) are the two predominant mineral elements, which are not only essential for plant growth and development in general but also play a key role in symbiotic N fixation in legumes. Legume plants have evolved complex signaling networks to respond to both external and internal levels of these macronutrients to optimize symbiotic N fixation in nodules. Inorganic phosphate (Pi) and nitrate (NO3 -) are the two major forms of P and N elements utilized by plants, respectively. Pi starvation and NO3 - application both reduce symbiotic N fixation via similar changes in the nodule gene expression and invoke local and long-distance, systemic responses, of which N-compound feedback regulation of rhizobial nitrogenase activity appears to operate under both conditions. Most of the N and P signaling and transport processes have been investigated in model organisms, such as Medicago truncatula, Lotus japonicus, Glycine max, Phaseolus vulgaris, Arabidopsis thaliana, Oryza sativa, etc. We attempted to discuss some of these processes wherever appropriate, to serve as references for a better understanding of the N and P signaling and transport during symbiosis.

The genome of a wild Medicago species provides insights into the tolerant mechanisms of legume forage to environmental stress.

BACKGROUND: Medicago ruthenica, a wild and perennial legume forage widely distributed in semi-arid grasslands, is distinguished by its outstanding tolerance to environmental stress. It is a close relative of commonly cultivated forage of alfalfa (Medicago sativa). The high tolerance of M. ruthenica to environmental stress makes this species a valuable genetic resource for understanding and improving traits associated with tolerance to harsh environments. RESULTS: We sequenced and assembled genome of M. ruthenica using an integrated approach, including PacBio, Illumina, 10×Genomics, and Hi-C. The assembled genome was 904.13 Mb with scaffold N50 of 99.39 Mb, and 50,162 protein-coding genes were annotated. Comparative genomics and transcriptomic analyses were used to elucidate mechanisms underlying its tolerance to environmental stress. The expanded FHY3/FAR1 family was identified to be involved in tolerance of M. ruthenica to drought stress. Many genes involved in tolerance to abiotic stress were retained in M. ruthenica compared to other cultivated Medicago species. Hundreds of candidate genes associated with drought tolerance were identified by analyzing variations in single nucleotide polymorphism using accessions of M. ruthenica with varying tolerance to drought. Transcriptomic data demonstrated the involvements of genes related to transcriptional regulation, stress response, and metabolic regulation in tolerance of M. ruthenica. CONCLUSIONS: We present a high-quality genome assembly and identification of drought-related genes in the wild species of M. ruthenica, providing a valuable resource for genomic studies on perennial legume forages.

The Medicago truncatula PIN2 auxin transporter mediates basipetal auxin transport but is not necessary for nodulation.

The development of root nodules leads to an increased auxin response in early nodule primordia, which is mediated by changes in acropetal auxin transport in some legumes. Here, we investigated the role of root basipetal auxin transport during nodulation. Rhizobia inoculation significantly increased basipetal auxin transport in both Medicago truncatula and Lotus japonicus. In M. truncatula, this increase was dependent on functional Nod factor signalling through NFP, NIN, and NSP2, as well as ethylene signalling through SKL. To test whether increased basipetal auxin transport is required for nodulation, we examined a loss-of-function mutant of the M. truncatula PIN2 gene. The Mtpin2 mutant exhibited a reduction in basipetal auxin transport and an agravitropic phenotype. Inoculation of Mtpin2 roots with rhizobia still led to a moderate increase in basipetal auxin transport, but the mutant nodulated normally. No clear differences in auxin response were observed during nodule development. Interestingly, inoculation of wild-type roots increased lateral root numbers, whereas inoculation of Mtpin2 mutants resulted in reduced lateral root numbers compared with uninoculated roots. We conclude that the MtPIN2 auxin transporter is involved in basipetal auxin transport, that its function is not essential for nodulation, but that it plays an important role in the control of lateral root development.

Negative gravitropic response of roots directs auxin flow to control root gravitropism.

Root tip is capable of sensing and adjusting its growth direction in response to gravity, a phenomenon known as root gravitropism. Previously, we have shown that negative gravitropic response of roots (NGR) is essential for the positive gravitropic response of roots. Here, we show that NGR, a plasma membrane protein specifically expressed in root columella and lateral root cap cells, controls the positive root gravitropic response by regulating auxin efflux carrier localization in columella cells and the direction of lateral auxin flow in response to gravity. Pharmacological and genetic studies show that the negative root gravitropic response of the ngr mutants depends on polar auxin transport in the root elongation zone. Cell biology studies further demonstrate that polar localization of the auxin efflux carrier PIN3 in root columella cells and asymmetric lateral auxin flow in the root tip in response to gravistimulation is reversed in the atngr1;2;3 triple mutant. Furthermore, simultaneous mutations of three PIN genes expressed in root columella cells impaired the negative root gravitropic response of the atngr1;2;3 triple mutant. Our work revealed a critical role of NGR in root gravitropic response and provided an insight of the early events and molecular basis of the positive root gravitropism.

A Remote cis-Regulatory Region Is Required for NIN Expression in the Pericycle to Initiate Nodule Primordium Formation in Medicago truncatula.

The legume-rhizobium symbiosis results in nitrogen-fixing root nodules, and their formation involves both intracellular infection initiated in the epidermis and nodule organogenesis initiated in inner root cell layers. NODULE INCEPTION (NIN) is a nodule-specific transcription factor essential for both processes. These NIN-regulated processes occur at different times and locations in the root, demonstrating a complex pattern of spatiotemporal regulation. We show that regulatory sequences sufficient for the epidermal infection process are located within a 5 kb region directly upstream of the NIN start codon in Medicago truncatula Furthermore, we identify a remote upstream cis-regulatory region required for the expression of NIN in the pericycle, and we show that this region is essential for nodule organogenesis. This region contains putative cytokinin response elements and is conserved in eight more legume species. Both the cytokinin receptor 1, which is essential for nodule primordium formation, and the B-type response regulator RR1 are expressed in the pericycle in the susceptible zone of the uninoculated root. This, together with the identification of the cytokinin-responsive elements in the NIN promoter, strongly suggests that NIN expression is initially triggered by cytokinin signaling in the pericycle to initiate nodule primordium formation.

Sodium-Related Adaptations to Drought: New Insights From the Xerophyte Plant Zygophyllum xanthoxylum.

Understanding the unusual physiological mechanisms that enable drought tolerance in xerophytes will be of considerable benefit because of the potential to identify novel and key genetic elements for future crop improvements. These plants are interesting because they are well-adapted for life in arid zones; Zygophyllum xanthoxylum, for example, is a typical xerophytic shrub that inhabits central Asian deserts, accumulating substantial levels of sodium (Na+) in its succulent leaves while growing in soils that contain very low levels of this ion. The physiological importance of this unusual trait to drought adaptations remains poorly understood, however. Thus, 2-week-old Z. xanthoxylum plants were treated with 50 mM NaCl (Na) for 7 days in this study in order to investigate their drought tolerance, leaf osmotic potential (Ψs) related parameters, anatomical characteristics, and transpiration traits. The results demonstrated that NaCl treatment significantly enhanced both the survivability and durability of Z. xanthoxylum plants under extreme drought conditions. The bulk of the Na+ ions encapsulated in plants was overwhelmingly allocated to leaves rather than roots or stems under drought conditions; thus, compared to the control, significantly more Na+ compared to other solutes such as K+, Ca2+, Cl-, sugars, and proline accumulated in the leaves of NaCl-treated plants and led to a marked decrease (31%) in leaf Ψs. In addition, the accumulation of Na+ ions also resulted in mesophyll cell enlargement and leaf succulence, enabling the additional storage of water; Na+ ions also reduced the rate of water loss by decreasing stomatal density and down-regulating stomatal aperture size. The results of this study demonstrate that Z. xanthoxylum has evolved a notable ability to utilize Na+ ions to lower Ψs, swell its leaves, and decrease stomatal aperture sizes, in order to enable the additional uptake and storage of water and mitigate losses. These distinctive drought adaption characteristics mean that the xerophytic plant Z. xanthoxylum presents a fascinating case study for the potential identification of important and novel genetic elements that could improve crops. This report provides insights on the eco-physiological role of sodium accumulation in xerophytes adapted to extremely arid habitats.

Physical Mutagenesis in Medicago truncatula Using Fast Neutron Bombardment (FNB) for Symbiosis and Developmental Biology Studies.

Medicago truncatula has been selected as a model species for legume molecular genetics and functional genomics studies. With the completion of the Medicago truncatula cv. Jemalong A17 genome sequencing, a major challenge is to determine the function of the large number of genes in the genome. Development of diverse mutant resources is crucial for gene functional studies. In the past years, M2 seeds from over 150,000 Medicago truncatula mutant lines in the Jemalong A17 background have been generated coordinately at the Noble Research Institute, USA, and the John Innes Centre, UK, using fast neutron bombardment (FNB) mutagenesis. These mutant resources have been used in screening and characterization of different categories of mutants including symbiotic nitrogen fixation, nodule development, and growth and patterning of leaf, stem, and root system architecture in the legume system. Here, we describe the detail procedure that has been used for screening of mutants derived from fast neutron bombardment mutagenesis in Medicago truncatula.

Functional Genomics and Genetic Control of Compound Leaf Development in Medicago truncatula: An Overview.

Diverse forms of leaves are present in nature. However, the regulatory mechanisms that underpin the development of diverse leaf forms remain enigmatic. The initiation of leaf primordia from the periphery of shoot apical meristem (SAM) requires downregulation of the class 1 knotted-like homeobox KNOXI proteins. In plants with simple leaves, this downregulation is permanent, consistent with leaves being determinant organs. In most of plants with compound leaves, the KNOXI proteins are reactivated in developing leaf primordia, and this reactivation is required for the development of compound leaves in these plants. Surprisingly, in Medicago truncatula and pea (Pisum sativum) that belong to the so-called inverted repeat-lacking clade (IRLC) of legume plants, the KNOXI proteins are not reactivated in leaf primordia and therefore not likely involved in the development of compound leaves in these plants. Instead, the legume FLORICAULA/LEAFY orthologues, UNIFOLIATA (UNI) and SINGLE LEAFLET1 (SGL1), are required for the initiation and development of lateral leaflet primordia in pea and M. truncatula plants, respectively. On the other hand, PALMATE-LIKE PENTAFOLIATA1 (PALM1) encoding a novel Cys(2)His(2) zinc finger transcription factor is required to suppress a morphogenetic activity at the leaf margin by negatively regulating SGL1 gene expression, and FUSED COMPOUND LEAF1 (FCL1) encoding a class M KNOX protein is required for the development of the leaf proximo-distal axis and organ boundary separation in M. truncatula. Thus, these recent studies have shown that SGL1/UNI, FCL1, and PALM1 provide a genetic framework for our understanding of compound leaf development in the legume plants.

Novel phosphate deficiency-responsive long non-coding RNAs in the legume model plant Medicago truncatula.

Emerging evidence indicates that long non-coding RNAs (lncRNAs) play important roles in the regulation of many biological processes. Inhibition of plant growth due to deficiency in soil inorganic phosphate (Pi) occurs widely across natural and agricultural ecosystems; however, we know little about the function of plant lncRNAs in response to Pi deficiency. To address this issue, we first identified 10 785 lncRNAs in the legume model species Medicago truncatula by sequencing eight strand-specific libraries. Out of these lncRNAs, 358 and 224 were responsive to Pi deficiency in the leaves and roots, respectively. We further predicted and classified the putative targets of those lncRNAs and the results revealed that they may be involved in the processes of signal transduction, energy synthesis, detoxification, and Pi transport. Finally, we functionally characterized three Phosphate Deficiency-Induced LncRNAs (PDILs) using their corresponding Tnt1 mutants. The results showed that PDIL1 suppressed degradation of MtPHO2, which encodes a ubiquitin-conjugating E2 enzyme regulated by miR399, while PDIL2 and PDIL3 directly regulated Pi transport at the transcriptional level. These findings demonstrate that PDILs can regulate Pi-deficiency signaling and Pi transport, highlighting the involvement of lncRNAs in the regulation of responses of plants to Pi deficiency.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants.

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

AUXIN RESPONSE FACTOR3 Regulates Compound Leaf Patterning by Directly Repressing PALMATE-LIKE PENTAFOLIATA1 Expression in Medicago truncatula.

Diverse leaf forms can be seen in nature. In Medicago truncatula, PALM1 encoding a Cys(2)His(2) transcription factor is a key regulator of compound leaf patterning. PALM1 negatively regulates expression of SGL1, a key regulator of lateral leaflet initiation. However, how PALM1 itself is regulated is not yet known. To answer this question, we used promoter sequence analysis, yeast one-hybrid tests, quantitative transcription activity assays, ChIP-PCR analysis, and phenotypic analyses of overexpression lines and mutant plants. The results show that M. truncatula AUXIN RESPONSE FACTOR3 (MtARF3) functions as a direct transcriptional repressor of PALM1. MtARF3 physically binds to the PALM1 promoter sequence in yeast cells. MtARF3 selectively interacts with specific auxin response elements (AuxREs) in the PALM1 promoter to repress reporter gene expression in tobacco leaves and binds to specific sequences in the PALM1 promoter in vivo. Upregulation of MtARF3 or removal of both PHANTASTICA (PHAN) and ARGONAUTE7 (AGO7) pathways resulted in compound leaves with five narrow leaflets arranged in a palmate-like configuration. These results support that MtARF3, in addition as an adaxial-abaxial polarity regulator, functions to restrict spatiotemporal expression of PALM1, linking auxin signaling to compound leaf patterning in the legume plant M. truncatula.

LeafletAnalyzer, an Automated Software for Quantifying, Comparing and Classifying Blade and Serration Features of Compound Leaves during Development, and among Induced Mutants and Natural Variants in the Legume Medicago truncatula.

Diverse leaf forms ranging from simple to compound leaves are found in plants. It is known that the final leaf size and shape vary greatly in response to developmental and environmental changes. However, changes in leaf size and shape have been quantitatively characterized only in a limited number of species. Here, we report development of LeafletAnalyzer, an automated image analysis and classification software to analyze and classify blade and serration characteristics of trifoliate leaves in Medicago truncatula. The software processes high quality leaf images in an automated or manual fashion to generate size and shape parameters for both blades and serrations. In addition, it generates spectral components for each leaflets using elliptic Fourier transformation. Reconstruction studies show that the spectral components can be reliably used to rebuild the original leaflet images, with low, and middle and high frequency spectral components corresponding to the outline and serration of leaflets, respectively. The software uses artificial neutral network or k-means classification method to classify leaflet groups that are developed either on successive nodes of stems within a genotype or among genotypes such as natural variants and developmental mutants. The automated feature of the software allows analysis of thousands of leaf samples within a short period of time, thus facilitating identification, comparison and classification of leaf groups based on leaflet size, shape and tooth features during leaf development, and among induced mutants and natural variants.

Increasing seed size and quality by manipulating BIG SEEDS1 in legume species.

Plant organs, such as seeds, are primary sources of food for both humans and animals. Seed size is one of the major agronomic traits that have been selected in crop plants during their domestication. Legume seeds are a major source of dietary proteins and oils. Here, we report a conserved role for the BIG SEEDS1 (BS1) gene in the control of seed size and weight in the model legume Medicago truncatula and the grain legume soybean (Glycine max). BS1 encodes a plant-specific transcription regulator and plays a key role in the control of the size of plant organs, including seeds, seed pods, and leaves, through a regulatory module that targets primary cell proliferation. Importantly, down-regulation of BS1 orthologs in soybean by an artificial microRNA significantly increased soybean seed size, weight, and amino acid content. Our results provide a strategy for the increase in yield and seed quality in legumes.

Negative gravitropism in plant roots.

Plants are capable of orienting their root growth towards gravity in a process termed gravitropism, which is necessary for roots to grow into soil, for water and nutrient acquisition and to anchor plants. Here we show that root gravitropism depends on the novel protein, NEGATIVE GRAVITROPIC RESPONSE OF ROOTS (NGR). In both Medicago truncatula and Arabidopsis thaliana, loss of NGR reverses the direction of root gravitropism, resulting in roots growing upward.

An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis.

In the nitrogen-fixing symbiosis between legume hosts and rhizobia, the bacteria are engulfed by a plant cell membrane to become intracellular organelles. In the model legume Medicago truncatula, internalization and differentiation of Sinorhizobium (also known as Ensifer) meliloti is a prerequisite for nitrogen fixation. The host mechanisms that ensure the long-term survival of differentiating intracellular bacteria (bacteroids) in this unusual association are unclear. The M. truncatula defective nitrogen fixation4 (dnf4) mutant is unable to form a productive symbiosis, even though late symbiotic marker genes are expressed in mutant nodules. We discovered that in the dnf4 mutant, bacteroids can apparently differentiate, but they fail to persist within host cells in the process. We found that the DNF4 gene encodes NCR211, a member of the family of nodule-specific cysteine-rich (NCR) peptides. The phenotype of dnf4 suggests that NCR211 acts to promote the intracellular survival of differentiating bacteroids. The greatest expression of DNF4 was observed in the nodule interzone II-III, where bacteroids undergo differentiation. A translational fusion of DNF4 with GFP localizes to the peribacteroid space, and synthetic NCR211 prevents free-living S. meliloti from forming colonies, in contrast to mock controls, suggesting that DNF4 may interact with bacteroids directly or indirectly for its function. Our findings indicate that a successful symbiosis requires host effectors that not only induce bacterial differentiation, but also that maintain intracellular bacteroids during the host-symbiont interaction. The discovery of NCR211 peptides that maintain bacterial survival inside host cells has important implications for improving legume crops.