Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system.

The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes-mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes-mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation.

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.

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.

The bs5 allele of the susceptibility gene Bs5 of pepper (Capsicum annuum L.) encoding a natural deletion variant of a CYSTM protein conditions resistance to bacterial spot disease caused by Xanthomonas species.

KEY MESSAGE: The bs5 resistance gene against bacterial spot was identified by map-based cloning. The recessive bs5 gene of pepper (Capsicum annuum L.) conditions a non-hypersensitive resistance trait, characterized by a slightly swollen, pale green, photosynthetically active leaf tissue, following Xanthomonas euvesicatoria infection. The isolation of the bs5 gene by map-based cloning revealed that the bs5 protein was shorter by 2 amino acids as compared to the wild type Bs5 protein. The natural 2 amino acid deletion occurred in the cysteine-rich transmembrane domain of the tail-anchored (TA) protein, Ca_CYSTM1. The protein products of the wild type Bs5 and mutant bs5 genes were shown to be located in the cell membrane, indicating an unknown function in this membrane compartment. Successful infection of the Bs5 pepper lines was abolished by the 6 bp deletion in the TM encoding domain of the Ca_CYSTM1 gene in bs5 homozygotes, suggesting, that the resulting resistance might be explained by the lack of entry of the Xanthomonas specific effector molecules into the plant cells.

Amino Acid Polymorphisms in the VHIID Conserved Motif of Nodulation Signaling Pathways 2 Distinctly Modulate Symbiotic Signaling and Nodule Morphogenesis in Medicago truncatula.

Legumes establish an endosymbiotic association with nitrogen-fixing soil bacteria. Following the mutual recognition of the symbiotic partner, the infection process is controlled by the induction of the signaling pathway and subsequent activation of symbiosis-related host genes. One of the protein complexes regulating nitrogen-fixing root nodule symbiosis is formed by GRAS domain regulatory proteins Nodulation Signaling Pathways 1 and 2 (NSP1 and NSP2) that control the expression of several early nodulation genes. Here, we report on a novel point mutant allele (nsp2-6) affecting the function of the NSP2 gene and compared the mutant with the formerly identified nsp2-3 mutant. Both mutants carry a single amino acid substitution in the VHIID motif of the NSP2 protein. We found that the two mutant alleles show dissimilar root hair response to bacterial infection. Although the nsp2-3 mutant developed aberrant infection threads, rhizobia were able to colonize nodule cells in this mutant. The encoded NSP2 proteins of the nsp2-3 and the novel nsp2 mutants interact with NSP1 diversely and, as a consequence, the activation of early nodulin genes and nodule organogenesis are arrested in the new nsp2 allele. The novel mutant with amino acid substitution D244H in NSP2 shows similar defects in symbiotic responses as a formerly identified nsp2-2 mutant carrying a deletion in the NSP2 gene. Additionally, we found that rhizobial strains induce delayed nodule formation on the roots of the ns2-3 weak allele. Our study highlights the importance of a conserved Asp residue in the VHIID motif of NSP2 that is required for the formation of a functional NSP1-NSP2 signaling module. Furthermore, our results imply the involvement of NSP2 during differentiation of symbiotic nodule cells.

The Medicago truncatula Vacuolar iron Transporter-Like proteins VTL4 and VTL8 deliver iron to symbiotic bacteria at different stages of the infection process.

The symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron, and questions remain regarding which transporters are involved. Here, we characterize two nodule-specific Vacuolar iron Transporter-Like (VTL) proteins in Medicago truncatula. Localization of fluorescent fusion proteins and mutant studies were carried out to correlate with existing RNA-seq data showing differential expression of VTL4 and VTL8 during early and late infection, respectively. The vtl4 insertion lines showed decreased nitrogen fixation capacity associated with more immature nodules and less elongated bacteroids. A mutant line lacking the tandemly-arranged VTL4-VTL8 genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was almost fully restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a moderate decrease in luminescence signal was observed in vtl4 mutant nodules and a strong decrease in 13U nodules. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. These data indicate that VTL8, the closest homologue of SEN1 in Lotus japonicus, is the main route for delivering iron to symbiotic rhizobia. We propose that a failure in iron protein maturation leads to early senescence of the bacteroids.

Suppression of NB-LRR genes by miRNAs promotes nitrogen-fixing nodule development in Medicago truncatula.

Plant genomes contain two major classes of innate immune receptors to recognize different pathogens. The pattern recognition receptors perceive conserved pathogen-associated molecular patterns and the resistance genes with nucleotide-binding (NB) and leucine-rich repeat (LRR) domains recognize specific pathogen effectors. The precise regulation of resistance genes is important since the unregulated expression of NB-LRR genes can inhibit growth and may result in autoimmunity in the absence of pathogen infection. It was shown that a subset of miRNAs could target NB-LRR genes and act as an important regulator of plant immunity in the absence of pathogens. Plants not only interact with pathogens, but they can also establish symbiotic interactions with microbes. Nitrogen-fixing symbiotic interaction and nodule formation of legumes may also require the suppression of host defence to prevent immune responses. We found that upon symbiotic interactions, miRNAs repressing NB-LRR expression are upregulated in the developing nodules of Medicago truncatula. Furthermore, we show that the suppression of the activity of the NB-LRR genes targeted by these miRNAs is important during nodule development. Our results suggest that the downregulation of NB-LRR resistance genes in the developing nodule produces a suitable niche that facilitates bacterial colonization and the development of an N-fixing nodule.

Functional conservation of CYCLOPS in crack entry legume Arachis hypogaea.

Root nodule symbiosis in legumes is established following interaction of compatible rhizobia that activates an array of genes, commonly known as symbiotic-pathway, resulting in nodule development. In model legumes, bacterial entry mainly occurs through infection thread involving the expression of transcription factor CYCLOPS/IPD3. Here we show the functional analysis of AhCYCLOPS in Arachis hypogaea where bacteria invade roots through epidermal cracks. Exploiting significant cross-species domain conservation, trans-complementation experiments involving ectopic expression of AhCYCLOPS in transgenic hairy-roots of Medicago truncatula ipd3 mutants resulted in functional complementation of Medicago nodules. Moreover, native promoter of AhCYCLOPS was sufficient for this cross-species complementation irrespective of the different modes of infection of roots by rhizobia and nodule ontology. To unravel the role of AhCYCLOPS during 'crack-entry' nodulation in A. hypogaea, RNAi of AhCYCLOPS was performed which resulted in delayed nodule inception followed by drastic reduction in nodule number on transgenic hairy-roots. The infection zone of a significant number of RNAi nodules showed presence of infected cells with enlarged nucleus and rod shaped undifferentiated bacteria. Expression analysis showed downregulation of several nodulation responsible effectors endorsing the compromised condition of RNAi roots. Together, the results indicated that AhCYCLOPS plays an important role in A. hypogaea nodule development.

Independent Regulation of Symbiotic Nodulation by the SUNN Negative and CRA2 Positive Systemic Pathways.

Plant systemic signaling pathways allow the integration and coordination of shoot and root organ metabolism and development at the whole-plant level depending on nutrient availability. In legumes, two systemic pathways have been reported in the Medicago truncatula model to regulate root nitrogen-fixing symbiotic nodulation. Both pathways involve leucine-rich repeat receptor-like kinases acting in shoots and proposed to perceive signaling peptides produced in roots depending on soil nutrient availability. In this study, we characterized in the M. truncatula Jemalong A17 genotype a mutant allelic series affecting the Compact Root Architecture2 (CRA2) receptor. These analyses revealed that this pathway acts systemically from shoots to positively regulate nodulation and is required for the activity of carboxyl-terminally encoded peptides (CEPs). In addition, we generated a double mutant to test genetic interactions of the CRA2 systemic pathway with the CLAVATA3/EMBRYO SURROUNDING REGION peptide (CLE)/Super Numeric Nodule (SUNN) receptor systemic pathway negatively regulating nodule number from shoots, which revealed an intermediate nodule number phenotype close to the wild type. Finally, we showed that the nitrate inhibition of nodule numbers was observed in cra2 mutants but not in sunn and cra2 sunn mutants. Overall, these results suggest that CEP/CRA2 and CLE/SUNN systemic pathways act independently from shoots to regulate nodule numbers.

NMR Metabolomics Defining Genetic Variation in Pea Seed Metabolites.

Nuclear magnetic resonance (NMR) spectroscopy profiling was used to provide an unbiased assessment of changes to the metabolite composition of seeds and to define genetic variation for a range of pea seed metabolites. Mature seeds from recombinant inbred lines, derived from three mapping populations for which there is substantial genetic marker linkage information, were grown in two environments/years and analyzed by non-targeted NMR. Adaptive binning of the NMR metabolite data, followed by analysis of quantitative variation among lines for individual bins, identified the main genomic regions determining this metabolic variability and the variability for selected compounds was investigated. Analysis by t-tests identified a set of bins with highly significant associations to genetic map regions, based on probability (p) values that were appreciably lower than those determined for randomized data. The correlation between bins showing high mean absolute deviation and those showing low p-values for marker association provided an indication of the extent to which the genetics of bin variation might be explained by one or a few loci. Variation in compounds related to aromatic amino acids, branched-chain amino acids, sucrose-derived metabolites, secondary metabolites and some unidentified compounds was associated with one or more genetic loci. The combined analysis shows that there are multiple loci throughout the genome that together impact on the abundance of many compounds through a network of interactions, where individual loci may affect more than one compound and vice versa. This work therefore provides a framework for the genetic analysis of the seed metabolome, and the use of genetic marker data in the breeding and selection of seeds for specific seed quality traits and compounds that have high commercial value.

NAD1 Controls Defense-Like Responses in Medicago truncatula Symbiotic Nitrogen Fixing Nodules Following Rhizobial Colonization in a BacA-Independent Manner.

Legumes form endosymbiotic interaction with host compatible rhizobia, resulting in the development of nitrogen-fixing root nodules. Within symbiotic nodules, rhizobia are intracellularly accommodated in plant-derived membrane compartments, termed symbiosomes. In mature nodule, the massively colonized cells tolerate the existence of rhizobia without manifestation of visible defense responses, indicating the suppression of plant immunity in the nodule in the favur of the symbiotic partner. Medicago truncatulaDNF2 (defective in nitrogen fixation 2) and NAD1 (nodules with activated defense 1) genes are essential for the control of plant defense during the colonization of the nitrogen-fixing nodule and are required for bacteroid persistence. The previously identified nodule-specific NAD1 gene encodes a protein of unknown function. Herein, we present the analysis of novel NAD1 mutant alleles to better understand the function of NAD1 in the repression of immune responses in symbiotic nodules. By exploiting the advantage of plant double and rhizobial mutants defective in establishing nitrogen-fixing symbiotic interaction, we show that NAD1 functions following the release of rhizobia from the infection threads and colonization of nodule cells. The suppression of plant defense is self-dependent of the differentiation status of the rhizobia. The corresponding phenotype of nad1 and dnf2 mutants and the similarity in the induction of defense-associated genes in both mutants suggest that NAD1 and DNF2 operate close together in the same pathway controlling defense responses in symbiotic nodules.

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.

Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant.

Host compatible rhizobia induce the formation of legume root nodules, symbiotic organs within which intracellular bacteria are present in plant-derived membrane compartments termed symbiosomes. In Medicago truncatula nodules, the Sinorhizobium microsymbionts undergo an irreversible differentiation process leading to the development of elongated polyploid noncultivable nitrogen fixing bacteroids that convert atmospheric dinitrogen into ammonia. This terminal differentiation is directed by the host plant and involves hundreds of nodule specific cysteine-rich peptides (NCRs). Except for certain in vitro activities of cationic peptides, the functional roles of individual NCR peptides in planta are not known. In this study, we demonstrate that the inability of M. truncatula dnf7 mutants to fix nitrogen is due to inactivation of a single NCR peptide, NCR169. In the absence of NCR169, bacterial differentiation was impaired and was associated with early senescence of the symbiotic cells. Introduction of the NCR169 gene into the dnf7-2/NCR169 deletion mutant restored symbiotic nitrogen fixation. Replacement of any of the cysteine residues in the NCR169 peptide with serine rendered it incapable of complementation, demonstrating an absolute requirement for all cysteines in planta. NCR169 was induced in the cell layers in which bacteroid elongation was most pronounced, and high expression persisted throughout the nitrogen-fixing nodule zone. Our results provide evidence for an essential role of NCR169 in the differentiation and persistence of nitrogen fixing bacteroids in M. truncatula.

The identification of novel loci required for appropriate nodule development in Medicago truncatula.

BACKGROUND: The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules. RESULTS: Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed. CONCLUSIONS: The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later stages associated with bacterial release and nodule function.

Medicago truncatula IPD3 is a member of the common symbiotic signaling pathway required for rhizobial and mycorrhizal symbioses.

Legumes form endosymbiotic associations with nitrogen-fixing bacteria and arbuscular mycorrhizal (AM) fungi which facilitate nutrient uptake. Both symbiotic interactions require a molecular signal exchange between the plant and the symbiont, and this involves a conserved symbiosis (Sym) signaling pathway. In order to identify plant genes required for intracellular accommodation of nitrogen-fixing bacteria and AM fungi, we characterized Medicago truncatula symbiotic mutants defective for rhizobial infection of nodule cells and colonization of root cells by AM hyphae. Here, we describe mutants impaired in the interacting protein of DMI3 (IPD3) gene, which has been identified earlier as an interacting partner of the calcium/calmodulin-dependent protein, a member of the Sym pathway. The ipd3 mutants are impaired in both rhizobial and mycorrhizal colonization and we show that IPD3 is necessary for appropriate Nod-factor-induced gene expression. This indicates that IPD3 is a member of the common Sym pathway. We observed differences in the severity of ipd3 mutants that appear to be the result of the genetic background. This supports the hypothesis that IPD3 function is partially redundant and, thus, additional genetic components must exist that have analogous functions to IPD3. This explains why mutations in an essential component of the Sym pathway have defects at late stages of the symbiotic interactions.

LIN, a novel type of U-box/WD40 protein, controls early infection by rhizobia in legumes.

The formation of a nitrogen-fixing nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. Based on its mutant phenotype, lumpy infections (lin), LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. We show that spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase is independent of LIN; thus, LIN is not necessary for nodule organogenesis. From this, we infer that LIN predominantly functions during rhizobial colonization and that the abortion of this process in lin mutants leads to a suppression of nodule development. Here, we identify the LIN gene in Medicago truncatula and Lotus japonicus, showing that it codes for a predicted E3 ubiquitin ligase containing a highly conserved U-box and WD40 repeat domains. Ubiquitin-mediated protein degradation is a universal mechanism to regulate many biological processes by eliminating rate-limiting enzymes and key components such as transcription factors. We propose that LIN is a regulator of the component(s) of the nodulation factor signal transduction pathway and that its function is required for correct temporal and spatial activity of the target protein(s).

Abscisic acid coordinates nod factor and cytokinin signaling during the regulation of nodulation in Medicago truncatula.

Nodulation is tightly regulated in legumes to ensure appropriate levels of nitrogen fixation without excessive depletion of carbon reserves. This balance is maintained by intimately linking nodulation and its regulation with plant hormones. It has previously been shown that ethylene and jasmonic acid (JA) are able to regulate nodulation and Nod factor signal transduction. Here, we characterize the nature of abscisic acid (ABA) regulation of nodulation. We show that application of ABA inhibits nodulation, bacterial infection, and nodulin gene expression in Medicago truncatula. ABA acts in a similar manner as JA and ethylene, regulating Nod factor signaling and affecting the nature of Nod factor-induced calcium spiking. However, this action is independent of the ethylene signal transduction pathway. We show that genetic inhibition of ABA signaling through the use of a dominant-negative allele of ABSCISIC ACID INSENSITIVE1 leads to a hypernodulation phenotype. In addition, we characterize a novel locus of M. truncatula, SENSITIVITY TO ABA, that dictates the sensitivity of the plant to ABA and, as such, impacts the regulation of nodulation. We show that ABA can suppress Nod factor signal transduction in the epidermis and can regulate cytokinin induction of the nodule primordium in the root cortex. Therefore, ABA is capable of coordinately regulating the diverse developmental pathways associated with nodule formation and can intimately dictate the nature of the plants' response to the symbiotic bacteria.

Dwarf plants of diploid Medicago sativa carry a mutation in the gibberellin 3-beta-hydroxylase gene.

In this paper we describe the identification of a gene, MsDWF1 coding for a putative gibberellin 3-beta-hydroxylase (GA3ox), whose natural mutation is conditioning a dwarf growth phenotype in Medicago sativa. The dwarf phenotype could not be complemented with grafting, which indicates that the bioactive gibberellin compound necessary for shoot elongation is immobile. On the contrary, exogenously added gibberellic acid restored normal growth. The genetic position of the Msdwf1 gene was mapped to linkage group 2 (LG2) and the physical location was delimited by map-based cloning using Medicago truncatula genomic resources. Based on the similar appearance and behavior of the dwarf Medicago sativa plants to the pea stem length mutant (le) as well as the synthenic map position of the two genes it was postulated that MsDWF1 and pea Le are orthologs. The comparison of wild type and mutant allele sequences of MsGA3ox revealed an amino acid change in a conserved position in the mutant allele, which most probably impaired the function of the enzyme. Our results indicate that the dwarf phenotype was the consequence of this mutation.

An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction.

Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor-induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.