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Key message The bs5 resistance gene against bacterial spot was identified by map-based cloning. Abstract 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.

SummaryIn the nodules of Inverted Repeat-Lacking Clade legumes, includingM. truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialised for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which about 700 are encoded in theM. truncatulagenome. Some of these NCR peptides, NCR169, NCR211 and NCR247, are essential for nitrogen-fixing symbiosis.The analysis of bacteroid and symbiotic host cell differentiation revealed that the symbiotic phenotype ofM. truncatulamutants,Mtsym19,Mtsym20and NF-FN9363, were defective likewise in the formerly studiedncrmutants,Mtdnf4-1andMtdnf7-2. The incomplete differentiation of bacteroids triggered premature senescence of rhizobia in the nitrogen fixation zones of mutant nodules.Mtsym19andMtsym20mutants are defective in the same peptide NCR-new35 and the lack ofNCR343is responsible for the ineffective symbiosis of NF-FN9363.The activity ofNCR-new35is significantly lower and limited to the transition zone of the nodule compared with other crucialNCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localize to the symbiotic compartment. Our discovery added two additional members to the group ofNCRgenes essential for nitrogen–fixing symbiosis inM. 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.

Hybrid tomato plants being heterozygous for certain induced loss-of-function mutations of the SP3D gene displayed fruit-yield heterosis. In order to investigate the possible occurrence of spontaneous mutations of that kind, the SP3D amplicon of 262 tomato seed bank accessions was deep sequenced at about 2000-fold coverage. Compared to the SL2.50 genome assembly, twenty-two novel SNPs in total were identified in the 652 bp upstream, 3817 bp coding and 613 bp downstream regions of the gene. Neither novel exonic mutations in the 262 accessions of this study nor possible loss-of-function mutations amongst the known exonic variants were identified in the SP3D gene of Solanum lycopersicum indicating that, very likely, no such spontaneous mutations occur in cultivated tomato. In wild tomato, however, there are missense variations in the coding region of SP3D, which might result in disabled SP3D proteins. This difference between cultivated and wild tomatoes might have reproductive-biological reasons, while in cultivated tomato missense SP3D mutations might have been eliminated during breeding because they could result in extremely late flowering phenotype. In the promoter region of the SP3D gene, two SNPs, of which one was novel, were identified in the − 62 and − 61 positions upstream of the ATG start codon. These positions were genotyped in the 262 accessions and, comparing our results with Ensembl Plants data, it could be concluded that the dinucleotide is a conserved CT in Solanum lycopersicum varieties, while it is either CA or TA in other Solanum species. This dinucleotide is in a predicted binding site of BELL homeodomain transcription factor(s), regulating meristem, fruit and inflorescence development, thus it might be an element of the regulatory cascade in which the SP3D gene is involved to regulate flowering in tomato. We presented a hypothesis that the SP3D and Single Flower Truss (SFT) genes might not be the same, as it is currently considered, and that SFT might be equivalent to the gene Solyc03g071710 encoding a putative Self-pruning interacting protein. This would logically explain both the absence of natural loss-of-function mutations in the SP3D gene and the absence of DNA lesions in the SP3D gene of sft mutant tomato.

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., Nitrogen‐fixing symbiotic interaction and nodule formation of legumes require the suppression of host defenses by differential expression of NB‐LRR‐regulating miRNAs to prevent immune responses.

SummaryThe symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron. The host plant is known to provide iron to developing bacteroids, but questions remain regarding which transporters are involved. Here, we characterize two Vacuolar Iron Transporter-Like (VTL) proteins in Medicago truncatula that are specifically expressed during nodule development. VTL4 is mostly expressed during early infection and the protein was localized to membranes and the infection thread. vtl4 mutants were delayed in nodule development. VTL8 is closely related to SEN1 in Lotus japonicus and expressed in the late stages of bacteroid differentiation. The VTL8 protein was localized to the symbiosome membrane. 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 restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a slight decrease in luminescence was observed in vtl4 mutants and a strong decrease in the 13U mutant. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. Taken together, these data indicate that VTL-type transporters are the main route for delivering iron to symbiotic rhizobia.

International audience; 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.

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.

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.

International audience; 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 truncatula DNF2 (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.

Nitrogen is a limiting factor for plant growth. Most crops obtain their nitrogen through the use of nitrogen-based fertilizers, which is costly, and also causes environmental pollution. Legumes, however, have the unique ability to fix atmospheric nitrogen through symbioses with nitrogen-fixing bacteria. Although legumes can be nodulated by indigenous soil bacteria, nitrogen fixation efficiency differs significantly depending on host and bacterial genotypes. Understanding the genetic mechanisms that underlie this specificity will allow for optimizing symbiotic partnerships with improved symbiotic performance. We report that specific nodule-specific cysteine-rich (NCR) peptides negatively regulate symbiotic persistence in a strain-specific manner in Medicago truncatula. This finding offers a strategy to improve nitrogen fixation efficiency through selection or manipulation of NCR alleles that favor specific bacterial strains.