Cécile Ben

Whole-genome nucleotide diversity, recombination, and linkage disequilibrium in the model legume Medicago truncatula

Medicago truncatula is a model for investigating legume genetics, including the genetics and evolution of legume–rhizobia symbiosis. We used whole-genome sequence data to identify and characterize sequence polymorphisms and linkage disequilibrium (LD) in a diverse collection of 26 M. truncatula accessions. Our analyses reveal that M. truncatula harbors both higher diversity and less LD than soybean (Glycine max) and exhibits patterns of LD and recombination similar to Arabidopsis thaliana. The population-scaled recombination rate is approximately one-third of the mutation rate, consistent with expectations for a species with a high selfing rate. Linkage disequilibrium, however, is not extensive, and therefore, the low recombination rate is likely not a major constraint to adaptation. Nucleotide diversity in 100-kb windows was negatively correlated with gene density, which is expected if diversity is shaped by selection acting against slightly deleterious mutations. Among putative coding regions, members of four gene families harbor significantly higher diversity than the genome-wide average. Three of these families are involved in resistance against pathogens; one of these families, the nodule-specific, cysteine-rich gene family, is specific to the galegoid legumes and is involved in control of rhizobial differentiation. The more than 3 million SNPs that we detected, approximately one-half of which are present in more than one accession, are a valuable resource for genome-wide association mapping of genes responsible for phenotypic diversity in legumes, especially traits associated with symbiosis and nodulation.

Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt

Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other crops. The model legume Medicago truncatula was used as a host for studying resistance and susceptibility to Verticillium albo-atrum. In addition to presenting well-established genetic resources, this wild plant species enables to investigate biodiversity of the response to the pathogen and putative crosstalk between disease and symbiosis. Symptom scoring after root inoculation and modelling of disease curves allowed assessing susceptibility levels in recombinant lines of three crosses between susceptible and resistant lines, in a core collection of 32 lines, and in mutants affected in symbiosis with rhizobia. A GFP-expressing V. albo-atrum strain was used to study colonization of susceptible plants. Symptoms and colonization pattern in infected M. truncatula plants were typical of Verticillium wilt. Three distinct major quantitative trait loci were identified using a multicross, multisite design, suggesting that simple genetic mechanisms appear to control Verticillium wilt resistance in M. truncatula lines A17 and DZA45.5. The disease functional parameters varied largely in lines of the core collection. This biodiversity with regard to disease response encourages the development of association genetics and ecological approaches. Several mutants of the resistant line, impaired in different steps of rhizobial symbiosis, were affected in their response to V. albo-atrum, which suggests that mechanisms involved in the establishment of symbiosis or disease might have some common regulatory control points.

The small RNA diversity from Medicago truncatula roots under biotic interactions evidences the environmental plasticity of the miRNAome

BackgroundLegume roots show a remarkable plasticity to adapt their architecture to biotic and abiotic constraints, including symbiotic interactions. However, global analysis of miRNA regulation in roots is limited, and a global view of the evolution of miRNA-mediated diversification in different ecotypes is lacking.ResultsIn the model legume Medicago truncatula, we analyze the small RNA transcriptome of roots submitted to symbiotic and pathogenic interactions. Genome mapping and a computational pipeline identify 416 miRNA candidates, including known and novel variants of 78 miRNA families present in miRBase. Stringent criteria of pre-miRNA prediction yield 52 new mtr-miRNAs, including 27 miRtrons. Analyzing miRNA precursor polymorphisms in 26 M. truncatula ecotypes identifies higher sequence polymorphism in conserved rather than Medicago-specific miRNA precursors. An average of 19 targets, mainly involved in environmental responses and signalling, is predicted per novel miRNA. We identify miRNAs responsive to bacterial and fungal pathogens or symbionts as well as their related Nod and Myc-LCO symbiotic signals. Network analyses reveal modules of new and conserved co-expressed miRNAs that regulate distinct sets of targets, highlighting potential miRNA-regulated biological pathways relevant to pathogenic and symbiotic interactions.ConclusionsWe identify 52 novel genuine miRNAs and large plasticity of the root miRNAome in response to the environment, and also in response to purified Myc/Nod signaling molecules. The new miRNAs identified and their sequence variation across M. truncatula ecotypes may be crucial to understand the adaptation of root growth to the soil environment, notably in the agriculturally important legume crops.

MtQRRS1, an R-locus required for Medicago truncatula quantitative resistance to Ralstonia solanacearum.

Ralstonia solanacearum is a major soilborne pathogen that attacks > 200 plant species, including major crops. To characterize MtQRRS1, a major quantitative trait locus (QTL) for resistance towards this bacterium in the model legume Medicago truncatula, genetic and functional approaches were combined. QTL analyses together with disease scoring of heterogeneous inbred families were used to define the locus. The candidate region was studied by physical mapping using a bacterial artificial chromosome (BAC) library of the resistant line, and sequencing. In planta bacterial growth measurements, grafting experiments and gene expression analysis were performed to investigate the mechanisms by which this locus confers resistance to R. solanacearum. The MtQRRS1 locus was localized to the same position in two recombinant inbred line populations and was narrowed down to a 64 kb region. Comparison of parental line sequences revealed 15 candidate genes with sequence polymorphisms, but no evidence of differential gene expression upon infection. A role for the hypocotyl in resistance establishment was shown. These data indicate that the quantitative resistance to bacterial wilt conferred by MtQRRS1, which contains a cluster of seven R genes, is shared by different accessions and may act through intralocus interactions to promote resistance.

Naturally occurring diversity helps to reveal genes of adaptive importance in legumes

Environmental changes challenge plants and drive adaptation to new conditions, suggesting that natural biodiversity may be a source of adaptive alleles acting through phenotypic plasticity and/or micro-evolution. Crosses between accessions differing for a given trait have been the most common way to disentangle genetic and environmental components. Interestingly, such man-made crosses may combine alleles that never meet in nature. Another way to discover adaptive alleles, inspired by evolution, is to survey large ecotype collections and to use association genetics to identify loci of interest. Both of these two genetic approaches are based on the use of biodiversity and may eventually help us in identifying the genes that plants use to respond to challenges such as short-term stresses or those due to global climate change. In legumes, two wild species, Medicago truncatula and Lotus japonicus, plus the cultivated soybean (Glycine max) have been adopted as models for genomic studies. In this review, we will discuss the resources, limitations and future plans for a systematic use of biodiversity resources in model legumes to pinpoint genes of adaptive importance in legumes, and their application in breeding.

Quantitative Resistance to Verticillium Wilt in Medicago truncatula Involves Eradication of the Fungus from Roots and Is Associated with Transcriptional Responses Related to Innate Immunity

Resistance mechanisms to Verticillium wilt are well-studied in tomato, cotton, and Arabidopsis, but much less in legume plants. Because legume plants establish nitrogen-fixing symbioses in their roots, resistance to root-attacking pathogens merits particular attention. The interaction between the soil-borne pathogen Verticillium alfalfae and the model legume Medicago truncatula was investigated using a resistant (A17) and a susceptible (F83005.5) line. As shown by histological analyses, colonization by the pathogen was initiated similarly in both lines. Later on, the resistant line A17 eliminated the fungus, whereas the susceptible F83005.5 became heavily colonized. Resistance in line A17 does not involve homologs of the well-characterized tomato Ve1 and V. dahliae Ave1 genes. A transcriptomic study of early root responses during initial colonization (i.e., until 24 h post-inoculation) similarly was performed. Compared to the susceptible line, line A17 displayed already a significantly higher basal expression of defense-related genes prior to inoculation, and responded to infection with up-regulation of only a small number of genes. Although fungal colonization was still low at this stage, the susceptible line F83005.5 exhibited a disorganized response involving a large number of genes from different functional classes. The involvement of distinct phytohormone signaling pathways in resistance as suggested by gene expression patterns was supported by experiments with plant hormone pretreatment before fungal inoculation. Gene co-expression network analysis highlighted five main modules in the resistant line, whereas no structured gene expression was found in the susceptible line. One module was particularly associated to the inoculation response in A17. It contains the majority of differentially expressed genes, genes associated with PAMP perception and hormone signaling, and transcription factors. An in silico analysis showed that a high number of these genes also respond to other soil-borne pathogens in M. truncatula, suggesting a core of transcriptional response to root pathogens. Taken together, the results suggest that resistance in M. truncatula line A17 might be due to innate immunity combining preformed defense and PAMP-triggered defense mechanisms, and putative involvement of abscisic acid.

Cell length instead of cell number becomes the predominant factor contributing to hypocotyl length genotypic differences under abiotic stress in Medicago truncatula

Hypocotyl elongation in the dark is a crucial process to ensure seedling emergence. It relies both on the cell number and cell length. The contribution of these two factors to the maximal hypocotyl length and the impact of environmental conditions on this contribution are not known. This is surprising considering the agronomic and economical importance of seedling emergence in crop establishment. Using 14 genotypes from a nested core collection representing Medicago truncatula (barrel medic) natural variation, we investigated how epidermal cell number and cell length contribute to hypocotyl length under optimal, low temperature (8°C) and water deficit (-0.50 MPa) conditions. Both cell number and length vary according to genotypes and contribute to maximal hypocotyl length differences between genotypes. This contribution, however, depends on growth conditions. Cell number is the major contributor under optimal conditions (60%) whereas cell length becomes the major determinant under stress. Maximal hypocotyl length is correlated with hypocotyl elongation rate under both stresses but not under optimal condition, revealing contrasted genotypes for cell elongation capacity under stress. To identify the genetic regulators determining cell number and cell length, quantitative trait loci (QTLs) were detected using a recombinant inbred lines population exhibiting segregation in maximal hypocotyl length. Two QTLs controlling cell number and three QTLs controlling cell length at low temperature were detected. One QTL for cell number and two for cell length were found to be associated with hypocotyl length under low temperature. This study provides new information to improve seedling emergence under abiotic stress.

Distinct Lotus japonicus Transcriptomic Responses to a Spectrum of Bacteria Ranging From Symbiotic to Pathogenic.

Lotus japonicus is a well-studied nodulating legume and a model organism for the investigation of plant-microbe interactions. The majority of legume transcriptome studies have focused on interactions with compatible symbionts, whereas responses to non-adapted rhizobia and pathogenic bacteria have not been well-characterized. In this study, we first characterized the transcriptomic response of L. japonicus to its compatible symbiont, Mesorhizobium loti R7A, through RNA-seq analysis of various plant tissues. Early symbiotic signaling was largely Nod factor-dependent and enhanced within root hairs, and we observed large-scale transcriptional reprogramming in nodule primordia and mature nitrogen-fixing nodules. We then characterized root transcriptional responses to a spectrum of L. japonicus interacting bacteria ranging from semi-compatible symbionts to pathogens. M. loti R7A and the semi-compatible strain Sinorhizobium fredii HH103 showed remarkably similar responses, allowing us to identify a small number of genes potentially involved in differentiating between fully and semi-compatible symbionts. The incompatible symbiont Bradyrhizobium elkanii USDA61 induced a more attenuated response, but the weakest response was observed for the foliar pathogen Pseudomonas syringae pv. tomato DC3000, where the affected genes also responded to other tested bacteria, pointing to a small set of common bacterial response genes. In contrast, the root pathogen Ralstonia solanacearum JS763 induced a pronounced and distinct transcriptomic pathogen response, which we compared to the results of the other treatments. This comparative analysis did not support the concept that an early defense-like response is generally evoked by compatible rhizobia during establishment of symbiosis.

Proteomics analysis of Medicago truncatula response to infection by the phytopathogenic bacterium Ralstonia solanacearum points to jasmonate and salicylate defence pathways

The infection of the model legume Medicago truncatula with Ralstonia solanacearum GMI1000 gives rise to bacterial wilt disease via colonisation of roots. The root and leaf responses to early infection (1 and 3 days post infection) were characterised to investigate the molecular mechanisms of plant resistance or susceptibility. A proteomics approach based on pools of susceptible and resistant recombinant inbred lines was used to specifically target the mechanisms for tolerance. Differential abundances were evidenced for proteins involved in defence (e.g., PR5, PR10, or Kunitz protease inhibitors) and signalling pathways (such as cyclophilin). R. solanacearum inoculation modifies expression levels of those genes, either in both genotypes (AOS1, LOX4, and proteinase inhibitors) or specifically in the resistant line (PR proteins). Exogenous application of salicylic acid (SA) enhanced tolerance to the bacteria, whereas methyl jasmonate (MeJA) enhanced short‐term tolerance then promoted disease in the susceptible ecotype, suggesting that they may mediate defence responses. Conversely, proteomics‐identified genes were also shown to be SA or MeJA responsive. This is the first description of differential response to R. solanacearum in M. truncatula. Our results suggest that root basal defence is activated at 1 dpi, together with the JA pathway. Specific resistance is then evidenced at three dpi, with the up‐regulation of SA‐dependent PR proteins.

Genetic determinism of reproductive fitness traits under drought stress in the model legume Medicago truncatula

Fitness traits that determine the reproductive ability of individuals and the persistence of populations are affected by drought stress. Medicago truncatula that commonly encounters drought stress in its natural area, and for which large natural diversity and genetic tools are available, is a suitable species to investigate genetic determinism of fitness traits under stress. In a common garden, three successive cycles of short drought stress were applied after flowering, during the reproductive stage that is the most susceptible to drought for that species. Ten genotypes derived from natural populations and a mapping population were used to investigate the genetic determinism of vegetative and reproductive traits as components of fitness. A large genetic variation was observed and transgressive genotypes (more resistant or more susceptible than the parental genotypes) were found in the mapping population. Fitness traits were reduced by 5–74% in drought condition compared to well-watered condition. The most affected characters were total pod number per plant and total pod weight per plant. A total of 49 QTL, explaining between 6 and 38% of phenotypic variation for vegetative and reproductive fitness traits, were detected on all chromosomes except chromosome 6. A major QTL for flowering date (R2 of 19 and 38%) that co-located with QTL for reproductive fitness traits were found on chromosome 7. In this study, no major QTL specific to drought-stressed or well-watered conditions were detected. We, thus, showed that QTL explaining fitness traits were numerous with small effects, in accordance with the genetic determinism of a complex trait.