Monitoring strategy of COVID-19 vaccination in dialysis patients based on a multiplex immunodot method: The CovidDial study.

INTRODUCTION: COVID-19 vaccine was demonstrated to be effective in dialysis patients, but boosters are mandatory due to a rapid waning of anti-spike antibodies. A vaccination strategy based on immunologic response might be useful to maintain a favorable risk-benefit balance in this vulnerable population. METHODS: CoviDial is an observational prospective study enrolling 121 dialysis patients to receive a 3-dose mRNA-1273 vaccine according to a uniform schedule. At baseline, months 1, 3, 6, 9, and 12, anti-spike antibodies against four epitopes (S1, S2, ECD-S1 + S2, RBD) were monitored with a multiplex immunodot enzymatic assay. Potential correlation between initial serologic response and subsequent COVID-19 infection was then assessed. RESULTS: Overall, 96.2% and 96.8% of patients had anti-RBD antibodies at 3 and 12 months, respectively. All antibodies titers significantly decreased at month 6 compared to month 3. Booster vaccine induced a robust serologic response at month 9, but with a waning 3 months later, particularly for anti-S2 (37.2 ± 3.3 vs. 61.3 ± 3.0, p < 0.0001) and anti-S1 + S2 antibodies (68.4 ± 3.3 vs. 88.4 ± 2.3, p = 0.0015). Fifteen patients were later tested positive for SARS-CoV-2. At month 3, mean titers of anti-RBD, anti-S1 + S2, and anti-S2 antibodies were lower in the subsequent SARS-CoV-2 infected cohort (71.57 ± 9.01 vs. 85.79 ± 2.61, p = 0.0131; 41.07 ± 7.96 vs. 61.68 ± 3.56, p = 0.0237; 13.79 ± 5.03 vs. 39.70 ± 3.86, p = 0.0096; respectively). CONCLUSION: Three doses of mRNA-1273 vaccine induce a robust but time-limited immunologic response in dialysis patients. Lower anti-spike antibodies titers after initial vaccination are associated with a higher risk to subsequently contract SARS-CoV-2, even beyond 6 months.

Rhizobium leguminosarum symbiovar viciae strains are natural wheat endophytes that can stimulate root development.

Although rhizobia that establish a nitrogen-fixing symbiosis with legumes are also known to promote growth in non-legumes, studies on rhizobial associations with wheat roots are scarce. We searched for Rhizobium leguminosarum symbiovar viciae (Rlv) strains naturally competent to endophytically colonize wheat roots. We isolated 20 strains from surface-sterilized wheat roots and found a low diversity of Rlv compared to that observed in the Rlv species complex. We tested the ability of a subset of these Rlv for wheat root colonization when co-inoculated with other Rlv. Only a few strains, including those isolated from wheat roots, and one strain isolated from pea nodules, were efficient in colonizing roots in co-inoculation conditions, while all the strains tested in single strain inoculation conditions were found to colonize the surface and interior of roots. Furthermore, Rlv strains isolated from wheat roots were able to stimulate root development and early arbuscular mycorrhizal fungi colonization. These responses were strain and host genotype dependent. Our results suggest that wheat can be an alternative host for Rlv; nevertheless, there is a strong competition between Rlv strains for wheat root colonization. In addition, we showed that Rlv are endophytic wheat root bacteria with potential ability to modify wheat development.

The pea branching RMS2 gene encodes the PsAFB4/5 auxin receptor and is involved in an auxin-strigolactone regulation loop.

Strigolactones (SLs) are well known for their role in repressing shoot branching. In pea, increased transcript levels of SL biosynthesis genes are observed in stems of highly branched SL deficient (ramosus1 (rms1) and rms5) and SL response (rms3 and rms4) mutants indicative of negative feedback control. In contrast, the highly branched rms2 mutant has reduced transcript levels of SL biosynthesis genes. Grafting studies and hormone quantification led to a model where RMS2 mediates a shoot-to-root feedback signal that regulates both SL biosynthesis gene transcript levels and xylem sap levels of cytokinin exported from roots. Here we cloned RMS2 using synteny with Medicago truncatula and demonstrated that it encodes a putative auxin receptor of the AFB4/5 clade. Phenotypes similar to rms2 were found in Arabidopsis afb4/5 mutants, including increased shoot branching, low expression of SL biosynthesis genes and high auxin levels in stems. Moreover, afb4/5 and rms2 display a specific resistance to the herbicide picloram. Yeast-two-hybrid experiments supported the hypothesis that the RMS2 protein functions as an auxin receptor. SL root feeding using hydroponics repressed auxin levels in stems and down-regulated transcript levels of auxin biosynthesis genes within one hour. This auxin down-regulation was also observed in plants treated with the polar auxin transport inhibitor NPA. Together these data suggest a homeostatic feedback loop in which auxin up-regulates SL synthesis in an RMS2-dependent manner and SL down-regulates auxin synthesis in an RMS3 and RMS4-dependent manner.

Diversification of cytokinin phosphotransfer signaling genes in Medicago truncatula and other legume genomes.

BACKGROUND: Legumes can establish on nitrogen-deprived soils a symbiotic interaction with Rhizobia bacteria, leading to the formation of nitrogen-fixing root nodules. Cytokinin phytohormones are critical for triggering root cortical cell divisions at the onset of nodule initiation. Cytokinin signaling is based on a Two-Component System (TCS) phosphorelay cascade, involving successively Cytokinin-binding Histidine Kinase receptors, phosphorelay proteins shuttling between the cytoplasm and the nucleus, and Type-B Response Regulator (RRB) transcription factors activating the expression of cytokinin primary response genes. Among those, Type-A Response Regulators (RRA) exert a negative feedback on the TCS signaling. To determine whether the legume plant nodulation capacity is linked to specific features of TCS proteins, a genome-wide identification was performed in six legume genomes (Cajanus cajan, pigeonpea; Cicer arietinum, chickpea; Glycine max, soybean; Phaseolus vulgaris, common bean; Lotus japonicus; Medicago truncatula). The diversity of legume TCS proteins was compared to the one found in two non-nodulating species, Arabidopsis thaliana and Vitis vinifera, which are references for functional analyses of TCS components and phylogenetic analyses, respectively. RESULTS: A striking expansion of non-canonical RRBs was identified, notably leading to the emergence of proteins where the conserved phosphor-accepting aspartate residue is replaced by a glutamate or an asparagine. M. truncatula genome-wide expression datasets additionally revealed that only a limited subset of cytokinin-related TCS genes is highly expressed in different organs, namely MtCHK1/MtCRE1, MtHPT1, and MtRRB3, suggesting that this "core" module potentially acts in most plant organs including nodules. CONCLUSIONS: Further functional analyses are required to determine the relevance of these numerous non-canonical TCS RRBs in symbiotic nodulation, as well as of canonical MtHPT1 and MtRRB3 core signaling elements.

The Medicago genome provides insight into the evolution of rhizobial symbioses.

Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.

Isonatric Dialysis Biofeedback in Hemodiafiltration with Online Regeneration of Ultrafiltrate in Hypertensive Hemodialysis Patients: A Randomized Controlled Study.

UNLABELLED: Dialysis biofeedback in hemodiafiltration with online regeneration of ultrafiltrate (HFR) could help to improve arterial hypertension. We evaluated the impact of isonatric HFR (HFR-iso) on hypertension control compared to conventional HFR. Forty-seven hemodialysis patients were included and randomized (ratio 2/1) HFR-iso versus HFR during 24 dialysis sessions. In the HFR-iso group (32 patients, 768 dialysis sessions), the predialytic systolic blood pressure (BP) decreased from S1 to S24 of 9 ± 20 mm Hg and increased of 5 ± 24 mm Hg in the HFR group (15 patients, 360 dialysis sessions), variation that differed between the 2 groups (x0394;S1-S24, p = 0.035; interaction group*time, p = 0.012). The diastolic BP (HFR-iso -3 ± 14 mm Hg vs. HFR 5 ± 13 mm Hg; p = 0.088), the DDD of antihypertensive treatment and the dry weight did not vary significantly during the study. Number of sessions complicated by symptomatic hypotension was similar in the 2 groups. HFR-iso improved BP control without increasing dialysis hypotension episodes. SHORT SUMMARY: In this multicenter, open-label, controlled, randomized study, we evaluated the impact of dialysis biofeedback in HFR on arterial hypertension compared to conventional HFR. We observed that HFR-iso improved arterial BP control without increasing dialysis hypotension episodes.

An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing.

Rhizobium-induced root nodules are specialized organs for symbiotic nitrogen fixation. Indeterminate-type nodules are formed from an apical meristem and exhibit a spatial zonation which corresponds to successive developmental stages. To get a dynamic and integrated view of plant and bacterial gene expression associated with nodule development, we used a sensitive and comprehensive approach based upon oriented high-depth RNA sequencing coupled to laser microdissection of nodule regions. This study, focused on the association between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti, led to the production of 942 million sequencing read pairs that were unambiguously mapped on plant and bacterial genomes. Bioinformatic and statistical analyses enabled in-depth comparison, at a whole-genome level, of gene expression in specific nodule zones. Previously characterized symbiotic genes displayed the expected spatial pattern of expression, thus validating the robustness of our approach. We illustrate the use of this resource by examining gene expression associated with three essential elements of nodule development, namely meristem activity, cell differentiation and selected signaling processes related to bacterial Nod factors and redox status. We found that transcription factor genes essential for the control of the root apical meristem were also expressed in the nodule meristem, while the plant mRNAs most enriched in nodules compared with roots were mostly associated with zones comprising both plant and bacterial partners. The data, accessible on a dedicated website, represent a rich resource for microbiologists and plant biologists to address a variety of questions of both fundamental and applied interest.

Combined genetic and transcriptomic analysis reveals three major signalling pathways activated by Myc-LCOs in Medicago truncatula.

Myc-LCOs are newly identified symbiotic signals produced by arbuscular mycorrhizal (AM) fungi. Like rhizobial Nod factors, they are lipo-chitooligosaccharides that activate the common symbiotic signalling pathway (CSSP) in plants. To increase our limited understanding of the roles of Myc-LCOs we aimed to analyse Myc-LCO-induced transcriptional changes and their genetic control. Whole genome RNA sequencing (RNA-seq) was performed on roots of Medicago truncatula wild-type plants, and dmi3 and nsp1 symbiotic mutants affected in nodulation and mycorrhizal signalling. Plants were treated separately with the two major types of Myc-LCOs, sulphated and nonsulphated. Generalized linear model analysis identified 2201 differentially expressed genes and classified them according to genotype and/or treatment effects. Three genetic pathways for Myc-LCO-regulation of transcriptomic reprogramming were highlighted: DMI3- and NSP1-dependent; DMI3-dependent and NSP1-independent; and DMI3- and NSP1-independent. Comprehensive analysis revealed overlaps with previous AM studies, and highlighted certain functions, especially signalling components and transcription factors. These data provide new insights into mycorrhizal signalling mechanisms, supporting a role for NSP1, and specialisation for NSP1-dependent and -independent pathways downstream of DMI3. Our data also indicate significant Myc-LCO-activated signalling upstream of DMI3 and/or parallel to the CSSP and some constitutive activity of the CSSP.

Human bone morphogenetic protein-7 does not counteract aristolochic acid-induced renal toxicity.

Aristolochic acids (AA) are nephrotoxic and profibrotic agents, leading to chronic kidney disease. As some controversial studies have reported a nephroprotective effect of exogenous recombinant human bone morphogenetic protein (rhBMP)-7 in several models of renal fibrosis, we investigated the putative effect of rhBMP-7 to prevent progressive tubulointerstitial damage after AA intoxication in vitro and in vivo. In vitro, the toxicity of AA on renal tubular cells was demonstrated by an increase in vimentin as well as a decrease in ß-catenin expressions, reflecting a dedifferentiation process. Increased fibronectin and interleukin-6 levels were measured in the supernatants. Enhanced α-SMA mRNA levels associated to decreased E-cadherin mRNA levels were also measured. Incubation with rhBMP-7 only prevented the increase in vimentin and the decrease in ß-catenin expressions. In vivo, in a rat model of AA nephropathy, severe tubulointerstitial lesions induced by AA after 10 and 35 days (collagen IV deposition and tubular atrophy), were not prevented by the rhBMP-7 treatment. Similarly, rhBMP-7 did not ameliorate the significant increase in urinary concentrations of transforming growth factor-ß. In summary, our in vitro data demonstrated a poor beneficial effect of rhBMP-7 to reverse cell toxicity while, in vivo, there was no beneficial effect of rhBMP-7. Therefore, further investigations are needed to confirm the exact role of BMP-7 in progressive chronic kidney disease.

Biochemical parameters after cholecalciferol repletion in hemodialysis: results From the VitaDial randomized trial.

BACKGROUND: The 2009 KDIGO (Kidney Disease: Improving Global Outcomes) chronic kidney disease-mineral and bone disorder clinical practice guideline suggests correcting 25-hydroxyvitamin D3 (25[OH]D) levels<30ng/mL in patients treated with maintenance hemodialysis, but does not provide a specific treatment protocol. STUDY DESIGN: 2-center, double-blind, randomized, 13-week, controlled trial followed by a 26-week open-label study. SETTING & PARTICIPANTS: 55 adult maintenance hemodialysis patients with 25(OH)D levels<30ng/mL were recruited from June 2008 through October 2009. INTERVENTION: Cholecalciferol, 25,000IU, per week orally versus placebo for 13 weeks, then 26 weeks of individualized cholecalciferol prescription based on NKF-KDOQI (National Kidney Foundation-Kidney Disease Outcomes Quality Initiative) guidelines. OUTCOMES: Primary end point was the percentage of patients with 25(OH)D levels≥30ng/mL at 13 weeks. Secondary outcomes included the percentage of patients with normal calcium, phosphorus, and intact parathyroid hormone (iPTH) blood levels. Safety measures included incidence of hypercalcemia and hypervitaminosis D. MEASUREMENTS: Blood calcium and phosphate were measured weekly; iPTH, 25(OH)D, 1,25-dihydroxyvitamin D3 (1,25[OH]2D), and bone turnover markers, trimonthly; fetuin A and fibroblast growth factor 23 (FGF-23) serum levels and aortic calcification scores were determined at weeks 0 and 39. RESULTS: The primary end point significantly increased in the treatment group compared with the placebo group (61.5% vs 7.4%; P<0.001), as well as 1,25(OH)2D levels (22.5 [IQR, 15-26] vs 11 [IQR, 10-15]pg/mL; P<0.001) and the proportion of patients achieving the target calcium level (76.9% vs 48.2%; P=0.03). Incidence of hypercalcemia and phosphate and iPTH levels were similar between groups. The second 26-week study phase did not significantly modify the prevalence of 25(OH)D level≥30ng/mL in patients issued from the placebo group. LIMITATIONS: Small size of the study population. CONCLUSIONS: Oral weekly administration of 25,000IU of cholecalciferol for 13 weeks is an effective, safe, inexpensive, and manageable way to increase 25(OH)D and 1,25(OH)2D levels in hemodialysis patients. Further evaluation of clinical end points is suggested.

High-density genome-wide association mapping implicates an F-box encoding gene in Medicago truncatula resistance to Aphanomyces euteiches.

• The use of quantitative disease resistance (QDR) is a promising strategy for promoting durable resistance to plant pathogens, but genes involved in QDR are largely unknown. To identify genetic components and accelerate improvement of QDR in legumes to the root pathogen Aphanomyces euteiches, we took advantage of both the recently generated massive genomic data for Medicago truncatula and natural variation of this model legume. • A high-density (≈5.1 million single nucleotide polymorphisms (SNPs)) genome-wide association study (GWAS) was performed with both in vitro and glasshouse phenotyping data collected for 179 lines. • GWAS identified several candidate genes and pinpointed two independent major loci on the top of chromosome 3 that were detected in both phenotyping methods. Candidate SNPs in the most significant locus (σ(A)²= 23%) were in the promoter and coding regions of an F-box protein coding gene. Subsequent qRT-PCR and bioinformatic analyses performed on 20 lines demonstrated that resistance is associated with mutations directly affecting the interaction domain of the F-box protein rather than gene expression. • These results refine the position of previously identified QTL to specific candidate genes, suggest potential molecular mechanisms, and identify new loci explaining QDR against A. euteiches.

A tandem array of CBF/DREB1 genes is located in a major freezing tolerance QTL region on Medicago truncatula chromosome 6.

BACKGROUND: Freezing provokes severe yield losses to different fall-sown annual legumes. Understanding the molecular bases of freezing tolerance is of great interest for breeding programs. Medicago truncatula Gaertn. is an annual temperate forage legume that has been chosen as a model species for agronomically and economically important legume crops. The present study aimed to identify positional candidate genes for a major freezing tolerance quantitative trait locus that was previously mapped to M. truncatula chromosome 6 (Mt-FTQTL6) using the LR3 population derived from a cross between the freezing-tolerant accession F83005-5 and the freezing-sensitive accession DZA045-5. RESULTS: The confidence interval of Mt-FTQTL6 was narrowed down to the region comprised between markers MTIC153 and NT6054 using recombinant F7 and F8 lines. A bacterial-artificial chromosome (BAC) clone contig map was constructed in an attempt to close the residual assembly gap existing therein. Twenty positional candidate genes including twelve C-repeat binding factor (CBF)/dehydration-responsive element binding factor 1 (DREB1) genes were identified from BAC-derived sequences and whole-genome shotgun sequences (WGS). CBF/DREB1 genes are organized in a tandem array within an approximately 296-Kb region. Eleven CBF/DREB1 genes were isolated and sequenced from F83005-5 and DZA045-5 which revealed high polymorphism among these accessions. Unique features characterizing CBF/DREB1 genes from M. truncatula, such as alternative splicing and large tandem duplication, are elucidated for the first time. CONCLUSIONS: Overall, twenty genes were identified as potential candidates to explain Mt-FTQTL6 effect. Their future functional characterization will uncover the gene(s) involved in freezing tolerance difference observed between F83005-5 and DZA045-5. Knowledge transfer for breeding improvement of crop legumes is expected. Furthermore, CBF/DREB1 related data will certainly have a large impact on research studies targeting this group of transcriptional activators in M. truncatula and other legume species.

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.

Genetic Variation in Host-Specific Competitiveness of the Symbiont Rhizobium leguminosarum Symbiovar viciae.

Legumes of the Fabeae tribe form nitrogen-fixing root nodules resulting from symbiotic interaction with the soil bacteria Rhizobium leguminosarum symbiovar viciae (Rlv). These bacteria are all potential symbionts of the Fabeae hosts but display variable partner choice when co-inoculated in mixture. Because partner choice and symbiotic nitrogen fixation mostly behave as genetically independent traits, the efficiency of symbiosis is often suboptimal when Fabeae legumes are exposed to natural Rlv populations present in soil. A core collection of 32 Rlv bacteria was constituted based on the genomic comparison of a collection of 121 genome sequences, representative of known worldwide diversity of Rlv. A variable part of the nodD gene sequence was used as a DNA barcode to discriminate and quantify each of the 32 bacteria in mixture. This core collection was co-inoculated on a panel of nine genetically diverse Pisum sativum, Vicia faba, and Lens culinaris genotypes. We estimated the relative Early Partner Choice (EPC) of the bacteria with the Fabeae hosts by DNA metabarcoding on the nodulated root systems. Comparative genomic analyses within the bacterial core collection identified molecular markers associated with host-dependent symbiotic partner choice. The results revealed emergent properties of rhizobial populations. They pave the way to identify genes related to important symbiotic traits operating at this level.

Developmental Modulation of Root Cell Wall Architecture Confers Resistance to an Oomycete Pathogen.

The cell wall is the primary interface between plant cells and their immediate environment and must balance multiple functionalities, including the regulation of growth, the entry of beneficial microbes, and protection against pathogens. Here, we demonstrate how API, a SCAR2 protein component of the SCAR/WAVE complex, controls the root cell wall architecture important for pathogenic oomycete and symbiotic bacterial interactions in legumes. A mutation in API results in root resistance to the pathogen Phytophthora palmivora and colonization defects by symbiotic rhizobia. Although api mutant plants do not exhibit significant overall growth and development defects, their root cells display delayed actin and endomembrane trafficking dynamics and selectively secrete less of the cell wall polysaccharide xyloglucan. Changes associated with a loss of API establish a cell wall architecture with altered biochemical properties that hinder P. palmivora infection progress. Thus, developmental stage-dependent modifications of the cell wall, driven by SCAR/WAVE, are important in balancing cell wall developmental functions and microbial invasion.

Aristolochic acid nephropathy: a worldwide problem.

Aristolochic acid nephropathy (AAN), a progressive renal interstitial fibrosis frequently associated with urothelial malignancies, was initially reported in a Belgian cohort of more than 100 patients after the intake of slimming pills containing a Chinese herb, Aristolochia fangchi. Although botanicals known or suspected to contain aristolochic acid (AA) were no longer permitted in many countries, several AAN cases were regularly observed all around the world. The incidence of AAN is probably much higher than initially thought, especially in Asia and the Balkans. In Asian countries, where traditional medicines are very popular, the complexity of the pharmacopoeia represents a high risk for AAN because of the frequent substitution of the botanical products by AA-containing herbs. In the Balkan regions, the exposure to AA found in flour obtained from wheat contaminated with seeds of Aristolochia clematitis could be responsible for the so-called Balkan-endemic nephropathy. Finally, despite the Food and Drug Administration's warnings concerning the safety of botanical remedies containing AA, these herbs are still sold via the Internet.

Whole-genome landscape of Medicago truncatula symbiotic genes.

Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies1, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.

Distribution of microsatellites in the genome of Medicago truncatula: a resource of genetic markers that integrate genetic and physical maps.

Microsatellites are tandemly repeated short DNA sequences that are favored as molecular-genetic markers due to their high polymorphism index. Plant genomes characterized to date exhibit taxon-specific differences in frequency, genomic location, and motif structure of microsatellites, indicating that extant microsatellites originated recently and turn over quickly. With the goal of using microsatellite markers to integrate the physical and genetic maps of Medicago truncatula, we surveyed the frequency and distribution of perfect microsatellites in 77 Mbp of gene-rich BAC sequences, 27 Mbp of nonredundant transcript sequences, 20 Mbp of random whole genome shotgun sequences, and 49 Mbp of BAC-end sequences. Microsatellites are predominantly located in gene-rich regions of the genome, with a density of one long (i.e., > or = 20 nt) microsatellite every 12 kbp, while the frequency of individual motifs varied according to the genome fraction under analysis. A total of 1,236 microsatellites were analyzed for polymorphism between parents of our reference intraspecific mapping population, revealing that motifs (AT)n, (AG)n, (AC)n, and (AAT)n exhibit the highest allelic diversity. A total of 378 genetic markers could be integrated with sequenced BAC clones, anchoring 274 physical contigs that represent 174 Mbp of the genome and composing an estimated 70% of the euchromatic gene space.

A rice calcium- and calmodulin-dependent protein kinase restores nodulation to a legume mutant.

The Medicago truncatula DMI3 gene encodes a calcium- and calmodulin-dependent protein kinase (CCaMK) that is necessary for the establishment of both rhizobial and mycorrhizal symbioses. The two symbiotic signaling pathways diverge downstream of DMI3; therefore, it has been proposed that legumes have evolved a particular form of CCaMK, acting like a switch able both to discriminate between rhizobial and mycorrhizal calcium signatures and to trigger the appropriate downstream signaling pathway. To test this hypothesis, we examined whether a CCaMK gene from a nonlegume species was able to restore the rhizobial symbiotic properties of a M. truncatula dmi3 mutant. Our results show that a CCaMK gene from rice can restore nodule formation, indicating that CCaMKs from nonlegumes can interpret the calcium signature elicited by rhizobial Nod factors and activate the appropriate downstream target. The nodules did not contain bacteria, which suggests that DMI3 is also involved in the control of the infection process.