Gabriella Endre

A receptor kinase gene regulating symbiotic nodule development

Leguminous plants are able to establish a nitrogen-fixing symbiosis with soil bacteria generally known as rhizobia. Metabolites exuded by the plant root activate the production of a rhizobial signal molecule, the Nod factor, which is essential for symbiotic nodule development. This lipo-chitooligosaccharide signal is active at femtomolar concentrations, and its structure is correlated with host specificity of symbiosis, suggesting the involvement of a cognate perception system in the plant host. Here we describe the cloning of a gene from Medicago sativa that is essential for Nod-factor perception in alfalfa, and by genetic analogy, in the related legumes Medicago truncatula and Pisum sativum. The identified ‘nodulation receptor kinase’, NORK, is predicted to function in the Nod-factor perception/transduction system (the NORK system) that initiates a signal cascade leading to nodulation. The family of ‘NORK extracellular-sequence-like’ (NSL) genes is broadly distributed in the plant kingdom, although their biological function has not been previously ascribed. We suggest that during the evolution of symbiosis an ancestral NSL system was co-opted for transduction of an external ligand, the rhizobial Nod factor, leading to development of the symbiotic root nodule.

Large‐scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190,000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R(1) lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14,000-16,000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500,000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.

Transcript Analysis of Early Nodulation Events in Medicago truncatula

Within the first 72 h of the interaction between rhizobia and their host plants, nodule primordium induction and infection occur. We predicted that transcription profiling of early stages of the symbiosis between Medicago truncatula roots and Sinorhizobium meliloti would identify regulated plant genes that likely condition key events in nodule initiation. Therefore, using a microarray with about 6,000 cDNAs, we compared transcripts from inoculated and uninoculated roots corresponding to defined stages between 1 and 72 h post inoculation (hpi). Hundreds of genes of both known and unknown function were significantly regulated at these time points. Four stages of the interaction were recognized based on gene expression profiles, and potential marker genes for these stages were identified. Some genes that were regulated differentially during stages I (1 hpi) and II (6–12 hpi) of the interaction belong to families encoding proteins involved in calcium transport and binding, reactive oxygen metabolism, and cytoskeleton and cell wall functions. Genes involved in cell proliferation were found to be up-regulated during stages III (24–48 hpi) and IV (72 hpi). Many genes that are homologs of defense response genes were up-regulated during stage I but down-regulated later, likely facilitating infection thread progression into the root cortex. Additionally, genes putatively involved in signal transduction and transcriptional regulation were found to be differentially regulated in the inoculated roots at each time point. The findings shed light on the complexity of coordinated gene regulation and will be useful for continued dissection of the early steps in symbiosis.

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., ≥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.

Comparative mapping between Medicago sativa and Pisum sativum

Comparative genome analysis has been performed between alfalfa ( Medicago sativa) and pea ( Pisum sativum), species which represent two closely related tribes of the subfamily Papilionoideae with different basic chromosome numbers. The positions of genes on the most recent linkage map of diploid alfalfa were compared to those of homologous loci on the combined genetic map of pea to analyze the degree of co-linearity between their linkage groups. In addition to using unique genes, analysis of the map positions of multicopy (homologous) genes identified syntenic homologs (characterized by similar positions on the maps) and pinpointed the positions of non-syntenic homologs. The comparison revealed extensive conservation of gene order between alfalfa and pea. However, genetic rearrangements (due to breakage and reunion) were localized which can account for the difference in chromosome number (8 for alfalfa and 7 for pea). Based on these genetic events and our increasing knowledge of the genomic structure of pea, it was concluded that the difference in genome size between the two species (the pea genome is 5- to 10-fold larger than that of alfalfa) is not a consequence of genome duplication in pea. The high degree of synteny observed between pea and Medicago loci makes further map-based cloning of pea genes based on the genome resources now available for M. truncatula a promising strategy.

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase1 Interacts with NORK and Is Crucial for Nodulation in Medicago truncatula

NORK in legumes encodes a receptor-like kinase that is required for Nod factor signaling and root nodule development. Using Medicago truncatula NORK as bait in a yeast two-hybrid assay, we identified 3-hydroxy-3-methylglutaryl CoA reductase 1 (Mt HMGR1) as a NORK interacting partner. HMGR1 belongs to a multigene family in M. truncatula, and different HMGR isoforms are key enzymes in the mevalonate biosynthetic pathway leading to the production of a diverse array of isoprenoid compounds. Testing other HMGR members revealed a specific interaction between NORK and HMGR1. Mutagenesis and deletion analysis showed that this interaction requires the cytosolic active kinase domain of NORK and the cytosolic catalytic domain of HMGR1. NORK homologs from Lotus japonicus and Sesbania rostrata also interacted with Mt HMGR1, but homologous nonsymbiotic kinases of M. truncatula did not. Pharmacological inhibition of HMGR activities decreased nodule number and delayed nodulation, supporting the importance of the mevalonate pathway in symbiotic development. Decreasing HMGR1 expression in M. truncatula transgenic roots by RNA interference led to a dramatic decrease in nodulation, confirming that HMGR1 is essential for nodule development. Recruitment of HMGR1 by NORK could be required for production of specific isoprenoid compounds, such as cytokinins, phytosteroids, or isoprenoid moieties involved in modification of signaling proteins.

Construction of an improved linkage map of diploid alfalfa (Medicago sativa)

Abstract An improved genetic map of diploid (2n=2x=16) alfalfa has been developed by analyzing the inheritance of more than 800 genetic markers on the F2 population of 137 plant individuals. The F2 segregating population derived from a self-pollinated F1 hybrid individual of the cross Medicago sativa ssp. quasifalcata ×Medicago sativa ssp. coerulea. This mapping population was the same one which had been used for the construction of our previous alfalfa genetic map. The genetic analyses were performed by using maximum-likelihood equations and related computer programs. The improved genetic map of alfalfa in its present form contains 868 markers (four morphological, 12 isozyme, 26 seed protein, 216 RFLP, 608 RAPD and two specific PCR markers) in eight linkage groups. Of the markers 80 are known genes, including 2 previously cytologically localized genes, the rDNA and the β-tubulin loci. The genetic map covers 754 centimorgans (cM) with an average marker density of 0.8/cM. The correlation between the physical and genetic distances is about 1000–1300 kilobase pairs per centiMorgan. In this map, the linkage relationships of some markers on linkage groups 6, 7, and 8 are different from the previously published one. The cause of this discrepancy was that the genetic linkage of markers displaying distorted segregation (characterized by an overwhelming number of heterozygous individuals) had artificially linked genetic regions that turned out to be unlinked. To overcome the disadvantageous influence of the excess number of heterozygous genotypes on the recombination fractions, we used recently described maximum-likelihood formulas and colormapping, which allowed us to exclude the misleading linkages and to estimate the genetic distances more precisely.

The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system

The fix‐2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf−/Fix−) is K+ sensitive and unable to adapt to alkaline pH in the presence of K+. Using directed Tn5 mutagenesis, we delimited a 6 kb genomic region in which mutations resulted in both Inf−/Fix− and K+‐sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA, B, C, D, E, F and G. The putative PhaABC proteins exhibit homology to the subunits of a Na+/H+ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton‐pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K+ efflux at alkaline pH after the addition of a membrane‐permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K+ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.

Six nodulation genes of nod box locus 4 in Rhizobium meliloti are involved in nodulation signal production: nodM codes for d-glucosamine synthetase

SummaryThe nucleotide sequence of the nod box locus n4 in Rhizobium meliloti was determined and revealed six genes organized in a single transcriptional unit, which are induced in response to a plant signal such as luteolin. Mutations in these genes influence the early steps of nodule development on Medicago, but have no detectable effect on Melilotus, another host for R. meliloti. Based on sequence homology, the first open reading frame (ORF) corresponds to the nodM gene and the last to the nodN gene of Rhizobium leguminosarum. The others do not exhibit similarity to any genes sequenced so far, so we designated them as nolF, nolG, nolH and nolI, respectively. We found that the n4 locus, and especially the nodM and nodN genes, are involved in the production of the root hair deformation (Had) factor. NodM exhibits homology to amidotransferases, primarily to the d-glucosamine synthetase encoded by the glmS gene of Escherichia coli. We demonstrated that in E. coli the regulatory gene nodD together with luteolin can activate nod genes. On this basis we showed that nodM complemented an E. coli glmS−mutation, indicating that nodM can be considered as a glmS gene under plant signal control. Moreover, exogenously supplied d-glucosamine restored nodulation of Medicago by nodM mutants. Our data suggest that in addition to the housekeeping glmS gene of R. meliloti, nodM as a second glmS copy provides glucosamine in sufficient amounts for the synthesis of the Had factor.

Construction of a bacterial artificial chromosome library of Medicago truncatula and identification of clones containing ethylene-response genes

Abstract To facilitate genome analysis and map-based cloning of symbiotic genes in the model legume Medicago truncatula, a bacterial artificial chromosome (BAC) library was constructed. The library consists of 30 720 clones with an average insert size of approximately 100 kb, representing approximately five haploid-genome equivalents. The frequency of BAC clones carrying inserts of chloroplast DNA was estimated to be 1.4%. Screening of the library with single- or low-copy genes as hybridization probes resulted in the detection of 1–12 clones per gene. Hybridization of the library with repeated sequences such as rDNA genes and transposon-like elements of M. truncatula revealed the presence of 60 and 374 BAC clones containing the two sequences, respectively. The BAC library was pooled for screening by polymerase chain reaction (PCR)-amplification. To demonstrate the utility of this system, we used primers designed from a conserved region of the ein3-like loci of Arabidopsis thaliana and isolated six unique BAC clones from the library. DNA gel-blot and sequence analyses showed that these ein3-like clones could be grouped into three classes, an observation consistent with the presence of multiple ein3-like loci in M. truncatula. These results indicate that the BAC library represents a central resource for the map-based cloning and physical mapping in M. truncatula and other legumes.