Pascal Ratet

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.

Medicago truncatula NIN Is Essential for Rhizobial-Independent Nodule Organogenesis Induced by Autoactive Calcium/Calmodulin-Dependent Protein Kinase

The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11∷GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules.

Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis

Abstract We describe a simple and efficient protocol for regeneration-transformation of two diploid Medicago lines: the annual M. truncatula R108-1(c3) and the perennial M. sativa ssp. falcata (L.) Arcangeli PI.564263 selected previously as highly embryogenic genotypes. Here, embryo regeneration of R108-1 to complete plants was further improved by three successive in vitro regeneration cycles resulting in the line R108-1(c3). Agrobacterium tumefaciens-mediated transformation of leaf explants was carried out with promoter-gus constructs of two early nodulins (MsEnod12A and MsEnod12B) and one late nodulin (Srglb3). The transgenic plants thus produced on all explants within 3–4 months remained diploid and were fertile. This protocol appears to be the most efficient and fastest reported so far for leguminous plants.

Cell and Molecular Biology of Rhizobium-Plant

Publisher Summary This chapter discusses the Cell and molecular biology of Rhizobium -plant interactions. Soil bacteria, referred to as rhizobia belonging to the genera Rhizobium , Bradyrhizobium , and Azorhizobium , have the unique ability to induce nitrogen-fixing nodules on the roots or stems of leguminous plants. Nodule development consists of several stages determined by different sets of genes both in the host and symbiont. At least at the very early steps of symbiosis, the bacterial and plant genes are activated consecutively by signal exchanges between the symbiotic partners. First, flavonoid signal molecules exuded by the host plant root induce the expression of nodulation ( nod, nol ) genes in Rhizobium in conjunction with the bacterial activator NodD protein. Then, in the second step, lipooligosaccharide Nod factors with various host-specific structural modifications are produced by the bacterial Nod proteins. The Nod factors induce various plant reactions, such as root hair deformation, initiation of nodule meristems, and induction of early nodulin genes, leading to nodule formation. Other classes of bacterial genes are required for successful infection and for nitrogen fixation. This chapter includes only the early events of communication between rhizobia and their host plants, that is, the perception of flavonoid signals by the bacteria, the production of Nod signals by rhizobia, and the early plant responses to the bacteria.

A GRAS-type transcription factor with a specific function in mycorrhizal signaling

Legumes establish mutualistic associations with mycorrhizal fungi and with nitrogen-fixing rhizobial bacteria. These interactions occur following plant recognition of Nod factor from rhizobial bacteria and Myc factor from mycorrhizal fungi. A common symbiosis signaling pathway is involved in the recognition of both Nod factor and Myc factor and is required for the establishment of these two symbioses. The outcomes of these associations differ, and therefore, despite the commonality in signaling, there must be mechanisms that allow specificity. In Nod factor signaling, a complex of GRAS-domain transcription factors controls gene expression downstream of the symbiosis signaling pathway. Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. Plants mutated in RAM1 are unable to be colonized by mycorrhizal fungi, with a defect in hyphopodia formation on the surface of the root. RAM1 is specifically required for Myc factor signaling and appears to have no role in Nod factor signaling. RAM1 regulates the expression of RAM2, a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. We conclude that mycorrhizal signaling downstream of the symbiosis-signaling pathway has parallels with nodulation-specific signaling and functions to promote mycorrhizal colonization by regulating cutin biosynthesis.

Vapyrin, a gene essential for intracellular progression of arbuscular mycorrhizal symbiosis, is also essential for infection by rhizobia in the nodule symbiosis of Medicago truncatula.

Intracellular invasion of root cells is required for the establishment of successful endosymbioses in legumes of both arbuscular mycorrhizal (AM) fungi and rhizobial bacteria. In both interactions a requirement for successful entry is the activation of a common signalling pathway that includes five genes required to generate calcium oscillations and two genes required for the perception of the calcium response. Recently, it has been discovered that in Medicago truncatula, the Vapyrin (VPY) gene is essential for the establishment of the arbuscular mycorrhizal symbiosis. Here, we show by analyses of mutants that the same gene is also required for rhizobial colonization and nodulation. VPY encodes a protein featuring a Major Sperm Protein domain, typically featured on proteins involved in membrane trafficking and biogenesis, and a series of ankyrin repeats. Plants mutated in this gene have abnormal rhizobial infection threads and fewer nodules, and in the case of interactions with AM fungi, epidermal penetration defects and aborted arbuscule formation. Calcium spiking in root hairs in response to supplied Nod factors is intact in the vpy-1 mutant. This, and the elevation of VPY transcripts upon application of Nod factors which we show to be dependent on NFP, DMI1, and DMI3, indicates that VPY acts downstream of the common signalling pathway.

Differentiation of Symbiotic Cells and Endosymbionts in Medicago truncatula Nodulation Are Coupled to Two Transcriptome-Switches

The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix− nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this “nodule-specific transcriptome” were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches.

Control of compound leaf development by FLORICAULA/LEAFY ortholog SINGLE LEAFLET1 in Medicago truncatula.

Molecular genetic studies suggest that FLORICAULA (FLO)/LEAFY (LFY) orthologs function to control compound leaf development in some legume species. However, loss-of-function mutations in the FLO/LFY orthologs result in reduction of leaf complexity to different degrees in Pisum sativum and Lotus japonicus. To further understand the role of FLO/LFY orthologs in compound leaf development in legumes, we studied compound leaf developmental processes and characterized a leaf development mutant, single leaflet1 (sgl1), from the model legume Medicago truncatula. The sgl1 mutants exhibited strong defects in compound leaf development; all adult leaves in sgl1 mutants are simple due to failure in initiating lateral leaflet primordia. In addition, the sgl1 mutants are also defective in floral development, producing inflorescence-like structures. Molecular cloning of SGL1 revealed that it encodes the M. truncatula FLO/LFY ortholog. When properly expressed, LFY rescued both floral and compound leaf defects of sgl1 mutants, indicating that LFY can functionally substitute SGL1 in compound leaf and floral organ development in M. truncatula. We show that SGL1 and LFY differed in their promoter activities. Although the SGL1 genomic sequence completely rescued floral defects of lfy mutants, it failed to alter the simple leaf structure of the Arabidopsis thaliana plants. Collectively, our data strongly suggest that initiation of lateral leaflet primordia required for compound leaf development involves regulatory processes mediated by the SGL1 function in M. truncatula.

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.

Alfalfa Root Flavonoid Production Is Nitrogen Regulated

Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.