Populations genomics analysis of legume host preference for specific rhizobial genotypes in the Rhizobium leguminosarum bv. viciae symbioses

Rhizobium leguminosarum bv. viciae establishes root nodule symbioses with several legume genera. Although most isolates are equally effective in establishing symbioses with all host genera, previous evidence suggests that hosts select specific rhizobial genotypes among those present in the soil. We have used population genomics to further investigate this observation. P. sativum, L. culinaris, V. sativa, and V. faba plants were used to trap rhizobia from a well-characterized soil, and pooled genomic DNAs from one-hundred isolates from each plant were sequenced. Sequence reads were aligned to the R. leguminosarum bv. viciae 3841 reference genome. High overall conservation of sequences was observed in all subpopulations, although several multigenic regions were absent from the soil population. A large fraction (16-22%) of sequence reads could not be recruited to the reference genome, suggesting that they represent sequences specific to that particular soil population. Although highly conserved, the 16S-23S rRNA gene region presented single nucleotide polymorphisms (SNPs) regarding the reference genome, but no striking differences could be found among plant-selected subpopulations. Plant-specific SNP patterns were, however, clearly observed within the nod gene cluster, supporting the existence of a plant preference for specific rhizobial genotypes. This was also shown after genome-wide analysis of SNP patterns

PoolSeq analysis of the selection of the Rhizobium genotypes by the legume host plant

Rhizobium leguminosarum establishes highly specific nitrogen-fixing symbioses. We have applied a Pool-Seq approach to study plant host selection of genotypes. Our results confirm, at the genomic level, previous observations regarding plant selection of specific genotype

Population genomics of host specificity in Rhizobium leguminosarum bv. viciae

Legumes establish a root-nodule symbiosis with soil bacteria collectively known as rhizobia. This symbiosis allows legumes to benefit from the nitrogen fixation capabilities of rhizobia and thus to grow in the absence of any fixed nitrogen source. This is especially relevant for Agriculture, where intensive plant growth depletes soils of useable, fixed nitrogen sources. One of the main features of the root nodule symbiosis is its specificity. Different rhizobia are able to nodulate different legumes. Rhizobium leguminosarum bv. viciae is able to establish an effective symbiosis with four different plant genera (Pisum, Lens, Vicia, Lathyrus), and any given isolate will nodulate any of the four plant genera. A population genomics study with rhizobia isolated from P. sativum, L. culinaris, V. sativa or V. faba, all originating in the same soil, showed that plants select specific genotypes from those available in that soil. This was demonstrated at the genome-wide level, but also for specific genes. Accelerated mesocosm studies with successive plant cultures provided additional evidence on this plant selection and on the nature of the genotypes selected. Finally, representatives from the major rhizobial genotypes isolated from these plants allowed characterization of the size and nature of the respective pangenome and specific genome compartments. These were compared to the different genotypes ?symbiotic and non-symbiotic?present in rhizobial populations isolated directly from the soil without plant intervention

Genomics of host specificity in the Rhizobium-legume simbiosis

Most Rhizobium leguminosarum bv. viciae isolates are able to specifically nodulate plants of any of four different legume genera: Pisum, Lens, Vicia, and Lathyrus. However, previous evidence suggests that some genotypes are more adapted to a given plant host than others, and that the plant host can select specific genotypes among those present in a given soil population. We have used a population genomics approach to confirm that this is indeed the case, and to analyze the specific genotypic characteristics that each plant host select

Optimizing Gateway™ technology (Invitrogen) to construct Rhizobium leguminosarum deletion mutants

The study of the role of different genes in Rhizobium leguminosarum requires the generation of mutants by homologous recombination. In this communication we describe a novel approach to obtain deletion mutants of genes in Rhizobium using Gateway TM Cloning technology (Invitrogen) and a new vector (pK18-attR), both conjugative and Rhizobium specific, that carries the recombination tails of Gateway system. This tool is a new alternative to the classic approach based on cloning using restriction enzymes. The first step consists of designing directed oligonucleotides with specific tails for isolating recombination fragments and a resistance marker cassette to an antibiotic by PCR. The three inserts are cloned by homologous recombination in three specific vectors, in a single step. The last step consists of multisite-directed recombination of the three donor vectors to the pK18-attR destination vector. After recombination, this vector loses the ccdB gene, whose expression results in synthesis of a DNA gyrase that is lethal to carrier cells and thus guarantees the effectiveness in obtaining clones that carry the homologous construction to the subsequent recombination in Rhizobiu

Efecto de los sistemas de Quorum Sensing sobre la eficiencia simbiótica de R. leguminosarum UPM791

Rhizobium leguminosarum bv viciae (Rlv) es una alfa-proteobacteria capaz de establecer una simbiosis diazotrófica con distintas leguminosas. Uno de los factores implicados en el establecimiento de la simbiosis es el sistema de comunicación intercelular conocido como Quorum Sensing (QS). Mediante este sistema, las bacterias actúan de manera coordinada en respuesta a cambios en la densidad de población a través de la producción y detección de señales extracelulares. El genoma de Rlv UPM791 contiene dos sistemas tipo luxRI mediados por señales de tipo N-acyl-homoserina lactonas (AHLs): el sistema rhiRI, codificado en el plásmido simbiótico, produce C6-HSL, C7-HSL y C8-HSL; y el sistema cinRI, localizado en el cromosoma, produce 3-OH-C14:1-HSL. Con el fin de analizar el significado y la regulación de los sistemas de QS en esta bacteria endosimbiótica se generaron mutantes defectivos en cada uno de los sistemas de QS, y se llevó a cabo un análisis detallado sobre la producción de AHLs y la simbiosis con plantas de guisante, veza y lenteja. El sistema rhiRI se necesita para un comportamiento simbiótico normal, dado que la mutación en rhiI reduce considerablemente la eficiencia simbiótica. rhiR es esencial para la fijación de nitrógeno en ausencia del plásmido pUPM791d. Asimismo, mutaciones en el sistema cinRIS mostraron también un importante efecto en simbiosis. El mutante ?cinRIS no produce la señal 3-OH-C14:1-HSL, y da lugar a nódulos blancos e inefectivos, carentes de bacteroides. El mutante ?cinI, incapaz de producir AHLs, no forma nódulos en ninguna de las leguminosas utilizadas. El análisis genético reveló que dicha mutación origina la inestabilización del plásmido simbiótico por un mecanismo dependiente de cinI que no ha sido aclarado. Los resultados obtenidos sugieren un papel relevante de los sistemas de Quorum Sensing de Rlv UPM791 en los primeros estadíos de la simbiosis, e indican la existencia de un modelo de regulación dependiente de QS significativamente distinto a los que se han descrito previamente en otras cepas de R. leguminosarum

MtYSL1: a putative metal transporter involved in Medicago truncatula-Sinorhizobium meliloti symbiotic interaction

Leguminous plants are able to grow under nitrogen-limiting conditions by establishing an endosymbiotic interaction with diazotrophic soil bacteria known as rhizobia. This interaction results in root structures, nodules, where rhizobia are differenciated to bacteroids and symbiotic nitrogen fixation (SNF) occurs (Van de Velde et al., 2006). Key enzymes involved in SNF require metals as cofactor to carry out their catalytic activity such as nitrogenase, leghemoglobin, cytochrome oxidase and superoxide dismutase. Previous results in Medicago truncatula showed that metals have to be provided to the bacteroids by the host legume, being released to the apoplast of zone II (infection/maturation zone) of the nodule (Rodriguez- Haas et al., 2013). It is known that Yellow Stripe-like (YSL) transporters mediate metal trafficking from the root to sink organs, however, no information of the role of these transporters in the context of SNF is available. Medicago truncatula YSL1 is a good candidate to mediate this transport, since it reaches an expression peak in these organs. MtYSL1 was localized around the vascular conducts of nodules and, in the root pericycle. MtYSL1 immunolocalization showed that it was embedded in the plasma membrane of non-infected cells surrounding the vessels. These results suggest a role of MtYSL1 in metal delivery to M. truncatula nodule

MtZIP6 is a novel metal transporter required for symbiotic nitrogen fixation in nodules of Medicago truncatula plants

Symbiotic nitrogen fixation (SNF) carried out by the interaction rhizobia-legumes takes place in legume root nodules. Many of the enzymes involved in SNF are metalloproteins that obtain their metal cofactor from the host plant. Metals reach the nodule through the vasculature, where they are released in the apoplast on the infection/differentiation zone (zone II) of the nodule (Rodriguez-Haas et al., 2013). From there, these oligonutrients have to cross a number of membranes to be used for metalloprotein synthesis (plasma membrane, endoplasmic reticulum, symbiosomes,..). Although several proteins have been suggested to mediate metal transport to the endosymbiotic nitrogen-fixing rhizobia (bacteroids), very little is known about transporters that mediate metal uptake from the apoplast. Recently, we have identified MtNramp1, the first iron transporter to mediate this uptake (Tejada-Jiménez et al., 2015). However, other transporters must mediate zinc, manganese or copper uptake from the nodule apoplast. Transcriptomic studies in Medicago truncatula revealed that MtZIP6, a ZIP family member, had a maximum of expression in the nodule. ZIP6 promoter::GUS fusions showed that MtZIP6 expression was confined to the nodule zone II, the region where metals have to be incorporated from the apoplast. These results were also validated by immunohistochemistry in nodule sections expressing MtZIP6 bound to 3xHA epitopes under the MtZIP6 promoter. This experiment showed that MtZIP6 is very likely localized in the plasma membrane and confirmed its expression in zone II of the nodule. Expression of MtZIP6 in Saccharomyces cerevisiae metal transport mutants showed MtZIP6 as a divalent metal importer, capable of transporting zinc, manganese, or iron. Loss of MtZIP6 function in M. truncatula knocked-down plants revealed a reduced plant and nodule size, and a reduced nitrogenase activity in comparison to control plants. Altogether these results suggest that MtZIP6 is an important element in the process of symbiotic nitrogen fixation by its ability to transport metals through the plasma membrane of the nodule cell

Quorum Sensing is essential for an effective symbiosis in R. leguminosarum UPM791.

The implications of Quorum Sensing in the establishment of a successful symbiosis of Rhizobium leguminosarum bv. viciae (Rlv) with legume plants are discussed in this work. In order to analyze the significance and regulation of the production of AHL signal molecules, mutants deficient in each of the two QS systems present in Rlv UPM791 were constructed. A detailed analysis of the effect of these mutations on growth, AHL production, biofilm formation and symbiosis with pea, vetch and lentil plants has been carried out

Metagenomic Anlaysis of microsymbiont selection by the legume plant host

Rhizobium leguminosarum bv.viciae is able to establish nitrogen-fixing symbioses with legumes of the genera Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants (Laguerre et al., Young et al.) provided evidence that different plant hosts are able to select different rhizobial genotypes among those available in a given soil. However, these studies were necessarily limited by the paucity of relevant biodiversity markers. We have now reappraised this problem with the help of genomic tools. A well-characterized agricultural soil (INRA Bretennieres) was used as source of rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were used as traps. Isolates from 100 nodules were pooled, and DNA from each pool was sequenced (BGI-Hong Kong; Illumina Hiseq 2000, 500 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered (FastQC, Trimmomatic), mapped against reference R. leguminosarum genomes (Bowtie2, Samtools), and visualized (IGV). An important fraction of the filtered reads were not recruited by reference genomes, suggesting that plant isolates contain genes that are not present in the reference genomes. For this study, we focused on three conserved genomic regions: 16S-23S rDNA, atpD and nodDABC, and a Single Nucleotide Polymorphism (SNP) analysis was carried out with meta / multigenomes from each plant. Although the level of polymorphism varied (lowest in the rRNA region), polymorphic sites could be identified that define the specific soil population vs. reference genomes. More importantly, a plant-specific SNP distribution was observed. This could be confirmed with many other regions extracted from the reference genomes (data not shown). Our results confirm at the genomic level previous observations regarding plant selection of specific genotypes. We expect that further, ongoing comparative studies on differential meta / multigenomic sequences will identify specific gene components of the plant-selected genotype