Nodulation Genes Research Articles

Mapping and identification of a Cicer arietinum NSP2 gene involved in nodulation pathway

For the first time the putative NSP2 gene in chickpea has been identified using pairs of NILs differing for the Rn1 / rn1 nodulation gene that was located in LG5 of chickpea genetic map. An intraspecific cross between the mutant non-nodulating genotype PM233, carrying the recessive gene rn1, and the wild-type CA2139 was used to develop two pairs of near-isogenic lines (NILs) for nodulation in chickpea. These pairs of NILs were characterized using sequence tagged microsatellite site (STMS) markers distributed across different linkage groups (LGs) of the chickpea genetic map leading to the detection of polymorphic markers located in LG5. Using this information, together with the genome annotation in Medicago truncatula, a candidate gene (NSP2) known to be involved in nodulation pathway was selected for mapping in chickpea. The full length sequence obtained in chickpea wild-type (CaNSP2) was 1,503bp. Linkage analysis in an F3 population of 118 plants derived from the cross between the pair of NILS NIL7-2A (nod)×NIL7-2B (non-nod) revealed a co-localization between CaNSP2 and Rn1 gene. These data implicate the CaNSP2 gene as a candidate for identity to Rn1, and suggest that it could act in the nodulation signaling transduction pathway similarly to that in other legumes species.

Phenotypic characteristics and genetic diversity of rhizobia nodulating soybean in Egyptian soils

Twenty rhizobial strains isolated from the root nodules of soybean (Glycine max L.) were collected from nine governorates representing different agro-climatic and soil conditions in Egypt. The strains were characterized using a polyphasic approach, including nodulation pattern, phenotypic characterization, 16S rDNA sequencing, nifH and nodA symbiotic genes sequencing, and rep-PCR fingerprinting. Symbiotic properties assay revealed that all local rhizobial strains showed a wide spectrum of prolific nodulation and a marked increase in plant growth parameters compared to the un-inoculated control. Complete sequencing of 16S rRNA demonstrated that, native soybean nodulating rhizobia are phylogenetically related to Bradyrhizobium, Ensifer and Rhizobium (syn. Agrobacterium) genera. Study of tolerance ability to environmental stresses revealed that local strains survived in a wide pH ranges (pH 5–11) and a few of them tolerated high acidic conditions (pH 4). Agrobacterium strains were identified as the highest salt-tolerant and were survived under 6% NaCl, however Ensifer strains were the uppermost heat-tolerant and can grow at 42 °C. Agrobacterium strains have been shown to harbor nifH and nodA genes similar to those in other fast growing soybean symbionts and were largely distinct from symbiotic genes of slow growing bradyrhizobia. The symbiotic effectiveness stability of Agrobacterium strains to nodulate soybean roots was confirmed using plant nodulation assay.

Genome sequence of the acid-tolerant Burkholderia sp. strain WSM2232 from Karijini National Park, Australia.

Burkholderia sp. strain WSM2232 is an aerobic, motile, Gram-negative, non-spore-forming acid-tolerant rod that was trapped in 2001 from acidic soil collected from Karijini National Park (Australia) using Gastrolobium capitatum as a host. WSM2232 was effective in nitrogen fixation with G. capitatum but subsequently lost symbiotic competence during long-term storage. Here we describe the features of Burkholderia sp. strain WSM2232, together with genome sequence information and its annotation. The 7,208,311 bp standard-draft genome is arranged into 72 scaffolds of 72 contigs containing 6,322 protein-coding genes and 61 RNA-only encoding genes. The loss of symbiotic capability can now be attributed to the loss of nodulation and nitrogen fixation genes from the genome. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

Rhizobium leguminosarumis the symbiont of lentils in the Middle East and Europe but not in Bangladesh

Lentil is the oldest of the crops that have been domesticated in the Fertile Crescent and spread to other regions during the Bronze Age, making it an ideal model to study the evolution of rhizobia associated with crop legumes. Housekeeping and nodulation genes of lentil-nodulating rhizobia from the region where lentil originated (Turkey and Syria) and regions to which lentil was introduced later (Germany and Bangladesh) were analyzed to determine their genetic diversity, population structure, and taxonomic position. There are four different lineages of rhizobia associated with lentil nodulation, of which three are new and endemic to Bangladesh, while Mediterranean and Central European lentil symbionts belong to the Rhizobium leguminosarum lineage. The endemic lentil grex pilosae may have played a significant role in the origin of these new lineages in Bangladesh. The presence of R. leguminosarum with lentil at the center of origin and in countries where lentil was introduced later suggests that R. leguminosarum is the original symbiont of lentil. Lentil seeds may have played a significant role in the initial dispersal of this Rhizobium species within the Middle East and on to other countries. Nodulation gene sequences revealed a high similarity to those of symbiovar viciae.

Calcium-Dependent Regulation of Genes for Plant Nodulation in Rhizobium leguminosarum Detected by iTRAQ Quantitative Proteomic Analysis

Rhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant-microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca(2+) involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the nod gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca(2+) chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca(2+) influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed.

From β- to α-Proteobacteria: The Origin and Evolution of Rhizobial Nodulation Genes nodIJ

Although many α- and some β-proteobacterial species are symbiotic with legumes, the evolutionary origin of nitrogen-fixing nodulation remains unclear. We examined α- and β-proteobacteria whose genomes were sequenced using large-scale phylogenetic profiling and revealed the evolutionary origin of two nodulation genes. These genes, nodI and nodJ (nodIJ), play key roles in the secretion of Nod factors, which are recognized by legumes during nodulation. We found that only the nodulating β-proteobacteria, including the novel strains isolated in this study, possess both nodIJ and their paralogous genes (DRA-ATPase/permease genes). Contrary to the widely accepted scenario of the α-proteobacterial origin of rhizobia, our exhaustive phylogenetic analysis showed that the entire nodIJ clade is included in the clade of Burkholderiaceae DRA-ATPase/permease genes, that is, the nodIJ genes originated from gene duplication in a lineage of the β-proteobacterial family. After duplication, the evolutionary rates of nodIJ were significantly accelerated relative to those of homologous genes, which is consistent with their novel function in nodulation. The likelihood analyses suggest that this accelerated evolution is not associated with changes in either nonsynonymous/synonymous substitution rates or transition/transversion rates, but rather, in the GC content. Although the low GC content of the nodulation genes has been assumed to reflect past horizontal transfer events from donor rhizobial genomes with low GC content, no rhizobial genome with such low GC content has yet been found. Our results encourage a reconsideration of the origin of nodulation and suggest new perspectives on the role of the GC content of bacterial genes in functional adaptation.

Signaling at the Root Surface: The Role of Cutin Monomers in Mycorrhization

Most vascular plants interact with arbuscular mycorrhizal fungi (AMF) and are thereby provided significant advantages in nutrient acquisition, especially phosphate. The widespread retention of this symbiosis in vascular plants is testament to its importance and ancient origins. This highlight focuses on the reports of two of the first genes identified through forward genetic screens for AMF symbiotic mutants: Required for Abuscular Mycorrhization 1 (RAM1) and RAM2, which respectively encode a GRAS transcription factor and a glycerol-3-phosphate acyl-transferase (GPAT) that are required for the formation of fungal entry structures (hyphopodia) on the root surface. This provides the first insights into a mycorrhization specific signaling pathway and reveals cutin monomers as a critical component of signaling in the mycorrhizal symbiosis. We discuss the proposed links between these genes, and the role of cutin and its precursors in interactions with AMF, and oomycete and fungal pathogens. Root colonization by AMF involves the detection of compounds found in host root exudates such as strigolactones. Strigolactones are carotenoid-derived terpenoids that serve as a rhizosphere signal to activate responses in AMF, including hyphal branching, and thereby promote mycorrhizal colonization of the root surface. This involves the fungus making contact with the plant epidermal cell wall and forming a hyphopodium which is a lobed hyphal contact point with the root that serves as the entry point of the fungus into the epidermis. The hyphopodium is a specialized structure similar to, but distinct from, the pathogenic appressorium. The fungus then passes through an epidermal cell and colonizes the root cortex by extensive intercellular hyphal growth and the formation of terminal intracellular structures called arbuscules. The arbuscule, which serves as the nutrient exchange interface in the symbiosis, is highly branched and is surrounded by plant plasma membrane. It has been known for some time that the evolutionary history of the mycorrhizal symbioses is closely intertwined with that of a second root endosymbiosis called nodulation. The signaling required for the initiation of nodulation in legumes involves several genes that are also essential for mycorrhization (Kistner et al., 2005). This common set of genes is usually referred to as the common signaling pathway. For clarity, throughout this manuscript, common signally pathway genes identified in model legumes will be given as Medicago truncatula/Lotus japonicus. These shared components comprise a signaling circuit, which includes the ion channel DMI1 (Does not Make Infections)/POLLUX, DMI2/SYMRK (SYMbiosis Receptor like Kinase) and the calcium calmodulin kinase DMI3/CCaMK (Catoira et al., 2000) and its substrate IPD3 (Interacting Protein of DMI3)/Cyclops. Downstream of this shared signaling pathway lie the genes regulating the transcriptional outputs, including the GRAS transcription factors NSP1 and NSP2, which interact to regulate expression of nodulation genes (Hirsch et al., 2009).

Alleviating the inhibitory effect of salinity stress on nod gene expression in Rhizobium tibeticum – fenugreek (Trigonella foenum graecum) symbiosis by isoflavonoids treatment

Rhizobia-legume symbiosis depends on molecular dialog, which involves the production of specific plant flavonoid compounds as signal molecules. Rhizobium tibeticum was recovered from the root nodule of fenugreek and identified by sequencing the 16S rRNA gene. The effect of salinity stress on nod gene expression was measured in terms of β-galactosidase activity. R. tibeticum containing Escherichia coli lacZ gene fusions to specific nodulation (nod) genes were used to determine β-galactosidase activity. Combination of hesperetin (7.5 µM) and apigenin (7.5 µM) significantly increased β-galactosidase activity more than the single application of hesperetin or apigenin. Preincubation of R. tibeticum with hesperetin and apigenin combination significantly alleviates the adverse effect of salinity on nod gene expression and therefore, enhances nodulation and nitrogen fixation of fenugreek.

The diversity of Rhizobia, Sinorhizobia and novel non-Rhizobial Paenibacillus nodulating wild herbaceous legumes

The objective of the present study was to isolate and characterize nodulating bacteria associated with wild legumes. For this purpose, we recovered twenty isolates from root nodules of five wild legume species: Melilotus alles, Melilotus officinalis, Trifolium pratense, Trifolium repens and Medicago sp. Most of the isolates were morphologically analogous with only few exceptions in colony shape, appearance and incubation time. All isolates were Gram negative except T.P2-4. Random amplification of polymorphic DNA showed genetic variation among isolates. The 16S rRNA sequence analysis revealed these isolates as Rhizobium, Sinorhizobium and Paenibacillus. Each of these was also screened for nod D and nod F genes with marked variation at these loci; however, the nucleotide sequence analysis confirmed the presence of nod genes. The assignment of strains to their hosts revealed a unique symbiotic association of Paenibacillus sp. nodulating T .pratense which is being reported here for the first time.

Isolation and Characterization of Mutant Sinorhizobium meliloti NodD1 Proteins with Altered Responses to Luteolin

NodD1, a member of the NodD family of LysR-type transcriptional regulators (LTTRs), mediates nodulation (nod) gene expression in the soil bacterium Sinorhizobium meliloti in response to the plant-secreted flavonoid luteolin. We used genetic screens and targeted approaches to identify NodD1 residues that show altered responses to luteolin during the activation of nod gene transcription. Here we report four types of NodD1 mutants. Type I (NodD1 L69F, S104L, D134N, and M193I mutants) displays reduced or no activation of nod gene expression. Type II (NodD1 K205N) is constitutively active but repressed by luteolin. Type III (NodD1 L280F) demonstrates enhanced activity with luteolin compared to that of wild-type NodD1. Type IV (NodD1 D284N) shows moderate constitutive activity yet can still be induced by luteolin. In the absence of luteolin, many mutants display a low binding affinity for nod gene promoter DNA in vitro. Several mutants also show, as does wild-type NodD1, increased affinity for nod gene promoters with added luteolin. All of the NodD1 mutant proteins can homodimerize and heterodimerize with wild-type NodD1. Based on these data and the crystal structures of several LTTRs, we present a structural model of wild-type NodD1, identifying residues important for inducer binding, protein multimerization, and interaction with RNA polymerase at nod gene promoters.

Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the Southern African crotalarioid clade Lotononis s.l.

The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The seasonally waterlogged habitat of Listia species may favour the development of symbiotic specificity.

An invasive Mimosa in India does not adopt the symbionts of its native relatives.

The large monophyletic genus Mimosa comprises approx. 500 species, most of which are native to the New World, with Central Brazil being the main centre of radiation. All Brazilian Mimosa spp. so far examined are nodulated by rhizobia in the betaproteobacterial genus Burkholderia. Approximately 10 Mya, transoceanic dispersal resulted in the Indian subcontinent hosting up to six endemic Mimosa spp. The nodulation ability and rhizobial symbionts of two of these, M. hamata and M. himalayana, both from north-west India, are here examined, and compared with those of M. pudica, an invasive species. Nodules were collected from several locations, and examined by light and electron microscopy. Rhizobia isolated from them were characterized in terms of their abilities to nodulate the three Mimosa hosts. The molecular phylogenetic relationships of the rhizobia were determined by analysis of 16S rRNA, nifH and nodA gene sequences. Both native Indian Mimosa spp. nodulated effectively in their respective rhizosphere soils. Based on 16S rRNA, nifH and nodA sequences, their symbionts were identified as belonging to the alphaproteobacterial genus Ensifer, and were closest to the 'Old World' Ensifer saheli, E. kostiensis and E. arboris. In contrast, the invasive M. pudica was predominantly nodulated by Betaproteobacteria in the genera Cupriavidus and Burkholderia. All rhizobial strains tested effectively nodulated their original hosts, but the symbionts of the native species could not nodulate M. pudica. The native Mimosa spp. in India are not nodulated by the Burkholderia symbionts of their South American relatives, but by a unique group of alpha-rhizobial microsymbionts that are closely related to the 'local' Old World Ensifer symbionts of other mimosoid legumes in north-west India. They appear not to share symbionts with the invasive M. pudica, symbionts of which are mostly beta-rhizobial.

RECENT PROGRESS IN THE ELUCIDATION OF THE FUNCTION OF nodA, B, AND C GENES OF RHIZOBIUM MELILOTI

ABSTRACT The common nodulation genes nodA, B, and C are highly conserved between different Rhizobium species and are required for the nodulation of legumes and non-legumes. The expression of these genes in Rhizobium meliloti is under both positive and negative control. Monospecific polyclonal antibodies were used to localize the NodA and NodB proteins in the cytosol of R. meliloti. These proteins are involved in the generation of small heat-stable compounds that stimulate the mitosis of different plant protoplasts. Our experiments suggest that the NodC transmembrane protein is not involved in the synthesis of these factors. Gene fusion experiments were used to define the membrane-anchor domain which is necessary for the insertion of the NodC protein into the membrane. A highly hydrophobic transmembrane-anchor domain was found near the carboxyl terminus, separating a large extracellular domain which contains a cystein-rich cluster from a short putative intracellular domain. The domain structure of the dime...

High NaCl Concentrations Induce the nod Genes of Rhizobium tropici CIAT899 in the Absence of Flavonoid Inducers

The nodulation (nod) genes of Rhizobium tropici CIAT899 can be induced by very low concentrations (micromolar to nanomolar range) of several flavonoid molecules secreted by the roots of leguminous plants under a number of different conditions. Some of these conditions have been investigated and appear to have a great influence on the concentration and the number of different Nod factors, which can induce root nodule primordia and pseudonodules in several leguminous plant roots. In one such condition, we added up to 300 mM NaCl to the induction medium of R. tropici CIAT899 containing the nod gene inducer apigenin. At the higher concentrations of NaCl, larger amounts and more different Nod factors were produced than in the absence of extra NaCl. To our surprise, under control conditions (300 mM NaCl without apigenin), some Nod-factor-like spots were also observed on the thin-layer plates used to detect incorporation of radiolabeled glucosamine into newly synthesized Nod factors. This phenomenon was further investigated with thin-layer plates, fusions of nod genes to the lacZ gene, high-performance liquid chromatography, mass spectrometry, and the formation of pseudonodules on bean roots. Here, we report that, in the absence of flavonoid inducers, high concentrations of NaCl induced nod genes and the production of Nod factors.

Rhizobium pisi sv. trifolii K3.22 harboring nod genes of the Rhizobium leguminosarum sv. trifolii cluster

The taxonomic status of the Rhizobium sp. K3.22 clover nodule isolate was studied by multilocus sequence analysis (MLSA) of 16S rRNA and six housekeeping chromosomal genes, as well as by a subsequent phylogenic analysis. The results revealed full congruence with the Rhizobium pisi DSM 30132T core genes, thus supporting the same taxonomic position for both strains. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence similarity to Rhizobium leguminosarum sv. trifolii, whereas the R. pisi DSM 30132Tnod genes were most similar to R. leguminosarum sv. viciae. The strains differed in the host range nodulation specificity, since strain K3.22 effectively nodulated red and white clover but not vetch, in contrast to R. pisi DSM 30132T, which effectively nodulated vetch but was not able to nodulate clover. Both strains had the ability to form nodules on pea and bean but they differed in bean cultivar specificity. The R. pisi K3.22 and DSM 30132T strains might provide evidence for the transfer of R. leguminosarum sv. trifolii and sv. viciae symbiotic plasmids occurring in natural soil populations.

Co‐invasion of South African ecosystems by an Australian legume and its rhizobial symbionts

AbstractAimTo determine and compare the taxonomic identity and diversity of root nodule and rhizospheric microbial symbionts associated with Acacia pycnantha Benth. in its native (Australian) and invasive (South African) ranges, and to establish whether these associations are general or host specific.LocationThe native range of A. pycnantha in Australia and invasive ranges in South Africa and Western Australia.MethodsBacteria were isolated from root nodules collected from 18 populations of A. pycnantha. Repetitive element polymerase chain reaction (REP‐PCR) fingerprinting was used to assess overall bacterial diversity and clustering. Molecular phylogenies for a subset of isolates representing major REP‐PCR clades were reconstructed using maximum parsimony and Bayesian phylogenetic analyses of the nuclear 16S–23S rRNA intergenic spacer (IGS), 16S rRNA, and the symbiotic nodA genes.ResultsTwelve clusters were identified from the REP‐PCR analysis; 11 included isolates from both the native range in Australia and invasive range in South Africa, while one cluster comprised only Australian isolates. Six rhizobial species were found in association with A. pycnantha: Bradyrhizobium japonicum, Rhizobium gallicum, R. lusitanum, R. miluonense, R. multihospitium and R. tropici. We also identified three plant‐growth promoting bacteria isolated from root nodules of A. pycnantha: Burkholderia caledonica, B. graminis and B. phytofirmans. Phylogenetic analysis of the IGS gene retrieved clades containing symbionts from both Australia and South Africa while others comprised only South African taxa, suggesting the introduction of bacterial lineages from Australia to South Africa. Our phylogeographic analysis of the nodA gene confirmed that A. pycnantha was co‐introduced with its symbionts to South Africa.Main conclusionsAcacia pycnantha is a promiscuous legume, associated with at least six different rhizobial symbionts, and forms associations with plant‐growth promoting rhizosphere bacteria from the genus Burkholderia. In the invasive range of A. pycnantha in South Africa, nodules contained some symbionts of South African origin while other symbionts appear to have been co‐introduced from Australia. Acacia pycnantha is associated with a wider suite of symbionts in its invasive than native range.

Cropping history affects nodulation and symbiotic efficiency of distinct hairy vetch (Vicia villosa Roth.) genotypes with resident soil rhizobia

Compatible rhizobia strains are essential for nodulation and biological nitrogen fixation (BNF) of hairy vetch (Vicia villosa Roth, HV). We evaluated how past HV cultivation affected nodulation and BNF across host genotypes. Five groups of similar HV genotypes were inoculated with soil dilutions from six paired fields, three with 10-year HV cultivation history (HV+) and three with no history (HV−), and used to determine efficiency of rhizobia nodulation and BNF. Nodulation was equated to nodule number and mass, BNF to plant N and Rhizobium leguminosarum biovar viceae (Rlv) soil cell counts using qPCR to generate an amplicon of targeted Rlv nodD genes. Both HV cultivation history and genotype affected BNF parameters. Plants inoculated with HV+ soil dilutions averaged 60 and 70 % greater nodule number and mass, respectively. Such plants also had greater biomass and tissue N than those inoculated with HV− soil. Plant biomass and tissue N were strongly correlated to nodule mass (r 2 = 0.80 and 0.50, respectively), while correlations to nodule number were low (r 2 = 0.50 and 0.31, respectively). Although hairy vetch rhizobia occur naturally in soils, past cultivation of HV was shown in this study to enhance nodulation gene-carrying Rlv population size and/or efficiency of rhizobia capable of nodulation and N fixation.

Establishment and survival of the South African legume Lessertia spp. and rhizobia in Western Australian agricultural systems

Background and aims The South African herbaceous legume species Lessertia capitata, L. diffusa, L. excisa L. incana and L. herbacea were introduced to Australia to assess plant establishment and survival, as well as the saprophytic ability of their root nodule bacteria (RNB). Methods Five Lessertia spp., were inoculated with selected RNB strains and were sown in five different agroclimatic areas of the Western Australian wheatbelt during 2007 and 2008. Plant population and summer survival were evaluated in situ. Soil samples and nodules from host plants were also taken from each site. The re-isolated rhizobia were RPO1-PCR fingerprinted and their partial dnaK and nodA genes were sequenced to confirm their identity. Results Plants achieved only poor establishment followed by weak summer survival. More than 83 % of the rhizobia re-isolated from Lessertia did not correlate with the original inoculants’ fingerprints, and were identified as Rhizobium leguminosarum. The nodA sequences of the naturalised strains were also

Single or Multiple Gene Silencing Directed by U6 Promoter-Based shRNA Vectors Facilitates Efficient Functional Genome Analysis in Medicago truncatula

Short hairpin RNA (shRNA) expression vectors are an effective means to deliver double-stranded RNA that mediates sequence-specific cleavage of target transcripts in RNA interference (RNAi). Previously, we constructed shRNA expression vectors based on U6 small nuclear RNA (snRNA) gene promoters of Medicago truncatula. To facilitate screening of shRNA candidates, a protoplast-based dual luciferase assay system was developed, which consists of a shRNA-expressing effector plasmid and a target gene-carrying reporter plasmid. RNAi efficiency of a given shRNA could be measured by comparing two luciferase activities derived from transcripts with an intact or interrupted 3′ untranslated region as a result of RNAi. Using a multiple U6-shRNA cassette plasmid that enables targeting of multiple genes simultaneously, the expression of two Δ1-pyrroline-5-carboxylate synthetase genes (MtP5CS1 and MtP5CS2) was silenced with a U6 plasmid carrying two 27-nucleotide-stem shRNA cassettes targeting each gene. The decreased transcript levels were accompanied by decreased protein levels and decreased free proline contents. To examine possible U6-shRNA-mediated RNAi in nodules, two remorin-encoding MtREM genes were targeted by single and double U6-shRNA plasmids. Silencing of MtREM2.2, an essential nodulation gene, MtREM1.1, an uncharacterized gene, or both inhibited nodule formation in transgenic roots. Moreover, M. truncatula roots transformed with a suppressor-overexpresser construct wherein MtREM2.2-targeting U6-shRNA and GmREM2.3-overexpressing cassettes were placed tandemly showed decreased MtREM2.2 transcripts yet survived nitrogen-free medium, indicating complementation of MtREM2.2 with the overexpressed soybean gene. These results demonstrate that U6 promoter-based shRNA-expressing plasmids are valuable for multiple gene functional analyses in protoplasts and nodules of M. truncatula.

Genetic diversity of indigenous rhizobial symbionts of the Lupinus mariae-josephae endemism from alkaline-limed soils within its area of distribution in Eastern Spain

The genomic diversity of a collection of 103 indigenous rhizobia isolates from Lupinus mariae-josephae (Lmj), a recently described Lupinus species endemic to alkaline-limed soils from a restricted habitat in Eastern Spain, was investigated by molecular methods. Isolates were obtained from soils of four geographic locations in the Valencia province that harbored the known Lmj plant populations. Using an M13 RAPD fingerprinting technique, 19 distinct RAPD profiles were identified. Phylogenetic analysis based on 16S rDNA and the housekeeping genes glnII, recA and atpD showed a high diversity of native Bradyrhizobium strains that were able to establish symbiosis with Lmj. All the strains grouped in a clade unrelated to strains of the B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. The phylogenetic tree based on concatenated glnII, recA and atpD gene sequences grouped the Lmj isolates in six different operational taxonomic units (OTUs) at the 93% similarity level. These OTUs were not associated to any specific geographical location, and their observed divergence predicted the existence of different Bradyrhizobium genomic species. In contrast, phylogenetic analysis of symbiotic genes based on nodC and nodA gene sequences, defined only two distinct clusters among the Lmj strains. These two Lmj nod gene types were largely distinct from nod genes of bradyrhizobia nodulating other Old World lupine species. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host.