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Rhizobia form specialized nodules on the roots of legumes (family Fabaceae) and fix nitrogen in exchange for carbon from the host plant. Although the majority of legumes form symbioses with members of genus Rhizobium and its relatives in class Alphaproteobacteria, some legumes, such as those in the large genus Mimosa, are nodulated predominantly by betaproteobacteria in the genera Burkholderia and Cupriavidus. The principal centers of diversity of these bacteria are in central Brazil and South Africa. Molecular phylogenetic studies have shown that betaproteobacteria have existed as legume symbionts for approximately 50 million years, and that, although they have a common origin, the symbiosis genes in both subclasses have evolved separately since then. Additionally, some species of genus Burkholderia, such as B. phymatum, are highly promiscuous, effectively nodulating several important legumes, including common bean (Phaseolus vulgaris). In contrast to genus Burkholderia, only one species of genus Cupriavidus (C. taiwanensis) has so far been shown to nodulate legumes. The recent availability of the genome sequences of C. taiwanensis, B. phymatum, and B. tuberum has paved the way for a more detailed analysis of the evolutionary and mechanistic differences between nodulating strains of alpha- and betaproteobacteria. Initial analyses of genome sequences have suggested that plant-associated Burkholderia spp. have lower G+C contents than Burkholderia spp. that are opportunistic human pathogens, thus supporting previous suggestions that the plant- and human-associated groups of Burkholderia actually belong in separate genera.

The oxygen diffusion resistance of Lupinus albus (L.) cv. Multolupa root nodules was increased by subjection to short-term stresses; lowering rhizosphere temperature from 25 to 16 °C (2 h), detopping plants (3 h), darkening plants (21 h) or exposing roots to 20 mol m-3 KN03 for 2, 4 or 6 d. Microscopic observations and measurements showed that this resulted in the area of open intercellular spaces within the inner cortex being reduced due to both cell expansion and increased production of an occluding glycoprotein. Electrophoretic and Western Blot analysis using the monoclonal antibodies MAC236 and MAC265 showed two distinct glycoprotein antigens with molecular weights of 240 and 135 kDa, respectively. Both antigens are localized within intercellular spaces of the inner cortex. The amount of glycoprotein was determined using either ELISA, with MAC265, or quantification of immunolabelling with MAC236. This immunolabelling also localized the glycoprotein within globules adhering to the inside of the inner cortical cell walls.

Soybean (Glycine max L. Merr) cv. Clarke plants inoculated with Bradyrhizobium japonicum strain RCR3407 were grown either in a greenhouse with a low irradiance (200-400 μmol m -2 s -1 ) or in a controlled-environment growth cabinet with a higher irradiance (600 μmol m -2 s -1 ). At 42 d plants were given a nitrogen-free nutrient solution containing 50 mol m -3 sodium chloride for 2 weeks and then allowed to recover from salt-stress for a further 2 weeks. Salt treatment reduced plant growth by at least half in both growth regimes, however, the controlled environment-grown (CEG) plants were five times larger than the greenhouse-grown (GG) plants in terms of dry weight and number/weight of nodules per plant, regardless of treatment

An enzyme-linked immunosorbant assay (ELISA) method based on a monoclonal antibody (MAC236) is described in which relative amounts of an intercellular glycoprotein were quantified in extracts of whole legume nodules. This glycoprotein has recently been shown to be an important component of the cortical oxygen diffusion barrier. The ELISA method is demonstrated on three examples of soybean (Glycine max L. Merr.) nodule systems which have been the subject of previously published investigations: (a), cv. Clarke inoculated with Bradyrhizobium japonicum RCR3442, nodulated root systems of which were subject to 10, 21 or 40% oxygen continuously for 28 d; (b), cv. Bragg and its supernodulating mutant derivative (nts382) inoculated with Bradyrhizobium japonicum USDA110; (c), cv. Clarke inoculated with Bradyrhizobium japonicum RCR3442 or RCR3407. ELISA results are related to oxygen diffusion characteristics defined in previous publications and show that increases in the amount of glycoprotein present correlated with increases in supra-ambient (40%) levels of rhizosphere pO2, in minimum gas diffusion resistance and in speed of diffusion barrier response. Area data of component parts of nodule inner cortices suggest that diffusion resistance control under sub-ambient (10%) oxygen levels also involves cell expansion. The amount of MAC236 antigen in nodules is affected by both host plant genotype and rhizobial strain and the latter also appears to be involved in determining the morphological development of the nodule inner cortex.

BacA is an integral membrane protein, the mutation of which leads to increased resistance to the antimicrobial peptides bleomycin and Bac7 1-35 and a greater sensitivity to SDS and vancomycin in Rhizobium leguminosarum bv. viciae , R. leguminosarum bv. phaseoli , and Rhizobium etli . The growth of Rhizobium strains on dicarboxylates as a sole carbon source was impaired in bacA mutants but was overcome by elevating the calcium level. While bacA mutants elicited indeterminate nodule formation on peas, which belong to the galegoid tribe of legumes, bacteria lysed after release from infection threads and mature bacteroids were not formed. Microarray analysis revealed almost no change in a bacA mutant of R. leguminosarum bv. viciae in free-living culture. In contrast, 45 genes were more-than 3-fold upregulated in a bacA mutant isolated from pea nodules. Almost half of these genes code for cell membrane components, suggesting that BacA is crucial to alterations that occur in the cell envelope during bacteroid development. In stark contrast, bacA mutants of R. leguminosarum bv. phaseoli and R. etli elicited the formation of normal determinate nodules on their bean host, which belongs to the phaseoloid tribe of legumes. Bacteroids from these nodules were indistinguishable from the wild type in morphology and nitrogen fixation. Thus, while bacA mutants of bacteria that infect galegoid or phaseoloid legumes have similar phenotypes in free-living culture, BacA is essential only for bacteroid development in indeterminate galegoid nodules.

•An extensive survey of nodulation in the legume genus Mimosa was undertaken in two major biomes in Brazil, the Cerrado and the Caatinga, in both of which there are high degrees of endemicity of the genus.•Nodules were collected from 67 of the 70 Mimosa spp. found. Thirteen of the species were newly reported as nodulating. Nodules were examined by light and electron microscopy, and all except for M. gatesiae had a structure typical of effective Mimosa nodules. The endosymbiotic bacteria in nodules from all of the Mimosa spp. were identified as Burkholderia via immunolabelling with an antibody against Burkholderia phymatum STM815.•Twenty of the 23 Mimosa nodules tested were shown to contain nitrogenase by immunolabelling with an antibody to the nitrogenase Fe- (nifH) protein, and using the δ15N (15N natural abundance) technique, contributions by biological N2 fixation of up to 60% of total plant N were calculated for Caatinga Mimosa spp.•It is concluded that nodulation in Mimosa is a generic character, and that the preferred symbionts of Brazilian species are Burkholderia. This is the first study to demonstrate N2 fixation by beta-rhizobial symbioses in the field., This work was funded by the Natural Environment Research Council, UK (NE/B505038/1). The collection and transfer of nodules were authorized by permit no. 007/2005 from the Ministério do Meio Ambiente, Brazil (IBAMA).

Bacterial infection of interior tissues of legume root nodules is controlled at the epidermal cell layer and is closely coordinated with progressing organ development. Using spontaneous nodulating Lotus japonicus plant mutants to uncouple nodule organogenesis from infection, we have determined the role of 16 genes in these two developmental processes. We show that host-encoded mechanisms control three alternative entry processes operating in the epidermis, the root cortex and at the single cell level. Single cell infection did not involve the formation of trans-cellular infection threads and was independent of host Nod-factor receptors and bacterial Nod-factor signals. In contrast, Nod-factor perception was required for epidermal root hair infection threads, whereas primary signal transduction genes preceding the secondary Ca2+ oscillations have an indirect role. We provide support for the origin of rhizobial infection through direct intercellular epidermal invasion and subsequent evolution of crack entry and root hair invasions observed in most extant legumes., Plant and bacteria symbiosis in some species results in the coordinate formation of nitrogen fixing nodules and infection of the plant host. Using mutant Lotus japonicus plants, Madsen and colleagues have determined the role of 16 different genes in these two processes.

Because they occur both underwater and in soil, we have studied the gas-exchange properties of nodules of N.plena. We report here on a part of this study in which the structure of nodules from aquatic and vermiculite-grown plants were compared, particular attention being paid to the cell layers external to the infected region (the nodule cortex) because, in other species, these are believed to be the site of an oxygen diffusion resistance mechanism

The aquatic legume Neptunia plena (L.) Benth. was grown in non-aerated water culture or vermiculite. Growth, nodulation, nitrogen fixation and nodule physiology were investigated

34 Pags.- 7 Figs.- 1 Tabl. The definitive version is available at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-8137, The activities and localizations of superoxide dismutases (SODs) were compared in root and stem nodules of the semi-aquatic legume Sesbania rostrata using gel-activity assays and immunogold labelling, respectively. Nodules were fixed by high-pressure freezing and dehydrated by freeze substitution. Stem nodules showed more total and specific SOD activities than root nodules because of the presence of chloroplastic CuZnSOD. Most of the total SOD activity of stem and root nodules resulted from 'cytosolic' CuZnSOD, localized in the cytoplasm and chromatin, and from MnSOD in the bacteroids and in the mitochondria of vascular tissue. FeSOD was present in nodule plastids and in leaf chloroplasts, and was found to be associated with chromatin. Superoxide production was detected histochemically in the vascular bundles and in the infected tissue of stem and root nodules, whereas peroxide accumulation was observed in the cortical cell walls and intercellular spaces, as well as within the infection threads of both nodule types. These data suggest a role of CuZnSOD and FeSOD in protecting nuclear DNA from reactive oxygen species and/or in modulating gene activity. The enhanced levels of CuZnSOD, MnSOD and superoxide production in vascular bundle cells are consistent with a role of CuZnSOD and superoxide in the lignification of xylem vessels, but also suggest additional functions in coping with superoxide production by the high respiratory activity of parenchyma cells., This work was supported by the Royal Society (UK), Ministerio de Educación y Ciencia-Fondos Europeos de Desarrollo Regional (AGL2005-01404 and AGL2008-01298) and Gobierno de Aragón (group A53). E.K.J. thanks the Royal Society (UK) and Gobierno de Aragón-Caja Inmaculada (Spain) for funding a sabbatical leave (‘Programa Europa’). M.C.R. was the recipient of a postdoctoral contract (Program I3P) of Consejo Superior de Investigaciones Científicas-Fondo Social Europeo.

International audience; During development of legume root nodules, rhizobia and their host plant cells undergo profound differentiation, which is underpinned by massive changes in gene expression in both symbiotic partners. Oxygen concentrations in infected and surrounding uninfected cells drop precipitously during nodule development. To assess what effects this has on plant and bacterial cell differentiation and gene expression, we used a leghemoglobin-RNA-interference (LbRNAi) line of Lotus japonicus, which is devoid of leghemoglobins and has elevated levels of free-oxygen in its nodules. Bacteroids in LbRNAi nodules showed altered ultrastructure indicating changes in bacterial differentiation. Transcript analysis of 189 plant and 192 bacterial genes uncovered many genes in both the plant and bacteria that were differentially regulated during nodulation of LbRNAi plants compared with the wild type (containing Lb and able to fix nitrogen). These included fix and nif genes of the bacteria, which are involved in microaerobic respiration and nitrogen fixation, respectively, and plant genes involved in primary and secondary metabolism. Metabolite analysis revealed decreased levels of many amino acids in nodules of LbRNAi plants, consistent with the defect in symbiotic nitrogen fixation of this line.

Free-living Sinorhizobium meliloti lpxXL and acpXL mutants lack lipid A very-long-chain fatty acids (VLCFAs) and have reduced competitiveness in alfalfa. We demonstrate that LpxXL and AcpXL play important but distinct roles in bacteroid development and that LpxXL is essential for the modification of S. meliloti bacteroid lipid A with VLCFAs.

International audience; Bacteria isolated from Mimosa nodules in Taiwan, Papua New Guinea, Mexico and Puerto Rico were identified as belonging to either the a-or b-proteobacteria. The b-proteobacterial Burkholderia and Cupriavidus strains formed effective sym-bioses with the common invasive species Mimosa diplotricha, M. pigra and M. pudica, but the a-proteobacterial Rhizobium etli and R. tropici strains produced a range of symbiotic phenotypes from no nodulation through ineffective to effective nodula-tion, depending on Mimosa species. Competition studies were performed between three of the a-proteobacteria (R. etli TJ167, R. tropici NGR181 and UPRM8021) and two of the b-rhizobial symbionts (Burkholderia mimosarum PAS44 and Cupriavidus taiwanensis LMG19424) for nodulation of these inva-sive Mimosa species. Under flooded conditions, B. mimosarum PAS44 out-competed LMG19424 and all three a-proteobacteria to the point of exclusion. This advantage was not explained by initial inoculum levels, rates of bacterial growth, rhizobia-rhizobia growth inhibition or individual nodulation rate. However, the competitive domination of PAS44 over LMG19424 was reduced in the presence of nitrate for all three plant hosts. The largest significant effect was for M. pudica, in which LMG19424 formed 57% of the nodules in the presence of 0.5 mM potassium nitrate. In this host, ammonium also had a similar, but lesser, effect. Comparable results were also found using an N-containing soil mixture, and environmental N levels are therefore suggested as a factor in the competitive success of the bacterial symbiont in vivo.

Infection thread–dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.

A glycoprotein which occludes intercellular spaces in the inner cortex of legume nodules may be involved in controlling oxygen diffusion into rhizobial-infected cells. Here we investigated this possibility by localizing the glycoprotein using monoclonal antibodies and immunogold labelling in nodulated roots of soybean cv. Clarke inoculated with Bradyrhizobium japonicum strain RCR3442 exposed to atmospheres with either 10, 21 (control) or 40% oxygen for 28d. Infected cells showed evidence of premature senescence when grown in above or below ambient pO2 particularly at 10% oxygen, although cortical cells appeared to be little altered by oxygen treatment. In the inner cortical cells, more glycoprotein was seen to be occluding intercellular spaces of those nodules subjected to 40% oxygen and less in those nodules exposed to 10% oxygen, when compared to controls. This observation, made at the light microscope level (using silver enhancement) was confirmed under the TEM using immunogold labelling. Therefore, it is suggested that intercellular space glycoprotein is one of the structural components of the diffusion resistance in the cortex of legume nodules.

Minchin, F. R., James, E. K., Becana, M. (2008). Oxygen diffusion, production of reactive oxygen and nitrogen species, and antioxidants in legume nodules. Nitrogen Fixation-Origins Applications and Research Progress, Nitrogen-fixing leguminous symbioses. (eds) Dilworth, M. J.James, E. K.Sprent, J. I.Newton, W. E. Springer, P. O. Box 17, 3300 AA Dordrecht, Netherlands, pp. 321-362

College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK; National Kaohsiung Marine University, Kaohsiung City 811, Taiwan; Department of Biology, University of York, York YO10 5YW, UK; EMBPRAPA-Agrobiologia, km 47, Seropedica, 23851-970, RJ, Brazil; EMBRAPA-Cedrrados, Planaltina, Brasilia, 73301-970, DF Brazil; Department of Plant Science, University of Oxford, Oxford, OXI 3RB, UK; Depto de Ciencias Agrarias e Ambientais, UESC, km 16, Ilheus 45662-000 BA, Brazil; Faculdade de Agronomia, UFMT, Cuiaba, 78060-900, MT, Brazil; LSTM, IRD/CIRAD/INRA/AGROM/UMII, 34398 Montpellier, France

International audience; Two rhizobial strains, Br3407 T and Br3405, were isolated from nitrogen-fixing nodules on the roots of Mimosa caesalpiniifolia, a legume tree native to Brazil. On the basis of 16S rRNA gene sequence similarities, both strains were shown previously to belong to the genus Burkholderia. A polyphasic approach, including DNA–DNA hybridizations, pulsed-field gel electrophoresis of whole-genome DNA profiles, whole-cell protein analyses, fatty acid methyl ester analysis and extensive biochemical characterization, was used to clarify the taxonomic position of these strains further; the strains are here classified within a novel species, for which the name Burkholderia sabiae sp. nov. is proposed. The type strain is strain Br3407 T (5LMG 24235 T 5BCRC 17587 T).

International audience; Aims Species of the genus Burkholderia, from the Betaproteobacteria, have been isolated from legume nodules, but so far they have only been shown to form symbioses with species of Mimosa, sub-family Mimosoideae. This work investigates whether Burkholderia tuberum strains STM678 (isolated from Aspalathus carnosa) and DUS833 (from Aspalathus callosa) can nodulate species of the South African endemic papilionoid genera Cyclopia (tribe Podalyrieae) and Aspalathus (Crotalarieae) as well as the promiscuous legume Macroptilium atropurpureum (Phaseoleae). † Methods Bacterial strains and the phylogeny of their symbiosis-related (nod) genes were examined via 16S rRNA gene sequencing. Seedlings were grown in liquid culture and inoculated with one of the two strains of B. tuberum or with Sinorhizobium strain NGR 234 (from Lablab purpureus), Mesorhizobium strain DUS835 (from Aspalathus linearis) or Methylobacterium nodulans (from Crotalaria podocarpa). Some nodules, inoculated with green fluor-escence protein (GFP)-tagged strains, were examined by light and electron microscopy coupled with immunogold labelling with a Burkholderia-specific antibody. The presence of active nitrogenase was checked by immunolabelling of nitrogenase and by the acetylene reduction assay. B. tuberum STM678 was also tested on a wide range of legumes from all three sub-families. † Key Results Nodules were not formed on any of the Aspalathus spp. Only B. tuberum nodulated Cyclopia falcata, C. galioides, C. genistoides, C. intermedia and C. pubescens. It also effectively nodulated M. atropurpureum but no other species tested. GFP-expressing inoculant strains were located inside infected cells of C. genistoides, and bac-teroids in both Cyclopia spp. and M. atropurpureum were immunogold-labelled with antibodies against Burkholderia and nitrogenase. Nitrogenase activity was also shown using the acetylene reduction assay. This is the first demonstration that a b-rhizobial strain can effectively nodulate papilioinoid legumes. † Conclusions Papilionoid legumes from widely different tribes can be nodulated by b-rhizobia, forming both indeterminate (Cyclopia) and determinate (Macroptilium) nodules.

The definitive version is available at http://apsjournals.apsnet.org/loi/mpmi, Superoxide dismutases (SODs) are metalloenzymes that play a primary role in the protection against oxidative stress in plants and other organisms. We have characterized four SOD genes in Lotus japonicus and have analyzed their expression in roots and four developmental stages of nodules. The expression of cytosolic CuZnSOD, at the mRNA, protein, and enzyme activity levels, decreases with nodule age, and the protein is localized in the dividing cells and infection threads of emergent nodules and in the infected cells of young nodules. The mitochondrial MnSOD was downregulated, whereas the bacteroidal MnSOD displayed maximal protein and enzyme activity levels in older nodules. Two additional genes, encoding plastidic (FeSOD1) and cytosolic (FeSOD2) FeSOD isoforms, were identified and mapped. The genes are located in different chromosomes and show differential expression. The FeSOD1 mRNA level did not change during nodule development, whereas FeSOD2 was upregulated. The distinct expression patterns of the SOD genes may reflect different regulatory mechanisms of the enzyme activities during nodule ontogeny. In particular, at the mRNA and activity levels, the virtual loss of cytosolic CuZnSOD in mature and old nodules, concomitant with the induction of FeSOD2, suggests that the two enzymes may functionally compensate each other in the cytosol at the late stages of nodule development., This work was supported by grants MERG-7-CT-2005-517605 and FP6-2003-INCO-DEV2-517617 from the European Commission and AGL2005-01404 from the Ministerio de Educación y Ciencia and Fondo Europeo de Desarrollo Regional. M. C. Rubio was the recipient of a Marie Curie Individual Fellowship of the European Commission (HPMF-CT-2002-0943) and a postdoctoral contract (I3P program) of CSIC-FSE.

Legume-Rhizobium symbiosis is an example of selective cell recognition controlled by host/non-host determinants. Individual bacterial strains have a distinct host range enabling nodulation of a limited set of legume species and vice versa. We show here that expression of Lotus japonicus Nfr1 and Nfr5 Nod-factor receptor genes in Medicago truncatula and L. filicaulis, extends their host range to include bacterial strains, Mesorhizobium loti or DZL, normally infecting L. japonicus. As a result, the symbiotic program is induced, nodules develop and infection threads are formed. Using L. japonicus mutants and domain swaps between L. japonicus and L. filicaulis NFR1 and NFR5, we further demonstrate that LysM domains of the NFR1 and NFR5 receptors mediate perception of the bacterial Nod-factor signal and that recognition depends on the structure of the lipochitin-oligosaccharide Nod-factor. We show that a single amino-acid variation in the LysM2 domain of NFR5 changes recognition of the Nod-factor synthesized by the DZL strain and suggests a possible binding site for bacterial lipochitin-oligosaccharide signal molecules. Udgivelsesdato: 2007-Sep-5

Legumes have an intrinsic capacity to accommodate both symbiotic and endophytic bacteria within root nodules. For the symbionts, a complex genetic mechanism that allows mutual recognition and plant infection has emerged from genetic studies under axenic conditions. In contrast, little is known about the mechanisms controlling the endophytic infection. Here we investigate the contribution of both the host and the symbiotic microbe to endophyte infection and development of mixed colonised nodules in Lotus japonicus. We found that infection threads initiated by Mesorhizobium loti, the natural symbiont of Lotus, can selectively guide endophytic bacteria towards nodule primordia, where competent strains multiply and colonise the nodule together with the nitrogen-fixing symbiotic partner. Further co-inoculation studies with the competent coloniser, Rhizobium mesosinicum strain KAW12, show that endophytic nodule infection depends on functional and efficient M. loti-driven Nod factor signalling. KAW12 exopolysaccharide (EPS) enabled endophyte nodule infection whilst compatible M. loti EPS restricted it. Analysis of plant mutants that control different stages of the symbiotic infection showed that both symbiont and endophyte accommodation within nodules is under host genetic control. This demonstrates that when legume plants are exposed to complex communities they selectively regulate access and accommodation of bacteria occupying this specialized environmental niche, the root nodule., Author Summary Plants have evolved elaborated mechanisms to monitor microbial presence and to control their infection, therefore only particular microbes, so called “endophytes,” are able to colonise the internal tissues with minimal or no host damage. The legume root nodule is a unique environmental niche induced by symbiotic bacteria, but where multiple species, symbiotic and endophytic co-exist. Genetic studies of the binary interaction legume-symbiont led to the discovery of key components evolved in the two partners allowing mutual recognition and nodule infection. In contrast, there is limited knowledge about the endophytic nodule infection, the role of the legume host, or the symbiont in the process of nodule colonisation by endophytes. Here we focus on the early stages of nodule infection in order to identify which molecular signatures and genetic components favour/allow endophyte accommodation, and multiple species co-existence inside nodules. We found that colonisation of Lotus japonicus nodules by endophytic bacteria is a selective process, that endophyte nodule occupancy is host-controlled, and that exopolysaccharides are key bacterial features for chronic infection of nodules. Our strategy based on model legume genetics and co-inoculation can thus be used for identifying mechanisms operating behind host-microbes compatibility in environments where multiple species co-exist.

International audience; • The ability of Burkholderia phymatum STM815 to effectively nodulate Mimosa spp., and to fix nitrogen ex planta , was compared with that of the known Mimosa symbiont Cupriavidus taiwanensis LMG19424. • Both strains were equally effective symbionts of M. pudica , but nodules formed by STM815 had greater nitrogenase activity. STM815 was shown to have a broader host range across the genus Mimosa than LMG19424, nodulating 30 out of 31 species, 21 of these effectively. LMG19424 effectively nodulated only nine species. GFP-marked variants were used to visualise symbiont presence within nodules. • STM815 gave significant acetylene reduction assay (ARA) activity in semisolid JMV medium ex planta , but no ARA activity was detected with LMG19424. 16S rDNA sequences of two isolates originally from Mimosa nodules in Papua New Guinea (NGR114 and NGR195A) identified them as Burkholderia phymatum also, with nodA , nodC and nifH genes of NGR195A identical to those of STM815. • B. phymatum is therefore an effective Mimosa symbiont with a broad host range, and is the first reported beta-rhizobial strain to fix nitrogen in free-living culture.

Fourteen strains were isolated from nitrogen-fixing nodules on the roots of plants of the genus Mimosa growing in Taiwan, Brazil and Venezuela. On the basis of 16S rRNA gene sequence similarities, all of the strains were previously shown to be closely related to each other and to belong to the genus Burkholderia. A polyphasic approach, including DNA–DNA reassociation, whole-cell protein analysis, fatty acid methyl ester analysis and extensive biochemical characterization, was used to clarify the taxonomic position of these strains: all 14 strains were classified as representing a novel species, for which the name Burkholderia mimosarum sp. nov. is proposed. The type strain, PAS44T (=LMG 23256T =BCRC 17516T), was isolated from Mimosa pigra nodules in Taiwan.

Root-nodule development in legumes is an inducible developmental process initially triggered by perception of lipochitin-oligosaccharide signals secreted by the bacterial microsymbiont. In nature, rhizobial colonization and invasion of the legume root is therefore a prerequisite for formation of nitrogen-fixing root nodules. Here, we report isolation and characterization of chemically induced spontaneously nodulating mutants in a model legume amenable to molecular genetics. Six mutant lines of Lotus japonicus were identified in a screen for spontaneous nodule development under axenic conditions, i.e., in the absence of rhizobia. Spontaneous nodules do not contain rhizobia, bacteroids, or infection threads. Phenotypically, they resemble ineffective white nodules formed by some bacterial mutants on wild-type plants or certain plant mutants inoculated with wild-type Mesorhizobium loti. Spontaneous nodules formed on mutant lines show the ontogeny and characteristic histological features described for rhizobia-induced nodules on wild-type plants. Physiological responses to nitrate and ethylene are also maintained, as elevated levels inhibit spontaneous nodulation. Activation of the nodule developmental program in spontaneous nodules was shown for the early nodulin genes Enod2 and Nin, which are both upregulated in spontaneous nodules as well as in rhizobial nodules. Both monogenic recessive and dominant spontaneous nodule formation (snf) mutations were isolated in this mutant screen, and map positions were determined for three loci. We suggest that future molecular characterization of these mutants will identify key plant determinants involved in regulating nodulation and provide new insight into plant organ development.

Nuclear-cytoplasmic partitioning and traffic between cytoplasmic and nuclear compartments are fundamental processes in eukaryotic cells. Nuclear pore complexes mediate transport of proteins, RNAs and ribonucleoprotein particles in and out of the nucleus. Here we present positional cloning of a plant nucleoporin gene, Nup133 , essential for a symbiotic signal transduction pathway shared by Rhizobium bacteria and mycorrhizal fungi. Mutation of Nup133 results in a temperature sensitive nodulation deficient phenotype and absence of mycorrhizal colonization. Root nodules developing with reduced frequency at permissive temperatures are ineffective and electron microscopy show that Rhizobium bacteria are not released from infection threads. Measurement of ion fluxes using a calcium-sensitive dye show that Nup133 is required for the Ca 2+ spiking normally detectable within minutes after application of purified rhizobial Nod-factor signal molecules to root hairs. Localization of NUP133 in the nuclear envelope of root cells and root hair cells shown with enhanced yellow fluorescent protein fusion proteins suggests a novel role for NUP133 nucleoporins in a rapid nuclear–cytoplasmic communication after host–plant recognition of symbiotic microbes. Our results identify a component of an intriguing signal process requiring interaction at the cell plasma membrane and at intracellular nuclear and plastid organelle-membranes to induce a second messenger.

Contains fulltext : 35876pub.pdf (Publisher’s version ) (Closed access) Laser photoacoustic spectroscopy continuously quantified the ethylene (C2H4) produced by strawberry flowers and fruits developing in planta. C2H4 was first detected as flower buds opened and exhibited diurnal oscillations (to approximately 200 pl flower(-1) h(-1)) before petal abscission. Exogenous application of silver thiosulphate (STS) to detached flowers inhibited petal abscission and flower senescence. In fruit, C2H4 production was maintained at a 'low level' (10-60 pl fruit(-1) h(-1)) until fruit expanded when levels increased in a diurnal pattern (to 200 pl fruit(-1) h(-1)). After expansion, C2H4 production declined to a low level until fruit attained the red-ripe stage for at least 24 h. After this time, C2H4 levels increased linearly (no diurnal fluctuation) to approximately 1 nL fruit(-1) h(-1). Twenty-four hours after the re-initiation of C2H4 production by red fruit, CO2 levels increased approximately three-fold, indicative of a respiratory climacteric. STS applied to fruits developing in planta and dissected fruit parts ex situ established that C2H4 production is regulated by negative feedback until fruits had expanded. The C2H4 produced by red-ripe fruit was regulated by positive feedback. Anti-1-amino-cyclopropane-1-carboxylic acid oxidase IgG localization identified immunoreactive antigens of 40 and 30 kDa (M-r) within the fruit achenes of expanding and red-ripe fruit. Analysis of dissected fruit showed that seed C2H4 accounts for 50% the C2H4 that is detectable from ripe fruit.

Summary • A total of 191 rhizobial isolates from the root nodules of three geographically separate populations of the invasive plant Mimosa pigra in Taiwan were examined using amplified rDNA restriction analysis, 16S rDNA sequences, protein profiles and ELISA. Of these, 96% were identified as Burkholderia and 4% as Cupriavidus taiwanensis. • The symbiosis-essential genes nodA and nifH were present in two strains of Burkholderia (PAS44 and PTK47), and in one of C. taiwanensis (PAS15). All three could nodulate M. pigra. • Light and electron microscopy studies with a green fluorescent protein transconjugant variant of strain PAS44 showed the presence of fluorescent bacteroids in M. pigra nodules. These bacteroids expressed the nifH protein, hence this is the first confirmation that Burkholderia is a genuine symbiont of legume nodules. • The predominance of Burkholderia in Taiwanese M. pigra suggests that this species may have brought its symbionts from its native South America, rather than entering into association with the Taiwanese Mimosa symbiont C. taiwanensis which so successfully nodulates Mimosa pudica and Mimosa diplotricha.

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.

Euan K. James, Wen-Ming Chen, Sergio M. De Faria, Jean L. SimoesAraujo, Roseangela Straliotto, Rosa M. Pitard, Jui-Hsing Chou, Yi-Ju Chou, Edmundo Barrios, Alan R. Prescott, Janet I. Sprent, J. Peter W. Young School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK. Laboratory of Microbiology, Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung City 811, Chinese Taiwan. EMBRAPA-Agrobiologia, km 47, Seropedica, 23851-970, RJ, Brazil. Tropical Soil Biology and Fertility (TSBF) Institute of CIAT, Centro Internacional de Agricultura Tropical (CIAT), A.A. 6713, Cali, Colombia. Department of Biology 3, University of York, P.O. Box 373, York YO10 5YW, UK.

For high-fidelity chromosome segregation, kinetochores must be properly captured by spindle microtubules, but the mechanisms underlying initial kinetochore capture have remained elusive. Here we visualized individual kinetochore-microtubule interactions in Saccharomyces cerevisiae by regulating the activity of a centromere. Kinetochores are captured by the side of microtubules extending from spindle poles, and are subsequently transported poleward along them. The microtubule extension from spindle poles requires microtubule plus-end-tracking proteins and the Ran GDP/GTP exchange factor. Distinct kinetochore components are used for kinetochore capture by microtubules and for ensuring subsequent sister kinetochore bi-orientation on the spindle. Kar3, a kinesin-14 family member, is one of the regulators that promote transport of captured kinetochores along microtubules. During such transport, kinetochores ensure that they do not slide off their associated microtubules by facilitating the conversion of microtubule dynamics from shrinkage to growth at the plus ends. This conversion is promoted by the transport of Stu2 from the captured kinetochores to the plus ends of microtubules.

A β-glucoronidase (GUS)-marked strain of Herbaspirillum seropedicae Z67 was inoculated onto rice seedling cvs. IR42 and IR72. Internal populations peaked at over 106 log CFU per gram of fresh weight by 5 to 7 days after inoculation (DAI) but declined to 103 to 104 log CFU per gram of fresh weight by 28 DAI. GUS staining was most intense on coleoptiles, lateral roots, and at the junctions of some of the main and lateral roots. Bacteria entered the roots via cracks at the points of lateral root emergence, with cv. IR72 appearing to be more aggressively infected than cv. IR42. H. seropedicae subsequently colonized the root intercellular spaces, aerenchyma, and cortical cells, with a few penetrating the stele to enter the vascular tissue. Xylem vessels in leaves and stems were extensively colonized at 2 DAI but, in later harvests (7 and 13 DAI), a host defense reaction was often observed. Dense colonies of H. seropedicae with some bacteria expressing nitrogenase Fe-protein were seen within leaf and stem epidermal cells, intercellular spaces, and substomatal cavities up until 28 DAI. Epiphytic bacteria were also seen. Both varieties showed nitrogenase activity but only with added C, and the dry weights of the inoculated plants were significantly increased. Only cv. IR42 showed a significant (approximately 30%) increase in N content above that of the uninoculated controls, and it also incorporated a significant amount of 15N2.

Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

utilizing naturally available N derived from biological nitrogen fixation (BNF) by free-living and plant-associated diazotrophs, and from the mineralization of soil N. This system of rice cultivation over thousand years has sustained population, particularly in Asia, which is currently growing at 1.8% a year. For higher yields to support a rapidly increasing population, however, additional N must be applied. During the Green Revolution, and since the 1960s, the application of chemical N fertilizers boosted rice yields by 100–200% to match the demands of the increasing human population. Although rice production has so far kept up with population growth, new studies suggest that an additional 30% on top of the current rice supply will be needed during the next 30 years [1]. There is increasing pressure on rice-growing resources, primarily because agricultural lands are shrinking in area owing to increased urbanization, soil erosion etc, and present trends suggest that tomorrow’s rice land will be under even greater pressure [2]. This is further exacerbated by the deleterious effects that modern agriculture itself has on environmental degradation, particularly those caused by excessive chemical fertilization. Rice production in the coming years is expected to lean toward more intensification in terms of increased cropping per year, and this will be facilitated by the greater use of high-input technologies. Further expansion and intensification of the irrigated rice area could greatly increase the application of nitrogenous fertilizers, which in turn may have further negative environmental consequences.

This is journal paper no. 12,643, Agricultural Research Division, University of Nebraska., Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH., This work was supported by the National Science Foundation (grant no. OSR-92552255) and the U.S. Department of Agriculture-Cooperative State Research Education and Extension Service (grant no. 95-37305-2441). Access to the BioCad workstation was provided by the Center for Biotechnology at the University of Nebraska, Lincoln, funded through the Nebraska Research Initiative. J.F.M. was the recipient of a postdoctoral contract from the Ministry of Education and Culture (Spain).

A polyclonal antibody raised against a purified glycosylated polygalacturonase-inhibiting protein (PGIP) from immature raspberry fruits (Johnston et al. 1994) was used in serological analysis of white lupin (Lupinus albus L. cv. Multolupa) leaf, root and nodule tissues. Western blots revealed a single protein band at 56 kDa in leaf extracts and two immunoreactive bands at 66 kDa and 112 kDa in root extracts. Nodule extracts revealed multiple bands ranging in molecular weight from 49 kDa to 125 kDa, the intensity of these bands being greater in Fix + than Fix − tissue. Light and transmission electron microscopy (TEM) coupled to immunogold-labelling of leaves showed that the antigens were primarily localised in intercellular depositions of electron-dense material in the spongy mesophyll and palisade cell layers, and also intracellularly, adjacent to the cell walls of epidermal cells. Sections of roots and nodules showed that the antigens were localised primarily within intercellular spaces in the root cortex and in the nodule inner-cortex and infected zone. In the case of nodules, labelling was also abundant within the cytoplasm of cortical cells, being particularly evident in 1–2 cell layers in the inner cortex; the labelling occurring within vesicles adjacent to intercellular spaces and cell walls, and also within nearby Golgi and endoplasmic reticulum. Enzyme-linked immunosorbent assay (ELISA) tests of nodule extracts showed the quantity of antigens to increase in reponse to supra-ambient oxygen concentrations. This suggests that, along with the glycoprotein recognised by MAC236 and MAC265 (Iannetta et al. 1995), PGIP (and/or other antigens recognised by the anti-PGIP IgG) have a potential role in the formation and operation of the nodule oxygen diffusion barrier.

16 páginas, 7 figuras., The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N2-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N2 fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N2-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii., This work was partly funded by CAPES grant 2127/09-1 (www.capes.gov.br) and by CSIC grant AGL2008-03360 (www.csic.es), which provided a JAE-predoc scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Endophytic N2-fixing bacteria from several genera can enter plants through natural openings (stomata, hydathodes), natural and artificial wounds (points of emergence of roots, via vegetative propagation techniques) and also intact plant tissues. They may penetrate to xylem vessels or intercellular spaces, but there is no evidence that they can survive in healthy plant cells. The nutrition of endophytic bacteria in these various locations is discussed. It is concluded that subtle interactions between the C and N metabolisms of both host and endophyte may lead to significant N2 fixation without causing undue burdens on the host. There are good prospects for improving these associations by interdisciplinary laboratory and field studies involving both microbiologists and plant scientists.

New Zealand became geographically isolated about 80 million years ago and this separation gave rise to a unique native flora including four genera of legume, Carmichaelia, Clianthus and Montigena in the Carmichaelinae clade, tribe Galegeae, and Sophora, tribe Sophoreae, sub-family Papilionoideae. Ten bacterial strains isolated from NZ Carmichaelinae growing in natural ecosystems grouped close to the Mesorhizobium huakuii type strain in relation to their 16S rRNA and nifH gene sequences. However, the ten strains separated into four groups on the basis of their recA and glnII sequences: all groups were clearly distinct from all Mesorhizobium type strains. The ten strains separated into two groups on the basis of their nodA sequences but grouped closely together in relation to nodC sequences; all nodA and nodC sequences were novel. Seven strains selected and the M. huakuii type strain (isolated from Astragalus sinicus) produced functional nodules on Carmichaelia spp., Clianthus puniceus and A. sinicus but did not nodulate two Sophora species. We conclude that rhizobia closely related to M. huakuii on the basis of 16S rRNA and nifH gene sequences, but with variable recA and glnII genes and novel nodA and nodC genes, are common symbionts of NZ Carmichaelinae.

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. The basic reaction is comprised of three steps: 1. Denaturation of the target DNA 2. Annealing of sequence specific primers 3. Template-specific elongation of these primers with a DNA polymerase and desoxynucleotides

Ethiopia remains a net importer of soybean partly due to low average yields which may be improved by inoculation with rhizobia and/or plant growth promoting rhizobacteria (PGPR). The functional characterisation of 231 rhizobacteria isolated from the rhizosphere of soybean grown in 102 soils collected from different pedo-climatic regions of Ethiopia was carried out. Isolates were initially characterised by Gram staining and then functionally for: indole-3-acetic acid production; phosphate solubilisation; growth on a nitrogen-free medium; and, resistance to the pathogenic fungus Fusarium oxysporum. A sub-set of 72 of the best performing isolates were tested in vitro for: production of bioprotectants; polysaccharide degradation; and their relative capacity to maintain growth in response to extremes of: temperature; pH; salinity; antibiotics; pesticides; and, heavy metals. Twenty isolates with the best PGPR potential were identified via 16S rRNA gene sequencing. Seventeen isolates were Gram-negative: Pseudomonas (7); Stenotrophomonas (5); Acinetobacter (3); Enterobacter (1); and Achromobacter (1). Gram-positive types were: Bacillus (2); and, Microbacterium (1). Of the six of the most promising PGPR tested on soybean plants, Achromobacter and Acinetobacter significantly enhanced soybean seed germination, seedling growth and plant vigour index compared to non-inoculated plants. Key words: Soybean, Ethiopia, plant growth promoting rhizobacteria, plant growth promoting rhizobacteria (PGPR), seedling vigour.

Nodulated white lupins (Lupinus albus L. cv. Multolupa) were subject to either darkening for 12 h, followed by 24 h recovery in light, or to 50% O2 for 30 min. For each treatment, nodules were harvested at intervals for analysis by light and electron microscopy and determination of glycoprotein content using Enzyme Linked Immunosorbent Assays (ELISA). This allowed for an analysis of the sequence of events causing an increase in intercellular space occlusion within the inner cortex. The temporal sequence in response to darkening appears to be: (1) an initial rapid increase in the detectable levels of intracellular glycoprotein, due to either a state change or de novo synthesis, (2) a concomitant increase in the volume of thickened cell walls, causing a reduction of intercellular space volume and (3) after 1-3 h a release of glycoprotein into the intercellular space network of the inner cortex, accompanied (and possibly spread) by the continued constriction of the spaces due to cell wall and cell content expansion. The results for exposure to 50% O2 showed a similar, but much more rapid, sequence of events, operating within 15-30 min. The main difference between the two sequences was the lack of expansion of thickened cell walls with increased pO2. Also, it was possible to detect glycoprotein within cell walls following exposure to 50% O2 but not following darkening. These observations are discussed in relation to proposed mechanisms for the operation of a variable oxygen diffusion barrier in legume nodules.