Symbiotic Performance of Diverse Frankia Strains on Salt-Stressed Casuarina glauca and Casuarina equisetifolia Plants

Symbiotic nitrogen-fixing associations between Casuarina trees and the actinobacteria Frankia are widely used in agroforestry in particular for salinized land reclamation. The aim of this study was to analyze the effects of salinity on the establishment of the actinorhizal symbiosis between C. glauca and two contrasting Frankia strains (salt sensitive; CcI3 vs. salt tolerant; CeD) and the role of these isolates in the salt tolerance of C. glauca and C. equisetifolia plants. We show that the number of root nodules decreased with increasing salinity levels in both plants inoculated with CcI3 and CeD. Nodule formation did not occur in seedlings inoculated with CcI3 and CeD, at NaCl concentrations above 100 and 200 mM, respectively. Salinity also affected the early deformation of plant root hairs and reduced their number and size. In addition, expression of symbiotic marker Cg12 gene, which codes for a subtilase, was reduced at 50 mM NaCl. These data suggest that the reduction of nodulation in C. glauca under salt stress is in part due to inhibition of early mechanisms of infection. We also show that prior inoculation of C. glauca and C. equisetifolia with Frankia strains CcI3 and CeD significantly improved plant height, dry biomass, chlorophyll and proline contents at all levels of salinity tested, depending on the Casuarina-Frankia association. There was no correlation between in vitro salt tolerance of Frankia strains and efficiency in planta under salt-stressed conditions. Our results strongly indicate that increased N nutrition, photosynthesis potential and proline accumulation are important factors responsible for salt tolerance of nodulated C. glauca and C. equisetifolia

A glutamate synthase mutant of Bradyrhizobium sp. strain ORS285 is unable to induce nodules on Nod factor-independent Aeschynomene species

International audienceAbstract The Bradyrhizobium sp. strain ORS285 is able to establish a nitrogen-fixing symbiosis with both Nod factor (NF) dependent and NF-independent Aeschynomene species. Here, we have studied the growth characteristics and symbiotic interaction of a glutamate synthase (GOGAT; gltD::Tn5 ) mutant of Bradyrhizobium ORS285. We show that the ORS285 gltD::Tn5 mutant is unable to use ammonium, nitrate and many amino acids as nitrogen source for growth and is unable to fix nitrogen under free-living conditions. Moreover, on several nitrogen sources, the growth rate of the gltB::Tn5 mutant was faster and/or the production of the carotenoid spirilloxanthin was much higher as compared to the wild-type strain. The absence of GOGAT activity has a drastic impact on the symbiotic interaction with NF-independent Aeschynomene species. With these species, inoculation with the ORS285 gltD::Tn5 mutant does not result in the formation of nodules. In contrast, the ORS285 gltD::Tn5 mutant is capable to induce nodules on NF-dependent Aeschynomene species, but these nodules were ineffective for nitrogen fixation. Interestingly, in NF-dependent and NF-independent Aeschynomene species inoculation with the ORS285 gltD::Tn5 mutant results in browning of the plant tissue at the site of the infection suggesting that the mutant bacteria induce plant defence responses

NodD1 and NodD2 Are Not Required for the Symbiotic Interaction of <i>Bradyrhizobium</i> ORS285 with Nod-Factor-Independent <i>Aeschynomene</i> Legumes - Fig 6

<p>Addition of the <i>nod</i>-gene inducing flavonoid naringenin delays nodulation of <i>A</i>. <i>indica</i> plants (A) Nodulation kinetics of <i>Bradyrhizobium</i> ORS285 (B) <i>Bradyrhizobium</i> ORS285 Δ<i>nod</i>D1 and (C) <i>Bradyrhizobium</i> ORS285 Δ<i>nod</i>A-J derivatives, on <i>A</i>. <i>indica</i> plants in the absence and presence of 20 μM of the <i>nod</i> gene inducing flavonoid naringenin. The average number of nodules per plant (n = 10) at various days post infection (dpi) is presented. (D) Acetylene reducing activity in <i>A</i>. <i>indica</i> plants inoculated with <i>Bradyrhizobium</i> ORS285 and ORS285 Δ<i>nod</i>D1 and Δ<i>nod</i>A-J derivatives in the absence and presence of 20 μM naringenin at 15 dpi. The average amount of produced ethylene per hour and per plant is indicated. Error bars represent standard deviations (n = 10).</p

Schematic representation of the genomic regions containing the <i>nod</i>D1 and <i>nod</i>D2 gene of <i>Bradyrhizobium</i> ORS285, respectively.

<p>Schematic representation of the genomic regions containing the <i>nod</i>D1 and <i>nod</i>D2 gene of <i>Bradyrhizobium</i> ORS285, respectively.</p

Nodulation kinetics of <i>Bradyrhizobium</i> ORS285 and Δ<i>nod</i>D1 and Δ<i>nod</i>D2 derivatives on <i>A</i>. <i>afraspera</i> plants.

<p>(A) The average number of nodules per plant (n = 10) at various days post infection (dpi) is presented. (B) Acetylene reducing activity in <i>A</i>. <i>afraspera</i> plants inoculated with <i>Bradyrhizobium</i> ORS285 and Δ<i>nod</i>D1 and Δ<i>nod</i>D2 derivatives at 14 dpi. The average amount of produced ethylene per hour and per plant is indicated. Error bars represent standard deviations (n = 10). (C) Mature nodules of <i>A</i>. <i>afraspera</i> plants inoculated with <i>Bradyrhizobium</i> ORS285 and Δ<i>nod</i>D1 and Δ<i>nod</i>D2 derivatives at 14 dpi. White arrows indicate the superficial outgrowth on nodules.</p

<i>Bradyrhizobium</i> ORS285 <i>nod</i> genes are not expressed upon contact with NF-independent <i>Aeschynomene</i> plants.

<p>(A) chromatogram of SepPak C18 bound material of root exudate from <i>A</i>. <i>afraspera</i> (absorbance measured at 295 nm). (B) chromatogram of SepPak C18 bound material of root exudate from <i>A</i>. <i>indica</i> (absorbance measured at 295 nm). The UV-VIS spectra of the numbered peaks as indicated in (A) and (B) can be found in the Supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157888#pone.0157888.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157888#pone.0157888.s002" target="_blank">S2</a> Figs). (C) dose dependence of root exudate / naringenin-induced β-galactosidase activity of the <i>nod</i>A-<i>lac</i>Z fusion in <i>Bradyrhizobium</i> ORS285 and (D) β-galactosidase activity of <i>Bradyrhizobium</i> ORS285::<i>nod</i>A-<i>lac</i>Z cells grown for three days in the presence of <i>A</i>. <i>afraspera</i> and <i>A</i>. <i>indica</i> plants, respectively. The results are from one representative experiment with three technical replicates for each experimental condition.</p

Symbiotic properties of a chimeric Nod-independent photosynthetic <em>Bradyrhizobium</em> strain obtained by conjugative transfer of a symbiotic plasmid

International audienceThe lateral transfer of symbiotic genes converting a predisposed soil bacteria into a legume symbiont has occurred repeatedly and independently during the evolution of rhizobia. We experimented the transfer of a symbiotic plasmid between Bradyrhizobium strains. The originality of the DOA9 donor is that it harbours a symbiotic mega-plasmid (pDOA9) containing nod, nif and T3SS genes while the ORS278 recipient has the unique property of inducing nodules on some Aeschynomene species in the absence of Nod factors (NFs). We observed that the chimeric strain ORS278-pDOA9* lost its ability to develop a functional symbiosis with Aeschynomene. indica and Aeschynomene evenia. The mutation of rhcN and nodB led to partial restoration of nodule efficiency, indicating that T3SS effectors and NFs block the establishment of the NF-independent symbiosis. Conversely, ORS278-pDOA9* strain acquired the ability to form nodules on Crotalaria juncea and Macroptillium artropurpureum but not on NF-dependent Aeschynomene (A. afraspera and A. americana), suggesting that the ORS278 strain also harbours incompatible factors that block the interaction with these species. These data indicate that the symbiotic properties of a chimeric rhizobia cannot be anticipated due to new combination of symbiotic and non-symbiotic determinants that may interfere during the interaction with the host plant

Complete Genome Sequence of Bradyrhizobium sp. Strain ORS3257, an Efficient Nitrogen-Fixing Bacterium Isolated from Cowpea in Senegal

International audienceHere, we report the complete genome sequence of Bradyrhizobium sp. strain ORS3257, which forms efficient symbioses with cowpea, peanut, or groundnut. These genomic data will be useful to identify genes associated with symbiotic performance and host compatibility on several legumes, including Aeschynomene species, with which a Nod-independent type III secretion system (T3SS)-dependent symbiosis can be established

Identification of type III effectors modulating the symbiotic properties of Bradyrhizobium vignae strain ORS3257 with various Vigna species

International audienceThe Bradyrhizobium vignae strain ORS3257 is an elite strain recommended for cowpea inoculation in Senegal. This strain was recently shown to establish symbioses on some Aeschynomene species using a cocktail of Type III effectors (T3Es) secreted by the T3SS machinery. In this study, using a collection of mutants in different T3Es genes, we sought to identify the effectors that modulate the symbiotic properties of ORS3257 in three Vigna species ( V. unguiculata , V. radiata and V. mungo ). While the T3SS had a positive impact on the symbiotic efficiency of the strain in V. unguiculata and V. mungo , it blocked symbiosis with V. radiata . The combination of effectors promoting nodulation in V. unguiculata and V. mungo differed, in both cases, NopT and NopAB were involved, suggesting they are key determinants for nodulation, and to a lesser extent, NopM1 and NopP1, which are additionally required for optimal symbiosis with V. mungo . In contrast, only one effector, NopP2, was identified as the cause of the incompatibility between ORS3257 and V. radiata . The identification of key effectors which promote symbiotic efficiency or render the interaction incompatible is important for the development of inoculation strategies to improve the growth of Vigna species cultivated in Africa and Asia

A Proteomic Approach of Bradyrhizobium/Aeschynomene Root and Stem Symbioses Reveals the Importance of the fixA Locus for Symbiosis

Rhizobia are soil bacteria that are able to form symbiosis with plant hosts of the legume family. These associations result in the formation of organs, called nodules in which bacteria fix atmospheric nitrogen to the benefit of the plant. Most of our knowledge on the metabolism and the physiology of the bacteria during symbiosis derives from studying roots nodules of terrestrial plants. Here we used a proteomics approach to investigate the bacterial physiology of photosynthetic Bradyrhizobium sp. ORS278 during the symbiotic process with the semi aquatical plant Aeschynomene indica that forms root and stem nodules. We analyzed the proteomes of bacteria extracted from each type of nodule. First, we analyzed the bacteroid proteome at two different time points and found only minor variation between the bacterial proteomes of 2-week- and 3-week-old nodules. High conservation of the bacteroid proteome was also found when comparing stem nodules and root nodules. Among the stem nodule specific proteins were those related to the phototrophic ability of Bradyrhizobium sp. ORS278. Furthermore, we compared our data with those obtained during an extensive genetic screen previously published. The symbiotic role of four candidate genes which corresponding proteins were found massively produced in the nodules but not identified during this screening was examined. Mutant analysis suggested that in addition to the EtfAB system, the fixA locus is required for symbiotic efficiency