Bacterial strains and plasmids.

a<p>The location of plasmid insertions (number of nucleotides after the start codon) is indicated between brackets.</p

Cytoskeleton modifications of HeLa cells infected with <i>S. meliloti</i> queuosine biosynthesis mutants.

<p>HeLa cells were incubated with <i>S. meliloti queC</i> (A, E), <i>queF</i> (B, F), <i>tgt</i> (C, G), <i>queA</i> (D, H), the wild type 1021 strain (I) and the <i>queF</i> complemented strain (GMI11186) (J) in 0.5% FCS culture medium alone (A–D, I, J) or supplemented with preQ1 (E–H). HeLa cells were stained with phalloidin-Texas red and observed by fluorescence microscopy 48 hpi. Scale bar: 10 µm.</p

Bacteria-induced cytoskeleton modifications of HeLa cells.

<p>HeLa cells untreated (A), inoculated with <i>S. meliloti</i> (B), <i>R. leguminosarum</i> (C), <i>A. caulinodans</i> (D), <i>C. taiwanensis</i> (E), <i>B. tuberum</i> (F), <i>C. crescentus</i> (G) and <i>E. coli</i> (H). HeLa cells were stained with phalloidin-Texas red and observed by fluorescence microscopy 48 hours after bacterial inoculation. Arrow: stress fiber.</p

The <i>S. meliloti</i> queuosine biosynthetic pathway.

<p>preQ₀: 7-cyano-7-deazaguanine, preQ1: 7-(aminomethyl)-7-deazaguanine, AdoMet: S-adenosyl-L-methionine, EpoxyQ: epoxyqueuosine, Q: queuosine. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056043#pone.0056043-IwataReuyl1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056043#pone.0056043-Reader1" target="_blank">[50]</a>.</p

Determination of GTPases activation state in bacteria-treated HeLa cells.

<p>(A) Representative pull down assays of active Cdc42, Rac1 and RhoA GTPases at 48 hpi in non-inoculated- (control), <i>S. meliloti</i> 1021- and <i>queF</i>-inoculated HeLa cells. (B) Quantification of pull down assays using ImageJ software. Means ± S.D. were calculated from three independent experiments for Cdc42-GTP and mean from two independent experiments for Rac1-GTP and RhoA-GTP. Results were normalized to the corresponding total protein. Statistical significance (P<0.001) is shown (*) with respect to the control. (C) Immunoprecipitation of active and total CdC42 from non-inoculated (control) HeLa cells or cells inoculated with live and heat-killed wild-type bacteria 48 hpi. (D). Kinetics of Cdc42 activation. Actin, total Cdc42, Rac1 and RhoA or active GTP-bound forms of Cdc42, Rac1 or RhoA were detected by immuno-blotting of SDS-PAGE gels.</p

Symbiotic phenotype of <i>S. meliloti</i> queuosine mutants.

<p>Dry weight of <i>M. truncatula</i> seedlings inoculated with <i>S. meliloti</i> 1021, different queuosine-deficient mutants and the <i>queF</i> complemented (GMI11186) strain at 40 dpi. Statistical significance (P<0.01) is shown with respect to strain 1021(*) and the <i>queF</i> mutant (^#), respectively. (B, C) Sections of <i>M. truncatula</i> 21–day old nodules induced by 1021 (B) and the <i>queF</i> isogenic mutant (C). (D, E) Electron micrographs of nodule cells infected with 1021 (D) or the <i>queF</i> mutant (E). <i>queF</i> mutant bacteroids are randomly organized within the infected cell whereas 1021 bacteroids show a radial organization. (Insert panel in E): arrows point to symbiosome membranes detached from <i>queF</i> bacteria (). Arrowhead, type 4/5 bacteroid. *, starch granules.</p

Model for symbiotic and mutagenic plasmid-driven evolution of rhizobia.

<p>Following horizontal transfer of a symbiotic plasmid to a soil bacterium, the recipient genome accumulates environment-induced mutations that lead to phenotypic diversification. The most beneficial variants are selected by the plant and clonally multiply within nodules before being released. Rounds of <i>ex planta</i> phenotypic diversification/plant selection/clonal multiplication may have driven the adaptation process <i>in natura</i>.</p

Distribution of plasmid <i>nodABC</i> (p<i>nod</i>) and plasmid <i>imuBC</i> (p<i>imuBC</i>) genes among α- and β-proteobacteria.

<p>Blue and yellow rectangles indicate presence and absence of genes in the corresponding genome, respectively, as assayed by BlastP analysis. Dark blue rectangles indicate <i>nodABC</i> and <i>imuBC</i> genes co-localized on the same plasmid. α- and β-proteobacteria are arranged according to their position on the core genome phylogeny. Species of the same genus are similarly colored. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942.s009" target="_blank">Table S3</a> for details.</p

Evolvability of <i>imuA2B2C2</i>⁺ populations.

<p>(A) Experimental evolution of <i>imuA2B2C2</i>⁺ and Δ<i>imuA2B2C2</i> nodulating chimeric <i>Ralstonia</i>. Each ancestor was evolved using serial <i>M.pudica</i>-bacteria co-culture cycles, either <i>ex planta-in planta</i> cycles of 21 days (nodule bacteria serving as inoculum in each cycle, red lines) or <i>ex planta</i> cycles of 7 days (rhizospheric bacteria serving as inoculum in each cycle, blue lines). For <i>ex planta</i> lineages, 7-day cycles were chosen since the mean time bacteria spent <i>ex planta</i> in the 16 cycle evolution experiment (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio-1001942-g001" target="_blank">Figure 1</a>) was estimated to be 7 days. Ancestors were antibiotic resistant derivatives of a <i>hrpG</i> mutant of GMI1000pRalta. Four to five independent lineages have been derived from each ancestor. SpeR, spectinomycin-resistant strain. KanR, kanamycin-resistant strain. (B) Relative <i>in planta</i> fitness of <i>imuA2B2C2</i>⁺ versus Δ<i>imuA2B2C2</i> populations following co-inoculations of final populations derived from <i>ex planta-in planta</i> lineages (red legend, all Ev-<i>imu</i>⁺(i) versus Ev-Δ<i>imu</i>(i) pairs) or from <i>ex planta</i> lineages (blue legend, all Ev-<i>imu</i>⁺(i′) versus Ev-Δ<i>imu</i>(i′) pairs). Nodule bacteria were counted 21 days after inoculation. *Indicates significant differences between ancestral <i>imu</i>⁺ clones and evolved <i>imu⁺</i> populations (t-test, <i>p</i><0.05). #Indicates significant differences between <i>Ev-imu</i>⁺(i) (evolved <i>ex planta-in planta</i>) and Ev-<i>imu⁺</i>(i′) (evolved <i>ex planta</i>) populations (t-test, <i>p</i><0.05). ‡Indicates significant differences between Ev-<i>imu</i>⁺ and Ev-Δ<i>imu</i> populations for each series of competition experiments (either Ev-<i>imu⁺</i>(i) versus Ev-Δ<i>imu</i>(i) or Ev-<i>imu⁺</i>(i′) versus Ev-Δ<i>imu</i>(i′), t-test, <i>p</i><0.001). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942.s005" target="_blank">Figure S5B and S5C</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942.s015" target="_blank">Data S3</a> for details. (C) Relative <i>ex planta</i> fitness of <i>imuA2B2C2</i>⁺ versus Δ<i>imuA2B2C2</i> populations following co-inoculations of final populations derived from <i>ex planta-in planta</i> lineages. Bacteria recovered from the Jensen medium were counted 7 days after inoculation in Gibson tubes containing <i>M. pudica</i> plants. <i>imuA2B2C2</i>⁺ ancestors better survived in Jensen-<i>Mimosa</i> than Δ<i>imuA2B2C2</i>, in accordance with results presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942.s002" target="_blank">Figure S2A</a>. ‡Indicates significant differences between <i>imu</i>⁺ and Δ<i>imu</i> populations for each series of competition experiments (either An-<i>imu</i>⁺(i) versus An-Δ<i>imu</i>(i) or Ev-<i>imu⁺</i>(i) versus Ev-Δ<i>imu</i>(i), t-test, <i>p</i><0.01). No significant difference was observed between ancestral <i>imu</i>⁺ clones and evolved <i>imu</i>⁺ populations. Raw data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942.s015" target="_blank">Data S3</a>.</p

Experimental evolution of <i>R. solanacearum</i> into <i>Mimosa</i> symbionts.

<p>(A) <i>R. solanacearum</i> GMI1000 containing the <i>C. taiwanensis</i> symbiotic pRalta plasmid was evolved under <i>M. pudica</i> selection pressure. In a first step, three spontaneous <i>M. pudica</i>-nodulating derivatives of <i>GMI1000</i> pRalta, CBM212, CBM349, and CBM356 (selection cycle), were selected using <i>M. pudica</i> as a trap <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942-Marchetti1" target="_blank">[24]</a>. Nine independent lineages have been then derived from CBM212 (A–C), CBM349 (G–I), and CBM356 (M, N, S) using serial <i>M. pudica</i>-bacteria co-culture cycles of 21 days (evolution cycles) <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942-Marchetti1" target="_blank">[24]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942-Guan1" target="_blank">[25]</a>. Green and red arrow heads indicate activation of nodulation and intracellular infection, respectively <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942-Marchetti1" target="_blank">[24]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942-Guan1" target="_blank">[25]</a>. Numbers between brackets indicate the total number of point mutations detected in each clone relative to its closest re-sequenced ancestor. Point mutations are available on the Microscope platform (<a href="https://www.genoscope.cns.fr/agc/microscope/expdata/evoProject.php" target="_blank">https://www.genoscope.cns.fr/agc/microscope/expdata/evoProject.php</a>). (B–D) Nodulation and infection have been dramatically improved over 16 evolution cycles. <i>In planta</i> fitness (B) and nodulation competitiveness (C) of final clones relative to their respective nodulating ancestors, following equal co-inoculation of each of the nine final/ancestral pairs. Nodule infectiveness (D) of final clones (Ev) as compared to ancestors (An). Graphs summarize data from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942-Guan1" target="_blank">[25]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942-Marchetti2" target="_blank">[26]</a>. *<i>p</i>-value (t-test) <0.05. (E) In each cycle, bacteria were inoculated in the Jensen plant medium. Root nodules, which appeared from 5 days after inoculation, were each induced by a single bacterial cell that subsequently multiplied within nodule tissue <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942-Gage1" target="_blank">[56]</a>. In the selection and evolution cycles bacteria spent ∼21 days and from a few days up to 14 days in the plant medium, respectively. Population sizes are estimates. gen., generations. Raw data are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001942#pbio.1001942.s013" target="_blank">Data S1</a>.</p