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

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

Bacterial strains and plasmids.

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

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

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

Physiological parameters of aposymbiotic vs. symbiotic <i>D. sansibarensis</i> in gnotobiotic conditions.

Wild-type colonized D. sansibarensis were inoculated by a O. dioscoreae R-71412 cell suspension (Orrella) or a sterile 0.4% NaCl solution (MOCK). Physiological parameters were measured using a hand-held optical meter after 4 weeks of growth in gnotobiotic conditions. Parameters measured include A. Chlorophyl content (Chl); B. Anthocyanins index, measured as a function of green light absorbed by the sample; C. Flavonoids index (Flav), measured as a function of UV light absorbed by the sample and D. Nitrogen Balance Index (NBI) is measured as the ratio of Chl and Flav and is an indicator of C/N allocation changes due to N-deficiency. Data from 2 independent experiments are shown separately. Data from mock-inoculated plants are shown in orange, and in blue for O. dioscoreae-inoculated plants. The distributions of values between the O. dioscoreae–or mock-inoculated plants are identical for each of the 4 parameters (Wilcoxon rank sum test p > 0.05). (PDF)</p

Bacterial species used in this study.

Hereditary, or vertically-transmitted, symbioses affect a large number of animal species and some plants. The precise mechanisms underlying transmission of functions of these associations are often difficult to describe, due to the difficulty in separating the symbiotic partners. This is especially the case for plant-bacteria hereditary symbioses, which lack experimentally tractable model systems. Here, we demonstrate the potential of the leaf symbiosis between the wild yam Dioscorea sansibarensis and the bacterium Orrella dioscoreae (O. dioscoreae) as a model system for hereditary symbiosis. O. dioscoreae is easy to grow and genetically manipulate, which is unusual for hereditary symbionts. These properties allowed us to design an effective antimicrobial treatment to rid plants of bacteria and generate whole aposymbiotic plants, which can later be re-inoculated with bacterial cultures. Aposymbiotic plants did not differ morphologically from symbiotic plants and the leaf forerunner tip containing the symbiotic glands formed normally even in the absence of bacteria, but microscopic differences between symbiotic and aposymbiotic glands highlight the influence of bacteria on the development of trichomes and secretion of mucilage. This is to our knowledge the first leaf symbiosis where both host and symbiont can be grown separately and where the symbiont can be genetically altered and reintroduced to the host.</div

Method developed to make aposymbiotic plants and re-introduce a bacterium of interest.

(A) Node cuttings were taken from adult plants and incubated for 8 hours in 5% PPM for initial sterilization. (B) Node cuttings were incubated in a mixture of liquid MS, antibiotics and PPM for 3 weeks. (C) After 3–4 weeks, a bulbil (b) with its root system became apparent. Multiple leaves have formed from the node and are providing sugars to the plant. (D) The bulbil grows its own stem (s) that uses gravitropism to grow up and after the emergence of 2 leaves, the apical bud becomes visible. (E) After confirmation of being aposymbiotic by crushing and plating out the newly developed acumen(s), the plant was re-inoculated with a bacterium of interest by dropping 2 μl of the bacterial suspension on the apical bud. (PDF)</p

Phenotypic differences between symbiotic (left) and aposymbiotic (right) <i>D</i>. <i>sansibarensis</i>.

A. Plants inoculated with O. dioscoreae or B. with a mock solution. C. Cross-section of D. sansibarensis gland stained with acridine red, chrysoidine and astra blue showing a dense, orange-colored mixture of mucus and bacteria filling the lumen of symbiotic glands, and D. glands of aposymbiotic plants; E. Adaxial side of leaves of symbiotic; F. aposymbiotic plants kept in gnotobiotic conditions. G. SEM cross-section of symbiotic and H. of aposymbiotic acumen. I. SEM detail picture of trichome cells in the acumen being colonized by bacteria or J. aposymbiotic. K. TEM of trichomes in the acumen, surrounded by bacteria in symbiotic glands or L. deteriorating in aposymbiotic glands. M. Close-ups TEM showing the endoplasmic reticulum, Golgi, and plastids in the trichomes; and N. being mostly empty and containing plastids.</p