Induction of<i>qsdA</i> gene transcription, NAHSL-breakdown and biocontrol activity of <b><i>R. erythropolis</i></b> in potato tubers.

<p>(A) <i>qsdA</i> gene transcription and biocontrol activity of the <i>R. erythropolis</i> BCA-<i>qsdA::gfp</i> against <i>P. atrosepticum</i> 6276 defective (<i>Pa</i>-QS<b>–</b>) or not (<i>Pa</i>-QS<b>+</b>) for NAHSL production were analyzed at 1, 2, 3 and 7 days after inoculation of <i>S. tuberosum</i> var. Allians tubers. For the controls, one of the two strains was replaced in the inoculum with a 0.9% NaCl solution. Asterisks indicate significantly less severe maceration symptoms in the presence of the BCA-<i>qsdA::gfp</i>, as assessed with the Mann and Whitney test (<i>α</i> = 0.05). The fluorescence of the BCA-<i>qsdA::gfp</i> was analyzed by confocal laser scanning microscopy. (B) The numbers of <i>P. atrosepticum</i> (black lines) and <i>R. erythropolis</i> (red lines) bacteria per unit weight (CFU/g fresh weight of potato tubers), and NAHSL concentration (ng/g of potato tubers; black and white bars) were determined for each condition in potato tubers. For lines and bars, each value is the mean of three replicates with the standard deviation indicated. NS, non-significant; NAHSL, <i>N</i>-acyl homoserine lactone.</p

NAHSL-breakdown and biocontrol activity of the<i>R.erythropolis qsdA</i> deletion mutant in potato tubers.

<p>(A) The <i>R. erythropolis</i> R138 wild-type (BCA) and the <i>R. erythropolis</i> R138 Δ<i>qsdA</i> (BCA-Δ<i>qsdA</i>) strains were compared for biocontrol activity against <i>P. atrosepticum</i> 6276 (<i>Pa</i>-QS+) 1, 2, 3 and 7 days after inoculation of potato tubers. For the controls, one or both strains were replaced in the inoculum with a 0.9% NaCl solution. Significant differences (Mann and Whitney test; <i>α</i> = 0.05) in maceration symptoms between infected tubers inoculated with the BCA or the BCA-Δ<i>qsdA</i> are indicated with an asterisk. (B) Population dynamics of <i>P. atrosepticum</i> and <i>R. erythropolis</i> bacteria (CFU/g fresh weight of potato tubers; black and red lines respectively), and NAHSL concentrations (ng/g of potato tubers; black and white bars) were determined for each condition in potato tubers. For lines and bars, each value is the mean of three replicates with the standard deviation indicated. NS, non-significant; NAHSL, <i>N</i>-acyl homoserine lactone.</p

Bacterial strains and plasmids.

<p>Km^R, Ap^R, Gm^R and Tc^R indicate resistance to kanamycin, ampicillin, gentamicin and tetracycline, respectively. NAHSL, <i>N</i>-acyl homoserine lactone; CFBP, Collection Française de Bactéries associées aux Plantes, Institut National de la Recherche Agronomique (INRA), Angers, France.</p

NAHSL-breakdown and biocontrol activity of the QsdA-expressing<i>E. coli</i> strain in potato tubers.

<p>(A) <i>E. coli</i> DH5α(pUC19) (<i>Ec</i>) and <i>E. coli</i> DH5α(pUC19-<i>qsdA</i>) (<i>Ec-qsdA</i>) were compared for biocontrol activity against <i>P. atrosepticum</i> 6276 (<i>Pa</i>-QS+) 1, 2, 3 and 7 days after inoculation of potato tubers. For the controls, one or both strains were replaced in the inoculum with a 0.9% NaCl solution. Significant differences (Mann and Whitney test; <i>α</i> = 0.05) in maceration symptoms between infected tubers inoculated with the <i>Ec</i> or the <i>Ec-qsdA</i> strains are indicated with an asterisk. (B) Population dynamics of <i>P. atrosepticum</i> and <i>E. coli</i> bacteria (CFU/g fresh weight of potato tubers; black and blue lines respectively), and NAHSL concentration (ng/g of potato tubers; black and white bars), were determined for each condition in potato tubers. For lines and bars, each value is the mean of three replicates with the standard deviation indicated. NS, non-significant; NAHSL, <i>N</i>-acyl homoserine lactone.</p

Virulence traits and origin of potato soft-rot pathogens.

*<p>The potential of each strain to induce a tuber soft-rot was evaluated in potato host plant seven days after infection by intra-medulla injection.</p>#<p>The potential of each strain to induce a hypersensitive response (HR) was evaluated in tobacco non host plant 24 hours after leaves infiltration.</p><p>CFBP, Collection Française de Bactéries Phytopathogènes, Institut National de la Recherche Agronomique, Angers, France.</p

Characterization of signaling molecules produced by potato soft-rot pathogens.

*<p>Production of different N-acyl homoserine lactones (NAHSL) was determined by thin-layer chromatography (TLC) and HPLC-MS/MS. For quantification, NAHSL were extracted from PGA minimal medium culture at late exponential phase (optimal production).</p>#<p>Autoinducer-2 (AI-2) activity was determined using biosensor <i>V. harveyi</i> BB170.</p>°<p>Production of 4-hydroxy-2-heptylquinoline (HHQ) and Pseudomonas quinolone signal (PQS) were determined by TLC.</p>†<p>Production of indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-butyric acid (IBA) and kynurenic acid (KA) were determined by HPLC-UV. For quantification, indolic compounds were extracted from M9 minimal medium supplemented with l-tryptophan culture at stationary growth phase (optimal production).</p>§<p>Production of γ-amino butyric acid (GABA) was determined by ELISA test.(+) positive detection by the biosensor; (−) not detected or below threshold.</p

Bacterial QS signal producers or biosensors, and plasmids.

<p>Gm^r and Tet^r indicate resistance to gentamicin and tetracycline, respectively.</p><p>NAHSL, N-acyl homoserine lactones; AI-2, Autoinducer-2; HHQ, 4-hydroxy-2-heptylquinoline; PQS, Pseudomonas quinolone signal.</p

Kinetics of IAA-like production measured in the supernatant of potato soft-rot <i>Dickeya</i> spp.

<p>Indolic compounds were quantified by a colorimetric method with Salkowski's reagent. Bacterial growth was monitored by measuring optical density (OD) at 580 nm. For each point, at least 3 independent cultures in M9 minimal medium supplemented with l-tryptophan (500 µg/ml) were analyzed, with standard deviation shown.</p

Kinetics of <i>metK</i>, <i>luxI</i>, <i>luxS</i> and <i>iaaM</i> gene expression involved in AI-1, AI-2 and IAA production by <i>D. chrysanthemi</i> strain CFBP 2048^T.

<p>AI-1 and AI-2 biosynthetic pathways are described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035176#pone-0035176-g003" target="_blank">Fig. 3</a>. A third signal, the indole-3-acetic acid (IAA) is synthesized by the indole-3-acetamine (IAM) pathway, in which l-tryptophan is first converted to IAM by the key enzyme of this pathway, the IaaM tryptophan-2-monooxygenase. IAM is then converted to IAA by IaaM hydrolase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035176#pone.0035176-Yang1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035176#pone.0035176-Spaepen1" target="_blank">[54]</a>. Abundances of <i>metK</i> (checkered bars), <i>luxI</i> (white bars), <i>luxS</i> (black bars) and <i>iaaM</i> (grey bars) mRNAs were determined by RT-PCR experiments on RNA extracts from cells grown in PGA minimal medium (<b>A</b>) or M9 minimal medium supplemented with l-tryptophan (500 µg/ml) (<b>B</b>) and harvested at mid-exponential phase (<b>1</b>), late exponential phase (<b>2</b>), during the transition from exponential to stationary phases (<b>3</b>) or early stationary growth phase (<b>4</b>) followed by electrophoresis on 1% (m/v) agarose gels. Results were expressed as a ratio: synthase transcripts vs. 16S transcripts. The corresponding kinetics of AI-1 and AI-2 activities and IAA production in the supernatant of potato soft-rot <i>Dickeya</i> strain are shown at the top.</p