<i>Pseudomonas fluorescens</i> strain MFE01 secretes Hcp-like and VgrG-like proteins.

<p>Concentrated supernatants of MFE01 cultures were analyzed by SDS-PAGE and Coomassie Blue staining. A band with an approximate molecular mass similar to that expected for an Hcp protein (≈21 kDa), indicated by an arrow, was observed in the early exponential growth phase and late exponential growth phase of cells grown at 28°C but not at 37°C. Mass spectrometry identified this major supernatant protein as an Hcp-like protein. The second arrow indicates a VgrG-like protein from cells in late exponential growth phase at 28°C, absent at 37°C, with an approximate molecular mass of ≈85 kDa (identification by mass spectrometry).</p

MFE01 protects against tuber soft-rot.

<p>This figure shows a soft-rot assay. (<b>A</b>) Coculture assay for MFE01 or MFE01<i>Δhcp2</i> (<i>Δhcp2</i>) and <i>Pectobacterium atrosepticum</i> (<i>P.a</i>), for 4 h at 28°C (ratio 5∶1, respectively) (<i>N</i> = 4 for each assay). * Indicates a significant difference in <i>P.a</i> CFU (<i>p</i>-value <0.05) when compared to the <i>P.a</i> control. (<b>B</b>) <i>Pectobacterium atrosepticum</i> (<i>P.a</i>) was incubated with or without <i>P. fluorescens</i> MFE01 or MFE01<i>Δhcp2</i> (<i>Δhcp2</i>) at a ratio of 1∶10, respectively. Each potato tuber was inoculated and incubated for 7 days at 25°C. Soft-rot diameter was monitored. The error bars indicate the standard error of the mean and the images shown are representative. Statistics were done by pairwise strain comparisons (non-parametric Mann-Whitney-two tailed Test): **<i>p</i>-value <0.01, ns no significant difference.</p

Rap immunity proteins from <i>Serratia marcescens</i> protect <i>E. coli</i> against MFE01.

<p>Recovery of viable <i>E. coli</i> cells harboring a pSUPROM vector conferring the constitutive production of various <i>Serratia marcescens</i> immunity proteins, Rap 1a, 1b, 2a or 2b, or an empty pSUPROM vector, as control. Cocultures were performed between the indicated <i>E. coli</i> strains and wild-type <i>P. fluorescens</i> MFE01 or MFE01<i>Δhcp2</i> (<i>Δhcp2</i>) (ratio 1∶5, respectively) at 28°C (<i>N</i> = 4 for each assay). * Indicates a significant difference in <i>E.coli</i> CFU (<i>p</i>-value <0.05) when compared to the <i>E.coli</i>/MFE0 assay. ns means no significant difference.</p

<i>P. fluorescens</i> MFE01 displays bacterial killing activity.

<p>For each assay, <i>N</i> = 4, and the error bars represent the standard error of the mean. (<b>A</b>) A quantitative coculture assay was performed for 4 h at 28°C. Different prey cells were incubated with or without <i>P. fluorescens</i> MFE01 or MFE01<i>Δhcp2 (Δhcp2)</i>, at a ratio of 1∶5, respectively. Statistics were done by pairwise strain comparisons (non-parametric Mann-Whitney-two tailed Test): *<i>p</i>-value <0.05, **<i>p</i>-value <0.02, ***<i>p</i>-value <0.002, ns no significant difference. <i>Sma : Serratia marcescens.</i> (<b>B</b>) <i>Pseudomonas fluorescens</i> MFP05 was incubated with or without wild-type MFE01 (ratio 1∶5) for 4 h at 37°C for coculture assays. (<b>C</b>) Quantitative assessment of the MFN1032 population cocultured for 4 h at 28°C with MFE01 or without MFE01. A filter with 0.22 µm pores was placed between MFE01 and MFN1032, to prevent physical contact.</p

Oligonucleotides used for this study.

<p>Oligonucleotides used for this study.</p

<i>hcp2</i> mutation inhibits VgrG secretion.

<p>Concentrated culture supernatants for wild-type MFE01 and mutant MFE01Δ<i>hcp2</i> were analyzed by SDS-PAGE and Coomassie Blue staining, for bacteria in stationary growth phase after incubation at 28°C. MFE01Δ<i>hcp2</i> displayed much lower levels of Hcp secretion (indicated by an arrow) than MFE01. The residual band was analysed by mass spectroscopy and identified as an Hcp-like protein. The second arrow indicates the absence of the band corresponding to the VgrG-like protein in MFE01Δ<i>hcp2</i> supernatant.</p

Plasmids and strains included in the study.

<p>Plasmids and strains included in the study.</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<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

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