Figure 1 Cell-associated hemolytic activity (cHA). Cell-associated hemolytic activity (cHA) was measured as described in the materials and methods. Results are mean values from at least three independent experiments. Standard deviation is shown. RBCs were incubated 1h at 37°C with MFN1032, MFY63, MFY70, MFY162, SBW25, C7R12, MF37 or DC3000 cultivated at 28°C (MOI of
1). The same panel of strains Bortezomib datasheet was tested on tobacco leaves to determine if these strains were able to induce HR. As illustrated in Figure 2, HR was only detected for C7R12 and DC3000. All clinical strains i.e., MFY63, MFY70, MFY162 and MFN1032 and two environmental strains, SBW25 and MF37, were unable to induce HR. Figure 2 Plant hypersensitive response (HR) assay. P. fluorescens strains, MFN1032, MFY63, MFY70, MFY162, SBW25, C7R12, MF37 and P. syringae DC3000, were infiltrated into Nicotiana tabacum cv. leaves. The leaves were evaluated for production of HR and were photographed after 48 h. This experiment
was repeated 2 times with similar results. P. fluorescens MFN1032 is virulent on Dictyostelium discoideum (D. discoideum) As described in Figure 3A, Klebsiella aerogenes (KA) (negative control for virulence), Pseudomonas aeruginosa PA14 (positive control for virulence), and MFN1032 were tested on D. discoideum. On a layer of KA, about one hundred lysis plaques were observed, corresponding this website to the zone where actively feeding and replicating D. discoideum have phagocytosed the bacteria. On a layer of PA14 or MFN1032 at 10%, no lysis plaque was detected. MFN1032 does indeed display a virulent phenotype on D. discoideum, either by evading D. discoideum killing, or by actively killing
amoebae. Then, our panel of strains was tested on D. discoideum (Figure 3B). Two strains, C7R12 and MF37 had a complete absence of D. discoideum growth inhibition (100% of D. discoideum remained). MFY63 and SBW25 were highly permissive for D. discoideum growth (90% and 75% of amoebae remained, respectively). MFY70 Dolichyl-phosphate-mannose-protein mannosyltransferase and MFY162 permitted the replication of about half of the D. discoideum (40% and 60% respectively). DC3000 had a slightly virulent phenotype on D. discoideum (20% of D. discoideum remained). In our panel, to small to be representative, D. discoideum growth inhibition above 50% was only observed for clinical or phytopathogenic strains of Pseudomonas. Figure 3 Virulence towards Dictyostelium discoideum. Approximately 100 D. discoideum cells were cultivated in SM-plates with the indicated proportion of Klebsiella aerogenes and Pseudomonas strains (10%). Plates were maintained at 22°C for 5 days. A: Pseudomonas aeruginosa PA14 (positive control), Klebsiella aerogenes (KA, negative control) and P. fluorescens MFN1032 virulence towards D. discoideum after 5 days. B: Virulence of different Pseudomonas strains at 10% against D. discoideum. These results were obtained by the ratio of the number of lysis plaques obtained with the negative control Klebsiella aerogenes (100% of amoebae remained).