PCR primers, rscSSBW25 (5′-ATGGAACCAATCGATCTGTTC-3′) and SBWrscU (5′-TCAGTGCCGTTCAAGCTC-3′), synthesized by Eurogentec (Angers, France), were designed to amplify rscSTU genes (2156 bp), a region of the rsp VS-4718 research buy cluster I of SBW25, corresponding to genes hrcSTU affected by hrpU-like operon disruption in MFN1030. PCR was carried out in a 50 μL reaction volume, in a MJ mini thermal cycler (Bio-rad laboratories incorporation, USA). Reaction

mixture contained 4 μL DNA, 0.5 μL Taq phusion polymerase (Biolabs, new England), 10 μL corresponding buffer, 4 μL primers (20 μM) and 4 μL deoxyribonucleoside triphosphate (2.5 mM). After initial denaturation for 10 seconds at 98°C, the reaction mixture was subjected to 30 cycles of 30 seconds at 98°C, 30 seconds at 49°C and 1 minute at 72°C, followed by a final 5 minutes extension at 75°C. Aliquots (10 μL) of the PCR products were https://www.selleckchem.com/products/NVP-AUY922.html analyzed by electrophoresis in 1% agarose gels, stained with ethidium bromide and photographed under UV illumination. PCR product was cloned with the pBBR1MCS-5(4,8KB) digested by Sma I [38]. This construction, pBBR-rscSTU (6,9 kb), was then introduced into Escherichia coli DH5α mcr cells by electroporation. White colonies were selected for their resistance to gentamycin (20 μg/mL). Plasmids were

isolated using the QIAprep Spin Miniprep Kit (Qiagen), checked by sequencing (beckman coulter genomics, Germany) and then transferred into the Escherichia coli conjugative strain S17.1. MFN1030 (tetracyclin resistant) cells were conjugated with S17.1 cells carrying the Tideglusib pBBR-rscSTU plasmid and strains were selected for their resistance to tetracycline (20 μg.mL-1) and gentamycin (20 μg.mL-1). The resulting strain was called MFN1030-pBBR-rscSTU. Bacterial strains and culture conditions The origin of each strain tested in this study can be found in Table 1. The bacteria were cultured in Luria Bertani PIK3C2G medium (LB) at optimum growth temperatures, i.e. 28°C for P. fluorescens (for MF37 origin, see [39]) and P. syringae DC3000

[40], 37°C for P. aeruginosa CHA or PA14 [41, 42] and Klesiella aerogenes[43], with shaking at 180 rpm. When necessary, 80 μg/mL Xgal, 20 μg/mL tetracycline, 20 μg/mL gentamycin or 30 μg/mL kanamycin were added. The bacterial density was determined by measuring optical density (OD) at 580 nm (Spectronic Unicam spectrophotometer). Acknowledgements This study was supported by grant from the Région Haute-Normandie. We thank INRA UR1282, infectiologie animale et santé publique, groupe “signalisation, portage et virulence bactérienne” for help with macrophage J774A.1 infection. We thank Azeddine Driouich and Sophie Bernard, Laboratoire de Glycobiologie et Matrice Extracellulaire Végétale (GlycoMEV), EA 4358, Université de Rouen, for help in tobacco assay. We thank Magalie Barreau for technical assistance and Christine Farmer and Victor Norris for linguistic support. References 1.