To further demonstrate promoter induction, the identified substra

To further demonstrate promoter induction, the identified substrates were tested in liquid cultures. Cells of Ea1189 harboring plasmid pBBR.acrD-Pro.egfp were incubated in LB broth supplemented with each substrate for 24 SC79 chemical structure hours, then harvested by centrifugation, resuspended in phosphate-buffered

saline, adjusted to an OD600 value of 0.1 and fluorescence determined. Apple plant material and inoculation procedures Apple plants (rootstock Malus MM106) were grown in a greenhouse at 20 to 25°C, 60% humidity, and 12 h photoperiod (15,000 lx). E. amylovora Ea1189 and its acrD mutant, grown on LB agar for 24 h, were resuspended and diluted to a cell density of 1 x 106 CFU/ml in sterile demineralized water. Apple plants were inoculated by CA4P manufacturer prick technique [52]. Each bacterial strain was inoculated into one

shoot of five single plants. A bacterial suspension (5 μl) was placed onto each wound on the shoot tip. Plants were monitored for symptom development daily. Survival of bacteria in plant tissue was examined by re-isolation of bacterial cells 1 and 5 day(s) after inoculation, respectively, from 1 cm of the shoot tip around the inoculation area. Ultimately, five wounds were pooled together, homogenized in 0.9% NaCl, serially diluted, and spread on LB agar plates. The experiment was repeated in triplicate. In order to analyze the abundance of acrA and acrD mRNA transcripts in E. amylovora Ea1189 during growth in apple rootstock MM106, total RNA was isolated from infected apple shoots 1, 4 and 7 day(s) post inoculation, respectively. Five individual wounds were pooled together, homogenized in 0.9% NaCl and centrifuged for 2 min at 4000 rpm. The supernatant was transferred to 15 ml killing buffer (20 mM Tris–HCl, pH 7.5; 20 mM NaN3) [53] and centrifuged for 20 min at 4000 rpm. The supernatant was decanted and the pellet frozen at -80°C for further RNA extraction. Virulence assay on immature pears Virulence of E. amylovora Ea1189 and its acrD mutant was determined 17-DMAG (Alvespimycin) HCl on immature pears (cv. ‘Bartlet’). Bacteria, grown at 28°C on LB agar plates for 24 h, were

resuspended and adjusted to an OD600 of 1.0 in sterile demineralized water for inoculation. Immature pear fruits were surface-sterilized and pricked with a sterile needle as described previously [54]. Wounds were inoculated with 5 × 106 CFU/ml and incubated in a humidified chamber at room temperature for 8 days. Disease symptoms were recorded by means of diameter of necrosis surrounding the infection site. Fruits were assayed in triplicates and the experiment was repeated twice. To analyze gene expression of E. amylovora Ea1189 during growth on pear fruits, immature fruits were cut in slices (approx. 0.5 cm). Five slices were inoculated with 100 μl of a bacterial suspension adjusted to an OD600 of 1.0 in sterile demineralized water.

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