in dH2O and mounted in glycerol. To stain fats and waxes, sections were incubated in 1% w/v Sudan IV for 10 min, rinsed in 50% v/v ethanol for 1 min and mounted on a slide in glycerol. Wild-type P. s. pv. aesculi was visualized using FITC conjugated anti-PRI1 antibodies (PRI, Wageningen, The Netherlands). Sections were washed in PBS for 10 min, incubated in 0.1 M hydroxyl-ammoniumchloride for 30 min and washed again in PBS for 5 min. Sections were blocked in 0.5% w/v Bovine Serum Albumin Fraction V (Sigma-Aldrich) for 30 min and rinsed twice with PBS for 15 min. Samples were incubated overnight at 4uC in anti-PRI1-FITC diluted 1:600 or 1:1200 in PBS, washed twice for 15 min and twice for 30 min in PBS. The sections were finally mounted on slides in glycerol Citifluor AF2 for microscopic observation. For vitality assessments liquid grown bacteria were stained with propidium iodide (Sigma) at a final concentration of 5 mM for 5 min in the dark. Image Acquisition and Processing Slides with anti-PRI1-FITC and GFP labelled bacteria were analysed with a Nikon Optiphot microscope under bright field and fluorescence conditions. For fluorescence a 100 W high pressure HB-10101AF mercury lamp (Nikon) and FITC filters were used. Images were acquired with a Kappa DX-20 camera. For smaller magnifications a Zeiss Discovery V12 stereomicroscope equipped with a JENOPTIK ProgRes C10+ camera was used. Bacterial clusters grown axenically were observed with a Zeiss Axiovert 200 M microscope connected to a Zeiss LSM510 META confocal scanning system using a 636 PlanApochromat 1.4 N.A. oil immersion objective. All images were processed with ImageJ 1.43 (http://rsbweb.nih.gov/ij/) and Photoshop 8 CS (Adobe systems Inc.). In vitro Production of Extracellular Matrix and Scanning Electron Microscopy To observe the production of extracellular material, P. s. pv. aesculi PD4818 and its pMP4655 harbouring derivative were cultured at 20uC in either LB or minimal medium containing 20 mM NaCl, 20 mM phosphate buffer, 10 mM (NH4)2 SO4, 5 mM MgSO4, 10 mM fructose and 10 mM mannitol (pH 6.1) [50,51]. After 2–3 days, cultures were observed by fluorescence microscopy as described above or prepared for scanning electron microscopy by fixing 1 mL samples with 3% v/v glutaraldehyde dissolved in the appropriate medium. Droplets of the sample were brought on Poly-L-lysine hydrobromide (Sigma) coated, circular cover slips (Menzel). After rinsing with water, the samples were dehydrated in series of acetone solutions and subsequently critical point dried with carbon dioxide using a BalTec CPD 030. Samples were sputter coated with 5 nm platinum in a dedicated preparation chamber (CT 1500 HF, Oxford Instruments) and analysed with a JEOL 6300F field emission scanning electron microscope. Images were digitally recorded using an Orion 6 PCI interface (Eli SPRL) and processed with Adobe Photoshop 8 CS. Characterisation of Extracellular Polysaccharides Cultures of 125 mL of P. s. pv. aesculi PD4818 were either grown in duplo in minimal medium for 5 days or in minimal medium for 3 days and then under salt stress (0.4 M NaCl in minimal medium) for 2 days. Cultures were centrifuged for 15 min (10,0006g, RT) and three volumes of ice cold ethanol were added to the supernatant to precipitate the extracellular material o/n at 4uC. The precipitate was collected after centrifugation (30 min, 10,0006g, 4uC) and subsequently dialyzed o/n (Medicell Visking, MWCO 12,000–14,000 Da) against dH2O. Four volumes of ice P. syringae in Aesculus, Infection and Control cold acetone were added to the retentate and after o/n precipitation at 4uC the extracellular material was collected by centrifugation for 30 min at 10,0006g (10uC). The solid material was washed with acetone and dried at 35uC. Commercial alginates, sodium alginate from Laminaria hyperborea (BDH Chem- icals) and Macrocystis pyrifera (Sigma-Aldrich), and the dried extracellular material were hydrolysed in 0.375 ml 72% w/v H2SO4 for 30 min at 30uC, continued in 1 M H2SO4 for 3 h at 100uC. The amount of uronic acids was determined using the m- hydroxydiphenyl assay as described by Blumenkrantz and Asboe- Hansen [52] and galacturonic acid was used as standard. The sugar composition was determined in more detail by High Performance Anion Exchange Chromatography (HPAEC) using an ICS-3000 Ion Chromatography HPLC system equipped with a CarboPac PA-1 column (26250 mm) in combination with a CarboPac PA guard column (2625 mm) and a pulsed electro- chemical detector in pulsed amperometric detection mode (Dionex). A flow rate of 0.3 mL min21 was used and the column was equilibrated with 16 mM NaOH. The following gradient was used: 0–26 min, 16 mM NaOH; 26–33 min, 16– 100 mM NaOH; 33–78 min 0–1 M sodium acetate in 100 mM NaOH; 78–83 min 1M sodium acetate in 100 mM NaOH. Before analysis samples were diluted in water and 2.5 mL/mL 0.1% w/v bromine phenol blue in ethanol was added to the samples. To adjust the pH, solid sodium carbonate was added until a clear magenta colour was obtained. Subse- quently the solution was filtered through a 0.45 mm PTFE filter. Rhamnose, fucose, mannitol, arabinose, glucose, xylose, galactose, sucrose, glucuronic acid and galacturonic acid (Sigma Aldrich) were used as standards for identification. Heat-treatment To test the efficacy of thermal treatment on active lesions, eight 4 year old A. hippocastanum saplings were inoculated with P. s. pv. aesculi PD4818-pMP4655 and two plants were inoculated with PBS. The saplings were maintained in climate cells as described above. Two months after inoculation half of the population was covered with plastic and incubated in a climate cell heated to 39uC for 48 h. Hereafter the plants were again incubated under the standard conditions. One day after treatment, expanded lesions were removed from all sapling stems, always excluding the original inoculation site (except for mock-inoculations). Samples were homogenized and put in an amount of PBS proportional to the weight of the sampled tissue. The equivalent of 0.5 mg tissue and a series of seven subsequent 10-fold dilutions were then spotted onto carbenicillin containing LB plates. Plates were incubated at 20uC for 4 days and then scored for bacterial growth. The identity of isolated bacteria was confirmed by fluorescence microscopy. Plant growth and appearance was monitored after heat-treatment for 2 months. Supporting Information Figure S1 Growth of two Pseudomonas syringae pv. aesculi strains and their GFP-expressing derivatives in LB. (PDF) Figure S2 High performance anion exchange chromatography of acid hydrolysed extracellular polysaccharide of Pseudomonas syringae pv. aesculi PD4818. (PDF) Table S1 List of predicted homologues of alginate biosynthesis and regulatory proteins in Pseudomonas syringae pv. aesculi 2250. (DOC) PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e39604