For comparison of the cma expression in vitro and in planta, PG4180 carrying plasmid pHW01 was used . Plasmid pHW01 contains a transcriptional fusion of a promoterless enhanced green fluorescent protein (egfp) gene to the cma promoter region of PG4180 in the broad-host range vector pBBR1MCS . The size of the cloned cma promoter region is 2.9 kb located at positions -717 to +2,135 with respect to the cmaA transcriptional start site .2.2. Plant material and inoculation proceduresSoybean plants (Glycine max (L.) Merr. cv. Maple Arrow) were grown in a greenhouse at 22 to 25 ��C, 60% humidity, with supplementary light for a 14-h photoperiod (350 ��E m-2 s-1). To study expression of the cma promoter in planta, P. syringae PG4180 carrying plasmid pHW01 was infiltrated into soybean leaves using a needleless syringe at an OD600 of 0?05 (approx.
5 �� 107 CFU per mL). Following infiltration, plants were transferred to growth chambers at 18 or 28 ��C. To recover bacteria after inoculation, 60 discs (7 mm diameter) containing infected leaf tissue were excised using a cork borer and macerated in 4 mL of isotonic NaCl. All greenhouse experiments were repeated at least three times to confirm reproducibility.2.3. Confocal laser scanning microscopyFluorescence of bacterial cells was detected with a confocal laser scanning microscope, DMR XE, type TCS NT (Leica) using a PL-Fluotar objective (�� 63; 1.32; numerical aperture, oil immersion). Images were analyzed with Leica software package TCS NT, version 1.5.451.2.4.
RNA isolation and spot blot analysisBacteria were cultured in HSC medium at 18 and 28 ��C until OD600 of 1?3 or until selected time points after temperature shift or addition of antibiotics. 15 mL of bacterial culture were mixed with an equal volume of chilled killing Dacomitinib buffer (20 mM Tris-HCl [pH 7.5], 20 mM NaN3) and subsequently centrifuged at 4 ��C for 15 min at 4,000 rpm.Total RNA was isolated from 5 mL of bacterial cells by acid phenol/chloroform extraction as described by Schenk et al. . Extracted RNA was analysed using an Agilent 2,100 Bioanalyzer. The yield of RNA was determined spectrophotometrically at 260 nm using an Ultrospec 2,100 pro UV/Visible Spectrophotometer (Amersham). Aliquots of total RNA (200 ng per dot) were transferred to positively charged nylon membranes (Pall) using the Minifold? I Spot-Blot System (Schleicher & Schuell BioScience) according to the manufacturer’s recommendations.
Successful transfer of the RNA was verified by reversible staining of the membrane with methylene blue . The digoxygenin-labelled specific RNA probes were synthesized by in vitro transcription using T7 RNA polymerase and specific PCR products as templates. Synthesis of the templates by PCR was performed using the following pairs of oligonucleotides:
The term radar is short for radio detection and ranging.