All the primers used in this study are listed in Table 1. The virulence of Xcc to cabbage was estimated after bacteria were introduced into the leaves by leaf clipping as described previously (Qian et al., 2005), and the lesion length was measured 14 days postinoculation. For measurements

of exopolysaccharide production, strains grown overnight in NYGB medium were washed and resuspended in 10 mM MgCl2 (OD600 nm=0.1). Aliquots (5 μL) of these bacterial suspensions were then added to 50 mL of TGM medium (1% tryptone, 0.5% yeast extract, 2% glycerol, 1% glucose, 0.07% K2HPO4 and 0.025% MgSO4·7H2O) Selleck NVP-BEZ235 and shaken for 48 h at 250 r.p.m. Exopolysaccharides were precipitated from culture supernatants by ethanol, dried and weighed as reported previously (Vojnov et al., 1998). Strains grown overnight in NYGB medium were washed and resuspended in 10 mM MgCl2 (OD600 nm=0.1). learn more To analyze swimming motility, 0.3% swimming agar TYGS (0.1% tryptone, 0.05% yeast extract, 0.1% NaCl and 1% glucose) plates were inoculated with 2-μL bacterial suspensions (OD600 nm=0.1) and incubated for 24 h. Agar (0.6%) plates were used to analyze swarming motility and were inoculated for 72 h. The diameters of the colony were measured 24 h or 72 h after incubation on swimming or swarming plates, respectively. The promoter of the gum gene cluster was

amplified by PCR using the primers listed in Table 1. The PCR product was cloned into pGEM-T Easy (Promega, Madison, WI) for sequence verification. It was then cloned into the upstream region of the βGUS (uidA) gene of the broad-host-range vector pL6GUS (Wang et al., 2007) digested with HindIII and BamHI. The plasmid was transformed into Xcc strain 8004 wild type and the ΔvemR mutant. The resulting strains were grown overnight in NYGB medium. Cells were collected by centrifugation and β-GUS

gene activity was assayed Gemcitabine as described elsewhere (Jefferson, 1987). The ability of Xcc to secrete extracellular enzymes was tested as described previously (Yang et al., 2009). Briefly, 2 μL of cells (OD600 nm=0.1) were inoculated onto NYGB plates containing skim milk (1%), starch (0.2%) or carboxymethylcellulose (0.5%) and incubated for 48 h. Protease activity was assessed by the appearance of clear zones surrounding the colonies on milk plates. Starch plates were stained with I2/KI (0.08 M/3.2 M) for 2 min, rinsed with water and clear zones were observed. Carboxymethylcellulose plates were stained with 0.1% Congo red for 5 min, rinsed with water and then washed twice with 1.0 M NaCl. To investigate whether VemR plays a role in Xcc pathogenesis, we created an in-frame deletion mutant ΔvemR in Xcc strain 8004. We then tested virulence in the ΔvemR mutant strain by measuring the lesion length by leaf clipping. The results showed that the ΔvemR mutant had significantly reduced virulence (Fig. 1b and c).