85% NaCl and plated; for SDS exposure, bacterial culture was treated with 0.1% SDS for Epacadostat 20 min; for sensitivity to hydrogen peroxide, bacterial culture was exposed

to 0.03% H2O2 for 20 min; for osmotic stress, bacterial culture was treated with 40% D-sorbitol for 40 min; for saline stress, bacterial culture was treated with 1.0 M NaCl for 20 min. Bacterial cells were serially diluted with NB medium and colony-forming units (cfu) were counted after being cultured on NA plates at 28°C for 48 h. Each test, plated in triplicate, was repeated three times with similar results. B Data shown are means and standard errors of three replicates from one representative experiment. Different letters in each data column indicate significant differences at P < 0.05 (Student's t-test). Mutation of gpsX has no impact on expression of virulence-related genes

Reduced virulence could result from down-regulation of key virulence genes. In order to test whether mutation of the gpsX gene affected the expression of virulence-related genes, quantitative reverse transcription-PCR Z-VAD-FMK price (QRT-PCR) assays were performed to monitor the expression profiles of six genes which were selected based on the alternated mutant phenotypes mentioned above. For total RNA preparation, the gpsX mutant and wild type strains were cultured to exponential phase in XVM2 medium that has been reported to mimic the environment of plant Thiamine-diphosphate kinase intercellular spaces [38]. The six target genes included one EPS biosynthesis gene (gumB), one LPS synthesis gene (rfbC), one catalase gene (katE), one TTSS component gene (hrcV), one TTSS regulator genes hrpX, and one TTSS effector gene (pthA). The results showed that none of the six genes was significantly differently expressed in the mutant 223 G4 (gpsX-) compared with wild-type strains when grown in XVM2 medium (Table 5), based on a student’s t-test (P < 0.05). Specifically, the primer set used for pthA is present

in pthA4 and its homologues pthA1, pthA2, and pthA3, but not in any other genes. Thus we refer it as pthA rather than differentiating them. The qRT-PCR result based on this primer should detect the expression of pthA4, pthA1, pthA2, and pthA3. It is very likely that pthA4, pthA1, pthA2, and pthA3 have similar gene expression pattern due to the same promoter sequences. The sequences are 100% identical in the 213 bp upstream of pthA4, pthA1, pthA2, and pthA3 including the predicated promoter region (data not shown). Consequently, the qRT-PCR result will represent the relative fold change in gene expression for pthA4, pthA1, pthA2, and/or pthA3 since it is relative fold change and not absolute expression value.