Figure 7. ssb_X is expressed in hrp-inducing conditions and regulated by HrpG and HrpX.

(A) Schematic map of a transcriptional fusion where the ssb_Xoc promoter of X. oryzae pv. oryzicola RS105 is fused to the gusA reporter gene. Upper panel shows pPIPAGUS containing the ssb_Xoc promoter and an imperfect PIP-box (TTCGC-N₁₉-TTCGT) fused with a promoter-less gusA gene. Lower panel shows pPIPBGUS with a mutated ssb_Xoc promoter (the first TT nucleotides replaced with AA) fused with gusA. (B) â-glucuronidase (GUS) activity in the hrp-inducing medium XOM3. Plasmids pPIPAGUS and pPIPBGUS were transferred into the wild-type RS105 and mutants RΔhrpG and RΔhrpX. The recombinant strains were then grown in hrp-inducing medium XOM3 for 16 h. GUS activity was determined by measuring the OD at 415 nm using ρ-nitrophenyl-â-D-glucuronide as a substrate. Data represent the mean ± SD of triplicate measurements. The different letters above each horizontal column indicate significant differences at P = 0.01 (t test). (C) Expression of ssb_Xoc in hrp-inducing and nutrient-rich media. Real-time quantitative RT-PCR was used to compare relative expression of ssb_Xoc in X. oryzae pv. oryzicola strains RS105, RΔhrpG, and RΔhrpX. RNA was isolated from strains grown in a nutrient-rich medium (NB) and the hrp-inducing medium (XOM3) for 16 h. The relative mRNA levels of ssb_Xoc in the hrpG and hrpX mutants were calculated with respect to the wild-type strain. Values given are the means ± SD of triplicate measurements from a representative experiment, and similar results were obtained in two other experiments. Different letters above horizontal columns represent significant differences at P = 0.01 using the Student’s t test. (D) Real-time RT-PCR evaluation of ssb_X expression in Xanthomonas species. Strains were grown at 28°C for 16 h in NB or one of the following hrp-inducing media: XOM3 for X. oryzae pv. oryzicola RS105 and X. oryzae pv. oryzae PXO99^A (Xiao et al. 2007), XVM2 for X. axonopodis pv. citri 306 & X. campestris pv. vesicatoria 85-10, and MMX for X. campestris pv. campestris 8004 (see methods). Relative mRNA quantitative of ssb_X was calculated with respect to the levels observed for wild-type strains grown in NB. Genes encoding 16S rRNA were used as internal controls. Data represent means ± SD of triplicate measurements (P = 0.01, t test).