Putative promoter region of type III effector gene avrAC(Xcc8004) in Xanthomonas campestris pv. campestris]

Xanthomonas campestris pv. campestris (Xcc) is the cause agent of black rot of crucifers. Xcc uses type III secretion system (T3SS) to deliver T3SS effectors (T3SEs) directly into host cells, where they play important roles in pathogenesis. Many identified T3SEs genes contain plant-inducible promoter(PIP) box and -10 box in their promoter regions. However, the relation among PIP-box, -10 box and -10 region, -35 region of the classic promoter is unclear, and the conservative characteristic of -10 box sequence is hardly reported. The aim of this study was to analyze the putative promoter region of T3SE gene avrAC(Xcc8004).Through 5' RACE, the transcriptional start site of avrAC(Xcc8004) was identified. Fusion PCR was introduced to generate the site-mutagenesis of -10 box for constructing the GUS fusion report strains.The 5' RACE results indicate that the transcription start site was A. After analysis, we found that -35 region was located 8 bp downstream of PIP-box, and -10 box was exactly overlapped with -10 region. The whole motif of PIP-box, -35 region, and -10 box was then counted as: TTCAC-N15-TTCGC-N8-TTGATG-N18-TACGTT. The GUS assay results demonstrate that the site-mutagenesis of -10 box caused a higher expression of avrAC(Xcc8004). The GUS activities in the mutant strains delta hrpX and delta hrpG were significantly lower than that in the wild type Xcc strains.PIP-box is tandem with -35 region, -10 box is just the same as -10 region, -10 box is important for the transcription of avrAC(Xcc8004), and HrpG and HrpX activate the expression of avrAC(X8004), despite of -10 box site-mutagenesis.