ted in triplicate on NA with appropriate antibiotics. Plates were incubated at 28uC for 34 days until single colonies could be counted. Bacterial numbers were calculated, and standard deviations were determined using colony counts from three triplicate spots in each of three samples per time point per inoculum. Experiments were repeated at least three times. Xanthomonas SSB Protein Acts as Harpins Promoter activity assays and quantitative real-time PCR. To construct a transcriptional fusion between the ssbX promoter and glucuronidase, the promoter region upstream of ssbX was amplified from the genomic DNA 21147071 of X. oryzae pv. oryzicola RS105 with the primer pair pssb-F/pssb-R. This PCR product was then fused with the promoterless gusA gene, which was obtained with primers gusA-F/gusA-R. The ssbX-gusA fusion was then cloned into pUFR034 at the EcoRI site, resulting in pPIPAGUS. In another experiment, a mutation was introduced into the PIP-box of the ssbX promoter using primers mpssb-F/pssb-R and fused with gusA, resulting in pPIPBGUS. For GUS activity assays, X. oryzae pv. oryzicola RS105 strain and hrp mutants were cultured in XOM3 to OD600 = 0.5. Bacterial cells were diluted and disrupted in sonication buffer. GUS activities were determined every 30 min over a 3-h time period by measuring absorbance with pnitrophenyl-D-glucuronide as the substrate. One unit was defined as 1 nmol of 4-methyl-umbelliferone produced per min per bacterium. For quantitative real-time PCR analysis, the bacteria were cultured as described for the GUS activity assay in this report or cultured in rice suspension cells as described by Li and her colleagues. Total RNA was extracted using Trizol reagent according to the manufacturer’s instructions. Total RNA was quantified by measuring the OD260/ OD280, and the quality was examined by gel electrophoresis. Before 936091-26-8 synthesis of the first stranded, total RNA was treated with RNase-free DNaseI to remove genomic DNA. Removal of contaminating DNA was confirmed by using extracted RNA as a template to amplify selected target genes using the primers listed in Results ssbXoc Encodes a Single-stranded DNA-binding Protein Eliciting HR in Tobacco Mutagenesis of hrpG or hrpX in X. oryzae pv. oryzicola abolishes the elicitation of HR in tobacco and pathogenicity in rice. Thus, we assumed that the expression of HR-eliciting genes, including hpa1, are also regulated by 
HrpG and HrpX. Using cDNA microarrays of X. oryzae pv. oryzicola strain RS105 and the hrpG 22440900 & hrpX mutants, we discovered that the expression of XOC_1514, which encodes a single-stranded DNAbinding protein , was positively regulated by HrpG and HrpX in pathogen-infected rice cells. This protein, which was designated SSBXoc, is rich in glycine but lacks cysteine residues; these are characteristics typical of the harpin protein family. To confirm this, we expressed ssbXoc in PVX vector pgR107, which is typically used to screen HR elicitors in tobacco. SSBXoc triggered HR in N. benthamiana that was similar to Hpa1 and Bax , suggesting that SSBXoc functions as a harpin in X. oryzae pv. oryzicola. Previously, we reported that the minimum concentration of Hpa1 for HR induction is 0.1 mM. To determine the concentration of SSBXoc required for HR induction, we overexpressed the protein in E. coli BL21 . Purified SSBXoc was infiltrated into tobacco at concentrations ranging from 0.01 to 50 mM. The minimum concentration of SSBXoc needed for HR induction in tobacco cv. Xanthi was 1.0
