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<p>Abstract</p> <p>Background</p> <p><it>Xanthomonas </it><it>oryzae </it>pv. <it>oryzae </it>(<it>Xoo</it>) is the causal agent of rice bacterial blight disease. <it>Xoo </it>produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that <it>Xoo </it>might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by <it>Xoo </it>and the factors influencing the signal production.</p> <p>Results</p> <p><it>Xoo </it>genome harbours an <it>rpf </it>cluster comprising <it>rpfB</it>, <it>rpfF</it>, <it>rpfC </it>and <it>rpfG</it>. The proteins encoded by these genes are highly homologous to their counterparts in <it>X. campestris </it>pv. <it>campestris </it>(<it>Xcc</it>), suggesting that <it>Xcc </it>and <it>Xoo </it>might use similar mechanisms for DSF biosynthesis and autoregulation. Consistent with <it>in silico </it>analysis, the <it>rpfF </it>mutant was DSF-deficient and the <it>rpfC </it>mutant produced about 25 times higher DSF-like activity than the wild type <it>Xoo </it>strain KACC10331. From the supernatants of <it>rpfC </it>mutant, we purified three compounds showing strong DSF-like activity. Mass spectrometry and NMR analysis revealed that two of them were the previously characterized DSF and BDSF; the third one was a novel unsaturated fatty acid with 2 double bonds and was designated as CDSF in this study. Further analysis showed that all the three DSF-family signals were synthesized via the enzyme RpfF encoded by <it>Xoo2868</it>. DSF and BDSF at a final concentration of 3 μM to the <it>rpfF </it>mutant could fully restore its extracellular xylanase activity and EPS production to the wild type level, but CDSF was less active than DSF and BDSF in induction of EPS and xylanase. DSF and CDSF shared a similar cell density-dependent production time course with the maximum production being detected at 42 h after inoculation, whereas the maximum production of BDSF was observed at 36 h after inoculation. When grown in a rich medium such as YEB, LB, PSA, and NYG, <it>Xoo </it>produced all the three signals with the majority being DSF. Whereas in nutritionally poor XOLN medium <it>Xoo </it>only produced BDSF and DSF but the majority was BDSF.</p> <p>Conclusions</p> <p>This study demonstrates that <it>Xoo </it>and <it>Xcc </it>share the conserved mechanisms for DSF biosynthesis and autoregulation. <it>Xoo </it>produces DSF, BDSF and CDSF signals in rich media and CDSF is a novel signal in DSF-family with two double bonds. All the three DSF-family signals promote EPS production and xylanase activity in <it>Xoo</it>, but CDSF is less active than its analogues DSF and BDSF. The composition and ratio of the three DSF-family signals produced by <it>Xoo </it>are influenced by the composition of culture media.</p>