AsnB, regulated by diffusible signal factor and global regulator Clp, is involved in aspartate metabolism, resistance to oxidative stress and virulence in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak in rice, which is a destructive disease worldwide. Xoc virulence factors are regulated by diffusible signal factor (DSF) and the global regulator Clp. In this study, we have demonstrated that asnB (XOC_3054), encoding an asparagine synthetase, is a novel virulence-related gene regulated by both DSF and Clp in Xoc. A sequence analysis revealed that AsnB is highly conserved in Xanthomonas. An asnB mutation in Xoc dramatically impaired pathogen virulence and growth rate in host rice, but did not affect the ability to trigger the hypersensitive response in nonhost (plant) tobacco. Compared with the wild-type strain, the asnB deletion mutant was unable to grow in basic MMX (-) medium (a minimal medium without ammonium sulphate as the nitrogen source) with or without 10 tested nitrogen sources, except asparagine. The disruption of asnB impaired pathogen resistance to oxidative stress and reduced the transcriptional expression of oxyR, katA and katG, which encode three important proteins responsible for hydrogen peroxide (H(2)O(2)) sensing and detoxification in Xanthomonas in the presence of H(2)O(2), and nine important known Xoc virulence-related genes in plant cell-mimicking medium. Furthermore, the asnB mutation did not affect extracellular protease activity, extracellular polysaccharide production, motility or chemotaxis. Taken together, our results demonstrate the role of asnB in Xanthomonas for the first time.

[Analysis of the flgD and figE genes regulated by diffusible signal factor in Xanthomonas oryzae pv. oryzicola].

OBJECTIVE: To investigate functions of flgDxoc and flgExoc genes regulated by diffusible signal factor (DSF) in Xanthomonas oryzae pv. oryzicola(Xoc)Rs105. METHODS: TheflgDxoc and flgExoc genes were amplified by PCR. We constructed deltaflgDxoc and deltaflgExoc, the deletion mutants from Rs105 by using double crossover method, and determined cell morphology, motility, pathogenicity in host rice and hypersensitive response (HR) in nonhost tobacco. We tested the differential expression of flgDxoc and flgExoc gene by reverse transcriptional polymerase chain reaction (RT-PCR) between the wide type and deltarpfFxoc (the deletion mutant of rpfFxoc gene, which could not produce DSF). RESULTS: We cloned flgDxoc and flgExoc from genomic DNA of Rs105. PCR and Southern blot analysis demonstrated that the flgDxoc and flgExoc genes were knocked out successfully. Both mutants were non-flagellated and significantly attenuated motility on the 0.3% semi-solid medium. The pathogenicity on rice were obviously attenuated in deltaflgDxoc and deltaflgExoc compared to the wild type. All the changes in mutant could be restored through the complementation. However, there was no significant difference in bacterial growth in MMX medium and induction of HR between mutant (deltaflgDxoc or deltaflgExoc) and the wild type. In addition, the results of RT-PCR demonstrated that the transcription level of flgDxoc and flgExoc were downregulated in deltarpfFxoc. CONCLUSION: This study showed that expressions of flgDxoc and flgExoc were positively regulated by DSF, and necessary for flagellar hook assembly and flagellar structure in Xoc. Meanwhile, FlgD and FlgE contributed to pathogen's virulence, motility and chemotaxis, but no differences at growth rate in MMX medium and HR in nonhost. In addition, our results provided molecular evidences that the contribution of DSF-type quorum sensing to pathogen's virulence might be, at least partially, dependent on bacterial flagellar in Xoc.