| Xanthomonas oryzae consists of two pathogenic variants, X. oryzae pv. oryzae （Xoo） and X. oryzae pv. oryzicola（Xoc）, which cause bacterial blight and bacterial steak （BLS） in rice（Oryza sativa） respectively. BLS constrains production of this staple crop in much of Asia and parts of Africa. Diffusible signal factor（DSF）-dependent quorum sensing （QS） system is an important component part of the global regulation networks in the Xanthomonas genus, which regulates multifaceted biological functions at the community level, such as biosynthesis of virulence factors, growth capacity, EPS, biofilm structure and other important biological functions. There were studies shows that the synthesis and conduction of DSF were closely related with rpf gene cluster and Clp. In Xcc, the regulation system also includes two transcription factors:a novel TetR-type transcription factor FhrR and zinc uptake regulator Zur which were regulated by Clp.In this study, we tried to investigate the functions of JhrR and zur which are similar to the regulators FhrR, Zur and asparagine synthetase B gene （asnB） of quorum sensing signal pathway in Xoc. And using real-time quantitative PCR method to analyze the status of the regulation of these genes in quorum sensing systems.1. The fhrR and zur genes were cloned by PCR. Then constructed mutant and the function recovered strain by using double crossover method, and determined cell morphology, motility, pathogenicity in host rice and hypersensitice response （HR） in nonhost tobacoo. For a further recognition of the two regulation factors JhrR and zur’s location in quorum sensing system, we build the over ecpression strain by using the ArpJF and Aclp strains, and measured the fhrR and zur expression levels in each strain by real-time quantitative PCR （qRT-PCR）. Compared with the wild type strain, there was no significant difference in bacterial growth in MMX medium but both mutants were significantly lower in motility as well as chemotaxis.The pathogenicity on rice was increased in ΔfhrR while which was attenuated in Azur, qRT-PCR showed that the transcription level of pathogentcity related genes were closely related with both fhrR and zur. qRT-PCR results showed that the transcription level of both fhrR and zur were down-regulated in Δclp, which suggested that fhrR and zur were positively regulated by quorum sensing system, and thus be inferred that the quorum sensing system regulated fhuR and zur genes expression to affect Xoc.2. the asnB gene was cloned by PCR. Then constructed mutant and the function recovered strain by using double crossover method, and determined cell morphology, motility, pathogentcity in host rice and hypersensitice response （HR） in nonhost tobacoo, proved that the deletion of asnB gene had seriously affected the pathogenicity and the absorptive capacity of nitrogen sources （Yin Fangqun’s graduation thesis）. We tested a variety of phenotypic determination and real-time quantitative PCR to research the functions of asnB gene and the pathogenic mechanism. The results showed that asnB gene was highly correlated with Xoc hydrogen peroxide tolerance, and qRT-PCR showed that the transcription level of antioxidant related genes and pathogentcity related genes were down-regulated in ΔasnB, which suggested that asnB gene might be affected pathogenic by the impact on the hydrogen peroxide tolerance. And, the asnB transcription level was ower than that in wild type strain, which suggested that fhrR and zur were positively regulated by DSF and Clp. On the other hand, we found xoc2999（asnB2） which encodes a second asparagine synthetase in Xoc genome and highly conserved in different species of Xanthomonas and shared33%of residues identity with asnB. We constructed ΔasnB2, the studies have shown that, there were no uniformity between asnB2and asnB, and the expression of asnB2has no correlation with asnB. |
