| Xanthomonas campestris pv.campestris(Xcc)is an important Gram-negative plant pathogen that infects almost all cruciferous plants,but there is still no effective prevention and treatment method.Understanding the signal transduction during the infection of this bacterium will facilitate new strategies of developing disease resistance and prevention.The current reporter system widely used in Xcc is based on ?-glucuronidase,which is time consuming,expensive,and impossible to detect directly on a plate.As a popular reporter system.P-galactosidase(LacZ)is a simple,rapid,low-cost,visible and stable method.We hope to utilize the feature of LacZ to study Xcc promoter activity but it can not be directly used because Xcc wild-type strain has LacZ activity.Therefore,it is necessary to create a strain that does not have LacZ activity for our purposes.The two-component system is the induction-response signaling device in most prokaryotes,which is the main regulatory system regulating various physiological functions including pathogenicity.Previous studies have found that Xcc RavA/RavR constitutes a two-component system.RavR belongs to the GGDEF-EAL protein family and is a bifunctional enzyme that synthesizes and decomposes C-di-GMP,while RavA acts as the cognate histidine kinase(HK)of RavR to regulate the phosphorylation level of RavR and affect the enzyme activities?To systematically analyze the pathways in which RavA/RavR regulates the expression of flagellin-related genes and explore the mechanism that C-di-GMP regulates bacterial movement,the focus of this study is on following aspects:(1)Bioinformatics analysis indicates that two protein families,GH2 and GH35,in Xcc may be galactosidase enzymes.Xcc encodes five GH2 proteins and three GH35 proteins.(2)The GH2 and GH35 deletion mutants were constructed.When all of these 8 genes were deleted.the galactosidase activity was completely lost complementary assays imply that XC1214 is the main galactosidase of Xcc.(3)The ravA and ravR mutants were constructed in Alac8 and the pathogenicity was analyzed.Results showed that ?ravA and ?ravR have decreased virulence than that of the wild type.We also analyzed some known bacterial pathogenic factors such as extracellular enzymes,extracellular polysaccharides and motility.Biochemical and phenotype test showed that the exopolysaccharide production of the mutants decreased,the ability to degrade starch and cellulose remained unchanged,and the cell motility was significantly weakened,suggesting that the lack of motility might be responsible for the pathogenicity reduction.(4)In order to answer why RavA/RavR regulates motility,we constructed the promoter-reporter fusion strains:?ravA-XC2245-lacZ,?ravA-XC2408-lacZ.?ravR-XC2245-lacZ,?ravR-XC2408-lacZ,wt-XC2245-lacZ,wt-XC2408-lacZ.LacZ activity analysis showed that the LacZ activities of AravR-XC2245-lacZ and AravA-XC2245-lacZ strains were significantly enhanced compared with the wild-type strains,indicating that the change of C-di-GMP content caused by the deletion of RavR and RavA regulates the synthesis of flagellum-related genes.In summary,this study constructed an Xcc galactosidase-deficient strain and used LacZ as a reporter gene to analyze the expression levels of flagella genes(XC2245 and XC2408)in wild type,ravR and ravA mutants.Our study provides the materials to further explore the molecular mechanisms and signal networks of RavR and RavA regulating bacterial movement?... |
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