| Xanthomonas campestris pv. campestris, referred Xcc, is an important plant pathogenic bacteria, which can infect all the worldwide cruciferous plants, causing severe plant disease in the world. Plant pathogenic bacteria can produce a virulence called type Ⅲeffectors, which can invade the host cell by the type Ⅲ secretion system, thus affecting the normal metabolism and defense reactions of the host, to help the growth of pathogens. Therefore, genome-wide screening and identification of the Xanthomonas campestris pv. campestris type Ⅲ effector was done in order to lay a foundation to further reveal of the pathogenic mechanism of Xanthomonas campestris pv. campestris.Currently, the transport and secretion of type Ⅲ effector in Xanthomonas campestris pv. campestris can be detected by in vivo and in vitro methods, in vitro testing is performed by cloning the promoter region and signal region of candidate gene into an expression vector, which carries a small immune identification tag （eg3×FLAG tag）. Under vitro culture conditions, the presence or absence of the fusion protein in the culture is detected by immunological methods （Western Blotting）. This high sensitive method can be applied to the detection of low secretion effectors. In vivo testing, effector protein transport is identified by detecting pathogens inducing allergic reactions （hypersensitive response） on the non-parasitic or resistance host or by immunochemical marker method. The sensitivity of detection system established by hypersensitive response characteristics is not high, so it does not apply to low secretion Ⅲ effectors identification. The immunochemical marker detection has higher detection sensitivity, and the current detection methods is to use the Bordetella pertussis calmodulin-dependent adenylate cyclase domain （CyaA） as a reporting signal, and quantitative detect the cAMP content through immune responses, so as to determine the transport of type Ⅲ effectors.In order to use both in vitro and in vivo two detection methods simultaneously detect Xanthomonas campestris pv. campestris type Ⅲ effector transit and secretion, the cyaA gene was cloned into the pJXG vector with3x FLAG to construct a reporter plasmid pJAA with3x FLAG and cyaA gene, and the promoter region and signal area of the identified type Ⅲeffectors XC1553was used to test whether the reporter plasmid can work. The recombinant plasmid pJAA1553was imported8004*and8004*ΔhrcV strain by tri-parental combination, and then XC1553secretion was detected by Western Blotting. The results showed that the XC1553secretion signal was detected in the8004*/pJAA1553extracellular proteins, and not detected in the negative control8004*ΔhrcV/pJAA1553extracellular protein. The cAMP levels in plants after infection of8004*/pJAA1553and8004*ΔhrcV/pJAA1553were quantitative detected by immunochemical marker detection, and it’s found the cAMP content in plants after8004*/pJAA1553infection was15times higher than that after8004*ΔhrcV/pJAA1553infection. The results showed that the reporter plasmid pJAA did work, and could be applied to detect the type Ⅲ effectors transport and secretion in Xanthomonas campestris pv. campestris. In the process of identifying effectors by Western Blotting, in order to increase the secretion of extracellular proteins, we explored induction conditions of extracellular protein. By two-dimensional electrophoresis, the Xcc extracellular proteins inducing ability of the MME and the XCM2medium were compared. It’s found that XCM2could induce more extracellular proteins, especially acidic extracellular proteins. Thereby the XCM2was determined as Xcc induction medium. |
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