Prokaryotic Expression And Its Binding With Amicarthiazol In Vitro Of SDHB From Strains Of Xanthomonas Axonopodis PV. Citri

Of all the agricultural pests and diseases that threated citrus crops, citrus bacterial canker disease(CBCD) is one of the most devastating,which caused by the bacterium Xanthomonas axonopodis pv.citri. Amicarthiazol(2-amino-4-methyl-carboxanniazol) is a bactericide containing both groups of carboxylamide fungicides. Amicarthiazol was applied not only to control basidiomycete diseases, but also to control bacterial disease, especially those caused by Xanthomonas spp. Preliminary studies in our laboratory showed that the mutation in SdhB subunit confers resitance of X. axonopodis pv.citri to amicarthiazol. The whole nucleotide sequences of SdhB gene was cloned from X.axonopodis pv.citri with different sensitivity to amicarthiazol. Then, the SdhB subunit was successfully expressed and purified from Rossatta (DE3) pLysS through prokaryotic expression vector pET30a(+). Finally the SdhB was used to bind with amicarthiazol in vitro in order to get the evidence that SdhB subunit conferred the resistance of X. axonopodis pv.citri against amicarthiazol. More details were shown:SdhB genes were cloned from one amicarthizol-sensitive isolate (X1) and three amicarthiazol-resistant isolates named as XA, XB and XC of Xanthomonas axonopodis pv.citri (Xac), and then inserted into prokaryotic expression vector pET30a(+), next transformed into the host strain Rossatta (DE3) pLysS. The recombinant strains were induced by IPTG(1mmol·L-1), and the proteins with SDS-PAGE electrophoresis analysis showed that the molecular weight of the proteins expressed in Escherichia coli was about35kDa, as expected. Compared the supernatant with the precipitate showed that SdhB subunit presented in inclusion bodies. After Western-blot, the expressed proteins had a specific reaction with the monoclonal antibody anti-His tag.SdhB of X. axonopodis pv.citri was expressed in E. coli in soluble form and purified by HisTrapTM HP Columns. The constructed recombination expression plasmid (pET30a+) was transformed into the hosts, Rossatta (DE3) pLysS and BL21(DE3), respectively. The induced fusion proteins were obtained and verified by SDS-PAGE and Western blot. In order to express the fusion protein greatly in soluble form, the inducing factors, including temperature(20℃), induction time(12h), IPTG (Isopropyl β-D-Thiogalactoside) concentration(0.6mM) and hosts(Rossatta), were screened. The positive clones which could express more fusion protein after screening inducing factors, however, the fusion proteins formed mainly in inclusion bodies. The molecular weight of fusion proteins were confirmed to be35kD by SDS-PAGE, which also showed specific reaction to anti-6xHis monoclonal antibody. After the optimization of imidazole concentration with binding buffer (20mM), wash buffer (75mM) and300mM Ellution buffer, the soluble fusion protein was purified and showed specific reaction by western-blot. The methods described here can be used to express and purify other recombinant proteins in soluble form in E. coli. The purified fusion tubulin can be used in the studies of SdhB-targeted drug resistant mechanisms as well as high throughout screening of new fungicide.The binding characteristics of amicarthiazol with SdhB subunit of different resistant-level strains in vitro were studied by DTNB-SH. We compared of the reaction of purified SdhB subunit (1.5ml-ml"1) with DTNB (lOμM), at a molar ratio (reagent/SH) of50:1in the presence or absence of amicarthiazol. Amicarthiazol significantly reduced the number of cysteine residues accessible to DTNB; there were4.62±0.33,6.88±0.43,10.60±1.00,12.26±0.45residues accessible per SdhB subunit in the presence of2.2uM amicarthiazol, respectively. The accessibility of the cysteine residues of SdhB subunit toward modification in association with amicarthiazol binding with sensitive strain was66.75%±2.39%, while the resistant strains XA, XB, XC achieved50.84%±3.13%,24.29%±7.17% and12.44%±4.07%, respectively. The differential affinity between amicarthiazol with SdhB led to strains with different sensitivity against amicarthiazol and this provided evidence for the conclusion, i.e. the SdhB subunit of X. axonopodis pv.citri was the target of amicarthiazol.