Xanthomonas 植物致病性基因HrpG、HrpB5及XC3070的表現與結晶研究

Abstract

Xanthomonas campestris pv. campestris (Xcc) is a gram-negative plant- pathogenic bacterium that hosts mainly in crucifers. When infected by this bacterium, hosts show syndrome of marginal leaf chlorosis, or rotten leafs. In Xcc, the hrp (hypersensitive reaction and pathogenicity) gene cluster are involved in the pathogenicity of host plants and induction of HRs in nonhost plant. When Xcc infects plants, its hrp gene cluster are activated. The structure and function associated with some of the hrp gene products were studied. In Xcc, HrpG is a key master regulator necessary for activating other hrp genes. It cpmprises 266 amino acids and belongs to a response regulator. The full-length HrpG was found to precipitate after cell lysis. To solve this problem, a hrpG (1-201) were cloned and purified successfully. A variety of crystallization condition were screened to obtain a crystal. However, the quality of the crystal was not good enough to obtain good X-ray diffraction data. The crystallization condition is being fine tuned. hrpB, the largest operon in the Xcc, could encode eight proteins (HrpB1-8), seven of which were soluble when expressed in E.coli. The HrpB5 were purified and the crystallization condition were screened for X-ray diffraction. A crystal was obtained and diffracted to a resolution of 2.5 A. To solve the phase problem, its Se-Met labeled derivative is being preparing. XAC3683 is a histidine kinase and was found to interact with HrpG in the XAC from a prior yeast two-hybrid experiment. Based on sequence comparison, XC3070 is a XAC3683 homologue in Xcc. Three different clones : the full-length, the N-terminus deleted XC3070 (115-630) and XC3070 (250-630) were constructed. The soluble form of XC3070 (115-630) was obtained. After purification, a crystal was obtained via in situ proteolysis. The Gel filtration, biacore or ITC experiments to determine the binding strength between XC3070 and XcHrpG will be tested.