| Xenocoumacin 1 (Xcn1), a benzopyranone derivative with amino acid and carboxylic acid hybrid moieties, is the major antimicrobial compound isolated from cultures of Xenorhabdus nematophila. Xcn1 has potent antiulcer activities and inhibits most gram-positive bacteria, partly gram-negative bacteria, and some pathogenic fungi. Xcn1 could strongly inhibit Phytophthora fungus compared to other agricultural pathogenic fungi. The research of its mode of action could not only provide theoretical basises for the designing, syhthesis and the structure modification of its derivatives in the near future, but also provide guidance on its application in the agriculatural disease control and therapy. In this paper, the mode of Xcn1 on Phytophthora capsici had been studied, while the Bacillus subtilis as model bacteria for the study of the mode of action for antibiotics also had been used in this study. This dissertation elucidates its mode of action against P. capsici and B. subtilis from the cellular, sub-cellular, and molecular levels.1. According to the characteristics of P. capsici causing plant diseases, the mode of action of Xcn1 was studied. It turned out that Xcn1 could not only inhibit the growth of the hypha, but also inhibit sporangium production, zoospore release, zoospore swimming, cystospore germination, and cystospore pathogenicity in different degrees.2. Under the stress of Xcn1, the growth rate of the hypha slowed down obviously, and colony morphology was loose. By optical microscopic examination, we revealed that P. capsici treated by Xcn1 showed increased hyphal branch spacing, significant plasmolysis, and condensed protoplast. Under transmission electron microscope, after treating P. capsici with Xcn1, we also saw apparent plasmolysis, condensed protoplast, and decreased electron density of cell wall.3. After the treatment of P. capsici hypha by Xcn1, the cell membrane permeability was not changed, indicating that cell membrane is not a primary target for Xcn1. Xcn1 weekly inhibited the aerobic respiration of hypha of P. capsici.4. Because of the genome of P. capsici is little known to us, so we studied the global gene expression profiles of B. subtilis which is a model in the antimicrobial mode of action study. We applied whole-genome DNA microarray technology to provide a global picture of the physiological response of B. subtilis cells exposed to Xcn1. Microarray data demonstrats that Xcn1 induction in B. subtilis led to the up-regulation of 480 genes and down-regulation of 479 genes more than 2 fold individually (q≤0.05). The Microarray analyses reveal that Xcn1 represses expression of genes mainly involved in amino acid biosynthesis, transport of oligopeptide, amino acid and iron ion, as well as aminoacyl-tRNA synthetases (AARSs) and class I heat-shock proteins, while up-regulated genes mainly involved in pyrimidine nucleotide biosynthesis and chemotaxis. Cluster analysis of the gene expression profiles of Xcn1 and 37 different antibiotics with known mechanisms discloses that Xcn1 has similar mode of action to protein synthesis inhibitors. These results indicate that Xcn1 is an inhibitor of arginyl-tRNA synthetase. The alterations in expression of six of effected genes, argG, ecsA, ppsA, ywpD, pyrB and yoaH, were analysed by real-time RT-PCR, and the results were consistent with those from microarray.To sum up, Xcn1 could inhibit differential stages in the life cycle of P. capsici including sporangium production, zoospore release, zoospore swimming, cystospore germination, and cystospore pathogenicity, lead to P. capsici obvious plasmolysis, cytoplasmic condensation and increased hyphal branch spacing, and affect the aerobic respiration of hypha of P. capsici at some extend; we predicted by genome microarray analysis of B. subtilis that Xcn1 is a protein synthesis inhibitor and its target is Arginyl-tRNA synthetase.
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