十字花科植物黑腐病菌菌黄素生物合成调控机理研究

The genus Xanthomonas is one of the most ubiquitous groups of plant-associated bacterial pathogens. Members of this genus have been shown to infect many economically important crops or plants, such as rice, cotton, soybean, banana and citrus. Xanthomonas infection causes serious loss in yield. A characteristic feature of the genus Xanthomonas is the production of yellow, membrane-bound pigments called xanthomonadin. Xanthomonadins are one of the most important virulence factors produced by Xanthomonas and have been shown to protect the pathogens from photooxidative damages, to enhance the pathogen's epiphytic survival on leaf surface, and to greatly promote the virulence on host plant. A diffusible factor (DF) has been reported to be implicated in the regulation of xanthomonadin production in Xanthomonas campestris. Our latest results have shown that DF is 3-hydroxybenzoic acid (3-HBA)and it is associated with diverse biological functions in Xanthomonas campestris. However, how DF is synthzied and how DF regulates xanthomonadins biosynthesis remains to be solved. In this proposal, we set out to investigate these important and interesting questions using the model baterial strain Xanthomonas campestris pv. campestris (Xcc), a causal agent of black rot in crucifers. Three specific aims will be conducted: (1)To characterize the enzymatic activity,catalytic mechanism and 3-D structure of a novel protein XanB2, which is essential for DF biosynthesis;(2)To establish DF biosynthetic pathway through deletion analysis of all the genes involved in the shikimate pathway, Tn5 mutagenesis of the whole geneome, and other genetic and biochemical approaches;(3)To investigate how DF is implicated in the regulation of xanthomonadins by C13-labelled DF feeding experiment, transcriptomic and proteomic analysis of Xcc wild type and xanB2 mutants; Our aims are to elucidate whether DF is acting as an intermediate or as a signal. If DF is acting as a signal, the putative DF receptor and the regulatory network will be elucidated. Unraveling how DF is synthesized in Xcc and how DF is implicated in the regulation of xanthomonadin biosynthesis is essential to not only understand the pathogenesis of the phytopathogen Xanthomonas, but also to facilitate the discovery of novel methods or biopesticides to control Xanthomonas infection.

黄单胞菌是一类革兰氏阴性植物病原细菌，能侵染水稻、油菜和柑橘等重要农经作物，导致严重减产。菌黄素是黄单胞菌产生一种附着在细胞外膜上的黄色色素，它是一种重要致病因子，能显著影响黄单胞菌在土壤环境和植物体表的附生能力以及致病性。菌黄素的生物合成受到一种调控因子DF的调控。我们实验室首次报道DF是3-羟基苯甲酸，在此基础上，本项目以野油菜黄单胞菌为模式菌株，从生物学和化学两个角度，(1)深入研究功能未知的DF合成相关蛋白XanB2的生化功能、酶促催化活性和催化机理；(2)通过全基因组突变分析、比较基因组分析和莽草酸途径基因敲除等方法，建立完整的DF生物合成途径)；(3）通过C13标记的DF喂饲实验、转录组学和蛋白组学分析等方法，研究DF调控菌黄素生物合成的分子机理和调控通路。预期的研究成果将居于国际领先水平，有助于阐明黄单胞菌致病机理等重大科学问题，为控制黄单胞菌危害提供理论依据。

黄单胞菌是一类重要农作物病原菌，它们产生大量的黄色色素，学名为菌黄素(xanthomonadins)。菌黄素不溶于水，附着在细胞膜外膜上因而使菌落呈黄色。菌黄素能有效保护黄单胞菌在土壤环境和叶表面的生存，显著促进黄单胞菌的致病性，因此，是一种重要的致病因子。本项目以野油菜黄单胞菌(Xcc)为模式菌株，主要从生物学和化学两个角度尝试回答三方面的问题：(1) 菌黄素合成调控因子DF生物合成途径和关键蛋白XanB2的酶活性及催化机理；(2) DF调控菌黄素生物合成的调控机理；(3) Xcc菌黄素的化学结构和生物合成机制。主要结果包括四方面：（1）初步阐明了DF的化学结构为3-羟基苯甲酸（3-HBA)和4-羟基苯甲酸(4-HBA)，3-HBA作为合成前体参与菌黄素的生物合成和功能，4-HBA既参与菌黄素生物合成，又参与辅酶Q生物合成；（2)系统阐明了3-HBA和4-HBA合成酶XanB2的生化功能和酶学特性；（3）克隆和鉴定了菌黄素生物合成基因簇，初步阐明了菌黄素生物通过新型2-型聚酮途径合成的分子机理；（4）克隆了黄单胞菌中负责降解4-HBA的基因簇，阐明了降解4-HBA的分子机理。这些结果为进一步研究黄单胞菌致病机理奠定了基础，同时也为研究绿色防控病原菌提供了靶标。