根癌农杆菌识别和传递宿主趋化信号的分子机理及其在调控致病性和转基因效率中的作用的研究

Agrobacterium tumefaciens is a plant pathogen that causes the crown gall tumor disease in various dicotyledonous plants. A. tumefaciens induces the tumor by transferring a segment of its Ti plasmid DNA (transferred DNA, or T-DNA) into the host genome and genetically transforming the host. A. tumefaciens has become to the most wildly used gene vector to deliver genetic material into plant because of its ability to transfer DNA into the host. However, wildly using of A. tumefaciens may take the risk of biosafety and ecology. Therefore, in order to minimize this risk, it is very important to seek for an approach that can regulate the pathogenicity and transgenesis ability of A. tumefaciens in nature, but not affect the transgenesis efficiency of A. tumefaciens in lab. With the implement of two CNNSF (Chinese National Natural Science Fund) research projects in our lab, we intensively and deeply investigated the molecular mechanism of A. tumefaciens-mediated T-DNA transform and know that regulating the recognition of A. tumefaciens to its natural host chemoattractants may realize the above-mentioned goal. Chemotaxis is the process by which motile bacteria are able to track the gradients of some chemicals in their environments and to move towards more favorable living environments. Bacteria use chemoreceptors to sense the chemoeffectors in their environments. A. tumefaciens has 20 genes that can encode chemoreceptors. It can sense many chemoeffectors, including the chemoattractants produced by plant host. In this project, we will mutate these 20 chemoreceptor-encoding genes one by one and construct their mutants. After we obtained these mutants, we will test the chemotactic responses of these mutants to the host chemoattractants to identify the chemoreceptors that can recognize the host chemoattractants. We will also unveil the molecular mechanism of A. tumefaciens to transduce the host chemotaxis signal, which will let us to idetify the key protein that can regulate the recognition and transduction of the host chemotaxis signal. By means of the function reformation of the key protein, we are able to investigate the role of the key protein in regulating the pathogenicity and transgenesis efficiency of A. tumefaciens so that we can find out an approach to use A. tumefaciens in the low risk of biosafety and ecology.

根癌农杆菌是一种能使许多双子叶植物发生冠瘿瘤疾病的病原细菌。它将自身的DNA转入宿主，使宿主发生遗传转化而致病。它的这种转基因能力使它成为应用最广泛的植物转基因工具。如何在不影响其作为植物转基因工具应用的情况下调控其在天然条件下的致病性和转基因能力，是防范因其被广泛使用而可能引起生物和生态安全风险的重要途径。我们在2项国家基金项目的资助下深入研究了农杆菌的转基因机理，认为调控宿主趋化信号的识别可实现上述目标。根癌农杆菌有20个能编码趋化受体的基因，能识别包括宿主趋化信号在内的多种趋化信号。本项目将对20个基因逐一突变，获得相应的突变体，检测这些突变体对宿主趋化信号的响应，确定识别宿主趋化信号的受体，阐明宿主趋化信号传递的机理，找出精准调控宿主趋化信号响应的关键蛋白。通过对关键蛋白的分子改造，研究其对根癌农杆菌致病性和转基因能力的调控作用，找到能够在安全风险更小的情况下利用根癌农杆菌的方法。

根癌农杆菌的致瘤诱导信号系统和趋化响应信号系统都是双组分信号系统，而且能够识别部分共同的信号物质。该资助项目的研究目的是搞清楚根癌农杆菌的这两个信号系统是否存在信号交互关系。为此，对根癌农杆菌趋化系统的信号传导机制进行了深入研究，发现其趋化系统有三个明显的特点：1）有20个趋化受体，分别属于4大类不同的受体，应该能够识别相当多化合物；2）含有2个偶联蛋白和2个趋化响应调节蛋白；3）没有终止趋化信号的去磷酸化酶CheZ。由于双组分信号系统的信号传递机制是响应调节蛋白的磷酸化和去磷酸化，因此，重点研究了2个趋化响应调节蛋白CheY1和CheY2的功能差异，并获得了一种能够显著增强根癌农杆菌趋化响应的CheY变异体。但，未找到两个信号系统可以直接相互磷酸化的证据。为了确定两个信号系统的信号交流是否可能发生在信号物质识别上，我们对根癌农杆菌的部分趋化受体的功能进行了鉴定。通过本项目的研究，鉴定了根癌农杆菌atu0514、atu0526、atu1912和atu2173等4个基因编码的趋化受体的生物学功能，其中atu0514基因编码的趋化受体参与多种不同类别的化学物质信号的识别和传导。同时，还发现编码细菌铁蛋白的atu2771基因和O-去甲基酶编码基因atu1420显著影响根癌农杆菌的致瘤性，并对这两个基因影响根癌农杆菌致瘤性的机制进行了深入研究，发现细菌铁蛋白改变根癌农杆菌胞内铁平衡和细胞的抗氧化性而影响其致瘤性， O-去甲基酶可以降解能诱导致瘤的信号物质——乙酰丁香酮。建立了根癌农杆菌的全基因代谢模型。由该资助项目所取得的研究结果发表了11篇SCI期刊学术论文，2篇中文核心期刊学术论文，授权2项中国发明专利。部分结果发表在高影响的国际顶级（top）期刊，Biotechnolgy Advances (SCI 影响因子17.68)和Molecular Plant Pathology。这些结果的发表，对细菌趋化系统的研究、化学受体生物学功能的鉴定和根癌农杆菌致瘤信号响应和识别机制的深入解析都具有重要理论意义，同时对如何通过改变根癌农杆菌的趋化响应和致瘤信号传导来调控根癌农杆菌的致瘤性也有实践指导意义。部分结果所发表的论文已经被引用34次。