第二信使c-di-GMP受体DGC3介导的内生蜡样芽孢杆菌生物防治小麦纹枯病的作用机制

Sharp eyespot of wheat is an important soil-borne disease of wheat worldwide. Control measures against this disease available at present are inefficient due to lack of wheat cultivars resistant to sharp eyespot and no efficient fungicide available. Biocontrol of sharp eyespot, as an alternative measure, have been investigated intensively. Bacillus cereus can form endospores that are tolerant to heat and desiccation, offer several advantages over other bacteria..The second messenger c-di-GMP is a broadly conserved intracellular signaling molecule that is important for controlling biofilm formation, adhesion, motility, virulence in diverse bacterial species. The intracellular levels of c-di-GMP are modulated by the opposite activity of diguanylate cyclases (DGCs), which contain GGDEF domains, and phosphodiesterases (PDEs), which contain EAL or HD-GYP domains. The presence of a variety of genes coding for GGDEF, EAL or GGDEF-EAL proteins in the genome of many bacterial species indicates that bacteria regulate c-di-GMP turnover in an extremely sophisticated manner. How microbes integrate multiple input signals to regulate critical biological processes is still largely unclear. .B.cereus B3-7 is an endophytic bacteria, isolated from wheat roots, which have biocontrol capacity against sharp eyespot of wheat. We have revealed that a degenerated GGDEF protein called DGC3 functioned as a c-di-GMP receptor which involves in biocontrol to sharp eyespot. To elucidate the function of DGC3 in the process of biocontrol. We constructed dgc3 deletion mutant（Δdgc3）and identified total of 72 differential expression proteins between B3-7 and its Δdgc3 mutant based on 2D electrophoresis. All of the 72 protein encoding genes were deleted and biocontrol activity of 72 gene deletion mutants were tested indivually. We found SbnB involved in biocontrol activity. No protein interaction found between DGC3 and SbnB. It was suggested that there existed a metabolism pathway between the c-di-GMP receptor DGC3 and SbnB，which mediated biocontrol of B3-7 to sharp eyespot of wheat. Based on research results previously, we would carry out co-immunoprecipitation, pull-down assay and Bacterial two-hybrid to characterize response regulator protein which binds DGC3 and relay signals to cellular processes and impact biocontrol activity of B3-7. We then would conduct biocontrol-related genes isolation from 16 biocontrol variant mutants characterized previously. We would analyze gene function so as to ascertain components of biocontrol pathway from DGC3 to SbnB. Next ,we would analyze the metabolic circuits of biocontrol related factors involved and carry out multigene deletion to disclose temporal and spatial expression patterns of genes in the process of biocontrol. We would assay the stability of mRNA and its encoding proteins of biocontrol gene to reveal the regulation patter caused by c-di-GMP levels. All the research findings will lay a foundation of development biocontrol agents.

利用内生细菌防治病害，可以克服根际细菌生物防治中易出现的不稳定现象。研究生防作用的代谢调控网络，有助于揭示生防机制。c-di-GMP是广泛存在于细菌中的一种第二信使信号分子，参与调节biofilm形成、运动性等多种基本生物学过程。前期我们证明了生防菌蜡样芽孢杆菌B3-7中存在c-di-GMP代谢网络，蛋白DGC3作为c-di-GMP受体参与介导B3-7对于小麦纹枯病生防作用中由DGC3到SbnB的信号传递，但是DGC3的效应蛋白和生防途径的中间组分未知。本研究鉴定DGC3的效应调节蛋白，研究DGC3和效应调节蛋白的互作方式，解析效应蛋白在生防中的作用。在此基础上，利用获得的16个生防变异突变体，分离生防相关基因并进行功能分析，确定生防途径的中间组分。通过上位性分析，解析不同组分的时空顺序，揭示DGC3介导的信号通路组成和c-di-GMP信号在B3-7生物防治中的作用。

小麦纹枯病是一种危害小麦生产的重要土传病害，目前生产上尚缺乏抗病品种和有效的防治方法。利用植物根际细菌及其代谢产物防治小麦土传病害，对于实现植物病害的绿色防控具有重要价值。生防细菌在植物根系的定殖是其发挥生防作用的前提，细菌形成生物薄膜（biofilm）是其定殖的有效方式。环二鸟苷酸（c-di-GMP）是细菌中广泛存在的一种第二信使信号分子，该分子的代谢在多种细菌中影响细菌的运动性和biofilm形成。有关c-di-GMP在生防细菌蜡样芽孢杆菌（Bacillus cereus）0-9 （另一编号B3-7）中是否以及如何影响菌株生态适应性和对小麦纹枯病的生防活性是本研究的中心。本项目基于前期研究发现的一个具有GGDEF结构域的蛋白（Dgc3）编码基因缺失突变体生防活性降低这一基础，研究发现Dgc3能够专一性结合c-di-GMP，形成二聚体，对biofilm形成、运动性、抗氧化胁迫，糖异生和抗菌物质产生等多种生理过程具有调控作用。蛋白Dgc3结合c-di-GMP后能够直接结合伴侣蛋白DnaK，维持0-9菌株中质粒的稳定存在，提高抗菌物质产生。Dgc3能够与超氧化物歧化酶SOD A1互作，提高细菌对于氧化胁迫的抗性，提高根系定殖能力；此外，在碳源缺乏时，Dgc3作用于三磷酸甘油醛脱氢酶GapB，提高细菌糖异生能力。Dgc3还能通过氧化还原转录调节因子Rex，维持细胞内氧化还原的平衡，提高在重金属胁迫条件下细菌的生存能力。Dgc3正调控生防菌0-9的N-乙酰转移酶基因表达，提高0-9菌株对于氨基糖苷类抗生素的抗性，可能参与与土壤土著微生物的竞争，提高在植物根际的生存能力。本研究还鉴定出8个与生防菌0-9生防活性和环境适应相关的基因，这些基因如何发挥作用，是进一步研究的重点。