N端规则途径在内生细菌变形斑沙雷氏菌336x生物防治小麦全蚀病中的作用

Take-all, caused by the fungus Gaeumannomyces graminis var.tritici, is the most important root disease of wheat worldwide. No efficient control measure to take-all of wheat is available owe to lack of resistant varieties and efficient pesticide at present. Biological control to take-all, as an alternative measure, have been investigated intensively. Traditionally, biological control of soil-borne plant disease was performed with rhizobacteria, which easily influenced by environmental factor such as temperature, water and other native microbes, and caused inefficient control results. Biological control to plant disease with endophytic bacteria could overcome defects of rhizobacteria and could increase control efficacy..The N-end rule pathway is a highly conserved process that operates in many different organisms. It relates the metabolic stability of a protein to its N-terminal amino acid. This pathway has been implicated in oxygen homeostasis, cell division, apoptosis, putrescine homeostasis, and other processes. In bacteria, such as Escherichia coli and Agrobacterium tumefaciens, four hydrophobic.N-terminal residues (Phe, Tyr, Trp, Leu) serve as N-end-rule degradation signals (N-degrons). The N-degrons of bacterial substrates are bound directly by ClpS, assembling into a ternary complex with the ClpAP AAA+ protease. The substrate is then transferred to ClpAP by an active-handoff mechanism involving remodeling of ClpS by ClpA. Whether N-end rule pathway existed in biocontrol bacteria is largely unknown up to now..Serratia proteamaculans 336x is an endophytic bacteria, isolated from wheat roots, which have biocontrol capacity against take-all of wheat. In previous research, we knock out genes of clpS and clpA, which encode adaptor protein ClpS and unfolding enzyme ClpA respectively and both proteins belong to components of N-end rule pathway in other bacteria. We found mutants of ΔclpS,ΔclpA and double gene mutant(ΔclpAΔclpS) loss biocontrol activities to take-all of wheat. We also demonstrated that purified ClpS protein could recognize N-end rule amino acid from E.coli and bind it. All the results suggested N-end rule pathway exists in biocontrol bacteria 336x, however, the action mechanism of N-end rule pathway in biocontrol bacteria 336x should be disclosed. To elucidate N-end rule pathway exists in biocontrol bacteria and to disclose the action mechanism of N-end rule pathway during biocontrol of bacteria 336x to take-all of wheat, We would carry out proteomic analysis using Two-dimensional Electrophoresis to acquired differently accumulated protein between wild type 336x and its double gene knock out mutant ΔclpAΔclpS. We identified N-end rule proteins after sequencing N end of accumulated protein. We then would conduct binding assay of N-end rule protein by adaptor ClpS after fusion N end amino acids of identified proteins into GFP in vitro and would conduct degradation assay of the fusion protein in vivo to reveal stability of N-end rule protein. Next, we would clone genes encoding N-end rule protein and analyze gene function. Last, we determine the relationship between the accumulation of N-end rule proteins and biocontrol abilities. All the research above would contribute to disclose the function of N-end rule pathway in biocontrol bacteria 336x.

利用内生细菌防治病害，可以克服根际细菌生物防治植物病害易出现的防效不稳定现象。研究生防细菌的代谢网络，有助于揭示生防机制。由衔接蛋白ClpS、去折叠酶ClpA和肽酶ClpP参与的N端规则途径在生物中保守存在，通过控制特异蛋白的有序降解调控多种生理学功能。前期我们发现在生防细菌变形斑沙雷氏菌336x中clpS和clpA两个基因参与生防菌336x对小麦全蚀病的生物防治，ClpS蛋白对于经典N端规则蛋白具有识别和结合作用。但是生防菌336x发挥生防过程中N端规则途径是否发挥作用以及通过控制哪些组分发挥作用尚不清楚。本研究鉴定生防过程中突变体中积累的N端规则蛋白，研究这些蛋白及其编码基因的生理功能以及N端规则蛋白积累对于细菌生防活性的影响，有助于解析N端规则在生防菌336x中的作用方式和揭示N端规则途径调控336x生防活性的作用机制。

内生细菌由于定殖于健康植物组织内部，生存环境相对稳定，因而在发挥生物防治作用时独具优势。蛋白质量控制系统是生物细胞中普遍存在的一套控制细胞内蛋白质稳定存在和有序周转的控制体系，对于细菌多种生理功能的发挥具有重要作用，通过ClpAP对蛋白质选择性降解的N端规则途径是该系统中维持蛋白动态平衡的重要机制，本研究以生防细菌变形斑沙雷氏菌和蜡样芽胞杆菌为研究对象，分析N端规则途径在生物防治中发挥的作用，鉴定N端规则底物蛋白及其编码基因的功能，解析内生细菌对小麦病害的生防机制。研究发现，蛋白质量控制系统成员编码基因clpA与N端规则途径衔接蛋白编码基因clpS缺失后，菌株生防能力下降，在突变株中检测共到148个差异积累量增加的蛋白，根据ClpS结合底物序列的保守性，推断共有27个N端规则底物蛋白，综合体内实验和体外实验，获得衔接蛋白ClpS的8种N端规则蛋白底物，同时在蜡样芽胞杆菌和变形斑沙雷氏菌出现的底物有两个，分别为磷酸二酯酶和3-磷酸甘油醛脱氢酶，研究发现，二者编码基因功能都参与细菌生物膜形成调控，前者ymdB通过抑制SinI/SinR系统，解除sinR对生物膜形成相关基因sipw、tasA、calY表达，促进胞外多糖、蛋白质这些生物膜组分的合成来发挥作用，而后者编码基因gapB通过调节下游基因lrgAB的表达活性，促进细胞自溶，释放更多的DNA参与生物膜形成，这种调控机制区别与典型的SinI/SinR系统。综上，本研究结论是， 在生防细菌蜡样芽胞杆菌和变形斑沙雷氏菌中，N端规则途径两种底物蛋白，通过不同的调控机制影响生物膜形成，进而发挥对小麦病害的生物防治作用。