大豆疫霉效应子PsCRN63调控植物先天免疫及细胞死亡的功能与作用机制研究

发布时间：2019-04-02 13:21

【摘要】：植物先天免疫在感受到来自病原微生物的分子后激活,这些分子包括保守的病原物或微生物相关分子模式(PAMPs/MAMPs)以及多样化的效应分子(effectors)。病原物相关分子模式PAMPs触发的免疫反应(PTI )和效应分子effectors诱导的免疫反应(ETI)是植物抵抗病原物侵染的两种有效武器,构成了植物的天然免疫系统。毒性病原菌可以分泌大量效应分子,这些效应蛋白可以在植物的胞外起作用,也可以转运到植物细胞并且在胞内起作用。效应分子的主要功能是抑制寄主免疫反应从而促进生长和繁殖,达到成功侵染的目的。但是绝大部分效应蛋白的生化功能和分子机制并不清楚。因此研究这些效应蛋白在致病过程中的功能和分子机制,将有助于我们深入理解病原菌致病机理和植物免疫机制。疫霉属病原菌能在植物上引起多种典型的植物病害,比如致病疫霉(Phytophthora infestans)引起的马铃薯晚疫病和大豆疫霉(Phytophthora sojae)导致的大豆根腐病每年都给世界范围内农业生产带来毁灭性的危害。大豆疫霉隶属于卵菌,这类病原菌虽然在形态上同真菌相似,但在进化上却和硅藻及蓝藻关系较近,所以现阶段针对于真菌设计的杀菌剂往往对大豆疫霉和其它卵菌无效。在卵菌的胞内效应分子中,RxLR(R代表精氨酸,L代表亮氨酸,X代表任意氨基酸)和CRN ( Crinkler)效应分子是最重要的两类。这两类效应分子是模块化的蛋白质:它们的N端含有保守的结构域(RxLR和dEER或LFLAK )并且能帮助效应蛋白转运到宿主细胞,而C端是多样化的功能域,在细胞内参与调节植物免疫反应。由于与已知蛋白缺乏序列相似性,因此很难预测这些效应蛋白的功能和作用机制。本文对大豆疫霉中的一个胞内效应子PsCRN63的功能进行了分析,并探究了其可能的毒性机制,获得的主要结果与结论如下:大豆疫霉效应分子PsCRN63与植物过氧化氢酶互作调控细胞死亡。先前的研究结果证明,大豆疫霉的效应分子PsCRN63 (起皱和坏死诱导蛋白)能够在植物中引起程序性细胞死亡(PCD, programmed cell death ),而PsCRN115可以阻断这一过程;然而两者对于病原菌的致病性都是必须的。这里,我们发现PsCRN63单独表达或者PsCRN63和PsCRN115共同表达都能够抑制烟草上的免疫反应;同时这两个胞内的效应分子都可以与来自烟草(Nicotiana enthamiana)和大豆(Glycine max)中的过氧化氢酶互作。进一步,我们发现当在植物中表达PsCRN63时,烟草中的过氧化氢酶NbCAT1 (N.benthamiana CATALASE1 )和大豆中的过氧化氢酶GmCAT1的蛋白质变得不稳定,而PsCRN115可以阻止这一变化。实验证明在烟草中瞬时表达PsCRN63可以导致过氧化氢(H2O2)的积累,PsCRN115则依然相反。最后,我们发现烟草中瞬时表达NbCAT1或者GmCAT1时可以特异性地缓解PsCRN63诱导的细胞死亡症状。因此,我们推测PsCRN63/115通过与过氧化氢酶互作干扰细胞内过氧化氢的内稳态,从而调控在植物上引起的细胞死亡。以上结果表明,大豆疫霉能够分泌两个效应分子,通过与过氧化氢酶的直接互作,来调控细胞死亡和过氧化氢的内稳态,进而克服寄主植物的免疫反应。大豆疫霉效应分子PsCRN63通过胞内二聚化调控植物先天免疫。在本研究中,我们发现,大豆疫霉的效应分子PsCRN63能够抑制病原物相关分子模式(PAMP)触发的免疫(PTI)的标记基因,flg22诱导的FRK1基因的表达。但是,PsCRN63不能抑制PTI上游信号通路的相关事件,包括flg22诱导的MAPK的激活以及BIK1的磷酸化,这表明它作用于MAPK级联反应的下游。PsCRN63转基因拟南芥植株对病原细菌丁香假单胞菌番茄致病变种(Pseudomonas syringae pathovar tomato, Pst)DC3000和病原卵菌辣椒疫霉(Phytophthoca capsici)的敏感性增强。另外,与野生型植株相比,PsCRN63转基因植株中flg22诱导的活性氧爆发和胼胝质沉积均受到抑制。同时,PTI途径相关基因的表达在PsCRN63转基因植株中也受到下调。有趣的是,我们发现PsCRN63蛋白的N端和C端能够通过反向连接的方式在植物细胞内发生互作,从而形成一个同源二聚体。另外,形成二聚体所需的N端和C端结构域在CRN效应分子中非常保守,这暗示着疫霉CRN效应分子的同源/异源聚合物的形成为其发挥生物学功能所必需。事实证明,二聚体的形成确为PsCRN63行使PTI抑制及细胞死亡诱导功能所必需。以上的研究结果能够增进我们对卵菌效应分子如何操纵植物免疫从而促进侵染的认识。
[Abstract]:The innate immunity of a plant is activated after a molecule from a pathogenic microorganism, which comprises a conserved pathogen or a microorganism-related molecular pattern (PMPs/ MMPs) and a variety of effector molecules. The immune response (PTI) and effector-induced immune response (ETI)-induced immune response (ETI), which are triggered by PMPs and effector-related molecular patterns, are two effective weapons for the plant to resist the infection of the pathogen, and constitute the natural immune system of the plant. The toxic pathogenic bacteria can secrete a large amount of effector molecules that can act outside the cell of the plant and can also be transported to the plant cells and function in the cell. The main function of the effector molecule is to inhibit the host immune response, thereby promoting growth and reproduction, and achieving the purpose of successful infection. However, the biochemical and molecular mechanisms of most effector proteins are not clear. Therefore, the study of the function and molecular mechanism of these effect proteins in the pathogenic process will help us to understand the pathogenic mechanism of the pathogenic bacteria and the mechanism of the plant immune. Phytophthora infestans can cause a variety of typical plant diseases on plants, such as Phytophthora infestans and Phytophthora sojae. Phytophthora sojae is a part of oomycetes, which is similar to the fungi in the form, but it has a close relationship with the diatom and the blue-green algae in the evolution, so the germicide designed for the fungus at the present stage is often ineffective for the Phytophthora sojae and other oomycetes. RxLR (R represents arginine, L represents leucine, X represents any amino acid) and CRN (Crinkler) effector molecules are the most important classes in the intracellular effector molecule of the oomycetes. These two types of effector molecules are modular proteins: their N-terminal contains a conserved domain (RxLR and dEOR or LFLAK) and can aid in the transport of effector proteins to the host cell, while the C-terminal is a diverse functional domain that is involved in the regulation of the plant immune response. Because of the lack of sequence similarity to known proteins, it is difficult to predict the function and the mechanism of action of these effector proteins. In this paper, the function of one intracellular effector PsCRN63 in Phytophthora sojae atricolor was analyzed and its possible toxicity mechanism was explored. The main results and conclusions were as follows: Previous studies have shown that the effects of Phytophthora sojae, PsCRN63 (creping and necrosis-inducing proteins), can cause programmed cell death in plants, while the PsCRN115 can block the process; however, both are essential to the pathogenicity of the pathogenic bacteria. Here, we found that the individual expression of the PsCRN63 or the co-expression of the PsCRN63 and the PsCRN115 can inhibit the immune response on the tobacco, and both intracellular effector molecules can interact with the catalase from the tobacco (Nicotiana enthamiana) and the soybean (Glycine max). Further, we have found that when the PsCRN63 is expressed in a plant, the protein of the catalase GmCAT1 (N. benthamiana CATAIL1) in the tobacco and the catalase GmCAT1 in the soybean becomes unstable, and the PsCRN115 can prevent this change. The experimental results show that the instantaneous expression of PsCRN63 in tobacco can lead to the accumulation of hydrogen peroxide (H2O2), and the PsCRN115 is still the opposite. Finally, we find that the transient expression of NbCAT1 or GmCAT1 in tobacco can specifically alleviate the cell death symptoms induced by PsCRN63. Therefore, we assume that PsCRN63/115 regulates the intracellular state of hydrogen peroxide within the cell by interacting with the catalase, thereby regulating the cell death induced on the plant. The results showed that Phytophthora sojae was able to secrete two effector molecules, and through direct interaction with catalase, the cell death and the internal steady state of hydrogen peroxide were regulated, and the immune response of host plants was overcome. Phytophthora sojae effect molecule PsCRN63 regulates the innate immunity of plants by intracellular dimerization. In this study, we found that the effect molecule PsCRN63 of Phytophthora sojae was able to inhibit the expression of the marker gene of the immune (PTI) triggered by the pathogen-related molecular pattern (PAMP), and the expression of the flg22-induced FRK1 gene. However, the PsCRN63 does not inhibit the related events of the signaling pathway upstream of the PTI, including the activation of the flg22-induced MAPK and the phosphorylation of BIK1, which indicates that it acts downstream of the MAPK cascade. The sensitivity of PsCRN63 transgenic Arabidopsis plants to the pathogenic bacteria of Pseudomonas syringae patovar tomas, Pst and Phytophthora capsici was enhanced. In addition, the active oxygen burst induced by flg22 in the PsCRN63 transgenic plant was inhibited compared to the wild-type plant. At the same time, the expression of PTI-related genes was also down-regulated in the transgenic plants of PsCRN63. Interestingly, we found that the N-and C-ends of the PsCRN63 protein were able to interact within the plant cells in a reverse-linked manner to form a homodimer. In addition, the N-and C-terminal domains required to form the dimer are very conserved in the CRN effector molecule, suggesting that the formation of the homologous/ heterologous polymer of the Phytophthora CRN effector molecule is necessary for its biological function. The formation of dimers has proven to be necessary for PsCRN63 to exercise PTI inhibition and cell death induction. The above results can improve our understanding of how to manipulate the plant to promote the infection.
【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S432.4

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