番茄抗病蛋白Sw-5b自抑制与激活的调控机理及亚细胞定位研究

发布时间：2018-05-15 15:57

本文选题：NB-LRR + Sw-5b　；参考：《南京农业大学》2016年博士论文

【摘要】：免疫受体蛋白(R蛋白)介导的对病原物效应蛋白的特异性识别并激活级联免疫反应是植物主要的防疫体系。NB-LRR类受体蛋白作为最大的一类R蛋白其功能结构域包括N端结构域、核苷酸结合结构域(NB-ARC)和C端富亮氨酸富集结构域(LRR)。基于N端结构域的不同分为TIR-NB-LRR和CC-NB-LRR。为了减少R蛋白过度激活带来的不必要成本,植物进化出了相应的机制来调控其自抑制和激活。番茄Sw-5b属于具有额外的N端结构域(NTD)的CC-NB-LRR类型R蛋白,但是额外的NTD、CC、NB-LRR如何调控其自抑制和激活的机制目前仍不清楚。此外,越来越多的研究表明,R蛋白的亚细胞定位在抗病信号通路中发挥着重要作用,然而带有额外N端结构域的植物R蛋白的亚细胞定位在抗病中的作用还没有开展过相关研究。本研究围绕两个重要科学问题,以番茄免疫受体蛋白Sw-5b和番茄斑萎病毒(Tomatospotted wilt virus,TSWV)移动蛋白NSm为研究模式,开展了以下两方面的研究:1.番茄免疫受体蛋白Sw-5b的自抑制和激活的多重调控机制。为了探究Sw-5b的自我调控机制,我们系统地剖析了 Sw-5b的各亚结构域的功能以及他们之间的关系。在农杆菌介导的本氏烟瞬时表达试验中,Sw-5b的NB-LRR片段能够介导依赖于NSm的细胞死亡,且这种诱发死亡的特性具有和全长Sw-5b 一样的特异性。将同源建模技术和定点突变实验相结合,发现保守的中心区域NB-ARC是激活NB-LRR的重要分子开关。基于以上研究,首先我们探讨了病毒激发子NSm不存在时,Sw-5b自抑制的调控机制。在瞬时表达试验中,NB-ARC自激活诱导细胞死亡,而NB-ARC与LRR 一起表达或单独表达NB-LRR则抑制细胞死亡,CC-NB-LRR或NTD-CC-NB-LRR单独表达不能诱导死亡,组成型激活突变位点S594A, D642E和D857V引入到NB-LRR中会导致其自激活,引入到CC-NB-LRR中自激活减弱,引入到NTD-CC-NB-LRR中不再产生自激活。免疫共沉淀试验发现:LRR, CC和NTD对NB-ARC的抑制作用与结构域之间的互作有关,LRR和NB-ARC发生物理互作,CC与LRR互作,而不与NB-ARC互作,NTD既可以和CC也可以和NB-LRR发生物理互作。因此,当病毒激发子NSm不存在条件下,LRR, CC和NTD的三层负调控作用使得Sw-5b维持在一个很稳定的自抑制状态;其次,我们探究了 Sw-5b激活的调控机制。在与NSm共同瞬时表达的试验中,NTD-CC-NB-LRR或其衍生突变体和NB-LRR 一样诱导产生很强的细胞死亡反应,而CC-NB-LRR及其衍生突变体只呈现很弱的细胞死亡反应。因此表明,当病毒激发子NSm存在情况下,LRR释放对NB-ARC的抑制,进一步NTD解除CC对NB-LRR的抑制,最后Sw-5b被完全激活。此外,我们还发现NB-LRR这一层自抑制和激活调控并不能抵抗TSWV病毒,在转基因植株的接病毒测试中,35S: NB-LRR转基因植株虽然对番茄斑萎病毒产生HR反应,但不具有系统抗性。而全长Sw-5b的转基因植株无论是35S启动子下或是本身启动子下都对番茄斑萎病毒表现抗性。在本试验基础上,我们提出如下假说:Sw-5b为了释放CC的负调控作用,进化出了额外NTD结构域来协调CC的作用,最终Sw-5b进化出了多重的调控机制来控制其自抑制和激活状态。重要的是,这种多层的调控机制,对于Sw-5b蛋白抗性功能的发挥是必须的。2.番茄免疫蛋白受体Sw-5b的亚细胞定位与功能的关系。大多数的R蛋白没有经典的核定位信号,因此最初人们认为这类受体主要定位在细胞质中。本研究首先测试Sw-5b的亚细胞定位,在其N端融合黄色荧光蛋白后,瞬时表达及转基因抗性测试发现,YFP-Sw-5b具有与Sw-5b一样诱导细胞死亡和诱导抗性的能力。共聚焦观察发现其在细胞质和细胞核及核仁都有分布。进一步探究Sw-5b进出核与功能的关系,我们在YFP-Sw-5b的N端融合外源的核输出信号(NES)将Sw-5b拉到细胞质,瞬时表达发现,完全的细胞质定位导致细胞死亡活性明显增强,但是,转基因植株接毒测试表明出核导致其失去了对TSWV的抗性;融合核定位信号(NLS)将Sw-5b全部拉到细胞核内,其诱导的细胞死亡明显减弱或消失但是却足以抵抗病毒。同样的方法改变NSm的亚细胞定位,发现NSm定位在细胞质才能诱导细胞死亡。此外,我们发现Sw-5b的核定位特性与抗性激活与否无关。P-loop保守基序突变破坏Sw-5b核苷酸结合构象后,YFP-Sw-5b诱导细胞死亡的功能丧失,但其核质分布没有受到影响。在重要分子伴侣SGT1被沉默的植株中,YFP-Sw-5b不能诱导细胞死亡,但依然能够进核。我们还发现Sw-5b的细胞核定位信号在N端的NTD结构域,其中YFP-NTD1 (51.7 kDa)和全长Sw-5b 一样核质都有分布,YFP-NTD2 (42kDa)能进核但不进核仁,细胞质定位信号在NB-LRR结构域。基于以上发现,我们认为Sw-5b与NSm的识别发生在细胞质,且Sw-5b介导的细胞死亡和抗病功能可以分开进行。
[Abstract]:The specific identification of the immune receptor protein (R protein) and the activation of the cascade immunoreaction are the.NB-LRR receptor proteins in the main epidemic prevention system of plants as the largest class of R proteins as the largest class of protein, including the N domain domain, the nucleotide binding domain (NB-ARC) and the C terminal rich leucine enriched domain (LRR). The different N terminal domains are divided into TIR-NB-LRR and CC-NB-LRR. in order to reduce the unnecessary cost of R protein overactivation. Plants have evolved a corresponding mechanism to regulate their self suppression and activation. Tomato Sw-5b belongs to the CC-NB-LRR type R protein with an additional N terminal domain (NTD), but the additional NTD, CC, how NB-LRR regulates its self suppression. The mechanism of the system and activation is still unclear. In addition, more and more studies have shown that the subcellular localization of R protein plays an important role in the disease resistance signaling pathway. However, the role of the subcellular localization of the plant R protein with the additional N terminal domain in the disease resistance has not been studied. This study revolves around two important scientific questions. On the basis of the model of tomato immune receptor protein Sw-5b and Tomatospotted wilt virus (TSWV) mobile protein NSm as the research model, the following two aspects were carried out: 1. the self inhibition and activation of the immune receptor protein Sw-5b of tomato, the multiple regulation mechanism. In order to explore the self-regulation mechanism of Sw-5b, we systematically analyzed Sw-5b The function of each subdomain and their relationship. In the Agrobacterium mediated instant expression test of Benedict's smoke, the NB-LRR fragment of Sw-5b can mediate cell death dependent on NSm, and this characteristic of inducing death has the same specificity as the full length Sw-5b. The central region, NB-ARC, is an important molecular switch to activate NB-LRR. Based on the above study, we first explored the regulation mechanism of Sw-5b self suppression when the virus elicitor NSm does not exist. In the transient expression test, NB-ARC induces cell death by self activation, and NB-ARC and LRR are expressed together or individually expressed in NB-LRR to inhibit cell death, CC-NB-LRR Or NTD-CC-NB-LRR alone expressed the inability to induce death, the constituent activation mutation site S594A, D642E and D857V were introduced into NB-LRR to cause their self activation, and the introduction into CC-NB-LRR to reduce self activation and no longer produce self activation in NTD-CC-NB-LRR. The immunoprecipitation test found that LRR, CC and NTD have inhibitory effects on NB-ARC and the domain. Interaction between LRR and NB-ARC, CC and LRR interact with each other instead of NB-ARC, and NTD can and CC can interact with NB-LRR in physical interaction. Therefore, the three layer negative regulation of LRR, CC and NTD makes a stable self suppression state of LRR, CC and NTD; secondly, we explore The regulation mechanism of Sw-5b activation. In the experiment with NSm's common transient expression, NTD-CC-NB-LRR or its derivative mutant and NB-LRR induced a strong cell death response, while CC-NB-LRR and its derived mutants only showed a very weak cell death response. Therefore, LRR release to NB-ARC in the presence of the pathogen NSm. Inhibition, further NTD relieves the inhibition of CC to NB-LRR, and finally Sw-5b is fully activated. In addition, we also found that the self inhibition and activation regulation of NB-LRR is not resistant to TSWV virus. In the test of transgenic plants, the 35S: NB-LRR transgenic plants do not have systemic resistance to the tomato Verticillium virus, although they produce HR response to lycopene Verticillium virus. The transgenic plants with full length of Sw-5b are resistant to tomato Verticillium virus either under 35S promoter or under their own promoter. On the basis of this experiment, we propose the following hypothesis: Sw-5b has evolved an additional NTD domain to coordinate the role of CC in order to release the negative regulation of CC, and finally the Sw-5b has evolved multiple regulatory mechanisms. It is important to control the self inhibition and activation state. It is important that this multi-layer regulation mechanism, which plays a role in the subcellular localization and function of the.2. tomato immune protein receptor Sw-5b, is a necessary function of the Sw-5b protein resistance function. Most of the R proteins have no classical nuclear location signals. In the cytoplasm, this study first tested the subcellular localization of Sw-5b. After the fusion of yellow fluorescent protein at its N end, transient expression and transgenic resistance test found that YFP-Sw-5b had the ability to induce cell death and induced resistance as Sw-5b. Confocal observation found that the cytoplasm, nucleus and nucleolus were distributed in cytoplasm and nucleus and nucleolus. Further exploration of Sw-5b In the relationship between nuclear and function, we fused the exogenous nuclear output signal (NES) at the N end of YFP-Sw-5b to pull the Sw-5b into the cytoplasm. Instantaneous expression found that the complete cytoplasmic localization led to a significant increase in cell death activity. However, the transgenic plant toxicity test showed that the nucleus induced its loss of resistance to TSWV; fusion nuclear positioning signal (NLS) We pull all Sw-5b into the nucleus, and the induced cell death obviously weakened or disappeared but was enough to resist the virus. The same method changed the subcellular location of NSm and found that NSm was located in the cytoplasm to induce cell death. In addition, we found that the nuclear location of Sw-5b is not related to the anti sexual activation of the.P-loop conservative radical mutation. After the combination of bad Sw-5b nucleotides binding conformation, YFP-Sw-5b induces cell death function loss, but its nuclear distribution is not affected. In the silent plants of the important molecular chaperone, YFP-Sw-5b can not induce cell death, but still can enter the nucleus. We also found the Sw-5b cell nucleus localization signal in the NTD domain of the N terminal, in which YFP-NTD1 (Sw-5b) 51.7 kDa) are all distributed as the whole Sw-5b nucleolus, YFP-NTD2 (42kDa) can enter the nucleus but not nucleolus, and the cytoplasmic localization signal is in the NB-LRR domain. Based on the above findings, we think that the identification of Sw-5b and NSm occurs in the cytoplasm, and Sw-5b mediated cell death and disease resistance can be carried out separately.

【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】：S432.23

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