S-腺苷甲硫氨酸脱羧酶1介导的多胺调控ODV RepA诱导HR的分子机制研究

The hypersensitive response (HR) is rarly reported for members of the destructive mastreviruses. We have investigated a HR-like response elicited in tobacco plants by RepA encoded by Oat dwarf virus (ODV). Using RepA as bait in a yeast two-hybrid screen, a RepA-interacting protein, S-adenosyl-methionine decarboxylase 1 (NbSAMDC1) that is involved in polyamine biosynthesis, was identified from an N.benthamiana cDNA library. Here, the RepA and NbSAMDC1 interaction will be further conformed in planta by BiFC and GST pull-down assays. In plants, polyamines (PA) are thought to play important roles in induction of hypersensitive cell death during pathogen attack. Hence, the effects of transient expression of RepA on accumulation of transcripts for polyamine biosynthetic enzymes and PA Levels will be firstly investigated in N. benthamiana leaves and apoplasts. To determine hydrogen peroxide that is produced through oxidation of polyamines whether plays a critical role in apoptosis, the accumulation of transcripts as well as the activity profile of amine oxidase during RepA-induced HR will be then examined. The relationship between PA and the RepA-induced HR will be further examined using inhibitors of polyamine biosynthesis and degradation or PA treatments. On the other hand, to make a better understanding of the mechanism of RepA-NbSAMDC1 interaction, the approximate domains of NbSAMDC1 and RepA involved in interactions and RepA-induced HR are performed using yeast two-hybrid and BiFC assays. In addition, biochemical analysis will be used to show whether RepA could attenuate the 26S proteasome-mediated degradation of NbSAMDC1 protein. Furthermore, effects of overexpressing NbSAMDC1 or loss of function of NbSAMDC1 on PA and hydrogen peroxide levels as well as RepA-induced HR will be investigated. To identify cross-talking between PA and other defense-signaling molecules involved response against RepA, we performed a genomewide expression analysis of modulated genes in the transgenic tobacco lines overexpressing NbSAMDC1 through RepA transient expression. The research work mentioned above will expanding our understanding of the molecular mechanism of NbSAMDC1 involved of polyamines signaling in ODV RepA-induced HR.

申请者前期研究发现ODV RepA是一个HR激发子，并且和多胺合成的关键因子S-腺苷甲硫氨酸脱羧酶1（NbSAMDC1）互作。在此基础上，本项目拟通过检测RepA表达引起的多胺代谢过程中相关基因、酶活性、多胺含量的变化，以及分析多胺代谢相关的抑制剂处理或外源施加多胺等对RepA诱导HR的影响，确定多胺作为防御信号分子参与调控RepA诱导HR；通过基因缺失突变等，明确RepA和NbSAMDC1互作的关键结构域并确定其对RepA诱导HR的影响；利用生化实验，解析RepA在26S蛋白酶体对NbSAMDC1蛋白降解中的作用；过量及抑制表达NbSAMDC1，明确其对植物体内多胺以及RepA诱导HR的影响；分析RepA在NbSAMDC1过表达植株上的表达谱表化，解析参与多胺调控RepA诱导HR的信号分子网络。本项目的实施将深入揭示NbSAMDC1介导的多胺调控ODV RepA诱导HR的分子机制。

申请者前期研究发现ODV RepA是一个HR激发子，并且和多胺合成的关键因子S-腺苷甲硫氨酸脱羧酶1（NbSAMDC1）互作。在此基础上，本项目通过检测不同时间点ODV RepA瞬时表达植株的多胺水平，证实ODV RepA诱导HR过程中引起腐胺和亚精胺水平显著上升，进一步通过多胺合成竞争性抑制剂MGBG和氨基胍处理以及外施腐胺、亚精胺和精胺，证实多胺作为信号分子参与调控ODV RepA诱导HR；通过酵母双杂交、Co-IP、BiFC和Pull-down等实验证实ODV RepA和多胺合成途径中的关键酶NbSAMDC1相互作用，并通过构建缺失突变体明确了NbSAMDC1与RepA互作的关键结构域；通过蛋白的稳定性分析实验证实NbSAMDC1蛋白易于被26S蛋白酶体降解系统降解，而RepA和NbSAMDC1的互作有助于维持NbSAMDC1蛋白的稳定性；通过VIGS和植物遗传转化证实过量及抑制表达NbSAMDC1会显著地调控植物体内多胺的水平，并影响ODV RepA诱导的HR；通过高通量表达谱测定发现ODV RepA诱导HR的过程中参与光合作用的大部分基因其表达都受到显著抑制，其中BiFC和酵母双杂实验证实电子传递链关键组分NbFDN1能够与NbTsip1蛋白相互作用，而VIGS沉默这两个基因能显著改变RepA诱导HR，初步实验证实NbFDN1通过调控NbTsip1的表达水平以及促进过氧化氢的积累调控ODV RepA诱导HR。