6K和P3N-PIPO蛋白调控烟草脉带花叶病毒复制和移动的分子机制

发布时间：2018-06-27 03:03

本文选题：马铃薯Y病毒属 + 病毒复制　；参考：《山东农业大学》2017年博士论文

【摘要】：马铃薯Y病毒属(Potyvirus)是最大植物病毒属,包含200多种病毒。该属病毒寄主范围广泛,发生普遍,给作物生产带来严重损失。种植抗病品种是防治作物病毒病最为经济有效的方式。但目前生产中缺乏有效的抗病毒品种。植物病毒需要借助寄主蛋白来完成侵染循环。沉默或敲除病毒依赖的寄主蛋白可以使植物产生抗性。同时,理解病毒蛋白的分子功能以及病毒与寄主互作的分子机制是研发新的抗病毒策略的前提。本研究揭示了马铃薯Y病毒属的烟草脉带花叶病毒(Tobacco vein banding mosaic virus,TVBMV)在复制和移动两个病毒侵染关键环节中与寄主的相互作用机制。主要研究结果如下:1、病毒寄主蛋白复合体6K1-6K2-PsbO1调控病毒复制的分子机制:第一6kDa蛋白(6K1)包含一个保守的RSD基序。丙氨酸扫描实验证明该基序中任何一个氨基酸的改变都使TVBMV复制的复制水平下降。亚细胞定位实验证明TVBMV 6K1单独瞬时表达时分布在整个细胞之中,然而在病毒侵染过程中被招募到叶绿体并与TVBMV复制相关第二6 kDa蛋白(6K2)和复制酶核内涵体b共定位。6K1能与6K2在体内相互作用并被6K2招募到位于叶绿体的病毒复制复合体。免疫共沉淀和质谱鉴定表明本氏烟光系统II放氧复合体蛋白(Photosystem II oxygen evolution complex protein of Nicotiana benthamiana,NbPsbO1)存在于6K1-6K2复合体上。NbPsbO1能与TVBMV 6K2蛋白互作但不与6K1互作。利用病毒诱导基因沉默降低NbPsbO1基因的表达抑制了马铃薯Y病毒属病毒TVBMV和马铃薯Y病毒基因组RNA和外壳蛋白在植株中的积累量,但并不影响马铃薯X病毒属的马铃薯X病毒基因组RNA和外壳蛋白的积累量。光系统II放氧复合体的另外两个元件NbPsbP1和NbPsbQ1不能与6K2互作,而且沉默NbPsbP1和NbPsbQ1基因不影响TVBMV复制。2、病毒寄主蛋白复合体P3N-PIPO-CI-NbDREPP调控TVBMV细胞间移动的作用机制:TVBMV的PIPO结构域由60个氨基酸位点组成。含有由7个、20个或43个氨基酸组成的PIPO结构域的TVBMV突变体丧失了细胞间移动和系统侵染能力。但这些突变体的复制水平与野生型TVBMV一致。本氏烟发育调控质膜蛋白(Developmentally regulated plasma membrane protein of N.benthamiana,NbDREPP)能与TVBMV P3N-PIPO和柱状内涵体蛋白CI互作。沉默NbDREPP基因抑制了TVBMV的细胞间移动和系统侵染。NbDREPP自身定位在细胞壁上,能与胞间连丝定位蛋白PDLP1共定位,而且能与P3N-PIPO和CI共定位于胞间连丝。早期分泌途径抑制剂Brefeldin A处理或瞬时过表达显性负抑制突变体ADP核糖基化因子1(ADP-ribosylation factor1)-T31N或分泌相关ras超家族相关基因1b(Secretion-associated and ras superfamily-related gene1b)-H74L都破坏了NbDREPP的胞间连丝定位。微丝骨架抑制剂Latrunculin B处理或过表达显性负抑制突变体肌球蛋白XI-2尾部结构域也破坏了NbDREPP的胞间连丝定位。3、通过新一代测序技术比较野生型TVBMV和弱毒突变体侵染植物后的转录组变化揭示了TVBMV的症状形成机制。辅助成分蛋白酶(helper component proteinase,HCpro)是TVBMV的RNA沉默抑制子。通过定点突变技术在TVBMV侵染性克隆pCamTVBMV-GFP(pCamT-WT)HCpro编码区引入突变,获得突变体pCamTVBMV-GFP-HCproD198K+DE250-251(pCamT-HCm)。这些氨基酸的改变破环了HCpro的RNA沉默抑制活性而且使T-HCm在本氏烟上的症状明显减轻。我们通过转录组测序比较了T-WT和T-HCm侵染本氏烟1天、2天和10天后的基因表达变化。在T-WT或T-HCm侵染后1天和2天,与蛋白翻译相关的基因上调表达;而与脂质代谢以及胞内刺激反应相关基因下调表达。在病毒接种后10天,T-WT的侵染抑制了光合合成相关基因的表达,而T-HCm的侵染对光合合成相关基因无明显影响。T-WT的侵染使RNA沉默相关途径的关键基因DCL2、DCL4、RDR1和AGO1上调表达;而T-HCm的侵染对这些基因的表达无影响。T-WT的侵染也使水杨酸和乙烯信号途径中的相关基因上调表达,但使茉莉酸信号途径的相关基因下调表达。T-WT和T-HCm的侵染差异调控了生长素信号转导途径中相关基因的表达,这与TVBMV引起的矮化症状有关。综上所述,TVBMV劫持寄主因子NbDREPP和NbPsbO1用于自身细胞间移动和复制,DREPP和PsbO1可以作为抗TVBMV的新靶标。TVBMV的侵染干扰了植物的卡尔文循环、叶绿素代谢以及生长素信号转导途径导致植物产生褪绿和矮化症状。
[Abstract]:Potato Y virus (Potyvirus) is the largest plant virus and contains more than 200 viruses. It has a wide range of host viruses and is widespread, causing serious loss to crop production. Planting resistant varieties is the most economical and effective way to prevent and control crop virus diseases. However, there are no effective antiviral varieties in production. Plant viruses need to be used. The host protein is the host protein to complete the infection cycle. The host protein that is silent or knocked out of the virus can cause resistance to plants. At the same time, understanding the molecular function of the virus protein and the molecular mechanism of the interaction between the virus and the host are the prerequisite for the development of the new antiviral strategy. This study revealed the tobacco vein zone mosaic virus (Tobacco) of the potato Y virus. Vein banding mosaic virus, TVBMV) interaction mechanism with the host in the replication and movement of two viral infection key links. The main results are as follows: 1, the molecular mechanism of viral replication by the host protein complex 6K1-6K2-PsbO1: the first 6kDa protein (6K1) package contains a conservative RSD motif. The alanine scanning experiment proved that The changes in any amino acid in the sequence reduced the replication level of TVBMV replication. The subcellular localization experiment showed that TVBMV 6K1 was distributed in the whole cell in a single transient expression. However, the chloroplasts were recruited during the virus infection process and were associated with the TVBMV replication related second 6 kDa egg white (6K2) and the replicating enzyme nucleus endosomal B to co localize.6K1. It can interact with 6K2 in the body and be recruited by 6K2 to the replication complex of the virus in the chloroplast. Immunoprecipitation and mass spectrometry identification show that the II oxygen complex protein (Photosystem II oxygen evolution complex protein of Nicotiana benthamiana) exists on the complex. Protein interaction but not interacted with 6K1. Using virus induced gene silencing to reduce the expression of NbPsbO1 gene inhibits the accumulation of genomic RNA and shell protein of potato Y virus TVBMV and potato Y virus, but does not affect the accumulation of genomic RNA and shell protein of potato X virus in potato X. The other two components of the II oxygen complex, NbPsbP1 and NbPsbQ1, cannot interact with 6K2, and the silence of NbPsbP1 and NbPsbQ1 genes does not affect TVBMV replicating.2, and the host protein complex P3N-PIPO-CI-NbDREPP regulates the mechanism of intercellular movement of TVBMV: TVBMV PIPO domain consists of 60 amino acid sites. 7, 20, or 43 contain The TVBMV mutant of the PIPO domain of the amino acid lost the intercellular movement and the ability to infect the system. However, the replication level of these mutants is the same as that of the wild type TVBMV. The plasma membrane protein (Developmentally regulated plasma membrane protein of N.benthamiana, NbDREPP) can be associated with TVBMV and columnar connotations. Somatic protein CI interacts. Silencing the NbDREPP gene inhibits the intercellular movement of TVBMV and the system infect.NbDREPP itself on the cell wall, can co localize with the cytoplasmin PDLP1, and can co locate with P3N-PIPO and CI in the intercellular plasmods. The early secretory pathway inhibitor Brefeldin A treatment or transient overexpression of dominant negative inhibition mutation ADP ribonucleic factor 1 (ADP-ribosylation factor1) -T31N or secretory related Ras superfamily associated gene 1B (Secretion-associated and RAS superfamily-related gene1b) -H74L both destroyed the localization of intercellular hyfiles. The domain also disrupted the NbDREPP intercellular localization.3. The transcriptional changes of the wild type TVBMV and the weakly toxic mutants revealed by a new generation sequencing technology revealed the mechanism of the TVBMV's symptom formation. The auxiliary component protease (helper component proteinase, HCpro) is a RNA silencing suppressor of TVBMV in RNA. The mutant pCamTVBMV-GFP-HCproD198K+DE250-251 (pCamT-HCm) was obtained by the introduction of mutation in the TVBMV infected clone pCamTVBMV-GFP (pCamT-WT) HCpro coding region. The changes of these amino acids changed the RNA silencing inhibitory activity of HCpro and reduced the symptoms of T-HCm on the tobacco. We compared T-WT and T-HCm infection through the sequence of transcriptional sequences. Gene expression changes in 1 days, 2 days and 10 days after 1 days. The genes related to protein translation were up-regulated at 1 and 2 days after T-WT or T-HCm infection. The genes related to lipid metabolism and intracellular stimulation were down regulated. The infection of T-WT inhibited the expression of photosynthetic related genes on the 10 day after the virus inoculation, and the infection of T-HCm The genes related to photosynthetic synthesis had no obvious effect on the infection of.T-WT, the key genes of RNA silencing related pathways, DCL2, DCL4, RDR1 and AGO1, were up-regulated, while T-HCm infection on these genes had no effect on the infection of.T-WT and related genes in salicylic acid and ethylene signal pathways, but related genes for jasmonic acid signaling pathways. The differential expression of.T-WT and T-HCm regulates the expression of related genes in the auxin signal transduction pathway, which is related to the dwarf symptoms caused by TVBMV. To sum up, TVBMV hijacking host factor NbDREPP and NbPsbO1 are used for intercellular migration and replication, and DREPP and PsbO1 can be interfered with the infection of.TVBMV of the new target for anti TVBMV. The Calvin cycle, chlorophyll metabolism and auxin signal transduction pathways cause the plants to produce chlorosis and dwarfing symptoms.
【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S432.41

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