【作者】 郑红英；

【导师】 侯明生； 陈剑平；

【作者基本信息】 华中农业大学 ， 植物病理学， 2011， 博士

【摘要】 芜菁花叶病毒(Turnip mosaic virus, TuMV)是马铃薯Y病毒属(genus Potyvirus)的确定成员,其分离物UK1能侵染拟南芥导致叶片黄化、植株矮化,侵染本氏烟引起严重的花叶、畸形等症状。TuMV-UK1编码的HC-Pro蛋白是症状决定因子,同时也是基因沉默的抑制子。为了更好的了解TuMV编码HC-Pro蛋白的功能及其在寄主上的致病机理,本研究对该蛋白的自身互作及其与拟南芥寄主因子的互作进行了深入研究。已报道马铃薯Y病毒属成员编码的HC-Pro蛋白自身可以发生相互作用,但采用酵母双杂交系统研究报道的不同病毒参与HC-Pro自身相互作用的关键功能区域并不一致。根据前人已报道的HC-Pro蛋白功能区,结合TuMV-UK1分离物的氨基酸序列,构建5个HC-Pro基因缺失突变体,对HC-Pro蛋白自身互作的功能区进行了分析。采用酵母双杂交及双分子荧光互补技术检测了HC-Pro蛋白及其突变体在酵母、本氏烟及昆虫细胞中的相互作用和共定位。研究表明TuMV HC-Pro蛋白中间段和C-末端是参与互作的区域,且C-末端258个氨基酸与HC-Pro完整蛋白的相互作用最强,而HC-Pro蛋白N-末端与蚜传相关的99个氨基酸不参与或仅有微弱的自身相互作用。双分子荧光互补实验在烟草细胞和昆虫细胞细胞质中都检测到不规则的黄色荧光聚集体,进一步证实了HC-Pro蛋白存在相互作用。这些研究还表明HC-Pro以形成二聚体或多聚体的形式行使其功能。进一步对TuMV-UK1编码的HC-Pro蛋白在烟草表皮细胞、原生质体及昆虫细胞中进行定位,荧光定位结果表明在烟草细胞质中检测到绿色荧光,且在内质网上有聚集的小点和聚集块,说明这些部位都有HC-Pro蛋白的存在,HC-Pro的大量积累可能会破坏正常内质网的结构。在昆虫细胞中表达也显示HC-Pro的定位主要分布在细胞质中。在前期以HC-Pro蛋白为诱饵,利用酵母双杂交从拟南芥cDNA文库中筛选与HC-Pro互作的寄主因子的基础上,对HC-Pro蛋白与筛选的寄主铁硫蛋白(RieskeFe/S)的相互作用作进一步研究。序列分析表明,Rieske Fe/S蛋白具有一个跨膜结构,成熟蛋白N-端少数氨基酸呈松散的排列,而C-端序列的氨基酸则呈相对有序的卷曲的紧密结构。酵母双杂交及BiFC都证实了HC-Pro蛋白与Rieske Fe/S蛋白的相互作用。通过酵母双杂交对HC-Pro蛋白与Rieske Fe/S全长蛋白及其互作的关键功能区进行鉴定,结果表明参与完整HC-Pro蛋白相互作用的是Rieske Fe/S蛋白的成熟蛋白,Rieske Fe/S蛋白N-末端的信号肽区域并不参与与HC-Pro蛋白相互作用,而与成熟的Rieske Fe/S蛋白互作的是HC-Pro蛋白中间段和C-末端功能区,HC-Pro蛋白N-末端与蚜传相关的功能区并不参与或仅与Rieske Fe/S蛋白有微弱的自身相互作用。BiFC的结果显示在本氏烟叶片细胞中HC-Pro蛋白与Rieske Fe/S蛋白相互作用的位点主要分布在细胞膜、细胞质等区域,呈不规则片状聚集。为了明确HC-Pro蛋白的表达是否影响Rieske Fe/S蛋白的细胞定位,对RieskeFe/S蛋白的定位及其与HC-Pro蛋白的共定位进行了研究。对Rieske Fe/S蛋白的定位研究表明该蛋白主要分布在烟草表皮细胞膜上,细胞质中,部分形成大的聚集体,最典型的是其在叶绿体边缘形成点状或块状的聚集。HC-Pro蛋白与Rieske Fe/S蛋白的共表达增强了Rieske Fe/S蛋白的表达量,但是对Rieske Fe/S蛋白的细胞定位改变并没有显著影响。这可能与HC-Pro蛋白具有抑制子功能,能增强外源基因的表达有关。为了研究Rieske Fe/S蛋白在寄主植物中的功能,对编码Rieske Fe/S蛋白的Pet C基因采用烟草脆裂病毒(Tobacco rattle virus, TRV)和马铃薯X病毒(Potato virusⅩ,PVX)介导其基因沉默,在拟南芥和烟草上都观察到相似的褪绿,黄化、矮化,植株长势弱,发育迟缓等现象。叶绿素荧光动力学参数的测定表明Pet C基因沉默的拟南芥与仅受TRV侵染的健康拟南芥叶片相比其Fo值升高,而Fv/Fm降低,说明该基因沉默后,叶片叶绿素含量降低。半定量检测结果显示基因沉默的拟南芥植株中PetC基因的mRNA量明显减少,转录水平降低约在30%-70%左右。而TuMV-UK1侵染拟南芥后常表现叶片黄化、花叶,植株矮化等症状,症状的出现可能与细胞内叶绿体的稳定性下降、结构破坏、叶绿素含量下降等因素相关。推测HC-Pro蛋白与Rieske Fe/S蛋白的相互作用可能阻断了Rieske Fe/S蛋白前体从细胞质向叶绿体的转运过程,从而降低了叶绿体内Rieske Fe/S蛋白的积累量,干扰了细胞色素b6/f复合体的正常组装,是导致TuMV侵染后寄主发生花叶、褪绿等症状的原因之一。更多还原

【Abstract】 Turnip mosaic virus (TuMV) is a definite member in the genus Potyvirus. TuMV naturally infects the plants belong to the Brassica family, but it can also infect and induce symptoms on Arabidopsis thaliana and Nicotiana benthamiana, which are now commonly used as the model system for study on the host-pathogen interaction. It has been reported that helper component-proteinase (HC-Pro) encoded by potyviruses is an important viral symptom determinant and a suppressor of RNA silencing. Previous studies had showed that HC-Pro can form dimer in-vitro and in infected plants, moreover the interaction of HC-Pro with plant cellular factors also had been reported. In this study, to gain better understanding on the molecular basis of HC-Pro function in the viral pathogencity, the interactions of TuMV (isolates UKl)-encoded HC-Pro with itself and an Arabidopsis host factor were investigated.Although there have been many studies reported the ability of HC-Pro to self interact, the yeast two hybrid (YTH) assay used to determine the HC-Pro domain required for its self-interaction showed somewhat conflicting results. Based on the previously reported mutagenesis studies on HC-Pro and sequence alignments of various HC-Pro of Potyviruses, five deletion mutants of HC-Pro were constructed and used to analysis the functional domain involved in its self-interaction. YTH and bimolecular fluorescence complementation (BiFC) assays were used to detect the self-interaction ability of HC-Pro and its deletion mutant derivatives. YTH and BiFC assays confirmed TuMV HC-Pro self interaction in yeast cells and plant cells or insect cells, respectively. Using YTH analysis, the central and C-terminal regions of HC-Pro were shown to be participated in the self-interactions, while in BiFC assay, all deletion mutants interacted with HC-Pro, including the N- terminal regions that did not show any interaction with HC-Pro in the YTH assay. The subcellular localization of TuMV HC-Pro was also studied by fluorescent fusion protein and was observed to be present in the cytoplasm and to form aggregates along the endoplasmic reticulum (ER). The over-expression of HC-Pro caused the morphological changes of the ER, which suggested that HC-Pro is associated with the ER.The previous work of our laboratory to screen for the potential host protein interactors with HC-Pro using the A. thaliana cDNA library has obtained several candidates protein interactors. One cDNA clone designated Pet C, which encodes the Rieske Fe/S protein was selected for further analysis. The sequence analysis of Rieske Fe/S protein suggested that it contains a single transmembrane helix, the N-terminal of mature protein includes a short loose amino acid sequence and the C-terminal hydrophilic domain provides the ligands for the [2 Fe-2S] cluster. Its interaction with HC-Pro was confirmed by using YTH and by BiFC in plant cells. Two truncated mutants of Pet C were then generated to determine the region required for binding of Rieske Fe/S protein to HC-Pro. The result showed that the mature protein of Rieske Fe/S protein ((56-229aa) is the binding site for HC-Pro, whereas the transit peptide is dispensable for the interaction with HC-Pro. Both the central (residues 101-300aa) and the C-terminal regions (residues 301-458aa) of HC-Pro can interact with the mature Rieske Fe/S protein, but the N-terminal region (residues 1-100aa), which is related to aphid transmission is dispensable for the interaction.To analysis whether the HC-Pro expression affects the subcellular localization of Rieske Fe/S protein, Rieske Fe/S was fused with GFP and transiently co-expressed with HC-Pro in plants. The observation of protein subcellular localization indicated that Rieske Fe/S:GFP protein accumulates in cytoplasm, partly forms aggregates, and especially punctuate spots and granular bodies around the chloroplast. Co-expression with HC-Pro did not change the subcellular localization of Rieske Fe/S protein significantly. However the expression of HC-Pro can enhance the expression of Rieske Fe/S, this may be because HC-Pro function as a viral silencing suppressor can increase the heterogeneous gene expression in plants.To analysis the function of Rieske Fe/S protein in plant, the cDNA fragment corresponding to Pet C gene of A. thaliana were inserted into Tobacco rattle virus (TRV) and Potato virus X (PVX) vectors and inoculated into Arabidopsis and N. benthamiana for virus-induced gene silencing (VIGS). The upper leaves of Arabidopsis and N. benthamiana systemically infected with TRV-Pet C or PVX-Pet C exhibited severe yellowing symptom. The plants become stunted and a substantial reduced of growth rate compared with TRV-or PVX-infected plants were observed. The analysis of chlorophyll fluorescence parameters revealed that the Fo values were increased, whereas the Fv/Fm values were decreased in TRV-Pet C-infected plants as compared with TRV-infected plants. Semi-quantitative RT-PCR result showed that the level of Pet C mRNA decreased about 30%-70% after VIGS. In TuMV-infected Arabidopsis, yellow mosaic and stunting are the most common symptoms, which maybe a consequence of the perturbation of the chloroplast structure and the degradation of chloroplast pigment. It is possible that the interaction of HC-Pro and Rieske Fe/S protein interferes with the import of the Rieske Fe/S protein into the chloroplast, so that the amount of Rieske Fe/S protein required for assembly of photosynthetic cytochrome b6/f complex could eventually be reduced, and in turn contribute to development of yellow mosaic symptom.更多还原