蚕豆萎焉病毒2感染豌豆叶细胞后引起的线粒体增生和聚集

应用超薄切片和免疫金标记电镜技术,结合体视学分析研究了受蚕豆萎焉病毒2(BBWV 2) 中国分离物B935侵染的豌豆(Pisum sativum)叶细胞中线粒体的异常变化。结果表明,感病细胞线粒体增生并聚集于细胞质的膜增生区周围,体积增大,形状畸变,一些线粒体内含有类结晶包涵体。病叶细胞与健康对照之间线粒体的体积密度(VV)、表面积密度(SV)、数密度(NV)等参数存在显著差异(P<0.01),而形状因子(PE)、周长指数(CI)、比表面积(RSV)等参数随不同病变阶段而有变化。在线粒体周围及线粒体之间的网格结构可被BBWV 2金标记抗体特异性标记．推断为正在组装的病毒粒子。子代病毒形成结晶体和管状体,有高密度的免疫金颗粒标记。上述研究结果提示BBWV 2 引起的细胞线粒体异常变化与病毒复制组装有关,聚集线粒体的外膜粘连面可能是病毒粒子组装部位,一些线粒体内的类结晶包涵体可能代表了某种蛋白质异常积累。     

PVY侵染后烟草营养成分的变化及其对介体烟蚜生长发育的影响

【目的】明确马铃薯Y病毒(potato virus Y,PVY)侵染后诱导的烟草营养成分的变化及其对烟蚜Myzus persicae生命特性的影响,旨在进一步解析PVY-烟草-烟蚜三者间的互作机制。【方法】通过蒽酮比色法和氨基酸自动分析仪测定了PVY不同侵染时期烟株体内的可溶性糖和游离氨基酸含量的变化;测定和比较了感病与健康烟草植株上烟蚜种群生长发育、成虫寿命、繁殖力和有翅蚜产生量的差异性。【结果】PVY侵染前、中、后期(分别为侵染后5,12和20 d)的烟草叶片中游离氨基酸的总量均显著高于健康烟草叶片。相较于健康烟草叶片,在PVY侵染前期的烟草叶片中,谷氨酸、脯氨酸、天冬氨酸、色氨酸、缬氨酸、赖氨酸和组氨酸的含量显著增加;PVY侵染中期,感病叶片中丝氨酸含量显著下降,谷氨酸、天冬氨酸、色氨酸、缬氨酸、亮氨酸、苯丙氨酸、精氨酸和组氨酸含量显著提高;PVY侵染后期,感病叶片中甘氨酸含量显著下降,谷氨酸、脯氨酸、天冬氨酸、苏氨酸、缬氨酸、亮氨酸、丙氨酸、苯丙氨酸、组氨酸、酪氨酸和精氨酸含量显著提高。在PVY侵染的前期和中期,感病叶片中的可溶性糖含量显著高于健康烟叶,而在侵染后期感病叶片中可溶性糖含量显著低于健康烟草叶片的。PVY侵染前期和中期的烟草叶片中总糖和总游离氨基酸的含量比值显著高于健康烟草叶片中的。在PVY侵染的烟草植株和健康烟草植株上取食的烟蚜其发育历期、若蚜历期、成蚜繁殖期、繁殖后期、寿命、烟蚜种群的内禀增长率、周限增长率和平均世代周期均无显著差异,但在感病烟草植株上取食的烟蚜成蚜繁殖前期显著缩短,其繁殖力和净生殖率显著提高。相较于健康烟草植株,在PVY侵染烟草植株上定殖的烟蚜种群有翅蚜发生的高峰期提前。【结论】PVY侵染前期和中期提高了寄主烟草的营养品质,从而提高了烟蚜的繁殖力。侵染后期烟草营养品质的下降,促使烟蚜种群有翅蚜的产生和扩散,从而有利于PVY自身的传播。     

蚕豆萎焉病毒2感染豌豆叶细胞后引起的线粒体增生和聚集

应用超薄切片和免疫金标记电镜技术,结合体视学分析研究了受蚕豆萎焉病毒2（BBWV2）中国分离物B935侵染的豌豆（Pisumsativum）叶细胞中线粒体的异常变化。结果表明,感病细胞线粒体增生并聚集于细胞质的膜增生区周围。体积增大,形状畸变,一些线粒体内含有类结晶包涵体。病叶细胞与健康对照之间线粒体的体积密度（Vv）、表面积密度（Sv）、数密度（Nv）等参数存在显著差异（P〈0．01）,而形状因子（PE）、周长指数（CI）、比表面积（Rsv）等参数随不同病变阶段而有变化。在线粒体周围及线粒体之间的网格结构可被BBWV2金标记抗体特异性标记。推断为正在组装的病毒粒子。子代病毒形成结晶体和管状体,有高密度的免疫金颗粒标记。上述研究结果提示BBWV2引起的细胞线粒体异常变化与病毒复制组装有关。聚集线粒体的外膜粘连面可能是病毒粒子组装部位。一些线粒体内的类结晶包涵体可能代表了某种蛋白质异常积累。     

一种基于过敏性反应机制的抗植物病毒侵染策略

基于植物的过敏性反应机制,构建了PVY Nib基因和来自于细菌Bacillus amy—loliquefaciens的一类Rnase基因Barnase基因的融合基因的植物表达载体。在此表达载体内两基因的拼接处,保留了原来PVY蛋白酶识别PVYNIb和CP蛋白剪切位点的七肽保守序列。通过农杆菌介导获得此融台基因的转基因烟草植株。病毒侵染试验表明,转基因植物在病毒侵染后,发病症状被改变。少部分转融合基因的植株对病毒侵染表现局部抗性。     

PVY和PVS病毒复合侵染对马铃薯光合及营养品质的影响

以2个马铃薯高代品系为实验材料,研究马铃薯病毒Y(PVY)和S(PVS)复合侵染对马铃薯叶片SPAD值、最大净光合速率、表观量子效率、气孔导度、暗呼吸速率、蒸腾速率、光补偿点、胞间CO_2浓度和饱和光强等光合参数,以及马铃薯块茎产量、干物含量、淀粉含量、还原糖含量、粗蛋白含量等产量品质指标的影响。结果表明:(1)田间受到PVY和PVS病毒复合侵染后,马铃薯品系2010-11植株的叶片PVS病毒含量显著低于品系34-2植株,2010-11植株的叶片PVY病毒含量同样低于品系34-2植株,但差异不显著。(2)与对照相比,感病后马铃薯品系34-2和2010-11的平均单株产量分别显著降低48.54%和19.98%,淀粉含量分别降低8.06%和3.38%,但不显著;粗蛋白含量分别显著升高14.05%和29.17%;还原糖含量分别显著降低11.11%和14.29%;34-2干物量含量显著降低6.9%,2010-11干物质含量略降低0.66%。(3)两品系感病植株的光合参数SPAD值分别显著降低13.37%、20.10%;最大净光合速率分别显著降低32.48%和4.54%;其它光合参数变化没有规律性。研究发现,PVY和PVS病毒混合侵染使马铃薯植株叶片叶绿素含量显著降低,进而影响光合作用的正常进行;病毒侵染使块茎中还原糖和淀粉的积累显著受到抑制,但能够促进块茎中粗蛋白的含量显著提高。     

烟草NtWRKY40在植物应答病毒侵染过程中的作用

WRKY转录因子家族在植物的抗病、抗逆反应中具有重要功能。已有研究表明,烟草花叶病毒(TMV)的侵染显著地诱导烟草Nt WRKY的表达,有必要进一步探明该基因在植物应答病毒侵染过程中的作用。采用PCR的方法克隆获得Nt WRKY cDNA,生物信息学分析结果显示,该基因属于WRKYⅡa亚族成员,与绒毛状烟草NtoWRKY40高度同源,命名为NtWRKY40。以此建立了过表达该基因的转基因烟草,并以TMV为毒源进行了转基因烟草和野生烟草的侵染实验,以观察NtWRKY40在烟草应答病毒侵染过程中的作用。实验结果表明,野生烟草在TMV侵染后9 d,NtWRKY40的表达量显著升高,而NtWRKY40过表达转基因烟草在病毒侵染后,病毒相关基因的表达高于野生型对照,与染病程度成正相关,说明过表达NtWRKY40增加了植株对病毒的敏感性,该基因为负调控因子。此外,为探索应用人工miRNA的抗病毒技术,以烟草天然miR167前体为骨架、马铃薯Y病毒(PVY)外壳蛋白基因的一段反向互补序列为成熟序列,构建了amiR167-PVY植物表达载体并转化烟草,以抑制PVY。对amiRNA转基因植株进行抗病毒实验的结果显示,amiR167-PVY能够部分抑制病毒基因的表达,转基因植株具有一定的抗病毒能力。     

马铃薯Y病毒蚜传辅助因子促进马铃薯X病毒长距离运输

采用PCR和定点突变法,对马铃薯Y病毒中国株系（Chyinese strain of potato Ypotyvirus,PVY-C)蚜传辅助成分（helper component proteinase,HC-Pro)基因中心区域的CCCT基序和PTK基序进行定点改造,获得了4种突变体。然后将突变体砍降到植物表达载体pBin438中,所得到的重组体通过根癌土壤杆菌（Agrobacterium tumefaciens(Smith et Townsend)Conn）介导法转了烟草（Nicotiana tabacum L.cv.K326).Southern blotting和Western blotting分析表明4种突变体已经成功整合到烟草的基因组中,并在蛋白水平上得到了表达。马铃薯X病毒（potato X potexvirus,PVX)对转基因烟草的攻毒实验表明,4种突变体均使PVY－C HYC－Prog严重丧失了促进PVX病毒粒子在寄主体内积累和提高PVX致病性的功能,说明CCCT、PTK基序为PVY－C HYC－Pro介导PVX／PVY协生作用所必需。同时证明了HC-Pro具有增强PVX在寄主体内长距离运输的功能。     

感染大丽菊花叶病毒的大丽菊细胞超微结构观察

本文描述了大丽菊花叶病毒(DaMV)感染大丽菊叶片细胞的超微结构变化。受侵细胞内形成切面直径约1.6—3.1μm的球状或卵球状缅胞贡内含体。病株细胞内过氧物酶体的数量显著增加。内含体周围较细胞其它部位有较多的核糖体。高尔基体及微泡。叶绿体,线粒体正常。     

马铃薯Y病毒HC-Pro中心区域在病毒协生作用中的主导地位

利用PCR方法获得了马铃薯病毒中国株系（PVY－C）HC－Pro基因的5个缺失突变体,构建了相应的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens)介导法转化了烟草品种K326(Nicotina tabacum cv.k326)。PCR和Southern blot分析证明了HC－Pro基因及其缺失突变体已整合到烟草基因组中,Western blot表明它们在转基因烟草中得到了表达。侵染性试验发现HC－Pro中心区域介导转基因烟草中PVC－C和黄瓜花叶病毒（CMV）、PVY－C和马铃薯X病毒（PVX）之间的协生作用,从而明确了PVY－C HC－Pro中心区域为病毒协生作用的功能区域。     

马铃薯Y病毒N株HC-pro基因的序列分析与表达

马铃薯Ｙ病毒(Potato virus Y,PVY)是马铃薯Ｙ病毒属(Potyvirus)的典型成员,是烟草、马铃薯和辣椒等茄科作物病毒病的主要病原之一,分布于世界各地.近年烟草上的 PVY在全国范围内特别是陕西、黄淮和东北烟区流行呈上升趋势,且以PVY坏死株系（PVY-N)为主,重病田发生率达40%以上,并常与烟草花叶病毒、黄瓜花叶病毒混合侵染,严重影响烟草品质造成巨大的经济损失.     

Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.     

Systemic Infection of Tobacco by Pepper Veinal Mottle Potyvirus (PVMV) Depends on the Presence of Potato Virus Y (PVY)

In single inoculations, both PVY and PVMV replicated in inoculated leaves of Nicotiana tabacum cv. ‘Xanthi nc’ plants, but only PVY infected the tobacco plants systemically, whereas PVMV caused localized infection. A mixed infection by the PVY-To72 and PVMV-type strains was experimentally realized in ‘Xanthi nc’ plants. In the presence of PVY, PVMV migrated systemically into the upper leaves of the tobacco plant, as was proved by back inoculation. It would appear that in tobacco, PVY acts as a “helper” virus, providing PVMV with the necessary component factor for migration. In extracts from the co–infected leaves. Immune Electron Microscopy (IEM) revealed phenotypic mixed particles which contained a mixture of coat proteins of PVY and PVMV. The role of the structural and functional interactions between the two viruses, which enable PVMV to migrate systemically in tobacco plants, is discussed.     

Interactions between pepper ringspot virus and cylindrical inclusions of two potyviruses

Immunoelectron microscopy showed that cylindrical inclusions (CI) of the potyvirus potato virus Y (PVY) bound in addition to their homologous virions those of a co-infecting rod-shaped virus, pepper ringspot virus (PRV), in infected Nicotiana benthamiana leaf cells. The latter virus does not code for cylindrical inclusions and is cl assified as a Tobravirus. Virions of PRV were scattered throughout the cell cytoplasm and not associated with mitochondria in PVY + PRV double infections. Binding of PRV to mitochondria was disrupted in PVY + PRV infected cells. In double infections with a second potyvirus, tobacco etch virus (TEV), and PRV in N. benthamiana cells, TEV-CI bound homologous TEV virions but did not bind PRV. In contrast to PVY + PRV infections, virions of PRV attached end-on to mitochondrial limiting membranes in PRV-only and in TEV + PRV double infections. The results are interpreted to mean that there are differences in the PRV virion binding sites of PVY-CI and TEV-CI. In previous reports, potyviral CI have been nondiscriminating in binding virions or capsid proteins of other co-infecting rod-shaped viruses.     

The participation of plant cell organelles in compatible and incompatible potato virus Y-tobacco and -potato plant interaction

Potyviruses replicate and express their genomes in the cytoplasm in closely related membranous structures such as the endoplasmic reticulum or in the vicinity of the ER. The present research demonstrates the participation of plant cell organelles based on ultrastructural examination of compatible and incompatible interactions in tobacco- and potato-potato virus Y (PVY) necrotic strains. In two interaction types, PVY^N Wi and PVY^NTN particles were documented inside cell nuclei. Virus cytoplasmic inclusions and particles were associated with nuclear envelope pore complexes. Moreover, the PVY capsid protein was immunolocalised in the cell nucleus and nucleolus. Our results for the first time show PVY particles and capsid proteins inside the mitochondrion in compatible interactions, whereas in hypersensitive responses these interactions were identified inside chloroplasts. The PVY particles attached to mitochondria caused association groups of these organelles. The ultrastructural analysis clearly demonstrated both the dynamics of the endoplasmatic reticulum in two types of PVY interactions and connections between PVY cytoplasmic inclusions and particles with its membranous structures. Moreover, we demonstrated strongly localised immunodetection of the PVY capsid protein on the surface and in the vicinity of ER in cases of hypersensitive response as well as in compatible interaction.     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

Cytopathology of satellite tobacco mosaic virus and its helper virus in tobacco

Ultrastructural responses of tobacco cells infected with a newly discovered satellite virus (STMV) that has an isometric morphology and is associated with rigid rodshaped tobacco mosaic virus (TMV) were studied in situ. In cells infected with TMV alone,TMV particles occurred as crystalline arrays in the cytoplasm and were usually associated with TMV-characteristic X bodies. In cells infected with both TMV and STMV, particles of STMV occurred only in cells that contained TMV particles, which suggests a correlation between the satellite and helper virus presence. However, the replication and/or accumulation sites of STMV appear to be independent from its helper virus. Unlike TMV particles, STMV particles were associated with several cytopathic structures such as granular inclusions, membranous vesicles of 50–80 nm, and myelin-like bodies which were all bounded by a single common membrane, No X bodies occurred in cells containing STMV particles, and the mitochondria possessed abnormal tubular structures containing flocculent material.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

EXPERIMENTS BEARING ON THE NATURE OF INTRACELLULAR INCLUSIONS IN PLANT VIRUS DISEASES

The intracellular changes resultant on infection with aucuba mosaic and Hy. III diseases are described and are compared with the cytological effects of tobacco mosaic virus. With the two former viruses, inclusion bodies are formed by the aggregation and fusion of minute particles which appear in the cytoplasmic stream. With tobacco mosaic disease an amoeba-like body is produced and this persists for some weeks before suddenly disappearing again. It is accompanied by striate material all of which ultimately fuses into one large body.
Attempts have been made to parallel these conditions in healthy cells of Solanaceous plants by treatment with substances known to coagulate protoplasm. Almost all the reagents used induced stimulation of the cytoplasmic stream similar to the initial sign of virus infection. With salts of molybdic acid, all the cytological abnormalities due to aucuba mosaic or Hy. III disease have been imitated. Treatment with lactic acid induces the formation of amoeboid bodies like the X-bodies of tobacco mosaic, but these bodies persist for only a few hours.
Attempts have also been made to inhibit the formation of inclusion bodies induced by several different diseases in a number of hosts but no success was obtained.
The experiments support the view that the intracellular inclusions of plant virus diseases are essentially products of the host cell.     

Ultrastructural events during hypersensitive response of potato cv. Rywal infected with necrotic strains of potato virus Y

Potato plants cv. Rywal with hypersensitivity gene Ny-1 infected with PVY^N or PVY^NTN reacted in local necroses 3 days after infection. Potato virus Y (PVY) particles were found in epidermis, mesophyll, phloem and xylem cells in inoculated leaves. Noncapsidated virus particles (without capsid protein) were observed already 10 h after infection by using electron microscopy in situ. Capsid protein on one terminus of noncapsidated virus particles was located 5 days after inoculation with the use of immunogold labeling method. Whereas cytoplasmic inclusions were observed for the first time 24 days after infection during hypersensitive response. Ultrastructural studies showed that ER may take part in PVY RNA replication and capsidation of Potyvirus particles. Observed cytopathological changes and virus particles indicate that cell nucleus and mitochondrion might participate in PVY life cycle. During hypersensitive response PVY particles were found in plasmodesmata as well as in phloem and xylem.