烟草花叶病毒和番茄花叶病毒在含N基因烟草上的症状差异是由运动蛋白基因决定的

烟草花叶病毒(TMV)和番茄花叶病毒(ToMV)是烟草花叶病毒属中关系最为密切的病毒, 但它们在含N基因烟草上产生的枯斑大小有明显的差异. 比较了TMV, ToMV及用ToMV运动蛋白基因(MP)精确置换TMV MP后获得的重组病毒T/OMP在不同寄主上的症状差异, 发现T/OMP在含N基因烟草上产生的枯斑大小与ToMV相似. 分析比较TMV, ToMV和T/OMP外壳蛋白和MP在植物体内的积累水平, 发现三者之间没有明显的差异, 而TMV和T/OMP在原生质体中的复制水平也没有差异. 比较TMV, ToMV和T/OMP接种后烟草体内防御相关酶(PAL, POD和PPO)的活性变化, 结果T/OMP和TMV所诱导酶的变化趋势基本一致, 而与ToMV有所差异, 因此认为MP基因功能的差异决定了TMV和ToMV在N基因烟草上枯斑的大小.     

番茄花叶病毒移动蛋白和番茄抗病基因Tm-2^2的互作诱导烟草转化体程序性细胞死亡

番茄的抗病基因Tm -2 2 与番茄花叶病毒 (ToMV)的移动蛋白MP基因是一对互作的基因 ,Tm- 2 2 基因和ToMV MP基因同时在烟草中表达 ,并分别获得单一基因整合的纯合转化体植株。病毒接种试验表明 ,Tm -2 2 基因转化体与Tm- 2 2 番茄对Tobamavirus病毒的特异抗性结果一致 ;Tm -2 2 转基因植株和ToMV MP转基因植株杂交试验及其农杆菌注射试验均证明 :(1)Tm -2 2 基因与ToMV- MP在转基因烟草上保持“基因对基因”的互作关系 ;(2 )在外源乙烯的参与下 ,ToMV的移动蛋白与Tm -2 2 基因编码蛋白的互作能够诱导转化体程序性细胞死亡。这一结果为今后研究Tm -2 2 与MP互作的分子机制奠定了基础。     

番茄花叶病毒移动蛋白和番茄抗病基因Tm-22的互作诱导烟草转化体程序性细胞死亡

番茄的抗病基因Tm22与番茄花叶病毒（ToMV）的移动蛋白MP基因是一对互作的基因,Tm22基因和ToMVMP基因同时在烟草中表达, 并分别获得单一基因整合的纯合转化体植株。病毒接种试验表明,Tm22基因转化体与Tm22番茄对Tobamavirus病毒的特异抗性结果一致;Tm22转基因植株和ToMVMP转基因植株杂交试验及其农杆菌注射试验均证明： （1）Tm22基因与ToMVMP在转基因烟草上保持“基因对基因"的互作关系; （2）在外源乙烯的参与下,ToMV的移动蛋白与Tm22基因编码蛋白的互作能够诱导转化体程序性细胞死亡。这一结果为今后研究Tm22与MP互作的分子机制奠定了基础。     

番茄花叶病毒单克隆抗体的制备及检测应用

用番茄花叶病毒(ToMV)免疫的BAL B/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代并分泌抗ToMV单克隆抗体(Mab)的杂交瘤细胞,其中2株能同时检测ToMV和烟草花叶病毒(TMV),各单克隆抗体腹水ELLSA效价在1∶32 000～1∶1 024 000之间。经TASELISA测定,4株单克隆抗体检测病汁液的稀释度均能达到1∶2 000倍以上。4株单克隆抗体与其他病毒无交叉反应。Westernblot分析表明,其中两株与ToMV176kD的外壳蛋白亚基有特异反应,而另两株无反应,推测它们是针对构象决定簇的抗体。     

酵母校正tRNA的提取及其对番茄花叶病毒增殖的抑制作用

从酵母(Saccharamyces cerevisiae SUP-6-1温度突变株,含SUPt RNATyr,SUFtRNAleo,SUPtRNAHis)和菠菜提取了总tRNA,经过BD-纤维素柱层析,得到部份纯化的amber终止密码校正tRNA,它们在兔网织细胞裂解液翻译体系中能促进番茄花叶病毒(ToMV)RNA183k抄读蛋白的合成。研究了酵母SUP6-l校正tRNA对ToMV在烟草中增殖的影响。在用酵母SUP6-l校正tRNA处理的植株顶部新生叶中病毒滴度接种3、5、11、15和20天后,分别是对照的3％、12％、3 8％、42％和67％。用校正tRNA处理的烟草植株中层叶片,在接种后11—20天明显低于对照。下层接种叶中的病毒滴度无显著差异。讨论了校正tRNA对ToMV增殖抑制的机理。     

用DNA杂交技术测定烟草花叶病毒与长叶车前花叶病毒的关系

从油菜上分离的属于烟草花叶病毒组的油菜花叶病毒15(YMV15)和从大白菜上分离到的白菜花叶病毒(ccMV)的外壳蛋白中,都含有组氨酸和甲硫氨酸,类似于长叶车前花叶病毒(RMV)。用YMV15一、RMV一和番茄花叶病毒(ToMV)突变体Nc广RNA的cDNA与Ymv_RNA、RMV—RNA和ccMV．RNA进行了同源和异源的分子杂交试验。 从分子杂交的R0t曲线和杂交产物的Tm值看,YMV-,和ccMV都不同于RMV,其饱和杂交率表明,ccMV与YMV,,有一定核苷酸序列的同源性,两者都与RMV没有核苷酸序列同源性,说明它们属于烟花叶病毒组的不同亚组。     

辽宁省番茄花叶病主要病毒和株系的研究

从辽宁南部及沈阳地区采集番茄花叶病样本111个(1985-1987),经鉴定得知主要毒原为番茄的烟草花叶病毒(TMV)占总调查的59.46％。黄爪花叶病毒(CMV)亦有发生仅占总调查的9.00％。还有一些系混合侵染(TMV和CMV)占21.62％。尚未认清毒原种的约占2.70％,接种无症的占7.20％。通过试验认为我省番茄上主要毒原为烟草花叶病毒,并进行了常规鉴定、提纯、电镜观察、光谱测定和血清测定等。对番茄上烟草花叶病毒16个分离物做了株系分化研究,按照Pelbam(1970,1972)〔14、15、16、17〕的抗性分类法应用番茄GCR一套品种接种观察,得知辽南及沈阳地区分布很广的番茄TMV分离物,均属TMV-O株系。故在番茄抗TMV育种中尚应考虑到抗1及2株系的能力。     

温度对番茄花叶病毒(TOMV)增殖及番茄叶片可溶性蛋白变化的影响

局部枯斑法测定结果表明,番茄GCR—267品系叶片内ToMV的含量仅为番茄GCR-26品系叶片内病毒含量的1/40—1/50;GCR-267品系含有的Tm-2~(?)基因对ToMV增殖的抑制作用不受温度变化的影响;而ToMV在GCR-26体内的增殖却依赖于环境温度,高温对它有部分的抑制作用。用非变性聚丙烯酰胺凝胶电泳和SDS—聚丙烯酰胺凝胶电泳分析上述两个品系在常温和高温下接种ToMV后其叶片可溶性蛋白的变化,结果发现在GCR-26品系中,ToMV的增殖与寄主体内新产生的14.2KD蛋白呈正相关,而在GCR—267品系中未检测到这种蛋白。我们推测14.2KD蛋白可能是一种能参与或促进ToMV增殖过程的温度敏感因子,称之为番茄“S”蛋白。     

TMVc的单克隆抗体及其对烟草花叶病毒组成员病毒间的鉴别

用烟草花叶病毒普通株(TMVc)纯病毒免疫BALB／c小鼠,取其脾细胞与小鼠骨髓瘤细胞sP2／0融合,通过2～8次有限稀释法克隆化获得10个能持续地分泌抗体的杂交瘤细胞株。所获抗体均属IgM。其中5个用来诱发高滴度腹水。用间接ELIsA、免疫电镜,中和侵染性试验证明所获单克隆抗体对TMVc有特异性。7A、。9G和N12C单克隆抗体对来自十字花科韵烟草花叶病毒组病毒YMVl5和TMV—B2无交叉反应;而对其它成员病毒如ToMV,ToMV．N14,TMV－L2有不同程度的交叉反应。7A~9G表现较强的异源特异性而12C却有明显的同源特异性。     

烟草花叶病毒运动蛋白cDNA的克隆及融合蛋白的表达

从烟草花叶病毒(TMV)中提取总RNA,通过反转录PCR (RTPCR) 扩增得到其运动蛋白(MP)的基因,将扩增产物克隆到pMD18T载体上。DNA序列分析表明,所得到的运动蛋白的基因全长为807bp (GenBank接受号AY300161), 与已发表TMV序列（GenBank登陆号为NC-001367）和同属的番茄花叶病毒（ToMV, GenBank登陆号为NC-002692相比核苷酸的同源性分别为98.0％和80.9％,氨基酸的同源性分别为99.1％和80.0％。 将目的片段亚克隆到表达载体pET30a上,并在大肠杆菌JM109中诱导表达,诱导9h 后,融合蛋白表达量最大。诱导后的工程菌超声后经SDSPAGE检测,融合蛋白以可溶形式存在。     

Ultrastructural Alteration of Host Plants Infected with Tomato Mosaic Virus

The ultrastructural aheration of two host plants infected with tomato mosaic virus (ToMV) were studies with transmission electron microscopy. A large number of virus particles were found being accumulated in different cells such as epidermis, parenchyma cells and vascular bundle cells of Lycopersicon esculentum Mill. grown at 25℃ Crystalline inclusions and paracrystal inclusions composed of ToMV particles were observed in the cytoplasm or vacuoles. Some muhivesicular bodies and myeloid bodies protming into the vacuole and vires-specific vesicles associated with the tonoplast were also observed. The ultrastructuml alteration of Nicotiana tabacum L. tv. Xanthinn was similar to that in tomato infected by ToMV grown at 25 cE. In addition to the aggregate inclusions described above, some cytoplasmic angularly-layered aggregates and abnormal chloroplasts with small peripheral vesicles were observed in the parenchyma cells. The densely stained amorphous material was seen in the cytoplasm of N. tabacum L. cv. Xanthiun grown at 35℃. No X- body was observed in the cytoplasm of the ToMV infected tomato and tobacco grown at 25℃ or 35℃. The authors' results suggest a significant difference between the cytopathological effects of ToMV and tobacco mosaic virus (TMV). These characteristic difference may be useful in the virus diagnosis and identification virus infections in plants.     

Two amino acid substitutions in the tomato mosaic virus 30-kilodalton movement protein confer the ability to overcome the Tm-2(2) resistance gene in the tomato.

The Tm-2(2) resistance gene is used in most commercial tomato cultivars for protection against infection with tobacco mosaic virus and its close relative tomato mosaic virus (ToMV). To study the mechanism of this resistance gene, cDNA clones encompassing the complete genome of a ToMV strain (ToMV-2(2)) that was able to break the Tm-2(2) resistance were generated. Chimeric full-length viral cDNA clones were constructed under the control of the cauliflower mosaic virus 35S RNA promoter, combining parts of the wild-type virus and ToMV-2(2). Using these clones in cDNA infection experiments, we showed that the 30-kDa movement protein of ToMV-2(2) is responsible for overcoming the Tm-2(2) resistance gene in the tomato. DNA sequence analysis revealed four amino acid exchanges between the 30-kDa proteins from wild-type ToMV and ToMV-2(2), Lys-130 to Glu, Gly-184 to Glu, Ser-238 to Arg, and Lys-244 to Glu. To clarify the involvement of the altered amino acid residues in the resistance-breaking properties of the ToMV-2(2) movement protein, different combinations of these amino acid exchanges were introduced in the genome of wild-type ToMV. Only one mutant strain which contained two amino acid substitutions, Arg-238 and Glu-244, was able to multiply in Tm-2(2) tomato plants. Both amino acid exchanges are found within the carboxy-terminal region of the movement protein, which displays a high variability among different tobamoviruses and has been shown to be dispensable for virus transport in tobacco plants. These observations suggest that the resistance conferred by the Tm-2(2) gene against ToMV depends on specific recognition events in this host-pathogen interaction rather than interfering with fundamental functions of the 30-kDa protein.     

Identification of a ribonucleoprotein intermediate of tomato mosaic virus RNA replication complex formation

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.     

Occurrence, Distribution and Relative Importance of Viruses Infecting Hot Pepper and Tomato in the Major Growing Areas of Ethiopia

Hot pepper and tomato fields in the main growing areas in the Rift Valley and the west of Ethiopia were surveyed for virus infections in 1994. A total of 286 samples from hot pepper and 222 samples from tomato plants and associated Datura stramonium L. and Nicandra physalodes Gaertn. weeds with symptoms suggestive of virus infections were collected and analysed using electron microscopy, serology and test plant reactions. Potato virus Y (PVY), Ethiopian pepper mottle virus (EPMV), pepper veinal mottle virus (PVMV) and tomato mosaic virus (ToMV) were detected in hot pepper samples while tomato samples were shown to be infected with tomato mild mottle virus (TMMV), PVY and ToMV. The most widespread and predominant viruses which also occurred frequently in mixed infections were PVY and EPMV in hot pepper and PVY and TMMV in tomato. TMMV was also found in many samples of D. stramonium and N. physalodes. ToMV was identified in only few samples from both crops in the Rift Valley by its characteristic particle morphology, serological properties and symptomatology. PVMV was found in hot pepper samples only from western Ethiopia, but no natural infection of tomato with this virus was revealed. This is the first report on the natural occurrence of TMMV in tomato, D. stramonium and N. physalodes, as well as of ToMV in hot pepper and tomato in Ethiopia.     

The Resistance Protein Tm-1 Inhibits Formation of a Tomato Mosaic Virus Replication Protein-Host Membrane Protein Complex

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3′ untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis.     

Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.     

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR

Aims: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. Methods and Results: A multiplex RT–PCR method consisting of one‐tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT–PCR detected up to 10^?6 dilution of total RNA extracted from infected leaves. Multiplex RT–PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. Conclusions: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. Significance and Impact of the Study: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.