A new aubergine disease caused by a whitefly‐borne strain of Tomato mild mottle virus (TomMMoV)

The aims of the present study were to further characterize the causal agent of a new viral disease of aubergines in Israel, first observed in 2003 and tentatively named eggplant mild leaf mottle virus (EMLMV) in a previous work, and to identify the vector responsible for its spread. The disease could be transmitted mechanically from infected source plants to healthy aubergines or laboratory test plants. Transmission electron microscopy (TEM) analysis of purified virus preparations indicated the presence of viral particles with a flexible filamentous morphology (approximately 720 nm long). TEM analysis of ultrathin sections prepared from infected leaf tissue revealed the presence of cytoplasmic inclusion bodies with pinwheel and crystalline structures, typical of those induced by potyviral infection. The viral coat protein subunit was shown to have a molecular weight of 37·5 kDa by SDS‐PAGE analysis. The viral particles reacted positively in western blot analysis with an antiserum against Tomato mild mottle virus (TomMMoV) from Yemen, described as a potyvirus, vectored by the aphid Myzus persicae. The current study describes some biological properties of EMLMV and presents evidence for its transmission by the whitefly Bemisia tabaci, but not by three aphid species. The taxonomic relationship between EMLMV and TomMMoV is discussed based on their biological characteristics and sequence analysis of their genomes. It is suggested that the Israeli EMLMV should be considered a distant strain of TomMMoV, designated TomMMoV‐IL, according to the present rules of Potyviridae molecular taxonomy.     

Biological and Molecular Characterization of a New Carlavirus Isolated from an Aconitum sp

ABSTRACT A new virus was isolated from symptomless Aconitum napellus plants. The virus, for which the name Aconitum latent virus (AcLV) is proposed, has flexuous particles 640 nm in length. The experimental host range was limited to Nicotiana clevelandii. Electron microscopy studies of ultrathin sections of infected A. napellus tissues revealed the presence of elongated virus particles. No inclusion bodies characteristic of potyvirus infection were observed. AcLV was purified from naturally infected A. napellus by cesium chloride step gradient centrifugation. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dissociated purified virus preparations, a major protein component with a molecular mass of 35 kDa was observed. Diagnostic antibodies that could specifically bind to virus particles were produced. The 5' terminus (620 nucleotides) of the viral RNA was cloned and sequenced. It comprised 71 nucleotides from the untranslated 5' terminus and 549 nucleotides of an open reading frame encoding 183 amino acids. Comparison of the predicted amino acid sequence with those of other plant viruses revealed 40 to 60% identity with several carlaviruses. Based on particle morphology, absence of inclusion bodies in ultrathin sections, the relative molecular weight of the coat protein, the nucleotide sequence, and predicted amino acid homology, it is suggested that this virus belongs to the carlavirus group.     

利用RNA介导的抗病性获得高度抗马铃薯Y病毒的转基因烟草

 以马铃薯Y病毒坏死株系(PVY^N)的RNA为模板,应用反转录-聚合酶链式反应(RT-PCR)方法,扩增出长度为801 bp的非翻译的马铃薯Y病毒外壳蛋白基因。将扩增的片段克隆到pBSK的BamHI和KpnI之间并进行了序列测定。用BamHI和KpnI从重组克隆载体上切下该基因并插入到质粒pROKII内得到植物表达载体pPVYCP。通过根癌农杆菌(LBA4404)介导的方法转化烟草NC89,经卡那霉素抗性筛选、PCR和Southern blot检测,获得82株转基因植株。Northern blot和Western blot分析表明,转基因植株只在RNA水平上得到了表达。抗病性试验表明转基因植株之间抗性水平存在着差异,其中有7株是对PVY^N高度抗病性的植株。转基因植株抗病特异性试验初步表明,对PVY^N表现高度抗病的植株对PVY^O也具有高度抗病性。     

The choice of target site is crucial in artificial miRNA-mediated virus resistance in transgenic Nicotiana tabacum

MicroRNAs (miRNAs) are negative regulators of gene expression via mRNA degradation or translational repression. The potential of artificial miRNAs (amiRNAs) as antiviral agents has been used in plant biotechnology. In this study, we designed eight amiRNAs derived from Potato Virus Y (PVY) Coat Protein RNA sequences and generated transgenic tobacco plants to express these amiRNAs to confer virus resistance against PVY. Together with transient assays, the hairpin amiRcp precursors that mimic natural miRNA precursor molecules proved to be effective in expressing amiRcps and silencing the target gene. Virus resistance assay revealed that not all amiRcps targeting viral CP sequence are equally effective in preventing PVY infection. Plants with amiRcp-8 targeting the 3′ end (nt735–nt754) region exhibited high virus resistance up to 64.69%. The amiRcp-6 harboring RNA sequence (nt567–nt586) induced the lowest percentages with only 17.05%. Besides, northern blots showed that there was a correlation between the resistance level and the accumulation of amiRNA expression. Furthermore, we used bioinformatics approach to predict the mRNA structure and found that targeting sequences of loose structure benefit to improve the virus resistance level. Our results indicate that the selection of appropriate target sequence is crucial for transgenic plant against virus and provide useful guideline for the design of pathogen-derived amiRNAs.     

马铃薯Y病毒复制酶基因不同位置cDNA区段介导对PVY的不同抗性

为探究靶序列位置对RNA介导的病毒抗性产生的影响,利用聚合酶链式反应(polymerase chain reaction,PCR)技术扩增马铃薯Y病毒(Potato virus Y,PVY)复制酶基因(nuclear inclusion b,NIb)不同位置的cDNA区段,反向插入双元载体pROKII中,构建了发夹RNA(hairpin RNA,hpR-NA)结构的植物表达载体。将构建的植物表达载体采用冻融法转入农杆菌LBA4404,叶盘法转化烟草NC89,获得转基因植株。攻毒试验表明:PVYNIb基因不同位置cDNA区段介导的对PVY的抗性存在显著差异;3′端1/2处和中间位置的序列可介导高水平的病毒抗性,抗性植株的比例在50%以上,而5′端、5′端1/2处和3′端的序列介导的抗性效率较低,抗性植株的比例仅为10%~30%。Northern杂交显示:抗病植株中RNA的积累量明显低于同类型的感病植株,抗性与RNA积累量呈负相关;抗病转基因植株中有siRNA存在,表明病毒抗性是由RNA介导的。     

侵染花椰菜的芜菁花叶病毒及RT-PCR扩增外壳蛋白基因研究

 从杭州市郊区及本校附近菜园地表现严重花叶症状的花椰菜上分离到一种线状病毒,接种6科31种鉴别寄主,能侵染5科22种植物。病组织超薄切片可见风轮状内含体和片层聚集状内含体。感病的芥菜叶片,用0.5mol/L磷酸钾缓冲液(pH7.5)匀浆后汁液加4% PEG-8000沉淀3h,5%~40%甘油梯度离心纯化病毒,可获得较高的产量。提纯的病毒均为一弯曲的线状颗粒,长度740nm左右。病毒提纯液于264nm处有一吸收峰,为典型的核蛋白紫外吸收。TuMV-H分离株抗血清包被的铜网能大量吸附病毒颗粒。用特异的PCR引物扩增其外壳蛋白基因,可得-0.9Kb的片段。     

马铃薯Y病毒侵染对叶绿体超微结构、光合和荧光参数的影响

 马铃薯Y病毒(PVY)侵染导致马铃薯叶片叶绿体个体变小,部分叶绿体的结构遭到破坏,淀粉粒的体积变小而粒数密度提高。随着病毒侵染,叶片中的叶绿素含量逐步减少,光合系统电子传递速率和净光合速率显著下降,而光系统Ⅱ的最大光化学效率则没有显著变化。PVY导致叶绿体结构破坏和碳同化酶活性下降,可能是造成光合作用下降的主要原因。     

华南番木瓜环斑病毒的鉴定、提纯与性质的初步研究

 为害华南番木瓜的病毒以番木瓜科(Caricaceae)和葫芦科(Cucurlitaceae)中若干植物为寄主,范围狭窄。病毒的稀释终点为10^-2~10^-3;热灭活点为50~55℃;体外存活期为8~16小时。病毒颗粒长约600~800nm、宽约10~15nm的略为弯曲的细丝。在感病番木瓜组织的细胞内,电镜观察到典型的风轮状内含体(pinwheel inclusion body)。病毒具有马铃薯Y病毒组成员的典型特征,可认为是国外报道的番木瓜环斑病毒的中国株系,初步命名为华南番木瓜环斑病毒。     

The movement of potato virus Y (PVY) in the vascular system of potato plants

Potato virus Y (PVY) is responsible for major viral diseases in most potato seed areas. It is transmitted by aphids in a non-persistent manner, and it is spread in potato fields by the winged aphids flying from an infected source plant to a healthy one. Six different PVY strains groups affect potato crops: PVY^C, PVY^N, PVY^O, PVY^N:O, PVY^NTN, and PVY^N-Wi. Nowadays, PVY^NTN and PVY^N-Wi are the predominant strains in Europe and the USA. After the infection of the leaf and accumulation of the virus, the virus is translocated to the progeny tubers. It is known that PVY^N is better translocated than PVY^O, but little is known about the translocation of the other PVY strains. The translocation of PVY occurs faster in young plants than in old plants; this mature plant resistance is generally explained by a restriction of the cell-to-cell movement of the virus in the leaves. The mother tuber may play an important role in explaining mature plant resistance. PVY is able to pass from one stem to the other stems of the same plant through the vascular system of the mother tuber, but it is unknown whether this vascular link between stems is permanent during the whole life of the plant. Two greenhouse trials were set up to study the spread of PVY in the vascular system of the potato plant. The PVY-susceptible cultivar Charlotte was used for both trials. It was demonstrated that all stems growing from a PVY-infected tuber will become infected sooner or later, and that PVY^N-Wi translocates more efficiently to progeny tubers than PVY^NTN. It was also demonstrated that the progressive decay of the mother tuber in the soil reduces the possibility for virus particles to infect healthy stems through the vascular system of the mother tuber. This new element contributes to a better understanding of the mechanism of mature plant resistance.     

A single amino Acid change in viral genome-associated protein of potato virus y correlates with resistance breaking in 'virgin a mutant' tobacco

ABSTRACT Tobacco cultivar Virgin A Mutant (VAM) is reported to have the recessive potyvirus resistance gene va. Varied levels of resistance were observed in VAM plants inoculated with Japanese potato virus Y (PVY) isolates. VAM was highly resistant to most of the PVY isolates tested and tolerant to three necrotic strain isolates of PVY-T. Based on data obtained from tissue printing and press blotting, the resistance appeared to be mainly at the level of cell-to-cell movement. PVY replicated in VAM proto-plasts, but the replication was 30% lower than in susceptible tobacco, suggesting that impairment of replication also contributes to resistance. To identify the viral gene product or products involved in VAM resistance, we isolated spontaneous resistance-breaking mutants by passing vein-banding (O strain) isolates several times through VAM plants. By comparing the amino acid sequences of the mutants with their original isolates, we identified a single amino acid substitution in the viral genome-associated protein (VPg) domain that is correlated with VAM resistance breaking. Together, these results suggest that, in addition to its role in replication, VPg plays an important role in the cell-to-cell movement of PVY.     

Hairpin-based virus resistance depends on the sequence similarity between challenge virus and discrete,highly accumulating siRNA species

Virus resistance can be effectively generated in transgenic plants by using the plant’s silencing machinery. To study the specificity of gene-silencing-based resistance, homozygous tobacco (Nicotiana tabacum L.) plants containing a 597-nt hairpin RNA construct of the Potato Virus Y (PVY) replicase sequence were challenged with a variety of PVY strains. The transgene-carrying tobacco line was immune to five potato PVY strains with high sequence similarity (88.3–99.5%) to the transgene. Infection with more distant tomato and pepper PVY field strains (86–86.8% sequence similarity) caused delayed symptom appearance in the transgenic tobacco. Transgene production of small interfering (si) RNA was detected by northern blot and measured using a custom-designed microarray for the detection of small RNAs. siRNA accumulation peaks were observed throughout the inverted-repeat transgene. In the resistance-breaking tomato and pepper strains there were nucleotide differences in the sequences correlated to siRNA transgene accumulation, indicating the role of siRNA specificity in resistance breaking. The log of transgene siRNA signal intensity increased with probe GC content, indicating that the accumulating siRNA molecules were GC-rich. Sequence similarity of highly accumulating siRNAs with the target virus strain appears to be important for both resistance and resistance-breaking characteristics.     

侵染药菊的一株病毒分离物的鉴定

 从安徽省药菊产区大面积受害显花叶症状的菊花上分离到-株病毒分离物(代号Chm-91)。Chm-91经人工接种能侵染10科30种植物;还可通过桃蚜(Myzus persicae)、萝卜蚜(Hyadaphiserysimi)和菊小长管蚜(Macrosiphoniella sanbornic)以非持久性方式传播。18种感染的植物种子不带毒;Chm-91致死温度65℃~70℃,稀释限点10^-2~10^-3,体外存活期4~5天。Chm-91粒体球状,直径约30nm;病组织超薄切片可见堆集状内含体。该分离物与番茄不孕病毒(TAV)抗血清呈阳性反应。Chm-91的标准紫外吸光度(A0.1% 260)为4.78,A260/A280比值为1.73;粒体平均分子量为1.835 X 106道尔顿,沉降系数为97.5 S;外壳蛋白由相同的亚基所组成,各亚基含236个氨基酸,亚基的分子量和等电点分别为25100(25K)和5.2;核酸类型为单链RNA,在粒体中的含量是16.9%,有四种大小不同组份,碱基百分比中鸟嘌呤(G)为23.3,腺嘌呤(A)为27.3,胞嘧啶(C)为19.4,尿嘧啶(u)为29.6。#br#根据上述生物学性状,血清学反应,粒体形态及理化特性等。作者认为该分离物隶属于TAv或者它的一个株系。这是为害药菊的一种病毒在我国首次报道。     

大豆花叶病毒(SMV)感染大豆品种“Lee”叶片细胞超微结构的研究

 本文对大豆花叶病毒(SMV-H-16)感染大豆品种"Lee"叶片细胞的超微结构进行了连续观察。研究结果证实:用SMV-H-16感染美国大豆品种"Lee"表现症状极轻微,易恢复。其超微结构特征如下:(1)病毒粒体出现于圆柱状和风轮状内含体之后。(2)风轮状内含体从细胞质中逐渐向液泡区伸进。(3)首先在细胞质和液泡膜附近,发现病毒粒体.(4)靠近细胞核处出现一些类似环孔膜板状结构。(5)感染28天后叶片细胞里病毒粒体被束缚在束丝中,附着在液泡膜的内圆面和叶绿体上,此时,风轮状内含体消失。     

Interaction between tobacco Ribulose-l,5-biphosphate Carboxylase/Oxygenase large subunit (RubisCO-LSU) and the PVY Coat Protein (PVY-CP)

A 54 kDa band (P54) was continually detected with the 30 kDa viral capsid protein (CP) on the SDS-PAGE migration profile of purified potato virus Y (PVY). P54 was observed following the use of two different procedures for the purification of the PVY from infected tobacco. It was a constitutively expressed tobacco protein. The analysis of the PVY preparation showed that P54 has aggregation properties and precipitates, thus pulling down the virus. We used an enzyme-linked immunosorbent assay (ELISA) to study the relationship between P54 and the PVY particles. We performed an inhibition test with monoclonal antibodies (mAb) directed against the PVY-CP, to show that these two components interact. This result was confirmed by western blot. The internal sequence of five major peptides, obtained by C-lysine endoprotease digestion of the P54 followed by HPLC separation, showed 100% homology with the large subunit of the ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO-LSU) of tobacco. MAb directed against RubisCO-LSU were produced and used to reveal the RubisCO-LSU/PVY complex in infected tobacco extracts. A phage library displaying random heptapeptides was used to isolate several peptides that specifically bound to the native form of the PVY. The sequences of thirty-three phage-displayed peptides, which bound specifically to this virus, present further discontinuous sequence homologies with the RubisCO-LSU. Five peptides (p1 to p5) corresponding to the RubisCO-LSU homologous regions were used for a bacterial two-hybrid system to confirm in vivo direct interactions between the selected RubisCO-LSU regions and the PVY-CP. We propose that the PVY-CP may be involved in the production of mosaics and yellowing symptoms in tobacco through its interaction with RubisCO-LSU.     

Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in northern Tunisia

The genetic structure of Potato virus Y (PVY) populations was investigated in naturally-infected pepper ( Capsicum annuum ) fields, collected at eight different localities in northern Tunisia, where 23% of the sampled plants were homozygous for the pvr2¹ recessive resistance allele, while the other plants carried the dominant susceptibility allele pvr2⁺ . Restriction fragment length polymorphism analysis at three PVY genome segments revealed a high level of viral diversity, with a majority of cases showing co-infection of individual plants by several PVY haplotypes and a strong genetic differentiation of viral populations collected in the different localities. Geographic distances affected the differentiation of PVY populations and isolation by distance among these populations was significant. However, the occurrence of the pvr2¹ resistance allele did not contribute to the structure of viral populations, suggesting that the virulence properties of the virus did not significantly affect its fitness. Consequently, greater deployment of the pvr2¹ gene would probably not be a suitable strategy to control PVY, and other resistance genes should be preferred.     

Impact of the potato inoculation date on potato virus Y load and viral distribution in daughter tubers at harvest

Aged plants are more difficult to infect than young plantlets. This modification of susceptibility is described as mature plant resistance (MPR). For potato virus Y (PVY), MPR is known to lead to low infection rates of plants inoculated at the postflowering stage and a decrease in the number of infected daughter tubers. However, the impact of inoculation date on the capacity of PVY to accumulate in daughter tubers has not been studied so far. Field and greenhouse experiments were carried out to better understand PVY epidemiology and to help potato growers to evaluate consequences of early/late infections on the quality of their crops. In field trials, potato plants (cv. Bintje) were covered by insectproof nets from planting to harvest except for a 14-day period to expose plants to natural PVY infections. Under controlled conditions, potato plants were mechanically inoculated with PVY at different dates from preflowering stages (early inoculations) to postflowering stage (late inoculations). At harvest, daughter tubers were individually collected and analysed to define proportions and viral load of infected tubers according to the time between virus inoculation and harvest. Our results showed that although the age of plants at the time of inoculation can modify their susceptibility to PVY infection, in return, early and late PVY inoculations lead to similar rates of infected tubers at the plant scale and equivalent viral accumulation in infected tubers. All together, these data revealed that both early/late infections are high risks for the sanitary quality of potato tubers.