Development of a stable attenuated double-mutant of tobacco mosaic virus for cross-protection

Tobacco(Nicotiana tabacum)and tomato(Solanum lycopersicum)are two major economic crops in China.Tobacco mosaic virus(TMV;genus Tobamovirus)is the most prevalent virus infecting both crops.Currently,some widely cultivated tobacco and tomato cultivars are susceptible to TMV and there is no effective strategy to control this virus.Cross-protection can be a safe and environmentally friendly strategy to prevent viral diseases.However,stable attenuated TMV mutants are scarce.In this study,we found that the substitutions in the replicase p126,arginine at position 196(R^(196))with aspartic acid(D),glutamic acid at position 614(E^(614))with glycine(G),serine at position 643(S^(643))with phenylalanine(F),or D at position 730(D^(730))with S,significantly reduced the virulence and replication of TMV.However,only the mutation of S^(643) to F reduced the RNA silencing suppression activity of TMV p126.A double-mutant TMV-E614G-S643F induced no visible symptom and was genetically stable through six successive passages in tobacco plants.Furthermore,our results showed that TMV-E614G-S643F double-mutant could provide effective protection against the wild-type TMV infection in tobacco and tomato plants.This study reports a promising mild mutant for cross-protection to control TMV in tobacco and tomato plants.

Agroinoculation as a Simple Way to Deliver a Tobacco Mosaic Virus- Based Expression Vector

烟草花叶病毒(TMV)表达载体30B是一个目前广泛应用的植物病毒表达载体,但用其生产外源蛋白时,必须先将它体外转录成RNA,才能被用来接种宿主植物。由于RNA体外转录费用昂贵、操作复杂,因此限制了30B表达载体的进一步应用。针对这一不足,我们用农杆菌接种法(agroinoculation)接种该病毒载体,即将30B cDNA置于花椰菜花叶病毒(CaMV)的35S启动子和终止子之间,再将整个表达框架插入到农杆菌T-DNA的左边界和右边界之内,构建成质粒p35S-30B,将转入该质粒的农杆菌注射到植物的叶片中,30B cDNA随T-DNA进入植物细胞后,被转录成可自我复制的RNA形式,进而发生系统侵染。为了检测此接种方式的可行性,绿色荧光蛋白(GFP)报告基因被克隆到p35S-30B中,构建成p35S-30B∶∶GFP,用含有该质粒的农杆菌进行注射操作。证实该病毒载体可通过简便的农杆菌接种法侵染Nicotiana benthamiana,在被接种植物的系统叶中,GFP的表达量可占植物总可溶蛋白的5.2％。

Effect of chitosan on tobacco mosaic virus(TMV) accumulation, hydrolase activity, and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun

The effect of chitosan on the development of infection caused by Tobacco mosaic virus(TMV) in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV(2 μg/mL) and chitosan(1 mg/mL) were lower in the early period of infection(3 days after inoculation), by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases(proteases, RNases) in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid(PTA)-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles(18 nm in diameter, 300 nm long), these suspensions contained abnormal(swollen, "thin" and "short") virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that "thin" virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.

Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus(TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000(PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 102 viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.

A TOM1 homologue is required for multiplication of Tobacco mosaic virus in Nicotiana benthamiana

The AtTOM1 gene of Arabidopsis thaliana had been shown to be essential for the efficient multiplication of Tobacco mosaic virus(TMV) in A.thaliana.In this study,we cloned an AtTOM1-like gene from Nicotiana benthamiana named as NbTOM1.Sequence alignment showed that NbTOM1 is closely related to AtTOM1 homologues of N.tabacum and Lycopersicon esculentum with 97.2% and 92.6% nucleotide sequence identities,respectively.Silencing of NbTOM1 by a modified viral satellite DNA-based vector resulted in complete inhibition of the multiplication of TMV in N.benthamiana.The result suggests that inhibition of NbTOM1 via RNA silencing is a potentially useful method for generating TMV-resistant plants.

Occurrence Regularity and Control of Tobacco Mosaic Virus (TMV) in Wuxi Tobacco Area of China in 2011

On the basis of general situation and new characteristics of tobacco mosaic virus （TMV） in Wuxi tobacco area in 2011, the paper expounded the objec-tive reasons of TMV via systemic investigation, field experiment and date sorting. Meanwhile, combined with mcteorological conditions and results of systemic inves-tigation, the study especially analyzed meteorological conditions, outbreak and prevalence regularity of TMV and control efficacy of chemical reagents against TMV. The results showed that climatic conditions were the main conditions affecting TMV. There were three occurrence peaks of TMV in 2011, as a result of meteorologi-cal conditions of the months from April to June. The peaks were concerned with a wide range of rainfall about half a month before, low temperature, high humidity and scant sunshine and temperature jump after rain. The results of control effects showed that the chemical reagents could obviously prevent TMV, but once tobacco plants were infected, spraying chemical reagents would not have effective control effect against TMV.

Analysis of Spatial Distribution Pattern of Tobacco Mosaic Virus (TMV) Based on Geostatistics

Using geostatistical method, the semi-variation function models of tobacco mosaic virus （TMV） in east-west and north-south directions were established, and the distribution pattern of TMV in large scale space was studied. The results showed that the distribution pattern of the disease in east-west and north-south directions belonged to linear model with abutment, and the spatial distribution pattern within the investigated areas was aggregated model. The spatial correlation distances in east-west and north-south directions were 29.953 4 and 47.813 8 km, and the spatial variabilities were 95.71% and 80.05%, respectively. This indicated that they had strong spatial correlation. Isoline map accessed by Kringing interpolation method could clearly reflect the spatial aggregated model.

Control Effects of Different Agents on Tobacco Mosaic Virus Disease

[Objectives] This study was conducted to screen out suitable agents for controlling tobacco mosaic virus disease and the best control period in Zhangzhou tobacco area, providing a theoretical basis for the control of virus diseases, thereby improving the quality of flue-cured tobacco and the income of tobacco farmers. [Methods] The effects on tobacco mosaic virus disease under the interaction between different agents and different application periods were investigated. The incidence of tobacco mosaic virus disease was investigated, and its control effect was analyzed. [Results] Different agents and different application periods had different control effects on tobacco mosaic virus disease. The incidence of tobacco mosaic virus disease: At 30 and 45 d after transplanting, the incidences of A2B1 treatment were the lowest, at 0.85%, 1.71%, respectively;and at 60 d after transplanting, the incidence of A3B1 treatment was the lowest, only 10.68%. The control effect: At 30 and 45 d after transplanting, A2B1 treatment had better control effects, reaching 79.39% and 73.06%, respectively. [Conclusions] 3% hypersensitive protein sprayed at 1 d before transplanting and 7 and 15 d after transplanting achieved the best effect, followed by 10% ningnanmycin sprayed at 1 d before transplanting and 7 and 15 d after transplanting. In tobacco production, it is recommended to apply 1 000 times dilution of 3% supersensitive protein microgranules for three times(at 1 d before transplanting and 7 and 15 d after transplanting), which can effectively prevent tobacco mosaic virus disease.

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth,development,and polyploidization.However,there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics.We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco,Nicotiana tabacum,using a methylation-sensitive amplified polymorphism(MSAP) technique.The results showed that methylation existed at a high level among tobacco accessions,among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic.A cluster analysis revealed distinct patterns of geography-specific groups.In addition,three polymorphic sites significantly related to tobacco mosaic virus(TMV) resistance were explored.This suggests that tobacco breeders should pay more attention to epigenetic traits.

Effects of mutated replicase and movement protein genes on attenuation of tobacco mosaic virus

Our previous reports showed that one opal mutation (UGA) and one ochre mutation (UAA) respectively located in the replicase and movement protein (MP) genes of the attenuated tomato mosaic virus K(ToMV-K) contribute to the viral attenuation. To explore a wider application of this attenuation pattern to other plant viruses, we have constructed three mutants which respectively contain one opal mutation of the replicase gene and/or one ochre mutation of the MP using PCR-mediated site-directed mutagenesis from a virulent tobacco mosaic virus isolated from China (TMV-Cv). Plant infection performed by in vitro transcripts revealed that the MP truncated mutant TMV-Cvmp and the replicase-MP truncated mutant TMV-Cvrase-mp were infectious on both local lesion (Nicotiana tabacum cv. Xanthi NC) and systemic (N. tabacum cv. K326) host plants, while the replicase truncated mutant TMV-Cvrase was non-infectious. The K326 plant infected by TMV-Cvrease-mp displayed on|y a little mild mosaic. By electronic microscopy (EM), plant re-inoculation, RNA Dot-blot, RT-PCR and sequencing we demonstrated that the progeny viruses of TMV-Cvmp and TMV-Cvrease-mp shared similar morphological character with TMV-Cv, owned the abilities to infect, replicate and propagate in the assayed plants, and maintained the mutated sites during infection. These data showed that both the opal and the ochre mutations are able to cooperatively induce the attenuated phenotypes of TMV-Cvrase-mp on plants, indicating that the mutation pattern of ToMV-K could be used to attenuate other virulent plant viruses.

Hyaluronic acid-based hydrogels with tobacco mosaic virus containing cell adhesive peptide induce bone repair in normal and osteoporotic rats

Tobacco mosaic virus(TMV)has been studied as a multi-functional agent for bone tissue engineering.An osteo-inductive effect of wild-type TMV has been reported,as it can significantly enhance the bone differentiation potential of bone marrow stromal cells both on a two-dimensional substrate and in a three-dimensional(3D)hydrogel system.A TMV mutant(TMV-RGD1)was created which featured the adhesion peptide arginyl-glycyl-aspartic acid(RGD),the most common peptide motif responsible for cell adhesion to the extracellular matrix,on the surface of the virus particle to enhance the bio-functionality of the scaffold material.We hypothesised that the incorporation of either wild-type TMV or TMV-RGD1 in the 3D hydrogel scaffold would induce bone healing in critical size defects of the cranial segmental bone.We have previously tested the virus-functionalised scaffolds,in vitro,with a hyaluronic acid-based system as an in-situ hydrogel platform for 3D cell encapsulation,culture,and differentiation.The results of these experiments suggested the potential of the virus-functionalised hydrogel to promote in vitro stem cell differentiation.The hydrogel-forming system we employed was shown to be safe and biocompatible in vivo.Here,we further explored the physiological responses regarding bone regeneration of a calvarial defect in both normal and osteoporotic ovariectomized rat models.Our results,based on histological analysis in both animal models,suggested that both wild-type TMV and TMV-RGD1 functionalised hydrogels could accelerate bone regeneration,without systemic toxicity,evaluated by blood counts.New bone formation was intensified by the incorporation of the RGD-mutant viral particles.This finding increased the potential for use of the rodshaped plant virus as a platform for the addition of powerful biofunctionality for tissue engineering applications.This study was approved by the Ethics Committee on Animal Use of the Zhenjiang Affiliated First People’s Hospital affiliated to Jiangsu University.

Self-assembly of anisotropic tobacco mosaic virus nanoparticles on gold substrate

A facile approach to assembled virus film with tunable structure is presented.Rod-like tobacco mosaic virus (TMV) was selected as the prototype in this study for its anisotropic structural feature.TMV can either "lie down" or "stand up" on gold substrate by tuning the solution pH.A quartz crystal microbalance with dissipation monitoring was used to monitor the pH-dependent self-assembly behavior of TMV nanoparticles,and atomic force microscopy and single molecule force spectroscopy further confirmed the different assembly structures.

Construction of RNAi Vector Which Resist Cucumber Mosaic Virus and Transformation of Tobacco

[Objective] Aimed to construct RNAi vector resistant to cucumber mosaic virus and transferred this vector into tobacco. [Method] RT-PCR method was used to amplify cucumber mosaic virus NS04 and process RNA2 gene sequen of tomato isolates. The analysis results of phylogenetic tree demonstrated that the sequence in RNA2 encoded CMV-2a had 98.0% and 96.5% homology with nucleotide and amino acid of DQ412731 isolate of Zhejiang,China. The replicase fragment in CMV RAN2 gene was taken as target sequence to construct pBi35SCR2 eukaryotic expression vector,then the expression vector was identified. Through agrobacterium-mediated method,the expression vector was transferred into tabacco and PCR method was used to check the transfer. The PCR results demonstrated that the experiment had successfully construct eukaryotic expression vector of pBi35SCR2 and the expression vector was successfully transferred into tabacco. [Conclusion] The obtained transgenic tobacco could be used as challenge test material in following experiment and provided foundation for studying processing tomato resist cucumber mosaic virus.

Establishment of Multiplex PCR for Three Virus of Potato

[Objective]The aim was to establish the multiplex PCR method for three virus of potato：PVA（potato virus A）,TMV（Tobacco mosaic virus）and PVY（potato virus Y）.[Method]According to the PVA,TMV and PVY sequences available in GenBank,pairs of primer were designed for establishing a multiplex PCR method,and constructing recombinant plasmid of target genes by PCR amplified of three viruses as reference standard simple to be used in sensitivity test;PVX（Potato virus X）,PVM（Potato virus M）,PVS（Potato virus S）,PVV（Potato virus V）and CMV（Cucumber mosaic virus）were used to carry out the specificity test and detection of 11 samples which were suspected of virus infected.[Result]The detection limit for PVA,TMV and PVY was 14,14 and 14 copies/ml,respectively.No cross-reactivity was observed with other viruses.Seven of 11 samples were infected by three viruses.[Conclusion]The multiplex PCR for PVA,TMV,PVY three viruses of potato was established successfully,which had provided basis for the detection technology of potato virus.

A bacterial protein Rhp-PSP inhibits plant viral proliferation through endoribonuclease activity

Plant virus causes massive crop losses globally.However,there is currently no effective measure to control plant viral disease.Previously,we identify an antiviral protein Rhp-PSP,produced by the bacterial Rhodopseudomonas palustris strain JSC-3b.In this study,we discover that the antiviral activity of Rhp-PSP relies on its endoribonuclease activity.Converting the arginine(R)residue at position 129 onto alanine(A)abolishs its endoribonuclease activity on coat protein(CP)RNA of tobacco mosaic virus(TMV),consequentially,compromises the antiviral activity of Rhp-PSP.Further investigation demonstrates that,the mutant Rhp-PSP^(R129A)is unable to form the homotrimer as the wild type,indicating the importance of quaternary junction for the endoribonuclease activity.Overexpression of Rhp-PSP in Nicotiana benthamiana significantly enhances the resistance against TMV of seedlings,while expression of Rhp-PSP^(R129A)did not,confirming that endoribonuclease activity is responsible for the antiviral activity of Rhp-PSP.In addition,foliar spray of Rhp-PSP solution on tomato and pepper plants significantly reduces the disease index of viral diseases,indicating that Rhp-PSP shows potential to develop antiviral agent in practice.

污水稳定塘菌-藻生态系统去除与灭活植物病毒TMV研究

了解污水稳定塘生态系统去除与灭活植物病毒的效率与机理具有重要的理论和实际意义.对模型植物病毒———烟草花叶病毒(tobaccomosaicvirus,TMV)的实验结果显示:稳定塘系统中的悬浮固体(SS)可在短时间内吸附TMV且达到一个饱和值.纯培养的枯草芽孢杆菌及小球藻对TMV的存活无明显不良影响,而光合细菌和稳定塘中的混合细菌群却对TMV具有灭活作用.当它们同藻类形成菌藻共生体系时,对TMV的灭活速率分别从对照的每天0 0404log10(枯斑数·mL-1)升高到0 0783log10(枯斑数·mL-1)和0 075log10(枯斑数·mL-1) 因此,在污水稳定塘生态系统中,由于病毒对悬浮固体的吸附以及菌藻共生体系中微生物的作用可有效地去除与灭活植物病毒,其中悬浮固体吸附病毒并非真正意义上的灭活,稳定塘中存在的细菌特别是光合细菌对植物病毒的灭活才是最主要的因素.

黄瓜上烟草花叶病毒的RT-PCR检测

根据烟草花叶病毒TMV的外壳蛋白基因序列设计、合成引物,以感病组织和健康组织总RNA为模板进行cDNA合成和PCR扩增,对2002年采集的75份黄瓜病毒病样本进行了检测,结果从感病组织中扩增出与预期的425bp大小一致的目标片段而健康组织无此扩增产物;29份材料检测到TMV,检出率达38.67%。

Anti-TMV Effects of Amaryllidaceae Alkaloids Isolated from the Bulbs of Lycoris radiata and Lycoricidine Derivatives

Fifteen known amaryllidaceae alkaloids were isolated from the bulbs of Lycoris radiata.Some of the compounds and lycoricidine derivatives had been screened for the activities against tobacco mosaic virus(TMV)by the conventional halfleaf method.Lycoricidine derivatives were also carried out the assay of effect on systemic infection of TMV by western-blot and RT-PCR analysis.The tested compounds showed moderate inactivation effect,whereas the lycoricidine derivatives showed good protective effect.The protective inhibitory activity of compounds L1(N-methyl-2,3,4-trimethoxyly-coricidine)(60.8%)and L3(N-methyl-2-methoxy-3,4-acetonidelycoricidine)(62.0%)was almost similar to the positive control,Ningnanmycin(66.4%).RT-PCR and Western-blot analysis displayed that compounds L1,L3,L5(N-allyl-2,3,4-triallyloxylycoricidine)exhibited antiviral activity,which was evidenced by reducing TMV-CP gene replication and TMV-CP protein expression.Additionally,defensive enzyme activities confirmed that compound L1 could increase the activity of PAL,POD,SOD to improve disease resistance of tobacco.

球状功能性烟草花叶病毒纳米颗粒的制备及表征

烟草花叶病毒(TMV)由于其良好的生物相容性、单分散性、多价性、低成本等优点,已作为功能材料的基本构筑单元应用于光电器件、组织工程、疫苗载体,无机纳米材料制备等研究领域。然而,相比于棒状的TMV,球状TMV纳米颗粒无核酸分子,抗环境影响能力更强,比表面积更大。本文利用蛋白质的热致变性原理,对经基因和化学改性后的棒状TMV如半胱氨酸突变体(TMV-Cys)、赖氨酸突变体(TMV-EPMK)和β-环糊精(β-CD)修饰的TMV(TMV-β-CD)进行热变性处理,探究其形成球型纳米颗粒(TMV-SNP)的能力及功能性。结果显示,改性后的TMV经历热变性后可得到形貌均一的球型纳米颗粒,且其暴露在纳米颗粒表面的功能基团Cys、Lys和β-CD仍具有反应活性。

Purification and Characterization of a New Ribosome Inactivating Protein from Cinchonaglycoside C-treated Tobacco Leaves

A new ribosome-inactivating protein （RIP） with a molecular weight of 31 kDa induced by Cinchonaglycoside C （1） designated CIP31, was isolated from tobacco leaves. Analysis of this protein sequence indicated that it belongs to the RIP family and it was distinct from the other plant RIPs reported previously at its N-terminal amino acid sequence. CIP31 can directly impair synthesis of coat protein （CP） of tobacco mosaic virus （TMV）, which resulted in inhibition of TMV long distance movement and multiplication in tobacco plants at concentrations of ng/mL. Furthermore, no toxicity was shown to the growth and fertility of the plants. CIP31 was synthesized only in the presence of Cinchonaglycoside C （1） and was independent of the salicylic acid （SA） signal pathway. We provided evidence for the SA-independent biological induction of resistance.