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.

Turnip mosaic virus pathogenesis and host resistance mechanisms in Brassica

Turnip mosaic virus(TuMV)is a devastating potyvirus pathogen that infects a wide variety of both cultivated and wild Brassicaceae plants.We urgently need more information and understanding of TuMV pathogenesis and the host responses involved in disease development in cruciferous crops.TuMV displays great versatility in viral pathogenesis,especially in its replication and intercellular movement.Moreover,in the coevolutionary arms races between TuMV and its hosts,the virus has evolved to co-opt host factors to facilitate its infection and counter host defense responses.This review mainly focuses on recent advances in understanding the viral factors that contribute to the TuMV infection cycle and the host resistance mechanism in Brassica.Finally,we propose some future research directions on TuMV pathogenesis and control strategies to design durable TuMV-resistant Brassica crops.

Reactions of Maruca Resistant Transgenic Cowpea to Cowpea Aphid-Borne Mosaic Virus

Cowpea (Vigna unguiculata L. [Walp.]) in one of the main grain legumes contributing to food security and poverty alleviation in Sub-Saharan Africa. To control the highly damaging legume pod borer Maruca vitrata F., transgenic cowpea lines expressing the insecticidal Cry1Ab Bt protein were developed. In this study, we evaluated the impact of Cry1Ab transgene expression on the susceptibility of four cowpea lines (named IT97K-T, IT98K-T, Gourgou-T and Nafi-T) and their respective non-transgenic near isogenic lines (IT97K, IT98K, Gourgou and Nafi) to Cowpea aphid-borne mosaic virus (CABMV) in greenhouse conditions. In a preliminary quality control test by enzyme-linked immunosorbent assay, the presence of Cry1Ab protein in transgenic seed lots ranged from 59% to 72%, with no significant differences among the lines (χ2 = 3.26;p = 0.35). Upon virus inoculation, all cowpea lines exhibited mosaic symptoms with similar severity between 7- and 11-day post-inoculation. No significant differences were observed in symptom severity. Significant differences were found between cowpea lines for time of symptom onset, virus accumulation in plants and days to 50% flowering. However, while comparing pairs of transgenic lines and corresponding non-transgenic lines, virus accumulation showed not significant differences whatever the pair. Time of symptom onset and days to 50% flowering did not also differ significantly between pairs of cowpea lines except Nafi/Nafi-T in which transgenic Nafi-T showed earlier symptoms (7.4 ± 0.7 vs. 8.9 ± 0.8 days post-inoculation) and shorter flowering time (37.3 ± 0.6 vs. 42 ± 1.7 days after sowing). Overall, these findings improve our understanding of the effects of Cry1Ab gene mediated genetic modification on cowpea infection by Cowpea aphid-borne mosaic virus, with potential implications for environmental safety assessment.

High-density SNP genetic linkage map construction and quantitative trait locus mapping for resistance to cucumber mosaic virus in tobacco(Nicotiana tabacum L.)

Genetic linkage maps are essential for studies of genetics, genomic structure, and genomic evolution, and for mapping quantitative trait loci (QTL). Identification of molecular markers and construction of genetic linkage maps in tobacco (Nicotiana tabacum L.), a classical model plant and important economic crop, have remained limited. In the present study we identified a large number of single nucleotide polymorphism (SNP) markers and constructed a high-density SNP genetic map for tobacco using restriction site-associated DNA sequencing. In 1216.30 Gb of clean sequence obtained using the Illumina HiSeq 2000 sequencing platform, 99,647,735 SNPs were identified that differed between 203 sequenced plant genomes and the tobacco reference genome. Finally, 13,273 SNP markers were mapped on 24 high-density tobacco genetic linkage groups. The entire linkage map spanned 3421.80 cM, with a mean distance of 0.26 cM between adjacent markers. Compared with genetic linkage maps published previously, this version represents a considerable improvement in the number and density of markers. Seven QTL for resistance to cucumber mosaic virus (CMV) in tobacco were mapped to groups 5 and 8. This high-density genetic map is a promising tool for elucidation of the genetic bases of QTL and for molecular breeding in tobacco.

Comparative transcriptome analysis reveals differential gene expression in resistant and susceptible tobacco cultivars in response to infection by cucumber mosaic virus

Cucumber mosaic virus(CMV) is one of the most severe viral diseases transmitted by aphids infecting Solanum crops in China, causing great losses of crop yields and income in rural communities.The tobacco cultivars NC82 and Taiyan 8 are closely related but differ in resistance to CMV.NC82 is susceptible to infection and Taiyan 8 is resistant, but the mechanisms underlying this difference in resistance are not clear.In this study, we conducted RNA sequencing to analyze changes in gene expression induced in the leaves of Taiyan 8 and NC82 upon systemic infection with CMV, compared with gene expression in the leaves of mock-inoculated plants.Leaves were sampled at one, three, eight, and 15 days after infection.In total, 3443 and 747 differentially expressed genes were identified in Taiyan 8 and NC82, respectively.Gene ontology and pathway enrichment analyses revealed that the different responses to CMV infection between cultivars were based on microtubulebased processes, pentose and glucuronate interconversions, plant–pathogen interaction,and hormone signal transduction pathways.Genes encoding pathogenesis-related proteins, disease-resistance proteins, lipoxygenase, cellulose synthase, an auxin response factor, and an ethylene receptor showed different expression patterns.The differences in gene expression following CMV infection likely contributed to the different resistance levels of these two tobacco cultivars.The comprehensive transcriptome dataset described here,which includes candidate response genes, will serve as a resource for further studies of the molecular mechanisms associated with tobacco defense responses against CMV.

GmTOC1b negatively regulates resistance to Soybean mosaic virus

Soybean(Glycine max)is a major oil and feed crop worldwide.Soybean mosaic virus(SMV)is a globally occurring disease that severely reduces the yield and quality of soybean.Here,we characterized the role of the clock gene TIMING OF CAB EXPRESSION 1b(GmTOC1b)in the resistance of soybean to SMV.Homozygous Gmtoc1b mutants exhibited increased tolerance to SMV strain SC3 due to the activation of programmed cell death triggered by a hypersensitive response.Transcriptome deep sequencing and RT-qPCR analysis suggested that GmTOC1b likely regulates the expression of target genes involved in the salicylic acid(SA)signaling pathway.GmTOC1b binds to the promoter of GmWRKY40,which encodes a protein that activates the expression of SA-mediated defense-related genes.Moreover,we revealed that the GmTOC1bH1 haplotype,which confers increased tolerance to SMV,was artificially selected in improved cultivars from the Northern and Huang-Huai regions of China.Our results therefore identify a previously unknown SMV resistance component that could be deployed in the molecular breeding of soybean to enhance SMV resistance.

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.

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.

Effects of Early Infection of Tobacco Mosaic Virus on Photosynthetic Proteins and Its Control

[Objectives] This study was conducted to explore the optimal period of TMV control and the physiological sites that interfere with TMV infection. [Methods] Proteome analysis was performed on the host tissues(tobacco plants) at different time periods of viral infection, to verify the changes in the expression of differential protein genes and N and PR1-a in the photosynthetic pathway and porphyrin metabolism and chlorophyll metabolism pathways in proteome; and the tobacco plants were treated with different preparations. [Results] The expression levels of N and PR1-a in the tobacco leaves treated with preparation B reached the highest level, and the effects on the expression levels of the differential protein genes were also the most significant. The control effects of the preparations were tested by the half-leaf method, and the results showed that preparation B had a significant control effect against the early infection of the virus.[Conclusions] This study lays a foundation for the prevention and control of tobacco mosaic virus on crops.

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.

Transgenic Tobacco Lines Expressing Yam Mosaic Virus Coat Protein-Derived dsRNA Are Resistant to Yam Mosaic Virus

Yam mosaic virus (YMV), a Potyvirus, is a highly destructive pathogen of yam accounting for yield losses up to 40%. Apart from causing significant reduction in tuber size and quality, it restricts international exchange of germplasms. It thus becomes crucial to get resistant or at least virus-free planting materials for farmers. This study was aimed at inducing resistance to YMV in tobacco by RNA silencing. An RNAi construct containing 161 bp fragment of YMV-coat protein (CP) gene was developed and used to produce transgenic tobacco lines expressing YMV-coat protein (CP) derived double stranded RNA (dsRNA) via Agrobacterium-mediated transformation. Of the eight T1 transgenic lines inoculated with YMV, six (L1, L2, L3, L5, L7 and L8) showed immunity to YMV as no symptoms were detected, whereas two (L4 and L10) exhibited high resistance with mild symptoms limited to inoculation portions. No virus could be detected in uninoculated new leaves of the transgenic lines after RT-PCR and qPCR analyses of YMV-coat protein (CP). The presence of small interfering RNAs in transgenic lines after virus challenge indicates that the resistance was acquired through RNA silencing.

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.

凤果花叶病毒上海分离物(Pepino Mosaic Virus,PepMV-Sh)的初步鉴定及其ELISA检测方法的建立

对上海市引种的凤果(Solanum muricatum)植株进行的病原物调查发现有较严重的凤果花叶病发生,经研究确认系病毒感染所致。从发病植物中提取得到长度主要为100-250nm,宽度约为10nm的短杆状病毒颗粒,SDS-PAGE电泳显示其外壳蛋白的主成分是20kD的蛋白质,变性琼脂糖凝胶电泳和琼脂糖凝胶电泳分析均得到一RNA条带。经比对,该病毒分离物与原产地的凤果花叶病毒特性有一定的差别,暂定名为凤果花叶病毒上海分离物(Pepino Mosaic Virus,PepMV-Sh),其植物病毒学分类地位待定。用纯化的凤果花叶病毒(PepMV)制备了抗血清,建立了ELISA检测方法,并应用于凤果组培苗及大棚植株中病毒病的检测。

几种血清学技术在浙贝母花叶病毒(Thunberg fritillary mosaic virus)检测中的应用

通过12%SDS-PAGE和5%～20%梯度SDS-PAGE二次制备电泳纯化浙贝母花叶病毒(Thunberg fritillary mosaic virus,TFMV)的外壳蛋白,4次免疫小鼠,获高纯度的抗血清。分别用Western blot、间接ELI SA、免疫扩散、火箭免疫电泳和对流免疫电泳5种免疫检测技术对TFMV样品进行了检测。结果表明,间接ELISA方法不能灵敏地检测新鲜病汁液中的病毒粒子,免疫扩散不能用于未变性病毒粒子的检测。Western blot灵敏度高,但操作复杂。火箭免疫电泳和对流免疫电泳均具有操作简单、灵敏度高、检测周期短的优点,但前者需抗血清的量大。5种免疫检测技术中,对流免疫电泳最适合浙贝母病样中TFMV的快速检测。

Tobacco Yellow dwarf Virus完整基因组上串联重复序列分布

利用自编计算机程序提取并展示Tobacco Yellow dwarf Virus完整基因组上串联重复序列分布特性。借助MATLAB软件和借用最优完全子图算法,提取并展示NCBI数据库中Tobacco Yellow dwarf Virus完整基因组(NC_003822.1)上串联重复序列分布特性。

Tobacco Vein Mottling Virus完整基因组上串联重复序列分布

利用自编计算机程序提取并展示Tobacco Vein Mottling Virus完整基因组上串联重复序列分布特性,从而为有效利用串联重复序列分布特性研究完整基因组的结构和功能、遗传和变异规律提供完备、细仔的数据支撑。

Five quinolizidine alkaloids with anti-tobacco mosaic virus activities from two species of Sophora

Three novel matrine-type alkaloids(1-3)and two unprecedented aloperine-type alkaloids(4 and 5)were isolated from the root of Sophora tonkinensis and the seeds of Sophora alopecuroides respectively.Notably,compound 1 possessed an unprecedented 6/5/6 tricyclic skeleton,while compounds 2 and 3 characterized by rare 6/6/5/6 tetracyclic system and 6/6/6/6/6 pentacyclic system respectively.Moreover,compound 4 possessed an unprecedented 6/7/6/6 tetracyclic core,and compound 5 characterized by rare 6/6/6/6tetracyclic skeleton.Their structures were elucidated by comprehensive spectroscopic data analysis and electronic circular dichroism(ECD)calculations.Biological tests indicated that compound 5 displayed significant anti-tobacco mosaic virus(TMV)activity compared with the positive control ningnanmycin.

Molecular characterization of Apple necrotic mosaic virus identified in crabapple(Malus spp.) tree of China

Apple necrotic mosaic virus(ApNMV) was identified in crabapple trees with mosaic symptoms from Zaozhuang, Shandong Province, China, by reverse transcription polymerase chain reaction(RT-PCR) analysis. The complete nucleotide sequences of one isolate from crabapple(ApNMV-Hai) and two isolates from apple(ApNMV-Hua and-Qu) were determined. The sizes of genomic RNA1, 2 and 3 of the three isolates differed from those of the previously reported isolate ApNMV-P126 from Japanese apple, especially RNA3. Compared with the nucleotide(nt) sequence of RNA3 in isolate P126, those in the Hai and Qu isolates were 7 and 33 nt shorter, respectively, and that of isolate Hua was 7 nt longer. Alignment analyses showed that these differences in size were mainly due to differences in the lengths of the 5′ untranslated region(UTR) and the UTR region between the ORFs encoding the movement protein and the coat protein. In the phylogenetic trees constructed using the full genomic sequences of RNA1, 2 and 3, the isolate Hai clustered into a group with the isolate Qu in the RNA1 tree, but formed an individual branch in the RNA2 and 3 trees. Three recombination events were identified in the nucleotide sequences of RNA1 and 2 among the isolates ApNMV-Hai,-Hua, and-Qu. This is the first report of the full genome sequence of ApNMV in crabapple.