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％。

Control Effects of Combination of Plant Induced Resistant Agents against Tobacco Mosaic Virus (TMV)

[ Objective] The paper was explore the control effects of combination of plant induced resistant agents against tobacco mosaic vires （TMV）. [ Method ] The control effects of 6 different combinations of plant induced resistant agents against TMV of flue-cured tobacco cultivar HangDa were studied under the environ- ment of simulated disease nursery. [ Result] The combination of 2 induced agents polypeptide-agent and 3-acetonyl-3-hydroxyoxindole （AHO） had good control effect against TMV, which could obviously delay the incidence time of TMV in infected tobacco plants. With water and Duxiao as control, their average control effects against TMV of tobacco plants during field period reached 69.64% and 43.25% after transplanting for 70 d. They also showed significant superiority accord- ing to Duncan＇s test （p = 0.05 ） in the aspects of plant height and leaf number, and the growth and development condition of leaves was good. Tobacco seedlings carrying TMV virus had no direct correlation with whether the symptoms performed, the seedlings carrying virus would perform symptom only when the incidence condition was suitable. The peak period for the incidence of TMV in seedlings carrying virus was during 19 d after transplanting. Spraying effective agents during nursery stage and field period, as well as promoting quick growth at the initial stage of tobacco seedlings after transplanting were the key measures to control its inci- dence. [ Conclusion] The study provided theoretical basis for preparing the control measures against TMV.

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.

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.

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.

CRISPR/CasRx-mediated resistance to Soybean mosaic virus in soybean

Soybean mosaic virus(SMV),an RNA virus,is the most common and destructive pathogenic virus in soybean fields.The newly developed CRISPR/Cas immune system has provided a novel strategy for improving plant resistance to viruses;hence,this study aimed to engineer SMV resistance in soybean using this system.Specifically,multiple sgRNAs were designed to target positive-and/or negative-sense strands of the SMV HC-Pro gene.Subsequently,the corresponding CRISPR/CasRx vectors were constructed and transformed into soybeans.After inoculation with SMV,39.02%,35.77%,and 18.70%of T_(1)plants were confirmed to be highly resistant(HR),resistant(R),and mildly resistant(MR)to SMV,respectively,whereas only 6.50%were identified as susceptible(S).Additionally,qRT-PCR and DAS-ELISA showed that,both at 15 and 30 d post-inoculation(dpi),SMV accumulation significantly decreased or was even undetectable in HR and R plants,followed by MR and S plants.Additionally,the expression level of the CasRx gene varied in almost all T_(1)plants with different resistance level,both at 15 and 30 dpi.Furthermore,when SMV resistance was evaluated in the T_(2)generation,the results were similar to those recorded for the T_(1)generation.These findings provide new insights into the application of the CRISPR/CasRx system for soybean improvement and offer a promising alternative strategy for breeding for resistance to biotic stress that will contribute to the development of SMV-immune soybean germplasm to accelerate progress towards greater soybean crop productivity.

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.

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.

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.

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.

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.

Virus Movement Protein Gene Mediated Resistance Against Cucumber Mosaic Virus Infection

Tobacco ( Nicotiana tabacum L.) “NC89” plants were transformed with deletion mutant of cucumber mosaic virus (CMV) movement protein (MP) gene and full_length CMV MP gene, respectively. The transformed plants were analyzed with polymerase chain reaction (PCR), PCR_Southern, Southern and Western blots. R 0 generation of the transgenic plants were inoculated with CMV. Five out of 10 lines of tobacco plants (BMPK) transformed with CMV MP deletion mutant gene showed high resistance to CMV infection and remained symptomless for up to 50 days post_inoculation. In contrast, tobacco plants (BMPR) transformed with full_length CMV MP gene did not show resistance to CMV infection. However, most of the infected full_length CMV MP gene transgenic plants recovered by showing none or very mild mosaic symptoms in 40 days post_inoculation. The results of R 1 generation of the BMPK transgenic plants tested under field conditions showed that all 5 lines of transgenic plants could delay the virus disease development.

Molecular Tagging of a New Resistance Gene to Maize Dwarf Mosaic Virus Using Microsatellite Markers

With joint analysis based on the parents, F 1, F 2 and backcrosses, the authors found that the resistance of the maize inbred line Huangzaosi to the maize dwarf mosaic virus strain B was conditioned by a major gene and polygene, and identified a new major gene. Bulked segregate and microsatellite analysis of a F 2 progeny from the combination of Huangzaosi×Mo17 were used to identify the resistance gene, mdm1(t), on the long arm of chromosome 6. This new resistance gene is tightly linked to and located between the microsatellite markers loci, phi077 and bnlg391. The linkage distances between phi077-mdm1(t) and mdm1(t)-bnlg391 are 4.74 centiMorgan (cM) and 6.72 cM respectively.

Molecular Characterization of a Chinese Soybean Mosaic Virus Isolate by RT_PCR, cDNA Sequence Analysis and Direct Expression of PCR Products in Bacteria

Soybean mosaic virus (SMV) causes one of the most severe viral diseases in soybean ( Glycine max L.) and is known to contain many pathogenically and serologically related isolates. In the present study, the authors have obtained cDNAs to all cistrons of a Chinese SMV isolate, SMV_ZK, by RT_PCR. By analysing the nucleotide and amino acid sequence of the HC_PRO, NIb and CP cistrons, it was found that SMV_ZK was highly homologous to the G2 strain of SMV, thus confirming the existence of G2_like isolates in soybean crop in China. The amplified cDNAs were directly cloned into a bacterial expression vector. With the exception of the P3 cistron, expression of the cDNAs of all other cistrons in bacteria gave rise to polypeptides of expected molecular weight. The expressed viral proteins were subsequently purified by gel elution. The preparation of viral_specific cDNAs and gene products will be useful in future functional study of the SMV genome.