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.

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.

Emergence of a new satellite RNA from cucumber mosaic virus isolate P1

The cucumber mosaic virus (CMV) isolate P1 caused very mild symptoms on many plant species. After serial passages by mechanical inoculation over five years, CMV P1 caused severe symptoms on several tobacco cultivars and tomato. A specific band of approximately 0.3 kb in length was amplified by RT PCR with primers synthesized based on reported CMV satellite RNA (satRNA) sequences. Sequence analysis showed there were two satRNAs (Sat P1 1 and Sat P1 2). Sat P1 1 contained 335 nucleotides, and Sat P1 2 contained 394 nucleotides. These two satRNAs shared 64% overall nucleotide sequence homology, and differences between the two satRNAs included mutations as well as deletions. Sat P1 1 was identical to a satRNA (Z96099) reported in 1995 in CMV P1. Based on differences in the sequence and secondary structure between these two satRNAs, we conclude that Sat P1 2 represents the emergence of a new satellite (necrotic satellite) from attenuated satRNA populations. The possible effect of the emergence of this new satRNA is discussed.

Study on Graft-induced Resistance to Cucumber Mosaic Virus in Pepper

To introduce CMV resistance to susceptible variety, interspecific grafting between d45-6 (Capsicum annuum) and LS1205 (C. baccatum) was conducted. The graft-progenies, G1 and G1S1 were obtained. Mechanical inoculation with CMV showed that disease index of G1 and G1S1 were significantly decreased, similar to that of the resistant stock. ELISA serological test indicated that resistance of LS1205 and the graft-progenies to CMV was caused by inhibition of virul replication. Further, peroxidase isozymatic analysis revealed that zymotypes of G1 were a summation of those found in the donor plants. This result perfectly fit the theory of gene transformation and reconfirmed the conclusion of mechanism of graft-induced variants.

A Review on the Pathogenicity of Cucumber Mosaic Virus

Cucumber mosaic virus （CMV）, a positive-sense single-stranded RNA virus, has a very wide host range and a worldwide distribution, and is considered as one of the most virulent plant viruses in the world. The CMV gene sequences, gene products, and their interaction with hosts have been extensively reported since it was discovered in 1916. With the development of high-throughput sequencing and proteomics, great progress has been made in the molecular mechanism of CMV pathogenesis in recent years. In this review, we introduce CMV-encoded proteins, CMV-related satellite RNAs and the roles of host factors in the pathogenesis of CMV, to provide a theoretical basis for future study of CMV.

A novel strategy to enhance resistance to Cucumber mosaic virus in tomato by grafting to transgenic rootstocks

Cucumber mosaic virus（CMV） can infect a wide range of host species. For the lacking of CMV resistant varieties of tomato, RNA interference（RNAi） can be used as a fast and effective method for the generation of transgenic resistant varieties. In this current study, five intron-spliced hairpin RNA（ihp RNA） plant expression vectors aimed at five genes of CMV have been constructed. Transgenic tomatoes were obtained by Agrobacterium tumefaciens-mediated transformation with expression vectors. Highly resistant generations of transgenic plants were employed as rootstocks and grafted onto non-transgenic tomatoes that resulted in the successful transfer of resistance to the scions. Using a novel method of plant cuttings for rootstock propagation, we obtained large quantities of disease-resistant material. Further, this method produces scions that can remain undetectable for transgenic resistance marker genes that may provide novel approaches to evade collective concerns about genetically-modified organism（GMO） biosafety.

Microarray-based identification of tomato microRNAs and time course analysis of their response to Cucumber mosaic virus infection

A large number of plant microRNAs (miRNAs) are now documented in the miRBase, among which only 30 are for Solanum lycopersicum (tomato). Clearly, there is a far-reaching need to identify and profile the expression of miRNAs in this important crop under various physiological and pathological conditions. In this study, we used an in situ synthesized custom microarray of plant miRNAs to examine the expression and temporal presence of miRNAs in the leaves of tomato plants infected with Cucumber mosaic virus (CMV). Following computational sequence homology search and hairpin structure prediction, we identified three novel tomato miRNA precursor genes. Our results also show that, in accordance with the phenotype of the developing leaves, the tomato miRNAs are differentially expressed at different stages of plant development and that CMV infection can induce or suppress the expression of miRNAs as well as up-regulate some star miRNAs (miRNA*s) which are normally present at much lower levels. The results indicate that developmental anomalies elicited by virus infection may be caused by more complex biological processes.

Recombination with coat protein transgene in a complementation system based on Cucumber mosaic virus (CMV)

In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an Nsi I site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants expressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination between the CMV vector and the CP transgene was proved by retro-transcriptional polymerase chain reaction (RT-PCR) and verified by DNA sequencing. Our results argue against the feasibility of the CMV-based replacement vector trans-complemented by the CP transgene, and at the same time, enlighten ways to improve the CMV-based expression vector and the biosafety of CMV CP-mediated virus resistant transgenic plants.

Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus

Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMMV)-based virus vectors have been constructed, SGP of the coat protein(CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site(TSS) as previous study identified by random amplification of c DNA ends(RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein(MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein(EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP.

An Attenuated Strain of Cucumber Green Mottle Mosaic Virus as a Biological Control Agent against Pathogenic Viral Strains

Cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus, causes a severe disease of cucurbits. In the Moscow region of Russian Federation, the incidence of infection on cucumber plants in greenhouses is high;however, the virus is poorly studied. In this work, the full-length genomes of two pathogenic MC-1 and MC-2 strains of CGMMV isolated from cucumber plants grown in greenhouses in the Moscow region and the attenuated VIROG-43M strain were sequenced. Comparison of VIROG-43M nucleotide sequence with those of the pathogenic strains revealed three missense mutations. Their role in attenuation is discussed. For the first time, in a number of trials conducted under laboratory conditions and in commercial greenhouses, the efficiency of the attenuated VIROG-43M strain as a biocontrol agent for cucumber plant protection resulting in significant yield gain was demonstrated. Phylogenetic analysis with 83 full-length CGMMV coat protein genes isolated in 16 different countries showed that Russian strains are related to isolates from Spain, Greece, USA and Israel.

Effects of Different Treatment Methods on Virus Elimination of Chrysanthemum morifolium 'Shenma'

[Objective]The paper was to explore the virus elimination technique of Chrysanthemum morifolium′Shenma′.[Method]With C.morifolium ′Shenma′as the test materials,the virus elimination effects of shoot-tip treatment,shoot-tip with virazole treatment and shoot-tip with heat treatment were used to study the effect of virus elimination in the paper.The shoot-tips with the lengths of 0.2-0.3,0.4-0.5,0.6-0.8 and 0.9-1.0 mm were cultured by the shoot-tip treatment.A randomized complete blocks design was conducted to screen the best shoot-tip length and virazole concentration by shoot-tip with virazole treatment.The test-tube seedling were cultured by the method of variable temperature at day and night and stepwise temperature rising.ELISA test was used to identify the elimination efficiencies.[Result]The shoot-tip with the length of 0.4-0.5 mm was the best in shoot-tip treatment;the combination of shoot-tip length of 0.4-0.5mm and virazole mass concentration of 10 mg/L was the best in shoot-tip with virazole treatment;the shoot-tip with the length of 0.4-0.5 mm stripped after heat treatment for 45d was the best in shoot-tip with heat treatment.[Conclusion]The study provides an effective way for virus elimination in C. morifolium′Shenma′.

Establishment of Multiplex RT-PCR Detection System for Three Viruses in Freesia

The specific primers were designed according to conserved sequences of coat protein （CP） gene of Freesia mosaic virus （FreMV）, Cucumber mosaic vi- rus （CMV） and Bean yellow mosaic virus （BYMV） published in GenBank, and a multiplex PCR protocol for simultaneous detection of these three viruses in freesia was developed. Three specific fragments were simultaneously amplified in a single PCR reaction. Their lengths were determined to be 340,628 and 212 bp, respec-tively. The sequence analysis indicated that three viruses shared at least 97% of homology with reference sequence. Sensitivity test showed that these three viruses could be detected out in the infected plant tissue greater than 10^-2 mg.

High-throughput Sequencing Reveals vsiRNAs Derived from Cucumber green mottle mosaic virus-infected Watermelon

Cucumber green mottle mosaic virus(CGMMV) is a member of the genus Tobamovirus, and is a serious pathogen of Cucurbitaceae crops. Virusderived small interfering RNAs(vsi RNAs), which are processed by Dicer-like and Argonaute proteins as well as RNA-dependent RNA polymerase,mediate the silencing of viral genomic RNA and host transcripts. To identify the CGMMV derived vsi RNAs and reveal interactions between CGMMV and watermelon host plant, deep sequencing technology was used to identify and characterize the vsi RNAs derived from CGMMV in infected watermelon plants in present study. A total of 10 801 368 vsi RNA reads representing 71 583 unique s RNAs were predicted in CGMMVinoculated watermelon plants. The CGMMV vsi RNAs were mostly 21 or 22 nt long. The majority of the CGMMV vsi RNAs(i.e., 91.7%) originated from the viral sense strand. Additionally, uracil was the predominant 5′-terminal base of vsi RNAs. Furthermore, the putative targets and functions of some of the CGMMV vsi RNAs were predicted and investigated. The results enhance our understanding of the interaction between CGMMV and the host watermelon and provide molecular basis for CGMMV resistance improvement in watermelon and other Cucurbitaceae crops.

The RNA-binding domain of DCL3 is required for long-distance RNAi signaling

Small RNA(sRNA)-mediated RNA silencing(also known as RNA interference,or RNAi)is a conserved mechanism in eukaryotes that includes RNA degradation,DNA methylation,heterochromatin formation and protein translation repression.In plants,sRNAs can move either cell-to-cell or systemically,thereby acting as mobile silencing signals to trigger noncell autonomous silencing.However,whether and what proteins are also involved in noncell autonomous silencing have not been elucidated.In this study,we utilized a previously reported inducible RNAi plant,PDSi,which can induce systemic silencing of the endogenous PDS gene,and we demonstrated that DCL3 is involved in systemic PDS silencing through its RNA binding activity.We confirmed that the C-terminus of DCL3,including the predicted RNA-binding domain,is capable of binding short RNAs.Mutations affecting RNA binding,but not processing activity,reduced systemic PDS silencing,indicating that DCL3 binding to RNAs is required for the induction of systemic silencing.Cucumber mosaic virus infection assays showed that the RNA-binding activity of DCL3 is required for antiviral RNAi in systemically noninoculated leaves.Our findings demonstrate that DCL3 acts as a signaling agent involved in noncell autonomous silencing and an antiviral effect in addition to its previously known function in the generation of 24-nucleotide sRNAs.

华南三省(自治区)部分西番莲种植区花叶病病原检测及EAPV多聚蛋白基因序列分析

西番莲(Passiflora edulis Sims.)是热带亚热带地区重要经济作物,我国福建、广东、广西、海南等地广泛种植[1]。调查发现,我国南方多地西番莲花叶病为害严重,症状表现为叶片花叶、黄化、斑驳、畸形,甚至整株褪绿,严重影响西番莲的产量和品质。

Effects of LaCl_3 on the growth and photosynthetic characteristics of Fny-infected tobacco seedlings

Plant growth, gas exchange characteristics, chlorophyll fluorescence, disease index, and disease prevention efficiency of LaCl3 in tobacco （Nicotiana glutinosa） plants infected by Fny-CMV （fny stain of cucumber mosaic virus） strain were determined. Leaf area, chloro-phyll and carotenoid contents, maximum photosynthesis rate, apparent quantum yield, and carboxylation efficiency dramatically decreased after 5 weeks post inoculation. The plants infected by Fny-CMV only presented much severer symptom than those infected in the presence of appropriate concentration of LaCl3. ETR （apparent rate of photosynthetic electron transport）, NPQ （nonphotochemical quenching）, qP （coef-ficient of photochemical quenching）, and yield （II） in infected tobacco plants obviously reduced in higher light intensity after 5 weeks post inoculation. And these fluorescence parameters in Fny-infected plants obviously reduced compared with those in the plants infected by Fny in the presence of LaCl3. Together with the growth status and disease index, it revealed that exogenously appropriate concentration of LaCl3 could significantly alleviate the damage of tobacco seedlings caused by Fny-CMV.

Three-dimensional reconstruction and comparison of vacuolar membranes in response to viral infection

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional(3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus(CMV) or tobacco necrosis virus A Chinese isolate(TNV-AC), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1 a and 2 a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast.Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant(+) RNA virus replication complexes.

Design, Synthesis, and Antiviral Activity of Novel Chalcone Derivatives Containing a Purine Moiety

To find new antiviral agents,novel chalcone derivatives containing a purine moiety were designed and synthesized by combining bioactive substructures.The antiviral activities of the derivatives against tobacco mosaic virus （TMV） and cucumber mosaic virus （CMV） were evaluated.Results showed that most of the derivatives showed antiviral activities.Compounds 3n and 3p exhibited excellent curative,protective and inactivation activities against TMV,with the EC50values of 452.4,416.2,241.2 and 438.7,418.6,261.7 μg·mL-1,respectively,which were better than those of ribavirin （585.8,436.0 and 268.7 μg·mL-1）.Compounds 3n and 3p showed remarkable curative and protective activities against CMV.Compound 3n showed a moderate affinity to TMV coat protein,with binding constant Ka and Kd values of 1.5 × 104 L·mol-1 and 79.8 μmol·L-1,respectively.These findings provided an important structural insight for further designs of highly active chalcone derivatives and a basis for further study on their mechanism of action.