一个双价锤头型核酶在体外对两种不同植物病毒RNA的专一切割作用

根据锤头核酶模型,设计合成了一个以黄瓜花叶病毒（ＣＭＶ） 外壳蛋白（ＣＰ） 亚基因组ＲＮＡ 为底物的锤头型核酶（ＲＺＣ） 。在证明它能有效切割该底物后,再将这个核酶与一个能专一性切割烟草花叶病毒（ＴＭＶ） 移动蛋白（ ＭＰ） 亚基因组ＲＮＡ 的锤头型核酶（ＲＺ１） 相互串联构成了一个双价核酶（ＲＺ１Ｃ） 。体外结果表明,这个双价核酶能与相应的单价核酶ＲＺ１ 和ＲＺＣ 一样专一而有效地切割ＣＭＶＣＰ和ＴＭＶ ＭＰＲＮＡ。     

Anti-sense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus

Three synthetic genes for the production of anti-sense RNA to different regions of the cucumber mosaic virus (CMV) genome were constructed using virus-derived double-stranded cDNA coupled to a promoter sequence from cauliflower mosaic virus. The genes were used to transform tobacco plants by a Ti plasmid vector. Transgenic plants obtained with the three constructs produced anti-sense RNA at different levels. Plants expressing each of the three anti-sense RNAs were inoculated with CMV and their sensitivity to the virus infection was compared with the non-transformed plants. Only one plant line which expressed relatively low levels of one of the anti-sense RNAs showed resistance to CMV but other plants expressing the same or the other two antisense RNAs had similar sensitivity to CMV infection as the non-transformed plants.     

应用RNAi技术培育抗TMV病毒转基因烟草

利用烟草花叶病毒(TMV)外壳蛋白基因构建RNAi干涉载体, 通过叶盘法转化至烟草K326 和龙江911两个栽培品种。对转基因株系的荧光定量PCR分析表明, 不同转基因株系的病毒RNA靶序列都得到一定程度的降解, 抗病性鉴定结果证实, 转基因K326和龙江911两个栽培品种的转基因材料分别有83％和90％转基因株系对TMV呈现免疫级抗性。     

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

Expression of a reporter gene is reduced by a ribozyme in transgenic plants

A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene as an indicator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentrations of ribozyme and target RNA were shown to be similar in the resulting single transformants. Direct evidence for a correlation between reduced npt gene expression and ribozyme expression was provided by crossing a plant containing only the ribozyme gene with a transgenic plant expressing the npt gene under control of the 35S promoter, i.e. the same promoter used to direct ribozyme expression. The expression of npt was reduced in all progeny containing both transgenes. Both steady-state levels of npt mRNA and amounts of active NPT enzyme are decreased. In addition, our data indicate that, at least in stable transformants, a large excess of ribozyme over target is not a prerequisite for achieving a significant reduction in target gene expression.     

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.     

The effect of structure in a long target RNA on ribozyme cleavage efficiency.

Inhibition of gene expression by catalytic RNA (ribozymes) requires that ribozymes efficiently cleave specific sites within large target RNAs. However, the cleavage of long target RNAs by ribozymes is much less efficient than cleavage of short oligonucleotide substrates because of higher order structure in the long target RNA. To further study the effects of long target RNA structure on ribozyme cleavage efficiency, we determined the accessibility of seven hammerhead ribozyme cleavage sites in a target RNA that contained human immunodeficiency virus type 1 (HIV-1) vif - vpr . The base pairing-availability of individual nucleotides at each cleavage site was then assessed by chemical modification mapping. The ability of hammerhead ribozymes to cleave the long target RNA was most strongly correlated with the availability of nucleotides near the cleavage site for base pairing with the ribozyme. Moreover, the accessibility of the seven hammerhead ribozyme cleavage sites in the long target RNA varied by up to 400-fold but was directly determined by the availability of cleavage sites for base pairing with the ribozyme. It is therefore unlikely that steric interference affected hammerhead ribozyme cleavage. Chemical modification mapping of cleavage site structure may therefore provide a means to identify efficient hammerhead ribozyme cleavage sites in long target RNAs.     

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection

RNA-interference (RNAi) silences gene expression by'guiding mRNA degradation in asequence-specific fashion.Small interfering RNA (siRNA),an intermediate of the RNAi pathway,has beenshown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells.Here,wereport that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) couldinhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-asso-ciated 126 kDa protein in intact plant tissue.Our results indicate that transiently expressed shRNA efficientlyinterfered with TMV infection.The interference observed is sequence-specific,and time-and site-dependent.Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumbermosaic virus (CMV),an unrelated tobamovirus.In order to interfere with TMV accumulation in tobaccoleaves,it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation.Ourresults support the view that RNAi opens the door for novel therapeutic procedures against virus diseases.We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expressioncould be employed as a potent antiviral treatment in plants.     

Transgenic tobacco plants expressing the bacterial mc gene resist virus infection

A bacterial rnc gene coding for a double-stranded RNA-dependent RNase III endoribonuclease and a mutant, rnc70, were expressed in tobacco plants. The RNase III protein produced in the transgenic plants was the same size as the bacterial protein. Expression of the wild-type gene could cause stunting in some plant lines, but not in others. Expression of the mutant protein did not affect normal growth and development of the transgenic plants. Transgenic plants of the R1 and R2 generations, expressing the wild type, as well as a mutant protein, were resistant to infection by three disparate RNA plant viruses with a divided genome but not against two viruses with a single-stranded RNA genome. Introduction of the rnc gene in crop plants may provide resistance to economically important virus diseases.     

The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants

As the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY), in transgenic tobacco plants. Tobacco lines expressing the foreign gene at varying levels were examined for resistance against TEV and PVY infection. There was a clear, direct correlation between the level of oryzacystatin message, inhibition of papain (a cysteine proteinase), and resistance to TEV and PVY in all lines tested. The inhibitor was ineffective against tobacco mosaic virus (TMV) infection because processing of this virus does not involve cysteine proteinases. These results show that plant cystatins can be used against different potyviruses and potentially also against other viruses, whose replication involves cysteine proteinase activity.     

Strategies for the suppression of peroxidase gene expression in tobacco. I. Designing efficient ribozymes

Five short hammerhead ribozymes (Rzs) were constructed and tested, using a range ofin vitro reaction conditions, for catalytic activity against the mRNA encoding the lignin-forming peroxidase (TPX) of tobacco. Although all 5 Rzs were shown to be able to cleave the RNA substrate, percentage cleavage varied with pre-denaturation of Rz and substrate, incubation temperature, length of incubation and ribozyme (Rz)-to-substrate ratio. One Rz with two catalytic units and 60 nucleotides of complementary sequence in 3 regions was shown to most efficiently cleave the substrate under allin vitro conditions tested. This ribozyme cleaved better than the two single ribozymes from which it was made. The superior cleaving ability of this Rz was shown to be due to the accessibility of the chosen target site and to the increased length of the hybridizing arms spanning this accessible region of the RNA.     

Milestones in the research on tobacco mosaic virus

Beijerinck's (1898) recognition that the cause of tobacco mosaic disease was a novel kind of pathogen became the breakthrough which eventually led to the establishment of virology as a science. Research on this agent, tobacco mosaic virus (TMV), has continued to be at the forefront of virology for the past century. After an initial phase, in which numerous biological properties of TMV were discovered, its particles were the first shown to consist of RNA and protein, and X-ray diffraction analysis of their structure was the first of a helical nucleoprotein. In the molecular biological phase of research, TMV RNA was the first plant virus genome to be sequenced completely, its genes were found to be expressed by cotranslational particle disassembly and the use of subgenomic mRNA, and the mechanism of assembly of progeny particles from their separate parts was discovered. Molecular genetical and cell biological techniques were then used to clarify the roles and modes of action of the TMV non-structural proteins: the 126 kDa and 183 kDa replicase components and the 30 kDa cell-to-cell movement protein. Three different TMV genes were found to act as avirulence genes, eliciting hypersensitive responses controlled by specific, but different, plant genes. One of these (the N gene) was the first plant gene controlling virus resistance to be isolated and sequenced. In the biotechnological sphere, TMV has found several applications: as the first source of transgene sequences conferring virus resistance, in vaccines consisting of TMV particles genetically engineered to carry foreign epitopes, and in systems for expressing foreign genes. TMV owes much of its popularity as a research mode to the great stability and high yield of its particles. Although modern methods have much decreased the need for such properties, and TMV may have a less dominant role in the future, it continues to occupy a prominent position in both fundamental and applied research.     

Leaf proteins in transgenic tobacco plants, expressing dual-stranded RNA in viral infection

Generation of transgenic tobacco plants, producing double-stranded RNA with no homology to tobacco genome sequences is reported. The RNA synthesis is mediated by a construct containing an inverted repeat of the pBR322 tetracycline-resistance gene fragment under control of the 35S CaMV promoter. Analysis of the resistance of transgenic plants to the tobacco mosaic virus revealed the changes in the protein spectra of the infected plants. The 25- and 30-kDa proteins found were not detected in the extracts of normal plants. Amino acid sequencing of the 30-kDa peptide with subsequent computer database search revealed the homology of this protein to the hydrolases belonging to the group of plant beta-glucanases. The role of the novel polypeptides in an increase of the resistance of transgenic plants to TMV, and also the possibility of the regulation of their expression by nonhomologous dsRNA are discussed.     

Expression of an extracellular ribonuclease gene increases resistance to Cucumber mosaic virus in tobacco

Background

The apoplast plays an important role in plant defense against pathogens. Some extracellular PR-4 proteins possess ribonuclease activity and may directly inhibit the growth of pathogenic fungi. It is likely that extracellular RNases can also protect plants against some viruses with RNA genomes. However, many plant RNases are multifunctional and the direct link between their ribonucleolytic activity and antiviral defense still needs to be clarified. In this study, we evaluated the resistance of Nicotiana tabacum plants expressing a non-plant single-strand-specific extracellular RNase against Cucumber mosaic virus.

Results

Severe mosaic symptoms and shrinkage were observed in the control non-transgenic plants 10 days after inoculation with Cucumber mosaic virus (CMV), whereas such disease symptoms were suppressed in the transgenic plants expressing the RNase gene. In a Western blot analysis, viral proliferation was observed in the uninoculated upper leaves of control plants, whereas virus levels were very low in those of transgenic plants. These results suggest that resistance against CMV was increased by the expression of the heterologous RNase gene.

Conclusion

We have previously shown that tobacco plants expressing heterologous RNases are characterized by high resistance to Tobacco mosaic virus. In this study, we demonstrated that elevated levels of extracellular RNase activity resulted in increased resistance to a virus with a different genome organization and life cycle. Thus, we conclude that the pathogen-induced expression of plant apoplastic RNases may increase non-specific resistance against viruses with RNA genomes.

     

Expression of recombinant trichosanthin,a ribosome-inactivating protein,in transgenic tobacco

Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.     

Cross protection in transgenic tobacco plants expressing a mild strain of tobacco mosaic virus

Summary Cross protection of plant viruses is a phenomenon in which plants infected with one strain of a virus are protected from the effects of superinfection by other related strains. Recently, we have succeeded in the introduction and expression of a cDNA copy of the tobacco mosaic virus (TMV) genomic RNA in transgenic tobacco plants. Using this system, we introduced a cDNA copy of a mild strain of TMV into tobacco plants. The transgenic plants did not develop any severe symptoms upon inoculation with a virulent TMV strain, indicating that these transgenic plants were cross protected against TMV infection. The system described here can be a useful model system to study the mechanism(s) of cross protection.     

Novel N gene-associated,temperature-independent resistance to the movement of tobacco mosaic virus vectors neutralized by a cucumber mosaic virus RNA1 transgene

The N gene conditions for resistance to Tobacco mosaic virus (TMV) but only below 28 degrees C. However, a TMV-based vector expressing green fluorescent protein (TMV-GFP) showed only limited movement at 33 degrees C in tobacco plants harboring the N gene and other genes cointrogressed from Nicotiana glutinosa. TMV-GFP moved efficiently in tobacco plants that either lacked these genes or that contained the N gene but were transgenic for RNA1 of Cucumber mosaic virus. These findings identified novel temperature-independent resistance to the movement of TMV-GFP which could be neutralized by a different viral transgene. Using the N gene and nahG gene-transgenic tobacco, we show that this novel resistance is manifested specifically by the N gene itself and operates via a pathway independent of salicylic acid.     

Ribozymes which cleave arenavirus RNAs: identification of susceptible target sites and inhibition by target site secondary structure.

The development of safe and effective antiviral agents has been a slow process, largely because of the difficulty in distinguishing between virus and host functions; materials toxic to the virus are frequently harmful also to the host in which the agent resides. Recently, techniques which target nucleic acid sequences as a means of reducing gene expression have emerged. This antisense armamentarium includes ribozymes, RNA enzymes which cleave other RNA molecules in a sequence-specific manner. We wish to assess the ability of ribozymes to control animal virus infection. Reasoning that the viruses most vulnerable to ribozyme intervention will be those whose complete life cycle is based on RNA (with no DNA stage), we have begun to develop ribozymes directed toward lymphocytic choriomeningitis virus (LCMV), the prototype of the arenavirus family. Using ribozymes of the hammerhead variety, we have identified several sites on the LCMV genome which can be efficiently cleaved in trans. The efficiency of cleavage is site dependent, and we demonstrate that secondary structure at the target site can abolish ribozyme cleavage. Computer-assisted analysis indicates that much of the LCMV genome may be involved in base pairing, which may render it similarly resistant to ribozyme attack. The few remaining open regions of LCMV lack a GUC target site, on which most studies to date have relied. Here we show that AUC, CUC, and AUU are alternative sites which can be cleaved by trans-acting ribozymes. This finding is important given the aforementioned restriction of available sites, imposed by secondary structure.     

Downregulation of a pathogen-responsive tobacco UDP-Glc:phenylpropanoid glucosyltransferase reduces scopoletin glucoside accumulation,enhances oxidative stress,and weakens virus resistance

Plant UDP-Glc:phenylpropanoid glucosyltransferases (UGTs) catalyze the transfer of Glc from UDP-Glc to numerous substrates and regulate the activity of compounds that play important roles in plant defense against pathogens. We previously characterized two tobacco salicylic acid- and pathogen-inducible UGTs (TOGTs) that act very efficiently on the hydroxycoumarin scopoletin and on hydroxycinnamic acids. To identify the physiological roles of these UGTs in plant defense, we generated TOGT-depleted tobacco plants by antisense expression. After inoculation with Tobacco mosaic virus (TMV), TOGT-inhibited plants exhibited a significant decrease in the glucoside form of scopoletin (scopolin) and a decrease in scopoletin UGT activity. Unexpectedly, free scopoletin levels also were reduced in TOGT antisense lines. Scopolin and scopoletin reduction in TOGT-depleted lines resulted in a strong decrease of the blue fluorescence in cells surrounding TMV lesions and was associated with weakened resistance to infection with TMV. Consistent with the proposed role of scopoletin as a reactive oxygen intermediate (ROI) scavenger, TMV also triggered a more sustained ROI accumulation in TOGT-downregulated lines. Our results demonstrate the involvement of TOGT in scopoletin glucosylation in planta and provide evidence of the crucial role of a UGT in plant defense responses. We propose that TOGT-mediated glucosylation is required for scopoletin accumulation in cells surrounding TMV lesions, where this compound could both exert a direct antiviral effect and participate in ROI buffering.