Synthesis of the tobacco mosaic virus in blocked spreading of infection

The possibility of infection of tobacco upper leaves with tobacco mosaic virus (TMV) was examined in experiments where the inoculum was imbibed through the cut stem. The inoculum used were: a) a preparation of a virus-specific informosome-like ribonucleoproteins (vRNP) isolated from TMV-infected plants; b) a TMV preparation; or c) a mixture of TMV and vRNP. Multiplication of TMV in upper leaves was observed in neither of the variants; nevertheless in the vascular tissue and/or probably in adjoining parenchymal cells, two kinds of RNA were synthesized: of mol. w. (1.1--1.3) X 10(6) and (0.6--0.8) X 10(6). These RNA were not found in healthy plants in the presence of actinomycin D. The synthesis of genomic TMV RNA is suppressed under these conditions. Thus, some kind of abortive TMV infection takes place under the condition of experimental inoculation of plants through a cut stem. Molecular hybridization with the DNA of recombinant plasmid containing a nucleotide sequence complementary to the 3'-portion of genomic TMV RNA proves that short RNAs synthesized under the abortive infection conditions are TMV-specific. The experiments with differential temperature treatment of N-gene-containing plants under abortive infection conditions suggest that necrotization is not necessarily induced by genomic TMV RNA synthesis.     

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.     

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.     

A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco

Ribozymes of the hammerhead class can be designed to cleave a target RNA in a sequence-specific manner and can potentially be used to specifically modulate gene activity. We have targeted the tobacco mosaic virus (TMV) genome with a ribozyme containing three catalytic hammerhead domains embedded within a 1 kb antisense RNA. The ribozyme was able to cleave TMV RNA at all three target sites in vitro at 25°C. Transgenic tobacco plants were generated which expressed the ribozyme or the corresponding antisense constructs directed at the TMV genome. Six of 38 independent transgenic plant lines expressing the ribozyme and 6 of 39 plant lines expressing the antisense gene showed some level of protection against TMV infection. Homozygous progeny of some lines were highly resistant to TMV; at least 50% of the plants remained asymptomatic even when challenged with high levels of TMV. These plants also displayed resistance to infection with TMV RNA or the related tomato mosaic virus (ToMV). In contrast, hemizygous plants of the same lines displayed only very weak resistance when inoculated with low amounts of TMV and no resistance against high inoculation levels. Resistance in homozygous plants was not overcome by a TMV strain which was altered at the three target sites to abolish ribozyme-mediated cleavage, suggesting that the ribozyme conferred resistance primarily by an antisense mechanism.     

Biological activities of hybrid RNAs generated by 3''-end exchanges between tobacco mosaic and brome mosaic viruses.

Sequences within the conserved, aminoacylatable 3' noncoding regions of brome mosaic virus (BMV) genomic RNAs 1, 2, and 3 direct initiation of negative-strand synthesis by BMV polymerase extracts and, like sequences at the structurally divergent but aminoacylatable 3' end of tobacco mosaic virus (TMV) RNA, are required in cis for RNA replication in vivo. A series of chimeric RNAs in which selected 3' segments were exchanged between the tyrosine-accepting BMV and histidine-accepting TMV RNAs were constructed and their amplification was examined in protoplasts inoculated with or without other BMV and TMV RNAs. TMV derivatives whose 3' noncoding region was replaced by sequences from BMV RNA3 were independently replication competent when the genes for the TMV 130,000-M(r) and 180,000-M(r) replication factors remained intact. TMV replicase can thus utilize the BMV-derived 3' end, though at lower efficiency than the wild-type (wt) TMV 3' end. Providing functional BMV RNA replicase by coinoculation with BMV genomic RNAs 1 and 2 did not improve the amplification of these hybrid genomic RNAs. By contrast, BMV RNA3 derivatives carrying the 3' noncoding region of TMV were not amplified when coinoculated with wt BMV RNA1 and RNA2, wt TMV RNA, or all three. Thus, BMV replicase appeared to be unable to utilize the TMV 3' end, and there was no evidence of intervirus complementation in the replication of any of the hybrid RNAs. In protoplasts coinoculated with BMV RNA1 and RNA2, the nonamplifiable RNA3 derivatives bearing TMV 3' sequences gave rise to diverse new rearranged or recombined RNA species that were amplifiable.     

RNA-dependent RNA Polymerase Activities in Tomato Plants Susceptible or Resistant to Tobacco Mosaic Virus

RNA-dependent RNA polymerase activities were measured in healthyand tobacco mosaic virus (TMV)-infected tomato plants, to investigatethe possibility that altered activity might be involved in theoperation of the Tm-I gene for resistance to TMV. Healthy, susceptibleand resistant plants had similar levels of enzyme activity.Infection with TMV strain 0, which is inhibited by Tm-I, causeda 2-fold increase in activity in susceptible plants but no increasein Tm-I plants. Infection with a number of strain 1 isolates,which overcome Tm-I resistance, led to a 2 to 4-fold increasein enzyme activity in resistant plants. RNA-dependent RNA polymerase, Tm-I resistance gene, tobacco mosaic virus, tomato, Lycopersicon esculentum     

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

TMV介导的外源蛋白在植物中表达

烟草花叶病毒(Tobaccomosaicvirus,TMV)为Tobamovirus代表成员,以此病毒介导的外源蛋白在植物中表达,经过了十几年的研究和不断完善,已被证实为一种有效的表达外源蛋白的途径.这项技术已经在医用活性多肽以及疫苗的研制、功能基因的鉴定、植物体内生物合成途径的研究等方面发挥越来越重要的作用.重点阐述了TMV基因组RNA的结构和分子生物学特征,并着重对重组载体的构建及其利用加以了论述.     

Genetically engineered transgenic plants with the domain 1 sequence of tobacco mosaic virus 126 kDa protein gene are completely resistant to viral infection

In many plant RNA viruses, Domains 1, 2 and 3 are conserved in replicase proteins. In order to examine the interference of viral replication by the Domain 1 sequence, we generated transgenic plants transformed with DNA corresponding to the Domain 1 sequence of the TMV 126 kDa protein. This DNA sequence includes the TMV RNA from nucleotides 1 to 2,149, which comprises both the 5'-untranslated and methyl transferase region. The transgenic plants obtained showed complete resistance to TMV infection. The presence of the Domain 1 sequence in the plants completely prevented local necrosis in Nicotiana tabacum cv. Xanthi nc, and any systemic development of symptoms in Nicotiana tabacum Xanthi upon TMV inoculation. Most transgenic plants sustained the conferred resistance even under TMV inoculum concentrations up to as high as 1,000 microg/ml. To detect any accumulation of TMV coat protein or viral RNA in infected transgenic plants, immunochemical tests and Northern blot analyses were carried out. Neither viral RNA or coat protein was detectable in the systemic leaves of the completely resistant transgenic plants, whereas they were accumulated in large quantities in all of the control plants. Because of the conservation of Domain 1 in many plant RNA viruses, the acquisition of resistance to virus infection using the Domain 1 sequence appears to be a very effective strategy for breeding of viral resistant plants.     

Expression of the tobacco mosaic virus movement protein alters starch accumulation inNicotiana benthamiana

Nicotiana benthamiana plants were transformed with the movement protein (MP) gene of tobacco mosaic virus (TMV), usingAgrobacterium-mediated transformation. Plants regenerated from the transformed cells accumulated 30-kDa MP and complemented the activity of TMV MP when infected with chimeric TMVs containing defective MR These transgenic plants displayed stunting, pale-green leaves, and starch accumulations, indicating that TMV MP altered the carbon partitioning for leaves involved in TMV cell-to-cell movement.     

Replication of tobacco mosaic virus RNA

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.     

The coat protein of potato virus X is a strain-specific elicitor of Rx1-mediated virus resistance in potato

The Rx1 gene in potato confers extreme resistance to potato virus X (PVX). To investigate the mechanism and elicitation of Rx resistance, protoplasts of potato cv. Cara (Rx1 genotype) and Maris Bard (rx1 genotype) were inoculated with PVX and tobacco mosaic virus (TMV). At 24 h post-inoculation in Maris Bard protoplasts there was at least 100-fold more PVX RNA than in protoplasts of Cara. TMV RNA accumulated to the same level in both types of protoplast. However, when the TMV was inoculated together with PVX the accumulation of TMV RNA was suppressed in the Cara (Rx1 genotype) protoplasts to the same extent as PVX. The Rx1 resistance also suppressed accumulation of a recombinant TMV in which the coat protein gene was replaced with the coat protein gene of PVX. It is therefore concluded that Rx1-mediated resistance is elicited by the PVX coat protein, independently of any other proteins encoded by PVX. The domain of the coat protein with elicitor activity was localized by deletion and mutation analysis to the structural core of a non-virion form of the coat protein.     

The Association of "Pathogenesis-related" Proteins with Viral Induced Necrosis in Nicotiana sylvestris

The association of “pathogenesis-related” (PR) proteins with protection from superinfection, systemic acquired resistance and production of localized necrotic lesions was examined with a system using tobacco mosaic virus (TMV) and Nicotiana sylvestris. Leaves of N. sylvestris with a mosaic from earlier inoculation with a systemically infecting strain of TMV (TMV-C) and control plants were challenged with a necrotizing strain of TMV (TMV-P), RNA of TMV-P and turnip mosaic virus (TuMV). TMV-P virions produced localized necrotic lesions only in the dark green areas of the mosaic of TMV-C infected plants. Both RNA of TMV-P and TuMV produced localized necrotic lesions in both light green and dark green areas of the mosaic of TMV-C infected plants. All three challenge inocula produced localized necrotic lesions in previously uninoculated plants. Six days after challenge inoculation proteins were extracted from separated dark green and light green mosaic leaf tissue, and leaf material from control plants. Proteins were separated by electrophoresis in a 5 % polyacrylamide spacer gel and 10 % polyacrylamide running gel. PR proteins were found in tissue where localized necrotic lesions were produced as a result of challenge inoculation, but not in tissue that was not superinfected. PR proteins were not found in light green or dark green mosaic leaf tissue as a result of TMV-C inoculation. No PR proteins were evident in protein extracts from light green tissue challenged with TMV-P, although PR proteins were produced in dark green tissue, where necrosis occurred, from the same leaves. Systemic acquired resistance (reduction in size of lesions formed by a challenge inoculation) to TuMV or RNA of TMV-P and PR protein concentration was measured at various times in light green areas of mosaic leaves where dark green areas of the mosaic leaves had been inoculated with TMV-P. No quantitative or temporal relationship between the onset of resistance and PR protein production was found. It is concluded that PR proteins are a result of pathogen induced necrosis and not significantly involved in the mechanism(s) of viral induced resistance.     

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.