Nucleotide sequence of Chinese rape mosaic virus (oilseed rape mosaic virus), a crucifer tobamovirus infectious on Arabidopsis thaliana

The complete nucleotide sequence of Chinese rape mosaic virus has been determined. The virus is a member of the tobamovirus genus of plant virus and is able to infect Arabidopsis thaliana (L.) Heynh systemically. The analysis of the sequence shows a gene array that seems to be characteristic of crucifer tobamoviruses and which is slightly different from the one most frequently found in tobamoviruses. Based on gene organization and on comparisons of sequence homologies between members of the tobamoviruses, a clustering of crucifer tobamoviruses is proposed that groups the presently known crucifer tobamovirus into two viruses with two strains each. A name change of Chinese rape mosaic virus to oilseed rape mosaic virus is proposed.Abbreviations 2-ME 2-mercaptoethanol - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - UTR untranslated region - MP movement protein - CP capsid protein - CRMV Chinese rape mosaic virus - TVCV turnip vein clearing virus - PaMMV paprika mild mottle virus - PMMV-I pepper mild mottle virus (Italian isolate) - PMMV-S pepper mild mottle virus (Spanish isolate) - ToMV tomato mosaic virus - TMV tobacco mosaic virus - TMGMV tobacco mild green mosaic virus - ORSV odontoglossum ringspot virus - SHMV sunn hemp mosaic virus - CGMMV cucumber green mottle mosaic virus - ORMV oilseed rape mosaic virus     

The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.     

Evolutionarily related Sindbis-like plant viruses maintain different levels of population diversity in a common host

The levels of population diversity of three related Sindbis-like plant viruses, Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), and Cowpea chlorotic mottle virus (CCMV), in infections of a common host, Nicotiana benthamiana, established from genetically identical viral RNA were examined. Despite probably having a common evolutionary ancestor, the three viruses maintained different levels of population diversity. CMV had the highest levels of diversity, TMV had an intermediate level of diversity, and CCMV had no measurable level of diversity in N. benthamiana. Interestingly, the levels of diversity were correlated to the relative host range sizes of the three viruses. The levels of diversity also remained relatively constant over the course of serial passage. Closer examination of the CMV and TMV populations revealed biases for particular types of substitutions and regions of the genome that may tolerate fewer mutations.     

The symptom difference induced by Tobacco mosaic virus and Tomato mosaic virus in tobacco plants containing the N gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaicvirus (ToMV) are members of the genus Tobamoviruswith a world-wide distribution, and cause severe dis-eases on many economically important crops. TMVand ToMV have very close relationship and both havessRNA genome with a length of about 6400 nucleo-tides, encoding at least three nonstructural proteinsand a 17.6 kD coat protein (CP). Both 126 kD and 183kD proteins function as components of replicase, andthe 30 kD protein is involved in viral ce…     

Modification of small RNAs associated with suppression of RNA silencing by tobamovirus replicase protein

Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.     

A tobamovirus infecting capsicum in Australia

A tobamovirus infection of Capsicum annuum is recorded for the first time in New South Wales, Australia. The causal virus is described and shown to differ from tobacco (TMV) and tomato (ToMV) mosaic viruses in its host reactions and serology. Seventeen capsicum cultivars were tested for reaction to the Australian isolate and ranked according to symptom severity. Field and glasshouse observations indicated that the virus causes a decrease in height and weight of plants, fruit malformation and leaf mosaic symptoms. It is proposed that the capsicum tobamovirus strains are sufficiently distinctive from TMV and ToMV to be grouped together and designated a separate virus: capsicum mosaic virus (CaMV).     

Proof by synthesis of Tobacco mosaic virus

Background

Synthetic biology is a discipline that includes making life forms artificially from chemicals. Here, a DNA molecule was enzymatically synthesized in vitro from DNA templates made from oligonucleotides representing the text of the first Tobacco mosaic virus (TMV) sequence elucidated in 1982. No infectious DNA molecule of that seminal reference sequence exists, so the goal was to synthesize it and then build viral chimeras.

Results

RNA was transcribed from synthetic DNA and encapsidated with capsid protein in vitro to make synthetic virions. Plants inoculated with the virions did not develop symptoms. When two nucleotide mutations present in the original sequence, but not present in most other TMV sequences in GenBank, were altered to reflect the consensus, the derivative synthetic virions produced classic TMV symptoms. Chimeras were then made by exchanging TMV capsid protein DNA with Tomato mosaic virus (ToMV) and Barley stripe mosaic virus (BSMV) capsid protein DNA. Virus expressing ToMV capsid protein exhibited altered, ToMV-like symptoms in Nicotiana sylvestris. A hybrid ORF6 protein unknown to nature, created by substituting the capsid protein genes in the virus, was found to be a major symptom determinant in Nicotiana benthamiana. Virus expressing BSMV capsid protein did not have an extended host range to barley, but did produce novel symptoms in N. benthamiana.

Conclusions

This first report of the chemical synthesis and artificial assembly of a plant virus corrects a long-standing error in the TMV reference genome sequence and reveals that unnatural hybrid virus proteins can alter symptoms unexpectedly.     

TOM1 family conservation within the plant kingdom for tobacco mosaic virus accumulation

The susceptibility factor TOBAMOVIRUS MULTIPLICATION 1 (TOM1) is required for efficient multiplication of tobacco mosaic virus (TMV). Although some phylogenetic and functional analyses of the TOM1 family members have been conducted, a comprehensive analysis of the TOM1 homologues based on phylogeny from the most ancient to the youngest representatives within the plant kingdom, analysis of support for tobamovirus accumulation and interaction with other host and viral proteins has not been reported. In this study, using Nicotiana benthamiana and TMV as a model system, we functionally characterized the TOM1 homologues from N. benthamiana and other plant species from different plant lineages. We modified a multiplex genome editing tool and generated a sextuple mutant in which TMV multiplication was dramatically inhibited. We showed that TOM1 homologues from N. benthamiana exhibited variable capacities to support TMV multiplication. Evolutionary analysis revealed that the TOM1 family is restricted to the plant kingdom and probably originated in the Chlorophyta division, suggesting an ancient origin of the TOM1 family. We found that the TOM1 family acquired the ability to promote TMV multiplication after the divergence of moss and spikemoss. Moreover, the capacity of TOM1 orthologues from different plant species to promote TMV multiplication and the interactions between TOM1 and TOM2A and between TOM1 and TMV-encoded replication proteins are highly conserved, suggesting a conserved nature of the TOM2A–TOM1–TMV Hel module in promoting TMV multiplication. Our study not only revealed a conserved nature of a gene module to promote tobamovirus multiplication, but also provides a valuable strategy for TMV-resistant crop development.     

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.     

Induction of a hypersensitive response by chimeric helicase sequences of tobamoviruses U1 and Ob in N-carrying tobacco

Recently, the helicase domain of the Tobacco mosaic virus (TMV)-U1 replicase proteins (designated MOREHEL:U1) was identified as the elicitor of the N gene-mediated hypersensitive response (HR) in tobacco. In this study, we used agroinfiltration to express the equivalent MOREHEL domain of the non-HR-inducing tobamovirus strain TMV-Ob. It appeared that this MOREHEL:Ob sequence did not elicit a HR in N gene-carrying tobacco. Both MOREHEL sequences were divided into eight subdomains, and chimeras of MOREHEL sequences from U1 and Ob were constructed. Expression of these chimeric MOREHEL sequences revealed that, in the TMV-U1 MOREHEL sequence, at least four domains involved in full HR induction were present. The presence of at least three of these four domains seems a minimal requirement for HR induction. Two additional domains may play a minor role in HR induction. To study the elicitor function of the chimeras during the TMV life cycle, chimeric MOREHEL domains were introduced into full-length TMV cDNA clones. These constructs, however, were unable to establish an infection in Nicotiana benthamiana or Nicotiana tabacum plants.     

Eukaryotic elongation factor 1A interacts with the upstream pseudoknot domain in the 3' untranslated region of tobacco mosaic virus RNA

The genomic RNA of tobacco mosaic virus (TMV), like that of other positive-strand RNA viruses, acts as a template for both translation and replication. The highly structured 3' untranslated region (UTR) of TMV RNAs plays an important role in both processes; it is not polyadenylated but ends with a tRNA-like structure (TLS) preceded by a conserved upstream pseudoknot domain (UPD). The TLS of tobamoviral RNAs can be specifically aminoacylated and, in this state, can interact with eukaryotic elongation factor 1A (eEF1A)/GTP with high affinity. Using a UV cross-linking assay, we detected another specific binding site for eEF1A/GTP, within the UPDs of TMV and crucifer-infecting tobamovirus (crTMV), that does not require aminoacylation. A mutational analysis revealed that UPD pseudoknot conformation and some conserved primary sequence elements are required for this interaction. Its possible role in the regulation of tobamovirus gene expression and replication is discussed.     

Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing

Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana benthamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. benthamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing.     

Tobacco mosaic virus and the study of early events in virus infections

In order to establish infections, viruses must be delivered to the cells of potential hosts and must then engage in activities that enable their genomes to be expressed and replicated. With most viruses, the events that precede the onset of production of progeny virus particles are referred to as the early events and, in the case of positive-strand RNA viruses, they include the initial interaction with and entry of host cells and the release (uncoating) of the genome from the virus particles. Though the early events remain one of the more poorly understood areas of plant virology, the virus with which most of the relevant research has been performed is tobacco mosaic virus (TMV). In spite of this effort, there remains much uncertainty about the form or constituent of the virus that actually enters the initially invaded cell in a plant and about the mechanism(s) that trigger the subsequent uncoating (virion disassembly) reactions. A variety of approaches have been used in attempts to determine the fate of TMV particles that are involved in the establishment of an infection and these are briefly described in this review. In some recent work, it has been proposed that the uncoating process involves the bidirectional release of coat protein subunits from the viral RNA and that these activities may be mediated by cotranslational and coreplicational disassembly mechanisms.     

RCY1-mediated resistance to Cucumber mosaic virus is regulated by LRR domain-mediated interaction with CMV(Y) following degradation of RCY1

RCY1, which encodes a coiled coil nucleotide-binding site leucine-rich repeat (LRR) class R protein, confers the hypersensitive response (HR) to a yellow strain of Cucumber mosaic virus (CMV[Y]) in Arabidopsis thaliana. Nicotiana benthamiana transformed with hemagglutinin (HA) epitope-tagged RCY1 (RCY1-HA) also exhibited a defense response accompanied by HR cell death and induction of defense-related gene expression in response to CMV(Y). Following transient expression of RCY1-HA by agroinfiltration, the defense reaction was induced in N. benthamiana leaves infected with CMV(Y) but not in virulent CMV(B2)-infected N. benthamiana leaves transiently expressing RCY1-HA or CMV(Y)-infected N. benthamiana leaves transiently expressing HA-tagged RPP8 (RPP8-HA), which is allelic to RCY1. This result suggests that Arabidopsis RCY1-conferred resistance to CMV(Y) could be reproduced in N. benthamiana leaves in a gene-for-gene manner. Expression of a series of chimeric constructs between RCY1-HA and RPP8-HA in CMV(Y)-infected N. benthamiana indicated that induction of defense responses to CMV(Y) is regulated by the LRR domain of RCY1. Interestingly, in CMV(Y)-infected N. benthamiana manifesting the defense response, the levels of both RCY1 and chimeric proteins harboring the RCY1 LRR domain were significantly reduced. Taken together, these data indicate that the RCY1-conferred resistance response to CMV(Y) is regulated by an LRR domain-mediated interaction with CMV(Y) and seems to be tightly associated with the degradation of RCY1 in response to CMV(Y).     

Replication in the phloem is not necessary for efficient vascular transport of tobacco mosaic tobamovirus

Plant viruses move systemically from one leaf to another via phloem. However, the viral functions needed for systemic movement are not fully elucidated. An experimental system was designed to study the effects of low temperature on the vascular transport of the tobacco mosaic tobamovirus (TMV). Vascular transport of TMV from lower inoculated leaves to upper non-inoculated leaves via a stem segment kept at low temperature (4 degrees C) was not affected. On the other hand, several experiments were performed on tobacco leaves to demonstrate that virus replication did not occur at the same temperature. The data suggest that replication of TMV in the phloem of wild-type tobacco plants is not necessary for the vascular transport of TMV, and that the virus moves with photoassimilates as suggested previously.     

Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus

The tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll-interleukin-1 receptor/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N-mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1-, EDS1-, and NPR1/NIM1- like genes in N-mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1-like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR-NBS-LRR class of resistance proteins. Requirement of Rar1- like gene for N-mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR-NBS-LRR and CC-NBS-LRR proteins. The TRV based VIGS approach as described here to study N-mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.     

A spontaneous mutation in the movement protein gene of brome mosaic virus modulates symptom phenotype in Nicotiana benthamiana.

Brome mosaic virus (BMV) is a positive-strand RNA virus with a multipartite genome that causes symptomless infection in Nicotiana benthamiana. We have isolated and characterized a strain of BMV that produced uniform vein chlorosis in systemically infected N. benthamiana. Analysis of pseudorecombinants constructed by exchanging RNA 1 and 2 and RNA 3 components between wild-type (non-symptom-inducing) and vein chlorosis-inducing strains of BMV indicated that the genetic determinant for the induction of the chlorotic phenotype is located on RNA 3. Sequence analysis of progeny RNA 3 recovered from symptomatic N. benthamiana plants revealed that vein chlorosis is due to the single nucleotide transition 887G-->887A, which changes the codon for Val-266 to Ile-266 in the movement protein gene. The mutation had no detectable effect on the accumulation of virus in either inoculated or systematically infected leaves of N. benthamiana. The vein chlorosis phenotype is the manifestation of the substitution of Ile-266 for Val-266 in the movement protein gene, since additional alterations in this region (a silent mutation, i.e., 887GUU889-->GUC, and an alteration of valine to phenylalanine, i.e., 887GUU889-->887UUU889) resulted in symptomless infections on N. benthamiana. The modulation of the symptom phenotype by the substitution of Ile-266 for Val-266 is specific for N. benthamiana, since neither movement nor the symptom phenotype in barley plants was affected.