A cucumber mosaic virus (CMV) RNA 1 transgene mediates suppression of the homologous viral RNA 1 constitutively and prevents CMV entry into the phloem.
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Resistance to Cucumber mosaic virus (CMV) in tobacco lines transformed with CMV RNA 1 is characterized by reduced virus accumulation in the inoculated leaf, with specific suppression of accumulation of the homologous viral RNA 1, and by the absence of systemic infection. We show that the suppression of viral RNA 1 occurs in protoplasts from resistant transgenic plants and therefore is not due to a host response activated by the cell-to-cell spread of virus. In contrast, suppression of Tobacco rattle virus vectors carrying CMV RNA 1 sequences did not occur in protoplasts from resistant plants. Furthermore, steady-state levels of transgene mRNA 1 were higher in resistant than in susceptible lines. Thus, the data indicate that sequence homology is not sufficient to induce suppression. Grafting experiments using transgenic resistant or susceptible rootstocks and scions demonstrated that the resistance mechanism exhibited an additional barrier to phloem entry, preventing CMV from moving a long distance in resistant plants. On the other hand, virus from susceptible rootstocks could systemically infect grafted resistant scions via the phloem. Analysis of viral RNA accumulation in the infected scions showed that the mechanism that suppresses the accumulation of viral RNA 1 at the single-cell level was overcome. The data indicate that this transgene-mediated systemic resistance probably is not based on a posttranscriptional gene-silencing mechanism. (+info)
Umbravirus-encoded movement protein induces tubule formation on the surface of protoplasts and binds RNA incompletely and non-cooperatively.
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Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed. The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast. The protein encoded by ORF4 was assessed also for RNA-binding properties. This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography. Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios. Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA. UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl. Probing the MP-RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed. (+info)
Differential effects of satellite RNA on the accumulation of cucumber mosaic virus RNAs and their encoded proteins in tobacco vs zucchini squash with two strains of CMV helper virus.
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The presence of cucumber mosaic virus (CMV) satellite RNA usually reduces the yield of accumulated helper virus, although more so in solanaceous than in cucurbit hosts. The accumulation of viral RNA and viral-encoded proteins of two strains of CMV (Fny- and Sny-) known to differ in their ability to support satellite RNA in zucchini squash was examined in squash and tobacco to determine the effect of satellite RNA on the accumulation of viral-associated components. In the absence of satellite RNA, Fny- and Sny-CMV showed similar levels of accumulation of RNA at 7 days postinoculation (p.i.), but by 14 days p.i. the Fny-CMV RNAs accumulated to lower levels than did both strains at 7 days p.i., in either host. The levels of accumulated Sny-CMV-encoded proteins were higher than those encoded by Fny-CMV in tobacco, but not squash plants, at 7 days p.i. At 14 days p.i., for Fny-CMV vs Sny-CMV, there were differences in the levels of accumulation of most CMV-encoded proteins in both hosts, more exacerbated in tobacco vs squash. The effect of satellite RNA was to intensify these differences; that is, by 7 days p.i., satellite RNA reduced the accumulation of Fny-CMV RNAs 1 and 2 and their encoded proteins in both tobacco and squash but had little or no effect on the accumulation of Sny-CMV RNAs or encoded proteins. By 14 days p.i., the levels of accumulation of all Fny-CMV RNAs and encoded proteins were severely reduced in both hosts, and the levels of accumulation of Sny-CMV RNAs 1 and 2 and their encoded proteins were also reduced in tobacco, but not squash. Sny-CMV did not support satellite RNA accumulation in squash plants or protoplasts. Satellite RNA did not appear to have a direct effect on the movement of either CMV strain. Rather, accumulation studies in tobacco protoplasts indicated that the difference in response of Fny-CMV vs Sny-CMV to satellite RNA in tobacco was due to the extent to which satellite RNA affected the levels of RNA 1, and to a lesser extent RNA 2, and their encoded proteins, 1a and 2a, both components of the CMV replicase. (+info)
Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement.
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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. (+info)
Evolutionary history of Cucumber mosaic virus deduced by phylogenetic analyses.
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Cucumber mosaic virus (CMV) is an RNA plant virus with a tripartite genome and an extremely broad host range. Previous evolutionary analyses with the coat protein (CP) and 5' nontranslated region (NTR) of RNA 3 suggested subdivision of the virus into three groups, subgroups IA, IB, and II. In this study 15 strains of CMV whose nucleotide sequences have been determined were used for a complete phylogenetic analysis of the virus. The trees estimated for open reading frames (ORFs) located on the different RNAs were not congruent and did not completely support the subgrouping indicated by the CP ORF, indicating that different RNAs had independent evolutionary histories. This is consistent with a reassortment mechanism playing an important role in the evolution of the virus. The evolutionary trees of the 1a and 3a ORFs were more compact and displayed more branching than did those of the 2a and CP ORFs. This may reflect more rigid host-interactive constraints exerted on the 1a and 3a ORFs. In addition, analysis of the 3' NTR that is conserved among all RNAs indicated that evolutionary constraints on this region are specific to the RNA component rather than the virus isolate. This indicates that functions other than replication are encoded in the 3' NTR. Reassortment may have led to the genetic diversity found among CMV strains and contributed to its enormous evolutionary success. (+info)
Heterologous movement protein strongly modifies the infection phenotype of cucumber mosaic virus.
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A hybrid virus (CMVcymMP) constructed by replacing the movement protein (MP) of cucumber mosaic cucumovirus (CMV) with that of cymbidium ringspot tombusvirus (CymRSV) was viable and could efficiently spread both cell to cell and long distance in host plants. The hybrid virus was able to move cell to cell in the absence of functional CP, whereas CP-deficient CMV was restricted to single inoculated cells. In several Chenopodium and Nicotiana species, the symptom phenotype of the hybrid virus infection was clearly determined by the foreign MP gene. In Nicotiana debneyi and Nicotiana tabacum cv. Xanthi, the hybrid virus could move systemically, contrary to CymRSV. (+info)
Inter- and intramolecular recombinations in the cucumber mosaic virus genome related to adaptation to alstroemeria.
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In four distinct alstroemeria-infecting cucumber mosaic virus (CMV) isolates, additional sequences of various lengths were present in the 3' nontranslated regions of their RNAs 2 and 3, apparently the result of intra- and intermolecular recombination events. Competition experiments revealed that these recombined RNA 2 and 3 segments increased the biological fitness of CMV in alstroemeria. (+info)
Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance.
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Transgene-induced post-transcriptional gene silencing (PTGS) results from specific degradation of RNAs that are homologous with the transgene transcribed sequence. This phenomenon, also known as cosuppression in plants and quelling in fungi, resembles RNA interference (RNAi) in animals. Indeed, cosuppression/quelling/RNAi require related PAZ/PIWI proteins (AGO1/QDE-2/RDE-1), indicating that these mechanisms are related. Unlike Neurospora crassa qde-2 and Caenorhabditis elegans rde-1 mutants, which are morphologically normal, the 24 known Arabidopsis ago1 mutants display severe developmental abnormalities and are sterile. Here, we report the isolation of hypomorphic ago1 mutants, including fertile ones. We show that these hypomorphic ago1 mutants are defective for PTGS, like null sgs2, sgs3, and ago1 mutants, suggesting that PTGS is more sensitive than development to perturbations in AGO1. Conversely, a mutation in ZWILLE/PINHEAD, another member of the Arabidopsis AGO1 gene family, affects development but not PTGS. Similarly, mutations in ALG-1 and ALG-2, two members of the C. elegans RDE-1 gene family, affect development but not RNAi, indicating that the control of PTGS/RNAi and development by PAZ/PIWI proteins can be uncoupled. Finally, we show that hypomorphic ago1 mutants are hypersensitive to virus infection, confirming the hypothesis that in plants PTGS is a mechanism of defense against viruses. (+info)