Cell-to-Cell, but not long-distance, spread of RNA silencing that is induced in individual epidermal cells.
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A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFPDeltaCP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in Nicotiana benthamiana. Mechanical inoculation of TCV-GFPDeltaCP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFPDeltaCP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFPDeltaCP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed. (+info)
Gaining insight into the role of serine 282 in B. napus FAE1 condensing enzyme.
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To gain some insight whether there is an absolute requirement for the serine 282 to yield a functional fatty acid elongase 1 condensing enzyme we have introduced point mutations in the FAE1 coding sequence which led to the substitution of serine 282 with several aliphatic or aromatic amino acids. The mutated FAE1 polypeptides were expressed in yeast. Gas chromatography analyses of the fatty acid methyl esters from yeast lysates and fatty acid elongase activity assays demonstrated that there is not an absolute requirement for serine at position 282 to yield a functional FAE1 condensing enzyme. (+info)
The avirulence domain of Cauliflower mosaic virus transactivator/viroplasmin is a determinant of viral virulence in susceptible hosts.
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Cauliflower mosaic virus (CaMV) transactivator/viroplasmin (Tav) is a multifunctional protein essential for basic replication of CaMV. It also plays a role in viral pathogenesis in crucifer and solanaceous host plants. Deletion mutagenesis revealed that N- and C-terminal parts of Tav are not essential for CaMV replication in transfected protoplasts. Two deletion mutants having only minimal defects in basic replication were infectious in turnips but only with highly attenuated virulence. This was shown to be due to delayed virus spread within the inoculated leaves and to the upper leaves. Unlike the wild-type virus, the mutant viruses successfully spread locally without inducing a host defense response in inoculated Datura stramonium leaves, but did not spread systemically. These results provide the first evidence that a Tav domain required for avirulence function in solanaceous plants is not essential for CaMV infectivity but has a role in viral virulence in susceptible hosts. (+info)
Complementation of Saccharomyces cerevisiae ccc2 mutant by a putative P1B-ATPase from Brassica napus supports a copper-transporting function.
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Copper transport across membranes plays an important role in plant growth and survival. P(1B)-type ATPases participate in transmembrane transport of copper in various organisms. A Brassica napus cDNA (BnRAN1) encoding a putative Cu(2+)-ATPase was cloned in this study. A complementation assay demonstrated that the protein encoded by this cDNA could functionally replace Ccc2p, a Saccharomyces cerevisiae Cu(2+)-ATPase, rescuing growth of ccc2 mutant under iron-limited conditions. Our results suggest that this rescue likely resulted from restoration of copper delivery, mediated by BnRAN1, to Fet3p. This study is amongst the first to demonstrate that a putative plant P(1B)-ATPase is functional and to examine its substrate specificity. (+info)
The import of phosphoenolpyruvate by plastids from developing embryos of oilseed rape, Brassica napus (L.), and its potential as a substrate for fatty acid synthesis.
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The plastidial phosphoenolpyruvate (PEP)/phosphate translocator (PPT) is expressed in the developing embryos of oilseed rape (Brassica napus L.). PEP can be imported by plastids isolated from embryos and used for fatty acid synthesis at rates that are sufficient to account for one-third of the rate of fatty acid synthesis in vivo. This provides the first experimental evidence for uptake of PEP and incorporation of carbon from it into fatty acids by plastids. PEP metabolism in isolated plastids is able to provide some of the ATP required for fatty acid synthesis. Expression of the PPT and related glucose 6-phosphate (Glc-6-P) translocator (GPT) is high in early embryo and leaf development and then declines. The marked decline in the abundance of PPT and GPT transcripts between the pre- and mid-oil accumulating stages of embryo development in B. napus does not correlate with the corresponding translocator activities, which both increase over the same period. This means that transcript abundance cannot be used to infer the activity of the translocators. (+info)
An important determinant of the ability of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein.
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Turnip mosaic virus (TuMV, genus Potyvirus, family Potyviridae) infects mainly cruciferous plants. Isolates Tu-3 and Tu-2R1 of TuMV exhibit different infection phenotypes in cabbage (Brassica oleracea L.) and Japanese radish (Raphanus sativus L.). Infectious full-length cDNA clones, pTuC and pTuR1, were constructed from isolates Tu-3 and Tu-2R1, respectively. Progeny virus derived from infections with pTuC induced systemic chlorotic and ringspot symptoms in infected cabbage, but no systemic infection in radish. Virus derived from plants infected with pTuR1 induced a mild chlorotic mottle in cabbage and infected radish systemically to induce mosaic symptoms. By exchanging genome fragments between the two virus isolates, the P3-coding region was shown to be responsible for systemic infection by TuMV and the symptoms it induces in cabbage and radish. Moreover, exchanges of smaller parts of the P3 region resulted in recombinants that induced complex infection phenotypes, especially the combination of pTuC-derived N-terminal sequence and pTuR1-derived C-terminal sequence. Analysis by tissue immunoblotting of the inoculated leaves showed that the distributions of P3-chimeric viruses differed from those of the parents, and that the origin of the P3 components affected not only virus accumulation, but also long-distance movement. These results suggest that the P3 protein is an important factor in the infection cycle of TuMV and in determining the host range of this and perhaps other potyviruses. (+info)
Repression and derepression of minus-strand synthesis in a plus-strand RNA virus replicon.
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Plus-strand viral RNAs contain sequences and structural elements that allow cognate RNA-dependent RNA polymerases (RdRp) to correctly initiate and transcribe asymmetric levels of plus and minus strands during RNA replication. cis-acting sequences involved in minus-strand synthesis, including promoters, enhancers, and, recently, transcriptional repressors (J. Pogany, M. R. Fabian, K. A. White, and P. D. Nagy, EMBO J. 22:5602-5611, 2003), have been identified for many viruses. A second example of a transcriptional repressor has been discovered in satC, a replicon associated with turnip crinkle virus. satC hairpin 5 (H5), located proximal to the core hairpin promoter, contains a large symmetrical internal loop (LSL) with sequence complementary to 3'-terminal bases. Deletion of satC 3'-terminal bases or alteration of the putative interacting bases enhanced transcription in vitro, while compensatory exchanges between the LSL and 3' end restored near-normal transcription. Solution structure analysis indicated that substantial alteration of the satC H5 region occurs when the three 3'-terminal cytidylates are deleted. These results indicate that H5 functions to suppress synthesis of minus strands by sequestering the 3' terminus from the RdRp. Alteration of a second sequence strongly repressed transcription in vitro and accumulation in vivo, suggesting that this sequence may function as a derepressor to free the 3' end from interaction with H5. Hairpins with similar sequence and/or structural features that contain sequence complementary to 3'-terminal bases, as well as sequences that could function as derepressors, are located in similar regions in other carmoviruses, suggesting a general mechanism for controlling minus-strand synthesis in the genus. (+info)
Biased hypermutagenesis associated with mutations in an untranslated hairpin of an RNA virus.
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The mutation frequency of Turnip crinkle virus can increase 12-fold without inducing error catastrophe. Lesions in a hairpin repressor frequently reverted and led to second-site alterations biased for specific mutations. These results suggest that the hairpin may also function as an RNA chaperone to properly fold the RNA-dependent RNA polymerase. (+info)