RNA-based silencing strategies in plants.

In plants, double-stranded RNA can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. Analyses of Arabidopsis mutants and plant viral suppressors of silencing are unraveling RNA-silencing mechanisms, which require common proteins in diverse organisms, and are assessing the role of methylation in transcriptional and posttranscriptional gene silencing.

HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal.

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs.

A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants.

Posttranscriptional gene silencing (PTGS) is an ancient eukaryotic regulatory mechanism in which a particular RNA sequence is targeted and destroyed. The helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS in plants. Using a yeast two-hybrid system, we identified a calmodulin-related protein (termed rgs-CaM) that interacts with HC-Pro. Here we report that rgs-CaM, like HC-Pro itself, suppresses gene silencing. Our work is the first report identifying a cellular suppressor of PTGS.

RNA viruses as inducers, suppressors and targets of post-transcriptional gene silencing.

Post-transcriptional gene silencing (PTGS) is a fundamental regulatory mechanism operating in diverse types of organisms, but the cellular components of the gene silencing machinery and the regulation of the process are not understood. Recent findings that cytoplasmically replicating RNA viruses act as both targets and inducers of PTGS has led to the idea that PTGS may have evolved as an anti-viral defense mechanism in plants. Consistent with this hypothesis, it has been found that certain plant viruses encode proteins that suppress PTGS. From a practical standpoint, an understanding of the mechanisms by which viruses regulate PTGS may well lead to better ways to control gene expression in plants. It is often desirable to overexpress selected beneficial genes or to silence detrimental ones in order to confer a particular phenotype. Induction of PTGS using RNA viruses as vectors or as transgenes provides a reliable and efficient way to interfere with the expression of a specific gene or with a family of genes. Conversely, expression of viral suppressors has significant potential to improve yields in technologies that use plants to express beneficial gene products. Given the antiviral nature of gene silencing in plants and the indications that PTGS is an ancient mechanism in eukaryotic organisms, understanding the phenomenon in plants could well lead to the development of anti-viral strategies in both plants and animals.

Plant viral suppressors of post-transcriptional silencing do not suppress transcriptional silencing.

Homology-dependent gene silencing is a regulatory mechanism that limits RNA accumulation from affected loci either by suppression of transcription (transcriptional gene silencing, TGS) or by activation of a sequence-specific RNA degradation process (post-transcriptional gene silencing, PTGS). The P1/HC-Pro sequence of plant potyviruses and the 2b gene of the cucumber mosaic virus have been shown to interfere with PTGS. The ability of these viral suppressors of PTGS to interfere with TGS was tested using the 271 locus which imposes TGS on transgenes under 35S or 19S promoters and PTGS on the endogenous nitrite reductase gene (Nii). Both P1/HC-Pro and 2b reversed PTGS of Nii genes in 271-containing tobacco plants, but failed to reverse TGS of 35S-GUS transgenes in the same plant. P1/HC-Pro expression from a transgene also failed to suppress either the initiation or maintenance of TGS imposed by the NOSpro-silencing locus, H2. These results indicate that PTGS and TGS operate through unlinked pathways or that P1/HC-Pro and 2b interfere at step(s) in PTGS that are downstream of any common components in the two pathways. The data suggest a simple assay to identify post-transcriptionally silenced transgenic lines with the potential to be stably converted to high expressing lines.

A viral suppressor of gene silencing in plants.

Gene silencing is an important but little understood regulatory mechanism in plants. Here we report that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing. The viral suppressor of silencing comprises the 5'-proximal region of the tobacco etch potyviral genomic RNA encoding P1, helper component-proteinase (HC-Pro) and a small part of P3, and is termed the P1/HC-Pro sequence. A reversal of silencing assay was used to assess the effect of the P1/HC-Pro sequence on transgenic tobacco plants (line T4) that are posttranscriptionally silenced for the uidA reporter gene. Silencing was lifted in offspring of T4 crosses with four independent transgenic lines expressing P1/HC-Pro, but not in offspring of control crosses. Viral vectors were used to assess the effect of P1/HC-Pro expression on virus-induced gene silencing (VIGS). The ability of a potato virus X vector expressing green fluorescent protein to induce silencing of a green fluorescent protein transgene was eliminated or greatly reduced when P1/HC-Pro was expressed from the same vector or from coinfecting potato virus X vectors. Expression of the HC-Pro coding sequence alone was sufficient to suppress virus-induced gene silencing, and the HC-Pro protein product was required for the suppression. This discovery points to the role of gene silencing as a natural antiviral defense system in plants and offers different approaches to elucidate the molecular basis of gene silencing.

Mutations in the region encoding the central domain of helper component-proteinase (HC-Pro) eliminate potato virus X/potyviral synergism.

Coinfection of tobacco plants with potato virus X (PVX) and any of several members of the potyvirus group causes a synergistic disease characterized by a dramatic increase in symptom severity correlated with a 3- to 10-fold increase in the accumulation of PVX in the first systemically infected leaves. We have recently shown that PVX/potyviral synergistic disease is mediated by expression of potyviral 5'-proximal sequences encoding P1, helper component-proteinase (HC-Pro), and a fraction of P3 (termed P1/HC-Pro sequence). Here we report the effect of mutations in this potyviral sequence on the induction of synergistic disease. Three transgenic tobacco lines expressing the tobacco etch potyvirus (TEV) P1/HC-Pro sequence with mutations within the P1 coding region were not impaired in their ability to mediate synergism when infected with PVX. In contrast, two of three transgenic lines with mutations in the HC-Pro coding region were unable to induce the synergistic increases in either symptom severity or PVX accumulation. Loss of synergistic function was associated with mutations within the region encoding the central domain of HC-Pro, while the ability to induce synergism was retained in a transgenic line expressing HC-Pro with an alteration in the amino-terminal "zinc-finger domain." In coinoculation experiments, a TEV mutant lacking the sequence encoding the zinc-linger domain of HC-Pro induced a typical synergistic response in interaction with PVX. The results indicate that the zinc-finger domain comprising the first 66 amino acid residues of HC-Pro is dispensable for induction of synergistic disease and transactivation of PVX multiplication, while regions within the central domain of HC-Pro are essential for both of these responses.

An eight-nucleotide sequence in the potato virus X 3' untranslated region is required for both host protein binding and viral multiplication.

Gel retardation and UV-cross-linking techniques were used to demonstrate that two tobacco proteins, with approximate molecular masses of 28 and 32 kDa, bind to a site within the 3' region of potato virus X (PVX) genomic RNA. The protein binding is specific, in that a 50-fold excess of unlabeled probe prevents formation of the complexes but no reduction is observed with a 2,000-fold molar excess of yeast tRNA. Complex formation is inhibited by poly(U) but is relatively unaffected by poly(A), poly(G), or poly(C-I). PVX RNA-host protein complex formation occurs in vitro at salt concentrations up to 400 mM. Deletion mapping indicates that the proteins bind within the 3' untranslated region (UTR) of PVX genomic RNA and that an 8-nucleotide U-rich sequence (5'-UAUUUUCU) is required for the binding. Deletion of the 8-nucleotide U-rich region from the 3' UTR of a sensitive PVX reporter virus that carries the luciferase gene in place of the PVX coat protein gene results in a more than 70,000-fold reduction in luciferase expression in tobacco protoplasts. RNA probes carrying the sequence GCGC in place of the central four contiguous uridines of the 8-nucleotide U-rich motif fail to bind host protein at detectable levels, and the same mutation, when introduced into the PVX reporter virus, eliminates viral multiplication. Mutations of 1 or 2 nucleotides within the same four uridines reduced both binding of host proteins and replication of reporter virus. These results indicate that the 8-nucleotide U-rich motif within the PVX 3' UTR is important for some aspect of viral multiplication and suggest that host protein binding plays a role in the process.

5' proximal potyviral sequences mediate potato virus X/potyviral synergistic disease in transgenic tobacco.

The interaction of potato virus X (PVX) and potato virus Y (PVY) in tobacco causes a synergistic disease characterized by a dramatic increase in symptom severity, a change in the regulation of PVX RNA replication, and an increase in accumulation of PVX. In this study we demonstrate that PVX also interacts synergistically with three other members of the potyvirus group of plant viruses, tobacco vein mottling virus (TVMV), tobacco etch virus (TEV), and pepper mottle virus. These synergisms resemble the classic PVX/PVY synergism with respect to both the increase in host response and the change in PVX replication. To determine if the induction of PVX/potyviral synergism requires potyviral genome replication per se or if the response is mediated by expression of one or more potyviral genes, we used tobacco plants stably transformed with various subsets of the TVMV genome. PVX infections of transgenic plants expressing the 5'-proximal region of the TVMV genome, including the protease-1, helper component protease, and protein-3 genes, result in symptoms resembling those of PVX/potyviral synergism. A similar synergistic-like response occurs when transgenic tobacco plants expressing the analogous but smaller region from the 5'-proximal region of the TEV genome were infected with PVX. Replication of PVX RNA is altered in transgenic plants expressing 5'-proximal sequences of either TVMV or TEV, and in a manner similar to that observed in double infections. These results indicate that replication of the potyviral genome is not required for PVX/potyviral synergism and that the response is mediated by expression of potyviral sequences which have been localized to the 5'-proximal third of the genomic RNAs of both TVMV and TEV.

The complete nucleotide sequence of prune dwarf ilarvirus RNA 3: implications for coat protein activation of genome replication in ilarviruses.

The complete nucleotide sequence of prune dwarf ilarvirus (PDV) RNA 3 has been determined from cloned viral cDNAs. The PDV RNA 3 is 2129 nucleotides and contains two large open reading frames (ORFs) separated by an intergenic region of 72 nucleotides. The 5' proximal ORF (ORF-1) is 882 nucleotides, encoding a gene product which shares homology with putative cell-to-cell movement proteins of related viruses, including tobacco streak virus (TSV) and alfalfa mosaic virus (AIMV). The downstream ORF (ORF-2) is 657 nucleotides and encodes a gene product which shares primary sequence homology and structural features with AIMV coat protein. Furthermore, when expressed in bacteria, this ORF produces a polypeptide which comigrates with authentic PDV coat protein and reacts with PDV coat protein antiserum. Hybridization data suggest that the genomic organization of PDV RNAs 3 and 4 is similar to that of TSV, the only other ilarvirus for which sequence information is published. The 3' untranslated region (UTR) of PDV RNA 3 is similar to that of TSV and AIMV, containing a potential stem-loop structure followed by the sequence AUGC, a structure which may signal binding of coat protein and activation of genome replication. However, a striking feature of the deduced PDV coat protein sequence is the absence of a "zinc-finger" motif thought to function in binding of the coat protein to the 3'-UTR in ilarviruses and AIMV. This result suggests that the zinc-finger motif is not a required aspect of coat protein activation of replication in ilarviruses.

The complete nucleotide sequence of pepper mottle virus genomic RNA: comparison of the encoded polyprotein with those of other sequenced potyviruses.

The complete nucleotide sequence of a pepper mottle virus isolate from California (PepMoV C) has been determined from cloned viral cDNAs. The PepMoV C genomic RNA is 9640 nucleotides excluding the poly(A) tail and contains a long open reading frame starting at nucleotide 168 and potentially encoding a polyprotein of 3068 amino acids. Comparison of the PepMoV C presumptive polyprotein with those of other sequenced members of the potyvirus group, including tobacco etch virus (TEV), tobacco vein mottling virus (TVMV), plum pox virus (PPV), and potato virus Y (PVY), allowed localization of putative protein cleavage sites. A similar analysis was used to determine the position of conserved viral protein-coding regions along the viral genomic RNA. These analyses confirm previous work indicating that genome organization is conserved among members of the genus Potyvirus. The localization of one PepMoV C gene product, the nuclear inclusion body protein a (NIa protein), was analyzed by expressing PepMoV cDNA deletion clones in bacteria and assaying for appearance of mature-sized coat protein, a cleavage product of the NIa protease. Comparative sequence analyses of the putative PepMoV polyprotein with those of TEV, TVMV, PPV, and PVY served to identify regions of the potyviral polyproteins which have diverged within the genus, as well as highly conserved protein features which may play an important functional role in the potyviral life cycle.

Evidence that pepper mottle virus and potato virus Y are distinct viruses: analyses of the coat protein and 3' untranslated sequence of a California isolate of pepper mottle virus.

Pepper mottle virus (PepMoV) is a member of the large and complex genus Potyvirus, and is classically distinguished from other members of the genus by differential host range and cytopathology as well as serology of the coat protein and cytoplasmic inclusion body proteins. Here we report the deduced amino acid sequence of the coat protein of a California potyvirus identified by a variety of classical methods as PepMoV (PepMoV C). Comparison of the 3' untranslated nucleic acid sequence and the deduced coat-protein amino acid sequence of the PepMoV C isolate with those of PVY and other potyviruses indicates that PepMoV C is sufficiently diverged to be considered a distinct virus species. Thus, comparative sequence analyses of the PepMoV C isolate support earlier serological and biological evidence that PepMoV and PVY are distinct viruses.

Replication of potato virus X RNA is altered in coinfections with potato virus Y.

Potato virus X (PVX) and potato virus Y (PVY) may coinfect tobacco to cause a classic synergistic disease. In the acute stage the disease is characterized by a dramatic increase in the accumulation of infectious PVX particles, with no corresponding increase or decrease in the accumulation of PVY. The accumulation of PVX genomic RNA and coat protein has been examined in doubly versus singly infected tobacco leaves. These experiments indicate that the levels of both viral components increase in doubly infected plants to about the same extent as the level of infectious PVX particles. The level of PVX subgenomic coat protein mRNA found associated with polyribosomes of synergistically infected plants is also increased to a similar extent. Pulse labelling experiments suggest that the increase in PVX coat protein is due to an increased rate of synthesis. The level of PVX (-) strand RNA template increases disproportionately in doubly infected tissue, to a level three times higher than that of the virion or its component parts. This result suggests that PVX/PVY synergism involves an alteration in the normal regulation of the relative levels of PVX (+) and (-) strand RNAs during viral replication.

The major protein from lipid bodies of maize. Characterization and structure based on cDNA cloning.

In plant seeds, the storage triacylglycerol is packed in discrete particles called lipid bodies which consist of a lipid core surrounded by a phospholipid monolayer with embedded proteins. We have cloned and sequenced a nearly full-length cDNA for the major protein (L3) associated with the lipid bodies of maize. The L3-cDNA clone was identified by hybrid-selected translation analysis and contains the complete 3' noncoding region and an open reading frame of 432 nucleotides. This open reading frame encodes a polypeptide with amino acid composition, hydrophobicity, and predicted protease digestion pattern which correlate well with those of the authentic L3 protein. Analyses of predicted secondary structure and local hydropathy of the deduced amino acid sequence suggest three structural domains in the protein. An internal domain of 72 contiguous hydrophobic or neutral amino acids is bounded at the amino-terminal side by a hydrophilic alpha-helix and on the carboxyl-terminal side by an amphipathic alpha-helix. The data suggest that L3 is uniquely suited to interact with both lipid and phospholipid moieties of the lipid body. A simple model for the topology of L3 on the lipid body is proposed. The unusual structure of the lipid body protein is discussed and compared to those of the two well-studied classes of lipid-associated proteins, apolipoproteins and intrinsic membrane proteins.

Characteristics and biosynthesis of membrane proteins of lipid bodies in the scutella of maize (Zea mays L.).

Storage lipid bodies, which are prominent organelles present in the storage tissues of most seeds, have not been subjected to intensive biochemical investigation. In the present studies the major proteins in lipid bodies isolated from eleven taxonomically diverse species were shown to be distinctly different, as revealed by SDS/polyacrylamide-gel electrophoresis. The lipid-body membrane of maize (Zea mays L.) contained three major proteins of low Mr (19,500, 18,000 and 16,500), and they were chosen for further study. They all had alkaline pI values and behaved as hydrophobic integral proteins, as shown by their resistance to solubilization after repeated washing, amino acid composition and partitioning in a Triton X-114 system. Labelling in vivo with [35S]methionine and translation in vitro using extracted RNA in a wheat-germ system showed that the proteins were synthesized during seed maturation and not germination. The proteins synthesized in vivo and in vitro exhibited no appreciable difference in their mobilities in two-dimensional gel electrophoresis (isoelectric focusing and molecular sieving). The most abundant protein, that of Mr 16,500, was shown to be synthesized predominantly, if not exclusively, by RNA derived from bound polyribosomes and not from free polyribosomes. The implication of the results on the biosynthesis of the lipid bodies is discussed.

Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing.

Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC-5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately cleaved at the 5' end of the 18S rRNA sequence, even though these transcripts contained neither the 3' end of mouse 18S rRNA nor any other downstream mouse sequences. Thus, cleavage at the 5' end of 18S rRNA is not dependent on long range interactions involving these downstream sequences.

Detection of genomic-length soybean mosaic virus RNA on polyribosomes of infected soybean leaves.

Soybean mosaic virus (SMV)-related RNAs were examined in both polyribosomal and nonpolyribosomal fractions of systemically infected soybean leaves. Viral RNAs were detected by Northern blot hybridization analysis using two cloned SMV-cDNAs representing different regions of the viral genome as hybridization probes. Genomic length SMV-RNA (Mr of 3.3 X 10(6] was found in specific association with EDTA-sensitive polyribosomes of infected leaves, indicating that it functions as a messenger RNA in these cells. A smaller SMV-related RNA (Mr of 1.6 X 10(6] was sometimes detected in the polyribosomal fraction; however, reconstruction experiments indicate that this RNA is a breakdown product of the genomic-length RNA, generated during cell fractionation or RNA extraction. Two other SMV-related RNAs with Mr of 2.0 and 0.78 X 10(6) were sometimes detected in infected cells and were not generated from genomic SMV-RNA or intact virus particles in reconstruction experiments. However, these RNAs were exclusively associated with the EDTA-resistant, nonpolyribosomal fraction of infected cells. These data suggest that genomic-length SMV-RNA is the only viral RNA which is translated in these infected plants.

Translation of soybean mosaic virus RNA in vitro: evidence of protein processing.

The genomic RNA of soybean mosaic virus (SMV), a member of the potyvirus group of plant viruses, was translated in both the wheat germ and reticulocyte cell free systems to identify some viral encoded proteins and as an approach to determining the translational strategy of the virus. The RNA was translated into the same specific set of 10 to 12 polypeptides in both in vitro systems. Immunological tests and peptide analyses indicate that six translation products are related to SMV coat protein, and one of these comigrated with coat protein during electrophoresis. Two other antigenically distinct classes of polypeptides were identified by their specific immunoprecipitation with antibody against the SMV cytoplasmic inclusion body protein or tobacco etch virus nuclear inclusion body protein. To determine if any products of the in vitro translation reactions resulted from proteolytic processing of a precursor molecule, translation reactions were carried out with amino terminal label N-formyl[35S]methionyl-tRNA(Met)i (f-Met), or with [35S]methionine, and the resultant products were compared. The putative SMV coat protein and a translation product related to the nuclear inclusion body protein were not labeled with f-Met indicating that they were generated by proteolytic processing at their amino termini. Consistent with this finding is the accumulation of new polypeptides of greater apparent molecular weight when amino acid analogs were present during translation.