Enhancement of RNA synthesis by promoter duplication in tombusviruses.

Replication of tombusviruses, small plus-strand RNA viruses of plants, is regulated by cis-acting elements present in the viral RNA. The role of cis-acting elements can be studied in vitro by using a partially purified RNA-dependent RNA polymerase (RdRp) preparation obtained from tombusvirus-infected plants, Virology 276, 279- 288). Here, we demonstrate that the minus-strand RNA of tombusviruses contains, in addition to the 3'-terminal minimal plus-strand initiation promoter, a second cis-acting element, termed the promoter proximal enhancer (PPE). The PPE element enhanced RNA synthesis by almost threefold from the adjacent minimal promoter in the in vitro assay. The sequence of the PPE element is 70% similar to the minimal promoter, suggesting that sequence duplication of the minimal promoter may have been the mechanism leading to the generation of the PPE. Consistent with this proposal, replacement of the PPE element with the minimal promoter, which resulted in a perfectly duplicated promoter region, preserved its enhancer-like function. In contrast, mutagenesis of the PPE element or its replacement with an artificial G/C-rich sequence abolished its stimulative effect on initiation of RNA synthesis in vitro. In vivo experiments are also consistent with the role of the PPE element in enhancement of tombusvirus replication. Sequence comparison of several tombusviruses and related carmoviruses further supports the finding that duplication of minimal promoter sequences may have been an important mechanism during the evolution of cis-acting elements in tombusviruses and related RNA viruses.

The RNA replication enhancer element of tombusviruses contains two interchangeable hairpins that are functional during plus-strand synthesis.

Replication of the RNA genomes of tombusviruses, which are small plus-sense RNA viruses of plants, may be regulated by cis-acting elements, including promoters and replication enhancers that are present in the RNA templates. Using a partially purified RNA-dependent RNA polymerase (RdRp) preparation (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), we demonstrate that the minus-strand templates of tombusviruses contain a replication enhancer, which can upregulate RNA synthesis initiating from the minimal plus-strand initiation promoter by 10- to 20-fold in an in vitro assay. Dissection of the sequence of the replication enhancer element revealed that the two stem-loop structures present within the approximately 80-nucleotide-long enhancer region have interchangeable roles in upregulating RNA synthesis. The single-stranded sequence located between the two stem-loops also plays an important role in stimulation of RNA synthesis. We also demonstrate that one of the two hairpins, both of which are similar to the hairpin of the minus-strand initiation promoter, can function as a promoter in vitro in the presence of short cytidylate-containing initiation sites. Overall, the in vitro data presented are consistent with previous in vivo results (D. Ray and K. A. White, Virology 256:162-171, 1999) and they firmly establish the presence of a replication enhancer on the minus-stranded RNA of tombusviruses.

Analysis of minimal promoter sequences for plus-strand synthesis by the Cucumber necrosis virus RNA-dependent RNA polymerase.

Tombusviruses are small, plus-sense, single-stranded RNA viruses of plants. A partially purified RNA-dependent RNA polymerase (RdRp) preparation of Cucumber necrosis virus (CNV), which is capable of de novo initiation of complementary RNA synthesis from either plus-strand or minus-strand templates, was used to dissect minimal promoter sequences for tombusviruses and their defective interfering (DI) RNAs. In vitro RdRp assay revealed that the core plus-strand initiation promoter included only the 3'-terminal 11 nucleotides. A hypothetical promoter-like sequence, which has been termed consensus sequence by Wu and White (1998, J. Virol. 72, 9897-9905), is recognized less efficiently by the CNV RdRp than the core plus-strand initiation promoter. The CNV RdRp can efficiently recognize the core plus-strand initiation promoter for a satellite RNA associated with the distantly related Turnip crinkle virus, while artificial AU- or GC-rich 3'-terminal sequences make poor templates in the in vitro assays. Comparison of the "strength" of minimal plus-strand and minus-strand initiation promoters reveals that the latter is almost twice as efficient in promoting complementary RNA synthesis. Template competition experiments, however, suggest that the minimal plus-strand initiation promoter makes an RNA template more competitive than the minimal minus-strand initiation promoter. Taken together, these results demonstrate that promoter recognition by the tombusvirus RdRp requires only short sequences present at the 3' end of templates.

Internal initiation by the cucumber necrosis virus RNA-dependent RNA polymerase is facilitated by promoter-like sequences.

Tombusviruses, small positive sense RNA viruses of plants, are replicated by the viral-coded RNA-dependent RNA polymerase (RdRp) in infected cells. An unusual feature of the tombusvirus RdRp that is partially purified from cucumber necrosis virus (CNV)-infected plants is the ability to initiate complementary RNA synthesis from several internal positions on minus-strand templates derived from DI RNAs ( Nagy and Pogany, 2000 ). In this study, we used template deletion, mutagenesis, and oligo-based inhibition of RNA synthesis to map the internal initiation sites observed with the in vitro CNV RdRp system. Comparing sequences around the internal initiation sites reveals that they have either (i) similar sequences to the core minus-strand initiation promoter; or (ii) similar structures to the core plus-strand initiation promoter. In addition, we find similarities among the internal initiation sites and the subgenomic RNA initiation sites. These similarities suggest that the mechanism of internal initiation is similar to initiation from the terminal core promoters or the putative subgenomic promoter sequences. We propose that internal initiation on full-length RNA templates may be important in defective interfering (DI) RNA formation/evolution by producing intermediate templates for RNA recombination in tombusviruses. This may explain why tombusviruses are frequently associated with DI RNAs.

Structural features and methylation patterns associated with paramutation at the r1 locus of Zea mays.

In paramutation, two alleles of a gene interact and, during the interaction, one of them becomes epigenetically silenced. The various paramutation systems that have been studied to date exhibit intriguing differences in the physical complexity of the loci involved. B and Pl alleles that participate in paramutation are simple, single genes, while the R haplotypes that participate in paramutation contain multiple gene copies and often include rearrangements. The number and arrangement of the sequences in particular complex R haplotypes have been correlated with paramutation behavior. Here, the physical structures of 28 additional haplotypes of R were examined. A specific set of physical features is associated with paramutability (the ability to be silenced). However, no physical features were strongly correlated with paramutagenicity (the ability to cause silencing) or neutrality (the inability to participate in paramutation). Instead, paramutagenic haplotypes were distinguished by high levels of cytosine methylation over certain regions of the genes while neutral haplotypes were distinguished by lack of C-methylation over these regions. These findings suggest that paramutability of r1 is determined by the genetic structure of particular haplotypes, while paramutagenicity is determined by the epigenetic state.

Molecular analysis of the Doppia transposable element of maize.

Doppia (Dop) transposable elements were first identified from element termini found in the upstream portions of certain alleles of the pl1 and r1 loci of maize. At the r1 locus, these Dop end sequences are present in a region called sigma, which functions as the promoter for the S genes of the R-r haplotype, and which is required for efficient epigenetic modification of the S genes during paramutation. In order to better understand the significance of the Dop element sequences at R-r, and to investigate the Dop-encoded products that might regulate r1 genes in this haplotype, we have cloned a more complete Dop element, Dop4. The Dop4 element can encode two proteins that have strong sequence similarity to the TnpA and TnpD proteins of the well characterized maize transposable element En/Spm. The DOPA protein, which is similar to TnpA of En/Spm, is shown to bind to short, subterminal repeat motifs located in the Dop element ends. Like TnpA, DOPA promotes intermolecular associations between DNA molecules. In contrast to the activity of TnpA, which is a transcriptional repressor of En/Spm, DOPA activates expression of reporter genes driven by either the Dop promoter or sigma in transient expression assays.

The structure and paramutagenicity of the R-marbled haplotype of Zea mays.

Paramutation is the meiotically heritable silencing of a gene that can occur in particular heterozygous combinations. The R-marbled (R-mb) haplotype is paramutagenic: it causes paramutable r1 haplotypes like R-r to become heritably silenced. R-mb was found to comprise three distinct r1 genes arranged as direct repeats. The most distal gene of R-mb, Scm, contains a novel transposable element, Shooter (Sho). Excision of the Sho element early in aleurone development results in the characteristic "marbled" aleurone pigmentation pattern conferred by R-mb. The effect of gene copy number on the paramutagenic strength of R-mb was tested. Paramutagenic strength of R-mb is directly correlated with r1 gene copy number. Paramutagenic strength of R-mb is directly correlated with r1 gene copy number. Paramutagenic strength of R-mb was not affected by removal, through crossing over, of the Sho transposon. Finally, R-mb does not appear to contain the transposable element, Doppia, which is associated with paramutability of R-r, and has been suggested to play a role in paramutagenicity of another paramutagenic haplotype, R-stippled.

Identification of senescence-associated genes from daylily petals.

The petals of daylily (Hemerocallis hybrid) have a genetically based program that leads to senescence and cell death ca. 24 h after the flower opens. In order to determine the components of this program, six cDNAs, whose levels increase during petal senescence, were isolated and sequenced and designated DSA3, 4, 5, 6, 12 and 15. All six DSAs are members of gene families and all but DSA5 and DSA6 have one to three other very similar genes. GenBank database homology searches indicate that DSA3 is most similar at the amino acid level to an in-chain fatty acid hydroxylase which is bound to cytochrome P450, DSA4 may be an aspartic proteinase, DSA5 is as yet unidentified, DSA6 is a putative S1-type nuclease, DSA12 is very similar to a cytochrome P450-containing allene oxide synthase, and DSA15 may be a fatty acid elongase. Except for DSA12, the genes are expressed at low levels in daylily roots. Levels of the DSA mRNAs in leaves are less than 4% of the maximum detected in petals, and there are no clear differences between younger and older leaves. With the exception of DSA4, accumulation of the DSA mRNAs is increased 3.2 to 43 times by a concentration of abscisic acid that causes premature senescence of the petals. The relationship of the putative DSA gene products to senescence and cell death of daylily petals is discussed.