Gail J. Pruss

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.

Escherichia coli DNA topoisomerase I mutants: Increased supercoiling is corrected by mutations near gyrase genes

Bacterial chromosomes and plasmid (pBR322) DNA from topoisomerase I-defective Escherichia coli strains have been characterized with respect to superhelical density. The topoisomerase I defect results in increased negative superhelical density of both the bacterial chromosome and pBR322. Thus topoisomerase I is involved in determining the level of supercoiling in bacteria. Three of the topoisomerase I-defective strains were studied carry secondary mutations that decrease superhelical density; these additional mutations are closely linked to the gyrB locus in two of the strains and to the gyrA locus in the third strain.

RNA Silencing and the Mobile Silencing Signal

RNA silencing is a sequence-specific RNA degradation mechanism that occurs in a broad range of eukaryotic organisms including fungi (quelling), animals (RNA interference [RNAi]), and plants (post-transcriptional gene silencing). In all these organisms, the process is triggered by double-stranded RNA

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.

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.

Small RNAs in viral infection and host defense

Small RNAs are the key mediators of RNA silencing and related pathways in plants and other eukaryotic organisms. Silencing pathways couple the destruction of double-stranded RNA with the use of the resulting small RNAs to target other nucleic acid molecules that contain the complementary sequence. This discovery has revolutionized our ideas about host defense and genetic regulatory mechanisms in eukaryotes. Small RNAs can direct the degradation of mRNAs and single-stranded viral RNAs, the modification of DNA and histones, and the inhibition of translation. Viruses might even use small RNAs to do some targeting of their own to manipulate host gene expression. This review highlights the current understanding and new insights concerning the roles of small RNAs in virus infection and host defense in plants.

The amplicon-plus system for high-level expression of transgenes in plants.

Many biotechnological applications require high-level expression of transgenes in plants. One strategy to achieve this goal was the production of potato virus X (PVX) “amplicon” lines: transgenic lines that encode a replicating RNA virus vector carrying a gene of interest. The idea was that transcription of the amplicon transgene would initiate viral RNA replication and gene expression, resulting in very high levels of the gene product of interest. This approach failed, however, because every amplicon transgene, in both tobacco and Arabidopsis thaliana, was subject to post-transcriptional gene silencing (PTGS). In PTGS, the transgene is transcribed but the transcripts fail to accumulate as a result of sequence-specific targeting and destruction. Even though the amplicon locus is silenced, the level of β-glucuronidase (GUS) activity in a PVX/GUS line is similar to that in some transgenic lines expressing GUS from a conventional (not silenced) GUS locus. This result suggested that the very high levels of expression originally envisioned for amplicons could be achieved if PTGS could be overcome and if the resulting plants did not suffer from severe viral disease. Here we report that high-level transgene expression can be achieved by pairing the amplicon approach with the use of a viral suppressor of PTGS, tobacco etch virus (TEV) helper component–proteinase (HC-Pro). Leaves of mature tobacco plants co-expressing HC-Pro and a PVX/GUS amplicon accumulate GUS to ∼3% of total protein. Moreover, high-level expression occurs without viral symptoms and, when HC-Pro is expressed from a mutant transgene, without detrimental developmental phenotypes.

DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis.

Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2–but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.

Two plant viral suppressors of silencing require the ethylene-inducible host transcription factor RAV2 to block RNA silencing.

RNA silencing is a highly conserved pathway in the network of interconnected defense responses that are activated during viral infection. As a counter-defense, many plant viruses encode proteins that block silencing, often also interfering with endogenous small RNA pathways. However, the mechanism of action of viral suppressors is not well understood and the role of host factors in the process is just beginning to emerge. Here we report that the ethylene-inducible transcription factor RAV2 is required for suppression of RNA silencing by two unrelated plant viral proteins, potyvirus HC-Pro and carmovirus P38. Using a hairpin transgene silencing system, we find that both viral suppressors require RAV2 to block the activity of primary siRNAs, whereas suppression of transitive silencing is RAV2-independent. RAV2 is also required for many HC-Pro-mediated morphological anomalies in transgenic plants, but not for the associated defects in the microRNA pathway. Whole genome tiling microarray experiments demonstrate that expression of genes known to be required for silencing is unchanged in HC-Pro plants, whereas a striking number of genes involved in other biotic and abiotic stress responses are induced, many in a RAV2-dependent manner. Among the genes that require RAV2 for induction by HC-Pro are FRY1 and CML38, genes implicated as endogenous suppressors of silencing. These findings raise the intriguing possibility that HC-Pro-suppression of silencing is not caused by decreased expression of genes that are required for silencing, but instead, by induction of stress and defense responses, some components of which interfere with antiviral silencing. Furthermore, the observation that two unrelated viral suppressors require the activity of the same factor to block silencing suggests that RAV2 represents a control point that can be readily subverted by viruses to block antiviral silencing.

DNA topoisomerase I mutants: Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon.