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.

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.

Topoisomerase I mutants: the gene on pBR322 that encodes resistance to tetracycline affects plasmid DNA supercoiling.

Plasmid pBR322 DNA isolated from topoisomerase I mutants of Escherichia coli and Salmonella typhimurium exhibits a distinctive supercoiling distribution characterized by an extremely heterogeneous distribution of linking numbers that contains highly negatively supercoiled topoisomers. Analysis of the supercoiling distributions of deletion and insertion derivatives of pBR322 shows that the presence of the gene on pBR322 encoding resistance to tetracycline is responsible for the unusual supercoiling distribution. Both an intact promoter and a portion of the remainder of the gene, but not the gene product, are required. However, no particular section of the gene outside the promoter appears to be necessary; only the size of the section remaining appears to be important. These observations suggest that transcription of this gene may be responsible for its effect on DNA supercoiling.

Effects of DNA gyrase inhibitors in Escherichia coli topoisomerase I mutants.

Relaxation of titratable supercoils in bacterial nucleoids was measured following treatment of topA mutants with coumermycin or oxolinic acid, inhibitors of DNA gyrase. Relaxation occurred after treatment of the mutants with either inhibitor. We detected no significant difference in relaxation between topA- and topA+ strains treated with coumermycin. This finding, together with previous observations, supports the idea that relaxation caused by coumermycin probably arises from the relaxing activity of gyrase itself. The source of DNA relaxation caused by oxolinic acid was not identified. Nucleoid supercoiling can be increased by adding oxolinic acid to a strain that carries three topoisomerase mutations: delta topA, gyrB225, and gyrA (Nalr) (S. H. Manes, G. J. Pruss, and K. Drlica, J. Bacteriol. 155:420-423, 1983). We found that this increase in supercoiling requires partial sensitivity to the drug and at the delta topA and gyrA mutations. Full resistance to oxolinic acid in the presence of the delta topA, gyrB225, and gyrA mutations was conferred by an additional mutation that maps at or near gyrB.

DNA supercoiling and suppression of the leu-500 promoter mutation.

top mutations (formerly supX) eliminate DNA topoisomerase I activity and suppress the leu-500 promoter mutation in Salmonella typhimurium (K. M. Overbye, S. K. Basu, and P. Margolin, Cold Spring Harbor Symp. Quant. Biol. 47:785-791, 1983). Sublethal doses of coumermycin which reduce intracellular levels of supercoiling activity in a top mutant eliminated suppression of the leu-500 mutation. This result provides evidence that increased DNA supercoiling suppresses the leu-500 promoter mutation in top mutants.

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.

DNA supercoiling in gyrase mutants.

Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%.

Inhibition of RNA synthesis by oxolinic acid is unrelated to average DNA supercoiling.

Oxolinic acid reduced RNA synthesis rates whether chromosome supercoiling decreased, increased, or remained unchanged. Thus, inhibition of RNA synthesis by oxolinic acid appears to involve factors other than average DNA supercoiling level. Coumermycin A1 caused RNA synthesis rates to increase or decrease roughly in parallel with DNA supercoiling.

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.