RNA Silencing in Plants

Features RNA RNA silencing in plants David Baulcombe Some scientific discoveries are made because someone had – tomato paste – to have been sold in Europe3. However, (University of Cambridge, UK) vision. The ‘someone’ could see a gap in understanding of the the mechanism of silencing in these plants could not be natural world and had insight, intuition or a combination easily explained. The experiments were designed in the of both that led to new understanding. Other, perhaps expectation that the transgene antisense RNA would bind most, advances may depend on an unexpected result as to the sense messenger RNA (mRNA) of an endogenous an indication that the current paradigm is incomplete or ripening gene. However, it seemed unlikely that such a Downloaded from http://portlandpress.com/biochemist/article-pdf/37/2/10/5405/bio037020010.pdf by guest on 26 September 2021 wrong. RNA silencing in plants is very definitely in the simple mechanism was involved because silencing could be latter category. None of the silencing pioneers set out to achieved with both sense and antisense transgenes3. discover a new level of genetic control in cells. They were In the virus resistance experiments, the original using transgenes to modify pigment production, tomato aim was to develop a type of genetic immunization by ripening or virus resistance in what Kuhn would have transformation of plants with transgenes containing all called “normal science”1. or part of a viral gene. We expected that that the encoded At first it was not obvious that there was a relationship protein would somehow disrupt the replication cycle of the between these different systems, although, in each instance, virus. These experiments were successful in that some of a transgene was unexpectedly and unpredictably silent. the lines were highly resistant against the virus4. However, In the flower colour experiments in petunia, the silencing there was an anomaly: the viral transgene was silent in the affected both the transgene and any endogenous genes with resistant lines and highly expressed in susceptible lines a similar sequence: there was co-ordinate suppression or (Figure 2). Normally, of course, one expects a transgenic ‘co-suppression’2. The flowers were white because neither trait to be associated with transgene expression, but, in the transgene nor the endogenous gene was sufficiently this instance, it was the opposite. The eventual unravelling active to support pigment production (Figure 1). of these various transgenic phenomena led to the The tomato experiments were designed to silence understanding that we have now of RNA silencing as a genes involved in fruit ripening using antisense RNA and central mechanism in genetic regulation. they were highly successful. The fruit from these plants were used for the first ever genetically modified (GM) food Viruses hold the key The mysterious observations prompted much speculation and, at one time, it seemed as though there were more review articles than primary research papers on this topic. However, after much head-scratching, we realized that we could explain these results very simply if a single silencing mechanism affected both transgene expression and virus resistance4. This was a useful insight because the viruses in our experiments inhabit the RNA world. They are packaged as RNA, translated as RNA and replicated as RNA. We concluded therefore that the silencing mechanism was Figure 1. Co-suppression in petunia. A normal petunia flower is targeted at RNA. It is for this reason that we started using purple due to pigments that are dependent on chalcone synthase the term ‘RNA silencing’6. (left). Introduction of a sense RNA chalcone synthase transgene It was striking that the resistance in our transgenic triggers RNA silencing. The silencing results in co-ordinate plants was highly specific for strains of virus that were closely suppression of the endogenous and transgene copies of chalcone similar to the transgene and that the transgene conferred synthase and the flowers are unpigmented (right). (Pictures resistance even if their RNA could not be translated into provided by and reproduced with permission of R. Jorgensen.) protein. On the basis of these findings, we speculated that Key words: antisense Abbreviations: miRNA, microRNA; siRNA, small interfering RNA; stRNA, short temporal RNA. RNA, co-suppression, RNA silencing, transgenes, viruses 10 April 2015 © Biochemical Society RNA Features there was an antisense RNA as the specificity determinant of the RNA-silencing mechanism6. It was an attractive hypothesis, at least in our minds, because it could explain the petunia and tomato results as well as our virus resistance data. However, the hypothesis did not have molecular or genetic support and the biological function of this RNA-silencing system was not obvious. To address these questions, we looked for the hypothetical antisense RNA and we set up a mutant screen to identify the genes encoding any proteins required for this RNA to have an effect. Andrew Hamilton joined my laboratory at this time Downloaded from http://portlandpress.com/biochemist/article-pdf/37/2/10/5405/bio037020010.pdf by guest on 26 September 2021 and he took on the antisense RNA challenge. His initial experiments failed to detect the predicted RNA, but, one evening, he stopped his gel electrophoresis early so that he could be in time to play football. The following day, the result Figure 2. RNA-mediated virus resistance. The leaves are from tobacco plants transformed was there in the autoradiograph: small antisense RNA at the with a gene from the RNA genome of potato virus X. The leaves are from plants that were: bottom of the gel in silencing lines, but not in the controls. not inoculated (far right) or inoculated with potato virus X. Susceptible plants showed mosaic These key molecules migrated rapidly in electrophoresis symptoms evident as light grey blotches in this black-and-white picture. Non infected and and, in his previous experiments, they had run off the end fully resistant plants had no mosaic and were uniformly grey. From 5 with permission. of the gel. I presented some of his data at a conference, but the autoradiographs were not pretty. One of my colleagues told me that I was damaging my reputation by presenting such ugly data and so we delayed publication for some time until we could get nice Northern blots7. The small RNAs are now known as small interfering RNAs (siRNAs). The mutant screens took even longer to set up, but they were successful, and, by 2000, we had a pretty good idea from work in several laboratories including my own that the mechanism involved antisense RNA and that RNA- dependent RNA polymerases were required to produce this antisense RNA8. Nature got there first A clue to the biological role of RNA silencing was its effectiveness in virus resistance. Perhaps we had stumbled on a process that normally protects plants against viruses? To test this idea, we designed experiments that demonstrated that transgene silencing has antiviral potential and that RNA silencing is induced as a normal consequence of virus infection9,10. A third line of evidence to support the role of silencing in natural protection against viruses was from the finding that virus-infected plants produced siRNAs corresponding Figure 3. RNA silencing as a virus defence system. These to the viral genome7 (Figure 3). In addition, we found that Nicotiana benthamiana plants were inoculated with potato plants without a functional RNA-silencing machinery virus X. The plants on the right are wild-type plants and they were hypersusceptible to virus infection11. Eventually, the exhibit mild symptoms characteristic of the disease due to this discovery that viruses encode suppressor proteins of RNA virus. The plant on the right is defective for an RNA-dependent silencing was the final support for our hypothesis12. We now RNA polymerase of the RNA-silencing pathway and it is had proof of RNA silencing in defence and of a counter- hypersusceptible to the virus. It is stunted and exhibits severe defence system encoded in the virus. Clearly, Nature had mosaic. Republished with permission from the American beaten us to the possibility of using RNA silencing to Society of Plant Biologists: Plant Physiology Schwach, F., Vaistij, protect plants against viral disease. F.E., Jones, L. and Baulcombe, D.C. 138 2005 11. April 2015 © Biochemical Society 11 Features RNA double-stranded RNA15. This double-stranded RNA could be formed by annealing of complementary RNA strands or by the action of an RNA-dependent RNA polymerase. An RNase III-like enzyme, Dicer, then produces small RNAs and a second nuclease known as Argonaute or Slicer then recruits one of the two strands. Slicer is the effector of RNA silencing. It uses the small RNA as a guide that base pairs to a target RNA and, in the simplest RNA-silencing pathway, it mediates RNA cleavage (Figure 4). Once these mechanisms were known, the link with the work of Ruvkun and Ambros was clear. Their stRNAs are now renamed microRNAs (miRNAs) and, like siRNAs, Downloaded from http://portlandpress.com/biochemist/article-pdf/37/2/10/5405/bio037020010.pdf by guest on 26 September 2021 they are produced by Dicer and they associate with Slicer16 (Figure 4). It transpires that both plant and animal cells have large populations of endogenous miRNAs. The miRNA pathway is therefore a variation of the defence pathway revealed by our work with transgenes and viruses, and RNA silencing is an important regulatory mechanism in gene expression of plants and animals. The common core RNA-silencing pathway in diverse organisms points clearly to the ancient origin of RNA silencing. One speculation is that this process could be a remnant of the RNA world before DNA became the primary genetic template. Perhaps the RNA-dependent RNA polymerases of RNA silencing are related to the Figure 4.

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