Nature Medicine Essay

COMMENTARY LASKER BASIC MEDICAL RESEARCH AWARD Of maize and men, or peas and people: case histories to justify plants and other model systems David Baulcombe One of the byproducts of molecular biology cork is altogether filled with air, and that air is has been support for the ‘model system’ con- perfectly enclosed in little boxes or cells distinct cept. All living organisms are based on the same from one another.”)2 (Fig. 1). Two hundred fifty genetic code, they have similar subcellular years later, Beijerinck discovered a contagium structures and they use homologous metabolic vivum fluidum in extracts of diseased tobacco pathways. So, mechanisms can be investigated plants that he later referred to as a virus3. using organisms other than those in which In contemporary science, a green alga— the knowledge will be exploited for practical Chlamydomonas reinhardtii—is a useful model benefit. Model systems are particularly use- in the analysis of kidney disease4. However, ful in the early discovery phase of a scientific in this article, I refer to the contribution of endeavor, and recent progress in biomedical plant biology to a family of mechanisms that I science has fully vindicated their use. Jacques refer to as RNA silencing. This topic has been Monod, for example, famously justified his reviewed comprehensively elsewhere5,6, so here work on a bacterial model system by stating I focus on personal experience and my view of that “what is true for Escherichia coli is also future potential from this work. true for elephants.” My fellow laureates, Victor Ambros and Gary Ruvkun, can defend the use The early history of RNA silencing in of the worm Caenorhabditis elegans as a good plants model system and so I will focus on plants. The most important development in plant Probably the best example I could use is biology in the 1980s was the introduction of that of Barbara McClintock, who used corn methods for transfer of DNA into the nuclear in her Nobel Prize–winning work1. Her iden- genome7. Transgenic techniques were devel- tification of mobile genetic elements has been oped as a routine research tool, and the new enormously influential in microbiology and technology prompted the introduction of Figure 1 Robert Hooke’s micrograph of cells immunology, and continues to bear on the genetic manipulation for crop improvement. in a sample of cork. Downloaded from http:// field of epigenetics that I refer to again below. The first generation of genetically modified www.nlm.nih.gov/exhibition/historicalanatomies/ She could have adapted Monod’s aphorism to plants used bacterial genes for herbicide tol- Images/1200_pixels/hooke_t11.jpg. refer to maize and men. Gregor Mendel would, erance or insect resistance8,9. There were also of course, have referred to peas and people. attempts from several laboratories, including structure would be formed between the mRNA There are many other historical examples, in my own, to develop virus resistance by trans- of the target gene and a transgenic antisense addition to McClintock’s and Mendel’s work, genic expression of virus-derived genes in the RNA. This duplex structure would then either that could justify the use of plants as models. host organism10–12. We were testing a concept block translation of the mRNA or initiate its Robert Hooke used his newly developed micro- known as parasite-derived resistance in which degradation by a nuclease specific for double- scope in 1664 to show that cork comprises the replication or spread of a disease agent stranded RNA. structures that he referred to as cells (“Our could be reduced by transgenic expression of Many of the early transgenic plants had the microscope informs us that the substance of its genes in a host. A third category of these desired traits, and they were the progenitors of early transgenic crops exploited an antisense transgenic crops that are now grown in large David Baulcombe is Royal Society Research approach to block expression of an endoge- areas of different countries. However, there Professor and Professor of Botany, Department of nous plant gene13. Crops were to be improved were anomalous phenotypes in some of the Plant Sciences, University of Cambridge, Downing in this approach by specific suppression of an transgenic lines. For example, the bacterial and Street, Cambridge CB2 3EA, UK. endogenous gene that limited agronomic per- viral transgenes were not always expressed8,9. e-mail: [email protected] formance. The idea was simple: a duplex RNA In the ‘antisense plants’, the suppression effect xx VOLUME 14 | NUMBER 10 | OCTOBER 2008 NATURE MEDICINE COMMENTARY that operates in a nucleotide sequence–specific ous now. My failure to make the connection manner. The silencing in cis would explain why was probably because the mechanism in worms the transgenes in certain lines were not abun- involved suppression of translation, whereas, dantly expressed. Correspondingly, the silenc- in plants, we were dealing with a process that ing in trans would account for the ability of decreased stability of the targeted RNA. these transgenes to silence endogenous genes Andrew Hamilton’s experiments further or viruses20,21. demonstrated that the production of small The hypothesis further proposed that the antisense RNA required transcription of the silencing mechanism operates at the RNA corresponding sense strand. This involvement level because the targeted viruses had an RNA of both RNA strands provided an obvious genome and did not have a DNA phase in their connection between RNA silencing in plants replication cycle. Originally, many of the silenc- and RNA interference in worms as revealed ing-related phenomena in transgenic plants by the famous Andrew Fire and Craig Mello 29 Richard Jorgensen Richard were referred to as ‘gene silencing’. However, 1998 paper in Nature . Once this connection Figure 2 Variegation of silencing in flowers at a seminar in Switzerland, I was interrupted was recognized, there was an opportunity for of petunia. The target of silencing by a sense by Ingo Potrykus—the inventor of ‘golden discoveries in plants to catalyze our under- transgene is chalcone synthase. The silencing rice’22—who pointed out that I was not talk- standing of animal systems and vice versa. It effect manifested as the absence of pigment ing about ‘gene silencing’; it was ‘RNA silenc- has been enormously satisfying to witness this is present in only part of the flower. Reprinted with permission of the American Society of ing’. I agreed and have since promoted ‘RNA exchange of information and the elucidation Plant Biologists. silencing’ as a generic term to cover a family of of variations on a molecular theme associated mechanisms involving silencing and RNA. with different types of silencing mechanism in was sometimes variegated—it was present on Support for the model came from a series diverse organisms5,6. some branches of the plant and not in others, of tests carried out by Jim English with geneti- Much current RNA silencing research inter- or it was absent in localized parts of a flower14. cally modified viruses20, and the remaining est is focused on endogenous small RNAs. In There was also a problem with some of the challenges were to find out about the details of plants, there are thousands of loci that produce sense RNA controls in these antisense experi- the mechanism and its biological role. We were these small RNAs30–32. A small proportion of ments. There was a silencing effect even when also interested in finding out whether the var- the endogenous small RNAs are similar to the transgene was designed to generate sense iegated silencing of transgenes and transcrip- the microRNAs of worms that were first dis- RNA that would not form a duplex with the tional silencing of promoter sequences could covered as short temporal RNAs by Ruvkun target mRNA15–17. In some examples, there was be accommodated in the model. and Ambros27,28. Most microRNAs are nega- variegation of this sense suppression as with the A central mystery in RNA silencing was its tive regulators, although a positive regulatory antisense plants (Fig. 2). Another strange pro- ability to act in a nucleotide sequence–specific microRNA has been recently described33. cess occurred when two transgenes carried the manner. The simplest explanation required The other small RNAs in plants are siRNAs, same promoter sequence: one of these trans- an antisense RNA that would guide a silenc- the double-stranded RNA precursors, which genes could silence the other through a process ing machinery to its target. In the transgenic are produced in various ways, including the operating at the transcriptional level18. experiments described above, this antisense annealing of complementary RNA, foldback In retrospect, I can see that these anomalies RNA would be present irrespective of whether of long inverted repeats and the action of challenged the existing paradigm of genetic the transgene was in a sense or an antisense RNA-dependent RNA polymerases on a single- regulation, but initially, I made the mistake of orientation. Andrew Hamilton, who had been stranded RNA30,31. thinking that they were due to multiple dis- interested in antisense RNA ever since his PhD It could be said that, now that we have the tinct mechanisms and that they were artifacts work on transgenic tomatoes23, joined the lab understanding of multiple variations on silenc- of transgenes. However, I became more inter- to carry out the search for this elusive molecu- ing mechanisms, RNA silencing research with ested when we characterized transgenic plants lar species. It is testimony to his persistence and a biomedical target should focus exclusively on manifesting parasite-derived resistance. The talent as a scientist that he eventually found people or at least on vertebrate experimental plants with the strongest viral resistance were them24. He tried several different methods, and systems.

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