RNA interference in crop plants

Makoto Kusaba
RNA interference (RNAi) is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in various organisms. Although RNAi was rst discovered in worms, related phenomena such as posttranscriptional gene silencing and coat protein mediated protection from viral infection had been observed in plants prior to this. In plants, RNAi is often achieved through transgenes that produce hairpin RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efciency and stability. It also offers advantages over mutation-based reverse genetics in its ability to suppress transgene expression in multigene families in a regulated manner.
Addresses Institute of Radiation Breeding, National Institute of Agrobiological Sciences, PO Box 3, Ohmiya-machi, Naka-gun, Ibaraki 319-2293, Japan e-mail: kusaba@affrc.go.jp

system have been developed for high-throughput analysis [3,4]. Recent studies have revealed the natural roles of RNAi and RNAi-related phenomena, including suppression of transposon activity, resistance to virus infection, post-transcriptional and post-translational regulation of gene expression, and epigenetic regulation of chromatin structure [5,6]. RNAi is expected to be of practical use in the genetic improvement of crop plants. Here, we focus on RNAi as a knockdown technology and its application to crop plants.

The discovery of RNAi and RNAi-related phenomena

RNAi was rst discovered as a gene-silencing phenomenon induced by dsRNA in worms [5,7] (Figure 1). Guo and Kemphues rst showed that injection of the sense or antisense RNA for a particular gene was able to suppress gene function in a sequence-specic manner. It was later shown by Fire and Mello that it was in fact contaminating dsRNA in the sense and antisense preparations that was the real inducer of gene suppression in the study; this phenomenon was termed RNAi [5]. Because dsRNA for introns did not show the RNAi effect, RNAi was thought to act in a post-transcriptional manner. RNAi-related phenomena had been demonstrated in plants before the discovery of RNAi by Guo and Kemphues. One of these phenomena is co-suppression, that is, gene silencing mediated by a sense transgene. In cosuppression, expression of the transgene itself is suppressed together with that of endogenous homologous genes. Co-suppression was subsequently shown to involve either transcriptional gene silencing (TGS) or post-transcriptional gene silencing (PTGS). Another example of an RNAi-related phenomenon is coat protein mediated protection (CPMP). Virus resistance is conferred by a sense coat protein transgene. Initially, protection was thought to be induced by the coat protein, but later it was shown that untranslatable coat protein transgenes could also confer virus resistance. Because CPMP was found to act post-transcriptionally, it was thought that CPMP and PTGS shared similar mechanisms. Hamilton and Baulcombe made a striking discovery: they showed that the appearance of a small RNA molecule of about 25 nucleotides (nt) with homology to the target gene of PTGS was associated with the PTGS phenotype. A similar molecule was later found in an in vitro RNAi system for Drosophila and was named small interfering RNA (siRNA) [5]. These observations strongly suggested that PTGS and RNAi shared the same suppression mechanism and raised the possibility that dsRNA is
Current Opinion in Biotechnology 2004, 15:139143

Current Opinion in Biotechnology 2004, 15:139143 This review comes from a themed issue on Plant biotechnology Edited by Takuji Sasaki and Paul Christou 0958-1669/$ see front matter 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2004.02.004

Abbreviations CPMP coat protein mediated protection dsRNA double-stranded RNA hpRNA hairpin RNA miRNA micro RNA PTGS post-transcriptional gene silencing RdRP RNA-dependent RNA polymerase RISC RNA-induced silencing complex RNAi RNA interference siRNA small interfering RNA UTR untranslated region VIGS virus-induced gene silencing

Introduction
RNA interference (RNAi) is a double-stranded RNA (dsRNA)-induced gene-silencing phenomenon that is conserved among various organisms, including animals and plants. Because of its high specicity and efcacy, it has been widely used as an efcient tool to analyze gene function. In worms and ies, genome-wide analysis based on complete genome sequences has already been performed using RNAi methods [13]. For studies with cultured cells, an automated RNAi screening system using assay plates and a microarray-based RNAi screening
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140 Plant biotechnology

Figure 1

Plants Coat protein mediated protection (1986)

Animals

Co-suppression (1990) Micro RNA (small temporal RNA) (1993) Virus-induced gene silencing (1995) The first description of RNAi (1995)

hpRNA transgene (1998) siRNA (1999), RdRP (1999)

RNAi (1998) in vitro RNAi (1999) RNA-induced silencing complex (2000) Dicer (2001)
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A chronological history of the early work on RNAi and RNAi-related phenomena.

Figure 2

PTGS Aberrant ssRNA

RdRP

generated during the PTGS process. It is thought that, in PTGS, aberrant single-stranded (ss) RNA transcribed from a transgene triggers the generation of dsRNA by RNA-dependent RNA polymerase (RdRP), and consequently the RNAi pathway is activated [7] (Figure 2). Interestingly, PTGS spreads systemically from the tissue where it was originally induced; the signaling molecule has not been identied, but is believed to be RNA [7].

The molecular mechanism of RNAi

hpRNA dsRNA pre-miRNA

Dicer

In vitro RNAi systems for Drosophila have revealed the detailed molecular mechanism of RNAi [5,8] (Figure 2). First, long dsRNA is recognized by a member of the RNase III family, Dicer, and digested into 21 nt siRNA duplexes. Each duplex is unwound and one of the two strands is incorporated, often preferentially, into the RNA-induced silencing complex (RISC). The antisense

RISC

Translation inhibition

mRNA cleavage
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The molecular mechanism of RNAi and RNAi-related phenomena in plants. PTGS involves the generation of dsRNA by RdRP. Micro RNA (miRNA) is an endogenous siRNA-like RNA known to be involved in the developmental regulation of gene expression in animals [5] and plants [35,36]. Its precursor (pre-miRNA) is a small hpRNA with bulges in its stem region. All dsRNA, hpRNA and pre-miRNA are processed by Dicer into 21 nt RNA duplexes and the unwound ssRNA is then incorporated into RISC. In plants, dsRNA and pre-miRNA can be processed by distinct DICER-LIKE proteins [37]. In animals, miRNA, which is partially complementary to mRNA, inhibits translation. In plants, like siRNA, miRNA cleaves mRNA despite a small number of mismatches with the target mRNA [11]. It should be noted, however, that some miRNAs also inhibit translation in plants as well as in animals [35]. www.sciencedirect.com

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strand of the siRNA then hybridizes to mRNA as a guide, and the RISC cleaves the mRNA near the center of the siRNA. The siRNA duplex consists of a 19 nt doublestranded region with 2 nt 30 overhangs. In Drosophila, mismatches between siRNA and the target mRNA greatly reduce the efciency of mRNA cleavage, particularly when these are located near the center of the siRNA [9,10]. It should be noted that in plants a small number of mismatches can be tolerated [11].

action of virus-encoded RdRP. If the virus genome contains a host plant gene, inoculation of the virus can trigger RNAi against the plant gene. Because this approach does not involve a transformation process, it might be suitable for the functional analysis of essential genes. Amplicon is a technology related to VIGS [12]. It uses a set of transgenes comprising virus genes that are necessary for virus replication and a target gene. Like VIGS, amplicon triggers RNAi but it can also overcome the problems of host-specicity of viruses.

RNAi as a tool for gene function analysis in plants

Although RNAi is not a knockout but a knockdown technology, its high efciency and ease of application make it applicable to genome-wide analysis of gene function. In plants, RNAi is often achieved by a transgene that produces hairpin RNA (hpRNA) with a dsRNA region [12]. Conventionally, antisense-mediated gene silencing has been widely used in the analysis of gene function in plants. Although antisense-mediated gene silencing is an RNAi-related phenomenon [13], hpRNA-induced RNAi has been shown to be much more efcient [14]. In an hpRNA-producing vector, the target gene is cloned as an inverted repeat spaced with an unrelated sequence and is driven by a strong promoter, such as the 35S CaMV promoter for dicots or the maize ubiquitin 1 promoter for monocots. When an intron is used as the spacer, which is essential for stability of the inverted repeat in Escherichia coli, the efciency becomes very high: almost 100% of transgenic plants show gene silencing [15,16]. However, the mechanism by which the intron increases silencing efciency remains unclear [17]. RNAi can be used against a vast range of targets; 30 and 50 untranslated regions (UTRs) as short as 100 nt could be efcient targets of RNAi. For genome-wide analysis of gene function, a vector for high-throughput cloning of target genes as inverted repeats, which is based on an LR Clonase reaction, has been constructed [16]. Another high-throughput RNAi vector is based on spreading of RNA targeting (also called transitive RNAi) from an inverted repeat of a heterologous 30 UTR [18]. For analysis of genes essential to plant viability, a chemically regulated RNAi system has also been developed [19]. Direct introduction of dsRNA or a plasmid producing hpRNA transiently by particle bombardment has been shown to induce RNAi in plants [20,21]. This approach is useful for the analysis of gene function in plants in cases where transgenic approaches that require stable transformation are more difcult. Virus-induced gene silencing (VIGS) is another approach often used to analyse gene function in plants [12]. RNA viruses generate dsRNA during their life cycle by the
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RNAi as a tool for the genetic improvement of crop plants

Trait stability from one generation to the next is essential for the genetic improvement of crop plants. Phenotype suppression by PTGS may be inherited unstably [22]. There are only a few reports describing the stability of hpRNA-induced RNAi. Phenotype suppression by hpRNA transgenes is inherited stably at least as far as the T5 generation in Arabidopsis [23]; no data are available beyond T5, but the transgene is expected to persist. The rice mutant line LGC-1 (Low Glutelin Content-1) was the rst commercially useful cultivar produced by RNAi [24]. It is a low-protein rice and is useful for patients with kidney disease whose protein intake is restricted. This dominant mutation produces hpRNA from an inverted repeat for glutelin, the gene for the major storage protein glutelin, leading to lower glutelin content in the rice through RNAi. Interestingly, this mutant was isolated in the 1970s, and the mutant trait appears to have been stable for over 20 generations. These examples suggest that the suppression of gene expression by hpRNAinduced RNAi would be inherited stably. RNAi induced by hpRNA does not require some of the genes or components involved in PTGS, including RdRP [25]. The reason why hpRNA-induced RNAi is inherited more stably than PTGS might be that hpRNA-induced RNAi does not require the generation of dsRNA mediated by RdRP for the suppression of gene expression. Downregulation can also be achieved through loss-offunction mutations. For rice, mutation-based reverse genetics and a gene targeting system are available [26,27]. The usefulness of gene targeting is discussed by S Iida in this issue. RNAi has some advantages over these systems, however. One of these is its applicability to multigene families and polyploids [28], as it is not straightforward to knockout a multigene family by the accumulation of mutations for each member of the family by conventional breeding, particularly if members of the family are tightly linked. In the example of LGC-1 discussed earlier, lowering of the glutelin content is achieved not by accumulation of loss-of-function of members of the glutelin multigene family (which comprises at least eight members, ve of which are clustered in a particular chromosomal region), but from a single RNAi-inducing locus [24] (M Kusaba, unpublished).
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Another advantage of RNAi lies in the ability to regulate the degree of suppression. Agronomic traits are often quantitative, and a particular degree of suppression of target genes may be required. Control of the level of expression of dsRNA through the choice of promoters with various strengths is thought to be useful in regulating the degree of suppression. However, the use of a weak promoter appears to result in a reduction in the frequency of suppression, rather than the induction of weak suppression [14]. An alternative approach is the use of sequences with various homologies to the target gene. In LGC-1, homology-dependent suppression by RNAi was observed [24]. Such homology dependency could result from the effectiveness of each siRNA to cleave target mRNA. The degree of suppression of a gene could be designed by using homologous genes isolated from closely or distantly related species that exhibit various homologies to the target gene. Such an approach could be applied to the improvement of various agronomic traits such as plant height [29] and organoleptic properties. The control of tissue-specic or stimuli-responsive suppression is another possible application of RNAi, as the choice of suitable promoters could enable such regulation. However, gene silencing, not only by PTGS but also by the direct introduction of dsRNA, is known to spread systemically [7,21]. This raises the possibility that when RNAi is induced in a particular tissue it might also suppress the target gene in other tissues where downregulation is not desired. A seed-specic promoter has been shown to be effective for suppressing constitutively expressed genes, but no data has yet been generated to demonstrate conclusively whether the suppression is conned to the seeds [23]. Lgc1 acts as a Mendelian factor in F2 seeds on a single F1 plant, suggesting that there is no transmission of the silencing signal among developing seeds [24]. Absence of plasmodesma between the seed and its surrounding tissues might affect the efciency of spread of the silencing signal. Alternatively, the signal might be excluded from seeds, as it is excluded from the shoot apex [30]. By such mechanisms, hpRNA-induced RNAi driven by a seed-specic promoter might confer seed-specic suppression; however, when other tissues, particularly where the PTGS signal travels easily, are specic targets of hpRNA-induced RNAi this specicity might be lost. In fact, systemic spread was observed in the chemically regulated RNAi system [19]. This potential problem could be overcome by the use of a virus protein that suppresses the systemic spread of the PTGS signal [31] or through knockout of a gene involved in the spread of the RNAi signal [32] (see also Update).

achieved by mutation-based reverse genetics, but its use is more limited than that of RNAi. Although the basic concept of the application of transgene-based RNAi to the genetic improvement of crop plants has been established, further feasibility studies are needed for its wider application.

Update
Recently, another inducible RNAi system in plants was reported [38]. In this ethanol-inducible system, no systemic spread of gene silencing was observed.

Acknowledgements
I would like to thank Ichiro Mitsuhara for useful discussions in the preparation of this article. This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome Project IP-1011).

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrman M, Ahringer J: Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 2000, 408:325-330. Gonczy P, Echeverri C, Oegema K, Coulson A, Jones SJM, Copley RR, Duperon J, Oegema J, Brehm M, Cassin E et al.: Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 2000, 408:331-336. Kiger AA, Baum B, Jones S, Jones MR, Coulson A, Echeverri C, Perrimon N: A functional genomic analysis of cell morphology using RNA interference. J Biol 2003, 2:27. Mousses S, Caplen NJ, Cornelison R, Weaver D, Basik M, Hautaniemi S, Elkahloun AG, Lotufo RA, Choudary A, Dougherty ER et al.: RNAi microarray analysis in cultured mammalian cells. Genome Res 2003, 13:2341-2347. Hannon GJ: RNA interference. Nature 2002, 418:244-251. Grewal SIS, Moazed D: Heterochromatin and epigenetic control of gene expression. Science 2003, 301:798-802. Waterhouse PM, Ming-Bo W, Lough T: Gene silencing as an adaptive defence against viruses. Nature 2001, 411:834-842.

2.

3.

4.

5. 6. 7. 8. 

gner G, Du T, Xu Z, Aronin N, Zamore PD: Schwarz DS, Hutva Asymmetry in the assembly of the RNAi enzyme complex. Cell 2003, 115:199-208. This paper provides the latest model of RNAi operation and gives important information for designing articial siRNAs. 9. Elbashir SM, Martinez J, Patkaniowska A, Lendeckedl W, Tuschl T: Functional anatomy of siRNAs for mediating efcient RNAi in Drosophila melanogaster embryo lysate. EMBO J 2001, 23:6877-6888.

10. Abdelgany A, Wood M, Beeson D: Allele-specic silencing of a pathogenic mutant acetylcholine receptor subunit by RNA interference. Hum Mol Genet 2003, 12:2637-2644. 11. Tang G, Reinhart BJ, Bartel DP, Zamore PD: A biochemical  framework for RNA silencing in plants. Genes Dev 2003, 17:49-63. This paper describes the peculiarity of micro RNA in plants using an in vitro RNAi system of wheat-germ extract. 12. Waterhouse PM, Helliwell CA: Exploring plant genomes by RNAinduced gene silencing. Nat Rev Genet 2003, 4:29-38. b H, Iglesias A, Tarina C, Bouldoires E, Meins FJ: 13. Serio FD, Scho Sense- and antisense-mediated gene silencing in tobacco is inhibited by the same viral suppressors and is associated with www.sciencedirect.com

Conclusions
Because RNAi is a very efcient knockdown technology in plants it is thought to be useful for genetic improvement, even in plants with low transformation efciencies [33,34]. Downregulation of a particular gene can be
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accumulation of small RNAs. Proc Natl Acad Sci USA 2001, 98:6506-6510. 14. Chuang C-F, Meyerowitz EM: Specic and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 2000, 97:4985-4990. 15. Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AG, Waterhouse PM: Total silencing by intron-spliced hairpin RNAs. Nature 2000, 407:319-320. 16. Wesley SV, Helliwell CA, Smith NA, Wang M-B, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA et al.: Construct design for efcient, effective and high-throughput gene silencing in plants. Plant J 2001, 27:581-590. 17. Helliwell C, Waterhouse P: Constructs and methods of high throughput gene silencing in plants. Methods 2003, 30:289-295. This paper provides tips for generating RNAi-inducing constructs using the most popular hpRNA vectors developed by the authors. 18. Brummell DA, Balint-Kurti P, Harpster MH, Palys JM, Oeller PW, Gutterson N: Inverted repeat of a heterologous 30 -untranslated region for high-efciency, high-throughput gene silencing. Plant J 2003, 33:798-800. 19. Guo H-S, Fei J-F, Xie Q, Chua N-H: A chemical-regulated inducible RNAi system in plants. Plant J 2003, 34:383-392. 20. Schweizer P, Pokorny J, Schulze-Lefert P, Dudler R: Doublestranded RNA interferences with gene function at the singlecell level in cereals. Plant J 2000, 24:895-903. te P, Leuenberger SA, Iglesias VA, Meins FJ: High 21. Klahre U, Cre molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants. Proc Natl Acad Sci USA 2002, 18:11981-11986. 22. Mitsuhara I, Shirasawa SN, Iwai T, Nakamura S, Honkura R, Ohashi Y: Release from post-transcriptional gene silencing by cell proliferation in transgenic tobacco plants: possible mechanism for noninheritance of the silencing. Genetics 2002, 160:343-352. 23. Stoutjesdijk PA, Singh SP, Liu Q, Hurlstone CJ, Waterhouse PA, Green AG: hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efcient and stable silencing. Plant Physiol 2002, 129:1723-1731. 24. Kusaba M, Miyahara K, Iida S, Fukuoka H, Takano T, Sassa H,  Nishimura M, Nishio T: Low glutelin content 1: a dominant mutation that suppresses the glutelin multigene family via RNA silencing in rice. Plant Cell 2003, 15:1455-1467. Report of the rst induced mutant in which involvement of the RNAi mechanism was demonstrated. Interesting as an example of RNAi in a multigene family. clin C, Boutet S, Waterhouse P, Vaucheret H: A branched 25. Be pathway for transgene-induced RNA silencing in plants. Curr Biol 2002, 12:684-688.

26. Miyao A, Tanaka K, Murata K, Sawaki H, Takeda S, Abe K, Shinozuka Y, Onosato K, Hirochika H: Target site specicity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell 2003, 15:1771-1780. 27. Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S: Efcient gene targeting by homologous recombination in rice. Nat Biotechnol 2002, 20:1030-1034. 28. Lawrence RJ, Pikaard CS: Transgene-induced RNA interference:  a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations. Plant J 2003, 36:114-121. This paper describes the usefulness of RNAi for genetic improvement of polyploids. 29. Sakamoto T, Morinaka Y, Ishiyama K, Kobayashi M, Itoh H, Kayano T, Iwahori S, Matsuoka M, Tanaka H: Genetic manipulation of gibberellin metabolism in transgenic rice. Nat Biotechnol 2003, 21:909-913. 30. Foster TM, Lough TJ, Emerson SJ, Lee RH, Bowman JL, Foster RLS, Lucas WJ: A surveillance system regulates selective entry of RNA into the shoot apex. Plant Cell 2002, 14:1497-1508. 31. Voinnet O, Lederer C, Baulcombe DC: A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 2000, 103:157-167. 32. Feinberg EH, Hunter CP: Transport of dsRNA into cells by the transmembrane protein SID-1. Science 2003, 301:1545-1547. 33. Liu Q, Singh SP, Green AG: High-stearic and high-oleic cottonseed oils produced by hairpin RNA-mediated posttranscriptional gene silencing. Plant Physiol 2002, 129:1732-1743. 34. Ogita S, Uefuji H, Yamaguchi Y, Koizumi N, Sano H:  Producing decaffeinated coffee plants. Nature 2003, 423:823. One of the most interesting examples of the application of transgenemediated RNAi to the genetic improvement of crop plants. 35. Aukerman MJ, Sakai H: Regulation of owering time and oral  organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 2003, 15:2730-2741. 36. Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC,  Weigel D: Control of leaf morphogenesis by microRNAs. Nature 2003, 425:257-263. 37. Finnegan EJ, Margis R, Waterhouse PM: Posttranscriptional gene silencing is not compromised in the Arabidopsis CARPEL FACTORY (DICER-LIKE1) mutant, a homolog of Dicer-1 from Drosophila. Curr Biol 2003, 13:236-240. rnke F: Temporal and 38. Chen S, Hous D, Sonnewald U, Bo spatial control of gene silencing in transgenic plants by inducible expression of double-stranded RNA. Plant J 2003, 36:731-740.

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