RNA Interference in Crop Plants

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RNA interference in crop plantsMakoto Kusaba

RNA interference (RNAi) is a post-transcriptional gene-silencingphenomenon induced by double-stranded RNA. It has been

widely used as a knockdown technology to analyze gene

function in various organisms. Although RNAi was first

discovered in worms, related phenomena such as post-

transcriptional 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 efficiency 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: [email protected]

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 protectiondsRNA double-stranded RNAhpRNA hairpin RNAmiRNA micro RNAPTGS post-transcriptional gene silencing

RdRP RNA-dependent RNA polymerase

RISC RNA-induced silencing complexRNAi RNA interferencesiRNA small interfering RNA

UTR untranslated region

VIGS virus-induced gene silencing

IntroductionRNA 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 specificity and efficacy, it

has been widely used as an efficient tool to analyze gene

function. In worms and flies, genome-wide analysis based

on complete genome sequences has already been per-

formed using RNAi methods [13]. For studies withcultured cells, an automated RNAi screening system

using assay plates and a microarray-based RNAi screening

system have been developed for high-throughput analysis[3,4]. Recent studies have revealed the natural roles of

RNAi and RNAi-related phenomena, including suppres-

sion 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-relatedphenomenaRNAi was first discovered as a gene-silencing phenom-

enon induced by dsRNA in worms [5,7] (Figure 1). Guo

and Kemphues first showed that injection of the sense or

antisense RNA for a particular gene was able to suppress

gene function in a sequence-specific 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 Kem-

phues. One of these phenomena is co-suppression, that is,gene silencing mediated by a sense transgene. In co-

suppression, expression of the transgene itself is sup-

pressed 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 con-

ferred by a sense coat protein transgene. Initially, protec-

tion was thought to be induced by the coat protein, but

later it was shown that untranslatable coat protein trans-

genes could also confer virus resistance. Because CPMP

was found to act post-transcriptionally, it was thought thatCPMP 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 targetgene 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

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generated during the PTGS process. It is thought that, in

PTGS, aberrant single-stranded (ss) RNA transcribedfrom a transgene triggers the generation of dsRNA by

RNA-dependent RNA polymerase (RdRP), and conse-

quently the RNAi pathway is activated [7] (Figure 2).

Interestingly, PTGS spreads systemically from the tissue

where it was originally induced; the signaling moleculehas not been identified, but is believed to be RNA [7].

The molecular mechanism of RNAiIn 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 siRNAduplexes. Each duplex is unwound and one of the two

strands is incorporated, often preferentially, into the

RNA-induced silencing complex (RISC). The antisense

Figure 1

Micro RNA (small temporal RNA) (1993)

RNAi (1998)in vitroRNAi (1999)RNA-induced silencing complex (2000)Dicer (2001)

Plants Animals

Current Opinion in Biotechnology

The first description of RNAi (1995)

Coat protein mediated protection (1986)

Co-suppression (1990)

Virus-induced gene silencing (1995)

hpRNA transgene (1998)

siRNA (1999), RdRP (1999)

A chronological history of the early work on RNAi and RNAi-related phenomena.

Figure 2

dsRNAhpRNA pre-miRNA

Dicer

RISC

RdRP

PTGS

Current Opinion in Biotechnology

Aberrant ssRNA

mRNA cleavageTranslation inhibition

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].

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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 double-

stranded region with 2 nt 30 overhangs. In Drosophila,

mismatches between siRNA and the target mRNA

greatly reduce the efficiency of mRNA cleavage, parti-cularly 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].

RNAi as a tool for gene function analysis inplantsAlthough RNAi is not a knockout but a knockdown tech-

nology, its high efficiency 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 efficient [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, whichis essential for stability of the inverted repeat in Escher-

ichia coli, the efficiency becomes very high: almost 100%

of transgenic plants show gene silencing [15,16]. How-

ever, the mechanism by which the intron increases silen-

cing efficiency remains unclear [17

]. RNAi can be usedagainst a vast range of targets; 30 and 50 untranslated

regions (UTRs) as short as 100 nt could be efficient

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, hasbeen 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 transfor-

mation are more difficult.

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

action of virus-encoded RdRP. If the virus genome con-

tains 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 oftransgenes comprising virus genes that are necessary

for virus replication and a target gene. Like VIGS, ampli-

con triggers RNAi but it can also overcome the problems

of host-specificity of viruses.

RNAi as a tool for the genetic improvementof crop plantsTrait 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 first 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 therice 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 hpRNA-

induced RNAi would be inherited stably. RNAi inducedby hpRNA does not require some of the genes or com-

ponents 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-of-

function 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 applicabilityto 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 mem-

bers of the glutelin multigene family (which comprises at

least eight members, five of which are clustered in a

particular chromosomal region), but from a single

RNAi-inducing locus [24] (M Kusaba, unpublished).

RNA interference in crop plants Kusaba 141



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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 regulatingthe 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 sup-

pression [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 fromclosely 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-specific or stimuli-responsive sup-

pression is another possible application of RNAi, as the

choice of suitable promoters could enable such regula-

tion. 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 alsosuppress the target gene in other tissues where down-

regulation is not desired. A seed-specific 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 isconfined to the seeds [23]. Lgc1 acts as a Mendelian factor

in F2 seeds ona singleF1 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 efficiency of

spread of the silencing signal. Alternatively, the signal

might be excluded from seeds, as it is excluded from theshoot apex [30]. By such mechanisms, hpRNA-induced

RNAi driven by a seed-specific promoter might confer

seed-specific suppression; however, when other tissues,

particularly where the PTGS signal travels easily, are

specific targets of hpRNA-induced RNAi this specificity

might be lost. In fact, systemic spread was observed in thechemically 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).

ConclusionsBecause RNAi is a very efficient knockdown technology

in plants it is thought to be useful for genetic improve-

ment, even in plants with low transformation efficiencies

[33,34]. Downregulation of a particular gene can be

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.

UpdateRecently, another inducible RNAi system in plants was

reported [38]. In this ethanol-inducible system, no sys-

temic spread of gene silencing was observed.

AcknowledgementsI would like to thank Ichiro Mitsuhara for useful discussions in thepreparation of this article. This work was supported by a grant from theMinistry of Agriculture, Forestry and Fisheries of Japan (Rice GenomeProject IP-1011).

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