RNA interference in crops

1. 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 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: kusaba@affrc.go.jp
Current Opinion in Biotechnology 2004, 15:139–143
This review comes from a themed issue on
Plant biotechnology
Edited by Takuji Sasaki and Paul Christou
0958-1669/$ – see front matter
ß 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 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 [1–3]. For studies with
cultured 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-related
phenomena
RNAi 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 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
www.sciencedirect.com Current Opinion in Biotechnology 2004, 15:139–143

2. 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 conse-
quently 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 identified, but is believed to be RNA [7].
The molecular mechanism of RNAi
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
Figure 1
Micro RNA (small temporal RNA) (1993)
RNAi (1998)
in vitro RNAi (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
].
140 Plant biotechnology
Current Opinion in Biotechnology 2004, 15:139–143 www.sciencedirect.com

3. 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 in
plants
Although 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, which
is 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 used
against 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, 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 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 of
transgenes 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 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 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 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 hpRNA-
induced RNAi would be inherited stably. RNAi induced
by 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 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 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
www.sciencedirect.com Current Opinion in Biotechnology 2004, 15:139–143

4. 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 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 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-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 also
suppress 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 is
confined 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 efficiency 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-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 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).
Conclusions
Because 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.
Update
Recently, another inducible RNAi system in plants was
reported [38]. In this ethanol-inducible system, no sys-
temic 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).
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