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GM Crops 1:4, 207-213; July/August/September/October 2010 © 2010 Landes Bioscience
REVIEW REVIEW
Introduction
RNA interference (RNAi) is a gene-silencing phenomenon,
whereby sequence specific RNA degradation takes place. The
RNA degradation process is triggered by introduction of dou-
ble stranded RNAs (dsRNA) via transgenes, which are further
cleaved by the enzyme Dicer to form duplexes of 21-nucleotides
(nt) with symmetric 2 nt 3' overhangs. These duplexes are com-
monly referred to as small interfering RNAs (siRNAs), which
mediates the degradation of mRNA, thus leading to suppression
or alteration of the gene expression.
RNAi is a scientific breakthrough with its most promising and
rapidly advancing frontiers in genetic improvement of crops. In
plants the RNAi technology has been employed successfully in
improvement of several plant species- by increasing their nutri-
tional value, overall quality and by conferring resistance against
pathogens and diseases. In this review, the main focus is on the
mechanism and application of RNAi as an efficient tool in plant
gene function analysis, plant metabolic engineering and reduc-
tion of specific allergens and toxic products in plants.
*Correspondence to: Karabi Datta; Email: krbdatta@yahoo.com
Submitted: 06/16/10; Revised: 08/16/10; Accepted: 08/17/10
Previously published online:
www.landesbioscience.com/journals/gmcrops/article/13344
DOI: 10.4161/gmcr.1.4.13344
RNA interference (RNAi) is a sequence-specific gene silencing
mechanism, triggered by the introduction of dsRNA leading
to mRNA degradation. It helps in switching the targeted
gene on and off, which might have a significant impact in
developmental biology. Discovery of RNAi represents one
of the most promising and rapidly advancing frontiers in
plant functional genomics and in crop improvement by plant
metabolic engineering and also plays an important role in
reduction of allergenicity by silencing specific plant allergens.
In plants the RNAi technology has been employed successfully
in improvement of several plant species- by increasing their
nutritional value, overall quality and by conferring resistance
against pathogens and diseases. The review gives an insight
to the perspective use of the technology in designing crops
with innovation, to bring improvement to crop productivity
and quality.
RNA interference in designing transgenic crops
Nusrat Ali, Swapan K. Datta and Karabi Datta*
Plant Molecular Biology and Biotechnology Laboratory; Department of Botany; University of Calcutta; Calcutta, India
Key words: RNAi, dicer, RISC, functional genomics, crop improvement
Abbreviations: RNAi, RNA interference; siRNA, small interfering RNA; dsRNA, double stranded RNA;
RISC, RNA-induced silencing complex
Discovery of RNAi
The discovery of RNAi, however, very accidental was unfolded by
a surprising observation made in petunias. In 1990, Jorgensen et
al.1 were trying to deepen the color of petunia flowers by upregu-
lation of the gene coding for chalcone synthase (pigment produc-
ing gene) under the control of constitutive 35s promoter. To their
surprise, instead of the expected deep purple color petunias the
flower appeared variegated or even white. Since the expression of
both the transgene and the homologous endogenous gene was sup-
pressed, the phenomenon was termed cosuppression. Later Guo
and Kemphues,2 while investigating the function of par-1 gene in
the nematode worm Caenorhabdites elegans showed that inject-
ing either sense or antisense RNA for the par-1 gene resulted in
its suppression. Further Andrew Fire3 and Craig Mello4 observed
that injection of the double stranded RNA (dsRNA) mixture i.e.,
both sense and antisense strand together resulted in more effi-
cient silencing of the target gene, than either strand alone. This
turned out to be the defining moment in RNAi research and
the effect came to be known as “RNA interference”. Since then,
work in this area has been booming and researchers in this field
believe that RNAi has greater potential over other posttranscrip-
tional regulators like antisense technology. As known already,
the action of RNAi relies upon an antisense mechanism, since
ultimately a single-stranded RNA molecule binds to the targeted
RNA and recruits a ribonuclease to degrade the targeted RNA.
However, in spite of the compelling similarity between antisense
and RNAi, there are several important differences. RNAi is a
naturally occurring phenomenon found exclusively in eukary-
otes as the oldest and most ubiquitous antiviral system, while the
majority of antisense RNAs are found in prokaryotes. This might
imply that by nature RNAi is more suitable than antisense RNAs
for silencing genes in eukaryotic cells. Further, self-amplification
and a “cell to cell” spreading of RNAi results in a long-lasting
suppression of the targeted gene in plants and worms, while anti-
sense RNA represents a rather transient suppression of the tar-
geted gene in prokaryotes.5
RNAi Machinery and Mechanisms
Components of the RNAi pathway. The RNAi pathway
involves two ribonuclease machines- firstly the ribonuclease III
enzyme Dicer that leads to cleavage of dsRNA into active siR-
NAs, thus initiating the RNAi pathway. The second one is the

208 GM Crops Volume 1 Issue 4
containing polyglutamine residues, PAZ and PIWI domains
characteristic of the Argonaute gene family members.18
Mechanism of action. RNA interference can be divided into
four stages: (1) Double stranded RNA cleavage by the Dicer,
(2) silencing complex (RISC) formation, (3) silencing complex
activation and (4) mRNA degradation.14 The initial step of
RNAi is connected with the delivery of dsRNA in the cell, which
is perfectly homologous, in sequence, to the target gene.19-21
The Dicer enzyme, recognizes the ds RNA,22-24 and further
processes dsRNA in an ATP dependent reaction into double
stranded siRNA 21–25 nucleotides in length, depending upon
the species.11,25 In the second step, the siRNAs produced by Dicer
are incorporated into a multicomponent nuclease complex- the
RNA-induced silencing complex (RISC), which is inactive in
this form to conduct RNAi.26 The third step involves unwinding
of the siRNA duplex in an ATP dependent process by a helicase
and further remodeling of the complex to create an active form of
the RISC.27 The final step includes the recognition and cleavage
of mRNA complementary to the siRNA strand present in RISC.
The target mRNA is cleaved into fragments of about 22 nucle-
otides long (Fig. 1). After the cleavage is complete, the RISC
departs and the siRNA can be used in a new cycle of mRNA
recognition and cleavage.28 One interesting feature encountered
in RNAi is its apparent catalytic nature. Though the cleavage of
dsRNA into small siRNAs results in some degree of amplifica-
tion, it is not sufficient to bring about continuous mRNA degra-
dation. Studies made by Lipardi et al.29 and Sijen et al.30 provides
very convincing genetic and biochemical evidence that RNA-
dependent RNA polymerase (RdRP) plays a very important role
in amplifying the RNAi effects. The enzyme RdRP uses siRNAs
as primers to generate new dsRNAs that can be further cleaved
into new siRNAs.31
RNAi as a Tool for Plant Functional Genomics
Functional genomics is an important aspect of genomics, where
the specific functions of genes and their vitality to an organism,
determining the function of all genes in a plant genome is very
challenging. For this purpose, insertional mutagenesis has been
a very efficient tool and has been extensively applied to the func-
tional characterization of plant genes. However, this approach
has limitations, arising from the randomness of mutagenesis,
which further restricts high-throughput reverse genetic analysis
of plants.32 Hence, silencing effect of RNAi is being exploited
for plant functional genomics, utilizing its ability to specifically
target the chosen gene. Further, the degree of gene silencing can
be varied in different transgenic lines by using the same ihpRNA
constructs.33 Moreover, the expression of ihpRNAs from induc-
ible promoters can control the extent and timing of gene silenc-
ing.34 All these characters make RNAi the technology of choice
for efficient plant gene function analysis.
The potential of RNAi in plant functional genomics has been
utilized in various plants. For example, Travella et al.35 intro-
duced dsRNA expressing constructs of two genes- phytoene
desaturase (PDS) and Ethylene Insensitive 2 (EIN2) into hexa-
ploid wheat (Triticum aestivum). The PDS is an enzyme involved
RNA-induced silencing complex (RISC),6 which brings about
the silencing effect together with its RNase H core enzyme
Argonaute.7-9 The RNase III family members shows specificity
for dsRNAs10 and ultimately cleaves them with 2 nt overhangs
at 3' and 5'-phosphate and 3'-hydroxyl termini.11 The Dicer12
is ATP dependent and contains four characteristic domains:
an amino terminal helicase domain, a PAZ (Piwi/Argonaute/
Zwille) domain, dual RNase III domains and a dsRNA binding
domain.13,14 The model for Dicer catalysis as described by Zhang
et al.15 suggests that the two RNase III domains in the Dicer
are associated in an intramolecular pseudo-dimer, thus creating
an active site. It has been shown that each domain cuts a single
strand of the duplex and generates one new terminus.15
The final step of the RNAi pathway is the nucleolytic
destruction of the target mRNA, which is achieved by the mul-
ticomponent protein complex RISC. Integral to this complex
is a member of the Argonaute family, which has the charac-
teristic nuclease activity responsible for mRNA target cleav-
age.7 Earlier, it has been already reported that the RISC has a
sequence specific nuclease activity which has siRNA as an inte-
gral component.16 The siRNAs associated to nuclease, actually
served as guides to target specific messages based upon sequence
recognition. Among the various Argonaute family members,
Argonaute 2 (AGO2)17 has been identified as an important
protein component of RISC. AGO2 is a 130 KDa protein
Figure 1. Mechanism of the RNAi pathway.