RNA interference: a promising technique for the improvement of traditional crops

RNA interference: a promising technique for the improvement oftraditional crops

RAJAN KATOCH & NEELAM THAKUR

Biochemistry Laboratory, Department of Crop Improvement, College of Agriculture, CSK Himachal Pradesh Krishi

Vishvavidyalaya, Palampur, India

AbstractRNA interference (RNAi) is a homology-dependent gene-silencing technology that involves double-stranded RNA directedagainst a target gene. This technique has emerged as powerful tool in understanding the functions of a number of genes in recentyears. For the improvement in the nutritional status of the plants and reduction in the level of antinutrients, the conventionalbreeding methods were not completely successful in achieving the tissue-specific regulation of some genes. RNAi has shownsuccessful results in a number of plant species for nutritional improvement, change in morphology and alteration in metabolitesynthesis. This technology has been applied mostly in genetic engineering of important crop plants, and till date there are noreports of its application for the improvement of traditional/underutilized crops. In this study, we discuss current knowledge ofRNAi function and concept and strategies for the improvement of traditional crops. Practical application. Although RNAi hasbeen extensively used for the improvement of popular crops, no attention has been given for the use of this technology for theimprovement of underutilized crops. This study describes the importance of use of this technology for the improvement ofunderutilized crops.

Keywords: RNAi, traditional crops, crop improvement, dsRNA, Dicer

Introduction

RNA interference (RNAi), which is also named as

post-transcriptional gene silencing (PTGS) or RNA

silencing, involves RNA degradation which was

believed to be an important defence against foreign

nucleic acids (Waterhouse et al. 2001). This phenom-

enon was initially discovered in plants and was thought

to function as part of a defence mechanism against

viruses (Ratcliff et al. 1997). Later, it emerged as a

common gene-silencing mechanism occurring in all

eukaryotes, including plants and animals. RNA

silencing is a homology-based process, which is

triggered by double-stranded RNA (dsRNA), leading

to the suppression of gene expression (Denli and

Hannon 2003). In plants, RNAi phenomenon was first

discovered during transformation of petunia to get

petunia flowers with darker purple hue by introducing

numerous copies of a gene that code for deep purple

flower, i.e. chalcone synthase (Chs A). Surprisingly,

some of the final plants yielded white or patchy flowers

and the transgene silenced the expression of both

homologous endogenous and introduced loci (Napoli

et al. 1990). At that moment the phenomenon was

termed as co-suppression, but it was Fire and Mello’s

work that observed the presence of dsRNA in

Caenorhabditis elegans that triggered silencing of

genes containing sequence homology to the dsRNA

(Montgomery and Fire 1998; Tabara et al. 1998).

They termed this unusual phenomenon of gene

silencing as ‘RNAi’. Their exhaustive work finally

revealed that both sense and antisense RNA are able to

silence gene expression, and dsRNA is involved in the

process of gene silencing and was a more efficient

elicitor of RNAi than either sense or antisense RNA

alone (Fire et al. 1998). It also became clear that the

silencing of RNA works in plants at three different

stages: (i) cytoplasmic silencing by dsRNA resulting in

ISSN 0963-7486 print/ISSN 1465-3478 online q 2012 Informa UK, Ltd.

DOI: 10.3109/09637486.2012.713918

Correspondence: Rajan Katoch, Biochemistry Laboratory, Department of Crop Improvement, College of Agriculture, CSK HPKV,Palampur – 176 062 (HP), India. Tel: 91-1894-232823. Fax: 91-1894-230434. E-mail: [email protected]

International Journal of Food Sciences and Nutrition,

March 2013; 64(2): 248–259

Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

the cleavage of mRNA, which is known as PGTS

(Hamilton and Baulcombe 1999); (ii) silencing of

endogenous mRNA by micro-RNAs (miRNAs),

which negatively regulate gene expression by base-

pairing to specific mRNA leading to RNA cleavage or

arrest of protein translation and (iii) RNA silencing

mediated by sequence-specific methylation of DNA

which ultimately results in suppression of transcription

known as transcriptional gene silencing. RNAi is a

complex phenomenon and silences genes through a

process in which a dsRNA or hairpin RNA (hpRNA)

molecule is broken into small interfering RNAs

(siRNAs) of 21–28 nt in length by an RNase III

Dicer. These siRNAs are then incorporated into a

RNA-induced silencing complex (RISC). Argonaute

proteins, which are the catalytic component of RISC,

use these siRNAs as a template to recognize and

degrade the complementary messenger RNA (Meister

and Tuschl 2004). The RNAi technology has now

become a technique of choice for understanding the

genes functions and their regulation associated with

different metabolic pathways controlling the import-

ant traits. After the refinement for several years, this

technique is now being successfully used in the

regulation of metabolic pathways both in animals

and in plants (Sinha 2010). There are now several

reports of manipulation of metabolic pathways in

plants for enhancement in the nutritional status and

reduction in the levels of antinutritional components

(Jagtap et al. 2011).

Plants are the one and only natural resource to

obtain different kinds of food material. They produce

different materials in accordance with its genetic

makeup, and many of the secondary metabolites

produced by the plants are in small ratios, which may

not be sufficient to meet our requirements. RNAi is

now one of the approaches for designing crop plants

for the nutritionally desirable products. This tech-

nique has now been implicated to improve the

nutritional status of different crops, which either

have low levels of nutrients or higher levels of

antinutrients. There exists good number of examples

in which the plants have been engineered for desired

products in appropriate proportions (Table I). The

regulation of gene functions in plants by the use of this

technology has now opened new vistas for improve-

ments of economically important plants and protec-

tion of plants from various biotic stresses. Till date

numerous success stories of RNAi technology in the

improvement of important traits in the crop plants

have been reported and few of them are summarized in

Table I. These successful results demonstrate that the

gene-silencing mechanism could be used to modulate

biosynthetic pathways in the economically important

plant species for obtaining a desired phenotype, which

is generally not possible by traditional plant breeding

Table I. RNAi-mediated improvement of traits in crop plants.

Engineered plant Gene targeted Trait developed References

Apple Mal d 1 Reduced allergens Zhu et al. (2003)

Coffee bean CaMxMt 1 Decaffeinated coffee Ogita et al. (2004)

Cotton ghSAD-1 and ghFAD2-1 Useful for cooking applications without the

need for hydrogenation

Liu et al. (2002)

Cotton d-Cadinene synthase gene Health benefits Sunilkumar et al. (2006)

Maize Gene for starch branching enzyme Maize quality improvement Chai et al. (2005)

Maize 22 kDa a-Zein High lysine kernel Segal et al. (2003)

Maize Lysine-ketogulutarate reductase High lysine kernel Houmard et al. (2007)

Maize opaque-2 Higher lysine content Segal et al. (2003)

Oilseed rape BP1 Improved photosynthesis Byzova et al. (2004)

Onion Lachrymatory factor synthase (LFS) Tearless onion Eady et al. (2008)

Onion LFS Reduced lachrymatory factor Eady et al. (2008)

Peanut Ara h2 Reduced allergend Dodo et al. (2008)

Potato SBE1 and SBE2 Increased amylase content Andersson et al. (2006)

Potato GBSS1 Reduced amylase content Heilersig et al. (2006)

Rice Lgc1 Health benefits Kusaba et al. (2003)

Rice OsBP-5 Reduced amylase content Zhu et al. (2003)

Rice qSW5 Increased weight Shomura et al. (2008)

Soybean GmFAD3 Reduced linoleic acid content Flores et al. (2008)

Sweet potato GBSS1 Reduced amylase content Otani et al. (2007)

Tobacco CHI Flower coloration Nishihara et al. (2005)

Tomato 1-Aminocyclo propane-1-carboxylate oxidase Reduced ethylene sensitivity Xiong et al. (2005)

Tomato DET1 Neutraceuticals Davuluri et al. (2005)

Tomato TDET1 Increased carotenoid content Davuluri et al. (2005)

Tomato Lyc e1 Reduced allergens Le et al. (2006)

Tomato ARF7 Seedless fruits De Jong et al. (2009)

Tomato AUCSIA Seedless fruits Molesini et al. (2009)

Tomato Chalcone synthase Seedless fruits Schijlen et al. (2007)

Wheat SBEIIa and SBEIIb Improved bowel functions Regina et al. (2006)

Wheat 1Dx5 Altered quality of flour Yue et al. (2008)

RNA interference for improvement of traditional crops 249

Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

practices. However, this technology remained to be

focused on the improvement of crop plants of high

commercial importance and there had been no reports

for the improvement of the traits in underutilized

crops, which have high nutritional value. RNAi

technology offers a high potential for the improvement

of such crop species, which are less in use due to

presence of one or a few antinutrients and thereby

have lost place in routine cuisine and industry. This

technology opens the gateway to the new frontiers

in the use of genetic manipulation to enhance the

nutritional value of such underutilized crops, ensuring

food security.

Basic mechanism of RNAi

The basic mechanism of RNAi machinery in the

plants has been presented in Figure 1. The technique

involves specific down-regulation or knockdown of

gene expression by dsRNA involving degradation of

a target mRNA. The process is triggered by dsRNA

precursors that vary in length and origin. These

dsRNAs are rapidly processed into shortRNAduplexes

of 21–28 nt in length, which then guide the recognition

and ultimately the cleavage or translational repression

of complementary single-stranded RNAs, such as

messenger RNAs or viral genomic/antigenomic

RNAs. These RNA-silencing mechanisms were

initially recognized as antiviralmechanisms that protect

organisms from RNA viruses or which prevent the

random integration of transposable elements (Ullu

et al. 2004). Later, it became known that specific

genes in plants and animals encode short forms of

dsRNA (Bartel 2004). In plants, miRNAs mainly

function as siRNAs that guide the cleavage of sequence-

complementary mRNAs. Naturally occurring dsRNA

can be produced by RNA-templated RNA polymeri-

zation (in viruses) or by hybridization of overlapping

transcripts (from repetitive sequences such as trans-

gene arrays or transposons). These dsRNAs give rise

to siRNAs or repeat-associated siRNA (rasiRNAs)

families, which generally function to guide mRNA

degradation or chromatin modification.

i) siRNAs: siRNAs mediate RNAi by down regulat-

ing target mRNAs through endonucleolytic

cleavage (Figure 1). These small RNAs (sRNAs)

originate from long dsRNA molecules which

are generally produced from RNA virus replica-

tion, convergent transcription of cellular genes

or mobile genetic elements, self-annealing tran-

scripts or experimental transfection process

(Jinek and Doudna 2009). It is the siRNA

structure and its sequence which determines

which one of the strand enters RISC as the

guide strand and which is excluded from RISC,

ultimately becoming passenger strand. It has

been observed that the siRNA strandwhose 50 end

is more weakly bound to the complementary

strand is more readily retained in RISC, and the

other strand is degraded (Khvorova et al. 2003;

Schwarz et al. 2003).

ii) miRNA: The endogenous mRNAs having com-

plementarity of ,20–50 base-pair inverted

repeats fold back on themselves to form dsRNA

hairpins, which are further processed into

miRNAs, which negatively regulate gene

expression by base-pairing to specific mRNAs,

resulting in either RNA cleavage or repression of

protein translation. These miRNAs are encoded

in the genome. In plants, miRNAs direct the

slicing of target messenger RNAs, much like

siRNAs, whereas in animals, miRNAs silence

target mRNAs without slicing. In this case,

these sRNAs are transcribed from endogenous

miRNA genes as primary transcripts, containing

,65–70-nt stem-loop structures. The hairpin

structure is excised in the nucleus by the Drosha–

DGCR8 complex to yield a precursor miRNA

(pre-miRNA). In the cytoplasm,Dicer cleaves the

pre-miRNA, producing a miRNA–miRNA*duplex (miRNA represents the guide strand

and miRNA* represents the passenger strand).

The guide strand is loaded onto an Argonuate

protein. From here, we understand that the

animal miRNAs are only partly complementary

siRNA

Formation of RISC complex

Interaction of RISC complex and target RNA

dsRNA

dsRNA binding proteins andDICER (ribonucleases)

ARGONAUTE (endonuclease with PAZ,MID and PIWI domains)Degradation of passenger strand

Binding of guide strand siRNA to targetmRNA

Target mRNAdegradation,

(Gene silencing)

RNAi effect

Figure 1. Mechanism of RNAi dsRNA is processed to obtain

21–23-bp siRNAs by an endonuclease known as Dicer. The

resulting siRNAs function as sequence-specific guides to direct

RISCs and guide complementary mRNAs through the RISC in

which they are degraded. Ultimatly, siRNA–RISC complex then

targets a sequence complementary to siRNA leading to the cleavage

of the target (mRNA).

R. Katoch and N. Thakur250

Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

to sequences in the 30 un-translated regions of

their target mRNAs; therefore, lack of perfect

complementarity prevents the target from being

sliced by the Argonuate protein. Furthermore,

some Argonuate proteins involved in the miRNA

pathway lack the catalytic residues needed for

slicer activity. The mechanism of miRNA-

mediated silencing is therefore thought to occur

by repression of target mRNA translation and

removal of mRNA poly (A) tails (that is dead-

enylation), which finally leads to mRNA degra-

dation (Jinek and Doudna 2009) contrary to

mRNA cleavage in case of dsRNA.

When compared with animals, higher plants

generate array of sRNAs with specialized functions.

The well studied plant Arabidopsis thaliana encodes

four different types of Dicer-like (DCL) proteins from

DCL1 to DCL4, 10 Argonuate proteins (component

of RISC catalytic core), six RNA-dependent RNA

polymerase and five dsRNA-binding proteins, which

participate in at least five different endogenous sRNA

pathways. The miRNAs are produced from their

corresponding imperfectly base-paired fold-back pre-

cursor by DCL1, whereas the other three DCL

proteins generate different types of endogenous

siRNA from perfectly dsRNAs, such as stress related

24-nt natural antisense transcript siRNA (natsiRNA)

by DCL2 (Borsani et al. 2005), 24-nt rasiRNA that

guide chromatin remodelling by DCL3 (Xie et al.

2005) and DCL4 generates 21-nt trans-acting siRNA

that has role in plant developments (Gasciolli et al.

2005). The siRNA generated by Dicer activity is

then incorporated into a different multicomponent

ribonuclease called RISC. In plants, short siRNAs

play a role in local RNA silencing, whereas the long

siRNAs are involved in systemic signalling and also

in RNA-directed DNA methylation (Zilberman et al.

2003). The long siRNA originated from repeat

sequences known as rasiRNA is associated with direct

DNA methylation of repetitive DNA sequence in the

plant genome (Chan et al. 2005).

RNAi and improvement of nutritional attributes

of plants

In plants, breeding of varieties with different traits

has been tremendously successful in improving the

nutritional status of food (Davies 2003), but because

of time consumption the limited genetic resources

of most of the crops have left small space for the

improvement of crop plants by these methods

(Hoisington et al. 1999). RNAi has now become an

alternative tool in the nutritional improvement of

important crop plants. The level of nutritionally

desired components has been successfully manipu-

lated in a variety of crop plants. The potential of

RNAi has also been recognized in reducing the levels

of undesirable components (antinutrients) in edible

plant parts by down-regulation of genes encoding

these components. This is therefore a versatile

approach for the improvement of a large number of

neglected/underutilized crops (Table II). These crops

are so called because their use has been limited

because of the presence of one or more antinutritional

factors (ANFs) including toxic amino acids, antigenic

proteins, digestive enzyme inhibitors, alkaloids, lec-

tins, glucocynolates, condensed tannins, mycotoxins

and so on which are generally associated with various

ailments, if consumed in large proportions. Because of

these reasons, the use of these crops has been

continuously reduced over time. On the other hand,

if the levels of antinutrient(s) in these crops are

manipulated, these crops have the potential to serve as

excellent source of nutrition. The applications of

RNAi technology have been largely associated with the

nutritional enrichment in the well-known crops, and

no information is available on the application of this

technology for the improvement of traditional crops.

Main approaches to suppress expression of

undesirable genes in crop plants

The developments in the RNAi technology have made

us understand the gene functions and their regulation

associated with different metabolic pathways control-

ling the important traits in plants. Two approaches

have been used for reducing the levels of undesirable

gene products (Tang et al. 2007):

1. Dominant gene silencing.

2. Recessive gene disruption.

In recessive gene disruption, the target sequence is

mutated to eliminate the expression or function of the

gene, whereas the dominant gene-silencing methods

induce either the destruction of the gene transcript or

the inhibition of transcription. RNAi, which is based

on dominant gene silencing, has been utilized as the

most powerful approach for the gene knockdown

effects (Smith et al. 2000). The practical advantage

related to dominant gene silencing is their manipu-

lation in a spatial and temporal manner by the use of

tissue-specific promoters (Smith et al. 2000). The

spatial and temporal expression of gene is extremely

important in manipulating genes as the plant part

consumed as food and which is the primary target for

the silencing is different from the other parts such as

roots and shoots, which are mainly related to plant

growth, development and productivity. In case gene

function is not controlled in a tissue-specific manner,

then gene-silencing effect may be transmitted to the

whole plant system. Therefore, the alteration in

expression of gene may be beneficial for improvement

of the edible part, but may affect the growth and

development of the whole plant. With the use of tissue-

specific promoters, the gene knockdown effect is

localized to specific tissues (Wesley et al. 2001;


Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

Sunilkumar et al. 2006; Tang et al. 2007). The

chemically inducible RNAi-silencing vectors have

revealed the temporal and spatial control of gene

silencing (Chen et al. 2003; Guo et al. 2003;

Wielopolska et al. 2005).

Constructs and transformation methods for

RNAi effects in underutilized crop plants

The RNAi effects in plants are mediated by expression

vectors which transcribe a self-complementary dsRNA

(Horiguchi 2004). The construction of plant RNAi

vectors is laborious work as sense and antisense target

sequences with a spacer sequence are required in a

single vector. Different types of constructs have been

reported from different research laboratories with

gene construct encoding intron-spliced RNA with

hairpin structure inducing PTGS with almost 100%

efficiency (Smith et al. 2000). pHELLSGATE had

been used as vector system to construct plant RNAi

vectors by using Gateway technology (Invitrogen). In

this system, PCR fragments with sense and antisense

orientations are amplified by using primers having

the recombinase recognition sites, attB1 and attB2

sequences (Wesley et al. 2001).The PCR products

are cloned into a plasmid containing attP1 and attP2

sites by a BP clonase. pHELLSGATE is directly used

for Agrobacterium-mediated transformation of plants.

Another vector known as pANDA vector developed

by Miki and Shimamoto (2004) using maize ubiquitin

promoter is being used successfully for high expression

of an RNAi cassette and a DNA fragment originated

from the beta-glucuronidase (GUS) gene. This vector

eases the overall cloning process. For cloning the

target DNA fragments, the Gateway technology is

used in the pANDA vector system in which a PCR

fragment containing CACC sequences at the 50 end of

forward primer is cloned into the pENTR/D-TOPO

vector supplied by Invitrogene, with a result that

these PCR fragments are flanked with two recombi-

nation sites, attL1 and attL2. An LR clonase

recombines this PCR fragment into two recombi-

nation sites (attR1 and attR2) of pANDA vector in

opposite directions. The pANDA vector is extensively

used in the Agrobacterium-mediated transformations

in plants. The Agrobacterium-mediated transformation

is a generally used transformation technique for

RNAi. Besides this, the direct introduction of

RNAi vectors via particle bombardment (Panstruga

et al. 2003) and electroporation (Akashi et al. 2004)

has also been employed in plant cells. Chuang and

Meyerowitz (2000) were the first to use this technique

in plants. These workers through dsRNAs triggered

efficient silencing of flower identity genes using

inverted repeats. This vector-based RNAi technology

was further improved, using an intron, instead of a

Table II. Common underutilized crops grown globally.

Cereal and pseudocereals Vegetable and pulse crops Root and tuber crops

Anaranthus spp. Alocasia spp. Amaranthus spp.

Basella alba Arracacia xanthorrhiza Chenopodium quinoa

Basella rubra Calathea allouia Digitaria exilis

Brassica carinata Canna edulis Echinochloa spp.

Canavalia spp. Colocasia esculenta Eleusine coracana

Celosia spp. Dioscorea spp. Fagopyrum esculentum

Chenopodium album Dioscorea spp. Panicum miliaceum

Corchorus spp. Dioscorea spp. Panicum miliare

Crambe cordifolia Harpagophytum procumbens Paspalum scrobiculatum

Crotalaria spp. Oxalis tuberosa Setaria italica

Curcuma spp. Pachyrhizus erosus Triticale

Emilia spp. Pachyrhizus erosus

Gymnandropsis synandra Plectranthus esculentus

Hibiscus sabdariffa Solenostemon rotundifolius

Ipmoea aquatica T. esculentum

Lablab purpureus Tropaeolum tuberosum

Lathyrus spp. Tylosema fassoglense

Macrotyloma uniflorum Ullucus tuberosus

Momordica spp. Vigna vexillata

Moringa oleifera Xanthosoma sagittifolium

Parkia biglobosa Xanthosoma spp.

Phytolocca acinosa

Psophocarpus tetragonolobus

Rorripa indica

Talinum triangulare

Vigna aconitifolia

Vigna angularis

Vernonia spp.

Vigna subterranea

Vigna umbellata

Voandzeia subterranea


Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

fragment of DNA, as the linker (Smith et al. 2000).

Different constructs now used for successful RNAi

effects usually have a spacer sequence between an

inverted repeat. The resulting transcript from these

constructs takes the shape of stem-loop or hairpin-like

structure and is inserted downstream of a constitutive

promoter (Hirai et al. 2007). Intron-containing

hpRNA-based vectors have been proven to be highly

efficient for plant RNAi-based gene silencing (Smith

et al. 2000). They have been shown to increase gene-

silencing efficiency by 90–100% (Wesley et al. 2001).

Therefore, in a hpRNA vector, the target gene is

cloned as an inverted repeat spaced with an intron and

is driven by either a strong whole plant promoter such

as the 35S cauliflower mosaic virus (CaMV) (dicots)

or the maize ubiquitin1 (monocots) or an organ-

specific silencing promoter (Tang et al. 2007). The

degree of silencing with these constructs was much

greater than that obtained using either co-suppression

or antisense constructs, thereby, it has become the

most popular method for genes silencing in plants

(Miki and Shimamoto 2004). In several studies,

intron-less hpRNA, antisense and co-suppression

constructs have been used in many of the gene/host

combinations (Sinha et al. 2008). The results have

revealed that ihpRNA constructs were effective,

with arm length ranging from 98 to 853 nt, giving

66–100% (average 30%) independent silenced trans-

formants (Wesley et al. 2001). Intron-free hpRNA

constructs gave 48–69% (average 58%) silenced

transformants, and conventional co-suppression or

antisense constructs gave 0–30% (average 13% and

12%, respectively) silenced transformants (Wesley

et al. 2001). However, the sequences to be used as

dsRNA trigger need to be selected carefully as long

hairpin-based siRNAs are more likely to generate a

diverse set of effective siRNA with an increased

potential for off-target effects.The PTGS pathway

targets dsRNA for degradation by DCL proteins in

a sequence-specific manner through the production

of small interfering (si)RNA. DCL2 is known to

cleave dsRNAs from replicating virus, and DCL3

cleaves dsRNAs derived from endogenous transcripts

through the activity of RNA-dependent RNA poly-

merases 2 and 6 (Dalmay et al. 2001). As described,

these siRNAs produced are incorporated into RISCs,

which guide cleavage of target RNAs.

Although siRNA-based RNAi vectors have shown

high specificity and efficiency, a new generation

miRNA-based RNAi vectors with high silencing

accuracy and fewer off-target effects are coming up,

which do not trigger protein kinase R (PKR)

pathways. In mammalian cells, PKR pathway causes

a non-specific cell death by long dsRNA produced by

siRNA-induced pathway (Marques and Williams

2005). These miRNAs have now been identified as

important regulators of gene expression in both plants

and animals. miRNAs are single-stranded RNAs of

20–24 nt in length, which are generated from

processing of longer pre-miRNA precursors by

DCL1 in A. thaliana (Bartel 2004). These miRNAs

are also recruited to the RISC complex. Through

RNA:RNA base-pairing, miRNAs direct RISC in a

sequence-specific manner to down regulate target

mRNAs in one of two ways. In animals, limited

miRNA:mRNA base-pairing results in translational

repression, whereas most plant miRNAs show

extensive base-pairing to, and guide cleavage of, their

target mRNAs (Rhoades et al. 2006). These miRNAs

are known to be important regulators of plant

developmental processes in A. thaliana. It is also well

established that the alteration of several nucleotides

within miRNA sequence does not affect its biogenesis

unless and until the initial base-pairing in stem-

loop structure of the precursor is changed (Herve et al.

2004). Different studies have shown that the alteration

of several nucleotides within an miRNA 21-nt

sequence does not affect its biogenesis (Vaucheret

et al. 2004). Therefore, it is possible to modify plant

miRNA sequence to target-specific transcripts, orig-

inally not under miRNA control.

This finding has made possible to use a modified

version of natural miRNA sequences which are known

as artificial miRNA (amiRNA). This amiRNA

technology was initially tested for gene knockdown in

human cell lines, and it was successfully employed to

down regulate gene expression without affecting the

expression of the other unrelated genes in transgenic

plants (Ossowski et al. 2008). The miRNA is around

21 nt endogenous regulator RNAs produced from

imperfectly base-paired hairpin precursor and pro-

cessed by DCL1 in plants (Voinnet 2009). The

amiRNA utilizes miRNA precursor as a backbone and

the stem region is substituted with sequences in several

base-pairs to gain new targeting ability (Ossowski et al.

2008). These amiRNAs targeting genes of interest

having being modified by modifying the endogenous

miRNA precursors have proved useful for down-

regulating gene expression in plants (Schwab et al.

2006; Brodersen et al. 2008).These plant amiRNAs

are expressed from vectors derived from precursors

of ath-miR159a, ath-miR164d, ath-miR174a, ath-

miR319a and Osa-miR528 (Liu et al. 2010). Animal

miRNAs typically cause translational arrest of target

mRNA that is only partially complementary to the

miRNA. Complementarity (position 2–8) to the seed

region of the miRNA is generally sufficient for effective

regulation, allowing an animal miRNA to control large

number of targets (Lewis et al. 2005; Schwab et al.

2006), whereas plant miRNAs have few (0–5)

mismatches to their targets and trigger local transcript

cleavage and subsequent degradation (Llave et al.

2002). The studies have shown that amiRNAs have

only limited autonomous effects and are, therefore,

quite suitable for gene-specific gene silencing in plants

(Wang et al. 2010).

During genetic transformations, use of tissue-

specific promoters is an effective way to avoid potential


Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

negative effects of gene silencing. Mainly, the

promoters have been classified into three categories:

(i) constitutive promoters, which are continuously

expressed in most or all tissues; (ii) spatiotemporal

promoters, which have stage-specific or tissue-specific

activity and (iii) inducible promoters, which are

regulated by external physical or chemical signals.

Constitutive promoters, such as the CaMV 35S

promoter (Odell et al. 1985), rice actin1 promoter

(McElroy et al. 1990) and maize ubiquitin1 promoter

(Chrisetnse et al. 1992), have been widely used in

plant genetic engineering. However, the constitutive

expression of transgenes could result in several

negative effects, including increasing metabolic bur-

den and food safety concerns (Conner et al. 2003;

Karlowski and Hirsch 2003). Many tissue-specific

promoters have now been identified, which result in

gene expression restricted to particular cells, tissues,

organs or developmental stages. GaMYB2 cotton

fibre-specific promoters drive gene expression specifi-

cally in glandular cells (head cells) and Arabidopsis

trichome-specific promoters, which also express in

glandular trichomes in transgenic tobacco (Shangguan

et al. 2008). The oil palm metallothionein promoter

with negative regulatory element (AGTTAGG) con-

fers fruit-specific expression (Omidvar et al. 2010).

The endosperm-specific promoter sequences have

been identified from the coconut (Cocos nucifera L.),

with expression in rice plants (Xu et al. 2010).

The identification of these tissue-specific promoters

makes the possibility of gene-silencing effect at a

specific site and thereby avoid pleiotripic side effects.

With these upcoming developments, RNAi is now

more refined for the better outputs in the modulation

of gene functions for dealing with ANFs present in

plant species.

Prospects of implication of RNAi for the

improvements of underutilized/neglected crops

In addition to the versatility of RNAi technique in

developing crop plant resistant to biotic stress caused

by insects, bacteria, viruses, fungi and nematods, it has

numerous potential to be used in nutritionally rich and

antinutrient-free varieties of less utilized/neglected

crops (Figure 2). This technology has the potential

for tissue-/organ-specific silencing, thereby it could

be utilized in the down-regulation of expression of

gene(s) for various ANFs in plant species (Figure 3).

Underutilized crops

Antimicrobialactivities, anticanceractivities, hypo-cholesterol activity,anti diabetic activityetc.

Rich source ofproteins, essentialamino acids,minerals, PUFA,energy value

Excellent source ofantioxidants,flavonoids,flavonols etc.

High quality food formillions of people

Genetic engineering withimplication of RNAitechnology formanagement of anti-nutritional factors

Safe food forconsumption

Main impediment inutilization of these

crops

Anti-nutrientsNutritionalexcellence

Pharmaceuticalimplications

Bioactivecompounds

Saponins, Tanins,flatulernce factors(oligosaccharides),Goitrogens, alkaloids, HCN,enzyme inhibitors(trypsin inhibitor),glucosinolates,cyanogenicglucosides,Mycotoxins, toxicamino acids etc.

Figure 2. Schematic representation of the potential of underutilized crops and the channel to overcome the problem of antinutrients for further

increasing their food value.


Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

The provision of nutritious and wholesome food

for poor and undernourished populations has been

a major challenge for the developing world. Acute

shortage, unreliable supply and elevated costs of

protein-rich foods of animal origin in the developing

and underdeveloped countries have always resulted in

the search for inexpensive and reliable alternative

sources of protein of plant origin. In the present times,

researchers throughout the world are focusing on

tapping natural wild and underutilized crops, which

have either remained unexplored or are underutilized

or only localized in a particular region mainly by poor

farming communities that derive their sustenance

and livelihood from such plants. For alleviating hunger

and to overcome malnutrition, there is an increased

demand in developing countries to explore under-

utilized legumes (Coulter et al. 1988; Chel-Guerrero

et al. 2002). Exploring wild/underutilized legumes is

of utmost significance for food security, meeting

nutritional requirements and agricultural develop-

ment. Underutilized crops constitute the lesser known

species in terms of trade and research, often well

adapted to marginal and stress conditions. They are

named not for the reason they are unsuitable for

consumption, but more precisely due to modern

consumption practices. Many of the known under-

utilized legumes possess adequate amounts of

protein, essential amino acids, polyunsaturated fatty

acids (PUFAs), dietary fibre and essential minerals

and vitamins comparable to other common legumes,

along with the presence of beneficial bioactive

compounds. Apart from this, these plants are also

adaptable to adverse environmental conditions and

can thrive under extreme stress conditions (Amubode

and Fetuga 1983; Sotelo et al. 1995; Bhat et al.

2008). Therefore, improvement of underutilized

crops holds promise to attain sustainability, profit-

ability and diversification.

Besides their commercial potential, many of the

underutilized crops also provide important environ-

mental usages, as they are adapted to marginal soil and

climate condition and have innate potential of

combating different stress factors in the environment.

The general decline of these crops may erode the

genetic base, preventing the use of distinctive useful

traits in crop adaptation and improvement. Therefore,

there is an urgent need for testing this fast-growing

technology in these crops for overcoming the problem

of toxic constituents to link them to the food supply

chain in an efficient way.

This in turn poses a challenge before the molecular

biologists to see the best possibility of using such a

promising technology to eliminate the harmful

compounds from the plant species, which otherwise

have high nutritive value. Tissue-/organ-specific

silencing approaches are the possible ways to achieve

targeted gene silencing without having any affect on

the normal life-cycle of the plant. Different successful

results with the implication of this technology have

been published during this decade, which also include

alteration in nutritional constituents in some plants,

lowering the level of antinutrients/toxins in edible

plant parts. Therefore, there are several success stories

for the improvement in the nutritional status of well-

known plant species; however, till date, there is no

report of study on underutilized crops. These crops

also include some of the leguminous plants such Vicia

faba (broad bean), Lathyrus sativus (grass pea), Vigna

angularis and so on, which have not been exploited

properly due to presence of some antinutrients,

despite the fact that they have good level of other

nutritionally desirable components. Generally, the

1. Analysis of metabolicpathways leading to anti

nutritional factors (ANFs)

6. Plant product without/reducedlevels of ANFs

Gene Silencing1. Constitutively2. Tissue specifically

dsRNA vector miRNA-like vectors

3. Development ofsuitable vector

2. Selection of target gene(s)

4. Delivery in to the plant

5. Screening and evaluation1. Metabolome analysis2. Transcriptome analysis3. Proteome analysis

Selection of promoter1.Constitutive promoter2.Tissue/organ specific promoter3.miRNA promoter

Figure 3. RNAi for elimination of ANFs from the plants. (1) The

first step involves the analysis of metabolic pathways leading to

production of ANFs. (2) The second step involves the selection of

probable gene(s). (3) The third step involves selection of vectors with

appropriate promoters for spatial and temporal regulation of gene(s)

using RNAi and miRNA technology. (4) The fourth step involves

delivery of RNAi vectors into the plant for silencing the targeted

gene leading to production of transgenic plants. (5) The fifth step is

the evaluation of silenced gene with different analysis. (6) In the last

step, the engineered plant will be screened and used for the purpose

without ANFs.


Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

occurrences of one or combination of antinutritional

components have often overshadowed the nutritional

superiority and neutraceutical value of these tra-

ditional crops.

The use of these crops is limited due to the presence

of unique antinutrients/toxic components which

obliterate their nutritive excellence. V. faba broad

beans contain the alkaloids vicine, isouramil and

convicine, which after their prolonged use may cause

‘favism’. RNAi is the potential approach for targeting

gene(s) responsible for the production of toxic

alkaloids. Another legume Lathyrus sativus (grass

pea) is harmless to humans in small quantities, but

continuous consumption of this crop causes ‘Neuro-

lathyrism’ due to neurotoxic amino acid, b-N-oxalyl-

L-a,b-diaminopropionic acid (ODAP). The crop is a

potential target for gene silencing responsible for the

production of ODAP which could make this non-

economical legume, a legume of high market value.

Likewise, other plants of this category are associated

with the presence of variable amounts of antinutrients,

majority of which include glucosinolates, cyanogenic

glucosides, tannins, saponins, phenolic compounds

and digestive enzyme inhibitors, which poses hurdle in

their use in routine cuisine. Therefore, some suitable

technology was desired from long which can address

the potential hurdles for the propagation and

utilization of these crops at commercial levels so that

these crops could contribute with their nutritive

potential to growing food demand with increasing

population pressure. On application of these novel

techniques of RNAi, we can to some extent ensure the

removal of naturally occurring toxic/antinutrients

compounds from the edible portion of the plant. The

main steps for RNAi-based gene silencing for the

elimination of ANFs in these categories of crops are

shown in Figure 2.

To emphasize the implication of this technique for

underutilized crops will somewhat ensure the nutri-

tional security for the people, especially in the

developing countries where the traditional crop is the

only cheap source of food. These people also face

consequences on health in the form of different

disorders due to continuous consumption of these

crops. Once the antinutrients level of these crops is

reduced, the nutritional excellence will attract other

group of consumers for their related health benefits.

The improvement in the nutritional status of the less

known but promising food crops with the RNAi

technology is sure to have global humanitarian and

economic implications globally. RNAi has been used

for a variety of applications as given in Table I, which

demonstrate that targeted gene silencing can be used

to modulate biosynthetic pathways in a specific tissue

in order to obtain a desired phenotype, which was

often not possible by traditional breeding. These

studies have in real sense opened the gateway to new

frontiers in the use of genetic manipulation to enhance

global food supply.

Limitations and of future prospects

Among several limitations, the most emphasized

limitation of this technology is the off-target effects

of siRNA, which may disrupt the functioning of

non-target gene it this gene has short regions of

similarity to the targeted gene. However, it is quite

clear from the fundamentals of this technique that

RNAi in plants is based on high levels of sequence

specificity and thereby off-target effects have the rare

possibility. Moreover, selection of the sequences to

be used as dsRNA trigger could be selected with care

as long hairpin-based siRNA, which would generate

a diverse set of effective siRNA. The reports of a

significant off-target effect in plants have been rare

(Xu et al. 2006). Since its advent, RNAi has become

the technology of choice for the scientists for

manipulating the plants for the desired traits with

enormous potential for the improvement of quality

traits of the plants.

With the ever increasing population, food security

in the present scenario is becoming the biggest

challenge to the global agriculture. The RNAi

technology has been continuously applied in the

recent past for generation of new crop quality traits

and plant protection from viruses, insects, nematodes

and other pathogens. Now we have better under-

standing of the endogenous gene-silencing mechan-

ism, providing knowledge and efficiency that can be

used to develop precisely targeted gene-silencing

approaches for production of crop plants without

significant levels of ANFs. The utilization of RNAi

technology with organ- or tissue-specific expression

has added features to this technique, which enhances

its application in the improvement of nutritional

status of plant. The manipulation of a plant regulatory

gene by this technique can simultaneously influence

the production of several phyto-nutrients generated

from independent biosynthetic pathways, and provide

a novel way of increasing food value with the possibility

of elimination of harmful compounds from the edible

plant parts with RNAi technology. The transgenic

plants as outcome of this technique would be cost-

effective by producing RNAi inducers throughout a

plant’s entire life (Zhao et al. 2008). The unique

quality of siRNA with mobile ability between the

cells and the organisms has enormous potential to be

tapped for the improvement of different crops. As this

technology ensures organ-/tissue-specific silencing,

thereby, its potential could be utilized in silencing

or down regulating the expression of several genes

simultaneously, which will enhance our ability to

have desired levels of traditional/underutilized crops.

As research to alter crops for their better performance

is underway, thereby many more deliverables are

expected from this technology for the nutritional

security and popularization of unconventional plant

species in coming times.


Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

Declaration of interest: The authors report no

conflicts of interest. The authors alone are responsible

for the content and writing of the paper.

References

Akashi H, Miyagishi M, Taira K. 2004. RNAi expression vectors in

plant cells. Methods Mol Biol 252:533–543.

Amubode FO, Fetuga BL. 1983. Proximate composition and

chemical assay of the methionine, lysine and tryptophan

concentration of some forest tree seeds. Food Chem 12:67–72.

Andersson M, Melander M, Pojmark P, Larsson H, Bulow L,

Hofvander P. 2006. Targeted gene suppression by RNA

interference: an efficient method for production of high-amylose

potato lines. J Biotechnol 123:137–148.

Bartel DP. 2004. MicroRNAs: genomics, biogenesis, mechanism,

and function. Cell 116:281–297.

Bhat R, Sridhar KR, Young C-C, Arun AB, Ganesh S. 2008.

Composition and functional properties of raw and electron

beam-irradiated Mucuna pruriens seeds. Int J Food Sci Technol

43:1338–1351.

Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK. 2005.

Endogenous siRNAs derived from a pair of natural cis-antisense

transcripts regulate salt tolerance in Arabidopsis. Cell 123:

1279–1291.

Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M,

Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O. 2008.

Widespread translational inhibition by plant miRNAs and

siRNAs. Science 320(5880):1185–1190.

Byzova M, Verduyn C, De Brouwer D, De Block M. 2004.

Transforming petals into sepaloid organs in Arabidopsis and

oilseed rape: implementation of the hairpin RNA-mediated

gene silencing technology in an organ-specific manner. Planta

218:379–387.

Chai XJ, Wang PW, Guan SY, Xu YW. 2005. Reducing the maize

amylopectin content through RNA interference manipulation.

J Plant Physiol Mol Biol 31:625–630, (in Chinese).

Chan SW, Henderson IR, Jacobsen SE. 2005. Gardening the

genome: DNA methylation in Arabidopsis thaliana. Nat Rev

Genet 6:351–360.

Chel-GuerreroL,Perez-FloresV,Bentacur-AnconaD,Davila-OrtizG.

2002. Functional properties of flours and protein isolates from

Phaseolus lunatus andCanavalia ensiformis seeds. JAgricFoodChem

50:584–591.

Chen S, Hofius D, Sonnewald U, Bornke F. 2003. Temporal and

spatial control of gene silencing in transgenic plants by inducible

expression of double-stranded RNA. Plant J 36:731–740.

Chrisetnse AH, Sharrock RA, Quail PH. 1992. Maize poly-

ubiqauitin genes: structure, thermal perturbation of expression

and transcript splicing and promoter activity following transfer

to protoplasts by extroporation. Plant Mol Biol 18:675–681.

Chuang C, Meyerowitz EM. 2000. Specific and heritable genetic

interference by double-stranded RNA in Arabidopsis thaliana.

Proc Natl Acad Sci USA 97:4985–4990.

Conner AJ, Glare TR, Nap JP. 2003. The release of genetically

modified crops into the environment. Part II. Overview of

ecological risk assessment. Plant J 33:19–46.

Coulter JB, Suliman GI, Omer MI, Macfarlane SB, Moody JB,

Hendrickse RG. 1988. Proteinenergy malnutrition in Northern

Sudan: clinical studies. Eur J Clin Nutr 42:787–796.

Dalmay T, Horsefield R, Braunstein TH, Baulcombe DC. 2001.

SDE3 encodes an RNA helicase required for post-transcriptional

gene silencing in Arabidopsis. EMBO J 20:2069–2078.

Davies WP. 2003. An historical perspective from the Green

Revolution to the gene revolution. Nut Rev 61:S124–S134.

Davuluri GR, Tuinen A, Fraser PD, Manfredonia A, Newman R,

Burgess D, et al. 2005. Fruit-specific RNAi-mediated suppres-

sion of DET1 enhances carotenoid and flavonoid content in

tomatoes. Nat Biotechnol 23:890–895.

De Jong M, Wolters-Arts M, Feron R, Mariani C, Vriezen WH.

2009. The Solanum lycopersicum auxin response factor 7

(SlARF7) regulates auxin signaling during tomato fruit set and

development. Plant J 5:160–170.

Denli AM, Hannon GJ. 2003. RNAi: an ever-growing puzzle.

Trends Biochem Sci 28:196–201.

Dodo HW, Konan KN, Chen FC, Egnin M, Viquez OM. 2008.

Alleviating peanut allergy using genetic engineering: the silencing

of the immunodominant allergen Ara h 2 leads to its significant

reduction and a decrease in peanut allergenicity. Plant

Biotechnol J 6:135–145.

Eady CC, Kamoi T, Kato M, Porter NG, Davis S, Shaw M, et al.

2008. Silencing onion lachrymatory factor synthase causes a

significant change in the sulfur secondary metabolite profile.

Plant Physiol 147:2096–2106.

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC.

1998. Potent and specific genetic interference by double-

stranded RNA in Caenorhabditis elegans. Nature 91:806–811.

Flores T, Karpova O, Su X, Zheng P, Bilyeu K, Sleper DA, et al.

2008. Silencing of GmFAD3 gene by siRNA leads to low

a-linolenic acids (18:3) of fad3-mutant phenotype in soybean

[Glycine max (Merr.)]. Transgenic Res 17:839–850.

Gasciolli V, Mallory AC, Bartel DP, Vaucheret H. 2005. Partially

redundant functions of Arabidopsis DICER-like enzymes and a

role for DCL4 in producing trans-acting siRNAs. Curr Biol 15:

1494–1500.

Guo HS, Fei JF, Xie Q, Chua NH. 2003. A chemical regulated

inducible RNAi system in plants. Plant J 34:383–392.

Hamilton AJ, Baulcombe DC. 1999. A species of small antisense

RNA in posttranscriptional gene silencing in plants. Science 286:

950–952.

Heilersig HJB, Loonen A, BergervoetM,Wolters AMA, Visser RGF.

2006. Post-transcriptional gene silencing of GBSSI in potato:

effects of size and sequence of the inverted repeats. Plant Mol

Biol 60:647–662.

Herve V, Vazquez F, Crete P, Bartel DP. 2004. The action of

ARGONAUTE1 in the miRNA pathway and its regulation by

the miRNA pathway are crucial for plant development. Genes

Dev 18:1187–1197.

Hirai S, Oka S, Adachi E, Kodama H. 2007. The effects of spacer

sequences on silencing efficiency of plant RNAi vectors. Plant

Cell Rep 26:651–659.

Hoisington D, Khairallah M, Reeves T, Ribaut JM, Skovmand B,

Taba S, Warburton M. 1999. Plant genetic resources: what can

they contribute toward increased crop productivity? Proc Natl

Acad Sci USA 96:5937–5943.

Horiguchi G. 2004. RNA silencing in plants: a shortcut to functional

analysis. Differentiation 72:65–73.

Houmard NM, Mainville JL, Bonin CP, Huang S, Luethy MH,

Malvar TM. 2007. High-lysine corn generated by endosperm

specific suppression of lysine catabolism using RNAi. Plant


Jagtap UB, Gaurav RG, Bapat VA. 2011. Role of RNA interference

in plant improvement. Naturwissenschaften 98:473–492.

Jinek M, Doudna JA. 2009. A three-dimensional view of the

molecular machinery of RNA interference. Nature 457:

405–412.

Karlowski WM, Hirsch AM. 2003. The over-expression of an alfalfa

RING-H2 gene induces pleiotropic effects on plant growth and

development. Plant Mol Biol 52:121–133.

Khvorova A, Reynolds A, Jayasena SD. 2003. Functional siRNAs

and miRNAs exhibit strand bias. Cell 115:209–216.

KusabaM,MiyaharaK, Iida S, FukuokaH,TakanoT, SassaH, et al.

2003. Low glutelin content1: a dominant mutation that suppresses

the Glutelin multigene family via RNA silencing in rice. Plant

Cell 15:1455–1467.

Llave C, Kasschau KD, Rector MA, Carrington JC. 2002.

Endogenous and silencing-associated small RNAs in plants.

Plant Cell 14:1605–1619.


Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

Le LQ, Mahler V, Lorenz Y, Scheurer S, Biemelt S, Vieths S,

Sonnewald U. 2006. Reduced allergenicity of tomato fruits

harvested from Lyc e 1-silenced transgenic tomato plants. J

Allergy Clin Immunol 118:1176–1183.

Lewis BP, Burge CB, Bartel DP. 2005. Conserved seed pairing,

often flanked by adenosines, indicates that thousands of human

genes are microRNA targets. Cell 120:15–20.

Liu Q, Singh SP, Green AG. 2002. High-stearic and high-oleic

cottonseed oils produced by hairpin RNA-mediated post-

transcriptional gene silencing. Plant Physiol 129:1732–1743.

Liu C, Zhang L, Sun J, Luo Y, WangMB, Fan YL, Wang L. 2010. A

simple artificial microRNA vector based on athmiR169d

precursor from Arabidopsis. Mol Biol Rep 37:903–909.

Marques JT, Williams BR. 2005. Activation of the mammalian

immune system by siRNAs. Nat Biotechnol 23:1399–1405.

McElroy D, ZhangWG, Cao J, Wu R. 1990. Isolation of an efficient

actin promoter for use in rice transformation. Plant Cell 2:

163–171.

Meister G, Tuschl T. 2004. Mechanism of gene silencing by double-

stranded RNA. Nature 431:343–349.

Miki D, Shimamoto K. 2004. Simple RNAi vectors for stable and

transient suppression of gene function in rice. Plant Cell Physiol

45:490–495.

Molesini B, Rotino GL, Spena A, Pandolfini T. 2009. Expression

profile analysis of early fruit development in iaaM-parthenocar-

pic tomato plants. BMC Res Notes 2:1–7.

Montgomery MK, Fire A. 1998. Double-stranded RNA as a

mediator in sequence-specific genetic silencing and co-suppres-

sion. Trends Genet 14:255–258.

Napoli C, Lemieux C, Jogensen R. 1990. Introduction of a chimeric

chalcone synthase gene into petunia results in reversible

co-suppression of homologous genes in trans. Plant Cell 2:

279–289.

Nishihara M, Nakatsuka T, Yamamuras S. 2005. Flavonoid

components and flower color change in transgenic tobacco

plants by suppression of chalcone isomerase gene. FEBS Lett

579:6074–6078.

Odell JT, Nagy F, ChuaNH. 1985. Identification of DNA sequences

required for activity of the cauliflower mosaic virus 35S

promoter. Nature 313:810–812.

Ogita S, Uefuji H, Morimoto M, Sano H. 2004. Application of

RNAi to confirm the obromine as the major intermediate for

caffeine biosynthesis in coffee plants with potential for con-

struction of decaffeinated varieties. Plant Mol Biol 54:931–941.

Omidvar V, Abdullah SNA, Izadfard A, Ho CL, Mahmood M.

2010. The oil palm metallothionein promoter contains a novel

AGTTAGG motif conferring its fruit-specific expression and is

inducible by abiotic factors. Planta 232:925–936.

Ossowski S, Schwab R, Weigel D. 2008. Gene silencing in plants

using artificial microRNAs and other small RNAs. Plant J 53:

674–690.

OtaniM,HamadaT,KatayamaK,KitaharaK,KimSH,TakahataY,

et al. 2007. Inhibition of the gene expression for granule-bound

starch synthase I by RNA interference in sweet potato plants.

Plant Cell Rep 26:1801–1807.

Panstruga R, KimMC, ChoMJ, Schulze-Lefert P. 2003. Testing the

efficiency of dsRNAi constructs in vivo: a transient expression

assay based on two fluorescent proteins. Mol Biol Rep 30:

135–140.

Ratcliff F, Harrison BD, BaulcombeDC. 1997. A similarity between

viral defense and gene silencing in plants. Science 276:

1558–1560.

Regina A, Bird A, Topping D, Bowden S, Freeman J, Barsby T, et al.

2006. High-amylose wheat generated by RNA interference

improves indices of large-bowel health in rats. Proc Natl Acad Sci

USA 103:3546–3551.

Rhoades J, Bartel MW, Bartel B. 2006. MicroRNAs and their

regulatory roles in plants. Annu Rev Plant Biol 57:19–53.

Schijlen EGWM, De Vos CHR, Martens S, Jonker HH, Rosin FM,

Molthoff JW, et al. 2007. RNA interference silencing of chalcone

synthase, the first step in the flavonoid biosynthesis pathway,

leads to parthenocarpic tomato fruits. Plant Physiol 144:

1520–1530.

Schwab R, Ossowski S, Riester M, Warthman N, Weigel D. 2006.

Highly specific gene silencing by artificial microRNAs in

Arabidopsis. Plant Cell 18:1121–1133.

Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD.

2003. Asymmetry in the assembly of the RNAi enzyme complex.

Cell 115:199–208.

Segal G, Song R, Messing J. 2003. A new opaque variant of maize

by a single dominant RNA-interference-inducing transgene.

Genetics 165:387–397.

Shangguan XX, Xu B, Yu ZX, Wang LJ, Chen XY. 2008. Promoter

of a cotton fiber MYB gene functional in trichomes of

Arabidopsis and glandular trichomes of tobacco. J Exp Bot 59:

3533–3542.

Shomura A, Izawa T, Ebana K, Ebitani T, Kanegae H. 2008.

Deletion in a gene associated with grain size increased yields

during rice domestication. Nat Genet 40:1023–1028.

Sinha SK. 2010. RNAi induced gene silencing in plants. Physiol Mol

Biol Plants 16:321–332.

Sinha SK, Dubey N, Sachdev A. 2008. Gene silencing constructs for

alteration of seed fatty acid composition by RNAi induced

transgene silencing. New Bot 35:23–32.

Smith NA, Singh SP, Wang MB, Stoutjesdijk P, Green A,

Waterhouse PM. 2000. Total silencing by intron-spliced hairpin

RNAs. Nature 407:319–320.

Sotelo A, Contreras E, Flores S. 1995. Nutritional value and content

of antinutritional compounds and toxics in ten wild legumes of

Yucatan Peninsula. Plant Foods Hum Nutr 47:115–123.

Sunilkumar G, Campbell LM, Puckhaber L, Stipanovic RD,

Rathore KS. 2006. Engineering cottonseed for use in human

nutrition by tissue-specific reduction of toxic gossypol. Proc Natl

Acad Sci USA 103:18054–18059.

Tabara H, Grishok A, Mello CC. 1998. RNAi in C. elegans: soaking

in the genome sequence. Science 282:430–431.

Tang G, Galili G, Zhuang X. 2007. RNAi and microRNA:

breakthrough technologies for the improvement of plant

nutritional value and metabolic engineering. Metabolomics 3:

357–369.

Ullu E, Tschudi C, Chakraborty T. 2004. RNA interference in

protozoan parasites. Cell Microbiol 6:509–519.

Voinnet O. 2009. Origin, biogenesis, and activity of plant

microRNAs. Cell 136:669–687.

Vaucheret H, Vazquez F, Crete P, Bartel DP. 2004. The action

of ARGONAUTE1 in the miRNA pathway and its regulation

by the miRNA pathway are crucial for plant development.

Genes Dev 18:1187–1197.

Wang X, Yang Y, Yu C, Zhou J, Cheng Y, Yan C, Chen J. 2010.

A highly efficient method for construction of rice artificial

micro-RNA vectors. Mol Biotehnol 46(3):211–218.

Waterhouse PM, Ming-Bo W, Lough T. 2001. Gene silencing as an

adaptive defense against viruses. Nature 411:834–842.

Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q,

et al. 2001. Constructs for efficient, effective and high

throughput gene silencing in plants. Plant J 27:581–590.

Wielopolska A, Townley H, Moore I, Waterhouse P, Helliwell C.

2005. A high-throughput inducible RNAi vector for plants. Plant


Xie Z, Allen E, Wilken A, Carrington JC. 2005. DICER-LIKE 4

functions in trans-acting small interfering RNA biogenesis and

vegetative phase change in Arabidopsis thaliana. Proc Natl Acad

Sci USA 102:12984–12989.

Xiong AS, Yao QH, Peng RH, Li X, Han PL, Fan HQ. 2005.

Different effects on ACC oxidase gene silencing triggered by

RNA interference in transgenic tomato. Plant Cell Rep 23:

639–646.

Xu P, Zhang Y, Kang L, Roossinck MJ, Mysore KS. 2006.

Computational estimation and experimental verification of


Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

off-target silencing during posttranscriptional gene silencing in

plants. Plant Physiol 142:429–440.

Xu L, Ye R, Zheng Y, Wang Z, Zhou P, Lin Y, Li D. 2010. Isolation

of the endosperm-specific LPAAT gene promoter from coconut

(Cocos nucifera L.) and its functional analysis in transgenic rice

plants. Plant Cell Rep 29:1061–1068.

Yue SJ, Li H, Li YW, Zhu YF, Guo JK, Liu YJ, et al. 2008.

Generation of transgenic wheat lines with altered expression

levels of 1Dx5 high-molecular weight glutenin subunit by RNA

interference. J Cereal Sci 47:153–161.

Zhao YY, Yang G, Wang-Pruski G, You MS. 2008. Phyllotreta

striolata (Coleoptera: Chrysomelidae): arginine kinase cloning

and RNAi-based pest control. Eur J Entomol 105:815–822.

Zhu Y, Cai XL, Wang ZY, HongMM. 2003. An interaction between

a MYC protein and an EREBP protein is involved in

transcriptional regulation of the rice Wx gene. J Biol Chem

278:47803–47811.

Zilberman D, Cao X, Jacobsen SE. 2003. ARGONAUTE4 control

of locus-specific siRNA accumulation and DNA and histone

methylation. Science 299:716–719.


Int J

Foo

d Sc

i Nut

r D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y D

alho

usie

Uni

vers

ity o

n 04

/26/

13Fo

r pe

rson

al u

se o

nly.

Documents

RNA interference: a promising technique for the improvement of traditional crops