Biotechnology Advances 28 (2010) 301–307

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Biotechnology Advances
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b i o t e c h a d v

Research review paper

A new mechanism in plant engineering: The potential roles of microRNAs in

molecular breeding for crop improvement
Qing Liu, Yue-Qin Chen ⁎
Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Sun Yat-sen University, Guangzhou 510275, PR China

a r t i c l e i n f o a b s t r a c t

Article history: MicroRNAs (miRNAs) are small, endogenous, noncoding RNAs that negatively modulate the expression of
Received 25 August 2009 genes by inhibiting translation or by promoting the degradation of target mRNAs. miRNAs are now known to
Received in revised form 30 December 2009 have greatly expanded roles in a variety of plant developmental processes, in signal transduction, and in the
Accepted 1 January 2010
response to environmental stress and pathogen invasion. Because of their ability to inactivate either specific
Available online 11 January 2010
genes or entire gene families, artificial miRNAs function as dominant suppressors of gene activity when
Keywords:
brought into a plant. Consequently, miRNA-based manipulations have emerged as promising new
miRNAs approaches for the improvement of crops. This includes the development of breeding strategies and the
Molecular breeding genetic modification of agronomic traits. Herein, we highlight new findings regarding the roles of miRNAs in
Crop improvement plant traits, and describe the current miRNA-based plant engineering approaches. Finally, we consider the
feasibility of modulating current approaches to address future challenges such as breeding programs to
increase crop yield.
© 2010 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
2. miRNAs as determinants of plant architecture and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
3. miRNAs in shoot morphogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
4. miRNAs in vascular and root development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
5. The response of miRNAs to phytohormones and stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
6. miRNAs in response to phytohormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
7. The use of miRNAs in stress responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
8. The modulation of miRNAs as an innovative approach to plant engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
9. Artificial miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
10. Target mimicry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
11. Challenges for improving the efficiency of miRNA-based approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
12. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306

1. Introduction
Increasing world population, the scarcity of arable lands and
water, global climate changes and the demand for bio-fuels as
replacement energy source have led to serious concerns about the
future sufficiency of the global production of food from crop plants
⁎ Corresponding author. Biotechnology Research Center, Sun Yat-sen University,
Guangzhou 510275, PR China. Tel.: + 86 20 84112739; fax: + 86 20 84036551.
E-mail address: lsscyq@mail.sysu.edu.cn (Y.-Q. Chen).
(Rosegrant and Cline, 2003; Brown and Funk, 2008). To overcome
these problems, new agricultural technologies will be needed to
ensure global food supply and security, in addition to water and land
conservation efforts. In particular, crop improvements that confer
tolerance to environmental stresses and soil viruses, and that provide
high yield, will be required (Takeda and Matsuoka, 2008).
Recent studies have revealed powerful and unexpected roles for
small interference RNAs (siRNAs) and microRNAs (miRNAs) in the
control of plant growth by the silencing of native genes. Although
both of these have pivotal roles in gene silencing, the actions of

0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2010.01.002
302 Q. Liu, Y.-Q. Chen / Biotechnology Advances 28 (2010) 301–307
miRNAs are more extensive and remarkable. miRNAs regulate almost
every aspect of plant growth and development, including leaf mor-
phogenesis and polarity, floral differentiation and development, root
initiation and development, vascular development, and the transition
from vegetative growth to reproductive growth (Jones-Rhoades et al.,
2006; Chuck et al., 2009). Even more intriguingly, it has been
discovered that miRNAs play a role in hormone signal transduction
(Liu and Chen, 2009), the response to environmental stress, and
pathogen invasion (Chen et al., 2004; Sunkar et al., 2006). These
regulatory miRNAs stimulated the idea of developing artificial
miRNAs that can silence specific gene(s). Such targeted gene silencing
could permit the direct molecular modulation of plant traits, which
could in turn be applied to the breeding of crop species. Here, we
summarize current knowledge regarding the roles of miRNAs in
various aspects of plant traits as well as the existing miRNA-based
approaches to plant engineering. We also consider recent advances
and the future challenges of putting these approaches to practical use
in breeding programs for the purpose of increasing crop yield.
2. miRNAs as determinants of plant architecture and development
Higher plants display a variety of architectures that are defined by
the degree of branching, internodal elongation, and shoot determinacy
(Wang and Li, 2008). The study of plant architecture has attracted
attention for centuries, as plant architecture has been found to correlate
with agronomical traits and the ability of plants to survive environ-
mental stress (Peng et al., 1999). During recent decades, studies of
Arabidopsis thaliana and crop plants such as rice (Oryza sativa) and
maize (Zea mays) have greatly strengthened our understanding of the
molecular genetic basis of plant architecture (Reinhardt and Kuhlemeier,
2002; Babb and Muehlbauer, 2003; Wang and Li, 2008). However,
studies of plant architecture and its influences on human existence
have a long history. Fortunately, recent studies have indicated that
miRNAs play a role in plant architecture, and this helps to enhance our
knowledge of the molecular mechanisms that are (at least partially)
responsible for plant architecture. This knowledge has allowed us to
pave the way to optimizing the architecture of crops through molecular
design and thereby improving grain productivity.
3. miRNAs in shoot morphogenesis
The shoot apical meristem (SAM), which determines the archi-
tecture of the aerial parts of a plant, generates all of the above-ground
components of a higher plant, including stems, leaves, and auxiliary
branches, by maintaining a dynamic balance between indeterminate
growth and differentiation (Wang and Li, 2008). To date, miR164 and
miR165/miR166 have been shown to participate in shoot morpho-
genesis by targeting NAC genes and Class III HD-ZIP genes, respectively
(Emery et al., 2003; Laufs et al., 2004; Mallory et al., 2004a; Baker
et al., 2005; Williams et al., 2005; Zhou et al., 2007). In addition, these
miRNAs have also been shown to regulate leaf development: miR164
negatively regulates leaf senescence by controlling the level of the
ORE1 transcript, and miR165/miR166-mediated HD-ZIP III acts as a
signal to specify leaf polarity (Kidner and Martienssen, 2004; Mallory
et al., 2004b; Timmermans et al., 2004; Nikovics et al., 2006; Peaucelle
et al., 2007; Kim et al., 2009). With respect to leaf development, it is
worth noting that miR319 is an miRNA that controls leaf morpho-
genesis and leaf senescence by regulating the plant-specific class II
TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors
(Palatnik et al., 2003; Ori et al., 2007; Efroni et al., 2008; Schommer
et al., 2008). Ectopic expression of miR319 leads to leaf curvature,
larger leaflets and the continuous growth of leaf margins (Palatnik
et al., 2003; Ori et al., 2007; Efroni et al., 2008). miR156 and miR172
are two important regulators that have been implicated in promoting
floral transitions through regulation of the SBP-box genes and AP2-like
genes, respectively (Lauter et al., 2005; Wu and Poethig, 2006;
Schwarz et al., 2008; Wang et al., 2009; Wu et al., 2009). miR159 has
also been found to regulate flowering time and anther development
through targeting GAMYB (an activator of LEAFY—an important factor
in floral development). Increased levels of miR159 cause a reduction
in the level of LEAFY transcripts, delay flowering and perturb anther
development (Achard et al., 2004). The involvement of miRNAs in
shoot morphogenesis is shown in Fig. 1.
4. miRNAs in vascular and root development
Plant vascular tissues, which originate from procambial cells, provide
a pathway for the transport of signaling molecules, water and nutrients
(Fukuda, 2004). The differentiation of xylem cells from procambial cells
is coupled with the expression level of HD-ZIP-III homeobox genes,
including PHB, PHV, REV, AtHB8 and AtHB15 in Arabidopsis (Fukuda,
2004). Recent studies have shown that both miR165 and miR166 are
generated to specifically target HD-ZIP-III mRNAs (Kim et al., 2005;
Williams et al., 2005; Zhou et al., 2007). miR165 or miR166 over-
expression causes a drastic reduction in the transcript levels of the HD-
ZIP III genes and results in prominent phenotypes. These phenotypes
include alterations of organ polarity, inhibition of vascular development
and aberrant differentiation of the interfascicular fibers (Zhou et al.,
2007). As the basic architecture of vascular systems and HD-ZIP III
mRNAs is conserved, the miRNA-mediated regulation of vascular de-
velopment may occur in all vascular plants (Kim et al., 2005).
Root system architecture (RSA) refers to the spatial configuration of
a root system in the soil, and it is used to describe the shape and
structure of the root systems (de Dorlodot et al., 2007). During recent
years, environmental stresses and global climate change have increas-
ingly curtailed crop yield, and manipulation of the RSA towards a
distribution of roots in the soil that optimizes water and nutrient uptake
is one way to approach this problem (de Dorlodot et al., 2007). The
genetic influences on RSA are governed by a series of regulatory factors,
including miRNAs (Guo et al., 2005). miR164 has been shown to play a
role in root development. Accordingly, Arabidopsis miR164a- and
miR164b-mutant plants expressed more lateral roots and led to the
accumulation of NAC1 mRNA, which transduces auxin signals for lateral
root emergence (Guo et al., 2005). miR166, a post-transcriptional
regulator that controls shoot branching in several plants, has also been
shown to affect legume root architecture through the post-transcrip-
tional regulation of several HD-ZIP III genes in roots and in nodules
(Boualem et al., 2008). Consequently, miR164 and miR166 have
emerged as promising tools for optimizing RSA for crop improvement.
The roles of miRNAs in plant architecture are summarized in Fig. 1.
5. The response of miRNAs to phytohormones and stress
Small-molecule hormones govern almost every aspect of the
biology of plant development, including organ formation and the
response to various environmental stresses. Biochemical and genetic
studies have identified many of the factors that mediate the hormonal
control of cell expansion, division, differentiation, and, ultimately,
death (Klee, 2003). More recently, studies have revealed that miRNAs
play extensive roles in the regulation of key components of hormone
signaling pathways and they are also involved in stress responses.
Therefore, they can be regarded as a promising candidate for trans-
gene engineering for stress resistance (Zhang et al., 2006).
6. miRNAs in response to phytohormones
Auxin regulates a host of plant developmental processes and controls
many aspects of plant growth by influencing Auxin Response Factor
(ARF) transcription levels (Hagen and Guilfoyle, 2002). At present,
miR160 and miR167 have been confirmed to regulate ARFs (Mallory
et al., 2005; Wang et al., 2005; Wu et al., 2006; Yang et al., 2006). miR167
controls the patterns of ARF6 and ARF8 expressions and regulates male
 Q. Liu, Y.-Q. Chen / Biotechnology Advances 28 (2010) 301–307
Fig. 1. A diagrammatic representation of the involvement of miRNAs in plant architecture.
and female reproduction, whereas miR160 cleaves ARF10, ARF16 and
ARF17 to affect many aspects of plant development. Freeing ARF17 or
ARF16 from regulation by miR160 results in dramatic morphological
mutants (Mallory et al., 2005; Wang et al., 2005; Wu et al., 2006; Yang
et al., 2006). Moreover, miR393 targets TIR1 and three other closely
related F-box proteins (AFB1, AFB2 and AFB3). These are all auxin
receptors and consequently target short-lived repressors of ARF
transcriptional activators for ubiquitin-mediated degradation in re-
sponse to auxin. This implies that miRNA can also target F-box proteins
and affect the ubiquitin (Ub) proteasome pathway (Dharmasiri et al.,
2005; Navarro et al., 2006). In addition, miR164 is induced by auxin to
clear NAC1 mRNA so as to reset auxin signals and to regulate lateral root
development (Guo et al., 2005). Recent studies have also indicated that
miR168 and miR169 play roles in auxin signaling. However, their
specific regulatory roles still need to be elucidated (Subramanian et al.,
2008; Liu et al., 2009).
Furthermore, several miRNAs have been shown to modulate other
hormone pathways. For example, miR393, miR397b, miR402, miR413
and miR159 are induced by abscisic acid (ABA), whereas miR167,
miR389a and miR319 are downregulated when plants are treated
with ABA (Sunkar and Zhu, 2004; Reyes and Chua, 2007; Liu et al.,
2009). The induction of miR159 by ABA has been shown to mediate
seed germination by directing the degradation of MYB33 and MYB101
mRNAs (Reyes and Chua, 2007). As ABA is a plant hormone that plays
a particularly important role in environmental stress, these miRNAs
are assumed to exert their functions on stress responses (Fedoroff
2002). Two other miRNAs, miR319 and miR166, have recently been
found to be downregulated by gibberellin (GA) (Liu et al., 2009).
Moreover, the expression levels of miR159 and miR394 decreased
when plants were subjected to ethylene treatment, whereas miR172
and miR319 were downregulated by 6-BA (Liu et al., 2009). Early
303
studies have reported that miR319 controls leaf development by
mediating TCPs, and it has recently been discovered that miR319
controls jasmonate biosynthesis and senescence to coordinate the
balance between leaf growth and leaf senescence. This observation
has extended our knowledge of the relationships between miRNAs
and plant hormone signaling, as it confirms that the function of
miRNA-targeted transcription factors is not limited to the modulation
of downstream hormonal responses; miRNAs can also regulate the
biosynthesis of hormones (Schommer et al., 2008).
7. The use of miRNAs in stress responses
Plants have to cope with a variety of environmental stresses
including drought, salinity, high and low temperatures, and pathogen
invasion. When they experience these stresses, a diverse set of
physiological, metabolic and defense systems are activated so that
they can survive to sustain growth (Valliyodan and Nguyen, 2006).
The tolerance or susceptibility to stress is a very complex issue.
Worldwide, stress is the primary cause of crop loss, causing average
losses in yield of more than 50% for the major crops (Boyer 1982).
Evidence has shown that miRNAs are involved in the response of
plants to stress and that they are promising candidates for transgene
engineering for stress resistance.
Previous reports have indicated that miR395 and miR399 are
induced when plants suffer from sulfur and phosphate starvation
(Jones-Rhoades and Bartel, 2004; Fujii et al., 2005; Kawashima et al.,
2009). miR395 targets the ATP sulfurylases APS1, APS3, and APS4 to
regulate sulfate metabolism (Jones-Rhoades and Bartel, 2004), whereas
miR399 has multiple target sites in the 5′ untranslated region (UTR) of a
gene that encodes a putative ubiquitin-conjugating enzyme (UBC) in A.
thaliana. During low-phosphate (Pi) stress, miR399 was highly induced
304 Q. Liu, Y.-Q. Chen / Biotechnology Advances 28 (2010) 301–307

and the amount of the target UBC mRNA was reduced. Accordingly,
transgenic plants that express miR399 constitutively accumulate more
Pi than the wild type (Fujii et al., 2005). miRNAs also respond to drought,
cold, salinity and oxidative stress (Sunkar and Zhu, 2004; Sunkar et al.,
2006; Yamasaki et al., 2007; Zhao et al., 2007). miR398 targets two
closely related Cu/Zn-SOD genes: cytosolic CSD1 and chloroplast-
localized CSD2. Transgenic plants that overexpress a miR398-resistant
form of CSD2 accumulate more CSD2 mRNA and are consequently much
more tolerant to high light levels, heavy metals, and other oxidative
stresses (Sunkar et al., 2006). Therefore, relieving the miR398-directed
suppression of CSD2 in transgenic plants is an effective new approach
for the improvement of plant productivity under oxidative stress
conditions (Sunkar et al., 2006).
Recent studies have shown that Helper component-proteinases
(HC-Pro), such as p69, promote the miRNA pathway. These studies also
showed that p69-expressing plants contained elevated levels of a Dicer
mRNA and miRNAs, and consequently that the miRNA-guided cleavage
of two host mRNAs was enhanced (Chen et al., 2004). miR393 is another
miRNA that contributes to antibacterial resistance by repressing auxin
signaling (Navarro et al., 2006). Bacterial PAMP downregulates auxin
signaling by targeting the auxin receptor transcripts TIR1, AFB1, AFB2
and AFB3 in a miR393-dependent manner, implicating auxin in disease
susceptibility and the miRNA-mediated suppression of auxin signaling
in resistance pathways (Navarro et al., 2006).
8. The modulation of miRNAs as an innovative approach to
plant engineering
Due to the significant and extensive roles of miRNAs in the
development of plants and animals through post-transcriptional gene
silencing (better known as RNA interference (RNAi)), knowledge
derived from existing antisense technology and gene engineering
approaches can be adapted to manipulate specific genes or the levels of
miRNAs in vivo. miRNA-based technologies that have been developed
to regulate the function of miRNA in plants are shown in Fig. 2. At
present, short-hairpin RNAs that are based on miRNA precursor
backbones (artificial miRNAs [amiRNAs]) are the most successful
expression cassettes that can induce highly specific gene silencing in
plants (Duan et al., 2008). The expression of amiRNA precursors can
generate mature amiRNAs that have high specificity, in a similar way
to natural miRNAs, and cleave their target genes (Alvarez et al., 2006;
Schwab et al., 2006). A schematic representation is shown in Fig. 2B.
Furthermore, target mimicry, which consists of a noncleavable RNA
that interacts nonproductively with a complementary miRNA, pro-
vides a new technique that can be used to inhibit the activity of specific
miRNAs (Franco-Zorrilla et al., 2007) (see Fig. 2C).
9. Artificial miRNAs
Previous reports have demonstrated that the alteration of several
nucleotides within the miRNA sequence does not affect its biogenesis
and that the positions of matches and mismatches within the
precursor stem loop remain unaffected. Because of these features,
artificial miRNAs can be created by exchanging the miRNA/miRNA*
sequence within the miRNA precursor genes (Vaucheret et al., 2004).
Additionally, genome-wide expression analyses in A. thaliana have
demonstrated that plant amiRNAs exhibit high specificity, in a similar
way to natural miRNAs, such that their sequences can easily be
optimized to knock down a single gene or several highly conserved
genes without affecting the expression of other genes (Schwab et al.,
2005; Schwab et al., 2006; Khraiwesh et al., 2008). Thus, when using
gene engineering to improve agronomical traits, amiRNAs can be

Fig. 2. RNA interference-based technologies that regulate miRNA function in plants. A, Following transcription, the pri-miRNA is processed by DCL1, possibly with the aid of HYL1 and
other factors, to an miRNA:miRNA duplex. The mature miRNA is incorporated into a silencing complex that includes AGO1, and it then mediates the degradation of mRNA or its
translational repression. B, amiRNA is engineered into an miRNA precursor using overlap PCR to replace the endogenous miRNA sequence. The pri-amiRNA is processed by DCL1 and
functions as a normal miRNA. C, Target mimicry uses noncleavable RNA that sequesters a complementary miRNA. Perfect sequence complementarity allows the target mimic to bind
to the miRNA and interfere with miRNA function.
 Q. Liu, Y.-Q. Chen / Biotechnology Advances 28 (2010) 301–307
designed to target any gene of interest with great specificity. For those
miRNAs that are positive regulators for agronomically important
traits, the specific reduction of their target genes would be desirable
so that crops could be bred with elite traits.
miRNAs and siRNAs can affect gene expression in animals and
plants by RNAi. Perfectly complementary siRNAs have been formed
from exogenous double-stranded RNAs and have been used widely in
animals and plants to cleave target mRNAs of interest (Elbashir et al.,
2001; Schwab et al., 2006). amiRNAs share many properties with
siRNAs, as both of them are produced from fold-back precursors to
guide gene silencing. However, differences still exist between them. In
particular, a diverse set of siRNAs can be produced from a complete
dsRNA. Thus, siRNAs not only target candidate RNAs but they can also
affect RNAs that are not perfectly complementary (these are generally
considered off-targets). A single species of small RNA is produced
from an amiRNA precursor, and an amiRNA sequence that has little
homology to any plant genes can be chosen to avoid off-target effects
(Duan et al., 2008; Khraiwesh et al., 2008). In addition, recent studies
have shown that transgene-derived or viral-induced siRNAs are able
to move from cell to cell, indicating that their functions are not cell
autonomous. miRNAs, however, are not mobile and act cell autono-
mously (Khraiwesh et al., 2008; Tretter et al., 2008). In conclusion,
gene silencing that is mediated by amiRNAs is controlled in a cell
autonomous manner, and the amiRNA-mediated method has no
environmental biosafety problems when applied in agriculture (Duan
et al., 2008). These advantages make amiRNAs desirable for use in
plant engineering.
Currently, amiRNAs have been used successfully as expression
cassettes to induce highly specific gene silencing, not only in higher
plants (A. thaliana, O. sativa, tomato and tobacco), but also in lower plants
(Physcomitrella patens) and unicellular organisms (Chlamydomonas)
(Alvarez et al., 2006; Schwab et al., 2006; Niu et al., 2006; Duan et al.,
2008; Khraiwesh et al., 2008; Warthmann et al., 2008; Molnar et al.,
2009; Zhao et al., 2009). The result is that custom-made, synthetic
miRNAs that are vectored by endogenous pre-miRNA backbones
produced phenocopies of multiple mutant combinations of genes that
are not naturally regulated by miRNA. This implies that amiRNAs can be
used to successfully target even genes that are not the natural targets of
endogenous miRNAs (Alvarez et al., 2006).
Apart from dicotyledons, amiRNAs have been successfully used for
the specific downregulation of genes in rice (Alvarez et al., 2006;
Warthmann et al., 2008). As yet, approximately 61% of the predicted
loci of rice genes have not been assigned, even putatively. This
problem can be tackled using directed gene silencing by the use of
amiRNA transgenes (Warthmann et al., 2008). amiRNAs can efficient-
ly silence one or more genes or specific alleles with great specificity,
even those genes that are essentially inaccessible to other reverse
genetic techniques, e.g., tandem duplicated genes (Warthmann et al.,
2008). Successful gene silencing by amiRNAs has been demonstrated
in both an indica and a japonica variety of rice. More importantly, the
transgenes were inherited stably and remained active in the progeny.
This remarkable characteristic paves the way for amiRNA-mediated
gene silencing to modulate agronomically important traits in varieties
that are used in modern breeding. The ultimate intention of this is to
improve the agronomic performance and nutritional value. Further-
more, because rice shares excellent gene co-linearity with other
monocots, an amiRNA-based gene breeding strategy in rice can also be
deployed to other species at large scales (Sallaud et al., 2004).
Finally, knowing that amiRNAs can specifically silence genes that
were not originally under miRNA control, and as RNA silencing in plants
is a natural defense system against foreign genetic elements, the use of
amiRNAs to engineer plants resistant to viruses has been extensively
explored (Schwab et al., 2006; Niu et al., 2006). For instance, amiRNAs
have been designed (based on an A. thaliana miR159 precursor) to
target viral mRNA sequences that encode two gene silencing suppres-
sors: P69 of the turnip yellow mosaic virus (TYMV) and HC-Pro of the
305
turnip mosaic virus (TuMV). The result was that transgenic A. thaliana
plants expressing amiR-P69159and amiR-HC-Pro159 became specifically
resistant to TYMV and TuMV, respectively, indicating that the
expression of amiRNAs can successfully confer specific TuMV resistance
(Niu et al., 2006). More importantly, the amiRNA-mediated virus
resistance occurs at the cellular level and is heritable, suggesting that
this approach should have broad applicability in the engineering of
multiple virus resistances in crop plants (Niu et al., 2006). Additionally,
an amiRNA that targets sequences that encode the silencing suppressor
2b of the cucumber mosaic virus (CMV) can efficiently inhibit 2b gene
expression and confer effective resistance to CMV infection in transgenic
tobacco plants (Qu et al., 2007; Duan et al., 2008). Taken together, these
results demonstrate that the expression of virus-specific artificial
miRNAs is an effective and practical new approach to engineering
resistance to CMV, TuMV, and (possibly) other plant viruses.
Based on these results, we conclude that amiRNAs have emerged as a
promising approach that can modulate agronomically important traits.
This is true for different varieties that are used in modern breeding, and
the ultimate intention is to improve agronomic performance and crop
yield. To generate specific desirable traits in crops, amiRNAs can be
developed that silence the expression of genes that both positively and
negatively regulate important agronomic traits. If sufficient effort is
directed at this approach, amiRNAs are highly likely to be used in the
molecular breeding of plants in the near-future.
10. Target mimicry
In plants, almost all miRNAs have significant complementarity to
their target genes and regulate their targets by directing mRNA cleavage
at single sites in the coding regions. This characteristic led to the strategy
of target mimicry, which is based on RNA that is not cleaved, but instead
sequesters the complementary miRNA (Franco-Zorrilla et al., 2007).
This specific counteractive function was found when studying Pi
homeostasis, a critical determinant of growth performance and one of
the processes that is regulated by miR399 (Fujii et al., 2005; Franco-
Zorrilla et al., 2007). Pi starvation induced the expression of miR399,
which guides the cleavage of PHO2 RNA (an E2 ubiquitin conjugase-
related protein that negatively affects shoot Pi content and Pi
remobilization) and IPS1 (INDUCED BY PHOSPHATE STARVATION 1)
RNA. IPS1 contains a region of complementarity with miR399, but the
pairing is interrupted by a mismatched loop at the expected miRNA
cleavage site (Fujii et al., 2005; Franco-Zorrilla et al., 2007). IPS1 RNA is
not cleaved, but sequesters miR399 instead. The overexpression of IPS1
results in an increased accumulation of PHO2 mRNA and, concomitantly,
in reduced Pi content of the shoots (Franco-Zorrilla et al., 2007). The
engineering of IPS1 to make it cleavable abolishes its inhibitory activity
on miR399. Thus, IPS1 is not cleaved by miR399, but it inhibits the effect
of miR399 on PHO2 mRNA using a strategy that is based on target
mimicry.
More significantly, the IPS1 family members represent an example of
natural target mimicry, and this mechanism can be exploited to inhibit
miRNAs other than miR399. As indicated above, miRNAs influence
almost every aspect of plant growth and development, and a diverse set
is directly involved in the modulation of agronomic traits (e.g., miR165/
miR166 in shoot branching and miR164 in lateral root formation). The
Arabidopsis REVALUTA (REV) gene has been identified as a key regulator
of apical meristem initiation. Mutations of this gene usually lead to
severely reduced branches at both the vegetative and reproductive
stages (Talbert et al., 1995; Wang and Li, 2006). This indicates that
REV is a positive regulator for branching, which is considered tillering
for grain-bearing in monocotyledonous plants, and it confers great
agronomical benefits by improving crop yields (Wang and Li, 2005). In
nature, miR165/miR166 negatively cleaves the ortholog of REV to
maintain its expression level. Here, target mimicry provides a feasible
method to counteract the cleavage function of miR165/miR166,
elevating the level of the ortholog of REV to increase the number of
306 Q. Liu, Y.-Q. Chen / Biotechnology Advances 28 (2010) 301–307
tillers, consequently improving the grain yield. Whereas, it has to be
mentioned that the counteraction of miR165/miR166 by target mimicry
might lead to the accumulation of other HD-ZIP III genes, such as the
orthologs of PHB and PHV. This poses a challenge that when using the
technique of target mimicry to counteract the cleavage function of a
miRNA which is validated to target a gene family, it would lead to the
elevated levels of the whole gene family but not a single gene. miR164
negatively modulates the homolog of NAC1 mRNA to repress lateral root
formation, and the reduced number of lateral roots influences the ability
of a plant to secure edaphic resources. This ultimately affects crop yields.
Engineering a noncleavable RNA to sequester miR164 would allow
plants to express more lateral roots, thereby securing sufficient edaphic
resources. Consequently, target mimicry might provide a novel tech-
nique that can be used to modulate the level of endogenous miRNAs and
thereby improve agronomic traits and crop yields. However, this is just a
speculation and requires further validation. In addition, there is still a
long way to determine how often this general principle is used in nature
and whether endogenous target mimics are normally restricted to
noncoding RNAs (Franco-Zorrilla et al., 2007).
11. Challenges for improving the efficiency of
miRNA-based approaches
Compared to the conventional targeted gene knockout approach, the
relatively new technique of using amiRNA appears to be at least as
effective and versatile as conventional hpRNAi. At the same time, it
promises greater specificity and safety (Ossowski et al., 2008). However,
it has been reported that the overall success rate of amiRNA-based gene
silencing is close to 75%, suggest that the design of amiRNAs has not
been optimized (Ossowski et al., 2008; Park et al., 2009). Thus, more
effort is required in the design of amiRNAs to improve the efficiency of
gene silencing. Recent work has shown that an amiRNA design that
incorporates perfect complementarity to a target gene could increase
the efficiency of gene silencing. The manipulation of the structure
and sequence of the amiRNA has also proven useful for enhancing
the effectiveness of amiRNA in plants. One example is the design of
amiRNAs that have perfect complementarity to the target gene and a
strong 5′ instability introduced by G–A mismatch (Park et al., 2009). In
addition, advances in the understanding of the biogenesis and action of
endogenous miRNAs, which has been a very active field of scientific
inquiry for a long time, should also allow further improvement and
increased sophistication in the application of the amiRNA approach
(Ossowski et al., 2008).
12. Concluding remarks
amiRNAs are designed by using an endogenous miRNA precursor as
the structural backbone and replacing the stem loop region with a
specific amiRNA sequence that is designed to specifically target a
selected gene(s) of interest (Ossowski et al., 2008; Park et al., 2009).
The expression of amiRNA precursors under the control of a strong
constitutive promoter in transgenic plants leads to an accumulation of
mature amiRNAs. These have been shown in genome-wide expression
analyses in Arabidopsis to exhibit high specificity, in a similar way to
natural miRNAs and to guide the RNA-induced silencing complexes
(RISCs) to cleave their target genes (Schwab et al., 2006). This
amiRNA-based gene silencing approach has proven to be functionally
effective in lower and higher plant species, and in Chlamydomonas
(unicellular algae). The application of this technique has been
facilitated by the development of an elaborate web-based platform,
the Web MicroRNA designer (see WMD at http://wmd2.weigelworld.
org). This platform allows for the automated design of amiRNAs
for genes of interest and, although it was initially implemented for
A. thaliana (Schwab et al., 2006), it has now been extended to N30
additional species for which genomic or extensive EST information is
available (Ossowski et al., 2008; Park et al., 2009). The next generation,
WMD2, has a better design (Warthmann et al., 2008). This tool allows
the optimization of small RNAs for maximal effectiveness and for the
selection of RNAs that have the highest specificity for the intended
target gene(s), so that the maximal specificity and effectiveness of
gene silencing are ensured (Ossowski et al., 2008). Moreover, great
efforts have been taken to generate amiRNAs for each Arabidopsis
gene, providing an invaluable resource for functional genomic studies
(Zhao et al., 2009).
More intriguingly, amiRNA-mediated transgenes are inherited
stably and remain active in the progeny. Because of this, and because
of the conservation and uniformity of action of miRNAs, amiRNAs should
be broadly applicable to the breeding of hybrid crops (Warthmann et al.,
2008). Recently, successful applications of amiRNAs to agronomically
relevant strains of japonica and indica rice have been reported
(Warthmann et al., 2008). A protocol for the design of rice amiRNA
constructs has been provided, and this can be adapted easily to other
crops. This makes the use of amiRNAs easy and rapid (Warthmann et al.,
2008). This approach can also be suitable for the validation of the
functions of putative genes that are responsible for advantageous
agronomical traits. Consequently, this will broaden the usage of
amiRNAs for the improvement of agronomic performance. Thus, an
amiRNA-based transgene strategy appears to be a promising method for
the identification of genes that are related to agronomical traits and that
will assist in their practical use in improving crops.
Acknowledgments
This research is supported by the key project of the National
Natural Science Foundation of China (No. U0631001), and funds from
Guangdong Province (2007A020300001-5 and 2009A020102001) and
the State Key Laboratory of Biocontrol (SKLBC09B08). We thank
Nature Publishing Group Language Editing, LLC for editing the text of
the manuscript.
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