Endogenous and Synthetic MicroRNAs Stimulate
Simultaneous, Efficient, and Localized Regulation
of Multiple Targets in Diverse Species
John Paul Alvarez,a,bIrena Pekker,aAlexander Goldshmidt,aEyal Blum,aZiva Amsellem,aand Yuval Esheda,1
aDepartment of Plant Sciences, Weizmann Institute of Science, Rehovot, 76100, Israel
bSchool of Biological Sciences, Monash University, Victoria 3800, Australia
Recent studies demonstrated that pattern formation in plants involves regulation of transcription factor families by
microRNAs (miRNAs). To explore the potency, autonomy, target range, and functional conservation of miRNA genes, a
systematic comparison between plants ectopically expressing pre-miRNAs and plants with corresponding multiple mutant
combinations of target genes was performed. We show that regulated expression of several Arabidopsis thaliana pre-
miRNA genes induced a range of phenotypic alterations, the most extreme ones being a phenocopy of combined loss of
their predicted target genes. This result indicates quantitative regulation by miRNA as a potential source for diversity in
developmental outcomes. Remarkably, custom-made, synthetic miRNAs vectored by endogenous pre-miRNA backbones
also produced phenocopies of multiple mutant combinations of genes that are not naturally regulated by miRNA. Arabidopsis-
based endogenous and synthetic pre-miRNAs were also processed effectively in tomato (Solanum lycopersicum) and
tobacco (Nicotiana tabacum). Synthetic miR-ARF targeting Auxin Response Factors 2, 3, and 4 induced dramatic trans-
formations of abaxial tissues into adaxial ones in all three species, which could not cross graft joints. Likewise, organ-
specific expression of miR165b that coregulates the PHABULOSA-like adaxial identity genes induced localized abaxial
transformations. Thus, miRNAs provide a flexible, quantitative, and autonomous platform that can be employed for reg-
ulated expression of multiple related genes in diverse species.
A major component of pattern formation in plants involves
complex interplay between transcription factors (TFs) expressed
in precisetemporal and spatialdomains andmodifiers thatactto
maintain and refine their expression boundaries. TFs are usually
expressed at low levels and guide the activity of many down-
stream effectors. Evolutionary expansion of TF families in plants
(Riechmann et al.,2000) hasmeant thatfunctional redundancyis
a common theme in plant genomes. The majority of recently
identified plant microRNAs (miRNAs) impose sequence-based
simultaneous downregulation of developmentally important TFs
(Llave et al., 2002a; Rhoades et al., 2002). Indirect evidence
suggests that plant miRNAs have the potential to act efficiently
to eliminate or clear cells of their target gene activities (Bartel,
2004). For instance, a large reduction in the activity of miRNA-
regulated targets, either at the RNA or protein level, is evident
upon ectopic miRNA expression (Aukerman and Sakai, 2003;
Palatnik et al., 2003; Achard et al., 2004; Chen, 2004; Li et al.,
2005; Schwab et al., 2005). Complementing these observations,
strong dominant phenotypes are induced by target genes upon
release of miRNA-guided regulation by mutations in their miRNA
mutations at the miRNA binding site results in much stronger
phenotypes than those obtained with native transcripts (re-
viewed in Chen, 2005). However, there is also evidence for
quantitative action of plant miRNAs in quenching, as opposed
to clearing, of the target gene activity. Thus, miRNA-resistant
mutants are inherited in an incompletely dominant manner and
are still subject to some miRNA-directed cleavage (Tang et al.,
2003; Mallory et al., 2004a), and mutations in miRNA genes can
result in increased levels of target gene expression (Baker et al.,
Despite molecular indications that miRNAs can be very effi-
cient, direct phenotypic evidence derived from comparing mul-
tiple mutant combinations with the effects of ectopic expression
of the corresponding miRNA is limited. Unlike animal miRNAs,
which simultaneously negatively regulate dozens of targets (Lim
et al., 2005), plant miRNAs appear to have more limited target
the same gene family or even members of a single monophyletic
clade of a larger family (Bartel and Bartel, 2003). These qualities
can be used to facilitate the comparison of multiple mutant
combinations with the effects of ectopic expression of the
corresponding miRNA. In the absence of such a comparison,
the prevailing model for high specificity and potency of plants
miRNAs has yet to be verified. For example, in one case, ectopic
1To whom correspondence should be addressed. E-mail yuval.eshed@
weizmann.ac.il; fax 972-8934-4181.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Yuval Eshed
WOnline version contains Web-only data.
Article, publication date, and citation information can be found at
The Plant Cell, Vol. 18, 1134–1151, May 2006, www.plantcell.org ª 2006 American Society of Plant Biologists
miR164 induced cotyledon fusion and meristem arrest, mimick-
ing the double mutant phenotype of two out of the five NAC
domain genes targeted by this miRNA (Aida et al., 1997; Jones-
Rhoades and Bartel, 2004; Laufs et al., 2004; Mallory et al.,
2004b; Baker et al., 2005). However, overexpression of the
miR165/6 failed to mimic the seedling arrest and production of a
cylindrical monocot-like radial shoot observed in multiple mu-
tants of the five miR165/6 PHABULOSA (PHB)-like targets
(Emery et al., 2003; Li et al., 2005; Prigge et al., 2005; Williams
et al., 2005a). The failure to recapitulate the phenotype in this
latter case may reflect the inability of strong 35S:miR165/6
embryos to survive after transformation. Such a proposal could
be confirmed by more precise viability-independent expression
of the miR165/6.
Deciphering the role of TFs and their miRNA regulators in
pattern formation can benefit greatly from spatial and temporal
can be bypassed and the autonomy of specific miRNA-induced
perturbations examined. Such tissue-specific silencing using
RNA interference (RNAi) has been successfully applied in plants
(Watson etal., 2005),but RNAi has alsobeen observed to induce
systemic spread of gene silencing (reviewed in Voinnet, 2005).
Notably, the common complexes involved in miRNA and short
interfering RNA (siRNA) biogenesis and processing raises the
possibility of a systemic component to miRNA-mediated regu-
lation. In support of such a role, different miRNAs have been
found in conducting phloem sap, a possible conduit for their
systemic spread (Yoo et al., 2004). However, while unequivocal
proof for long distance translocation of silencing signals was
perceived through grafting experiments between silenced and
nonsilenced transgenic tobacco (Palauqui et al., 1997), no such
evidence has been provided for miRNA signals. Moreover, in
mature Arabidopsis thaliana tissues, there is a strict overlap
a sensor construct, suggesting that this miRNA maintains strict
spatial autonomy (Parizotto et al., 2004).
Many plant miRNAs appear to have a long coevolutionary
history with their targets, extending back to moss and lycopods
(Floyd and Bowman, 2004; Axtell and Bartel, 2005). The pairing
structure of the miRNA and nearly perfect complementary
miRNA* can be highly conserved. Though sequence conserva-
tion also occurs outside the domain of the miRNA and its
complement, it is the general predicted structure of the pre-
miRNA foldback, rather than its sequence per se, that is con-
served between the distantly related Arabidopsis and rice (Oryza
sativa) (Reinhart et al., 2002). This raises the question whether
pre-miRNAs retain functional conservation between distant spe-
cies. Such conservation would involve efficient miRNA biogen-
esis, including correct spatial recognition and processing of
the miRNA/miRNA* from the pre-miRNA foldback structure
(reviewed in Chen, 2005). Likewise, the coevolutionary history
of miRNA and their targets also raises the question of whether
unique characteristics define particular mRNAs as targets.
Evidence from metazoans suggests that this is not the case, as
the miRNA sequence and its complement in the foldback struc-
2005). Similarly, analysis in Arabidopsis demonstrated that the
miRNA and its complement miRNA* domains of the pre miR171
backbone could be substituted to produce a novel miRNA that
successfully targeted green fluorescent protein (GFP) (Parizotto
et al., 2004). However, the capability and efficacy of the manip-
ulated pre-miRNA in targeting multiple endogenous genes not
normally targeted by miRNA regulation has yet to be examined.
that can be induced by miRNAs, we have chosen an experimen-
tal platform that uses sets of target genes for which complete
conventional mutants are also available. Furthermore, miRNA
ectopic expression has been brought under the control of in vivo
constitutive ortissue-specificectopic expression ofpre-miRNAs
guided by RNA Pol II promoters. This system enabled us to
demonstrate that miRNAs have the potential to impose full or
partial phenocopies of multiple mutants in several independent
assays. These observations enabled us to expand the range of
miRNA control by custom-designed pre-miRNAs that can stim-
ulate phenocopy of mutations in genes not naturally regulated
in this manner. Endogenous and synthetic Arabidopsis pre-
miRNAs were functionally conserved in tomato (Solanum lyco-
persicum) and tobacco (Nicotiana tabacum), and tissue-specific
miRNA misexpression produced phenotypes that are limited to
the area of expression. Hence, these data suggest that miRNA
activities are quantitative and, at least for distances greater than
few cells, do not act outside of their domain of expression.
Precise Ectopic Expression of Endogenous miRNA Can
Phenocopy Multiple Mutant Combinations
Plant miRNAs, such as miR164, have been ectopically ex-
pressed using the 35S promoter directly or through chemical
induction driving the stem foldback that constitutes the pre-
miRNA with various 59 and 39 endogenous additions (Figure 1A;
Guo et al., 2005). We wished to examine the ability of precise
potential. Transactivation of protein-encoding genes using the
LhG4-OP system has been effective in driving strong, specific
expression and obviating early deleterious effects of gene over-
expression (Moore et al., 1998). To assay the capacity of this
system to express pre-miRNAs, genomic fragments containing
the pre-miRNA foldback flanked by short 59 and 39 pre-miRNA
sequences were cloned behind an OP array followed by a TATA
box (see Supplemental Table 1 online). In the case of OP:
miR164b, the resulting T2 lines (see Methods for selection and
scoring of transgenic lines; summarized in Table 1) were trans-
activated using the PHB:LhG4 promoter, which is expressed in
the developing cotyledon primordia and throughout the apical
meristem (Figure 1C) to provide PHB?miR164b F1s (Figure
1E; ? denotes transactivation). Expression of miR164 under this
promoter mimicked the fused cotyledon and meristem arrest
phenotype of the cup-shaped cotyledon1 (cuc1) and cuc2 dou-
ble mutant, two of miR164’s six targets (Figure 1D). These ob-
servations indicate that expression of pre miR164b using the
transactivation systemcanefficientlydeplete cellsoftarget gene
To extend these observations to other Arabidopsis miRNAs,
we expressed OP:miR165b using the promoter of one of its
Mutant Phenocopy with MicroRNA 1135
Figure 1. miRNAs Can Quantitatively Regulate Multiple Transcripts Simultaneously and Phenocopy Their Combined Loss of Function.
(A) A scheme of an endogenous pre-miRNA. The red and blue fragments will be cleaved by DICER-LIKE1 (DCL1) to generate the miRNA and miRNA*,
(B) A 10-d-old wild-type seedling.
(C) The promoter of PHB drives GFP expression throughout the shoot apex in wild-type heart-stage embryos.
1136The Plant Cell
target genes, the PHB:LhG4 driver. miR165/6 target the five
PHB-like genes that redundantly promote meristem establish-
ment and maintenance as well as differentiation of lateral organs
and the vasculature. phb phavoluta (phv) revoluta (rev) triple
mutants or loss of all of the five PHB-like genes result in seedling
arrest after the production of a cylindrical monocot-like radial
shoot in which the apical meristem activity is abolished (Figure
1F; Emery et al., 2003; Prigge et al., 2005). However, previous
overexpression studies with 35S:miR165 resulted in variable
seedling phenotypes in which the most extreme plants had small
leaves with some polarity defects (Li et al., 2005). By contrast,
PHB?miR165b plants gave rise to radial seedlings, phenocopy-
ing the multiple mutant combination (Figure 1G). These results
illustrate that specific miRNA expression can abolish target gene
activity and demonstrate the potential potency of precise pre-
miRNA misexpression as a vehicle for simultaneous downregu-
lation of multiple members of the same gene family.
35S-Driven miRNAs Can Faithfully Mimic
It is likely that the inefficiency of 35S:miR165a in producing a
expression in early stage embryos and their importance in
embryogenesis. Auxin Response Factor6 (ARF6) and ARF8 are
the only predicted targets of miR167, and arf6 arf8 double
mutants are viable, late flowering, have dark green leaves, and
exhibit unexpanded 2nd and 3rd whorl floral organs (Nagpal
et al., 2005). To assay whether the 35S promoter can effectively
drive a miRNA-mediated reduction in these genes, we assayed
35S:pre miR167a transformants. Out of 20 independent T1
plants, eight had a similar phenotype to that reported for arf6
arf8 double mutants both vegetatively and in flowers, while the
remainder had a range of weaker phenotypes (Figure 1H, Table
1). No additional features than described for the double mutants
were noticed. Thus, depending on the miRNA and its targets,
elevated ectopic miRNA expression by the constitutive 35S
promoter can specifically reduce multiple target gene activities
to levels that parallel that of multiple loss-of-function mutants.
Precise Expression of miRNAs Can Reveal Novel
Embryonic expression of either miR164 or miR165 resulted
in a seedling phenocopy of multiple mutants in the correspond-
ing target genes. While cuc1 cuc2 seedlings can be rescued
using tissue culture, phb phv rev or plants mutant for all five
PHB-like mutant genes do not develop beyond the seedling
stage. Because the CUC-like and PHB-like genes are active
throughout plant development, the use of tissue-specific miRNA
expression could reveal functions of these genes later in plant
development. To examine the utility of this approach, trans-
was performed using the promoter of the flower meristem gene
As shown in Figures 1I and 1J, the expression mediated by
AP1 promoter initiates transcript accumulation throughout
young floral meristems. In AP1?miR164b plants, the sepals
are completely fused, petals are absent, and stamens exhibit
fusion to each other and the gynoecium (cf. Figures 1L and 1K).
This phenotype is similar, albeit more severe than that observed
1997). In AP1?miR165b plants, only radial filamentous struc-
tures were observed in the place of flowers, consistent with the
miR165 effectively eliminating flower meristem function (Figure
1M). These findings are consistent with the observed seedling
phenotypes, indicating that the PHB-like genes are essential for
embryo and flower meristem maintenance. These observations
demonstrate that the transactivation system can be effectively
usedinconjunction withmiRNA-mediated lossof geneactivity in
specific cells at any stage of the plant’s life cycle.
Low Levels of miRNA Expression or High Levels of
Inefficient miRNA Suggest That Plant miRNAs Can
Act in a Quantitative Fashion
Tissue-specific transactivation of pre-miRNAs identified lines
producing a loss-of-function phenocopy but also uncovered
OP:miRNA lines that induced mild phenotypes (Table 1). For
Figure 1. (continued).
(D) Arabidopsis cuc1 cuc2 double mutant seedlings.
(E) F1 seedlings of OP:miR164b transactivated by PHB:LhG4.
(F) A monocot-like phv phb rev triple mutant seedling.
(G) A monocot-like PHB?miR165b seedling of comparable age.
(H) Whole shoot and flower (inset) of 35S:miR167a plant next to same age wild type display identical alterations found in arf6 arf8 double mutants (cf.
with Nagpal et al., 2005).
(I) Scanning electron micrograph of wild-type flowering apex.
(J) A cross section through AP1?HP-GFP flowering apex with expression throughout emerging flower meristems.
(K) Wild-type flower.
(L) Fused sepals and absent petals in AP1?miR164b flower.
(M) Flowering apex and filamentous flowers (inset) of strong AP1?miR165b plant.
(N) Normal sepals, radial petals, distorted stamens, and multiple carpels in a weak AP1?miR165b flower.
(O) Sequence alignment of the wild type and phv-1d mutant with corresponding miR165b and miR165bm6.
(P) Seedling expressing ANT?miR165b#4 results in radialized cotyledons and aborted meristem.
(Q) and (R) Adaxial surface of wild-type (Q) and ANT?miR165bm6 (R) leaves. Note the adaxial outgrowths of the transgenic leaf (arrow).
(S) Normal sepals, distorted stamens, and multiple carpels in a strong ANT?miR165bm6 flower.
FM, flower meristem; IM, inflorescence meristem; P, petal. Bars ¼ 3 mm in (B), (F), and (G) and 20 mm in (C), (I), and (J).
Mutant Phenocopy with MicroRNA1137
example, whena weak OP:miR165b line was transactivated with
the AP1:LhG4 promoter, the growth of the petals and stamens
was markedly affected and additional carpels were formed
(Figure 1N). Associations between phenotype strength and tran-
scription levels have been shown previously for ectopic miR164
and miR166g expression (Laufs et al., 2004; Williams et al.,
2005a). Likewise, assaying both the weak and strong OP:
miR165b lines with the same promoters revealed consistent
phenotype strengths restricted to the promoter’s expression
domain. These observations imply that when expressed in a
discrete group of cells, miR165 can act in a quantitative fashion.
We hypothesized that quantitative action of miRNA may
also be achieved by the generation of miRNA with lower homol-
ogy. This was based on the observation that mRNA of the
dominant phv-1d miRNA-resistant mutation was still cleaved
(our unpublished data; Tang et al., 2003). The phv-1d molecular
lesion is a G-to-A transition in the miR165/6 target region
opposite position 6 from the 59 end of the miR165/6 (Figure
Table 1. Endogenous and Synthetic miRNAs Examined in This Study
Target Gene Function and
Frequency and Range of
Phenotypic Responses in
NAC domain TFs
CUC1, CUC2, NAC1,
Embryonic meristem establishment,
organ separation, and lateral root
outgrowth (Figure 1D)
PHB-like class III
PHV, CNA, REV,
Meristem establishment and
maintenance, adaxial differentiation
of lateral organs, and vasculature
patterning (Figure 1F)
As for miR165b
As for miR165b
As for miR165b As for miR165b
As for miR165b but with reduced
homology toits targets
As for miR165b
Promotion of flowering and
flower organ maturation
(Nagpal et al., 2005)
Abaxial differentiation of lateral organs,
vascular development, flowering,
and cell growth (Figure 2D)
B3 domain TFs
NGA1, NGA2, NGA3, NGA4
Lateral organ growth
(Figures 3E and 3H)5
Weak homology with
As for miR-NGAa
As for miR-NGAa
Frequent silencing was observed in subsequent generations for the lines marked with asterisks.
aThe 10OP:miRNA lines were examined upon transactivation with a promoter LhG4 driver. Combinations with specific promoters are described in the
text. The 35S:miRNA lines were scored directly as T1s and showed consistent phenotypes upon cross with the wild type.
bPhenotype relative to loss-of-function phenocopy (close resemblance is strong).
cPhenotype scoring is relative to other transgenic lines carrying the same construct because corresponding mutants are not available.
1138The Plant Cell
Thus, miR165bm6 (C-to-U substitution in position 6 of the
miR165b) mimics the corresponding position of the phv-1d
mutation in the miRNA rather than in the target. The efficacy of
the modified miRNA was demonstrated by its ability to restore
wild-type morphology to the phv-1d mutant plants (see Supple-
mental Figure 1 online). The AINTEGUMENTA:LhG4 (ANT) pro-
moter is expressed in the primordia of all above-ground organs.
ANT?miR165b plants exhibited a strong phenotype of radial-
ized cotyledons and an aborted apical meristem (Figure 1P). By
contrast, ANT?miR165m6 plants had a weak, abaxialized phe-
notype, consistent with inefficient miR165bm6 action. The ad-
axial surface of the leaves developed localized outgrowths (no
effect was observed on the abaxial leaf surface), while in the
flowers, the stamens locules were reversed and the gynoecium,
like that of weak AP1?miR165b lines, had additional carpels
(Figures 1Q to 1S). A similar floral phenotype was observed of
AP1?miR165m6 plants (seeSupplemental Figure 1online). The
mild abaxialized phenotype of OP:miR165bm6 plants and that of
the weak OP:miR165b lines argues that miRNAs can act in a
quantitative fashion depending on the pairing quality and quan-
tity of the miRNA with respect to its target mRNAs.
General Strategy for Developing Synthetic miRNA on the
Backbone of Native miRNA
Ectopic expression of endogenous pre-miRNAs allowed con-
trolled downregulation of multiple coregulated miRNA targets.
This prompted us to explore the possibility to design synthetic
miRNA that will target genes not normally regulated by miRNA.
We used the pre miR164b backbone as a template to approach
these questions based on the consistency and efficacy of
miR164b in the overexpression analysis. In addition, this back-
bone contains a bulge at position 4 of the conceptualized Dicer-
generated miRNA/miRNA* duplex in the desired miRNA strand
(marked red in Figure 2B). Asymmetric instability in this hybrid is
believed to help differentiate between the miRNA and miRNA*
in the RNA-induced silencing complex (RISC) (Schwarz et al.,
2003), thus theoretically helping define the miRNA (upper strand
in pre-miRNA, Figure 2B) irrespective of the sequence intro-
duced. As initial gene targets for synthetic miRNA regulation, we
selected the abaxial promoting ARF3 and ARF4 along with
ARF2, for which respective mutations have been described (Li
et al., 2004; Pekker et al., 2005; Okushima et al., 2005b; Schruff
et al., 2006). Importantly, these ARFs contain common, con-
served sequences, which are the basis for negative coregulation
by evolutionary conserved transacting siRNAs (ta-siRNAs),
siR2141 and siR2142 (Allen et al., 2005; Williams et al., 2005b).
The target sequences of these ta-siRNAs (two sites in ARF3/
ETTIN [ETT] and ARF4 and one in ARF2) are conserved in
Arabidopsis, tomato, and monocotyledonous plants and sug-
gest that ARF2 may have overlapping functions with ARF3/ETT
and ARF4 (Figure 2A; Pekker et al., 2005; Williams et al., 2005b).
Exploiting this, we designed a miRNA that should directly target
these three ARFs and ensured that it began with U, like most
plant miRNAs. As shown in the bottom line of Figure 2B,
mismatches were introduced into the miRNA complementary
assuming that bulges in the miR164 backbone contain essential
recognition and processing information. The miR-ARF2/3/4
(hereafter referred to as miR-ARF) was integrated into the pre
miR164b backbone by direct gene synthesis (Figure 2B, right
or OP promoter.
Synthetic miRNA Efficiently Regulates ARF2, 3, and 4,
Which Are Naturally Regulated by Transacting siRNAs
Arabidopsis ett/arf3 plants are characterized by a loss of abaxial
identity in the gynoecium, while in ett/arf3 arf4 double mutants,
all lateral organs exhibit reduced abaxial identity and outgrowths
on the abaxial surface of their leaves (Figure 2C; Pekker et al.,
2005). Triple ett/arf3 arf4 arf2 mutants are unavailable due to the
tight genetic linkage between arf4 and arf2 (Okushima et al.,
2005a). Strikingly, all 35S:miR-ARF Arabidopsis transformants
exhibited phenotypes consistent with an effective targeting of
the three ARF genes. Plants with a strong phenotype resembled
ett/arf3 arf4 double mutants but had more outgrowths on the
abaxial leaf surface (Figures 2C to 2E, Table 1) as well as an
increased floral organ number and frequently radialized stamens
(Figures 2F to 2H). Similar to arf2 single mutants, these 35S:miR-
ARF plants were also late flowering (Ellis et al., 2005; Okushima
et al., 2005b).
A Synthetic miRNA Born on a Stem-Loop Precursor
Efficiently Guides RISC Complexes
Weisolated RNAfromplants of a35S:miR-ARF line withastrong
phenotype and verified by RNA gel blot analysis that miR-ARF
was efficiently processed from its precursor by identifying its
21-nucleotide miRNA, which was comparable in size with the
endogenous miR164 (Figure 2I). After processing, miRNAs enter
the RISC complex and guide cleavage of their target transcripts,
inducing their degradation and/or interference with translation
(Llave et al., 2002b; Aukerman and Sakai, 2003; Chen, 2004). To
examine the mode of action of the synthetic miRNA, we assayed
the relative abundance of the full-length ARF transcripts using
RNA gel blot analysis. For all three, a significant but incomplete
reduction in RNA levels was observed in 35S:pre miR-ARF
Arabidopsis plants (Figure 2J).
Cleavage of the three ARF genes by the synthetic miR-ARF
was investigated using 59 RNA ligase-mediated rapid amplifica-
tion of cDNA ends (RLM-RACE) analysis (Kasschau et al., 2003).
Endogenous ta-siRNA–directed cleavage (siR2141and siR2142)
of ARF transcripts has been shown in Arabidopsis (Allen et al.,
2005; Williams et al., 2005b). However, a much greater abun-
dance of cleaved products of all three ARF genes was observed
in 35S:miR-ARF plants than in wild-type Arabidopsis (Figure 2K;
see Supplemental Figure 2 online). In wild-type plants, we could
detect products at relatively low amounts from the ta-siRNA–
mediated cleavage at both ARF3/ARF4 A and ARF3/ARF4 B
sites using primers downstream of the B site (primer b, Figure
2K). For 35S:miR-ARF plants, we managed to detect only B-site
cleavage products with primer B, but using a different primer
(primer a between the A and B sites for ARF4), we amplified
abundant products from cleavage at site A (Figure 2K). In the
35S:miR-ARF plants, sequence analysis demonstrated that
Mutant Phenocopy with MicroRNA1139
Figure 2. Custom-Designed Synthetic miRNAs Efficiently Regulate ta-siRNA Targets.
1140 The Plant Cell
cleavage in the target mRNA occurred consistently between
nucleotides 10 and 11 at miR-ARF complementary sites(marked
in Figure 2L), while much more dispersed cleavage products
were amplified from the wild type (see Supplemental Figure 2
online; Allen et al., 2005). We therefore conclude that miRNAs
generated by a stem-loop precursor guide the RISC complexes
more specifically than ta-siRNA–mediated cleavage.
Synthetic miRNA Efficiently Regulates the NGATHA
Multigene Family Not Normally Associated with
are normally regulated by various RISC-based small RNA regu-
lation, we next targeted the NGATHA (NGA) genes for which
there is no evidence of this form of regulation.
The NGA clade is composed of four genes (NGA1-4); all are
closely related to the RAV-like proteins in the structure of the B3-
DNA binding domain but do not have an AP2 domain (Figure 3A).
Like ett mutations, mutants in nga1 phenotypically enhance the
kanadi1 mutant phenotype in the gynoecium (Bowman et al.,
2002; nga1 was renamed from howard). We identified NGA1 and
found that all four NGA genes redundantly regulate lateral organ
growth (as will be described elsewhere). However, only minor
morphological alterations are apparent in any of the single gene
mutants. A single consensus stretch of 21 nucleotides within the
B3 domain was identified that could constitute a common
synthetic miRNA target site in the four NGA-like genes of
Arabidopsis (Figure 3B). This 21-nucleotide sequence fulfilled
the available criteria of allowable mismatches in number and
position as well as low free energy characterizing endogenous
plant miRNAs (Figure 3B; Allen et al., 2005; Schwab et al., 2005).
The 21-nucleotide specificity was confirmed in the genome via
miRNAs. The designed 21 nucleotides were introduced into the
miR164a and miR164b backbones to generate miR-NGAa and
miR-NGAb, respectively (Figure 3C).
nga1 nga2 nga3 nga4 quadruple mutant plants have shorter
and wider leaves than the wild type, sepals and petals are broad,
and petals are slightly green-yellow. Style development is se-
verely impaired, with disruptions in the coordinated growth that
normally seal the two-carpel gynoecium. Consequently, the
gynoecium remains distally unfused, with distinctive projections
emanating from the top of the valves (cf. Figures 3E and 3H with
NGAb showed phenotypic alterations approaching that of the
nga1 nga2 nga3 nga4 quadruple mutant plants (Figures 3F to 3I,
Table 1). These results show that miRNA-dependent control can
be extended to other multigene families.
NGA RNA Analysis Detects Cleavage Products and
Differential Reduction of NGA RNA
We asked whether the molecular processing of NGA RNA by
miR-NGA is similar to RISC-mediated processes. An RNA gel
blot from RNA extracted from plants of a line with a strong
phenotype confirmed the presence of the 21-nucleotide syn-
thetic miR-NGA, and RLM-RACE analysis identified miRNA-
directed cleavage products (Figures 3K and 3L). No evidence of
NGA-like cleavage products was observed in RNA from wild-
type inflorescences. Notably, thepredominant cleavage position
as determined by direct sequencing of the RLM-RACE products
was identical in all four NGA genes and was comparable to a
the miR-NGA (as marked in Figure 3B). However, though RNA
levels of NGA1 were reduced in 35S:miR-NGAa plant inflores-
cences, very mild reduction in the levels of NGA3 was detected
(Figure 3M). This could be due to attenuation of protein transla-
tion or a transcriptional autofeedback loop.
We next asked whether miR-NGA could ameliorate the phe-
notype produced by overexpression of the NGA1 gene. The
CAB3:LhG4 (chlorophyll a/b binding promoter) line drives ex-
pression throughout photosynthetic tissues, and CAB3?NGA1
Figure 2. (continued).
(A) Sequence alignment of the ta-siRNA binding sites in Arabidopsis ARF2, ARF3, and ARF4, the endogenous ta-siRNAs, and a designed miR-ARF
sequence with better homology to all target sites. The A and B sites are designated according to Allen et al. (2005). Mismatches are marked red and G-U
(B) Predicted folding and dicing of the pre miR164b backbone before (left) and after (right) replacement of miR164 with the miR-ARF sequence.
(C) Abaxial side of wild-type (a), ett-1 arf4-1 (b), and 35S:miR-ARF (c) leaves.
(D) Bolting shoot of ett-1 arf4-1 plant.
(E) Bolting shoot of 35S:miR-ARF plant.
(F) to (H) Flowers of wild-type (F), ett-1 arf4-1 (G), and 35S:miR-ARF plants (H). Note the gradual increase in the number of sepals, stamen radialization,
and decrease in petal width. sp, sepal; p, petal; st, stamen.
(I) Detection of miR-ARF and miR164 in wild-type and 35S:miR-ARF plants by RNA gel blot analysis. Both miRNAs are the same ;21 nucleotides.
(J) Reduced levels of full-length transcripts of the three ARF genes in 35S:miR-ARF plants.
(K) A scheme of ARF4 cDNA with primers used for RLM-RACE detection. Gel images showing RLM-RACE–detected wild-type and 35S:miR-ARF (miR-
ARF) cleavage products at sites A and B detected using either primer a or b, where A is the expected gel position for a product cleaved at site A, and B is
the expected gel position of product cleaved at site B. In 35S:miR-ARF, amplification products are more prevalent relative to the wild type. In addition,
amplification of a product cleaved at site A could only be obtained with the a primer (gels for PCR products of ARF2 and ARF3 cleavage analysis are
shown in Supplemental Figure 2 online).
(L) Summary of cleavage analysis by direct sequencing of RLM-RACE products (see Supplemental Figure 2 for details) and product cloning of the three
ARF genes. Cleavage analysis for 35S:miR-ARF plants is shown, and the dispersed cleavage products of the wild type are shown in Supplemental
Figure 2 online and in Allen et al. (2005). DS, direct sequencing.
Mutant Phenocopy with MicroRNA1141
Figure 3. Custom-Designed Synthetic miRNA Efficiently and Specifically Codownregulate Non-Native Small RNA Targets.
1142 The Plant Cell
plants are dwarfed with small leaves (Figure 3J). However, in
plants where both NGA1 and miR-NGA were cotransactivated
by CAB3:LhG4 (CAB3?NGA1 and miR-NGAa, Figure 3J), plant
stature and leaf growth resembled that of CAB3?miR-NGAa
plants (a near wild-type phenotype; data not shown), indicating
that the levels of NGA protein from NGA gene overexpression
were efficiently reduced by miR-NGA.
Thus, custom-designed miRNAs that follow a few basic rules
of pairing are capable of simultaneously and efficiently down-
regulating multiple transcripts that are not naturally regulated by
small RNAs and induce morphological changes that match
computer predictions for target specificity.
Arabidopsis Pre-miRNAs Can Effectively Regulate
To investigate the functional conservation of Arabidopsis pre-
miRNA processing in distantly related species, we first intro-
ducedOP:miR164binto tomato,a distantlyrelated plant species
that shows conserved miR164 targets (N. Ori, personal commu-
nication). The progenies of two selected 35S?miR164b lines
had fused cotyledons (Figure 4A), a similar phenotype to the
1996). Thus, misexpression of pre miR164b resulted in uniform,
heritable, and stable lines capable of simultaneous downregu-
lation of multiple members of the same gene family both in
Arabidopsis and in tomato. We also expressed the OP:miR165b
in tomato plants whose homologous target genes are also
conserved (our analysis) to explore whether this functional con-
servation of pre-miRNAs in other species is a general phenom-
enon. The At FILAMENTOUS FLOWER (FIL) promoter drives
expression throughout tomato leaf primordia before becoming
abaxially restricted (Lifschitz et al., 2006). FIL?miR165b plants
had abaxialized, filamentous leaves consistent with miR165b
function in downregulating the adaxial-promoting PHB-like
genes in tomato (Figures 4B and 4C). The abaxial nature of the
filamentous leaves was evident by the prevalence of long linear
trichomes and the absence of short globular ones, a typical
composition of abaxial leaf epidermis (Reinhardt et al., 2005).
Subsequently, we tested whether the synthetic pre miR-ARF
would also function in other species. 35S:miR-ARF tobacco and
tomato transformants exhibited phenotypes consistent with the
specific transformations of abaxial cell types into adaxial ones
observed in 35S:miR-ARF Arabidopsis plants. The leaves of 35S:
miR-ARF tomato plants had small, misshapen, up-curled leaflets
that were darker green and developed outgrowths on their abaxial
surface (Figure 4D). The inflorescence structure approximatedthat
of the wild type, but in the flowers, the number of sepals was
increased, sepals andpetals were narrowerand shorter, andwhite
rather than green carpels were topped by thick, green style/
stigmatic tissue (Figures 4E to 4H). Such redistribution of gynoe-
cium celltypes is similar to single mutations in ett/arf3in Arabidop-
sis, where style tissues are expanded basally (Figures 4L and 4M).
Similarly, 35S:miR-ARF tobacco lines had small, up-curling
leaves that, in more severe lines, developed outgrowths on the
it finally occurred in severe lines, the corolla remained largely
green. Strikingly, very pronounced abaxial outgrowths devel-
oped around the entire corolla circumference, mostly in its distal
third (Figure 4J). However, even in the most severe lines, the
adaxial (inner) side of the corolla surface was unchanged. As in
4K). These results confirm and extend previous suggestions on
the central role of the three ARFs in promotion of abaxial identity
to the remotely related Solanaceae.
To associate the 35S:miR-ARF phenotype of tomato and
tobacco with miR-ARF activity, 59 RLM-RACE was used. As
with Arabidopsis, tomato ARF3/ETT has two presumptive ta-
siRNA target sites and, thus, two miR-ARF target sites (Figure
5A). In 35S:miR-ARF tomato plants, we detected much higher
levels of cleavage at both A and B sites than in wild-type tomato
plants (Figure 5B). Notably, the predominant cleavage position
within the target site was shifted one nucleotide 39 relative to that
observed in Arabidopsis (Figure 5C). These observations sug-
gest that the pre miR-ARF is functionally conserved in tomato. In
tobacco, we only tested miR-ARF activity at the A site of the
ARF3/ETT homolog by cleavage analysis. However, the level of
cleaved transcript detected was again greater than that ob-
served in wild-type tobacco plants and occurred predominantly
at the same position within the target domain that was observed
in tomato (Figures 5B and 5C).
Figure 3. (continued).
(A) A phylogenetic tree of the NGA-like proteins and their closest Arabidopsis homologs. Tree was constructed with the ;120 amino acids that
constitute the B3 domain as shown in Supplemental Figure 3 online. ETT was included as an outgroup, and numbers represent bootstrap percentage
from 1000 trials.
(B) A general scheme of NGA1/2/3/4 transcripts, outlining the position of a consensus sequence aligned. A synthetic miRNA has 0 to 2 mismatches with
all four, and its conceptual dicing from pre miR164b backbone is illustrated. The arrow above pileup denotes cleavage point as described below.
(C) Predicted folding and dicing of the pre miR164a backbone before (left) and after (right) replacement of miR164 with the miR-NGA sequence.
(D) to (F) Young seedling of wild-type (D), nga1-1 nga2-1 nga3-1 nga4-1 quadruple mutant (E), and 35S:miR-NGAa (F) plants. Note the angular leaf
blade of the mutants compared with the round leaf blade of the wild type. cd, cotyledons.
(G) to (I) Inflorescence and pre-anthesis flower (insets) of wild-type (G), nga1-1 nga2-1 nga3-1 nga4-1 quadruple mutant (H), and 35S:miR-NGAa (I)
plants. Note the broad yellowish petals and the protruding distal portion of the unfused gynoecium (gy).
(J) Cotransactivation of NGA1 and miR-NGAa by CAB3 promoter eliminates dwarfism induced by ectopic NGA1 with the same promoter line (inset).
(K) Detection of miR-NGA in wild-type and 35S:miR-NGAa plants by RNA gel blot analysis.
(L) RLM-RACE detection of cleaved products of the four NGA-like transcripts in 35S:miR-NGAa (miR-NGA) plants but not wild-type plants.
(M) RNA gel blot analysis reveals differential reduction in RNA levels of NGA1 and NGA3 in wild-type and 35S:miR-NGAa plants.
Mutant Phenocopy with MicroRNA1143
Use of the 35S:miR-NGAa construct in tomato resulted in a
fruit phenotype, and miR-NGA–mediated cleavage in the tomato
NGA-like transcripts (NGA-like/BI934304 and BI934637) was
detected, albeit at low levels (see Supplemental Figure 3 online).
In this case, it is likely that mismatches in the critical 59 region
of the miR-NGA relative to the target mRNAs obviate efficient
Cell-Autonomous Effects of miRNA Expression
It has been suggested that miRNA activities might not be cell-
autonomous (e.g., short-range movement of miR166 was sug-
gested to account for restriction of the PHB-like activities from
the abaxial side of emerging organ primordial) (Juarez et al.,
2004; Kidner and Martienssen, 2004). The potency of miRNA
target regulation and its efficacy across species allowed exam-
ination of cell autonomy by the different criteria of tissue spec-
ificity and gross systemic spread. To examine the autonomy
of miRNA action, we specifically expressed pre miR166g, pre
miR165a, or pre miR165b in the 2nd and 3rd whorl floral organ
primordia of Arabidopsis using an AP3 driver line (AP3:LhG4;
Figure 6A, Table 1). As expected, in AP3?miR165/6 plants, pet-
als and stamens became completely radialized, a typical mor-
Figure 4. Arabidopsis Pre-miRNA Backbones Induce Homologous Mutant Phenotypes Heterologously.
(A) Wild-type and F1 tomato seedlings of OP:miR164b transactivated by 35S:LhG4. The top and bottom insets are close-ups of the upper portion of the
(B) and (C) Upper part of wild-type (B) and FIL?miR165b (C) tomato shoots. Leaves in (C) are short and radial, and floral organs are narrow. l, leaf; f,
flower; rl, radialized leaf.
(D) Abaxial side of wild-type and 35S:miR-ARF leaves. A close-up of 35S:miR-ARF leaf at the right illustrates the abaxial-specific outgrowths found
along the veins.
(E) to (H) Flowers ([E] and [F]) and carpels ([G] and [H]) of wild-type ([E] and [G]) and 35S:miR-ARF ([F] and [H]) tomato plants. Note the thin sepals and
petals, the short style, and the thickened green stigma of the transformant. st, style; sg, stigma.
(I) to (K) Comparison of leaves (I), flowers (J), and carpels (K) of wild-type (left) and 35S:miR-ARF tobacco plants. Gradual effects from weak to strong
(right) are notable in independent T1 plants. As in tomato, abaxial leaf outgrowths are evident along the veins. Corolla outgrowths are external (abaxial)
(L) and (M) Gynoecium of wild-type (L) and ett-1 (M) Arabidopsis plants. As in tomato and tobacco, stigmatic tissue in the mutant is expanded basally
while style length is reduced.
1144 The Plant Cell
In the upper part of the stamens, guard cells that are typically
restricted to the abaxial connective tissue were present all
around (Figure 6D). In contrast with the 2nd and 3rd whorl
organs, normal gynoecia were formed (Figure 6C), suggesting
autonomous restriction imposed on miR165/6 activity or, alter-
natively, insensitivity of the gynoecium. Thus, we expressed pre
miR165b by the gynoecium-specific promoter of CRABS CLAW
(CRC) that drives expression throughout the gynoecium valve
anlagen before becoming abaxially restricted (Figure 6B). In
CRC?miR165b plants, a thin gynoecium was formed, without
adaxial placenta and ovules, while the stamens and petals were
unaffected (Figure 6E). Thus, expression of pre miR165/6 in cells
of organ primordia bordering other primordia provided no evi-
dence for expanded miRNA activity.
Thestriking phenotypeoftomatoandtobacco plantsexpress-
ing 35S:pre miR-ARF provided an opportunity to phenotypically
assay for systemic translocation of this miRNA. In six out of six
reciprocal grafts between strong 35S:miR-ARF and wild-type
tomato, both rootstock and scion maintained autonomous mor-
phology over 6 months (Figure 6F). Likewise, the dramatic
morphological effects of ectopic miR-ARF expression did not
cross graft barriers in two out of two reciprocal tobacco grafts
between 35S:miR-ARF and the wild type, even though graft
union supported viable scions for over 6 months (Figures 6G to
6I). As long-range movement often follows source to sink flow,
we repeatedly defoliated the wild-type shoots after complete
graft union was obtained in both tomato and tobacco (arrows in
Figures 6F and 6G). After 2 months of such repeated defoliation,
all new emerging leaves and subsequently floral primordia had a
wild-type appearance. Thus, using morphological criteria, no
support for short or long-range translocation of miRNA-induced
alterations was observed.
Pattern formation of plant organs often involves regulation of TF
families by miRNAs. In this study, we have systematically dem-
onstrated that miRNA can simultaneously quench the activity of
their multiple predicted TF targets to levels matched by conven-
tional loss-of-function mutations. However, no morphological
alterations beyond the computational predictions for target
specificity were encountered, suggesting very limited and pre-
cise targeting. The efficient silencing potential was not restricted
miRNA backbones were efficiently processed to induce homol-
ogous loss-of-function phenotypes in distantly related tomato
and tobacco. Finally, we have demonstrated that miRNA activ-
ities can be quantitative, do not cross graft barriers, and at least
for short distances, exert autonomous effects.
Gene Silencing with miRNAs
Functional overlap (redundancy) presents a difficult logistic
obstacle for in vivo characterization of gene function in general
and more so in plants that in many cases have undergone
multiple cycles of polyploidization and subsequent selective
gene loss (Moore and Purugganan, 2005). Here, we show that
miRNAs can potentially abolish simultaneously the activities of
all of their targets to levels matched by conventional loss-of-
function mutations. Endogenous pre-miRNA–encoding genes of
Arabidopsis could be modified to specifically target (sequence
allowing) selected gene families. The range of phenotypic per-
turbations, even at the high levels of ectopic expression, did not
exceed that of corresponding multiple mutant combinations in
Figure 5. Induction of Solanaceae Target Cleavage by Arabidopsis-
Based Synthetic Pre-miRNAs.
(A) Sequence alignment of tomato ARF2/3/4, tobacco ETT/ARF3, and
miR-ARF. The nucleotides not in gray are predicted wobbles.
(B) Gel images showing RLM-RACE detection of tomato and tobacco
ARF3 cleavage products from wild-type and 35S:miR-ARF (miR-ARF)
plants. Primer position follows the ARF4 design in Figure 2K. Cleavage
products at sites A and B were detected using either primer a or b, where
A is the expected gel position for a product cleaved at site A, and B is the
expected gel position of product cleaved at site B.
(C) Cleavage point mapping of tomato and tobacco ARF3. Arrows mark
cleavage sites in sequenced, cloned products, where arrow size corre-
sponds with frequency of clones obtained (also given). Arrows above
each sequence are for clones obtained from the wild type, and those
below are from 35S:miR-ARF plants. The number of clones matching the
predicted target region out of total number of sequenced clones is
shown in parentheses.
Mutant Phenocopy with MicroRNA1145
the predicted target genes, suggesting that off-targets are not
significantly affected. It was also possible to reduce the com-
plementarity of the miRNA to its targets and to produce weaker
of targets by their corresponding miRNA were formulated earlier
(Allen et al., 2005; Schwab et al., 2005). Following these basic
rules, we could custom design synthetic miRNAs that were as
efficient as endogenous ones in simultaneous downregulation of
multiple targets, and all are members of the same gene family.
Using such sequence-specific design, it should be possible to
specifically target unique isoforms generated via alternative
splicing or target multiple homologs ordered in tandem. Such
application might be highly significant for plant genomes; it is
estimated that 29% of the rice genes are organized in clusters of
gene families (International Rice Genome Sequencing Project,
2005) and are not amenable for functional analysis via classical
Most of the miRNAs and gene family targets found in plants
have a long coevolutionary history. Nearly all highly conserved
miRNAs and corresponding gene target sites are identical in
plants (Floyd and Bowman, 2004; Axtell and Bartel, 2005).
Comparison of the pre-miRNA genes between Arabidopsis and
other species shows some conservation beyond the miRNA and
Figure 6. miRNA Activities Are Spatially Restricted and Do Not Cross Graft Joints.
(A) Transverse section of an Arabidopsis flower showing promoter AP3-mediated expression of GFP limited to petals and stamen.
(B) Longitudinal section of same age Arabidopsis flower showing that promoter CRC-mediated expression of GFP is limited to carpels.
(C) Flower of AP3?miR165b plant with radial petals and stamens but normal gynoecium.
(D) Close-up of radial stamens with normally abaxial-restricted guard cells scattered all around (arrows).
(E) Flower of CRC?miR165b plant with thin gynoecium and normal stamens.
(F) A graft of 35S:miR-ARF on wild-type tomato. Picture was taken 4 months after grafting and after two rounds of defoliation (arrows) of wild-type
leaves. Newly initiating shoots remain normal. White box shows graft union.
(G) to (I) A graft of the wild type on 35S:miR-ARF tobacco (G). Here, too, defoliation (arrows) of wild-type leaves did not stimulate miRNA-derived
phenotype on wild-type shoot. The white box shows the graft union. Upon flowering, wild-type acceptor shoots were normal (H) even though 35S:miR-
ARF donor flowers are highly distinct (I).
sp, sepal; p, petal; st, stamen; gy, gynoecium; rp, radial petal; rst, radial stamen. Bars ¼ 50 mm in (A) and (B) and 100 mm in (C) and (D).
1146 The Plant Cell
et al., 2002; Floyd and Bowman, 2004). Using phenotypic and
molecular assays, we found that pre-miRNAs were processed
efficiently when the miRNA sequence and its complement were
substituted in Arabidopsis and that these synthetic Arabidopsis
miRNAs and their native counterparts functioned in distantly
related Solanaceae species. Recent work in mammal cells sug-
gests that the miRNA RISC loading complex processes the pre-
miRNA directly, unwinding and loading the mature miRNA to
Argonaute2 of the RISC (Maniataki and Morelatos, 2005). Thus,
basic, conserved structural elements of the pre-miRNA second-
ary structure may be sufficient for miRNA RISC loading complex
recognition and processing, and these are maintained in the
manipulations we performed.
Endogenous plant miRNAs and the synthetic miRNAs we
tested have a high degree of homology to their targets and
mediated genesuppression primarily throughcleavage. Inmeta-
zoans, miRNA-mediated cleavage also occurs where the miRNA
(Yekta et al., 2004). Using appropriate computational analyses,
this effective and stable mechanism for gene targeting can be
broadly applied in all organisms with miRNA machinery.
RNAi versus miRNA Silencing
stranded RNA strands cleaved to short siRNAs, while miRNAs
are processed from precursors containing mismatches and
bulges. We assume that it is those mismatches that provide
structural information for Dicer activities guiding precise cleav-
the perfect match stems, a mixture of mers is produced unless
phased first by precise guiding cleavage (Allen et al., 2005).
Moreover, even though a single miRNA molecule is generated
per one pre-miRNA, efficient downregulation of multiple targets
with matching sequence homology can be achieved. This result
is in contrast with the long stem-loop RNAi vectors or cosup-
pression lines where downregulation of a specific transcript only
is commonly evident. For example, Chuang and Meyerowitz
of perfect homologies with the homologous CAULIFLOWER
(CAL) gene, no ap1 cal-like plants were observed. Likewise,
RNAi-targeted suppression of ARF2 in Arabidopsis (Li et al.,
2004) and ARF4 in tomato (Jones et al., 2002) resulted in a
specific single mutant phenotype, in contrast with our results.
Keeping in mind that double-stranded RNA is also amplified and
dependent on the activities of RNA-dependent RNA polymerase
(RdRp), stoichiometric effects of miRNAs appear to be much
more potent (Sijen et al., 2001).
Quantitative versus Qualitative Regulation by miRNAs
Most plant miRNAs target TFs that play critical roles in morpho-
genesis. In animals, recent studies suggest that miRNAs and
their targets are expressed in adjacent, largely nonoverlapping
domains through transcriptional control (Stark et al., 2005).
Under this scenario, miRNAs function to confer robustness on
gene expression boundaries. In plants, the evidence of miRNA
function paints a more complex picture. It has been proposed
that miRNAs act to clear cells of their target gene activities as
supported bythe strongdevelopmental phenotypes of dominant
miRNA-resistant mutations (Rhoades et al., 2002). Such a mode
of action is attractive for modeling formation and stabilization of
sharp boundaries between adjacent expression domains. How-
ever, observations of miR165/6 and miR164 expression and
action relative to their respective PHB-like and CUC-like targets
suggest a more quantitative role for the miRNA through damp-
ening down target gene expression levels in cells coexpressing
both (McConnell et al., 2001; Bao et al., 2004; Baker et al., 2005;
Li et al., 2005; Williams et al., 2005a). In both cases of miRNA/
target pairs, increased target levels were observed when either
the target was resistant to its miRNA or when endogenous
miRNAs were reduced by mutation. Since zonation of the plant
apical meristem into central and peripheral domains is an ongo-
domains ofboth CUC-like andPHB-likemultiple familymembers
likely reflects a more fluid regulation of these genes by their
corresponding miRNAs. Similarly, during leaf morphogenesis,
cell position rather than lineage is important until relatively late in
leaf development (reviewed in Scheres, 2001). Therefore, proper
leaf morphogenesis involves a continual interplay between
adaxial-promoting PHB-like and abaxial-promoting ARF2/3/4
TFs, each class with supporting small RNA regulation (miR165/6
and siR2141/2, respectively) to reinforce cell identity. In such a
scenario, the ta-siRNAs and miR165/6 may be coexpressed
process of acquiring and translating positional information.
Our experiments with endogenous pre miR165b, pre miR164b,
and pre miR167a overexpression indicate that plant miRNAs
have the potential to act in a quantitative fashion. High and/or
precise pre-miRNA expression of these miRNAs mimicked mul-
tiple mutants of the target genes, demonstrating that these plant
miRNAs have the potential to clear cells of their target gene
activities. However, weak overexpression elicited mild pheno-
types only, presumably by reducing and not eliminating target
gene function. Thus, it appears that a broad range of plant
miRNAs can act quantitatively, depending on expression levels.
The weak abaxialized phenotype obtained from strong specific
overexpression of the miR165bm6 with reduced homology to its
PHB-like gene transcripts (Figure 1; see Supplemental Figure
1 online) also suggests that miRNA target mismatches are a po-
tential source for inefficiency in miRNA action. A striking feature
of endogenous plant miRNAs is the evolutionary conservation of
miRNA target mismatches, even between the critical 59 region of
the miRNA and its target (Axtell and Bartel, 2005). The synthetic
miR-ARF and miR-NGA we assayed had perfect homology
to target regions in ETT/ARF4 and NGA2, respectively. From
the simple perspective of thermodynamic rules of pairing, this
regulation of these targets. While we cannot say whether perfect
miRNA target homology is the most efficient form of these or any
other miRNAs, our results suggest that perfect-match miRNAs
are as efficient as native ones. If a perfect target match is the
most efficient form of a miRNA, the evolutionary conservation of
mismatches suggests a selection for miRNA inefficiency. One
Mutant Phenocopy with MicroRNA1147
possibility is that an inefficient miRNA, combined with variable
target gene outputs, which can be translated into a morphoge-
netic gradient. Thus, it can be speculated that while plant
miRNAs have the capacity to clear cells of target gene activities,
they act endogenously to both clear cells of their targets and to
dampen their expression, depending on the miRNA and devel-
Local versus Systemic Regulation by miRNA
The quantitative nature of miRNA regulation and the transient
nature of the pattern formation process do not support a role for
long-distance miRNA transport. In agreement, we could not find
any evidence for translocation of miR-ARF effects across graft
barriers in tomato and tobacco. In Arabidopsis, we also failed to
find phenotypic evidence of miRI65a/b ormiR166g movingsmall
cellular distances in the developing flower. Still, a context-
dependent short-range movement (one to two cells) of selected
miRNAs in particular domains cannot be ruled out, a feature
common to other macromolecules shown to traffic from cell to
cell (Gallagher et al., 2004; Kurata et al., 2005). DCL4 activities
are required for cell-to-cell spread of transgene-born silencing
signals(Dunoyer etal.,2005),suggesting thatsiRNAsdo provide
the spreading sequence-based information agent. Significantly,
mutations in DCL4 orin components of the RdRP machinery that
abolish systemic silencing spread do not affect patterning. Thus,
miRNAs and siRNAs might not serve as equal substrates for
trafficking. siRNAs contrast with miRNAs in having a perfect
match with their target mRNA. In this respect, one possible role
for mismatches between miRNAs and their targets is that they
act as a means to avoid the miRNA acting as a template for the
RdRP silencing machinery. The miR-ARF, which has a perfect
match to ARF3/ETT and ARF4 of tomato and ARF3/ETT of to-
bacco, failed to elicit a systemic silencing response, suggesting
that the miRNA/target mismatches may not function in this
process. Thus, regulated expression of synthetic miRNA can
allow for targeting of multiple transcripts for simultaneous sector
The Arabidopsis thaliana plants described are all in the Landsberg erecta
background. Plants were grown under 18 h of cool white fluorescent light
at 208C. M82 tomato (Solanum lycopersicum) and Samsun tobacco
(Nicotiana tabacum) plants were grown in greenhouse conditions with
temperatures ranging between 18 and 258C. The nga1 mutation was
identified in a gymnos kanadi background (Bowman et al., 2002). Map-
based identification of At2g46870 as the gene mutant in nga1 will be
described elsewhere. nga1 has a premature stop codon at Gln-203.
nga2-1 (At3g61970) (a kind gift from Venkatesan Sundaresan) contains a
Ds insertion at þ142 from the first Met codon within the B3 domain-
encoding region. nga3-1 (At1g01030) has a T-DNA insertion (Garlic/
Sail_232_E10) at position þ238 from the first Met codon, likewise within
the B3-encoding domain, and nga4-1 (At4g01500) (also a kind gift from
Venkatesan Sundaresan) has a DS insertion þ112 from the first Met
codon, again within the B3 domain. nga1-1 nga2-1 nga3-1 nga4-1 qua-
druple mutant plants were produced by conventional breeding. In the F2,
at an approximate ratio of 1:256, and their genotype was verified using
PCR (see Supplemental Table 3 online for primer details).
We used a classic wedge-shaped/slit grafting technique with the site of
union wrapped by Parafilm. Plants were kept for 2 to 3 d in the shade and
for 7 d in 80% humidity provided by plastic bags. Success exceeds 90%.
Pre-miRNA Clones and Plant Transformation
For isolation of pre-miRNA–containing sequences, the genomic DNA
flanking the predicted stem-loop of different, annotated pre-miRNAs was
amplified using PCR (see supplemental tables online for primers and
details). Details on the stem-loop and short 59 and 39 ends are summa-
rized in Supplemental Table 1 online. The pre miR165bm6 and pre miR-
NGAa synthetic genes were generated by assembly PCR, as detailed in
NGAb synthetic genes were synthesized by Epoch Biolabs and DNA 2.0,
theWeb-based mfoldprogram (Zuker,2003). Aftersequenceverification,
the pre-miRNA clones were cloned behind an OP array (10OP-TATA-
BJ36) or 35S promoter (ART7) and transferred into the binary pMLBART
(for Arabidopsis) or pART27 (for tomato and tobacco) vectors (Eshed
et al., 2001). A 6-kb PHB promoter and a 6.1-kb At FIL promoter were
PCR amplified with the primers in Supplemental Table 2 online. FIL, PHB,
and 35S promoters were subcloned in front of LhG4 (Moore et al., 1998)
and subsequently cloned into the pART27 or pMLBART binary vector.
Other transactivation driver lines were described earlier (Pekker et al.,
2005). Cotyledon transformation in tomato and leaf disc transformationin
tobacco were performed according to McCormick (1991) and Horsch
the floral dip method, and BASTA-resistant transformants were selected
Evaluation of Transgenic Lines
Two types of evaluation of transgenic lines were performed. Primary
35S:miRNA T1 lines were scored directly as having a strong, intermedi-
ate, or weak phenotype. Selected 2 to 10 lines (fertility allowing) were
backcrossed (BC) with the wild type, and a minimum of 30 BC1 progeny
were scored to assay phenotype severity and heritability. Unless other-
wise mentioned, originalphenotypes were faithfully transmitted, and only
lines with a single T-DNA insert locus were used for molecular analyses.
transformants, 10 to 30 T1 plants were crossed to homozygous promoter:
LhG4 lines, and a minimum of 30 promoter?miRNA F1 plants per cross
were scored. Eachprimary linewasdesignated asstrong, intermediate, or
weak on the basis of the morphology of the F1 progenies. Only 10OP:pre-
miRNA lines with single, unlinked T-DNA insert loci were used for further
experiments. In all cases, unless otherwise mentioned, strong lines were
used for morphological and molecular characterizations.
RNA Isolation and Analysis
Total RNA was extracted using TRI Reagent (Sigma-Aldrich)according to
the manufacturer’s instructions. High molecular weight RNA was nor-
malized by spectrophotometry to 20 mg/lane. Radiolabeled probes for
Probes consisted of the 39 (to the stop codon) 1049 bp (ETT), 1497 bp
(ARF4), 1459 bp (ARF2), 617 bp (NGA1), and 828 bp (NGA3) of these
1148 The Plant Cell
genes annotated cDNAs. Equivalent loading of samples was monitored
by detection of 28S and 18S RNA in all gels prior to blot transfer.
Low molecular weight RNA was purified with an RNeasy plant mini kit
(Qiagen), resolved on a 17% polyacrylamide-urea gel, transferred to a
Zeta-Probe GT membrane (Bio-Rad), and probed with a32P end-labeled
oligonucleotide, complementary to the mature miRNAs (see Supplemen-
talTable4online).Equivalentloading ofsampleswas shownbydetection
of 5S RNA in all gels prior to blot transfer.
Isolation of Tomato and Tobacco cDNAs
The complete coding sequences of the ARF2, ARF3/ETT, and ARF4
genes of tomato were cloned using primers designed from the alignment
of different tomato EST sequences in GenBank. A partial ARF3/ETT gene
of N. tabacum was isolated using homology with the tomato ARF3/ETT
gene (see supplemental tables online for primer details). Young leaves,
apices, and flowers were harvested, and total mRNA was extracted from
vegetative and reproductive shoot tips using Trizol reagent. cDNA tem-
plate was synthesized from 1 mg of total RNA using Superscript II reverse
transcriptase (Invitrogen) or PowerScript RT enzyme (Clontech).
RACE Analysis of Cleaved miRNA Target Genes
Cleavage sites in the miRNA target genes were mapped using RLM-
RACE, a modified 59 RACE procedure as described by Kasschau et al.
(2003), using the GeneRacer (Invitrogen) protocol coupled with nested
gene-specific primers ;200 to 400 nucleotides downstream of the
predicted miRNA target site (see Supplemental Table 5 online). The
PCR products werepurified and directly sequenced or cloned into pDrive
(Qiagen) or pCRII (Sigma-Aldrich) and sequenced.
Microscopy and Confocal Imaging
Tissue preparation and histological analyses were performed according
to Pekker et al. (2005). Scanning electron microscopy was performed
using an XL30 ESEM FEG microscope (FEI). For confocal imaging, tissue
Fluorescence was observed by an Olympus CLSM500 microscope
with an argon laser at 488 nm for excitation and 505 to 525 nm for GFP
Tomato ARF sequence data from this article can be found in the
GenBank/EMBL data libraries under accession numbers DQ340254 (Sl
for tobacco are DQ340258 (Nt ARF2), and DQ340256 and DQ340257 (Nt
The following materials are available in the online version of this article.
Supplemental Table 1. Sequence Parameters of Pre-miRNA.
Supplemental Table 2. Primers for PCR-Mediated Cloning.
Supplemental Table 3. Primers for PCR-Mediated Genotyping of
NGATHA2-4 Insertion Alleles.
Supplemental Table 4. Oligonucleotides for miRNA Detection by
RNA Gel Blot Analysis and Primers for RLM-RACE.
Supplemental Table 5. Gene-Specific Primers for RLM-RACE.
Supplemental Figure 1. Design of and Additional Phenotypes
Induced by miR165bm6.
Supplemental Figure 2. Analysis of Cleavage Products of Arabidop-
sis ARF Genes.
Supplemental Figure 3. NGA-Like Gene Alignment and Cleavage
Analysis in Tomato.
We thank Eugenia Klein and the electron microscopy facility for help
with scanning electron microscopy, Raya Eilam for help with tissue
preparation techniques, and Vladimir Kiss for assistance with confocal
laser scanning microscopy. We also thank Venkatesan Sundaresan,
Michael Lenhard, the SIGNAL collection of Syngenta Seeds, and the
ABRC stock center for providing plasmids and plant material. We
thank John Bowman, Robert Fluhr, Naomi Ori, and members of Y.E.’s
lab for comments and discussions and Detlef Weigel for sharing
unpublished results. This work was made possible with funding from
Grants 3328-02 from the U.S.–Israel Binational Agricultural Research
and Development Fund and 386-02 from the Israel Science Foundation.
J.P.A. is an honorary research fellow of the School of Biological
Sciences (Monash University). Y.E. is an incumbent of the Judith and
Martin Freedman Career Development Chair.
Received December 25, 2005; revised March 10, 2006; accepted March
20, 2006; published April 7, 2006.
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