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The Plant Cell, Vol. 28: 272–285, February 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.
REVIEW
RNAi in Plants: An Argonaute-Centered View
Xiaofeng Fanga,b and Yijun Qia,b,1
a Center
for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China
Center for Life Sciences, Beijing 100084, China
b Tsinghua-Peking
Argonaute (AGO) family proteins are effectors of RNAi in eukaryotes. AGOs bind small RNAs and use them as guides to
silence target genes or transposable elements at the transcriptional or posttranscriptional level. Eukaryotic AGO proteins
share common structural and biochemical properties and function through conserved core mechanisms in RNAi pathways,
yet plant AGOs have evolved specialized and diversified functions. This Review covers the general features of AGO proteins
and highlights recent progress toward our understanding of the mechanisms and functions of plant AGOs.
INTRODUCTION
RNAi refers to a conserved gene silencing process mediated by
small RNAs (sRNAs). It has emerged as one of the most important
mechanisms in regulating gene expression and repressing transposable elements (TEs) at the transcriptional or posttranscriptional
level in eukaryotes.
sRNAs are processed from stem-loop-structured or perfect long
double-stranded RNAs by Dicer or DICER-LIKE (DCL) proteins. In
plants, several sRNA species that differ in biogenesis and functions
have been characterized. The predominant sRNA species include
microRNAs (miRNAs) generated by DCL1 (Reinhart et al., 2002;
Kurihara and Watanabe, 2004; Qi et al., 2005), trans-acting small
interfering RNAs (ta-siRNAs) processed by DCL4 (Peragine et al.,
2004; Vazquez et al., 2004; Gasciolli et al., 2005; Xie et al., 2005), and
heterochromatic siRNAs (hc-siRNAs) produced by DCL3 (Xie et al.,
2004; Qi et al., 2005; Henderson et al., 2006). Additional types of
sRNAs including natural antisense siRNAs (Borsani et al., 2005), long
siRNAs (Pontes et al., 2006), long miRNAs (lmiRNAs) (Wu et al.,
2010), double-strand-break (DSB)-induced sRNAs (diRNAs) (Wei
et al., 2012), and DCL-independent siRNA (sidRNAs) (Ye et al., 2016)
have also been discovered. While miRNAs, ta-siRNAs, and natsiRNAs mediate posttranscriptional gene silencing (PTGS),
hc-siRNAs, lmiRNAs, and sidRNAs direct DNA methylation thus
inducing transcriptional gene silencing (TGS).
Despite their diversity, all characterized sRNAs associate with
ARGONAUTE (AGO) family proteins to form the core of RNAinduced silencing complexes (RISCs). sRNAs function as guides
to direct RISCs to RNA or DNA targets through base-pairing
(Peters and Meister, 2007; Vaucheret, 2008). Eukaryotic AGOs can
be phylogenetically classified into two major groups: AGO and
PIWI subfamilies (Carmell et al., 2002; Hutvagner and Simard,
2008). AGO subfamily proteins are present in a wide range of
organisms with varied gene copy numbers, whereas PIWI subfamily proteins are absent in plants (Cerutti and Casas-Mollano,
2006).
1 Address
correspondence to [email protected].
www.plantcell.org/cgi/doi/10.1105/tpc.15.00920
In plants, the roles of AGOs in PTGS and TGS have been well
established. Recent studies have revealed new functions of plant
AGO proteins and provided further insights into the mechanisms
of AGO action. In this Review, we summarize the general features
of AGOs and mechanisms of RISC assembly and action, and
discuss recent progress in understanding the roles of AGOs in
plants.
ORIGIN AND DIVERSITY OF AGO FAMILY PROTEINS
The AGO family was named after AGO1 in Arabidopsis thaliana, loss
of which leads to tubular shaped leaves resembling small squids
(Argonautus) (Bohmert et al., 1998). AGOs were later found to be
present in bacteria, archaea, and eukaryotes, suggesting that they
have an ancient origin (Cerutti and Casas-Mollano, 2006). The
number of AGO family members in different species varies greatly:
There is only one AGO protein in fission yeast (Schizosaccharomyces
pombe), five in fly (Drosophila melanogaster), eight in humans
(Homo sapiens), and 27 in worm (Caenorhabditis elegans). In plants,
the AGO family has expanded during evolution. The green algae
Chlamydomonas reinhardtii has three AGOs (Schroda, 2006; Zhao
et al., 2007), while moss (Physcomitrella patens) has six (Axtell et al.,
2007; Arif et al., 2013). The number of AGOs further increases in
flowering plants: 10 in Arabidopsis (Carmell et al., 2002; Morel et al.,
2002), 15 in poplar (Populus trichocarpa) (Zhao et al., 2015), 17 in
maize (Zea mays) (Qian et al., 2011; Zhai et al., 2014), and 19 in rice
(Oryza sativa) (Kapoor et al., 2008). Based on phylogenetic relationships, plant AGO proteins can be grouped into three major
clades named after Arabidopsis AGOs: AGO1/5/10, AGO2/3/7, and
AGO4/6/8/9 (Figure 1). It is noteworthy that rice and maize have
evolved an AGO18 subclade that falls into the AGO1/5/10 clade.
STRUCTURAL FEATURES OF AGO FAMILY PROTEINS
Our initial structural insights into AGO proteins came from crystal
structures of domains of eukaryotic AGOs or entire prokaryotic
AGOs (Hall, 2005; Jinek and Doudna, 2009). In the past few years,
high-resolution structures of human Ago2 (Elkayam et al., 2012;
Schirle and MacRae, 2012) and budding yeast (Kluyveromyces
Mechanisms and Functions of Plant Argonautes
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Figure 1. Phylogenetic Analysis of Plant AGO Family Proteins.
The protein sequences of selected AGOs were obtained from JGI Phytozome (http://phytozome.jgi.doe.gov) and aligned using ClustalW. The neighborjoining tree was constructed using the MEGA6.0 software. Bootstrap values (10,000 replicates) are presented near each branch. Abbreviations for selected
species are as follows: Physcomitrella patens, Pp; Arabidopsis thaliana, At; Populus trichocarpa, Pt; Zea mays, Zm; Oryza sativa, Os.
polysporus) Ago (Nakanishi et al., 2012) with guide RNA were
solved, greatly deepening our understanding about the structures
and functions of eukaryotic AGOs.
All eukaryotic AGOs are characterized by the presence of four
domains: a variable N-terminal (N) domain and conserved PAZ
(PIWI-ARGONAUTE-ZWILLE), MID (middle), and PIWI domains
(Tolia and Joshua-Tor, 2007). The function of the N domain is less
understood. Structural analysis of Thermus thermophilus Ago
suggests that the N domain may block the propagation of guidetarget pairing beyond position 16 (Wang et al., 2009). In vitro
biochemical studies suggest that the N domain of human Agos is
required for sRNA duplex unwinding (Kwak and Tomari, 2012) and
important for cleavage activity (also known as slicer activity)
(Hauptmann et al., 2013). It remains elusive how the N domain
contributes to these activities. The PAZ domain harbors an OB
(oligonucleotide/oligosaccharide binding) fold that enables AGO
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proteins to bind single-stranded nucleic acids (Lingel et al., 2003;
Song et al., 2003; Yan et al., 2003). By bending the 39 end of the
guide strand into a specific binding pocket, the PAZ domain binds
and anchors sRNAs (Lingel et al., 2004; Ma et al., 2004). In addition, the PAZ domain contributes to slicer-independent unwinding of the duplexes (Gu et al., 2012). A rigid loop in the MID
domain, termed the nucleotide specificity loop, specifically recognizes the 59 nucleotide of sRNAs (Frank et al., 2010, 2012),
accounting for the binding preferences of different AGO proteins
for sRNAs with different 59-nucleotides. The MID-PIWI interface
contains a basic binding pocket that helps to bind and anchor the
59 phosphate of sRNAs (Parker et al., 2005). The PIWI domain
adopts an RNase H-like fold and enables some, but not all, AGO
proteins to cleave target RNAs complementary to the bound
sRNAs (Song et al., 2004; Rivas et al., 2005). A catalytic triad (AspAsp-His/Asp, DDH/D) is generally thought to be responsible for
slicer activity (Liu et al., 2004; Song et al., 2004; Rivas et al., 2005).
More recently, structural analysis with budding yeast Ago suggests that slicer activity requires a catalytic tetrad with an additional invariant Glu, rather than a triad (Nakanishi et al., 2012).
To date, no crystal structures of entire plant AGOs are available.
Nevertheless, given that all solved eukaryotic AGO structures are
highly similar, and crystal structures of the MID domains from
Arabidopsis AGOs resemble that of human Ago2 (Frank et al.,
2010, 2012), it seems reasonable to base our understanding of
plant AGO structures on solved structures of other eukaryotic
AGOs. Moreover, intensive analyses of Arabidopsis ago mutants
have also provided important insights into the structures and
functions of plant AGOs (Poulsen et al., 2013).
LOADING OF SMALL RNAs INTO AGO FAMILY PROTEINS
AGO proteins accommodate different sRNA species to perform
specialized functions. The majority of sRNAs, including miRNAs
and siRNAs, are produced as duplexes from precursors by Dicer
or DCL proteins and subsequently loaded onto AGO proteins. The
so-called passenger strand of the duplex is selectively displaced
and degraded, while the guide strand is retained to form the
mature RISC.
Earlier biochemical studies in fly demonstrated that Dicer-2
(Dcr-2) and its partner protein R2D2 are involved in loading of
sRNAs into Ago2 (Liu et al., 2003; Lee et al., 2004; Pham et al.,
2004; Tomari et al., 2004b). The Dcr-2-R2D2 heterodimer is also
capable of sensing the thermodynamic asymmetry of sRNA duplexes, thereby determining which strand is to be retained in the
mature RISC (Tomari et al., 2004a). However, Dicer and its partner
proteins are not essential for the assembly of fly Ago1-RISC
(Kawamata et al., 2009) and human Ago2-RISC (Kanellopoulou
et al., 2005; Murchison et al., 2005; Betancur and Tomari, 2012). In
Arabidopsis, the DCL1 partner protein HYPONASTIC LEAVES1
contributes to strand selection from miRNA duplexes by AGO1
(Eamens et al., 2009; Manavella et al., 2012), the mechanism of
which remains to be determined.
There is a growing body of evidence indicating that the molecular chaperone heat shock protein 90 (HSP90) and its
cochaperones participate in sRNA loading onto AGO proteins.
HSP90 binds to a set of folded proteins and undergoes conformational changes to promote ATP hydrolysis (Röhl et al., 2013). In
fly, the HSC70/HSP90 chaperone machinery associates with
Ago1 and Ago2 and is required for the incorporation of sRNAs into
RISCs (Iwasaki et al., 2010; Miyoshi et al., 2010; Iwasaki et al.,
2015). It has been proposed that the chaperone machinery
supports a loading-competent state of fly Ago2, allowing it to
accept siRNA duplexes from Dcr-2-R2D2 (Miyoshi et al., 2010;
Iwasaki et al., 2015). In plants, studies using tobacco BY-2 lysate
demonstrated that HSP90 binds to Arabidopsis AGO1 and AGO4
and promotes the loading of sRNA duplexes (Iki et al., 2010; Ye
et al., 2012). The cochaperones of HSP90 are capable of modulating the ATP-hydrolyzing rate of HSP90, recruiting client proteins, or playing chaperone roles on their own (Röhl et al., 2013).
Biochemical studies in plants revealed that the cochaperone
Cyclophilin 40/SQUINT (CYP40/SQN) in tobacco and Arabidopsis
associates with AGO1 in an HSP90-dependent manner and facilitates RISC assembly (Earley and Poethig, 2011; Iki et al., 2012).
In line with this, inactivation of SQN impairs miRNA activity in
Arabidopsis (Smith et al., 2009).
Loading of sRNAs into AGOs can be a regulatory point for their
activity. A genetic screen identified SAD2/EMA1, an importin
b family member originally implicated in nuclear import of proteins,
as a negative regulator of miRNA loading into AGO1 in Arabidopsis
(Wang et al., 2011a). However, it remains to be determined whether
SAD2/EMA1 directly prevents the loading of miRNAs or indirectly
controls miRNA loading through regulating the cytoplasmic- nuclear
shuttling of proteins with roles in miRNA loading.
For proper functioning, sRNAs need to be correctly sorted into
specific AGO complexes. In fly, the structure of the duplexes
appears to be a major determinant for sorting of miRNAs and
siRNAs into Ago1 and Ago2, respectively (Tomari et al., 2007;
Czech et al., 2009). In plants, the identity of the 59 nucleotide plays
a key role in sorting of an sRNA into a specific AGO (Mi et al., 2008;
Montgomery et al., 2008; Takeda et al., 2008). Arabidopsis AGO1
preferentially binds miRNAs with a 59 U, AGO2 favors siRNAs with
a 59 A, AGO5 has a bias toward sRNAs with a 59 C, whereas AGO4
primarily associates with hc-siRNAs bearing a 59 A (Mi et al., 2008;
Montgomery et al., 2008; Takeda et al., 2008).
Arabidopsis miR390 and miR165/166 have 59 A and 59 U, respectively. However, they are preferentially recruited by AGO7
and AGO10, respectively, suggesting that additional mechanisms
exist to regulate sRNA sorting (Montgomery et al., 2008; Zhu et al.,
2011). It has been shown that the 59 U is necessary but not sufficient for miR166 loading into AGO10 (Zhu et al., 2011), and it
appears that specific mispairings and pairings in the miR166/
miR166* duplex determine its predominant association with
AGO10 (Zhu et al., 2011). In vitro experiments revealed that the
59 A, the central mismatch within the miRNA duplex as well as
cleavage-dependent removal of miR390* strand collectively determines the binding preference of AGO7 for miR390 (Endo et al.,
2013). Recently, it was found that miRNA duplex structure also
contributes to sorting of certain miRNAs into AGO1 or AGO2 in
Arabidopsis and both the QF-V motif and DDDE catalytic tetrad
within the PIWI domain are important for the recognition of the
structure (Zhang et al., 2014).
In rice, in addition to 21-nucleotide canonical miRNAs (cmiRNAs) that are generated by DCL1, there is a class of 24-nucleotide
lmiRNAs that are processed by DCL3 (Wu et al., 2010). Most of the
cmiRNAs initiate with 59 U and are incorporated into AGO1 clade
Mechanisms and Functions of Plant Argonautes
proteins (Wu et al., 2009), following the 59 nucleotide-directed
loading rule. However, regardless of the identities of their 59 nucleotides, DCL3-dependent lmiRNAs are recruited by AGO4 clade
proteins. The specificity of sRNA sorting between AGO1 and AGO4
clade proteins is unlikely to be determined by the length of the
sRNAs, as both AGO1 and AGO4 clade proteins have comparable
binding affinities for 21- and 24-nucleotide sRNAs (Wu et al., 2010).
These observations suggest that the enzymatic machinery underlying the biogenesis of cmiRNAs and lmiRNAs could dominate
over the 59 terminal nucleotide to funnel their respective products
into AGO1 and AGO4 clade proteins, respectively.
These studies collectively suggest that the specificity of sRNA
sorting into AGOs is determined by the identity of 59 nucleotide
and the structure of the sRNA duplex as well as a signal emanating
from the upstream biogenesis machinery. The mechanisms underlying the recognition of sRNA duplex structure and biogenesis
machinery by AGOs and/or loading factors remain to be investigated.
ACTIONS OF PLANT AGO COMPLEXES
AGO/sRNA complexes have different modes of action (Figure 2).
Their functions in gene regulation at the posttranscriptional level
through endonucleolytic cleavage and/or translational inhibition and
at the transcriptional level through directing DNA methylation have
been well established (Law and Jacobsen, 2010; Rogers and Chen,
2013). More recent studies have revealed that some AGOs have
additional functions. These include Arabidopsis AGO10 and rice
AGO18 that act as decoys of specific miRNAs (Zhu et al., 2011; Wu
et al., 2015) and AGO2 that mediates DSB repair (Wei et al., 2012; Ba
and Qi, 2013). Below we discuss the actions and mechanisms of
AGOs in association with different classes of sRNAs.
Endonucleolytic Cleavage and Translational Inhibition
AGO1 (Baumberger and Baulcombe, 2005; Qi et al., 2005), AGO2
(Carbonell et al., 2012), AGO4 (Qi et al., 2006), AGO7 (Montgomery
et al., 2008), and AGO10 (Ji et al., 2011; Zhu et al., 2011) have been
demonstrated to have slicer activity that catalyzes sRNA-directed
endonucleolytic cleavage of target RNAs. AGO1 is one of the bestcharacterized AGO family members in plants. AGO1 predominantly
binds miRNAs (Baumberger and Baulcombe, 2005; Qi et al., 2005).
Early studies showed that plant miRNAs display a high degree of
sequence complementarity to their target mRNAs and direct
target mRNA cleavage (Llave et al., 2002; Rhoades et al., 2002;
Tang et al., 2003) whereas animal miRNAs are partially complementary to their targets and regulate gene expression via translational inhibition and/or mRNA destabilization (Lewis et al., 2003;
Humphreys et al., 2005; Pillai et al., 2005). This led to the assumption that the degree of complementarity between an sRNA
and its target serves as a switch between mRNA cleavage and
translational inhibition. However, later studies showed that many
plant miRNAs are capable of repressing translation (Aukerman
and Sakai, 2003; Chen, 2004; Gandikota et al., 2007; Brodersen
et al., 2008; Li et al., 2013), arguing against this assumption.
The mechanism underlying the choice between mRNA cleavage and translational inhibition by AGO1-miRISC remains to be
elucidated. Emerging evidence suggests that the subcellular
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compartmentalization of AGO1-miRISC may play a role in such
choice. Genetic studies have implicated a role for the microtubule
network (Brodersen et al., 2008) and processing (P) bodies
(Brodersen et al., 2008; Yang et al., 2012) in miRNA-mediated
translational repression. More recently, an elegant study showed
that miRNAs inhibit translation of target mRNAs on the endoplasmic
reticulum (Li et al., 2013). Altered Meristem Program1 (AMP1), an
integral membrane protein that associates with AGO1, is required for
miRNA-mediated translational repression (Li et al., 2013). Intriguingly, these components that are required for translational repression are dispensable for miRNA-directed target mRNA cleavage
(Brodersen et al., 2008; Yang et al., 2012; Li et al., 2013), raising the
possibility that these two processes take place in different cellular
compartments. The deficiency of isoprenoids synthesis, which is
essential for a variety of pathways including the production of
membrane sterols, impairs miRNA-mediated cleavage of at least
some target transcripts, suggesting that membrane association of
AGO1 is important for mRNA cleavage (Brodersen et al., 2012). The
identity of the subcellular compartment where miRNA-mediated
RNA cleavage occurs remains to be revealed.
Sequestration
Sequestration has emerged as a common scenario of gene
regulation in eukaryotes. For instance, noncoding RNAs can act as
decoys to sequester miRNAs and downregulate their activities
(Franco-Zorrilla et al., 2007).
Two plant AGO proteins, AGO10 in Arabidopsis and AGO18 in
rice, have been demonstrated to compete with AGO1 for specific
miRNAs and abrogate their activities through sequestration (Zhu
et al., 2011; Wu et al., 2015). Arabidopsis AGO10 predominantly
binds miR165/166, thereby preventing their loading into AGO1
and the subsequent repression of HD-ZIP III, targets of miR165/
166 (Zhu et al., 2011). Rice AGO18 is induced by virus infection and
preferentially associates with miR168 that targets AGO1 (Wu et al.,
2015). This association diminishes miR168-directed cleavage of
AGO1, thus increasing the levels of AGO1 accumulation and
antiviral defense (Wu et al., 2015). In agreement with their roles as
decoys for miRNAs, sRNA binding but not slicing activity is required for the functions of AGO10 in Arabidopsis shoot apical
meristem development and AGO18 in rice antiviral defense (Zhu
et al., 2011; Wu et al., 2015). These findings suggest that Arabidopsis
AGO10-bound miR165/166 and rice AGO18-bound miR168 are not
proficient in cleaving target mRNAs, although both AGOs contain the
catalytic triad for slicing activity. This raises an interesting question as
to how their catalytic activities are repressed when they act as
decoys for miRNAs. Several possibilities have been envisioned: (1)
the intrinsic catalytic activities of Arabidopsis AGO10 and rice
AGO18 are weaker than AGO1; (2) the catalytic activities are inhibited
by yet to be identified interacting proteins; and (3) miRNAs bound by
these two AGOs are different from those bound by AGO1 in subcellular compartmentalization, preventing their access to target
mRNAs (Zhu et al., 2011; Wu et al., 2015).
DNA Methylation
DNA methylation is an epigenetic modification that plays critical
roles in regulating gene expression, repressing transposons, and
maintaining genome stability. In plants, DNA methylation can be
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Figure 2. Modes of Action of Representative Plant AGO Proteins.
(A) AGO1 binds miRNAs or ta-siRNAs and cleaves target mRNAs.
(B) AGO1 binds miRNAs and inhibits the translation of target mRNAs.
(C) Arabidopsis AGO10 and rice AGO18 act as decoys for miR165/166 and miR168, respectively.
(D) AGO4 binds hc-siRNAs or lmiRNAs and mediates DNA methylation.
(E) AGO2 binds diRNAs and mediates DSB repair.
mediated by AGO4-bound hc-siRNAs through a specialized RNAi
pathway termed RNA-directed DNA methylation (RdDM). RdDM
entails the generation of double-stranded RNAs (dsRNAs) by
coordinated activities of RNA Polymerase IV (Pol IV) and RNAdependent RNA polymerase 2 (RDR2) (Xie et al., 2004; Herr et al.,
2005; Kanno et al., 2005; Onodera et al., 2005; Li et al., 2008; He
et al., 2009; Haag et al., 2012). dsRNAs are then processed by
DCL3 to produce 24-nucleotide hc-siRNAs (Xie et al., 2004).
Recent studies suggest that Pol IV/RDR2 products are mainly 30
to ;40 nucleotides in length and on average only one siRNA could
be produced from each precursor (Blevins et al., 2015; Zhai et al.,
2015b). hc-siRNAs are exported into the cytoplasm, where they
are loaded into AGO4, and mature AGO4/siRNA complexes are
selectively transported into the nucleus, which may provide
a regulatory point prior to the effector stage of RdDM (Qi et al.,
2006; Ye et al., 2012). The AGO4/siRNA complexes are recruited
to target loci via base-pairing with scaffold transcripts generated
by RNA polymerase Pol V (Pontier et al., 2005; Mosher et al., 2008;
Wierzbicki et al., 2008, 2009). The recruitment of AGO4 to chromatin may also be facilitated by its interaction with the GW/WGrich extensions of NRPE1 (the largest subunit of Pol V) (Li et al.,
2006; Pontes et al., 2006; El-Shami et al., 2007; Li et al., 2008) and
KTF1/SPT5L (a transcription elongation factor) (Bies-Etheve et al.,
2009; He et al., 2009). The conserved GW/WG motif, defining an
“Ago hook,” is widely present in factors implicated in AGO actions
(Azevedo et al., 2011; Garcia et al., 2012; Pontier et al., 2012).
AGO4/siRNA complexes further recruit Domain Rearranged
Methyltransferase 2 (DRM2), a key de novo methyltransferase, to
methylate target DNA (Cao and Jacobsen, 2002; Zhong et al.,
2014).
In addition to 24-nucleotide hc-siRNAs, RDR6-dependent
21-nucleotide siRNAs that are produced from TAS loci can also be
recruited by AGO4 to direct DNA methylation at TAS loci in
Arabidopsis (Wu et al., 2012). In rice, 24-nucleotide lmiRNAs
generated by coordinated actions of DCL1 and DCL3 are associated with AGO4 to direct DNA methylation in trans at the miRNA
target genes, resulting in TGS in some cases (Wu et al., 2010). In
addition to AGO4 that functions in the canonical RdDM pathway,
AGO2 and AGO6 can also mediate de novo DNA methylation by
recruiting 21- to 22-nucleotide siRNAs, which are processed from
Pol II/RDR6-dependent dsRNAs by DCL2 and DCL4 (Pontier et al.,
2012; Marí-Ordóñez et al., 2013; Nuthikattu et al., 2013).
Recently, a class of sidRNAs has been identified in Arabidopsis
(Ye et al., 2016). These sidRNAs are present as ladders of ;20 to
60 nucleotides in length, associated with AGO4, and capable of
directing DNA methylation. The production of sidRNA is dependent on distributive 39-59 exonucleases. It was proposed that
sidRNAs are the initial triggers of de novo DNA methylation (Ye
et al., 2016).
DSB Repair
DSB is the most deleterious form of DNA damage and poses
a major threat to genome stability if not properly repaired (Ciccia
and Elledge, 2010). A class of ;21-nucleotide diRNAs were found
to play an essential role in DSB repair in Arabidopsis and
Mechanisms and Functions of Plant Argonautes
mammalian cells (Wei et al., 2012; Gao et al., 2014). In Arabidopsis,
diRNAs are produced from the vicinity of DSB sites in Pol
IV-dependent manner. However, diRNAs are recruited by AGO2
and do not mediate DSB repair through directing DNA methylation
at the DSB sites because DRM2 is dispensable for DSB repair (Wei
et al., 2012). In humans, the diRNA effector protein Ago2 forms
a complex with the repair protein Rad51 and is required for the
recruitment of Rad51 to DSB sites, suggesting that guided by
diRNAs, Ago2 promotes Rad51 accumulation at DSBs to facilitate
repair (Gao et al., 2014). Whether the AGO2/diRNA complexes in
Arabidopsis function through similar mechanism remains to be
investigated.
BIOLOGICAL ROLES OF PLANT AGO PROTEINS
The AGO family has expanded during plant evolution (Singh et al.,
2015), leading to the functional diversification of plant AGOs (Table 1).
Specialization of AGOs in different sRNA pathways and biological
processes can be attributed to their intrinsic biochemical properties, spatiotemporal expression patterns, and protein and/or
sRNA partners with which they interact.
AGO1/5/10 Clade
AGO1 is the effector protein for miRNAs and ta-siRNAs (Vaucheret
et al., 2004; Baumberger and Baulcombe, 2005; Qi et al., 2005; Mi
et al., 2008). Guided by miRNAs and ta-siRNAs, AGO1 regulates
the expression of genes that are involved in numerous developmental and physiological processes (Fei et al., 2013; Rogers
and Chen, 2013). AGO1 also functions in defense against some
viruses upon loading with virus-derived siRNAs (vsiRNAs) (Morel
et al., 2002; Zhang et al., 2006; Qu et al., 2008; Takeda et al., 2008;
Wang et al., 2011b).
Rice contains four AGO1 homologs (AGO1a, AGO1b, AGO1c,
and AGO1d). Knockdown of these AGO1s led to pleiotropic developmental phenotypes in rice (Wu et al., 2009), due to the abrogation of miRNA activity. Profiling of sRNAs associated with
AGO1a, AGO1b, and AGO1c revealed that most miRNAs are
evenly sorted into AGO1a, AGO1b, and AGO1d, whereas a subset
of miRNAs are preferentially incorporated into or excluded from
one of the AGO1s (Wu et al., 2009), suggesting both redundant and
specialized functions for AGO1s in recruiting miRNAs. Rice
AGO1s also bind vsiRNAs and confer resistance to viral diseases
(Wu et al., 2015).
Arabidopsis AGO10 (previously known as ZLL or PNH) and
AGO1 are highly similar in their protein sequences but differ in their
expression patterns and developmental functions. In contrast to
the ubiquitous expression of AGO1, AGO10 is expressed in
provasculature, adaxial leaf primordia, and the meristem
(Moussian et al., 1998; Lynn et al., 1999). AGO10 plays important
roles in the maintenance of undifferentiated stem cells of shoot
apical meristem (SAM) (Moussian et al., 1998; Lynn et al., 1999)
and the establishment of leaf polarity (Liu et al., 2009). As mentioned above, AGO10 specifically sequesters miR165/166 and
abrogates their activities (Zhu et al., 2011). A recent study showed
that AGO10 quenches the activity of miR165/166 that moves into
AGO10-expressing zone, thus releasing the expression of HD-ZIP
III family genes to regulate meristem development (Zhou et al.,
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2015). In addition to sequestering miR165/166, AGO10 may
regulate meristem development through mediating the translational inhibition of multiple miRNA target genes (Brodersen et al.,
2008; Mallory et al., 2009) and act partly through miR172 to
promote floral determinacy (Ji et al., 2011). AGO10 can also bind
vsiRNAs and cooperate with AGO1 to have modest antiviral effect
in inflorescences (Garcia-Ruiz et al., 2015).
Rice has one homolog of AGO10 that is strongly expressed in
the provascular region of the leaf primordia around SAM and plays
an important role in rice SAM maintenance and leaf development
(Nishimura et al., 2002), suggesting a conserved function between
Arabidopsis and rice AGO10s. It remains to be examined whether
rice AGO10 also functions as a miRNA decoy.
The expression of Arabidopsis AGO5 is confined to the somatic
cells around megaspore mother cells as well as in the megaspores, and a semidominant ago5 mutant is defective in the initiation of megagametogenesis (Tucker et al., 2012), suggesting
that a somatic sRNA pathway mediated by AGO5 promotes
megagametogenesis. AGO5 preferentially binds sRNAs with
a 59 C that are derived from intergenic sequences (Mi et al., 2008),
the function of which is largely unexplored. AGO5 can also bind
vsiRNAs of Cucumber mosaic virus (Takeda et al., 2008), suggesting a role in antiviral defense. A recent study showed that
AGO5 is induced by Potato virus X infection and cooperates with
AGO2 to restrict Potato virus X infection (Brosseau and Moffett,
2015).
MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1 or AGO5c), one
of the five AGO5 homologs in rice, is expressed specifically in
germ cells and has specific functions in the development of
premeiotic germ cells and meiosis progression during sporogenesis (Nonomura et al., 2007). Like AGO5 in Arabidopsis, MEL1/
AGO5c predominantly associated with 21-nucleotide phased
siRNAs (phasiRNAs) that are mainly derived from intergenic regions and initiate with a 59 C (Komiya et al., 2014). The function of
MEL1/phasiRNA complexes remains obscure, yet the cytoplasmic
localization of MEL1 implies a potential role of MEL1/phasiRNA in
posttranscriptional regulation of genes.
AGO18 has evolved as a subclade conserved in monocots.
AGO18 expression is barely detectable in rice seedlings but is
highly induced by viral infection (Wu et al., 2015). Deficiency of
AGO18 leads to increased susceptibility to viral infection, whereas
its overexpression enhances resistance to viral diseases in rice.
AGO18 only binds very low levels of vsiRNAs but preferentially
recruits miR168 (Wu et al., 2015). As mentioned above, it has been
shown that AGO18 confers viral resistance by competing with
AGO1 for miR168 to upregulate AGO1 upon viral infection (Wu
et al., 2015).
Maize encodes two AGO18 subclade proteins (AGO18a and
AGO18b). AGO18b is predominantly expressed in the tapetal
and meiotic cells (Zhai et al., 2014), which matches the distribution
and timing of 24-nucleotide meiotic phasiRNAs (Zhai et al., 2015a).
This implies that AGO18b might be an effector protein for the
24-nucleotide phasiRNAs.
AGO2/3/7 Clade
Arabidopsis AGO2 plays a major role in antiviral defense and has
been shown to be required for resistance to a broad spectrum of
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Table 1. Biological Roles of AGO Proteins in Plants
sRNAs
Bound
AGO Name
Locus ID
Function
Reference
AtAGO1
AT1G48410
miRNAs
ta-siRNAs
vsiRNAs
Plant development, antiviral defense
miRNAs
vsiRNAs
miRNAs
vsiRNAs
miRNAs
siRNAs
vsiRNAs
phasiRNAs
miR165/166
miR172
vsiRNAs
Plant development, antiviral defense
(Bohmert et al., 1998; Morel et al., 2002;
Vaucheret et al., 2004; Baumberger and
Baulcombe, 2005; Qi et al., 2005; Zhang
et al., 2006)
(Wu et al., 2009, 2015)
OsAGO1a
Os02g45070
OsAGO1b
Os04g47870
Plant development, antiviral defense
(Wu et al., 2009, 2015)
OsAGO1c
AtAGO5
Os02g58490
AT2G27880
Plant development
Megagametogenesis, antiviral defense
(Wu et al., 2009)
(Takeda et al., 2008; Tucker et al., 2012;
Brosseau and Moffett, 2015)
(Nonomura et al., 2007; Komiya et al., 2014)
(Moussian et al., 1998; Lynn et al., 1999; Liu
et al., 2009; Ji et al., 2011; Zhu et al.,
2011; Garcia-Ruiz et al., 2015; Zhou
et al., 2015)
(Nishimura et al., 2002)
(Wu et al., 2015)
(Zhai et al., 2015a)
(Harvey et al., 2011; Jaubert et al., 2011;
Wang et al., 2011b; Zhang et al., 2011;
Wei et al., 2012)
OsAGO5c
AtAGO10
Os03g58600
AT5G43810
OsAGO10
OsAGO18
ZmAGO18b
AtAGO2
Os06g39640
Os07g28850
GRMZM2G457370
AT1G31280
AtAGO3
AtAGO7
AT1G31290
AT1G69440
OsAGO7
ZmAGO7
AtAGO4
Os03g33650
GRMZM5G892991
AT2G27040
miR390
miR390
hc-siRNAs
ta-siRNAs
OsAGO4a
Os01g16870
OsAGO4b
Os04g06770
AtAGO6
AT2G32940
OsAGO16
AtAGO8
AtAGO9
ZmAGO9
Os07g16224
AT5G21030
AT5G21150
GRMZM2G141818
lmiRNAs
hc-siRNAs
lmiRNAs
hc-siRNAs
hc-siRNAs
siRNAs
ta-siRNAs
lmiRNAs
n.d.
siRNAs
n.d.
n.d.
miR168
n.d.
diRNAs
vsiRNAs
miR393*
n.d.
miR390
Germ cell division
SAM development, antiviral defense
SAM maintenance and leaf development
Antiviral defense
Tapetum and germ cell development
DSB repair, antiviral defense, antibacterial
immunity
n.d.
ta-siRNA biogenesis, phase transition,
antiviral defense
ta-siRNA biogenesis, SAM development
ta-siRNA biogenesis, leaf development
DNA methylation, antibacterial immunity,
defense against DNA viruses
DNA methylation
(Hunter et al., 2003; Adenot et al., 2006;
Montgomery et al., 2008; Qu et al., 2008)
(Nagasaki et al., 2007)
(Douglas et al., 2010)
(Zilberman et al., 2003; Li et al., 2006;
Pontes et al., 2006; Qi et al., 2006; Agorio
and Vera, 2007; Raja et al., 2008; Wu
et al., 2012)
(Wu et al., 2010; Wei et al., 2014)
DNA methylation
(Wu et al., 2010; Wei et al., 2014)
DNA methylation
(Zheng et al., 2007; Havecker et al., 2010;
Wu et al., 2012; Nuthikattu et al., 2013;
Duan et al., 2015; McCue et al., 2015)
(Wu et al., 2010)
n.d.
n.d.
Gamete cell specification
Somatic cell specification
(Olmedo-Monfil et al., 2010)
(Singh et al., 2011)
At, Arabidopsis thaliana; Os, Oryza sativa; Zm, Zea mays. n.d., not determined.
plant viruses (Harvey et al., 2011; Jaubert et al., 2011; Wang
et al., 2011b). AGO2 binds vsiRNAs with 59 terminal A (Wang
et al., 2011b), and the catalytic activity of AGO2 is essential for its
role in antiviral defense (Carbonell et al., 2012). Consistent with
these findings, biochemical experiments using cytoplasmic
extracts of evacuolated tobacco protoplasts revealed that AGO2
loaded with synthetic vsiRNAs can target viral RNAs for cleavage, thereby inhibiting viral replication (Schuck et al., 2013).
AGO2 also binds miR393b* to silence a Golgi-localized gene
MEMB12 likely via translational repression, resulting in exocytosis of antimicrobial pathogenesis-related protein PR1 and
increased antibacterial activity (Zhang et al., 2011). Moreover,
the expression of AGO2 can be induced by g-irradiation that
causes DNA lesions (Culligan et al., 2004), and it acts as an
effector of diRNAs to facilitate DSB repair as mentioned above
(Wei et al., 2012).
AGO3 has a close phylogenetic relationship and genomic
location to AGO2. AGO3 binds siRNAs derived from Potato
spindle tuber viroid (Minoia et al., 2014), and AGO3 programmed
with exogenous siRNAs can cleave viral RNAs in vitro (Schuck
et al., 2013). Thus far, no biological functions have been demonstrated for AGO3.
Arabidopsis AGO7 (also known as ZIP) specifically binds
miR390 (Montgomery et al., 2008), which targets TAS3 transcripts
Mechanisms and Functions of Plant Argonautes
for the biogenesis of tasiRNAs (Allen et al., 2005; Adenot et al.,
2006; Axtell et al., 2006; Fahlgren et al., 2006; Howell et al., 2007).
TAS3 tasiRNAs target several AUXIN RESPONSE FACTOR genes
that are involved in the regulation of developmental timing and
lateral organ development (Williams et al., 2005; Adenot et al.,
2006; Fahlgren et al., 2006; Garcia et al., 2006; Hunter et al., 2006;
Montgomery et al., 2008). The function of AGO7 in ta-siRNA
biogenesis is conserved in rice and maize (Axtell et al., 2006;
Nagasaki et al., 2007; Douglas et al., 2010).
AGO4/6/8/9 Clade
AGO4 was first discovered in a genetic screen for mutants with
released silencing of the SUPERMAN (SUP) locus, and a mutation
in AGO4 leads to reduced asymmetric DNA methylation levels at
SUP (Zilberman et al., 2003). AGO4 was later demonstrated to be
the effector protein for hc-siRNAs that direct DNA methylation
genome-wide through the RdDM pathway (Pontes et al., 2006; Qi
et al., 2006). AGO4 is required for resistance to Pseudomonas
syringae infection (Agorio and Vera, 2007). However, loss of other
RdDM pathway components does not impair resistance to this
pathogen, suggesting that AGO4 likely functions independently of
the RdDM pathway in mediating bacterial resistance. AGO4 is also
involved in plant defense against DNA viruses. In tissues that have
recovered from infection, DNA of Beet curly top virus L22 mutant is
hypermethylated and host recovery requires AGO4 (Raja et al.,
2008), suggesting that DNA methylation mediated by AGO4 is
a mechanism used by plants to defend against DNA viruses.
Rice encodes four AGO4 homologs (AGO4a, AGO4b, AGO15,
and AGO16) (Kapoor et al., 2008). AGO4a, AGO4b, and AGO16
can bind lmiRNAs and 24-nucleotide siRNAs that are mainly
produced from miniature inverted repeat TEs and other TEs (Wu
et al., 2010). Knocking down AGO4a and AGO4b affects GA and
BR homeostasis-related genes and causes dwarfism phenotype,
likely through affecting DNA methylation mediated by the
24-nucleotide siRNAs associated with miniature inverted repeat
TEs (Wei et al., 2014).
AGO6 was first identified in a screen for suppressors of ros1-1
(Zheng et al., 2007). Mutation of AGO6 partially suppresses TGS in
the DNA demethylase mutant ros1-1 and affects DNA methylation
levels in several RdDM target loci (Zheng et al., 2007). AGO6
appears to differ from AGO4 in binding preference for siRNAs
produced from different loci, which may be attributed to their
different spatiotemporal expression and interaction with target
loci (Havecker et al., 2010). AGO4 is ubiquitously expressed
throughout plant, whereas AGO6 is predominantly expressed in
shoot and root apical meristems and in tissues enriched for dividing cells but not in mature leaves (Zheng et al., 2007; Havecker
et al., 2010; Eun et al., 2011). A recent study provided additional
insights into the nonredundant roles of AGO4 and AGO6 (Duan
et al., 2015). This work showed that AGO6 and AGO4 are both
required for DNA methylation at most of their target loci, indicating
that they are mutually dependent. It was also shown that AGO4
and AGO6 differ in their subnuclear colocalization and in interactions with Pol II and Pol IV, and it was proposed that they may act
sequentially to mediate DNA methylation (Duan et al., 2015). AGO6
also binds RDR6-dependent 21- to 22-nucleotide siRNAs generated from transcriptionally active TEs and is thought to trigger
279
the initiation of de novo TE DNA methylation (Nuthikattu et al.,
2013; McCue et al., 2015), which may provide a link between PTGS
and TGS of TEs.
AGO8 and AGO9 are encoded by two neighboring loci and
highly similar in sequences. Sequence alignment showed that
AGO8 contains a deletion of one of the central strands in the
b-sheet of the PIWI domain, presumably precluding correct
folding of this protein (Poulsen et al., 2013). AGO8 expression has
not been detected in any tissues examined thus far and has been
proposed to be a pseudogene (Kapoor et al., 2008). AGO9 is
expressed in cytoplasmic foci of somatic companion cells. AGO9
preferentially interacts with 24-nucleotide siRNAs derived from
TEs and silences TEs in female gamete and their accessory cells
likely in a non-cell-autonomous manner, thereby repressing germ
cell fate in somatic cells (Olmedo-Monfil et al., 2010). In maize, the
AGO9 homolog (named AGO104) is specifically expressed in the
somatic cells around the precursors of the gametic cells but acts to
repress somatic fate in germ cells (Singh et al., 2011).
CONCLUSION
Since the discovery of AGO1 in Arabidopsis, the founding member
of the AGO family, much has been learned about the diverse biological functions of plant AGOs and the mechanisms through
which they act, yet many interesting questions remain unanswered. miRNAs and hc-siRNAs are produced in the nucleus but
loaded into AGOs in the cytoplasm. Are the biogenesis and
loading of these sRNAs two separate processes or are they
coupled? How does the sRNA biogenesis machinery or a signal
emanating from the biogenesis machinery affect sRNA loading
into AGOs? As elaborated above, some AGOs can act in different
modes. For instance, AGO1 can mediate endonucleolytic
cleavage and translational repression; AGO10 can function in
translational repressing mediated by some miRNAs but acts as
a decoy for specific miRNAs. It remains obscure how these distinct
modes of action of a single AGO are regulated or coordinated.
AGOs likely need interacting cofactors to perform their functions.
However, few AGO-interacting proteins have been identified.
Finding cofactors of AGOs remains to be a major challenge. It
will be also exciting to investigate whether AGOs have novel
functions through mechanisms independent of the canonical
RNAi pathways.
Despite major advances in understanding the functions of
Arabidopsis AGOs, AGOs in other plants, including important crop
species, are poorly characterized. Study of AGOs in other plants is
needed to deepen our understanding about the conservation and
diversification of AGO function and may reveal novel functions of
AGOs.
METHODS
Phylogenetic Analysis
Alignment of protein sequences was performed with ClustalW in Mega
6.0 with default parameters. The alignment is shown in Supplemental Data
Set 1. Phylogenetic analysis was performed using Mega 6.0 (Tamura et al.,
2013). The evolutionary history was inferred using the Minimum Evolution
method (Nei, 1992). The neighbor-joining algorithm (Saitou and Nei, 1987)
was used to generate the tree with bootstrap values (10,000 replicates)
280
The Plant Cell
(Felsenstein, 1985). The evolutionary distances were computed using the
Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the
units of the number of amino acid substitutions per site (Supplemental Data
Set 2). Pairwise deletion was used to eliminate gaps in pairwise sequence
comparisons.
Supplemental Data
Supplemental Data Set 1. Alignment of Sequences of AGO Proteins
in Selected Plant Species.
Supplemental Data Set 2. Pairwise Matrix of the Evolutionary
Distances between AGO Proteins in Selected Plant Species.
ACKNOWLEDGMENTS
This review is dedicated to the memory of Professor Biao Ding, a great
mentor, inspirer, colleague, and friend who will be deeply missed by all
those who had the privilege of knowing and working with him. Our research
is supported by grants from National Natural Science Foundation of China
(Grants 31330042, 31421001, and 31225015) to Y.Q. We apologize to
colleagues whose work could not be fully cited owing to space limits.
AUTHOR CONTRIBUTIONS
Both authors contributed to the writing of this article.
Received October 28, 2015; revised December 29, 2015; accepted February 10, 2016; published February 11, 2016.
REFERENCES
Adenot, X., Elmayan, T., Lauressergues, D., Boutet, S., Bouché, N.,
Gasciolli, V., and Vaucheret, H. (2006). DRB4-dependent TAS3
trans-acting siRNAs control leaf morphology through AGO7. Curr.
Biol. 16: 927–932.
Agorio, A., and Vera, P. (2007). ARGONAUTE4 is required for resistance to Pseudomonas syringae in Arabidopsis. Plant Cell 19:
3778–3790.
Allen, E., Xie, Z., Gustafson, A.M., and Carrington, J.C. (2005).
MicroRNA-directed phasing during trans-acting siRNA biogenesis
in plants. Cell 121: 207–221.
Arif, M.A., Frank, W., and Khraiwesh, B. (2013). Role of RNA interference (RNAi) in the moss Physcomitrella patens. Int. J. Mol. Sci.
14: 1516–1540.
Aukerman, M.J., and Sakai, H. (2003). Regulation of flowering time
and floral organ identity by a microRNA and its APETALA2-like
target genes. Plant Cell 15: 2730–2741.
Axtell, M.J., Jan, C., Rajagopalan, R., and Bartel, D.P. (2006). A
two-hit trigger for siRNA biogenesis in plants. Cell 127: 565–577.
Axtell, M.J., Snyder, J.A., and Bartel, D.P. (2007). Common functions for diverse small RNAs of land plants. Plant Cell 19: 1750–
1769.
Azevedo, J., Cooke, R., and Lagrange, T. (2011). Taking RISCs with
Ago hookers. Curr. Opin. Plant Biol. 14: 594–600.
Ba, Z., and Qi, Y. (2013). Small RNAs: emerging key players in DNA
double-strand break repair. Sci. China Life Sci. 56: 933–936.
Baumberger, N., and Baulcombe, D.C. (2005). Arabidopsis ARGONAUTE1
is an RNA Slicer that selectively recruits microRNAs and short interfering
RNAs. Proc. Natl. Acad. Sci. USA 102: 11928–11933.
Betancur, J.G., and Tomari, Y. (2012). Dicer is dispensable for
asymmetric RISC loading in mammals. RNA 18: 24–30.
Bies-Etheve, N., Pontier, D., Lahmy, S., Picart, C., Vega, D., Cooke,
R., and Lagrange, T. (2009). RNA-directed DNA methylation requires an AGO4-interacting member of the SPT5 elongation factor
family. EMBO Rep. 10: 649–654.
Blevins, T., Podicheti, R., Mishra, V., Marasco, M., Wang, J.,
Rusch, D., Tang, H., and Pikaard, C.S. (2015). Identification of
Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de
novo DNA methylation in Arabidopsis. eLife 4: e09591.
Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., and
Benning, C. (1998). AGO1 defines a novel locus of Arabidopsis
controlling leaf development. EMBO J. 17: 170–180.
Borsani, O., Zhu, J., Verslues, P.E., Sunkar, R., and Zhu, J.K.
(2005). Endogenous siRNAs derived from a pair of natural cisantisense transcripts regulate salt tolerance in Arabidopsis. Cell 123:
1279–1291.
Brodersen, P., Sakvarelidze-Achard, L., Bruun-Rasmussen, M.,
Dunoyer, P., Yamamoto, Y.Y., Sieburth, L., and Voinnet, O.
(2008). Widespread translational inhibition by plant miRNAs and
siRNAs. Science 320: 1185–1190.
Brodersen, P., Sakvarelidze-Achard, L., Schaller, H., Khafif, M.,
Schott, G., Bendahmane, A., and Voinnet, O. (2012). Isoprenoid
biosynthesis is required for miRNA function and affects membrane
association of ARGONAUTE 1 in Arabidopsis. Proc. Natl. Acad. Sci.
USA 109: 1778–1783.
Brosseau, C., and Moffett, P. (2015). Functional and genetic analysis
identify a role for Arabidopsis ARGONAUTE5 in antiviral RNA silencing. Plant Cell 27: 1742–1754.
Cao, X., and Jacobsen, S.E. (2002). Role of the Arabidopsis DRM
methyltransferases in de novo DNA methylation and gene silencing.
Curr. Biol. 12: 1138–1144.
Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Gilbert, K.B.,
Montgomery, T.A., Nguyen, T., Cuperus, J.T., and Carrington,
J.C. (2012). Functional analysis of three Arabidopsis ARGONAUTES
using slicer-defective mutants. Plant Cell 24: 3613–3629.
Carmell, M.A., Xuan, Z., Zhang, M.Q., and Hannon, G.J. (2002). The
Argonaute family: tentacles that reach into RNAi, developmental control,
stem cell maintenance, and tumorigenesis. Genes Dev. 16: 2733–2742.
Cerutti, H., and Casas-Mollano, J.A. (2006). On the origin and
functions of RNA-mediated silencing: from protists to man. Curr.
Genet. 50: 81–99.
Chen, X. (2004). A microRNA as a translational repressor of APETALA2 in
Arabidopsis flower development. Science 303: 2022–2025.
Ciccia, A., and Elledge, S.J. (2010). The DNA damage response:
making it safe to play with knives. Mol. Cell 40: 179–204.
Culligan, K., Tissier, A., and Britt, A. (2004). ATR regulates a G2phase cell-cycle checkpoint in Arabidopsis thaliana. Plant Cell 16:
1091–1104.
Czech, B., Zhou, R., Erlich, Y., Brennecke, J., Binari, R., Villalta, C.,
Gordon, A., Perrimon, N., and Hannon, G.J. (2009). Hierarchical
rules for Argonaute loading in Drosophila. Mol. Cell 36: 445–456.
Douglas, R.N., Wiley, D., Sarkar, A., Springer, N., Timmermans,
M.C., and Scanlon, M.J. (2010). ragged seedling2 encodes an
ARGONAUTE7-like protein required for mediolateral expansion, but
not dorsiventrality, of maize leaves. Plant Cell 22: 1441–1451.
Duan, C.G., et al. (2015). Specific but interdependent functions for
Arabidopsis AGO4 and AGO6 in RNA-directed DNA methylation.
EMBO J. 34: 581–592.
Eamens, A.L., Smith, N.A., Curtin, S.J., Wang, M.B., and Waterhouse,
P.M. (2009). The Arabidopsis thaliana double-stranded RNA binding
protein DRB1 directs guide strand selection from microRNA duplexes.
RNA 15: 2219–2235.
Mechanisms and Functions of Plant Argonautes
Earley, K.W., and Poethig, R.S. (2011). Binding of the cyclophilin 40
ortholog SQUINT to Hsp90 protein is required for SQUINT function
in Arabidopsis. J. Biol. Chem. 286: 38184–38189.
Elkayam, E., Kuhn, C.D., Tocilj, A., Haase, A.D., Greene, E.M.,
Hannon, G.J., and Joshua-Tor, L. (2012). The structure of human
argonaute-2 in complex with miR-20a. Cell 150: 100–110.
El-Shami, M., Pontier, D., Lahmy, S., Braun, L., Picart, C., Vega, D.,
Hakimi, M.A., Jacobsen, S.E., Cooke, R., and Lagrange, T.
(2007). Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related
components. Genes Dev. 21: 2539–2544.
Endo, Y., Iwakawa, H.O., and Tomari, Y. (2013). Arabidopsis
ARGONAUTE7 selects miR390 through multiple checkpoints during
RISC assembly. EMBO Rep. 14: 652–658.
Eun, C., Lorkovic, Z.J., Naumann, U., Long, Q., Havecker, E.R.,
Simon, S.A., Meyers, B.C., Matzke, A.J., and Matzke, M. (2011).
AGO6 functions in RNA-mediated transcriptional gene silencing in
shoot and root meristems in Arabidopsis thaliana. PLoS One 6:
e25730.
Fahlgren, N., Montgomery, T.A., Howell, M.D., Allen, E., Dvorak,
S.K., Alexander, A.L., and Carrington, J.C. (2006). Regulation of
AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr. Biol. 16:
939–944.
Fei, Q., Xia, R., and Meyers, B.C. (2013). Phased, secondary, small
interfering RNAs in posttranscriptional regulatory networks. Plant
Cell 25: 2400–2415.
Felsenstein, J. (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783–791.
Franco-Zorrilla, J.M., Valli, A., Todesco, M., Mateos, I., Puga, M.I.,
Rubio-Somoza, I., Leyva, A., Weigel, D., García, J.A., and PazAres, J. (2007). Target mimicry provides a new mechanism for
regulation of microRNA activity. Nat. Genet. 39: 1033–1037.
Frank, F., Hauver, J., Sonenberg, N., and Nagar, B. (2012). Arabidopsis Argonaute MID domains use their nucleotide specificity loop
to sort small RNAs. EMBO J. 31: 3588–3595.
Frank, F., Sonenberg, N., and Nagar, B. (2010). Structural basis for
59-nucleotide base-specific recognition of guide RNA by human
AGO2. Nature 465: 818–822.
Gandikota, M., Birkenbihl, R.P., Höhmann, S., Cardon, G.H.,
Saedler, H., and Huijser, P. (2007). The miRNA156/157 recognition element in the 39 UTR of the Arabidopsis SBP box gene SPL3
prevents early flowering by translational inhibition in seedlings.
Plant J. 49: 683–693.
Gao, M., et al. (2014). Ago2 facilitates Rad51 recruitment and DNA
double-strand break repair by homologous recombination. Cell Res.
24: 532–541.
Garcia, D., Collier, S.A., Byrne, M.E., and Martienssen, R.A. (2006).
Specification of leaf polarity in Arabidopsis via the trans-acting
siRNA pathway. Curr. Biol. 16: 933–938.
Garcia, D., Garcia, S., Pontier, D., Marchais, A., Renou, J.P.,
Lagrange, T., and Voinnet, O. (2012). Ago hook and RNA helicase motifs underpin dual roles for SDE3 in antiviral defense and
silencing of nonconserved intergenic regions. Mol. Cell 48: 109–
120.
Garcia-Ruiz, H., Carbonell, A., Hoyer, J.S., Fahlgren, N., Gilbert,
K.B., Takeda, A., Giampetruzzi, A., Garcia Ruiz, M.T., McGinn,
M.G., Lowery, N., Martinez Baladejo, M.T., and Carrington, J.C.
(2015). Roles and programming of Arabidopsis ARGONAUTE proteins during Turnip mosaic virus infection. PLoS Pathog. 11:
e1004755.
Gasciolli, V., Mallory, A.C., Bartel, D.P., and Vaucheret, H. (2005).
Partially redundant functions of Arabidopsis DICER-like enzymes
281
and a role for DCL4 in producing trans-acting siRNAs. Curr. Biol. 15:
1494–1500.
Gu, S., Jin, L., Huang, Y., Zhang, F., and Kay, M.A. (2012). Slicingindependent RISC activation requires the argonaute PAZ domain.
Curr. Biol. 22: 1536–1542.
Haag, J.R., Ream, T.S., Marasco, M., Nicora, C.D., Norbeck, A.D.,
Pasa-Tolic, L., and Pikaard, C.S. (2012). In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and
RDR2 for dsRNA synthesis in plant RNA silencing. Mol. Cell 48:
811–818.
Hall, T.M. (2005). Structure and function of argonaute proteins.
Structure 13: 1403–1408.
Harvey, J.J., Lewsey, M.G., Patel, K., Westwood, J., Heimstädt, S.,
Carr, J.P., and Baulcombe, D.C. (2011). An antiviral defense role of
AGO2 in plants. PLoS One 6: e14639.
Hauptmann, J., Dueck, A., Harlander, S., Pfaff, J., Merkl, R., and
Meister, G. (2013). Turning catalytically inactive human Argonaute
proteins into active slicer enzymes. Nat. Struct. Mol. Biol. 20: 814–
817.
Havecker, E.R., Wallbridge, L.M., Hardcastle, T.J., Bush, M.S.,
Kelly, K.A., Dunn, R.M., Schwach, F., Doonan, J.H., and
Baulcombe, D.C. (2010). The Arabidopsis RNA-directed DNA
methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22: 321–334.
He, X.J., Hsu, Y.F., Zhu, S., Wierzbicki, A.T., Pontes, O., Pikaard,
C.S., Liu, H.L., Wang, C.S., Jin, H., and Zhu, J.K. (2009). An effector
of RNA-directed DNA methylation in arabidopsis is an ARGONAUTE
4- and RNA-binding protein. Cell 137: 498–508.
Henderson, I.R., Zhang, X., Lu, C., Johnson, L., Meyers, B.C.,
Green, P.J., and Jacobsen, S.E. (2006). Dissecting Arabidopsis
thaliana DICER function in small RNA processing, gene silencing
and DNA methylation patterning. Nat. Genet. 38: 721–725.
Herr, A.J., Jensen, M.B., Dalmay, T., and Baulcombe, D.C. (2005).
RNA polymerase IV directs silencing of endogenous DNA. Science
308: 118–120.
Howell, M.D., Fahlgren, N., Chapman, E.J., Cumbie, J.S., Sullivan,
C.M., Givan, S.A., Kasschau, K.D., and Carrington, J.C. (2007).
Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/
DICER-LIKE4 pathway in Arabidopsis reveals dependency on miRNAand tasiRNA-directed targeting. Plant Cell 19: 926–942.
Humphreys, D.T., Westman, B.J., Martin, D.I., and Preiss, T. (2005).
MicroRNAs control translation initiation by inhibiting eukaryotic
initiation factor 4E/cap and poly(A) tail function. Proc. Natl. Acad.
Sci. USA 102: 16961–16966.
Hunter, C., Sun, H., and Poethig, R.S. (2003). The Arabidopsis heterochronic gene ZIPPY is an ARGONAUTE family member. Curr.
Biol. 13: 1734–1739.
Hunter, C., Willmann, M.R., Wu, G., Yoshikawa, M., de la Luz
Gutiérrez-Nava, M., and Poethig, S.R. (2006). Trans-acting siRNAmediated repression of ETTIN and ARF4 regulates heteroblasty in
Arabidopsis. Development 133: 2973–2981.
Hutvagner, G., and Simard, M.J. (2008). Argonaute proteins: key
players in RNA silencing. Nat. Rev. Mol. Cell Biol. 9: 22–32.
Iki, T., Yoshikawa, M., Meshi, T., and Ishikawa, M. (2012). Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants.
EMBO J. 31: 267–278.
Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M.C., MatsumotoYokoyama, E., Mitsuhara, I., Meshi, T., and Ishikawa, M. (2010).
In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Mol. Cell 39: 282–291.
Iwasaki, S., Kobayashi, M., Yoda, M., Sakaguchi, Y., Katsuma, S.,
Suzuki, T., and Tomari, Y. (2010). Hsc70/Hsp90 chaperone
282
The Plant Cell
machinery mediates ATP-dependent RISC loading of small RNA
duplexes. Mol. Cell 39: 292–299.
Iwasaki, S., Sasaki, H.M., Sakaguchi, Y., Suzuki, T., Tadakuma, H.,
and Tomari, Y. (2015). Defining fundamental steps in the assembly
of the Drosophila RNAi enzyme complex. Nature 521: 533–536.
Jaubert, M., Bhattacharjee, S., Mello, A.F., Perry, K.L., and
Moffett, P. (2011). ARGONAUTE2 mediates RNA-silencing antiviral defenses against Potato virus X in Arabidopsis. Plant Physiol.
156: 1556–1564.
Ji, L., et al. (2011). ARGONAUTE10 and ARGONAUTE1 regulate the
termination of floral stem cells through two microRNAs in Arabidopsis. PLoS Genet. 7: e1001358.
Jinek, M., and Doudna, J.A. (2009). A three-dimensional view of the
molecular machinery of RNA interference. Nature 457: 405–412.
Kanellopoulou, C., Muljo, S.A., Kung, A.L., Ganesan, S., Drapkin,
R., Jenuwein, T., Livingston, D.M., and Rajewsky, K. (2005).
Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev. 19: 489–501.
Kanno, T., Huettel, B., Mette, M.F., Aufsatz, W., Jaligot, E.,
Daxinger, L., Kreil, D.P., Matzke, M., and Matzke, A.J. (2005).
Atypical RNA polymerase subunits required for RNA-directed DNA
methylation. Nat. Genet. 37: 761–765.
Kapoor, M., Arora, R., Lama, T., Nijhawan, A., Khurana, J.P., Tyagi,
A.K., and Kapoor, S. (2008). Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNAdependent RNA Polymerase gene families and their expression
analysis during reproductive development and stress in rice. BMC
Genomics 9: 451.
Kawamata, T., Seitz, H., and Tomari, Y. (2009). Structural determinants of miRNAs for RISC loading and slicer-independent unwinding. Nat. Struct. Mol. Biol. 16: 953–960.
Komiya, R., Ohyanagi, H., Niihama, M., Watanabe, T., Nakano, M.,
Kurata, N., and Nonomura, K. (2014). Rice germline-specific Argonaute MEL1 protein binds to phasiRNAs generated from more
than 700 lincRNAs. Plant J. 78: 385–397.
Kurihara, Y., and Watanabe, Y. (2004). Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc. Natl. Acad. Sci.
USA 101: 12753–12758.
Kwak, P.B., and Tomari, Y. (2012). The N domain of Argonaute drives
duplex unwinding during RISC assembly. Nat. Struct. Mol. Biol. 19:
145–151.
Law, J.A., and Jacobsen, S.E. (2010). Establishing, maintaining and
modifying DNA methylation patterns in plants and animals. Nat.
Rev. Genet. 11: 204–220.
Lee, Y.S., Nakahara, K., Pham, J.W., Kim, K., He, Z., Sontheimer,
E.J., and Carthew, R.W. (2004). Distinct roles for Drosophila
Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell
117: 69–81.
Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P., and
Burge, C.B. (2003). Prediction of mammalian microRNA targets.
Cell 115: 787–798.
Li, C.F., Henderson, I.R., Song, L., Fedoroff, N., Lagrange, T., and
Jacobsen, S.E. (2008). Dynamic regulation of ARGONAUTE4 within
multiple nuclear bodies in Arabidopsis thaliana. PLoS Genet. 4: e27.
Li, C.F., Pontes, O., El-Shami, M., Henderson, I.R., Bernatavichute,
Y.V., Chan, S.W., Lagrange, T., Pikaard, C.S., and Jacobsen, S.E.
(2006). An ARGONAUTE4-containing nuclear processing center
colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126: 93–106.
Li, S., et al. (2013). MicroRNAs inhibit the translation of target mRNAs
on the endoplasmic reticulum in Arabidopsis. Cell 153: 562–574.
Lingel, A., Simon, B., Izaurralde, E., and Sattler, M. (2003). Structure
and nucleic-acid binding of the Drosophila Argonaute 2 PAZ domain. Nature 426: 465–469.
Lingel, A., Simon, B., Izaurralde, E., and Sattler, M. (2004). Nucleic
acid 39-end recognition by the Argonaute2 PAZ domain. Nat. Struct.
Mol. Biol. 11: 576–577.
Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson, J.M.,
Song, J.J., Hammond, S.M., Joshua-Tor, L., and Hannon, G.J.
(2004). Argonaute2 is the catalytic engine of mammalian RNAi.
Science 305: 1437–1441.
Liu, Q., Rand, T.A., Kalidas, S., Du, F., Kim, H.E., Smith, D.P., and
Wang, X. (2003). R2D2, a bridge between the initiation and effector
steps of the Drosophila RNAi pathway. Science 301: 1921–1925.
Liu, Q., Yao, X., Pi, L., Wang, H., Cui, X., and Huang, H. (2009). The
ARGONAUTE10 gene modulates shoot apical meristem maintenance and establishment of leaf polarity by repressing miR165/166
in Arabidopsis. Plant J. 58: 27–40.
Llave, C., Xie, Z., Kasschau, K.D., and Carrington, J.C. (2002).
Cleavage of Scarecrow-like mRNA targets directed by a class of
Arabidopsis miRNA. Science 297: 2053–2056.
Lynn, K., Fernandez, A., Aida, M., Sedbrook, J., Tasaka, M.,
Masson, P., and Barton, M.K. (1999). The PINHEAD/ZWILLE
gene acts pleiotropically in Arabidopsis development and has
overlapping functions with the ARGONAUTE1 gene. Development
126: 469–481.
Ma, J.B., Ye, K., and Patel, D.J. (2004). Structural basis for overhangspecific small interfering RNA recognition by the PAZ domain. Nature 429: 318–322.
Mallory, A.C., Hinze, A., Tucker, M.R., Bouché, N., Gasciolli, V.,
Elmayan, T., Lauressergues, D., Jauvion, V., Vaucheret, H., and
Laux, T. (2009). Redundant and specific roles of the ARGONAUTE
proteins AGO1 and ZLL in development and small RNA-directed
gene silencing. PLoS Genet. 5: e1000646.
Manavella, P.A., Hagmann, J., Ott, F., Laubinger, S., Franz, M.,
Macek, B., and Weigel, D. (2012). Fast-forward genetics identifies
plant CPL phosphatases as regulators of miRNA processing factor
HYL1. Cell 151: 859–870.
Marí-Ordóñez, A., Marchais, A., Etcheverry, M., Martin, A., Colot,
V., and Voinnet, O. (2013). Reconstructing de novo silencing of an
active plant retrotransposon. Nat. Genet. 45: 1029–1039.
McCue, A.D., Panda, K., Nuthikattu, S., Choudury, S.G., Thomas,
E.N., and Slotkin, R.K. (2015). ARGONAUTE 6 bridges transposable element mRNA-derived siRNAs to the establishment of DNA
methylation. EMBO J. 34: 20–35.
Mi, S., et al. (2008). Sorting of small RNAs into Arabidopsis argonaute
complexes is directed by the 59 terminal nucleotide. Cell 133: 116–127.
Minoia, S., Carbonell, A., Di Serio, F., Gisel, A., Carrington, J.C.,
Navarro, B., and Flores, R. (2014). Specific argonautes selectively
bind small RNAs derived from potato spindle tuber viroid and attenuate viroid accumulation in vivo. J. Virol. 88: 11933–11945.
Miyoshi, T., Takeuchi, A., Siomi, H., and Siomi, M.C. (2010). A direct
role for Hsp90 in pre-RISC formation in Drosophila. Nat. Struct. Mol.
Biol. 17: 1024–1026.
Montgomery, T.A., Howell, M.D., Cuperus, J.T., Li, D., Hansen,
J.E., Alexander, A.L., Chapman, E.J., Fahlgren, N., Allen, E., and
Carrington, J.C. (2008). Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133: 128–141.
Morel, J.B., Godon, C., Mourrain, P., Béclin, C., Boutet, S.,
Feuerbach, F., Proux, F., and Vaucheret, H. (2002). Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional
gene silencing and virus resistance. Plant Cell 14: 629–639.
Mosher, R.A., Schwach, F., Studholme, D., and Baulcombe, D.C.
(2008). PolIVb influences RNA-directed DNA methylation independently
of its role in siRNA biogenesis. Proc. Natl. Acad. Sci. USA 105:
3145–3150.
Mechanisms and Functions of Plant Argonautes
Moussian, B., Schoof, H., Haecker, A., Jürgens, G., and Laux, T.
(1998). Role of the ZWILLE gene in the regulation of central shoot
meristem cell fate during Arabidopsis embryogenesis. EMBO J. 17:
1799–1809.
Murchison, E.P., Partridge, J.F., Tam, O.H., Cheloufi, S., and
Hannon, G.J. (2005). Characterization of Dicer-deficient murine
embryonic stem cells. Proc. Natl. Acad. Sci. USA 102: 12135–
12140.
Nagasaki, H., Itoh, J., Hayashi, K., Hibara, K., Satoh-Nagasawa,
N., Nosaka, M., Mukouhata, M., Ashikari, M., Kitano, H.,
Matsuoka, M., Nagato, Y., and Sato, Y. (2007). The small interfering RNA production pathway is required for shoot meristem
initiation in rice. Proc. Natl. Acad. Sci. USA 104: 14867–14871.
Nakanishi, K., Weinberg, D.E., Bartel, D.P., and Patel, D.J. (2012).
Structure of yeast Argonaute with guide RNA. Nature 486: 368–374.
Nei, A.R.M. (1992). A simple method for estimating and testing minimum evolution trees. Mol. Biol. Evol. 9: 945–967.
Nishimura, A., Ito, M., Kamiya, N., Sato, Y., and Matsuoka, M.
(2002). OsPNH1 regulates leaf development and maintenance of the
shoot apical meristem in rice. Plant J. 30: 189–201.
Nonomura, K., Morohoshi, A., Nakano, M., Eiguchi, M., Miyao, A.,
Hirochika, H., and Kurata, N. (2007). A germ cell specific gene of
the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice. Plant Cell
19: 2583–2594.
Nuthikattu, S., McCue, A.D., Panda, K., Fultz, D., DeFraia, C.,
Thomas, E.N., and Slotkin, R.K. (2013). The initiation of epigenetic
silencing of active transposable elements is triggered by RDR6 and
21-22 nucleotide small interfering RNAs. Plant Physiol. 162: 116–
131.
Olmedo-Monfil, V., Durán-Figueroa, N., Arteaga-Vázquez, M.,
Demesa-Arévalo, E., Autran, D., Grimanelli, D., Slotkin, R.K.,
Martienssen, R.A., and Vielle-Calzada, J.-P. (2010). Control of
female gamete formation by a small RNA pathway in Arabidopsis.
Nature 464: 628–632.
Onodera, Y., Haag, J.R., Ream, T., Costa Nunes, P., Pontes, O.,
and Pikaard, C.S. (2005). Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120: 613–622.
Parker, J.S., Roe, S.M., and Barford, D. (2005). Structural insights
into mRNA recognition from a PIWI domain-siRNA guide complex.
Nature 434: 663–666.
Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H.L., and Poethig,
R.S. (2004). SGS3 and SGS2/SDE1/RDR6 are required for juvenile
development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev. 18: 2368–2379.
Peters, L., and Meister, G. (2007). Argonaute proteins: mediators of
RNA silencing. Mol. Cell 26: 611–623.
Pham, J.W., Pellino, J.L., Lee, Y.S., Carthew, R.W., and
Sontheimer, E.J. (2004). A Dicer-2-dependent 80s complex
cleaves targeted mRNAs during RNAi in Drosophila. Cell 117:
83–94.
Pillai, R.S., Bhattacharyya, S.N., Artus, C.G., Zoller, T., Cougot, N.,
Basyuk, E., Bertrand, E., and Filipowicz, W. (2005). Inhibition of
translational initiation by Let-7 microRNA in human cells. Science
309: 1573–1576.
Pontes, O., Li, C.F., Costa Nunes, P., Haag, J., Ream, T., Vitins, A.,
Jacobsen, S.E., and Pikaard, C.S. (2006). The Arabidopsis
chromatin-modifying nuclear siRNA pathway involves a nucleolar
RNA processing center. Cell 126: 79–92.
Pontier, D., Yahubyan, G., Vega, D., Bulski, A., Saez-Vasquez, J.,
Hakimi, M.A., Lerbs-Mache, S., Colot, V., and Lagrange, T.
(2005). Reinforcement of silencing at transposons and highly
283
repeated sequences requires the concerted action of two distinct
RNA polymerases IV in Arabidopsis. Genes Dev. 19: 2030–2040.
Pontier, D., et al. (2012). NERD, a plant-specific GW protein, defines
an additional RNAi-dependent chromatin-based pathway in Arabidopsis. Mol. Cell 48: 121–132.
Poulsen, C., Vaucheret, H., and Brodersen, P. (2013). Lessons on
RNA silencing mechanisms in plants from eukaryotic argonaute
structures. Plant Cell 25: 22–37.
Qi, Y., Denli, A.M., and Hannon, G.J. (2005). Biochemical specialization within Arabidopsis RNA silencing pathways. Mol. Cell 19:
421–428.
Qi, Y., He, X., Wang, X.J., Kohany, O., Jurka, J., and Hannon, G.J.
(2006). Distinct catalytic and non-catalytic roles of ARGONAUTE4 in
RNA-directed DNA methylation. Nature 443: 1008–1012.
Qian, Y., Cheng, Y., Cheng, X., Jiang, H., Zhu, S., and Cheng, B.
(2011). Identification and characterization of Dicer-like, Argonaute
and RNA-dependent RNA polymerase gene families in maize. Plant
Cell Rep. 30: 1347–1363.
Qu, F., Ye, X., and Morris, T.J. (2008). Arabidopsis DRB4, AGO1,
AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proc. Natl. Acad.
Sci. USA 105: 14732–14737.
Raja, P., Sanville, B.C., Buchmann, R.C., and Bisaro, D.M. (2008).
Viral genome methylation as an epigenetic defense against geminiviruses. J. Virol. 82: 8997–9007.
Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., and
Bartel, D.P. (2002). MicroRNAs in plants. Genes Dev. 16: 1616–
1626.
Rhoades, M.W., Reinhart, B.J., Lim, L.P., Burge, C.B., Bartel, B.,
and Bartel, D.P. (2002). Prediction of plant microRNA targets. Cell
110: 513–520.
Rivas, F.V., Tolia, N.H., Song, J.J., Aragon, J.P., Liu, J., Hannon,
G.J., and Joshua-Tor, L. (2005). Purified Argonaute2 and an siRNA
form recombinant human RISC. Nat. Struct. Mol. Biol. 12: 340–349.
Rogers, K., and Chen, X. (2013). Biogenesis, turnover, and mode of
action of plant microRNAs. Plant Cell 25: 2383–2399.
Röhl, A., Rohrberg, J., and Buchner, J. (2013). The chaperone
Hsp90: changing partners for demanding clients. Trends Biochem.
Sci. 38: 253–262.
Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:
406–425.
Schirle, N.T., and MacRae, I.J. (2012). The crystal structure of human
Argonaute2. Science 336: 1037–1040.
Schroda, M. (2006). RNA silencing in Chlamydomonas: mechanisms
and tools. Curr. Genet. 49: 69–84.
Schuck, J., Gursinsky, T., Pantaleo, V., Burgyán, J., and Behrens,
S.E. (2013). AGO/RISC-mediated antiviral RNA silencing in a plant
in vitro system. Nucleic Acids Res. 41: 5090–5103.
Singh, M., Goel, S., Meeley, R.B., Dantec, C., Parrinello, H.,
Michaud, C., Leblanc, O., and Grimanelli, D. (2011). Production
of viable gametes without meiosis in maize deficient for an ARGONAUTE
protein. Plant Cell 23: 443–458.
Singh, R.K., Gase, K., Baldwin, I.T., and Pandey, S.P. (2015). Molecular evolution and diversification of the Argonaute family of
proteins in plants. BMC Plant Biol. 15: 23.
Smith, M.R., Willmann, M.R., Wu, G., Berardini, T.Z., Möller, B.,
Weijers, D., and Poethig, R.S. (2009). Cyclophilin 40 is required for
microRNA activity in Arabidopsis. Proc. Natl. Acad. Sci. USA 106:
5424–5429.
Song, J.J., Liu, J., Tolia, N.H., Schneiderman, J., Smith, S.K.,
Martienssen, R.A., Hannon, G.J., and Joshua-Tor, L. (2003). The
crystal structure of the Argonaute2 PAZ domain reveals an RNA
284
The Plant Cell
binding motif in RNAi effector complexes. Nat. Struct. Biol. 10:
1026–1032.
Song, J.J., Smith, S.K., Hannon, G.J., and Joshua-Tor, L. (2004).
Crystal structure of Argonaute and its implications for RISC slicer
activity. Science 305: 1434–1437.
Takeda, A., Iwasaki, S., Watanabe, T., Utsumi, M., and Watanabe,
Y. (2008). The mechanism selecting the guide strand from small
RNA duplexes is different among argonaute proteins. Plant Cell
Physiol. 49: 493–500.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S.
(2013). MEGA6: Molecular Evolutionary Genetics Analysis version
6.0. Mol. Biol. Evol. 30: 2725–2729.
Tang, G., Reinhart, B.J., Bartel, D.P., and Zamore, P.D. (2003). A
biochemical framework for RNA silencing in plants. Genes Dev. 17:
49–63.
Tolia, N.H., and Joshua-Tor, L. (2007). Slicer and the argonautes.
Nat. Chem. Biol. 3: 36–43.
Tomari, Y., Du, T., Haley, B., Schwarz, D.S., Bennett, R., Cook,
H.A., Koppetsch, B.S., Theurkauf, W.E., and Zamore, P.D.
(2004b). RISC assembly defects in the Drosophila RNAi mutant
armitage. Cell 116: 831–841.
Tomari, Y., Du, T., and Zamore, P.D. (2007). Sorting of Drosophila
small silencing RNAs. Cell 130: 299–308.
Tomari, Y., Matranga, C., Haley, B., Martinez, N., and Zamore, P.D.
(2004a). A protein sensor for siRNA asymmetry. Science 306: 1377–
1380.
Tucker, M.R., Okada, T., Hu, Y., Scholefield, A., Taylor, J.M., and
Koltunow, A.M. (2012). Somatic small RNA pathways promote the
mitotic events of megagametogenesis during female reproductive
development in Arabidopsis. Development 139: 1399–1404.
Vaucheret, H. (2008). Plant ARGONAUTEs. Trends Plant Sci. 13: 350–358.
Vaucheret, H., Vazquez, F., Crété, P., and Bartel, D.P. (2004). The
action of ARGONAUTE1 in the miRNA pathway and its regulation by
the miRNA pathway are crucial for plant development. Genes Dev.
18: 1187–1197.
Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli,
V., Mallory, A.C., Hilbert, J.L., Bartel, D.P., and Crété, P. (2004).
Endogenous trans-acting siRNAs regulate the accumulation of
Arabidopsis mRNAs. Mol. Cell 16: 69–79.
Wang, W., Ye, R., Xin, Y., Fang, X., Li, C., Shi, H., Zhou, X., and Qi,
Y. (2011a). An importin b protein negatively regulates microRNA
activity in Arabidopsis. Plant Cell 23: 3565–3576.
Wang, X.B., Jovel, J., Udomporn, P., Wang, Y., Wu, Q., Li, W.X.,
Gasciolli, V., Vaucheret, H., and Ding, S.W. (2011b). The 21nucleotide, but not 22-nucleotide, viral secondary small interfering
RNAs direct potent antiviral defense by two cooperative argonautes in
Arabidopsis thaliana. Plant Cell 23: 1625–1638.
Wang, Y., Juranek, S., Li, H., Sheng, G., Wardle, G.S., Tuschl, T.,
and Patel, D.J. (2009). Nucleation, propagation and cleavage of
target RNAs in Ago silencing complexes. Nature 461: 754–761.
Wei, L., Gu, L., Song, X., Cui, X., Lu, Z., Zhou, M., Wang, L., Hu, F.,
Zhai, J., Meyers, B.C., and Cao, X. (2014). Dicer-like 3 produces
transposable element-associated 24-nt siRNAs that control agricultural traits in rice. Proc. Natl. Acad. Sci. USA 111: 3877–3882.
Wei, W., Ba, Z., Gao, M., Wu, Y., Ma, Y., Amiard, S., White, C.I.,
Rendtlew Danielsen, J.M., Yang, Y.G., and Qi, Y. (2012). A role for
small RNAs in DNA double-strand break repair. Cell 149: 101–112.
Wierzbicki, A.T., Haag, J.R., and Pikaard, C.S. (2008). Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135: 635–648.
Wierzbicki, A.T., Ream, T.S., Haag, J.R., and Pikaard, C.S. (2009).
RNA polymerase V transcription guides ARGONAUTE4 to chromatin. Nat. Genet. 41: 630–634.
Williams, L., Carles, C.C., Osmont, K.S., and Fletcher, J.C. (2005). A
database analysis method identifies an endogenous trans-acting
short-interfering RNA that targets the Arabidopsis ARF2, ARF3, and
ARF4 genes. Proc. Natl. Acad. Sci. USA 102: 9703–9708.
Wu, J., et al. (2015). Viral-inducible Argonaute18 confers broad-spectrum
virus resistance in rice by sequestering a host microRNA. eLife 4: 4.
Wu, L., Mao, L., and Qi, Y. (2012). Roles of dicer-like and argonaute
proteins in TAS-derived small interfering RNA-triggered DNA
methylation. Plant Physiol. 160: 990–999.
Wu, L., Zhang, Q., Zhou, H., Ni, F., Wu, X., and Qi, Y. (2009). Rice
microRNA effector complexes and targets. Plant Cell 21: 3421–
3435.
Wu, L., Zhou, H., Zhang, Q., Zhang, J., Ni, F., Liu, C., and Qi, Y.
(2010). DNA methylation mediated by a microRNA pathway. Mol.
Cell 38: 465–475.
Xie, Z., Allen, E., Wilken, A., and Carrington, J.C. (2005). DICER-LIKE 4
functions in trans-acting small interfering RNA biogenesis and
vegetative phase change in Arabidopsis thaliana. Proc. Natl. Acad.
Sci. USA 102: 12984–12989.
Xie, Z., Johansen, L.K., Gustafson, A.M., Kasschau, K.D., Lellis,
A.D., Zilberman, D., Jacobsen, S.E., and Carrington, J.C. (2004).
Genetic and functional diversification of small RNA pathways in
plants. PLoS Biol. 2: E104.
Yan, K.S., Yan, S., Farooq, A., Han, A., Zeng, L., and Zhou, M.M.
(2003). Structure and conserved RNA binding of the PAZ domain.
Nature 426: 468–474.
Yang, L., Wu, G., and Poethig, R.S. (2012). Mutations in the GWrepeat protein SUO reveal a developmental function for microRNAmediated translational repression in Arabidopsis. Proc. Natl. Acad.
Sci. USA 109: 315–320.
Ye, R., Chen, Z., Lian, B., Rowley, M.J., Xia, N., Chai, J., Li, Y., and
He, X.-J., Wierzbicki, A.T., and Qi, Y. (2016). A Dicer-independent
route for biogenesis of siRNAs that direct DNA methylation in
Arabidopsis. Mol. Cell. 61: 222–235
Ye, R., Wang, W., Iki, T., Liu, C., Wu, Y., Ishikawa, M., Zhou, X., and
Qi, Y. (2012). Cytoplasmic assembly and selective nuclear import of
Arabidopsis Argonaute4/siRNA complexes. Mol. Cell 46: 859–870.
Zhai, J., Zhang, H., Arikit, S., Huang, K., Nan, G.L., Walbot, V.,
and Meyers, B.C. (2015a). Spatiotemporally dynamic, cell-typedependent premeiotic and meiotic phasiRNAs in maize anthers. Proc.
Natl. Acad. Sci. USA 112: 3146–3151.
Zhai, J., et al. (2015b). A one precursor one siRNA model for Pol
IV-dependent siRNA biogenesis. Cell 163: 445–455.
Zhai, L., Sun, W., Zhang, K., Jia, H., Liu, L., Liu, Z., Teng, F., and
Zhang, Z. (2014). Identification and characterization of Argonaute
gene family and meiosis-enriched Argonaute during sporogenesis
in maize. J. Integr. Plant Biol. 56: 1042–1052.
Zhang, X., Niu, D., Carbonell, A., Wang, A., Lee, A., Tun, V., Wang,
Z., Carrington, J.C., Chang, C.E., and Jin, H. (2014). ARGONAUTE
PIWI domain and microRNA duplex structure regulate small RNA
sorting in Arabidopsis. Nat. Commun. 5: 5468.
Zhang, X., Yuan, Y.R., Pei, Y., Lin, S.S., Tuschl, T., Patel, D.J., and
Chua, N.H. (2006). Cucumber mosaic virus-encoded 2b suppressor
inhibits Arabidopsis Argonaute1 cleavage activity to counter plant
defense. Genes Dev. 20: 3255–3268.
Zhang, X., Zhao, H., Gao, S., Wang, W.C., Katiyar-Agarwal, S.,
Huang, H.D., Raikhel, N., and Jin, H. (2011). Arabidopsis Argonaute 2
regulates innate immunity via miRNA393(∗)-mediated silencing of a Golgilocalized SNARE gene, MEMB12. Mol. Cell 42: 356–366.
Zhao, K., Zhao, H., Chen, Z., Feng, L., Ren, J., Cai, R., and Xiang, Y.
(2015). The Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families in Populus trichocarpa: gene structure, gene expression, phylogenetic analysis and evolution. J. Genet. 94: 317–321.
Mechanisms and Functions of Plant Argonautes
Zhao, T., Li, G., Mi, S., Li, S., Hannon, G.J., Wang, X.J., and Qi, Y.
(2007). A complex system of small RNAs in the unicellular green
alga Chlamydomonas reinhardtii. Genes Dev. 21: 1190–1203.
Zheng, X., Zhu, J., Kapoor, A., and Zhu, J.K. (2007). Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J. 26: 1691–1701.
Zhong, X., Du, J., Hale, C.J., Gallego-Bartolome, J., Feng, S.,
Vashisht, A.A., Chory, J., Wohlschlegel, J.A., Patel, D.J., and
Jacobsen, S.E. (2014). Molecular mechanism of action of plant
DRM de novo DNA methyltransferases. Cell 157: 1050–1060.
Zhou, Y., Honda, M., Zhu, H., Zhang, Z., Guo, X., Li, T., Li, Z., Peng,
X., Nakajima, K., Duan, L., and Zhang, X. (2015). Spatiotemporal
sequestration of miR165/166 by Arabidopsis Argonaute10
285
promotes shoot apical meristem maintenance. Cell Reports 10:
1819–1827.
Zhu, H., Hu, F., Wang, R., Zhou, X., Sze, S.H., Liou, L.W., Barefoot,
A., Dickman, M., and Zhang, X. (2011). Arabidopsis Argonaute10
specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145: 242–256.
Zilberman, D., Cao, X., and Jacobsen, S.E. (2003). ARGONAUTE4
control of locus-specific siRNA accumulation and DNA and histone
methylation. Science 299: 716–719.
Zuckerkandl, E., and Pauling, L. (1965). Evolutionary divergence
and convergence in proteins. In Evolving Genes and Proteins,
V. Bryson and H.J. Vogel, eds (New York: Academic Press), pp.
97–166.
RNAi in Plants: An Argonaute-Centered View
Xiaofeng Fang and Yijun Qi
Plant Cell 2016;28;272-285; originally published online February 11, 2016;
DOI 10.1105/tpc.15.00920
This information is current as of June 18, 2017
Supplemental Data
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References
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