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, Chinab Tsinghua-Peking Center for Life Sciences, Beijing 100084, China

Argonaute (AGO) family proteins are effectors of RNAi in eukaryotes. AGOs bind small RNAs and use them as guides tosilence target genes or transposable elements at the transcriptional or posttranscriptional level. Eukaryotic AGO proteinsshare 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 proteinsand highlights recent progress toward our understanding of the mechanisms and functions of plant AGOs.

INTRODUCTION

RNAi refers to a conserved gene silencing process mediated bysmall RNAs (sRNAs). It has emerged as one of the most importantmechanisms in regulating gene expression and repressing trans-posable elements (TEs) at the transcriptional or posttranscriptionallevel in eukaryotes.

sRNAs are processed from stem-loop-structured or perfect longdouble-stranded RNAs by Dicer or DICER-LIKE (DCL) proteins. Inplants, several sRNA species that differ in biogenesis and functionshave been characterized. The predominant sRNA species includemicroRNAs (miRNAs) generated by DCL1 (Reinhart et al., 2002;Kurihara and Watanabe, 2004; Qi et al., 2005), trans-acting smallinterfering RNAs (ta-siRNAs) processed by DCL4 (Peragine et al.,2004;Vazquezet al., 2004;Gasciolli et al., 2005; Xie et al., 2005), andheterochromatic siRNAs (hc-siRNAs) produced by DCL3 (Xie et al.,2004; Qi et al., 2005; Henderson et al., 2006). Additional types ofsRNAs includingnaturalantisensesiRNAs (Borsanietal., 2005), longsiRNAs (Pontes et al., 2006), long miRNAs (lmiRNAs) (Wu et al.,2010), double-strand-break (DSB)-induced sRNAs (diRNAs) (Weiet al., 2012), andDCL-independent siRNA (sidRNAs) (Yeet al., 2016)have also been discovered. While miRNAs, ta-siRNAs, and nat-siRNAs mediate posttranscriptional gene silencing (PTGS),hc-siRNAs, lmiRNAs, and sidRNAs direct DNA methylation thusinducing transcriptional gene silencing (TGS).

Despite their diversity, all characterized sRNAs associate withARGONAUTE (AGO) family proteins to form the core of RNA-induced silencing complexes (RISCs). sRNAs function as guidesto direct RISCs to RNA or DNA targets through base-pairing(PetersandMeister, 2007;Vaucheret, 2008).EukaryoticAGOscanbe phylogenetically classified into two major groups: AGO andPIWI subfamilies (Carmell et al., 2002; Hutvagner and Simard,2008). AGO subfamily proteins are present in a wide range oforganisms with varied gene copy numbers, whereas PIWI sub-family proteins are absent in plants (Cerutti and Casas-Mollano,2006).

In plants, the roles of AGOs in PTGS and TGS have been wellestablished. Recent studies have revealed new functions of plantAGO proteins and provided further insights into the mechanismsof AGO action. In this Review, we summarize the general featuresof AGOs and mechanisms of RISC assembly and action, anddiscuss recent progress in understanding the roles of AGOs inplants.

ORIGIN AND DIVERSITY OF AGO FAMILY PROTEINS

The AGO family was named after AGO1 inArabidopsis thaliana, lossof which leads to tubular shaped leaves resembling small squids(Argonautus) (Bohmert et al., 1998). AGOs were later found to bepresent in bacteria, archaea, and eukaryotes, suggesting that theyhave an ancient origin (Cerutti and Casas-Mollano, 2006). Thenumber of AGO family members in different species varies greatly:There isonlyoneAGOprotein infissionyeast (Schizosaccharomycespombe), five in fly (Drosophila melanogaster), eight in humans(Homosapiens), and27 inworm (Caenorhabditiselegans). In plants,the AGO family has expanded during evolution. The green algaeChlamydomonas reinhardtii has three AGOs (Schroda, 2006; Zhaoet al., 2007),whilemoss (Physcomitrella patens) has six (Axtell et al.,2007; Arif et al., 2013). The number of AGOs further increases inflowering plants: 10 inArabidopsis (Carmell et al., 2002;Morel et al.,2002), 15 in poplar (Populus trichocarpa) (Zhao et al., 2015), 17 inmaize (Zeamays) (Qian et al., 2011; Zhai et al., 2014), and 19 in rice(Oryza sativa) (Kapoor et al., 2008). Based on phylogenetic rela-tionships, plant AGO proteins can be grouped into three majorcladesnamedafterArabidopsisAGOs:AGO1/5/10,AGO2/3/7,andAGO4/6/8/9 (Figure 1). It is noteworthy that rice and maize haveevolved 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 crystalstructures of domains of eukaryotic AGOs or entire prokaryoticAGOs (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

1 Address correspondence to qiyijun@tsinghua.edu.cn.www.plantcell.org/cgi/doi/10.1105/tpc.15.00920

The Plant Cell, Vol. 28: 272–285, February 2016, www.plantcell.org ã 2016 American Society of Plant Biologists. All rights reserved.

polysporus) Ago (Nakanishi et al., 2012) with guide RNA weresolved, greatly deepening our understanding about the structuresand functions of eukaryotic AGOs.

All eukaryotic AGOs are characterized by the presence of fourdomains: 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 lessunderstood. Structural analysis of Thermus thermophilus Ago

suggests that the N domain may block the propagation of guide-target pairing beyond position 16 (Wang et al., 2009). In vitrobiochemical studies suggest that the N domain of human Agos isrequired for sRNAduplex unwinding (Kwak andTomari, 2012) andimportant for cleavage activity (also known as slicer activity)(Hauptmann et al., 2013). It remains elusive how the N domaincontributes to these activities. The PAZ domain harbors an OB(oligonucleotide/oligosaccharide binding) fold that enables AGO

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 neighbor-joining treewas constructed using theMEGA6.0 software. Bootstrap values (10,000 replicates) are presented near each branch. Abbreviations for selectedspecies are as follows: Physcomitrella patens, Pp; Arabidopsis thaliana, At; Populus trichocarpa, Pt; Zea mays, Zm; Oryza sativa, Os.

Mechanisms and Functions of Plant Argonautes 273

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 theguide strand into a specific binding pocket, the PAZdomain bindsand anchors sRNAs (Lingel et al., 2004; Ma et al., 2004). In ad-dition, the PAZ domain contributes to slicer-independent un-winding of the duplexes (Gu et al., 2012). A rigid loop in the MIDdomain, termed the nucleotide specificity loop, specifically rec-ognizes the 59 nucleotide of sRNAs (Frank et al., 2010, 2012),accounting for the binding preferences of different AGO proteinsfor sRNAs with different 59-nucleotides. The MID-PIWI interfacecontains a basic binding pocket that helps to bind and anchor the59 phosphate of sRNAs (Parker et al., 2005). The PIWI domainadopts an RNase H-like fold and enables some, but not all, AGOproteins to cleave target RNAs complementary to the boundsRNAs (Song et al., 2004; Rivas et al., 2005). A catalytic triad (Asp-Asp-His/Asp, DDH/D) is generally thought to be responsible forslicer activity (Liu et al., 2004; Song et al., 2004; Rivas et al., 2005).More recently, structural analysis with budding yeast Ago sug-gests that slicer activity requires a catalytic tetrad with an addi-tional invariant Glu, rather than a triad (Nakanishi et al., 2012).

To date, no crystal structures of entire plant AGOsare available.Nevertheless, given that all solved eukaryotic AGO structures arehighly similar, and crystal structures of the MID domains fromArabidopsis AGOs resemble that of human Ago2 (Frank et al.,2010, 2012), it seems reasonable to base our understanding ofplant AGO structures on solved structures of other eukaryoticAGOs. Moreover, intensive analyses of Arabidopsis agomutantshave also provided important insights into the structures andfunctions of plant AGOs (Poulsen et al., 2013).

LOADING OF SMALL RNAs INTO AGO FAMILY PROTEINS

AGO proteins accommodate different sRNA species to performspecialized functions. The majority of sRNAs, including miRNAsand siRNAs, are produced as duplexes from precursors by Diceror DCLproteins and subsequently loadedontoAGOproteins. Theso-called passenger strand of the duplex is selectively displacedand degraded, while the guide strand is retained to form themature RISC.

Earlier biochemical studies in fly demonstrated that Dicer-2(Dcr-2) and its partner protein R2D2 are involved in loading ofsRNAs into Ago2 (Liu et al., 2003; Lee et al., 2004; Pham et al.,2004; Tomari et al., 2004b). The Dcr-2-R2D2 heterodimer is alsocapable of sensing the thermodynamic asymmetry of sRNA du-plexes, thereby determining which strand is to be retained in themature RISC (Tomari et al., 2004a). However, Dicer and its partnerproteins are not essential for the assembly of fly Ago1-RISC(Kawamata et al., 2009) and human Ago2-RISC (Kanellopoulouet al., 2005;Murchison et al., 2005; Betancur and Tomari, 2012). InArabidopsis, the DCL1 partner protein HYPONASTIC LEAVES1contributes to strand selection from miRNA duplexes by AGO1(Eamens et al., 2009; Manavella et al., 2012), the mechanism ofwhich remains to be determined.

There is a growing body of evidence indicating that the mo-lecular chaperone heat shock protein 90 (HSP90) and itscochaperones participate in sRNA loading onto AGO proteins.HSP90 binds to a set of folded proteins and undergoes confor-mational changes to promote ATP hydrolysis (Röhl et al., 2013). In

fly, the HSC70/HSP90 chaperone machinery associates withAgo1 andAgo2 and is required for the incorporation of sRNAs intoRISCs (Iwasaki et al., 2010; Miyoshi et al., 2010; Iwasaki et al.,2015). It has been proposed that the chaperone machinerysupports a loading-competent state of fly Ago2, allowing it toaccept siRNA duplexes from Dcr-2-R2D2 (Miyoshi et al., 2010;Iwasaki et al., 2015). In plants, studies using tobacco BY-2 lysatedemonstrated that HSP90 binds to Arabidopsis AGO1 and AGO4and promotes the loading of sRNA duplexes (Iki et al., 2010; Yeet al., 2012). The cochaperones of HSP90 are capable of mod-ulating the ATP-hydrolyzing rate of HSP90, recruiting client pro-teins, or playing chaperone roles on their own (Röhl et al., 2013).Biochemical studies in plants revealed that the cochaperoneCyclophilin 40/SQUINT (CYP40/SQN) in tobaccoandArabidopsisassociates with AGO1 in an HSP90-dependent manner and fa-cilitates RISC assembly (Earley andPoethig, 2011; Iki et al., 2012).In line with this, inactivation of SQN impairs miRNA activity inArabidopsis (Smith et al., 2009).Loading of sRNAs into AGOs can be a regulatory point for their

activity. A genetic screen identified SAD2/EMA1, an importinb familymemberoriginally implicated innuclear importofproteins,asanegative regulatorofmiRNA loading intoAGO1 inArabidopsis(Wang et al., 2011a). However, it remains to be determined whetherSAD2/EMA1 directly prevents the loading of miRNAs or indirectlycontrolsmiRNA loading through regulating thecytoplasmic-nuclearshuttling 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 duplexesappears to be a major determinant for sorting of miRNAs andsiRNAs into Ago1 and Ago2, respectively (Tomari et al., 2007;Czech et al., 2009). In plants, the identity of the 59 nucleotide playsa key role in sorting of an sRNA into a specificAGO (Mi et al., 2008;Montgomery et al., 2008; Takeda et al., 2008). Arabidopsis AGO1preferentially bindsmiRNAswith a 59U, AGO2 favors siRNAswitha 59A, AGO5 has a bias toward sRNAswith a 59C,whereas AGO4primarily associateswith hc-siRNAs bearing a 59A (Mi et al., 2008;Montgomery et al., 2008; Takeda et al., 2008).Arabidopsis miR390 and miR165/166 have 59 A and 59 U, re-

spectively. However, they are preferentially recruited by AGO7andAGO10, respectively, suggesting that additionalmechanismsexist to regulate sRNAsorting (Montgomery et al., 2008; Zhuet al.,2011). It has been shown that the 59 U is necessary but not suf-ficient for miR166 loading into AGO10 (Zhu et al., 2011), and itappears that specific mispairings and pairings in the miR166/miR166* duplex determine its predominant association withAGO10 (Zhu et al., 2011). In vitro experiments revealed that the59 A, the central mismatch within the miRNA duplex as well ascleavage-dependent removal of miR390* strand collectively de-termines the binding preference of AGO7 for miR390 (Endo et al.,2013). Recently, it was found that miRNA duplex structure alsocontributes to sorting of certain miRNAs into AGO1 or AGO2 inArabidopsis and both the QF-V motif and DDDE catalytic tetradwithin the PIWI domain are important for the recognition of thestructure (Zhang et al., 2014).In rice, in addition to 21-nucleotide canonical miRNAs (cmiR-

NAs) that are generated byDCL1, there is a class of 24-nucleotidelmiRNAs that are processedbyDCL3 (Wuet al., 2010).Most of thecmiRNAs initiate with 59 U and are incorporated into AGO1 clade

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proteins (Wu et al., 2009), following the 59nucleotide-directedloading rule. However, regardless of the identities of their 59 nu-cleotides, DCL3-dependent lmiRNAs are recruited by AGO4 cladeproteins. The specificity of sRNA sorting between AGO1 and AGO4clade proteins is unlikely to be determined by the length of thesRNAs, as both AGO1 and AGO4 clade proteins have comparablebinding affinities for 21- and 24-nucleotide sRNAs (Wu et al., 2010).These observations suggest that the enzymatic machinery un-derlying the biogenesis of cmiRNAs and lmiRNAs could dominateover the 59 terminal nucleotide to funnel their respective productsinto AGO1 and AGO4 clade proteins, respectively.

These studies collectively suggest that the specificity of sRNAsorting into AGOs is determined by the identity of 59 nucleotideand the structure of the sRNAduplex aswell as a signal emanatingfrom the upstream biogenesis machinery. The mechanisms un-derlying the recognition of sRNA duplex structure and biogenesismachinery by AGOs and/or loading factors remain to be in-vestigated.

ACTIONS OF PLANT AGO COMPLEXES

AGO/sRNA complexes have different modes of action (Figure 2).Their functions in gene regulation at the posttranscriptional levelthroughendonucleolytic cleavageand/or translational inhibitionandat the transcriptional level through directing DNA methylation havebeen well established (Law and Jacobsen, 2010; Rogers and Chen,2013). More recent studies have revealed that some AGOs haveadditional functions. These include Arabidopsis AGO10 and riceAGO18 that act as decoys of specificmiRNAs (Zhu et al., 2011; Wuet al., 2015) andAGO2 thatmediatesDSB repair (Wei et al., 2012;Baand Qi, 2013). Below we discuss the actions and mechanisms ofAGOs 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 (Montgomeryet al., 2008), andAGO10 (Ji et al., 2011; Zhu et al., 2011) havebeendemonstrated to have slicer activity that catalyzes sRNA-directedendonucleolytic cleavageof targetRNAs.AGO1 isoneof thebest-characterizedAGO familymembers inplants. AGO1predominantlybindsmiRNAs (BaumbergerandBaulcombe,2005;Qietal., 2005).Early studies showed that plant miRNAs display a high degree ofsequence complementarity to their target mRNAs and directtarget mRNA cleavage (Llave et al., 2002; Rhoades et al., 2002;Tang et al., 2003) whereas animal miRNAs are partially comple-mentary to their targets and regulate gene expression via trans-lational inhibition and/or mRNA destabilization (Lewis et al., 2003;Humphreys et al., 2005; Pillai et al., 2005). This led to the as-sumption that the degree of complementarity between an sRNAand its target serves as a switch between mRNA cleavage andtranslational inhibition. However, later studies showed that manyplant miRNAs are capable of repressing translation (Aukermanand Sakai, 2003; Chen, 2004; Gandikota et al., 2007; Brodersenet al., 2008; Li et al., 2013), arguing against this assumption.

The mechanism underlying the choice between mRNA cleav-age and translational inhibition by AGO1-miRISC remains to beelucidated. Emerging evidence suggests that the subcellular

compartmentalization of AGO1-miRISC may play a role in suchchoice. Genetic studies have implicated a role for the microtubulenetwork (Brodersen et al., 2008) and processing (P) bodies(Brodersen et al., 2008; Yang et al., 2012) in miRNA-mediatedtranslational repression. More recently, an elegant study showedthatmiRNAs inhibit translationof targetmRNAson the endoplasmicreticulum (Li et al., 2013). Altered Meristem Program1 (AMP1), anintegralmembraneprotein thatassociateswithAGO1, is required formiRNA-mediated translational repression (Li et al., 2013). In-triguingly, these components that are required for translational re-pressionaredispensable formiRNA-directed targetmRNAcleavage(Brodersen et al., 2008; Yang et al., 2012; Li et al., 2013), raising thepossibility that these two processes take place in different cellularcompartments. The deficiency of isoprenoids synthesis, which isessential for a variety of pathways including the production ofmembrane sterols, impairs miRNA-mediated cleavage of at leastsome target transcripts, suggesting that membrane association ofAGO1 is important for mRNA cleavage (Brodersen et al., 2012). Theidentity of the subcellular compartment where miRNA-mediatedRNA cleavage occurs remains to be revealed.

Sequestration

Sequestration has emerged as a common scenario of generegulation ineukaryotes.For instance,noncodingRNAscanactasdecoys 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 specificmiRNAs and abrogate their activities through sequestration (Zhuet al., 2011; Wu et al., 2015). Arabidopsis AGO10 predominantlybinds miR165/166, thereby preventing their loading into AGO1and the subsequent repression of HD-ZIP III, targets of miR165/166 (Zhuetal., 2011).RiceAGO18 is inducedbyvirus infectionandpreferentially associateswithmiR168 that targetsAGO1 (Wuetal.,2015). This association diminishes miR168-directed cleavage ofAGO1, thus increasing the levels of AGO1 accumulation andantiviral defense (Wu et al., 2015). In agreement with their roles asdecoys for miRNAs, sRNA binding but not slicing activity is re-quired for the functions of AGO10 in Arabidopsis shoot apicalmeristem development and AGO18 in rice antiviral defense (Zhuetal., 2011;Wuetal., 2015).Thesefindingssuggest thatArabidopsisAGO10-boundmiR165/166 and riceAGO18-boundmiR168 are notproficient incleavingtargetmRNAs,althoughbothAGOscontain thecatalytic triad forslicingactivity.This raisesan interestingquestionasto how their catalytic activities are repressed when they act asdecoys for miRNAs. Several possibilities have been envisioned: (1)the intrinsic catalytic activities of Arabidopsis AGO10 and riceAGO18areweaker thanAGO1; (2) thecatalyticactivitiesare inhibitedby yet to be identified interacting proteins; and (3)miRNAsboundbythese two AGOs are different from those bound by AGO1 in sub-cellular compartmentalization, preventing their access to targetmRNAs (Zhu et al., 2011; Wu et al., 2015).

DNA Methylation

DNA methylation is an epigenetic modification that plays criticalroles in regulating gene expression, repressing transposons, andmaintaining genome stability. In plants, DNA methylation can be

Mechanisms and Functions of Plant Argonautes 275

mediated byAGO4-bound hc-siRNAs through a specializedRNAipathway termed RNA-directed DNA methylation (RdDM). RdDMentails the generation of double-stranded RNAs (dsRNAs) bycoordinated activities of RNA Polymerase IV (Pol IV) and RNA-dependent RNA polymerase 2 (RDR2) (Xie et al., 2004; Herr et al.,2005; Kanno et al., 2005; Onodera et al., 2005; Li et al., 2008; Heet al., 2009; Haag et al., 2012). dsRNAs are then processed byDCL3 to produce 24-nucleotide hc-siRNAs (Xie et al., 2004).Recent studies suggest that Pol IV/RDR2 products are mainly 30to;40nucleotides in length andonaverageonly one siRNAcouldbe produced from each precursor (Blevins et al., 2015; Zhai et al.,2015b). hc-siRNAs are exported into the cytoplasm, where theyare loaded into AGO4, and mature AGO4/siRNA complexes areselectively transported into the nucleus, which may providea regulatory point prior to the effector stage of RdDM (Qi et al.,2006; Ye et al., 2012). The AGO4/siRNA complexes are recruitedto target loci via base-pairing with scaffold transcripts generatedbyRNApolymerasePol V (Pontier et al., 2005;Mosher et al., 2008;Wierzbicki et al., 2008, 2009). The recruitment of AGO4 to chro-matin may also be facilitated by its interaction with the GW/WG-rich 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) andKTF1/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 AGOactions(Azevedo et al., 2011; Garcia et al., 2012; Pontier et al., 2012).AGO4/siRNA complexes further recruit Domain RearrangedMethyltransferase 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 areproduced fromTAS loci canalsoberecruited by AGO4 to direct DNA methylation at TAS loci inArabidopsis (Wu et al., 2012). In rice, 24-nucleotide lmiRNAsgenerated by coordinated actions of DCL1 and DCL3 are asso-ciated with AGO4 to direct DNAmethylation in trans at themiRNAtarget genes, resulting in TGS in some cases (Wu et al., 2010). Inaddition to AGO4 that functions in the canonical RdDM pathway,AGO2 and AGO6 can also mediate de novo DNA methylation byrecruiting 21- to 22-nucleotide siRNAs, which are processed fromPol II/RDR6-dependentdsRNAsbyDCL2andDCL4 (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 to60 nucleotides in length, associated with AGO4, and capable ofdirecting DNA methylation. The production of sidRNA is de-pendent on distributive 39-59 exonucleases. It was proposed thatsidRNAs are the initial triggers of de novo DNA methylation (Yeet al., 2016).

DSB Repair

DSB is the most deleterious form of DNA damage and posesa major threat to genome stability if not properly repaired (Cicciaand Elledge, 2010). A class of;21-nucleotide diRNAswere foundto play an essential role in DSB repair in Arabidopsis and

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.

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mammaliancells (Wei et al., 2012;Gaoetal., 2014). InArabidopsis,diRNAs are produced from the vicinity of DSB sites in PolIV-dependent manner. However, diRNAs are recruited by AGO2anddonotmediateDSB repair throughdirectingDNAmethylationat theDSBsitesbecauseDRM2 isdispensable forDSB repair (Weiet al., 2012). In humans, the diRNA effector protein Ago2 formsa complex with the repair protein Rad51 and is required for therecruitment of Rad51 to DSB sites, suggesting that guided bydiRNAs,Ago2promotesRad51accumulation atDSBs to facilitaterepair (Gao et al., 2014). Whether the AGO2/diRNA complexes inArabidopsis function through similar mechanism remains to beinvestigated.

BIOLOGICAL ROLES OF PLANT AGO PROTEINS

The AGO family has expanded during plant evolution (Singh et al.,2015), leading to the functionaldiversificationofplantAGOs (Table1).Specialization of AGOs in different sRNA pathways and biologicalprocesses can be attributed to their intrinsic biochemical prop-erties, spatiotemporal expression patterns, and protein and/orsRNA partners with which they interact.

AGO1/5/10 Clade

AGO1 is theeffectorprotein formiRNAsand ta-siRNAs (Vaucheretet al., 2004; Baumberger andBaulcombe, 2005; Qi et al., 2005;Miet al., 2008). Guided by miRNAs and ta-siRNAs, AGO1 regulatesthe expression of genes that are involved in numerous de-velopmental and physiological processes (Fei et al., 2013; Rogersand Chen, 2013). AGO1 also functions in defense against someviruses upon loading with virus-derived siRNAs (vsiRNAs) (Morelet 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 de-velopmental phenotypes in rice (Wu et al., 2009), due to the ab-rogation of miRNA activity. Profiling of sRNAs associated withAGO1a, AGO1b, and AGO1c revealed that most miRNAs areevenly sorted intoAGO1a, AGO1b, andAGO1d,whereas a subsetof miRNAs are preferentially incorporated into or excluded fromoneof theAGO1s (Wuetal., 2009), suggestingboth redundantandspecialized functions for AGO1s in recruiting miRNAs. RiceAGO1s also bind vsiRNAs and confer resistance to viral diseases(Wu et al., 2015).

Arabidopsis AGO10 (previously known as ZLL or PNH) andAGO1arehighly similar in their protein sequencesbut differ in theirexpression patterns and developmental functions. In contrast tothe ubiquitous expression of AGO1, AGO10 is expressed inprovasculature, adaxial leaf primordia, and the meristem(Moussian et al., 1998; Lynn et al., 1999). AGO10 plays importantroles in the maintenance of undifferentiated stem cells of shootapical meristem (SAM) (Moussian et al., 1998; Lynn et al., 1999)and the establishment of leaf polarity (Liu et al., 2009). As men-tioned above, AGO10 specifically sequesters miR165/166 andabrogates their activities (Zhu et al., 2011). A recent study showedthat AGO10 quenches the activity of miR165/166 that moves intoAGO10-expressing zone, thus releasing theexpressionofHD-ZIPIII family genes to regulate meristem development (Zhou et al.,

2015). In addition to sequestering miR165/166, AGO10 mayregulate meristem development through mediating the trans-lational inhibition ofmultiplemiRNA target genes (Brodersen et al.,2008; Mallory et al., 2009) and act partly through miR172 topromote floral determinacy (Ji et al., 2011). AGO10 can also bindvsiRNAs and cooperatewith AGO1 to havemodest antiviral effectin inflorescences (Garcia-Ruiz et al., 2015).Rice has one homolog of AGO10 that is strongly expressed in

theprovascular region of the leaf primordia aroundSAMandplaysan important role in rice SAMmaintenance and leaf development(Nishimura et al., 2002), suggesting a conserved functionbetweenArabidopsis and rice AGO10s. It remains to be examined whetherrice AGO10 also functions as a miRNA decoy.The expression of ArabidopsisAGO5 is confined to the somatic

cells around megaspore mother cells as well as in the mega-spores, and a semidominant ago5 mutant is defective in the ini-tiation of megagametogenesis (Tucker et al., 2012), suggestingthat a somatic sRNA pathway mediated by AGO5 promotesmegagametogenesis. AGO5 preferentially binds sRNAs witha 59C that are derived from intergenic sequences (Mi et al., 2008),the function of which is largely unexplored. AGO5 can also bindvsiRNAs of Cucumber mosaic virus (Takeda et al., 2008), sug-gesting a role in antiviral defense. A recent study showed thatAGO5 is induced by Potato virus X infection and cooperates withAGO2 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 ingerm cells and has specific functions in the development ofpremeiotic germ cells and meiosis progression during sporo-genesis (Nonomura et al., 2007). LikeAGO5 inArabidopsis,MEL1/AGO5c predominantly associated with 21-nucleotide phasedsiRNAs (phasiRNAs) that are mainly derived from intergenic re-gions and initiate with a 59 C (Komiya et al., 2014). The function ofMEL1/phasiRNA complexes remains obscure, yet the cytoplasmiclocalization of MEL1 implies a potential role of MEL1/phasiRNA inposttranscriptional regulation of genes.AGO18 has evolved as a subclade conserved in monocots.

AGO18 expression is barely detectable in rice seedlings but ishighly induced by viral infection (Wu et al., 2015). Deficiency ofAGO18 leads to increasedsusceptibility to viral infection,whereasits overexpression enhances resistance to viral diseases in rice.AGO18 only binds very low levels of vsiRNAs but preferentiallyrecruitsmiR168 (Wuet al., 2015). Asmentioned above, it has beenshown that AGO18 confers viral resistance by competing withAGO1 for miR168 to upregulate AGO1 upon viral infection (Wuet al., 2015).Maize encodes two AGO18 subclade proteins (AGO18a and

AGO18b). AGO18b is predominantly expressed in the tapetalandmeiotic cells (Zhai et al., 2014),whichmatches thedistributionand timingof24-nucleotidemeioticphasiRNAs (Zhaietal., 2015a).This implies that AGO18b might be an effector protein for the24-nucleotide phasiRNAs.

AGO2/3/7 Clade

Arabidopsis AGO2 plays a major role in antiviral defense and hasbeen shown to be required for resistance to a broad spectrum of

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plant viruses (Harvey et al., 2011; Jaubert et al., 2011; Wanget al., 2011b). AGO2 binds vsiRNAs with 59 terminal A (Wanget al., 2011b), and the catalytic activity of AGO2 is essential for itsrole in antiviral defense (Carbonell et al., 2012). Consistent withthese findings, biochemical experiments using cytoplasmicextractsof evacuolated tobaccoprotoplasts revealed thatAGO2loaded with synthetic vsiRNAs can target viral RNAs for cleav-age, thereby inhibiting viral replication (Schuck et al., 2013).AGO2 also binds miR393b* to silence a Golgi-localized geneMEMB12 likely via translational repression, resulting in exo-cytosis of antimicrobial pathogenesis-related protein PR1 andincreased antibacterial activity (Zhang et al., 2011). Moreover,

the expression of AGO2 can be induced by g-irradiation thatcauses DNA lesions (Culligan et al., 2004), and it acts as aneffector 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 Potatospindle tuber viroid (Minoia et al., 2014), andAGO3programmedwith exogenous siRNAs can cleave viral RNAs in vitro (Schucket al., 2013). Thus far, no biological functions have been demon-strated for AGO3.Arabidopsis AGO7 (also known as ZIP) specifically binds

miR390 (Montgomery et al., 2008),which targetsTAS3 transcripts

Table 1. Biological Roles of AGO Proteins in Plants

AGO Name Locus IDsRNAsBound Function Reference

AtAGO1 AT1G48410 miRNAs Plant development, antiviral defense (Bohmert et al., 1998; Morel et al., 2002;Vaucheret et al., 2004; Baumberger andBaulcombe, 2005; Qi et al., 2005; Zhanget al., 2006)

ta-siRNAsvsiRNAs

OsAGO1a Os02g45070 miRNAs Plant development, antiviral defense (Wu et al., 2009, 2015)vsiRNAs

OsAGO1b Os04g47870 miRNAs Plant development, antiviral defense (Wu et al., 2009, 2015)vsiRNAs

OsAGO1c Os02g58490 miRNAs Plant development (Wu et al., 2009)AtAGO5 AT2G27880 siRNAs Megagametogenesis, antiviral defense (Takeda et al., 2008; Tucker et al., 2012;

Brosseau and Moffett, 2015)vsiRNAsOsAGO5c Os03g58600 phasiRNAs Germ cell division (Nonomura et al., 2007; Komiya et al., 2014)AtAGO10 AT5G43810 miR165/166 SAM development, antiviral defense (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; Zhouet al., 2015)

miR172vsiRNAs

OsAGO10 Os06g39640 n.d. SAM maintenance and leaf development (Nishimura et al., 2002)OsAGO18 Os07g28850 miR168 Antiviral defense (Wu et al., 2015)ZmAGO18b GRMZM2G457370 n.d. Tapetum and germ cell development (Zhai et al., 2015a)AtAGO2 AT1G31280 diRNAs DSB repair, antiviral defense, antibacterial

immunity(Harvey et al., 2011; Jaubert et al., 2011;

Wang et al., 2011b; Zhang et al., 2011;Wei et al., 2012)

vsiRNAsmiR393*

AtAGO3 AT1G31290 n.d. n.d.AtAGO7 AT1G69440 miR390 ta-siRNA biogenesis, phase transition,

antiviral defense(Hunter et al., 2003; Adenot et al., 2006;

Montgomery et al., 2008; Qu et al., 2008)OsAGO7 Os03g33650 miR390 ta-siRNA biogenesis, SAM development (Nagasaki et al., 2007)ZmAGO7 GRMZM5G892991 miR390 ta-siRNA biogenesis, leaf development (Douglas et al., 2010)AtAGO4 AT2G27040 hc-siRNAs DNA methylation, antibacterial immunity,

defense against DNA viruses(Zilberman et al., 2003; Li et al., 2006;

Pontes et al., 2006; Qi et al., 2006; Agorioand Vera, 2007; Raja et al., 2008; Wuet al., 2012)

ta-siRNAs

OsAGO4a Os01g16870 lmiRNAs DNA methylation (Wu et al., 2010; Wei et al., 2014)hc-siRNAs

OsAGO4b Os04g06770 lmiRNAs DNA methylation (Wu et al., 2010; Wei et al., 2014)hc-siRNAs

AtAGO6 AT2G32940 hc-siRNAs 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)

siRNAsta-siRNAs

OsAGO16 Os07g16224 lmiRNAs n.d. (Wu et al., 2010)AtAGO8 AT5G21030 n.d. n.d.AtAGO9 AT5G21150 siRNAs Gamete cell specification (Olmedo-Monfil et al., 2010)ZmAGO9 GRMZM2G141818 n.d. Somatic cell specification (Singh et al., 2011)

At, Arabidopsis thaliana; Os, Oryza sativa; Zm, Zea mays. n.d., not determined.

278 The Plant Cell

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 severalAUXINRESPONSEFACTORgenesthat are involved in the regulation of developmental timing andlateral 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-siRNAbiogenesis 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 withreleased silencing of theSUPERMAN (SUP) locus, and amutationin AGO4 leads to reduced asymmetric DNA methylation levels atSUP (Zilberman et al., 2003). AGO4 was later demonstrated to bethe effector protein for hc-siRNAs that direct DNA methylationgenome-wide through the RdDMpathway (Pontes et al., 2006; Qiet al., 2006). AGO4 is required for resistance to Pseudomonassyringae infection (Agorio and Vera, 2007). However, loss of otherRdDM pathway components does not impair resistance to thispathogen, suggesting thatAGO4 likely functions independently oftheRdDMpathway inmediatingbacterial resistance.AGO4 isalsoinvolved in plant defense against DNAviruses. In tissues that haverecovered from infection,DNAofBeetcurly topvirusL22mutant ishypermethylated and host recovery requires AGO4 (Raja et al.,2008), suggesting that DNA methylation mediated by AGO4 isa 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 AGO16can bind lmiRNAs and 24-nucleotide siRNAs that are mainlyproduced from miniature inverted repeat TEs and other TEs (Wuet al., 2010). Knocking down AGO4a and AGO4b affects GA andBR homeostasis-related genes and causes dwarfism phenotype,likely through affecting DNA methylation mediated by the24-nucleotide siRNAs associated with miniature inverted repeatTEs (Wei et al., 2014).

AGO6 was first identified in a screen for suppressors of ros1-1(Zhenget al., 2007).MutationofAGO6partially suppressesTGS intheDNAdemethylasemutant ros1-1 and affectsDNAmethylationlevels in several RdDM target loci (Zheng et al., 2007). AGO6appears to differ from AGO4 in binding preference for siRNAsproduced from different loci, which may be attributed to theirdifferent spatiotemporal expression and interaction with targetloci (Havecker et al., 2010). AGO4 is ubiquitously expressedthroughout plant, whereas AGO6 is predominantly expressed inshoot and root apical meristems and in tissues enriched for di-viding cells but not in mature leaves (Zheng et al., 2007; Haveckeret al., 2010; Eun et al., 2011). A recent study provided additionalinsights into the nonredundant roles of AGO4 and AGO6 (Duanet al., 2015). This work showed that AGO6 and AGO4 are bothrequired for DNAmethylation atmost of their target loci, indicatingthat they are mutually dependent. It was also shown that AGO4and AGO6 differ in their subnuclear colocalization and in inter-actionswithPol II andPol IV,and itwasproposed that theymayactsequentially tomediateDNAmethylation (Duanetal., 2015).AGO6also binds RDR6-dependent 21- to 22-nucleotide siRNAs gen-erated from transcriptionally active TEs and is thought to trigger

the initiation of de novo TE DNA methylation (Nuthikattu et al.,2013;McCueetal., 2015),whichmayprovidea linkbetweenPTGSand TGS of TEs.AGO8 and AGO9 are encoded by two neighboring loci and

highly similar in sequences. Sequence alignment showed thatAGO8 contains a deletion of one of the central strands in theb-sheet of the PIWI domain, presumably precluding correctfolding of this protein (Poulsen et al., 2013).AGO8 expression hasnot been detected in any tissues examined thus far and has beenproposed to be a pseudogene (Kapoor et al., 2008). AGO9 isexpressed in cytoplasmic foci of somatic companion cells. AGO9preferentially interacts with 24-nucleotide siRNAs derived fromTEs and silences TEs in female gamete and their accessory cellslikely in a non-cell-autonomousmanner, thereby repressing germcell fate in somatic cells (Olmedo-Monfil et al., 2010). Inmaize, theAGO9 homolog (named AGO104) is specifically expressed in thesomaticcellsaround theprecursorsof thegameticcellsbutacts torepress somatic fate in germ cells (Singh et al., 2011).

CONCLUSION

Since thediscoveryofAGO1 inArabidopsis, the foundingmemberof the AGO family, much has been learned about the diverse bi-ological functions of plant AGOs and the mechanisms throughwhich they act, yet many interesting questions remain unan-swered. miRNAs and hc-siRNAs are produced in the nucleus butloaded into AGOs in the cytoplasm. Are the biogenesis andloading of these sRNAs two separate processes or are theycoupled? How does the sRNA biogenesis machinery or a signalemanating from the biogenesis machinery affect sRNA loadinginto AGOs? As elaborated above, some AGOs can act in differentmodes. For instance, AGO1 can mediate endonucleolyticcleavage and translational repression; AGO10 can function intranslational repressing mediated by some miRNAs but acts asadecoy forspecificmiRNAs. It remainsobscurehowthesedistinctmodes 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. Itwill be also exciting to investigate whether AGOs have novelfunctions through mechanisms independent of the canonicalRNAi pathways.Despite major advances in understanding the functions of

ArabidopsisAGOs,AGOs inotherplants, including importantcropspecies, are poorly characterized. StudyofAGOs in other plants isneeded to deepen our understanding about the conservation anddiversification of AGO function and may reveal novel functions ofAGOs.

METHODS

Phylogenetic Analysis

Alignment of protein sequences was performed with ClustalW in Mega6.0 with default parameters. The alignment is shown in Supplemental DataSet 1. Phylogenetic analysis was performed usingMega 6.0 (Tamura et al.,2013). The evolutionary history was inferred using the Minimum Evolutionmethod (Nei, 1992). The neighbor-joining algorithm (Saitou and Nei, 1987)was used to generate the tree with bootstrap values (10,000 replicates)

Mechanisms and Functions of Plant Argonautes 279

(Felsenstein, 1985). The evolutionary distances were computed using thePoisson correction method (Zuckerkandl and Pauling, 1965) and are in theunits of thenumberof aminoacidsubstitutionsper site (SupplementalDataSet 2). Pairwise deletion was used to eliminate gaps in pairwise sequencecomparisons.

Supplemental Data

Supplemental Data Set 1. Alignment of Sequences of AGO Proteinsin Selected Plant Species.

Supplemental Data Set 2. Pairwise Matrix of the EvolutionaryDistances between AGO Proteins in Selected Plant Species.

ACKNOWLEDGMENTS

This review is dedicated to the memory of Professor Biao Ding, a greatmentor, inspirer, colleague, and friend who will be deeply missed by allthosewhohad the privilege of knowing andworkingwith him.Our researchis supported by grants fromNational Natural Science Foundation of China(Grants 31330042, 31421001, and 31225015) to Y.Q. We apologize tocolleagues 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 Feb-ruary 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 TAS3trans-acting siRNAs control leaf morphology through AGO7. Curr.Biol. 16: 927–932.

Agorio, A., and Vera, P. (2007). ARGONAUTE4 is required for re-sistance 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 biogenesisin plants. Cell 121: 207–221.

Arif, M.A., Frank, W., and Khraiwesh, B. (2013). Role of RNA in-terference (RNAi) in the moss Physcomitrella patens. Int. J. Mol. Sci.14: 1516–1540.

Aukerman, M.J., and Sakai, H. (2003). Regulation of flowering timeand floral organ identity by a microRNA and its APETALA2-liketarget genes. Plant Cell 15: 2730–2741.

Axtell, M.J., Jan, C., Rajagopalan, R., and Bartel, D.P. (2006). Atwo-hit trigger for siRNA biogenesis in plants. Cell 127: 565–577.

Axtell, M.J., Snyder, J.A., and Bartel, D.P. (2007). Common func-tions for diverse small RNAs of land plants. Plant Cell 19: 1750–1769.

Azevedo, J., Cooke, R., and Lagrange, T. (2011). Taking RISCs withAgo hookers. Curr. Opin. Plant Biol. 14: 594–600.

Ba, Z., and Qi, Y. (2013). Small RNAs: emerging key players in DNAdouble-strand break repair. Sci. China Life Sci. 56: 933–936.

Baumberger, N., and Baulcombe, D.C. (2005). Arabidopsis ARGONAUTE1is an RNA Slicer that selectively recruits microRNAs and short interferingRNAs. Proc. Natl. Acad. Sci. USA 102: 11928–11933.

Betancur, J.G., and Tomari, Y. (2012). Dicer is dispensable forasymmetric 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 re-quires an AGO4-interacting member of the SPT5 elongation factorfamily. 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 ofPol IV and RDR2-dependent precursors of 24 nt siRNAs guiding denovo DNA methylation in Arabidopsis. eLife 4: e09591.

Bohmert, K., Camus, I., Bellini, C., Bouchez, D., Caboche, M., andBenning, C. (1998). AGO1 defines a novel locus of Arabidopsiscontrolling 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 cis-antisense 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 andsiRNAs. Science 320: 1185–1190.

Brodersen, P., Sakvarelidze-Achard, L., Schaller, H., Khafif, M.,Schott, G., Bendahmane, A., and Voinnet, O. (2012). Isoprenoidbiosynthesis is required for miRNA function and affects membraneassociation of ARGONAUTE 1 in Arabidopsis. Proc. Natl. Acad. Sci.USA 109: 1778–1783.

Brosseau, C., and Moffett, P. (2015). Functional and genetic analysisidentify a role for Arabidopsis ARGONAUTE5 in antiviral RNA si-lencing. Plant Cell 27: 1742–1754.

Cao, X., and Jacobsen, S.E. (2002). Role of the Arabidopsis DRMmethyltransferases 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 ARGONAUTESusing slicer-defective mutants. Plant Cell 24: 3613–3629.

Carmell, M.A., Xuan, Z., Zhang, M.Q., and Hannon, G.J. (2002). TheArgonaute 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 andfunctions of RNA-mediated silencing: from protists to man. Curr.Genet. 50: 81–99.

Chen, X. (2004). A microRNA as a translational repressor of APETALA2 inArabidopsis 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 G2-phase 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). Hierarchicalrules 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 anARGONAUTE7-like protein required for mediolateral expansion, butnot dorsiventrality, of maize leaves. Plant Cell 22: 1441–1451.

Duan, C.G., et al. (2015). Specific but interdependent functions forArabidopsis 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 bindingprotein DRB1 directs guide strand selection from microRNA duplexes.RNA 15: 2219–2235.

280 The Plant Cell

Earley, K.W., and Poethig, R.S. (2011). Binding of the cyclophilin 40ortholog SQUINT to Hsp90 protein is required for SQUINT functionin 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 humanargonaute-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 evolution-arily conserved ARGONAUTE-binding platforms in RNAi-relatedcomponents. Genes Dev. 21: 2539–2544.

Endo, Y., Iwakawa, H.O., and Tomari, Y. (2013). ArabidopsisARGONAUTE7 selects miR390 through multiple checkpoints duringRISC 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 inshoot 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 ofAUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects de-velopmental timing and patterning in Arabidopsis. Curr. Biol. 16:939–944.

Fei, Q., Xia, R., and Meyers, B.C. (2013). Phased, secondary, smallinterfering RNAs in posttranscriptional regulatory networks. PlantCell 25: 2400–2415.

Felsenstein, J. (1985). Confidence limits on phylogenies: An ap-proach 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 Paz-Ares, J. (2007). Target mimicry provides a new mechanism forregulation of microRNA activity. Nat. Genet. 39: 1033–1037.

Frank, F., Hauver, J., Sonenberg, N., and Nagar, B. (2012). Arabi-dopsis Argonaute MID domains use their nucleotide specificity loopto sort small RNAs. EMBO J. 31: 3588–3595.

Frank, F., Sonenberg, N., and Nagar, B. (2010). Structural basis for59-nucleotide base-specific recognition of guide RNA by humanAGO2. Nature 465: 818–822.

Gandikota, M., Birkenbihl, R.P., Höhmann, S., Cardon, G.H.,Saedler, H., and Huijser, P. (2007). The miRNA156/157 recogni-tion element in the 39 UTR of the Arabidopsis SBP box gene SPL3prevents early flowering by translational inhibition in seedlings.Plant J. 49: 683–693.

Gao, M., et al. (2014). Ago2 facilitates Rad51 recruitment and DNAdouble-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-actingsiRNA 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 heli-case motifs underpin dual roles for SDE3 in antiviral defense andsilencing 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 pro-teins 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

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). Slicing-independent 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 ac-tivities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV andRDR2 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 ofAGO2 in plants. PLoS One 6: e14639.

Hauptmann, J., Dueck, A., Harlander, S., Pfaff, J., Merkl, R., andMeister, G. (2013). Turning catalytically inactive human Argonauteproteins 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., andBaulcombe, D.C. (2010). The Arabidopsis RNA-directed DNAmethylation argonautes functionally diverge based on their ex-pression 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 effectorof RNA-directed DNA methylation in arabidopsis is an ARGONAUTE4- 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 Arabidopsisthaliana DICER function in small RNA processing, gene silencingand 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. Science308: 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 miRNA-and 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 eukaryoticinitiation 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 het-erochronic gene ZIPPY is an ARGONAUTE family member. Curr.Biol. 13: 1734–1739.

Hunter, C., Willmann, M.R., Wu, G., Yoshikawa, M., de la LuzGutiérrez-Nava, M., and Poethig, S.R. (2006). Trans-acting siRNA-mediated repression of ETTIN and ARF4 regulates heteroblasty inArabidopsis. Development 133: 2973–2981.

Hutvagner, G., and Simard, M.J. (2008). Argonaute proteins: keyplayers in RNA silencing. Nat. Rev. Mol. Cell Biol. 9: 22–32.

Iki, T., Yoshikawa, M., Meshi, T., and Ishikawa, M. (2012). Cyclo-philin 40 facilitates HSP90-mediated RISC assembly in plants.EMBO J. 31: 267–278.

Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M.C., Matsumoto-Yokoyama, E., Mitsuhara, I., Meshi, T., and Ishikawa, M. (2010).In vitro assembly of plant RNA-induced silencing complexes facili-tated 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

Mechanisms and Functions of Plant Argonautes 281

machinery mediates ATP-dependent RISC loading of small RNAduplexes. 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 assemblyof the Drosophila RNAi enzyme complex. Nature 521: 533–536.

Jaubert, M., Bhattacharjee, S., Mello, A.F., Perry, K.L., andMoffett, P. (2011). ARGONAUTE2 mediates RNA-silencing antivi-ral defenses against Potato virus X in Arabidopsis. Plant Physiol.156: 1556–1564.

Ji, L., et al. (2011). ARGONAUTE10 and ARGONAUTE1 regulate thetermination of floral stem cells through two microRNAs in Arabi-dopsis. PLoS Genet. 7: e1001358.

Jinek, M., and Doudna, J.A. (2009). A three-dimensional view of themolecular 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 dif-ferentiation 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 DNAmethylation. 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, organi-zation and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymerase gene families and their expressionanalysis during reproductive development and stress in rice. BMCGenomics 9: 451.

Kawamata, T., Seitz, H., and Tomari, Y. (2009). Structural determi-nants of miRNAs for RISC loading and slicer-independent un-winding. 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 Ar-gonaute MEL1 protein binds to phasiRNAs generated from morethan 700 lincRNAs. Plant J. 78: 385–397.

Kurihara, Y., and Watanabe, Y. (2004). Arabidopsis micro-RNA bio-genesis 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 drivesduplex unwinding during RISC assembly. Nat. Struct. Mol. Biol. 19:145–151.

Law, J.A., and Jacobsen, S.E. (2010). Establishing, maintaining andmodifying 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 DrosophilaDicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell117: 69–81.

Lewis, B.P., Shih, I.H., Jones-Rhoades, M.W., Bartel, D.P., andBurge, C.B. (2003). Prediction of mammalian microRNA targets.Cell 115: 787–798.

Li, C.F., Henderson, I.R., Song, L., Fedoroff, N., Lagrange, T., andJacobsen, S.E. (2008). Dynamic regulation of ARGONAUTE4 withinmultiple 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 centercolocalized with Cajal bodies in Arabidopsis thaliana. Cell 126: 93–106.

Li, S., et al. (2013). MicroRNAs inhibit the translation of target mRNAson the endoplasmic reticulum in Arabidopsis. Cell 153: 562–574.

Lingel, A., Simon, B., Izaurralde, E., and Sattler, M. (2003). Structureand nucleic-acid binding of the Drosophila Argonaute 2 PAZ do-main. Nature 426: 465–469.

Lingel, A., Simon, B., Izaurralde, E., and Sattler, M. (2004). Nucleicacid 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., andWang, X. (2003). R2D2, a bridge between the initiation and effectorsteps of the Drosophila RNAi pathway. Science 301: 1921–1925.

Liu, Q., Yao, X., Pi, L., Wang, H., Cui, X., and Huang, H. (2009). TheARGONAUTE10 gene modulates shoot apical meristem mainte-nance and establishment of leaf polarity by repressing miR165/166in 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 ofArabidopsis miRNA. Science 297: 2053–2056.

Lynn, K., Fernandez, A., Aida, M., Sedbrook, J., Tasaka, M.,Masson, P., and Barton, M.K. (1999). The PINHEAD/ZWILLEgene acts pleiotropically in Arabidopsis development and hasoverlapping functions with the ARGONAUTE1 gene. Development126: 469–481.

Ma, J.B., Ye, K., and Patel, D.J. (2004). Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Na-ture 429: 318–322.

Mallory, A.C., Hinze, A., Tucker, M.R., Bouché, N., Gasciolli, V.,Elmayan, T., Lauressergues, D., Jauvion, V., Vaucheret, H., andLaux, T. (2009). Redundant and specific roles of the ARGONAUTEproteins AGO1 and ZLL in development and small RNA-directedgene 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 identifiesplant CPL phosphatases as regulators of miRNA processing factorHYL1. Cell 151: 859–870.

Marí-Ordóñez, A., Marchais, A., Etcheverry, M., Martin, A., Colot,V., and Voinnet, O. (2013). Reconstructing de novo silencing of anactive 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 transpos-able element mRNA-derived siRNAs to the establishment of DNAmethylation. EMBO J. 34: 20–35.

Mi, S., et al. (2008). Sorting of small RNAs into Arabidopsis argonautecomplexes 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 selectivelybind small RNAs derived from potato spindle tuber viroid and at-tenuate viroid accumulation in vivo. J. Virol. 88: 11933–11945.

Miyoshi, T., Takeuchi, A., Siomi, H., and Siomi, M.C. (2010). A directrole 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., andCarrington, J.C. (2008). Specificity of ARGONAUTE7-miR390 in-teraction and dual functionality in TAS3 trans-acting siRNA forma-tion. Cell 133: 128–141.

Morel, J.B., Godon, C., Mourrain, P., Béclin, C., Boutet, S.,Feuerbach, F., Proux, F., and Vaucheret, H. (2002). Fertile hypo-morphic ARGONAUTE (ago1) mutants impaired in post-transcriptionalgene 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 independentlyof its role in siRNA biogenesis. Proc. Natl. Acad. Sci. USA 105:3145–3150.

282 The Plant Cell

Moussian, B., Schoof, H., Haecker, A., Jürgens, G., and Laux, T.(1998). Role of the ZWILLE gene in the regulation of central shootmeristem cell fate during Arabidopsis embryogenesis. EMBO J. 17:1799–1809.

Murchison, E.P., Partridge, J.F., Tam, O.H., Cheloufi, S., andHannon, G.J. (2005). Characterization of Dicer-deficient murineembryonic 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 in-terfering RNA production pathway is required for shoot meristeminitiation 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 min-imum 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 theshoot 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 ofthe ARGONAUTE family is essential for the progression of pre-meiotic mitosis and meiosis during sporogenesis in rice. Plant Cell19: 2583–2594.

Nuthikattu, S., McCue, A.D., Panda, K., Fultz, D., DeFraia, C.,Thomas, E.N., and Slotkin, R.K. (2013). The initiation of epigeneticsilencing of active transposable elements is triggered by RDR6 and21-22 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 offemale 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 medi-ates siRNA and DNA methylation-dependent heterochromatin for-mation. Cell 120: 613–622.

Parker, J.S., Roe, S.M., and Barford, D. (2005). Structural insightsinto 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 juveniledevelopment and the production of trans-acting siRNAs in Arabi-dopsis. Genes Dev. 18: 2368–2379.

Peters, L., and Meister, G. (2007). Argonaute proteins: mediators ofRNA silencing. Mol. Cell 26: 611–623.

Pham, J.W., Pellino, J.L., Lee, Y.S., Carthew, R.W., andSontheimer, E.J. (2004). A Dicer-2-dependent 80s complexcleaves 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 oftranslational initiation by Let-7 microRNA in human cells. Science309: 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 Arabidopsischromatin-modifying nuclear siRNA pathway involves a nucleolarRNA 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

repeated sequences requires the concerted action of two distinctRNA polymerases IV in Arabidopsis. Genes Dev. 19: 2030–2040.

Pontier, D., et al. (2012). NERD, a plant-specific GW protein, definesan additional RNAi-dependent chromatin-based pathway in Arabi-dopsis. Mol. Cell 48: 121–132.

Poulsen, C., Vaucheret, H., and Brodersen, P. (2013). Lessons onRNA silencing mechanisms in plants from eukaryotic argonautestructures. Plant Cell 25: 22–37.

Qi, Y., Denli, A.M., and Hannon, G.J. (2005). Biochemical speciali-zation 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 inRNA-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, Argonauteand RNA-dependent RNA polymerase gene families in maize. PlantCell 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 si-lencing 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 gem-iniviruses. J. Virol. 82: 8997–9007.

Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B., andBartel, 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. Cell110: 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 siRNAform recombinant human RISC. Nat. Struct. Mol. Biol. 12: 340–349.

Rogers, K., and Chen, X. (2013). Biogenesis, turnover, and mode ofaction of plant microRNAs. Plant Cell 25: 2383–2399.

Röhl, A., Rohrberg, J., and Buchner, J. (2013). The chaperoneHsp90: changing partners for demanding clients. Trends Biochem.Sci. 38: 253–262.

Saitou, N., and Nei, M. (1987). The neighbor-joining method: a newmethod for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406–425.

Schirle, N.T., and MacRae, I.J. (2012). The crystal structure of humanArgonaute2. Science 336: 1037–1040.

Schroda, M. (2006). RNA silencing in Chlamydomonas: mechanismsand 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 plantin 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). Productionof viable gametes without meiosis in maize deficient for an ARGONAUTEprotein. Plant Cell 23: 443–458.

Singh, R.K., Gase, K., Baldwin, I.T., and Pandey, S.P. (2015). Mo-lecular evolution and diversification of the Argonaute family ofproteins 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 formicroRNA 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). Thecrystal structure of the Argonaute2 PAZ domain reveals an RNA

Mechanisms and Functions of Plant Argonautes 283

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 sliceractivity. Science 305: 1434–1437.

Takeda, A., Iwasaki, S., Watanabe, T., Utsumi, M., and Watanabe,Y. (2008). The mechanism selecting the guide strand from smallRNA duplexes is different among argonaute proteins. Plant CellPhysiol. 49: 493–500.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S.(2013). MEGA6: Molecular Evolutionary Genetics Analysis version6.0. Mol. Biol. Evol. 30: 2725–2729.

Tang, G., Reinhart, B.J., Bartel, D.P., and Zamore, P.D. (2003). Abiochemical 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 mutantarmitage. Cell 116: 831–841.

Tomari, Y., Du, T., and Zamore, P.D. (2007). Sorting of Drosophilasmall 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., andKoltunow, A.M. (2012). Somatic small RNA pathways promote themitotic events of megagametogenesis during female reproductivedevelopment 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 bythe 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 ofArabidopsis 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 microRNAactivity 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 21-nucleotide, but not 22-nucleotide, viral secondary small interferingRNAs direct potent antiviral defense by two cooperative argonautes inArabidopsis 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 oftarget 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 producestransposable element-associated 24-nt siRNAs that control agri-cultural 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 forsmall RNAs in DNA double-strand break repair. Cell 149: 101–112.

Wierzbicki, A.T., Haag, J.R., and Pikaard, C.S. (2008). Noncoding tran-scription by RNA polymerase Pol IVb/Pol V mediates transcriptional si-lencing 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 chroma-tin. Nat. Genet. 41: 630–634.

Williams, L., Carles, C.C., Osmont, K.S., and Fletcher, J.C. (2005). Adatabase analysis method identifies an endogenous trans-actingshort-interfering RNA that targets the Arabidopsis ARF2, ARF3, andARF4 genes. Proc. Natl. Acad. Sci. USA 102: 9703–9708.

Wu, J., et al. (2015). Viral-inducible Argonaute18 confers broad-spectrumvirus resistance in rice by sequestering a host microRNA. eLife 4: 4.

Wu, L., Mao, L., and Qi, Y. (2012). Roles of dicer-like and argonauteproteins in TAS-derived small interfering RNA-triggered DNAmethylation. Plant Physiol. 160: 990–999.

Wu, L., Zhang, Q., Zhou, H., Ni, F., Wu, X., and Qi, Y. (2009). RicemicroRNA 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 4functions in trans-acting small interfering RNA biogenesis andvegetative 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 inplants. 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 GW-repeat protein SUO reveal a developmental function for microRNA-mediated 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., andHe, X.-J., Wierzbicki, A.T., and Qi, Y. (2016). A Dicer-independentroute for biogenesis of siRNAs that direct DNA methylation inArabidopsis. Mol. Cell. 61: 222–235

Ye, R., Wang, W., Iki, T., Liu, C., Wu, Y., Ishikawa, M., Zhou, X., andQi, Y. (2012). Cytoplasmic assembly and selective nuclear import ofArabidopsis 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-type-dependent 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 PolIV-dependent siRNA biogenesis. Cell 163: 445–455.

Zhai, L., Sun, W., Zhang, K., Jia, H., Liu, L., Liu, Z., Teng, F., andZhang, Z. (2014). Identification and characterization of Argonautegene family and meiosis-enriched Argonaute during sporogenesisin 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). ARGONAUTEPIWI domain and microRNA duplex structure regulate small RNAsorting in Arabidopsis. Nat. Commun. 5: 5468.

Zhang, X., Yuan, Y.R., Pei, Y., Lin, S.S., Tuschl, T., Patel, D.J., andChua, N.H. (2006). Cucumber mosaic virus-encoded 2b suppressorinhibits Arabidopsis Argonaute1 cleavage activity to counter plantdefense. 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 2regulates innate immunity via miRNA393(∗)-mediated silencing of a Golgi-localized 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 poly-merase gene families in Populus trichocarpa: gene structure, gene ex-pression, phylogenetic analysis and evolution. J. Genet. 94: 317–321.

284 The Plant Cell

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 greenalga Chlamydomonas reinhardtii. Genes Dev. 21: 1190–1203.

Zheng, X., Zhu, J., Kapoor, A., and Zhu, J.K. (2007). Role of Arabi-dopsis AGO6 in siRNA accumulation, DNA methylation and tran-scriptional 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., andJacobsen, S.E. (2014). Molecular mechanism of action of plantDRM 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). Spatiotemporalsequestration of miR165/166 by Arabidopsis Argonaute10

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 Argonaute10specifically sequesters miR166/165 to regulate shoot apical meri-stem development. Cell 145: 242–256.

Zilberman, D., Cao, X., and Jacobsen, S.E. (2003). ARGONAUTE4control of locus-specific siRNA accumulation and DNA and histonemethylation. Science 299: 716–719.

Zuckerkandl, E., and Pauling, L. (1965). Evolutionary divergenceand convergence in proteins. In Evolving Genes and Proteins,V. Bryson and H.J. Vogel, eds (New York: Academic Press), pp.97–166.

Mechanisms and Functions of Plant Argonautes 285

DOI 10.1105/tpc.15.00920; originally published online February 11, 2016; 2016;28;272-285Plant Cell

Xiaofeng Fang and Yijun QiRNAi in Plants: An Argonaute-Centered View

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