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Roles of Plant Small RNAsin Biotic Stress ResponsesVirginia Ruiz-Ferrer and Olivier VoinnetInstitut de Biologie Moleculaire des Plantes du CNRS, UPR2357, 67084 Strasbourg Cedex,France; email: olivier.voinnet@ibmp-ulp.u-strasbg.fr

Annu. Rev. Plant Biol. 2009. 60:485–510

The Annual Review of Plant Biology is online atplant.annualreviews.org

This article’s doi:10.1146/annurev.arplant.043008.092111

Copyright c© 2009 by Annual Reviews.All rights reserved

1543-5008/09/0602-0485$20.00

Key Words

RNA silencing, innate immunity, virus, bacteria, suppressors

AbstractA multitude of small RNAs (sRNAs, 18–25 nt in length) accumulatein plant tissues. Although heterogeneous in size, sequence, genomicdistribution, biogenesis, and action, most of these molecules mediaterepressive gene regulation through RNA silencing. Besides their rolesin developmental patterning and maintenance of genome integrity,sRNAs are also integral components of plant responses to adverseenvironmental conditions, including biotic stress. Until recently, an-tiviral RNA silencing was considered a paradigm of the interactionslinking RNA silencing to pathogens: Virus-derived sRNAs silence vi-ral gene expression and, accordingly, viruses produce suppressor pro-teins that target the silencing mechanism. However, increasing evidenceshows that endogenous, rather than pathogen-derived, sRNAs also havebroad functions in regulating plant responses to various microbes. Inturn, microbes have evolved ways to inhibit, avoid, or usurp cellularsilencing pathways, thereby prompting the deployment of counter-counterdefensive measures by plants, a compelling illustration of theneverending molecular arms race between hosts and parasites.

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Contents

BUILDING BLOCKS OF THEMAIN RNA-SILENCINGPATHWAYS IN PLANTS . . . . . . . . . 486Processors and Operators of RNA

Silencing in Arabidopsis . . . . . . . . . . 486Major Cellular RNA-Silencing

Pathways of Arabidopsis . . . . . . . . . . 488INDUCTION OF

RNA SILENCING DURINGDEFENSE RESPONSES. . . . . . . . . . 488Small RNAs Produced from

Pathogen-Derived NucleicAcids . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

Endogenous Small RNAs WhoseExpression Is Altered byPathogens . . . . . . . . . . . . . . . . . . . . . . 491

OPERATING RNA SILENCINGIN PLANT-PATHOGENINTERACTIONS . . . . . . . . . . . . . . . . 496Antiviral RNA-Induced Silencing

Complexes and Their PossibleActivities . . . . . . . . . . . . . . . . . . . . . . . 497

Effecting miRNA, nat-siRNA, andlsiRNA Defensive Functions. . . . . 499

Role of AGO4 in AntibacterialDefense: A Link toRNA-Directed DNAMethylation? . . . . . . . . . . . . . . . . . . . 500

SUPPRESSION, AVOIDANCE,AND USURPATION OF RNASILENCING BYPATHOGENS . . . . . . . . . . . . . . . . . . . . 500Plant Viruses . . . . . . . . . . . . . . . . . . . . . . 500RNA Silencing Suppression

by Other Pathogens . . . . . . . . . . . . . 503HOST RESPONSES TO

SILENCING SUPPRESSIONBY PATHOGENS. . . . . . . . . . . . . . . . . 505R Proteins and Direct Targeting

of VSR and BSRIntegrity/Function . . . . . . . . . . . . . . 505

Altering the Levels of EndogenousSmall RNAs That RegulateAntiviral or AntibacterialComponents . . . . . . . . . . . . . . . . . . . . 505

Sentinel Small RNAs Generatedat Complex R Gene Loci . . . . . . . . 505

Effectors: virulencefactors injected orsecreted into host cellsby pathogens such usbacteria, fungi,nematodes, or insects

BUILDING BLOCKS OF THEMAIN RNA-SILENCINGPATHWAYS IN PLANTS

Processors and Operators of RNASilencing in Arabidopsis

Plant RNA-silencing phenomena share fourconsensus biochemical steps: (a) induction bydouble-stranded RNA (dsRNA), (b) dsRNAprocessing into 18–25-nt small RNA (sRNA),(c) 2′-O-methylation of sRNA, and (d ) sRNAincorporation into effector complexes thatassociate with partially or fully complementarytarget RNA or DNA. dsRNA might derive di-rectly from virus replication, inverted repeats,or convergent transcription of transgenes ortransposons. dsRNA formation may also begenetically programmed at endogenous loci

that produce transcripts with internal stem-loop structures. Alternatively, dsRNA maybe synthesized by one of six RNA-dependentRNA polymerases (RDR1–6) that copy single-stranded RNA (ssRNA). RDR templatesinclude mRNAs with aberrant features ortranscripts produced by a putative plant-specific RNA polIV, whose subunit NRPD1ais found at certain methylated loci (reviewed inReferences 11 and 14) (Figure 1a).

In Arabidopsis, the dsRNA is processed intospecifically sized sRNA duplexes by one offour Dicer-like (DCL1–4) proteins. DCL1synthesizes 18–21-nt-long sRNA, whereasthe products of DCL2, DCL3, and DCL4are 22 nt, 24 nt, and 21 nt long, respectively(78). dsRNA processing, called dicing, is facil-itated by one of five dsRNA-binding proteins

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RNA Pol II

MIRNA gene

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RNA-dependent RNA polymerases: RDR1–6

2

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(A)nRNA Pol II

NAT pair

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Inverted repeat

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Dicer-like proteins: DCL1–4dsRNA-binding proteins: HYL1, DRB2–5

DCL

CH3

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Cytosol

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(A)n

NRPD1a and/or RNA Pol II

aberrant RNA

19–25 nt

AGO

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RNA cleavage

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Repression of translation

CH3

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DNA cytosine and/orhistone methylation

DRB

ARGONAUTE proteins:AGO1–10

or

a dsRNA production d sRNA function

b

sRNA processing

c sRNA stabilization & export

HEN1

CH3

HST NRPD1b

Figure 1Consensus steps of Arabidopsis RNA silencing pathways. (a) Various sources of double-stranded RNA (dsRNA) and (b) its processinginto small RNAs (sRNAs) by one of four Dicer-like proteins (DCLs) assisted by dsRNA-binding proteins. (c) HUA ENHANCER 1(HEN1)-mediated sRNA stabilization and export by HASTY (HST). (d ) sRNA operation. Selected strands of sRNA duplexes guideARGONAUTE (AGO)-containing RNA-induced silencing complex (RISC) to target RNAs (1) for endonucleolytic cleavage,(2) for translation repression, or (3) to target chromatin for cytosine and/or histone methylation together with NRPD1b.

(HYPONASTIC 1 or HYL1 and DRB2–5) thatinteract with specific DCLs (Figure 1b). Upondicing, the sRNA 3′ overhanging ends are 2′-O-methylated by the methyltransferase HUAENHANCER 1 (HEN1) (83), which protectsthem from oligouridylation and degradation(Figure 1c). Stabilized sRNA duplexes are thenretained nuclearly for chromatin-level activitiesor exported cytoplasmically, possibly via theexportin-5 homolog HASTY (HST), for post-transcriptional gene silencing (PTGS). One se-lected sRNA strand incorporates one or severalRNA-induced silencing complexes (RISC) thatscan the cell for complementary nucleic acidsto execute their function. sRNA-directed RISCactivities include (a) RNA endonucleolytic

cleavage (slicing) at the center of sRNA-targethybrids, (b) translational repression throughunknown mechanisms, and (c) DNA cytosineand/or histone methylation (Figure 1d ) withthe assistance of Pol IV subunit b (NRPD1b).Eukaryotic RISCs invariably include anARGONAUTE (AGO) protein. AGOs containa sRNA-binding PAZ domain and a PIWI do-main with catalytic residues conferring endonu-cleolytic activity to those RISCs programmedto slice RNA. Among the ten predicted Ara-bidopsis family members (AGO1–10), roles forAGO1, AGO4, AGO6, and AGO7 in sRNA-directed silencing have been established, and aslicer activity has been demonstrated for AGO1and AGO4 (reviewed in References 11 and 14).

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RNA interference(RNAi): RNAsilencing process bywhich exogenousdsRNA directs theposttranscriptionalsilencing ofhomologous genes

Transfer DNA(T-DNA): DNA oftumor-inducing (Ti)plasmids of somespecies of plantbacteria such asA. tumefaciens andA. rhizogenes

Major Cellular RNA-SilencingPathways of Arabidopsis

High-throughput cloning and sequencingshow that the Arabidopsis sRNA repertoire islargely dominated by short-interfering RNAs(siRNAs) that act mostly at the chromatin leveland by microRNAs (miRNAs) (29, 63). Syn-thesis of other classes of cellular sRNA, someof which are covered in specific sections ofthis review, occurs through combinations of themiRNA and siRNA pathways detailed below.

microRNA pathway. Most MIRNA genesare intronic or intergenic RNA pol II tran-scription units, whose expression frequentlyexhibits high tissue specificity and/or sensi-tivity to external stimuli including microbialchallenges. MIRNA transcription yields aprimary miRNA transcript (pri-miRNA) thatforms an imperfect fold-back structure. Thepri-miRNA is processed into a stem-loop pre-cursor (premiRNA) and then diced as a duplexcontaining the mature miRNA and a labilepassenger strand called miRNA∗ (reviewed inReference 36). The forkhead-associated do-main protein DAWDLE (DDL) is requiredfor pri-RNA accumulation (82), whereasboth HYL1 and the zinc-finger proteinSERRATE (SE) are required for pri-miRNA-to-premiRNA processing. Most ArabidopsismiRNAs are matured in subnuclear bodies byDCL1, although a few appear to be DCL4dependent (29, 63). Upon HEN1-mediated2′O-methylation, the mature miRNA strandis selectively incorporated into AGO1-containing or AGO10-containing RISCs topromote slicing or translational repressionof target transcripts (10) (Figure 5a, right).miRNA function is believed to be cytoplasmic,following HST-mediated transport of miRNA-RISCs or miRNA duplexes from the nucleus.More than 180 Arabidopsis MIRNA loci havebeen identified, representing nearly 80 miRNAfamilies, many of which are important for plantdevelopment. Some miRNA are also inducedor repressed by abiotic and biotic stresses (36),as detailed in this review.

Short-interfering RNA pathway. Among theendogenous siRNA pathways of Arabidopsis,the heterochromatic pathway largely domi-nates by the sheer amount and sequence di-versity of sRNA it produces at transposonloci and DNA repeats. RDR2, RNA pol IV,and DCL3 cooperatively generate the largestbulk of heterochromatin-associated 24-ntsiRNAs. These siRNAs incorporate into AGO4or AGO6 and guide cytosine methylation inall sequence contexts, a landmark of RNA-directed DNA methylation (RdDM). RdDM isalso accompanied by histone modifications, in-cluding deacetylation and methylation. Hete-rochromatic siRNAs are often referred to as cis-acting siRNAs because they affect the genomicloci that produce them, which often results intheir transcriptional gene silencing (TGS; re-viewed in Reference 13). Other siRNAs pro-duced at discrete endogenous loci act in transto direct PTGS of mRNAs notably involvedin developmental phase changes and organ po-larity. These trans-acting siRNAs (ta-siRNAs)are produced upon miRNA-guided cleavage ofnoncoding primary transcripts that are thenconverted into dsRNA by RDR6. The dsRNAis sequentially diced by DCL4 in a phased re-action that can be carried out by DCL2 whenDCL4 is genetically inactivated (reviewed inReference 14). DCL4 and DCL2 also redun-dantly mediate dsRNA-mediated RNA inter-ference (RNAi) used for experimental geneknockdown in plants (25).

INDUCTION OFRNA SILENCING DURINGDEFENSE RESPONSES

This section discusses the genetic pathwaysunderlying RNA silencing activation by plantparasites. Some sRNAs are produced directlyfrom pathogen-derived nucleic acids, includ-ing viral RNA and Agrobacterium tumefacienstransfer DNAs (T-DNAs). Other pathogensand pests induce modification of cellularsRNA profiles, which in turn impact expres-sion of regulators or effectors of host defensepathways.

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Small RNAs Produced fromPathogen-Derived Nucleic Acids

Viruses. sRNA cloning and high-throughputsequencing in virus-infected plants identifiedfold-back structures within single-strandedviral transcripts as major dsRNA sourcesfrom DNA viruses, RNA viruses, satellites, andviroids. Replication intermediates (RIs) synthe-sized by viral-encoded replicases also accountfor dsRNA production by some RNA viruses(32, 33, 47), whereas converging transcriptionof nuclear transcripts is a major dsRNA sourcefrom DNA geminiviruses (16). dsRNA arisingdirectly from structural features or replicationof viral genomes generates primary viral smallRNA (vsRNA), in contrast to secondary vsRNAproduced by host-encoded RDRs that convertviral ssRNA into dsRNA. Arabidopsis RDR6and RDR1 [the latter is induced by salycilicacid (SA), a defense-signaling compound com-monly produced during pathogen infections]play major and probably redundant antiviralroles (21, 23, 61*). These functions requirecofactors, including the putative RNA helicaseSILENCING DEFECTIVE 3 (SDE3) andthe coiled-coil domain protein SUPPRESSOROF GENE SILENCING 3 (SGS3), necessaryfor RDR6-mediated suppression of RNA andDNA virus accumulation (7, 19, 50, 51). Theheterochromatic RDR2 is additionally re-quired for silencing DNA viruses and the RNAvirus Tobacco rattle virus (TRV) (23, 62). HostRDR activities are stimulated by RNAs lackingquality control marks, such as a 5′-cap or a 3′-polyA tail, features frequently exhibited by viralRNAs (reviewed in Reference 75). Additionally,primary vsRNAs may promote RDR activitiesby generating uncapped or polyA− RNAfragments upon RISC-mediated slicing of viraltranscripts or by acting as primers for RDR-directed dsRNA synthesis (45) (Figure 2). RDRfunctions are also required for amplifying a sys-temic silencing response that contributes im-munizing tissues that are yet to be infected (seeNon-Cell-Autonomous Silencing in Plants).

These intricate dsRNA-generating path-ways make it difficult to distinguish the rel-

NON-CELL-AUTONOMOUS SILENCINGIN PLANTS

Unlike the miRNA pathway, the DCL4-dependent siRNA path-way is not cell autonomous in plants: 21-nt siRNAs or their longdsRNA precursors move between cells through plasmodesmataand over long distances through the phloem. RDRs involved inPTGS, most notably RDR6, play a crucial role in this movementprocess by amplifying signal molecules or their precursors, thusensuring a potent and sustained response throughout plants. Al-though silencing spread has been studied mostly in the context oftransgene silencing, it has also been observed indirectly duringvirus infections, where it likely constitutes the systemic compo-nent of antiviral RNA silencing. For instance, viral suppressors ofRNA silencing (VSR)-deficient viruses accumulating in vascularbundles fail to unload into neighboring cells. Although virus-free,these cells exhibit sequence-specific resistance to secondary infec-tion, a phenomenon alleviated in the Arabidopsis loss-of-functiondcl4 mutant. Thus, cell-to-cell spread and amplification ofDCL4-dependent silencing signals likely immunize tissues justahead of the infection. Phloem-mediated silencing spread be-tween distant organs has also been demonstrated with movement-deficient and replication-proficient recombinant viruses, whichpromote systemic silencing responses in noninoculated tissues.As in the case of cell-to-cell movement, phloem-mediated trans-port of RNA silencing also likely has antiviral roles because itis precluded by the CMV-encoded VSR 2b. Moreover, silenc-ing amplification upon vascular transport probably helps im-munize recipient tissues, because RDR6 activities that enabledetection/amplification of long-distance transgene silencing ex-clude many plant viruses from meristems in apical growing points.

Satellite: a subviralagent composed ofnucleic acid thatdepends on thecoinfection of a hostcell with a helper ormaster virus for itsmultiplication

ative importance of primary versus secondaryvsRNA synthesis in antiviral defense, whichmoreover might vary from one virus to an-other. Thus, TRV-derived vsRNA are nearlyeliminated in triple rdr1 rdr2 rdr6 mutants, in-dicating that these molecules originate mostlyfrom dicing of RDR products rather than fromintrinsic dsRNA features of the TRV genome(23). Another difficulty in distinguishing pri-mary and secondary vsRNA contribution to an-tiviral silencing is that both derive from simi-lar DCL activities. Hence, DCL4 and DCL2act redundantly upstream (primary vsRNAs)and downstream (secondary siRNAs) of RDR6

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or(A)n

CH3

(A)n

(A)n

DCL3 DRBX

or(A)n

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?

24nt

Episome

DNA

(A)n

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SDE3SGS3

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(A)n

AGO1

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40S &

Replicase

DCL4

DCL2

21nt 22nt

(A)n

AGO1 Amplification

CytosolNucleusDCL4

DCL2

??RDR1

SA

Geminivirus

RNA virus

?

DRB4

DRBXDRBX

DRB4

DRB4

DRBX

+

VAMP?

SA

Systemicdefenseresponses

Silencingsignal

Figure 2Antiviral silencing in Arabidopsis. Dicer-like4 (DCL4) primarily processes viral double-stranded RNA (dsRNA) into 21-nt viral smallRNAs (vsRNAs). If suppressed by viruses, DCL4 function is substituted by DCL2, which generates 22-nt vsRNAs. ARGONAUTE1(AGO1) is presented as the major antiviral AGO that mediates slicing and possibly translational repression of viral RNA. However,other AGO paralogs, including AGO7, are likely to be involved. DNA virus genomes (here shown as geminivirus) undergoDNA/histone methylation in the nucleus by DCL3-dependent vsRNAs. Primary vsRNAs are amplified into secondary vsRNAs byRNA-dependent RNA polymerase 6 (RDR6) and the salycilic acid (SA)-induced RDR1. Cellular and systemic SA production might beinduced upon possible recognition of an as yet uncharacterized viral-associated molecular pattern (VAMP). Aberrant viral mRNAs canenter RNA-dependent RNA polymerase (RDR1, RDR2, RDR6) pathways independently of primary vsRNA synthesis. Alternatively,vsRNA might act as primers for RDRs. An amplified, DCL4-dependent silencing signal (possibly the 21-nt product of DCL4) movesthrough the plasmodesmata to immunize neighboring cells. DRB, dsRNA binding; SGS, SUPPRESSOR OF GENE SILENCING;SDE, SILENCING DEFECTIVE; RI, replication intermediate.

Viroid: anautonomouslyreplicatingplant-specific subviralpathogen that consistsof a short stretch ofhighly complementary,circular, and ssRNAwithout an ORF

and RDR1 action during RNA virus infec-tions (23, 45) (Figure 2). DCL2 can be con-sidered a DCL4 surrogate because its antivi-ral role is evident only if the action of DCL4is genetically compromised or suppressed bydedicated proteins called viral suppressors ofRNA-silencing (VSRs) (20, 21). The miRNA-specific enzyme DCL1, although not impli-cated in a major way in processing dsRNA from

RNA viruses, modulates DCL4 expression neg-atively such that antiviral silencing is exacer-bated in dcl1 hypomorphic mutants (61). Thus,as yet unidentified DCL1-dependent miRNAscould target DCL4 transcripts for degrada-tion or negatively control transcription factorsrequired for DCL4 expression. Consistently,DCL4 transcript levels are enhanced in Ara-bidopsis deprived of the miRNA effector protein

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AGO1 (61). All four Arabidopsis DCLs arecritically involved in vsRNA production fromDNA viruses (gemini- and pararetroviruses;7, 46). Notably, DCL3-dependent, 24-nt-longvsRNA may dampen viral transcription byinducing chromatin condensation of nuclear vi-ral episomes and minichromosomes (62). Opti-mal vsRNA production requires DRBs, amongwhich DRB4 facilitates synthesis of DCL4-dependent vsRNA from RNA and DNA viruses(31, 61). vsRNA duplexes are then stabilized byHEN1 (7, 42).

In vertebrates, viral dsRNA is perceivedas a pathogen-associated molecular pattern(PAMP) through dedicated Toll-like recep-tors (TLRs). Activated TLRs signal down-stream innate immune responses, which,unlike RNA silencing, are not sequence specific(40). Although the existence of plant dsRNA-stimulated immune receptors remains enig-matic, the expression of several antiviral silenc-ing components is induced by virus-triggeredplant hormones, notably SA (2), which is alsotypically produced upon recognition of micro-bial PAMPs and virulence factors (71). Thus,detection of viral signatures other than dsRNAcould potentially exacerbate antiviral silencingby promoting a generalized, hormone-basedimmune reaction. The catalytic triad GDDfound in all RNA virus replicases (54) mightconstitute one of these putative viral-associatedmolecular patterns (VAMPs) (Figure 2). Wefinally note that global changes incurred byviruses to cellular sRNAs, including miRNAsthat might control nonsilencing-based antiviraldefense pathways, have so far eluded character-ization in plants.

Agrobacterium tumefaciens. In crown galldisease, A. tumefaciens transferred T-DNAsintegrate into plant genomes to express onco-genes. Bacteria then thrive on the resultingtumors by metabolizing nutrients producedby dedicated T-DNA-encoded enzymes (72).Although oncogene-free T-DNAs are widelyexploited for plant transformation, transgenesare often poorly expressed owing to an RNA-silencing phenomenon that recapitulates a

Replicationintermediate (RI):long dsRNAintermediate thoughtto be produced by viralreplicases ofpositive-strand RNAviruses

Salycilic acid (SA): aphytohormoneinvolved in plantdefense against insectsand pathogens; hasintrinsic antimicrobialproperties; is the activeprinciple of aspirin

Pathogen-associatedmolecular pattern(PAMP): signaturemolecules that areindispensable tomicrobial growth;includelipopolisaccharides,flagellin, elongationfactor Tu, cold-shockproteins,peptidoglycans, anddsRNA

host defense reaction preventing expressionof tumor-inducing T-DNAs of virulent bac-teria (26). Hence, siRNAs with sequencesof T-DNA-encoded oncogenes accumulatein tobacco leaves infected with virulentA. tumefaciens. Production of these 21-nt-longsiRNAs likely involves DCL4 and the upstreamaction of (at least) RDR6, because rdr6 loss-of-function mutants are more susceptible toA. tumefaciens than WT Arabidopsis (Figure 3).Presumably, RDR6 converts uncapped orpolyA− RNAs produced from integrated orepisomal T-DNA into dsRNA substratesfor DCL4. T-DNA arrays with head-to-tailconfiguration, frequently found at genomicinsertion sites, might also contribute to siRNAproduction by generating dsRNA directly(26).

Endogenous Small RNAs WhoseExpression Is Altered by Pathogens

Natural antisense and long siRNAs.Defense responses to bacteria, fungi, andoomycetes can be conceptually separatedinto PAMP-triggered immunity (PTI) andeffector-triggered immunity (ETI) (see Non-host Resistance, PAMP-Triggered Immunity,and Effector-Triggered Immunity). The linkbetween ETI and RNA silencing was discov-ered by comparing global sRNA profiles withthe expression of known pathogen-regulatedgenes in Arabidopsis. A natural antisenseRNA (nat-siRNA) called nat-siRNAATGB2is specifically induced upon recognition ofPseudomonas syringae effector AvrPt2 by thecognate Arabidopsis disease resistance (R)protein RPS2 (39). nat-siRNAATGB2 de-rives from the overlapping region of a pairof natural antisense (NAT) transcripts: aRab2-like small GTP-binding protein gene,ATGB2, and a constitutively expressed PPR(pentatricopeptide repeats) protein–like gene,PPRL. nat-siRNAATGB2 synthesis presum-ably requires ATGB2 transcriptional inductionthrough a mechanism involving the plasmamembrane protein NDR1, which is requiredfor RPS2-mediated resistance (Figure 4a,

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2b

P0

U

Saturation Inaccessibility

HEN1

122

Degradation

CH3

CH3

UUU

UUU

3' uridylation

a

2b

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2b

siRNA

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AGO1AGO1

AGO1

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dcl1

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PIWI

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Mid

PAZ

PIWI

ND

QR

3'

5'

3'3'

3'3'

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3'

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Col-0

rdr6

Figure 3Viral suppressors of RNA silencing (VSRs) from RNA viruses and silencing usurpation strategies. (a) P122-mediated block of HUAENHANCER 1 (HEN1) function resulting in viral small RNA (vsRNA) uridylation and degradation. (b) Upper: P19 acts as a vsRNAcaliper that binds specifically to 21-bp duplexes. Lower: The hook-like structures of 2b can interact with long and short double-strandedRNA (dsRNA). (c) 2b and P0 inhibit ARGONAUTE1 (AGO1) activity through direct interaction with PAZ, PIWI, and ND domains(2b) or just PAZ and ND domains (P0). (d ) Efficient dicing of viroid genomes might generate large amounts of vdsRNAs with 5′-Utermini. These may saturate AGO1, preventing its productive use of other vdRNAs, which may, in any case, be largely inert owing tothe strong secondary structures of viroid target RNA that prevent RNA-induced silencing complex (RISC) action. (e) Left:Agrobacterium tumefaciens stab-inoculated stems of Dicer-like1 (dcl1) mutants are immune to tumor formation compared with WTArabidopsis (Ler). Middle: Root inoculation assays unravel the hypersusceptibility of the Arabidopsis RNA-dependent RNA polymerase6(rdr6) mutant to A. tumefaciens compared with WT Col0 plants (Col-0). Right: Tumors developing on GFP-silenced Nicotianabenthamiana plants appear bright green under UV illumination, demonstrating RNA-silencing suppression in proliferating cells.

left). nat-siRNAATGB2 biogenesis entails acomplex series of reactions whose precise orderis unspecified, involving DCL1, HYL1, andHEN1 (miRNA pathway); RDR6 and SGS3(siRNA amplification); and the RNA pol IV

subunit NRPDla (heterochromatic silencing)(39) (Figure 4a, right).

A class of siRNAs that is atypical in size (39–41-nt long), dubbed long siRNAs (lsiRNAs),also accumulates in biotically stressed

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Arabidopsis. Among these, lsiRNA-1 is specificto RPS2-mediated resistance. Similarly to nat-siRNAATGB2, lsiRNA-1 production dependson a NAT pair in which induction of a receptor-like kinase (RLK) transcript requires RPS2activation by bacterial AvrPt2. lsiRNA-1 isproduced at the overlap between RLK and the3′-UTR of the antisense transcript RAP (37).The biogenesis of lsiRNA-1 is similar, albeitnot identical, to that of siRNAATGB2 becauseit involves the additional action of DCL4,ARGONAUTE 7 (AGO7), and NRPD1b, butnot of RDR6 and SGS3, again through an asyet unspecified order of events (Figure 4b,left). Mutations in RDR6 and HYL1, but notin silencing components not required for theirbiogenesis, compromise RPS2-mediated resis-tance, suggesting a role for nat-siRNAATGB2and lsiRNA-1 in AvrPt2-specified ETI (37).An outstanding issue pertains to the natureof signaling cascades that specifically induceATGB2 or RLK to act as major on/off switchesto control nat-siRNA and lsiRNA production,respectively (Figure 4b, right). The AvrPt2-RPS2 interaction likely involves additionalnat-siRNA or lsiRNAs, and more generally,many other nat-si or lsiRNAs might orches-trate ETI and PTI (see below) against a widerange of pathogens. More than 3000 ArabidopsissRNAs match >60% of protein-coding NATpairs (77). Furthermore, more than 30% ofthe Arabidopsis genome can produce transcriptsfrom both sense and antisense strands (80),some of which might be transcriptionallyinduced by biotic stresses to form dsRNAsubstrates for DCLs.

microRNAs. Arabidopsis miR393 was the firstsRNA implicated in bacterial PTI (52). MIR393transcription is induced by the flagellin-derivedPAMP peptide, flg22, to target mRNAs en-coding the F-box auxin receptor transport in-hibitor response 1 (TIR1) and related proteins(Figure 5a). Enhanced miR393 accumulationwas similarly found during sRNA profilingin Arabidopsis challenged with P. syringae pv.tomato (Pst) DC3000 hrcC, which lacks a func-tional type-III secretion system required for vir-

NONHOST RESISTANCE, PAMP-TRIGGEREDIMMUNITY, AND EFFECTOR-TRIGGEREDIMMUNITY

Plants have evolved multiple obstacles to protect themselvesfrom pathogen attacks, including (a) nonhost resistance viaphysical barriers, (b) PAMP-triggered immunity (PTI), and(c) effector-triggered immunity (ETI). The first obstacle accountsfor plant resistance to a majority of pathogens: deployment ofwaxy cuticles and thickened cell walls or deprivation of fac-tors required for pathogen growth. Successful pathogens thenencounter the PTI defense layer, orchestrated by transmem-brane pattern-recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs). The best-characterizedplant PAMP-PRR interaction involves recognition of a highlyconserved 22-amino-acid epitope (flg 22) in the N terminus ofbacterial flagellin by the leucine-rich-repeat receptor-like kinaseFLAGELLIN-SENSING 2 (FLS2). PAMP recognition triggersa cascade of reactions known as basal defense, which involves acti-vation of mitogen-activated protein (MAP) kinases, production ofreactive oxygen species and nitric oxide, cell wall reinforcement,and salycilic acid (SA) synthesis and signaling. On occasion, po-tent basal defense might stop pathogen attack, resulting in non-host resistance. However, many pathogens can overcome PTIby delivering virulence factors (effectors), which suppress basaldefense signaling, into plant cells. As a counter-counterdefensiveresponse to PTI suppression, plants deploy resistance (R) proteinsthat recognize specific pathogen effectors (avirulence or Avr pro-teins) or modifications incurred by pathogen effectors to host de-fense components. Specific R-mediated recognition of Avrs thentriggers ETI signaling, thought to be quantitatively stronger thanPTI signaling. ETI often culminates in a form of programmedcell death called the hypersensitive response (HR) and is accom-panied by a potent SA-mediated systemic defense response.

Type-III secretionsystem: a proteinsecretion apparatusused by somegram-negative bacteriato inject virulencefactors (or effectors)into the cytoplasm ofhosts

ulence (29) (see Nonhost Resistance, PAMP-Triggered Immunity, and Effector-TriggeredImmunity). Accordingly, constitutive overex-pression in a tirl-1 mutant background of amiR393-resistant TIR1 paralog enhanced sus-ceptibility to Pst DC3000, whereas bacterialgrowth was reduced in miR393-overexpressinglines (52). Pst DC3000 hrcC infection up-regulated many additional miRNAs unre-lated to auxin signaling, suggesting a globalcontribution of the miRNA pathway to PTI

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Figure 4Contribution of Arabidopsis small RNA (sRNA) to race-specific resistance against Pseudomonas syringae carrying avRpt2 mediated byRPS2. (a) The 22-nt-long natural antisense RNA (nat-siRNA) ATGB2 produced at the ATGB2-PPRL overlap specificallydownregulates the expression of PPRL, a possible negative regulator of defense. (b) In a related scheme, the 40-nt long siRNA1(lsiRNA1) produced at the receptor-like kinase (RLK)/RAP overlap contributes to PPRL mRNA decay through VARICOSE (VCS) andTRIDENT (TDT). The precise order of action of the RNA silencing components required for nat-siRNA and lsiRNA generation[right side of (a) and (b), respectively] remains to be formally established. AGO7, ARGONAUTE7; BAK1, BRI1-associatedreceptor-kinase 1; DCL1, Dicer-like1; FLS2, FLAGELLIN-SENSING 2; HEN1, HUA ENHANCER 1; HYL1, HYPONASTIC 1;NDR, non–race specific disease resistance; NRPD1a or b, Pol IV subunit a or b; Pst, P. syringae pv. tomato; RDR6, RNA-dependentRNA polymerase6; SDE3, SILENCING DEFECTIVE 3; SGS3, SUPPRESSOR OF GENE SILENCING 3.

(29) (Figure 5a). Indeed, growth and symptomsfrom normally nonvirulent Pst DC3000 hrcCwere appreciably, albeit not completely, res-cued in miRNA-deficient dcl1, but not in siRNA

pathway mutants of Arabidopsis (53). More-over, dcl1 also sustained growth from bacte-ria not infecting Arabidopsis, unraveling strongmiRNA contributions to nonhost resistance

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Figure 5microRNAs (miRNAs) in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). (a) Left panel: Under lowmiR393 levels, the transport inhibitor response 1 (TIR1) and related F-box proteins signal to increase the ubiquitin-mediateddegradation of auxin/indole-3-acetic acid (Aux/IAA) factors, promoting Aux-responsive gene expression and suppression of defense.Middle panel: Upon flagellin elicitation of FLAGELLIN-SENSING 2 (FLS2), MIR393 is transcriptionally activated, leading tosuppression of TIR1 mRNA and protein production. The ensuing Aux/IAA accumulation reduces Aux-responsive gene expression,enhancing PTI. Right panel: Factors required for miR393 biogenesis and action. (b) A balanced output of miRNA action during PTI.AGO1, ARGONAUTE1; ARF, auxin response factor; BAK1, BRI1-associated receptor-kinase 1; DCL1, Dicer-like1; DDL,DAWDLE; HEN1, HUA ENHANCER 1; HYL1, HYPONASTIC 1; Pst, P. syringae pv. tomato; R, resistance; RLK, receptor-likekinase; SCF, Skp, Cullin, F-box containing; SE, SERRATE.

(see Nonhost Resistance, PAMP-Triggered Im-munity, and Effector-Triggered Immunity).Nonetheless, Pst DC3000 hrcC growth was con-sistently higher in hen1 than in dcl1 mutants(53). Because HEN1 affects all known Ara-bidopsis silencing pathways, other cellular sRNAclasses might thus orchestrate PTI in addi-tion to miRNAs, including as yet unidenti-fied lsiRNAs and nat-siRNAs, whose biogenesis

Nonhost resistance:the resistance observedwhen all members of aplant species exhibitresistance to allmembers of a givenpathogen species

also relies heavily on DCL1 and HEN1 (37, 39).High-throughput sequencing identified sev-eral DC3000 hrcC-downregulated, rather than-upregulated, miRNAs, among which miR825targets three potential positive PTI regulators(29, 38). This result could explain why rescueof Pst DC3000 hrcC growth was only partial indcl1 and hen1 mutants, because reduced miRNAaccumulation in both backgrounds would

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Jasmonic acid ( JA):a phytohormoneinvolved in plantdefense against someplant pathogens, aswell as wounding,growth inhibition,senescence, and leafabscission

elevate the levels of miR825 (and possibly ofother miRNA) targets to promote defense.Downregulation of 10 out of 11 largemiRNA families of loblolly pine (Pinus taeda)—including seven pine-specific families—was alsoobserved in galls induced by the rust fungusCronartium quercuum (43). Remarkably, mostvalidated targets of the pine-specific familiesencode defense factors, including R proteinsand RLKs; others include orthologs of highlyconserved miRNAs that repress organ devel-opment. Nearly all mRNA targets accumu-lated highly in healthy stem tissues surround-ing galls, suggesting that miRNA suppressionactivates growth- and resistance-related genesto restrict fungal growth (43). Therefore, themiRNA involvement in PTI and basal defenseduring compatible interactions probably entailsa balanced output of (a) induction of miRNA-mediated repression of negative defense regu-lators and (b) repression of miRNA-mediatedrepression of positive effectors of defense(Figure 5b).

Some miRNAs facilitate symbiotic and par-asitic plant-bacterium interactions: Restrictionby miR169 of the MtHAP2-1 transcriptionfactor to nodule meristematic zones facili-tates differentiation of nitrogen-fixing cellsin Rhizobium-inoculated Medicago roots (18).Likewise, miR166 expression in root vascularand apical regions promotes nodule differentia-tion (9). Whereas the RDR6-DCL4-dependentpathway restricts Agrobacterium growth, suc-cessful tumor development requires miRNApathway integrity, because Arabidopsis hen1 andhypomorphic dcl1 mutants are immune tocrown gall formation (26) (Figure 3e). Spe-cific host miRNAs might be indispensable fordifferentiation of cells required for tumor vas-cularization or for elimination of putative tu-mor suppressors, for example. Alternatively,T-DNA-encoded miRNAs may act as viru-lence factors, similar to miRNAs producedby some mammalian-infecting DNA viruses(reviewed in Reference 60). These examplesunravel highly complex interactions that linkplant microbes to miRNAs and the extraor-

dinary variety of their possible outcomes. Asfor lsiRNAs and nat-siRNAs, a major chal-lenge ahead will be to determine which sig-naling pathways and regulatory DNA elementsmodulate, positively or negatively, specific ar-rays of MIRNA genes during various bioticstresses.

NaRDR1-dependent sRNAs in herbivoreattacks. A central role in herbivore resis-tance was identified for the Nicotinana attenu-ata RDR1 (NaRDR1) ortholog: Insect oral se-cretions and SA or jasmonate ( JA)—hormonescommonly produced in response to herbi-vore attack—strongly enhance NaRDR1 ex-pression (56). Comparative sRNA profiling inWT versus NaRDR1-knockdown plants be-fore and after herbivore elicitation identifiedNaRDR1-specific siRNAs with predicted tar-gets involved in SA and JA signaling (57). More-over, NaRDR1 knockdown severely altered in-duced transcript accumulation of most targetstested, and exogenous JA application restoredinsect resistance. Thus, NaRDR1-dependentsiRNAs seem key to JA signaling and probablycontribute to NaRDR1 homeostasis throughpositive feedback. NaRDR1 might control avast array of additional traits because only 6%overlap was found between global sRNA pro-files of WT versus NaRDR1-knockdown plants(57). Although lack of genomic information inN. attenuata precludes identification of theNaRDR1 templates involved, these resultsclearly implicate RDR1 beyond mere antiviraldefense in Nicotianae.

OPERATING RNA SILENCINGIN PLANT-PATHOGENINTERACTIONS

This section illustrates how host proteins op-erate RNA silencing, notably as part of RISCs.This section also highlights original gene ex-pression regulatory schemes in which RNAsilencing provides safeguards against constitu-tive defense activation, thereby reducing fitnesscosts to plants.

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Antiviral RNA-Induced SilencingComplexes and Their PossibleActivities

Plant recombinant viruses whose genomes in-corporate fragments of host transcripts inducesymptoms that mimic those of knockdown mu-tations in corresponding mRNAs (65). There-fore, vsRNAs can inhibit expression of com-plementary RNA in trans upon loading intoAGO(s). Consistently, Arabidopsis plants withknockdown mutations in the miRNA effectorAGO1 are hypersusceptible to RNA virus in-fections (49). Although generally interpretedas evidence for an AGO1-associated antivi-ral RISC, this hypersusceptibility could alsobe partially contributed to by reduced activ-ities of cellular miRNAs that normally con-trol negative regulators of antiviral defense.Studies of Cymbidium ringspot virus (CyRSV)provide perhaps the most compelling supportfor an AGO1-associated antiviral RISC. Thus,CyRSV-derived vsRNAs and cellular miRNAscofractionate into two protein complexes likelycorresponding to free AGO1 and partially orfully assembled RISC (58). Moreover, a simi-larly sized complex containing vsRNAs from aCyRSV-related virus exhibits virus sequence–preferential and ssRNA-specific nuclease ac-tivity (55), and AGO1 immunoprecipitatesisolated from Cucumber mosaic virus (CMV)-infected Arabidopsis contain CMV-derivedvsRNAs (84).

Nevertheless, immunoprecipitations withAGO2 and AGO5 yield similar results (68), yettheir role in antiviral defense has never beenestablished; AGO2 even lacks catalytic residuespotentially required for viral RNA slicing. Se-lective sRNAs loading into specific AGOs seemstrongly (albeit not entirely) influenced by their5′ terminal nucleotide (44, 48, 68). Thus, miR-NAs with predominant 5′-Us are frequentlyloaded into AGO1, whereas most AGO2- andAGO5-associated sRNAs have 5′-G and 5′-Utermini, respectively. Assuming similar rulesapply to vsRNAs and viroid-derived sRNAs(vdsRNAs), many vdsRNAs might thus incor-porate AGOs with little or no intrinsic an-

tiviral activity. Second, vs/vdsRNA allocationto plant AGOs might vary extensively fromone virus to another, or even between viralstrains, owing to differences in vsRNA popu-lations and 5′-nucleotide polymorphisms. Byextension, some plant AGOs might stronglyinhibit specific virus subsets, but not others.This hypothesis might partly explain the re-current difficulties in identifying single agomutations with broad antiviral silencing de-fects in Arabidopsis. Moreover, susceptibility as-says usually involve viruses that produce VSRswhose effects are redundant with those of muta-tions in DCLs, RDRs, and AGOs. Hence, onlywith P38-deficient Turnip crinkle virus (TCV )(P38 is the TCV-encoded VSR) was the roleof DCL2 and DCL4 and AGO1 in restrict-ing TCV accumulation clearly appreciated(Figure 6), as was the milder AGO7 con-tribution to this process (20, 61). Interest-ingly, P38-deficient TCV with a foreign GFPRNA insert becomes far more sensitive toRDR6 and AGO7 antiviral activities, suggest-ing that AGO7 might specifically incorporatesecondary, rather than primary, vsRNAs (61).Therefore, distinct RISCs might simultane-ously operate in virus-infected cells.

Antiviral AGO-mediated silencing isthought to occur mainly via vsRNA-directedslicing. However, global viral RNA levelsrather than site-specific endonucleolyticcleavage events are usually measured ex-perimentally. Yet, viral RNA accumulationdepends ultimately upon the rate of viralprotein synthesis (e.g., replicase), of viralRNA decay and, possibly, of slicing, layersof gene regulation that are all influenced bysRNA activities in plants and animals (10,28). Repression of viral protein synthesiscertainly deserves careful attention, especiallywith vsRNAs derived from imperfect hairpinswithin mRNAs (47), because they would beonly partially complementary to the otherarm of the stem and could, therefore, favortranslational inhibition of targeted transcripts,as with imperfectly matched plant and animalmiRNAs (4, 24). Moreover, even sRNAs with100% target complementary (for example,

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Figure 6Redundancy between viral suppressor of RNA silencing (VSR) function and RNA silencing mutations.(a) Schematics of WT Turnip crinkle virus (TCV) (expressing the P38 VSR) and TCV-�P38, in which theP38 open reading frame is replaced by that of the GFP, used to image viral accumulation. (b) TCV-�P38primary lesions remain small in inoculated leaves of WT plants. They expand, however, in leaves of dcl4mutants and become confluent in leaves of dcl4 dcl2 double mutant plants, in which viral systemic movementis also specifically restored. This simple rescue experiment identifies Dicer-like2 (DCL2) and DCL4 asgenetic targets of P38.

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those directing experimental RNAi) can haveextensive translational inhibitory effects underappropriate circumstances, possibly includingviral infections (10). Nonetheless, vsRNAloading into AGOs does not guarantee theirfunctionality, because sRNA efficacy is largelyinfluenced by RISC target site accessibility,which is potentially inhibited by viral or hostprotein binding or local secondary RNAstructures (3). The latter is well illustratedby viroids, whose tight, rod-like, and circularRNA genome is amenable to dicing but largelyresistant to RISC (34) (Figure 3d ).

AGOs also exert chromatin-level antivi-ral effects against nuclear genomes of DNAviruses. Hence, the RdDM pathway likely ac-counts for methylation of geminivirus DNAand histone H3 lysine 9, because ArabidopsisRdDM mutants are hypersusceptible to gemi-nivirus infection (62). Moreover, accumulationof VSR-deficient geminivirus is specifically res-cued in ago4 mutants. Geminivirus infectionproduces large amounts of DCL3-dependent,24-nt-long vsRNA, and similarly sized endoge-nous siRNAs direct nuclear RdDM throughAGO4 (62). Therefore, AGO4 contributesto heterochromatinization of geminiviral epi-somes, probably as does AGO6, because itacts redundantly in endogenous RdDM (85)(Figure 2).

Effecting miRNA, nat-siRNA, andlsiRNA Defensive Functions

Bacterial- and fungal-induced miRNAs likelyoperate through AGO1. This is the casefor miR393, which prevails in PTI againstP. syringae by targeting the auxin receptor TIR1and paralogs (29, 52). Because TIR1 interactswith and degrades Aux/IAA proteins, miR393induction increases the cellular Aux/IAA avail-ability to repress auxin response factors (ARFs)through heterodimerization and therebyinhibit auxin-responsive gene expression(Figure 5a). AGO1-loaded miR160 andmiR167, both induced by Pst DC3000 HrcC,target mRNAs of additional ARF familymembers (29). Therefore, AGO1-mediated

suppression of auxin signaling seems inte-gral to bacterial PTI, suggesting that auxinpromotes disease susceptibility through mech-anisms awaiting characterization. DuringETI through AvRpt2-elicitation of RPS2,nat-siRNAATGB2 suppresses in cis accu-mulation of a constitutive PPRL transcript(39), but the AGO involved, if any, is cur-rently unknown (Figure 4a). Because PPRLoverexpression attenuates disease resistance,PPRL may normally regulate negatively theRPS2-mediated response. Similarly, RPS2-dependent elicitation of lsiRNA1 productioninduces degradation in cis of constitutivelyexpressed RAP transcripts produced at theRLK-RAP NAT pair (Figure 4b). rap mutantsdisplay enhanced resistance to virulent andavirulent P. syringae, suggesting that the RAP-encoded RNA-domain protein negativelyregulates RPS2-mediated resistance throughunspecified mechanisms (37). The lsiRNA1pathway probably involves the biogenesisrather than the action of AGO7. Indeed, RAPturnover appears to be slicing independent butrequires the decapping factors VARICOSE(VCS) and TRIDENT (TDT) and the pres-ence of the lsiRNA1-complementary site in theRAP 3′-UTR, indicating sequence specificity(37) (Figure 4b).

Analyses of miRNAs in PTI and of NAT-derived siRNAs in ETI reveal a consensual generegulation pattern, whereby pathogen-inducedsRNAs repress constitutively expressed nega-tive regulators of defense. This modus operan-dus likely reflects an adaptation against pro-longed defense activation, which considerablyreduces plant fitness (70). In this context, oneobvious advantage of using sRNAs lies in thenecessity to deplete cells of mRNA and proteinpools of negative regulators already present atthe time of pathogen elicitation. A second antic-ipated advantage is reversibility. Indeed, plantsRNA action has a widespread translational re-pression component (10), which can be reversedwithin minutes, at least in mammalian cells (6).Similar reversibility in silencing mediated bymicrobe-induced sRNAs in plants would ensurethat translation of negative regulators resumes

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rapidly once the burst of defense responses haselapsed. The compatible interaction betweenpine and C. quercuum illustrates an alternativeyet related regulation mode (43), where defensepathway components might be repressed con-stitutively by specific miRNAs in healthy tissuesbut activated upon stress-induced inhibition ofthose miRNAs (Figure 5b).

Role of AGO4 in AntibacterialDefense: A Link to RNA-DirectedDNA Methylation?

Pst DC3000 induces DNA hypomethylationof genomic loci, including pericentromericrepeats and retrotransposons, as well as decon-densation of chromocenters in infected Ara-bidopsis tissues (59). The ago4-2 mutant, isolatedin a forward genetic screen, also displays en-hanced susceptibility to virulent Pst DC3000,to avirulent Pst DC3000 carrying the effec-tor avrRpml, and to the nonhost P. syringaepv. phaseolicola. In fact, all ago4 alleles tested—including loss-of-function alleles—exhibit thisphenotype, suggesting the involvement of het-erochromatic siRNAs (1). However, none ofthe factors normally associated with AGO4function in RdDM, most prominently DCL3,RDR2, and chromomethylase 3 (CMT3), couldbe linked to the ago4-enhanced susceptibil-ity phenotype (1). Although this could reflectfunctional redundancy among RdDM factors,a novel AGO4 function might also contributeto disease resistance independently of RdDMor, indeed, of sRNAs.

SUPPRESSION, AVOIDANCE,AND USURPATION OF RNASILENCING BY PATHOGENS

The neverending molecular arms race betweenparasites and their hosts is also a feature of an-timicrobial silencing. This section highlightssome of the sophisticated mechanisms by whichviruses or pathogenic bacteria may actively sup-press, evade, or sometimes usurp host RNA si-lencing pathways to cause disease.

Plant Viruses

Viral suppressors of RNA silencing. Awidespread viral counterdefensive strategyagainst RNA silencing is the deployment ofVSRs, which are highly diverse in sequence,structure, and activity (reviewed in Reference22). Single VSRs may target multiple pointsin RNA silencing pathways; viruses with largegenomes may encode several functionally dis-tinct proteins to achieve this effect. Whereasillustrations of VSR action abound, we pro-vide molecularly well-characterized examplesof proteins that each inhibit at least one step inthe antiviral silencing pathway. We start withinhibition of host-directed dsRNA productionby the cytoplasmic V2 protein of Tomato yel-low leaf curl virus (TYLCV, DNA virus). V2binds to and colocalizes with the tomato or-tholog of SGS3, which is required for RDR6function (30). Disruption of binding preventsV2’s VSR activity, suggesting that V2 inter-action with SGS3 compromises its function(Figure 7a). Studies of the Cauliflower mosaicvirus (CaMV, DNA genome) P6 protein showhow viruses impinge upon dsRNA processinginto vsRNAs (31). Although most P6 moleculesaggregate into cytoplasmic inclusion bodies (vi-roplasms), a small, nuclear fraction is essentialto CaMV infectivity. Transgenic P6 expressionin Arabidopsis is genetically equivalent to inacti-vating the nuclear protein DRB4, which facili-tates synthesis by DCL4 of 21-nt-long vsRNAs.Moreover, P6 is detected in DRB4 immuno-precipitates isolated from CaMV-infected cells,suggesting not only genetic, but also physicalinteractions between the two factors in the nu-cleus (31) (Figure 7b). Several VSRs inhibitHEN1-mediated 2′O-methylation of vsRNAduplexes, including the P122 kDa replicase(Figure 3a) of Tobacco mosaic virus (TMV, RNAvirus) (42).

Direct sequestration of vsRNA duplexesis one mode of action of the P19 protein oftombusviruses and 2b protein of cucumoviruses(RNA viruses). P19 uses an extended β-sheetsurface and a small α-helix to form a caliper-like structure for binding and measuring

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Figure 7Modes of action of DNA virus–encoded viral suppressors of RNA silencing (VSRs). (a) Two strategies deployed by geminivirusesagainst RNA silencing: (i ) inhibition of RNA-dependent RNA polymerase6 (RDR6) function through SUPPRESSOR OF GENESILENCING 3 (SGS3) interference by cytoplasmic V2 and (ii ) indirect suppression of RNA-directed DNA methylation (RdDM)through inhibition of the methyl cycle by nuclear L2. (b) Free cytoplasmic P6 molecules produced by Cauliflower mosaic virus (CaMV)interact with importin (αβ) and are translocated to the nucleus to inhibit dsRNA binding 4 (DRB4) function through physicalinteractions.

the characteristic length of vsRNAs to pref-erentially sequester DCL4-dependent, 21-bpduplexes (73) (Figure 3b). By contrast, 2b usesa pair of hook-like structures that interact morepromiscuously with long and short dsRNA(73). Additionally, CMV 2b binds AGO1 andblocks slicing without interfering with sRNAloading in vitro (84). Although seeminglycontradictory, these two antisilencing 2bactivities are reconcilable, because 2b’s affinity

for dsRNA is weak compared with that ofP19. Thus, its interaction with AGO1 couldincrease 2b local concentrations and enhancespecific binding to vsRNAs (Figure 3b). Thepolerovirus (RNA viruses) VSR, P0, contains anF-box-like domain that interacts with compo-nents of the SKP1-Cullin-F box (SCF) familyof ubiquitin ligases. Because P0 binds AGO1and promotes its decay, direct P0-mediatedubiquitination of AGO1 might induce its 26S

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proteasome–dependent degradation (8). How-ever, the P0-AGO1 interaction maps outsidethe F-box domain, and the 26S proteasomeinhibitor MG132 does not affect P0-mediatedAGO1 turnover (5). P0 is possibly a dominant-negative inhibitor of a host F-box protein thatregulates AGO1 homeostasis, or it may directerroneous AGO1 ubiquitination patternsthat interfere with RISC function/assembly(Figure 3c). The geminivirus-encoded L2protein illustrates indirect inhibition of RdDMof viral DNA. L2 interacts with and inhibitsadenosine kinase (ADK), which is requiredfor production of the methyl group donorS-adenosyl methionine (SAM), such that ADK-deficient plants display global methylationdefects, consistent with their hypersuscep-tibility to geminivirus infection (62, 76)(Figure 7a).

Other viral strategies to suppress, avoid, orusurp RNA silencing. Because HEN1 andAGO1 are shared components of multiple en-dogenous silencing pathways, VSRs that af-fect those factors are likely to broadly interferewith cellular sRNA functions. Indeed, VSR-overexpressing plants often exhibit develop-mental anomalies typical of miRNA pathwaymutants, which are usually considered to beside effects of primary inhibition of vsRNA-directed antiviral silencing (15, 27). However,VSR expression in these plants occurs in amuch broader tissue range than in natural in-fections. Second, miRNAs and other cellularsRNAs may regulate innate antiviral responsesunrelated to RNA silencing (akin to fungal andbacterial PTI); their inhibition may thus reflectdeliberate viral strategies. For instance, DCL1,which represses transcript accumulation of an-tiviral DCL4 (61), is itself negatively regulatedby miR162 (79). Viral inhibition of miRNAfunctions could thus augment DCL1 levels andactivity, resulting in reduced DCL4 accumu-lation and, hence, attenuated antiviral silenc-ing. Assuming the 5′ nt bias for sRNA sort-ing into specific AGOs applies to vsRNAs (44,48, 68), viral production of large amounts ofvsRNA with 5′-terminal U could possibly satu-

rate AGO1 to impair its action in antiviral de-fense and possibly in innate immunity mediatedby endogenous sRNAs. This might be partic-ularly true of viroids, whose genomes are effi-ciently diced but largely immune to RISC (34)(Figure 3d ). Titration of DCL activities likelyexplains how Red clover necrotic mosaic virus(RCNMV, RNA virus) might use replicatingRNA rather than dedicated proteins to in-hibit silencing (69). Because many plant virusesreplicate into cytoplasmic membranous com-partments, this raises the question as to howtheir genome is accessed by DCLs, whichseem nearly exclusively nuclear in Arabidopsis,DCL2 aside (78). Membrane-associated repli-cation might thus allow viral evasion rather thansuppression of silencing. Evasion might alsooccur through acquisition of mutations withinvsRNA target sites owing to error-prone viralreplication. Hence, recombinant Plum pox virus(PPV, RNA virus) containing target sites of cel-lular miRNAs rapidly evaded their inhibitoryeffects by mutating the seed region essential formiRNA-target interactions (67).

Plant viruses might also usurp RNA silenc-ing, as do mammalian DNA viruses, whichencode their own miRNAs to regulate viralgene expression and/or target transcripts of an-tiviral, proapoptotic, or antiproliferating hostfactors (reviewed in Reference 60). A paral-lel in plants is provided by vsRNAs producedfrom an extensive stem-loop structure withinthe CaMV-encoded 35S RNA (Figure 7a), ofwhich several exhibit near-perfect complemen-tarity to Arabidopsis mRNAs that are effectivelydownregulated during infection (46). vsRNA-mediated downregulation of cellular mRNAsis likely often fortuitous, but some targetingevents might be positively selected, for instanceif they affect host defense factors. Althoughseemingly contradictory to the deployment ofVSR (to inhibit RNA silencing), studies ofthe CaMV-encoded P6 protein illustrate howthe two processes might coexist within cells(31). Indeed, by inhibiting the DCL4 cofac-tor DRB4, P6 reduces but does not eliminateDCL4 function, resulting in 21-nt vsRNA lev-els low enough to dampen antiviral defense

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but high enough to target host gene expression(Figure 7a).

RNA Silencing Suppressionby Other Pathogens

Agrobacterium infections illustrate an indirectform of bacterial-mediated silencing suppres-sion. Although the RDR6/DCL4-dependentsiRNA pathway initially limits T-DNA onco-gene expression, primary tumors become pro-gressively resistant to this process owing tocompromised DCL4-mediated siRNA process-ing (Figure 3e). Silencing suppression seemsindependent of T-DNA-encoded proteins orbacterial virulence factors, because it is recapit-ulated in calli derived from A. tumefaciens–freesilenced tissues (26). Therefore, Agrobacterium-induced cell proliferation and/or dedifferen-tiation likely promotes a general block onsiRNA production. Although as yet unchar-acterized, the underlying molecular processesmust be highly specific, because miRNAaccumulation/activity is not affected in primarytumors, consistent with the strict requirementfor the miRNA pathway in crown gall disease(26).

The partial rescue of P. syringae type-III se-cretion mutants in miRNA- but not siRNA-deficient Arabidopsis demonstrates a key rolefor host miRNAs in PTI. A corollary, there-fore, is that some bacterial virulence factorsinjected into host cells through the type-IIIapparatus to suppress PTI should also sup-press the miRNA pathway. This idea was vali-dated in a study providing examples of DC3000effector proteins (named bacterial suppres-sors of RNA-silencing, BSRs) that inhibitmiRNA transcription, biogenesis/stability, oractivity (53) (Figure 8a). Transcriptional sup-pression of PAMP-responsive MIRNA genes,such as flg22-induced MIR393, was demon-strated with AvrPtoB. Suppression was inde-pendent of AvrPtoB’s E3-ligase activity, as isthe case for AvrPtoB-mediated suppression of

PAMP-responsive protein-coding genes thatmediate PTI. This result indicates a general in-terference with PTI signaling at or downstreamof the FLS22 receptor-like kinase (53).

A somewhat less trivial scenario is re-quired to explain how the DC3000 effec-tor AvrPto broadly suppresses accumulation ofPTI-related and PTI-unrelated miRNAs, pre-sumably by inhibiting DCL1-mediated pro-cessing (53). The fact that mutations prevent-ing AvrPto plasma membrane localization alterits antimiRNA activity is indeed intriguing be-cause AvrPto’s membrane anchoring is requiredfor its virulence function through inhibition ofthe kinase activities of the PAMP receptors, in-cluding FLS2. Although this result could sug-gest the existence of an as yet uncharacterizedmembranous pool of miRNA-processing fac-tors, an alternative explanation lies in the in-teraction of AvrPto with the plasma membraneprotein BAK1 (BRI1-associated receptor kinase1) (66). BAK1 is a shared signaling partner ofFLS2 and of BRI1 (brassinosteroid-insensitive1), the receptor of the phytohormone brassinos-teroid, which is required for development (17).Constitutive AvrPto expression indeed gener-ates developmental phenotypes indistinguish-able from those of Arabidopsis bri1 mutants, in-dicating inhibition of brassinosteroid signaling(66). One possible explanation for the AvrPtoeffects on miRNA biogenesis is therefore thatbrassinosteroids are constitutively required foroptimal expression of some miRNA processingfactors*.

Suppression of miRNA activity was docu-mented with the bacterial effector HOPT1-1, which inhibits cleavage or translational re-pression of several endogenous miRNA targets,mimicking the effects of Arabidopsis ago1 mutantalleles. Thus, HOPT1-1 might directly interactwith AGO1 or with a component of AGO1-RISC (53). Bacterial suppression of RNA si-lencing in ETI was also suggested to explainthe unusual size of At-lsiRNA1, produced uponRPS2 elicitation by Pst DC3000 AvRpt2 (37).

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Pst

(A)n

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(A)n

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AGO1

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AGO1 R

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+Other silencingcomponents

SA

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Sentinel sRNA

R1 R2SNC1

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R R

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Figure 8Host counter-counterdefense strategies against silencing suppression. (a) The effects of viral suppressors of RNA silencing (VSRs) orbacterial suppressors of RNA-silencing (BSRs) [here P. syringae pv. tomato (Pst) effectors] might be detected by dedicated resistance (R)proteins guarding key effectors of RNA-silencing pathway components. R protein activation might induce a hypersensitive response(HR) and salycilic acid (SA) production, leading to exacerbated RNA silencing and systemic defense responses. (b) SNC1 is one ofseveral R genes present at the RPP4 locus that share extensive sequence homology (orange). Endogenous 21-nt siRNAs or sentinelsiRNAs produced by antisense transcription at Suppressor of nprl-1, constitutive 1 (SNC1) and operating through ARGONAUTE1(AGO1) coordinately cosuppress the R genes in the locus. (c) Inhibition of AGO1 function by VSR or BSR prevents the action ofsentinel short-interfering RNAs (siRNAs) and releases inhibition of RPP4-like gene expression, potentially promoting defense againstthe VSR- or BSR-producing pathogens. BAK1, BRI1-associated receptor-kinase 1; DCL, Dicer-like; DRB4, dsRNA binding 4; FLS2,FLAGELLIN-SENSING 2; RDR1, RNA-dependent RNA polymerase1; vsRNA, viral small RNA.

Although likely synthesized by DCL1 (whichtypically produces miRNAs in the 19–24-ntsize range) the 30-nt-long at-lsiRNA1 mayarise from AvRpt2-mediated interference withDCL1 or interacting partners in the miRNAprocessing machinery, including the dsRNA-binding protein HYL1.

RNA silencing suppression by pathogensother than viruses, P. syringae, and Agrobac-terium awaits demonstration. However, given

the broad involvement of endogenous sRNAs inbiotic stress responses and the availability of ro-bust silencing reporter systems (25, 53), effectorproteins injected or secreted into host cells byfungi, nematodes, or insects will probably soonbe trivially identified as silencing inhibitors.Key issues will be to identify the mode of actionand targets of these suppressors and, perhapsmore importantly, to understand their spatialand temporal dynamics of expression/action in

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authentic infections rather than nonbiologicalexpression systems.

HOST RESPONSES TOSILENCING SUPPRESSIONBY PATHOGENS

This final section illustrates how silencing sup-pression by pathogens might be perceived andreacted against by plants. Although less welldocumented or understood, the deployment ofcounter-counterdefensive measures to accom-modate VSR or BSR detrimental effects prob-ably underlies the proliferation of plant antimi-crobial silencing components and also explains,in turn, the remarkable fluidity of genes en-coding VSR (and possibly BSR), presumablyrequired for their constant adaptation to thevarious host responses to silencing suppression.

R Proteins and Direct Targeting ofVSR and BSR Integrity/Function

Plants exploit rapidly evolving R proteins tomonitor or guard the integrity of specific hostdefense components called guardees, which arethe primary targets of a pathogen’s virulencefactors (35). During ETI, changes in the statusof guardees usually result in R proteins trig-gering host defense reactions. These reactionssometimes culminate in a form of programmedcell death called the hypersensitive response(HR) and are accompanied by phytohor-mone release (see Nonhost Resistance, PAMP-Triggered Immunity, and Effector-TriggeredImmunity). VSR and BSR exert their virulencefunctions at least partly through suppressionof RNA silencing. Therefore, some R genesmay have evolved to specifically sense the mod-ifications incurred by pathogens to antiviraland antimicrobial silencing components, par-ticularly if these components are shared be-tween multiple defense pathways (e.g., AGO1).An R-based system to counteract VSR or BSReffects could also be advantageous in furtherpromoting phytohormone-mediated stimula-tion of RNA-silencing components, as seenwith SA- or JA-mediated induction of RDR1during defense (21, 56) (Figure 8a). Although

no experimental proof supports these ideas asyet, strikingly, several VSR are known to triggerR-gene-dependent HR in specific hosts and, inone case, VSR mutations that compromised si-lencing suppression also compromised HR (41,64). Whether the same applies to bacterial ef-fector alleles known to escape R protein recog-nition during ETI awaits characterization.

Hosts could also directly neutralize silenc-ing suppressors through activities that degradeor relocate them into inappropriate subcellularcompartments. The former probably explainsthe failure of specific alleles of the CMV-encoded 2b protein to accumulate in Ara-bidopsis, possibly because of allele-dependentproteolysis (84). The latter is suggested by thenuclear relocation of the tombusviral P19 pro-tein caused by host-encoded P19-interactingALY proteins (12). The varying efficacy ofhost-directed degradation/relocation of micro-bial silencing suppressors and polymorphismamong these factors could thus contribute todifferences in viral or microbial susceptibilitybetween plant ecotypes or species.

Altering the Levels of EndogenousSmall RNAs That Regulate Antiviralor Antibacterial Components

Together with miR825, miR162 and miR168are downregulated upon elicitation of PTI byPst DC3000 HrcC (29). This phenomenon ishighly significant, because miR162 and miR168normally suppress DCL1 and AGO1, the ma-jor processor and effector of the miRNApathway, respectively (74, 79). Thus, bacterialPAMP recognition might elevate DCL1 andAGO1 cellular levels, potentially resulting inenhanced PTI. Suppression of miRNA process-ing or AGO1 function by bacterial effectors orVSR is similarly predicted to enhance DCL1and AGO1 levels during ETI and antiviraldefense.

Sentinel Small RNAs Generatedat Complex R Gene Loci

R genes are constantly acquiring new speci-ficity through high recombination rates,

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which typically results in complex arrays ofsilencing-prone gene clusters. For instance,the Arabidopsis Columbia RPP4 locus (namedRPP5 in the Landsberg ecotype) containsseven TIR-nucleotide binding site (NBS)-leucine-rich repeat (LRR) class R genes, ofwhich two, RPP4 and SNC1 (for Suppressorof nprl-1, constitutive 1), confer resistance tothe bacterium P. syringae and the oomyceteHyaloperonospora parasitica, respectively (81).These R genes are interspersed with twonon-R genes and three related sequences thatare coordinately regulated by transcriptionalactivation and RNA silencing. EndogenoussiRNAs indeed accumulate at the RPP4 locus,possibly originating through annealing ofsense and antisense transcripts detected at thisregion. SNC1 mRNA levels are elevated in dcl4and ago1 mutants (defective in processing andactivity of 21-nt-long siRNAs, respectively)

suggesting that SNC1 and the other highlyrelated RPP4 genes are coordinately cosup-pressed by siRNAs (81) (Figure 8c*, upper). Asexplained earlier, reversible posttranscriptionalsilencing of resistance-related genes might berequired to reduce the fitness costs of consti-tutive defense activation. However, an addedadvantage is anticipated from the observationthat expression of helper component proteinase(HcPro), a potyviral VSR, enhances steady-state expression of SNC1, presumably becauseHcPro inhibits the action of DCL4-dependentsiRNAs, notably found at the RPP4 locus(81) (Figure 8c). Put into a broader contextof plant-microbe interactions, these resultssuggest that sRNA involved in dampening Rgene expression may directly sense silencingsuppression caused by pathogens and stimulatean immediate and global enhancement ofdefense.

SUMMARY POINTS

1. Plant parasites activate RNA silencing through at least two different mechanisms: (a) byproducing their own small RNAs (sRNAs) (viruses and A. tumefaciens) or (b) by alteringendogenous sRNA levels in plant hosts.

2. sRNAs regulate gene expression by mediating mRNA degradation, translational inhibi-tion, or promoting chromatin modifications.

3. Many sRNAs are induced or repressed in response to pathogen attack. These sRNAscontribute to basal and race-specific defense responses upon their incorporation intoeffector complexes containing ARGONAUTE proteins.

4. Healthy plants may use cellular sRNAs to reversibly attenuate expression of manyresistance-related genes, thereby reducing the fitness costs of constitutive defense ac-tivation.

5. Classical hormonal responses to pathogens, notably based on salycilate and jasmonate,appear to be intimately linked to the operation of antimicrobial RNA silencing.

6. To counteract the effects of sRNA, pathogens produce virulence factors, called suppres-sors of RNA silencing, that inhibit various steps of the RNA silencing machinery. Themode of action and cellular targets of viral and bacterial suppressors of RNA silencing(VSR and BSR) are being progressively elucidated.

7. Viruses and pathogenic bacteria may also evade or sometimes usurp host RNA silencingpathways to cause disease.

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8. Plants can sense the modifications incurred by pathogens to RNA silencing componentsand respond through different strategies. These strategies possibly include resistanceprotein-mediated recognition of VSR/BSR and ensuing enhancement of innate immuneresponses, transcriptional or posttranscriptional stimulation of RNA silencing compo-nents, or the direct use of cellular sRNA as sensors or sentinels against aggressions.

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity of thisreview.

ACKNOWLEDGMENTS

The authors are supported by a grant from the Bettencourt Foundation for Life Sciences, agrant from European Union–integrated project SIROCCO (Silencing RNAs: Organisers andCoordinators of Complexity in Eukaryotic Organisms; LSHG-CT-2006-037900), a starting grantfrom the European Research Council (ERC, Frontiers of RNAi, 210890) to O.V., and a EuropeanUnion Marie Curie fellowship (041419) to V.R.F.

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Annual Review ofPlant Biology

Volume 60, 2009Contents

My Journey From Horticulture to Plant BiologyJan A.D. Zeevaart � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Roles of Proteolysis in Plant Self-IncompatibilityYijing Zhang, Zhonghua Zhao, and Yongbiao Xue � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

Epigenetic Regulation of Transposable Elements in PlantsDamon Lisch � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �43

14-3-3 and FHA Domains Mediate Phosphoprotein InteractionsDavid Chevalier, Erin R. Morris, and John C. Walker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �67

Quantitative Genomics: Analyzing Intraspecific Variation UsingGlobal Gene Expression Polymorphisms or eQTLsDan Kliebenstein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �93

DNA Transfer from Organelles to the Nucleus: The IdiosyncraticGenetics of EndosymbiosisTatjana Kleine, Uwe G. Maier, and Dario Leister � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 115

The HSP90-SGT1 Chaperone Complex for NLR Immune SensorsKen Shirasu � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 139

Cellulosic BiofuelsAndrew Carroll and Chris Somerville � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 165

Jasmonate Passes Muster: A Receptor and Targetsfor the Defense HormoneJohn Browse � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 183

Phloem Transport: Cellular Pathways and Molecular TraffickingRobert Turgeon and Shmuel Wolf � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 207

Selaginella and 400 Million Years of SeparationJo Ann Banks � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 223

Sensing and Responding to Excess LightZhirong Li, Setsuko Wakao, Beat B. Fischer, and Krishna K. Niyogi � � � � � � � � � � � � � � � � � � � � 239

Aquilegia: A New Model for Plant Development, Ecology, andEvolutionElena M. Kramer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 261

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AR375-FM ARI 25 March 2009 16:41

Environmental Effects on Spatial and Temporal Patterns of Leafand Root GrowthAchim Walter, Wendy K. Silk, and Ulrich Schurr � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 279

Short-Read Sequencing Technologies for Transcriptional AnalysesStacey A. Simon, Jixian Zhai, Raja Sekhar Nandety, Kevin P. McCormick,Jia Zeng, Diego Mejia, and Blake C. Meyers � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 305

Biosynthesis of Plant Isoprenoids: Perspectives for MicrobialEngineeringJames Kirby and Jay D. Keasling � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 335

The Circadian System in Higher PlantsStacey L. Harmer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 357

A Renaissance of Elicitors: Perception of Microbe-AssociatedMolecular Patterns and Danger Signals by Pattern-RecognitionReceptorsThomas Boller and Georg Felix � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 379

Signal Transduction in Responses to UV-B RadiationGareth I. Jenkins � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 407

Bias in Plant Gene Content Following Different Sorts of Duplication:Tandem, Whole-Genome, Segmental, or by TranspositionMichael Freeling � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 433

Photorespiratory Metabolism: Genes, Mutants, Energetics,and Redox SignalingChristine H. Foyer, Arnold Bloom, Guillaume Queval, and Graham Noctor � � � � � � � � � � � 455

Roles of Plant Small RNAs in Biotic Stress ResponsesVirginia Ruiz-Ferrer and Olivier Voinnet � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 485

Genetically Engineered Plants and Foods: A Scientist’s Analysisof the Issues (Part II)Peggy G. Lemaux � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 511

The Role of Hybridization in Plant SpeciationPamela S. Soltis and Douglas E. Soltis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 561

Indexes

Cumulative Index of Contributing Authors, Volumes 50–60 � � � � � � � � � � � � � � � � � � � � � � � � � � � 589

Cumulative Index of Chapter Titles, Volumes 50–60 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 594

Errata

An online log of corrections to Annual Review of Plant Biology articles may be found athttp://plant.annualreviews.org/

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