Role of Small RNAs in Host-Microbe Interactions · PY48CH11-Jin ARI 29 April 2010 19:41 R E V I E W...

PY48CH11-Jin ARI 29 April 2010 19:41

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Role of Small RNAs inHost-Microbe InteractionsSurekha Katiyar-Agarwal1,∗ and Hailing Jin2,∗

1Department of Plant Molecular Biology, University of Delhi South Campus,New Delhi 110021, India; email: [email protected] of Plant Pathology and Microbiology, Center for Plant Cell Biology, Institutefor Integrative Genome Biology, University of California, Riverside, California 92521;email: [email protected]

Annu. Rev. Phytopathol. 2010. 48:11.1–11.22

The Annual Review of Phytopathology is online atphyto.annualreviews.org

This article’s doi:10.1146/annurev-phyto-073009-114457

Copyright c© 2010 by Annual Reviews.All rights reserved

0066-4286/10/0908/0001$20.00

Key Words

miRNAs, siRNAs, dicer, argonaute, RNA silencing suppressors

Abstract

Plant defense responses against pathogens are mediated by activationand repression of a large array of genes. Host endogenous small RNAsare essential in this gene expression reprogramming process. Here,we discuss recent findings of pathogen-regulated host microRNAs(miRNAs) and small interfering RNAs (siRNAs) and their roles inplant-microbe interaction. We further introduce small RNA pathwaycomponents, including Dicer-like proteins (DCLs), double-strandedRNA (dsRNA) binding protein, RNA-dependent RNA polymerases(RDRs), small RNA methyltransferase HEN1, and Argonaute (AGO)proteins, that contribute to plant immune responses. The strategies thatpathogens have evolved to suppress host small RNA pathways are alsodiscussed. Collectively, host small RNAs and RNA silencing machin-ery constitute a critical layer of defense in regulating the interaction ofpathogens with plants.

11.1

Review in Advance first posted online on May 5, 2010. (Changes may still occur before final publication online and in print.)

Changes may still occur before final publication online and in print.

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INTRODUCTION

Small RNAs are 20 to 40 nucleotide (nt)-longnoncoding RNA molecules present in most eu-karyotic organisms that regulate gene expres-sion in a sequence-specific manner either tran-scriptionally or posttranscriptionally (5, 11, 98,99). On the basis of their biogenesis and precur-sor structure, small RNAs are placed in two dis-tinct groups: microRNAs (miRNAs) and smallinterfering RNAs (siRNAs). Small RNAs regu-late a multitude of biological processes in plants,including development, metabolism, mainte-nance of genome integrity, immunity againstpathogens, and abiotic stress responses. In-creasing evidence suggests that small RNAsplay a critical role in regulating the interactionof pathogens with plants.

SMALL RNA BIOGENESISPATHWAYS IN PLANTS

Small RNA pathways in plants have been bestcharacterized in the model plant Arabidopsis,and seminal pieces of work involving bothforward and reverse genetic screens have

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 1Endogenous small RNA pathways in Arabidopsis. (a) microRNA (miRNA) pathway. miRNAs are generated by transcription ofnoncoding miRNA genes by RNA Pol II. The primary miRNAs possess stem-loop structures that are acted upon by DCL1-HYL1-SEprotein complex. DDL protein is known to be involved in the formation of precursor miRNA (pre-miRNA). The DCL1-HYL1complex further processes pre-miRNA into 21 nucleotide (nt) miRNAs. The miRNA (miRNA:miRNA∗) duplexes are then methylatedat their 3′ ends by HEN1. These methylated miRNAs are transported into cytoplasm by an exportin homolog, or HASTY (HST). Themature miRNA is incorporated into the RNA-induced silencing complex (RISC) containing AGO1 protein. The RISC is recruited tothe target gene on the basis of sequence complementarity and AGO1 represses gene expression either by mRNA degradation ortranslational repression. (b) transacting small interfering RNA (ta-siRNA) pathway. The process of TAS precursor is triggered by anmiRNA–mediated cleavage. The resulting 5′ fragment (in case of TAS1a-c and TAS2) and 3′ fragment (in case of TAS3) act astemplates for the formation of long double-stranded RNA (dsRNA) by concerted action of RDR6 and SGS3. These long dsRNAs arethen recognized by DCL4-DRB4 complex and cut into phased 21 nt small RNAs that undergo further methylation by HEN1. Theta-siRNAs is incorporated into a RISC containing AGO7 (in case of TAS3) or unidentified AGO (in case of TAS1 and 2), which resultsin target cleavage. (c) natural antisense transcript-derived siRNA (nat-siRNA) pathway. Natural antisense transcripts produced by Pol IIform dsRNA within their overlapping regions. The dsRNAs are processed by DCL1 and/or DCL2 into siRNAs that target antisensetranscripts through an unidentified AGO protein containing. RDR6-SGS3, together with Pol IV form an amplification loop togenerate more nat-siRNAs, which reinforce the cleavage of antisense transcript. (d) heterochromatic siRNA (hc-siRNA) pathway.Transcription of heterochromatic regions, repeat regions or transposons by Pol II and/or Pol IV results in the formation ofsingle-stranded RNA (ssRNA), which is converted into dsRNA by the action of RDR2. This dsRNA is processed into predominantly24 nt long hc-siRNAs by DCL3. These 24 nt siRNAs associate with AGO4 (or AGO6, or AGO9) through an adaptor protein KTF1 toform a RNA-directed DNA methylation (RdDM) effector complex that directly or indirectly recruits proteins involved inheterochromatin formation, including DRM2, DRD1, and DMS3, to the hc-siRNA target loci. (e) long siRNA (lsiRNA) pathway.lsiRNAs are generated by DCL1 from coding or noncoding genes, or overlapping regions of antisense transcription, or dsRNAs fromthe action of Pol IV and RDRs. lsiRNAs are methylated by HEN1 and repress the expression of target genes by guiding mRNAdecapping mediated by DCP2 (decapping 2) and VCS (Varicose) and 5′-3′ RNA decay mediated by exoribonuclease XRN4.

delineated the cellular proteins that are in-volved in biogenesis and function of miRNAsand siRNAs. In this section, we present a briefsummary of different kinds of small RNA path-ways known in Arabidopsis (Figure 1). For de-tailed information, please refer to other reviews(11, 44, 47, 53, 66, 92, 98, 99, 107, 115).

miRNA Pathway

miRNAs are derived from the transcripts ofmiRNA genes generated by RNA PolymeraseII. The primary miRNA (pri-miRNA) tran-script forms a fold-back structure, which is pro-cessed into a stem-loop precursor known as pre-cursor miRNA (pre-miRNA). A protein namedDAWDLE (DDL) has been proposed to playan important role in miRNA biogenesis by re-cruiting predominantly DICER-like protein 1(DCL1) to pri-miRNA for downstream pro-cessing (110). The pre-miRNA is acted upon byDCL1 together with HYL1 (HYPONASTICLEAVES 1) and SE (SERRATE) to form thesmall RNA duplex. The small RNA duplex isthen methylated at the 3′ ends by HEN1 (HUA

11.2 Katiyar-Agarwal · Jin

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RISC: RNA-inducedsilencing complex

PAMP: pathogenassociated molecularpattern

PTI: PAMP-triggered immunity

ETI: effector-triggered immunity

ENHANCER 1) and is exported to the cyto-plasm by an exportin homolog, HST (HASTY).Mature miRNA is preferentially incorporatedinto AGO1 (or AGO10) and guides the com-plex to the target mRNA for cleavage or trans-lational inhibition on the basis of sequencecomplementarity.

siRNA Pathways

In contrast to miRNAs that are derivedfrom imperfectly base-paired hairpin loopstructures, siRNAs are derived from perfectlypaired double-stranded RNA (dsRNA) pre-cursors. These dsRNA precursors are derivedeither from antisense transcription or by theaction of a cellular RNA-dependent RNApolymerase (RDR). Four different types ofsiRNAs are known in plants: trans-actingsiRNAs (ta-siRNAs), natural antisense tran-scripts (NATs)-derived siRNAs (nat-siRNAs),heterochromatic siRNAs (hc-siRNAs) orrepeat-associated siRNAs (ra-siRNAs), andlong siRNAs (lsiRNAs). RNA Pol II transcribesnoncoding TAS genes, and the long primarytranscript products are initially cleaved bymiRNAs loaded with RNA-induced silencingcomplexes (RISCs), resulting in a 5′ fragmentor a 3′ fragment. These fragments then actas template for synthesis of a complementarystrand by the concerted action of RDR6 andSGS3 (46, 99). The resulting dsRNA moleculeis acted upon by DCL4 and DRB4 to triggerthe subsequent production of ta-siRNAs (37,46). nat-siRNAs are produced from the overlapregions of sense and antisense transcripts ofcis-NATs. A significant proportion of most eu-karyotic genomes encode overlapping cis-NATgenes, which have the potential to generatesiRNAs when base pairing between senseand antisense transcripts occurs. Though nat-siRNAs have been shown to play an importantfunction in both abiotic and biotic stresses (8,55), their roles in other plant processes remainsto be investigated. The cellular components in-volved in production of nat-siRNAs are DCL1and/or DCL2, HYL1, and HEN1 (8, 55). Thenat-siRNAs studied also partially depend on

RDR6, SGS3, and Pol IV (8, 55). hc-siRNAsor ra-siRNAs are usually 24 nts in length andare primarily derived from transposons, repeatelements, and heterochromatin regions. Theirbiogenesis is dependent on the DCL3-RDR2-Pol IV pathway (65, 66, 78). hc-siRNAs orra-siRNAs function in the RNA-dependentDNA methylation (RdDM) pathway by me-diating DNA methylation and/or histonemodification at the target sites. In additionto 21 to 24 nt siRNAs, a class of lsiRNAs inthe size range of 30 to 40 nt was discovered(54). The biogenesis of lsiRNAs is dependenton DCL1, HYL1, HEN1, AGO7, HST andpartially dependent on RDR6 and Pol IV (54).AtlsiRNA-1 is induced by bacterial pathogenPseudomonas syringae and triggers silencing ofthe target by destabilizing the target mRNAthrough decapping and 5′-3′ degradation (54).

HOST ENDOGENOUS SMALLRNAS IN PLANT-MICROBEINTERACTIONS

Plants have evolved multiple layers of de-fense in response to pathogen attacks, andbacterial pathogens provide useful exam-ples of how pathogens are encountered atvarious levels. The preliminary interactionbetween the pathogen and host is responsiblefor pathogen-associated molecular pattern(PAMP)-triggered immunity (PTI) in theplants (15, 52). Bacteria counteract PTIby secreting and injecting effector proteinsinto plant cells, which leads to suppressionof PTI. Host plants, in turn, have evolvedresistance components such as resistance (R)proteins that can recognize effectors and eliciteffector-triggered immunity (ETI) (15, 52).

An important role for small RNAs was firstdemonstrated in plant growth and develop-ment (63, 74). There is increasing evidence,however, that small RNAs are involved in reg-ulating plant responses to adverse conditions,including biotic stresses (13, 51). Antiviral de-fense involving virus-derived small RNAs is animportant example of an interaction betweenplant and pathogen that is mediated by small


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TTSS: type IIIsecretion system

RNAs. However, these small RNAs are derivedfrom viruses and are therefore exogenous inorigin. Unlike viruses that replicate inside thehost cell, bacteria, fungi, and other microbesinteract with plants without undergoing DNAor RNA replication and transcription insidethe plant cell. In these interactions, hostendogenous small RNAs play an importantrole in counteracting these pathogens. Recentreports have shown that plant-endogenoussmall RNAs, including miRNAs and siRNAs,are integral regulatory components of plantdefense machinery against bacteria and fungi.

miRNAs

A number of miRNAs have been linked to bi-otic stress respsonses in plants. In this sectionwe discuss the role of these miRNAs in plantswhen infected by different types of pathogenssuch as bacteria, virus, and fungi. Moreover, itis now known that these small RNAs are alsoimportant in regulating plant-microbe interac-tion during nitrogen fixation by Rhizobium andtumor formation by Agrobacterium.

Bacterial infection. In Arabidopsis, the firstmiRNA discovered to play a role in defenseagainst pathogens was miR393 (70). miR393 isinduced by a bacterial flagellin-derived PAMP,flg 22. miR393 negatively regulates auxinsignaling by targeting auxin receptors TIR1(transport inhibitor response 1), AFB2 (auxinsignaling F-box protein 2), and AFB3. How-ever, a TIR1 paralog, AFB1, was found to bepartially resistant to miR393-mediated cleav-age because of extra mismatches in the miRNAtarget site. Transgenic lines expressing Myc-AFB1 in tir1-1 background were more sus-ceptible to virulent Pseudomonas syringae pv.tomato strain DC3000 (Pst DC3000) and dis-played enhanced disease symptoms. To de-termine whether miR393 is involved in race-specific resistance, these transgenic plants wereinoculated with avirulent Pst DC3000 carry-ing an effector gene avrRpt2. Bacterial growthin transgenic lines expressing Myc-AFB1 didnot differ from non-transformed plants evenat 4 days postinoculation (dpi). These data

suggest that miR393 has a role in impartingbasal resistance but not race-specific resistance.Induction of miR393 was further confirmedby Fahlgren et al. (31) when they carried outdeep sequencing of Arabidopsis leaves at 1 h and3 h postinoculation (hpi) with a nonpathogenicstrain Pst DC3000 hrcC−, with a mutation inthe type III secretion system (TTSS). Relativeto uninfected leaves, miR393 was induced ten-fold in the infected leaves at 3 hpi. Overexpres-sion of miR393a from a strong constitutive pro-moter resulted in lower levels of TIR1 mRNAin transgenic lines, and these lines exhibited re-stricted bacterial growth. Besides miR393, twoother miRNA families, miR160 and miR167,were also up-regulated at 3 hpi. These miRNAstarget members of the auxin-response factor(ARF) family of transcription factors that arealso involved in auxin signaling (85). Thus,in response to bacterial infection, plants sup-press multiple components of the auxin sig-naling pathways. Another miRNA, miR825,which is predicted to target remorin, zinc fingerhomeobox family protein, and frataxin-relatedprotein, was also elevated during Pst hrcC −

infection (31).The interaction between the plant and

another bacterium, Agrobacterium tumefaciens,is of general interest because of the widespreaduse of A. tumefaciens for transferring genes intoplant genomes. Pruss et al. (79) showed thatan oncogenic strain of A. tumefaciens inducedmiR393 at the infiltrated zones, whereasthe disarmed strain (i.e., a strain lackingtumor-inducing properties) did not inducemiR393. Interestingly, the levels of miR393and miR167, which repress the auxin signalingpathway, were greatly reduced in tumors in-duced by A. tumefaciens infection. Derepressionof the auxin signaling pathway promotes tumorgrowth. Moreover, roots and stems of miRNA-deficient mutants, dcl1 and hen1, were immuneto A. tumefaciens infection (28). All of theseresults indicate a possible role of miR393 andmiR167 in regulating auxin responses in tumortissue. However, the levels of all other miRNAsstudies were moderately altered in tumor tissue.All these results demonstrate the importance


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of miR393 and miR167 in coordinating theinteraction between plants and A. tumefaciens.

Recognition of pathogens by plants can ini-tiate an oxidative burst resulting in enhancedabundance of reactive oxygen species (ROS)(25, 58, 72, 94, 104). Whereas miR398 is down-regulated in response to oxidative stress im-posed by high Cu2+ or high Fe3+ in Arabidop-sis, the levels of the target mRNAs, Cu/Znsuperoxide dismutatses 1 and 2 (CSD1 andCSD2), increase significantly (91). To investi-gate effects of microbe infection on the levelsof miR398 and its targets, plants were infectedwith both virulent Pst DC3000 and avirulentPst DC3000 carrying effector avrRpt2 or avr-Rpm1 (48). miR398 was downregulated by in-fection with Pst (avrRpm1) and Pst (avrRpt2)but was unaffected by Pst DC3000 infiltration.miR398 cleaves its target mRNA, thereby de-creasing the levels of CSD1 and CSD2. DuringPst (avrRpm1) infection, CSD1 but not CSD2exhibited strong up-regulation in transcript lev-els. The differential response of CSD2 mRNAto diverse stress conditions suggests the exis-tence of multiple mechanisms that are eitherdependent or independent of miR398-guidedregulation in plants.

Fungal infection. RNA silencing is a robuststrategy developed by plants to defend againstpathogens, including fungi. Posttranscriptionalgene silencing (PTGS) was shown to affectfungal resistance in Arabidopsis in studies em-ploying the RNA silencing mutants sgs2, sgs3,ago7, dcl4, nrpd1a, and rdr2, which exhibited en-hanced susceptibility to Verticillium strains (30).

Lu et al. (61) tested whether small RNAsare involved in the infection of loblolly pineby the endemic rust fungus, Cronartium quer-cuum f. sp. fusiforme. Infection with this fun-gus causes fusiform rust disease, which is char-acterized by stem and/or branch galls. SmallRNAs were cloned from the developing xylemof pine, and 26 miRNAs belonging to four con-served and seven loblolly pine-specific miRNAfamilies were identified. Using small RNA ex-pression profiling, miRNAs involved in diseasedevelopment were delineated and compared in

uninfected pine trees and trees infected with thefusiform rust fungus. The transcript levels forthese 11 families of miRNAs were unchanged inroots and in stems above the galls but transcriptlevels for 10 of these miRNA families were sig-nificantly suppressed in the galled stem. Thesereduced levels of miRNAs in galled stems rel-ative to healthy stems were correlated with in-creased levels of their target transcripts relativeto healthy stems. Interestingly, although the ex-pression of these miRNAs was unchanged in thestem above the gall, their target transcripts weresignificantly up-regulated in the stem above thegall as compared with healthy stems. This resultsuggests that fungal infection at the gall prob-ably immunizes the uninfected stem and mayprovide protection ahead of the spreading in-fection. Taken together, these data highlightthe complexity of plant-microbe interactionsmediated by small RNAs in the galled tissueand the tissue surrounding the gall. The signalsresponsible for the upregulation of defense re-sponsive genes in the uninfected tissue aroundthe gall remain to be identified.

Viral infection. As discussed earlier, hostmiRNAs respond to attack by pathogenicfungi and bacteria. This prompts the ques-tion of whether host miRNAs respond to vi-ral infection. Two miRNAs, bra-miR158 andbra-miR1885, were greatly up-regulated whenBrassica rapa was infected by Turnip mosaicvirus (TuMV) (42). The induction of bra-miR158 and bra-miR1885 is highly specific toTuMV infection because infection of B. rapaand B. napus with Cucumber mosaic virus, To-bacco mosaic virus, or the fungal pathogen Scle-rotinia sclerotiorum resulted in no such change.The putative target for bra-miR1885 is pre-dicted to be a member of the TIR-NBS-LRRclass of disease-resistant proteins. It is sug-gested that bra-miR1885 is a novel miRNAgenerated from gene-duplication events fromthe TIR-NBS-LRR class of proteins. Un-derstanding the mechanism of plant defenseresponses against viruses will require theidentification of additional miRNAs that areregulated by viral infection.


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Symbiotic nitrogen fixation. Nitrogen fixa-tion in soybean and other legumes is the resultof a symbiotic association between the legumi-nous plant and rhizobial bacteria. This mutu-ally beneficial association involves the exchangeof chemical signals leading to the formationof specialized nitrogen-fixing structures knownas root nodules (17). Understanding and elu-cidating this symbiotic association will requirethe identification of the molecular determi-nants and their regulators at different stagesof the interaction leading to nodule develop-ment. Two strategies have been employed foridentifying small RNAs that could participatein this interaction: One involves the identifi-cation of conserved miRNAs in soybean basedon the homology of known miRNAs in otherplant species, and the other involves the useof high-throughput sequencing and cloning

small RNAs differentially expressed in soybeanroots inoculated with the bacterium Bradyrhi-zobium japonicum compared to mock-inoculatedroots (90). Approximately 55 families ofmiRNAs were identified, of which 35 werefound to be novel. Further expression analy-sis of B. japonicum–responsive miRNAs revealedthat miR168 and miR172 were transiently up-regulated up to 3 hpi but were downregulatedto basal levels by 12 hpi. Although miR159 andmiR393 exhibited significant up-regulation at3 hpi, miR160 and miR169 showed downreg-ulation. Rhizobial infection changes the lev-els of miR160, miR393, miR164, and miR168,which target ARFs (ARF10, ARF16, andARF17), TIR1, NAC1, and AGO1, respec-tively. These results strongly support the roleof auxin homeostasis/signaling in nodulation(Figure 2).

mtr-miR396iii

mtr-miR166iiigma-miR168i

gma-miR172i

gma-miR159i

gma-miR393i

gma-miR160i

gma-miR169i

mtr-miR169iii

mtr-miR107iii

mtr-miR162iii

mtr-miR398iii

mtr-miR166iii

gma-miR167ii

gma-miR172ii

gma-miR396ii

gma-miR399ii

gma-miR1507ii

gma-miR1508ii

gma-miR1509ii

gma-miR1510ii

Root(nitrogen-limitingconditions)

a Inoculated root(recognition andattachment)

b Infection thread andbacteroid development(meristem establishment)

c Mature nodule(nitrogen fixing)

d

Figure 2Small RNAs regulate rhizobia-legume symbioses, resulting in the nodule development for nitrogen fixation.Different steps in the nodule formation are shown along with the miRNAs predicted to be involved atspecific steps. (a) The interaction of nitrogen-starved plants with rhizobial bacteria results in the exchange ofchemical signals as plants secrete flavonoids and bacteria produce lipochitooligosaccharides. (b) Therecognition of signals results in the attachment of bacterial cells to root hairs. (c) Changes in ionic equilibriumleads to the deformation of root hairs and transcription of nodulation-specific genes. Curling of root hairs toengulf bacteria results in the formation of infection thread that transports bacteria deep into the root tissuefollowed by bacteroid development. (d) Within 2 to 3 weeks postinoculation mature nitrogen-fixing nodulesare formed. Superscripts i, ii, and iii represent the miRNAs identified by Subramanian et al. (90), Wang et al.(101), and Lelandais-Briere et al. (59), respectively. mtr-Medicago truncatula; gma-Glycine max.


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To understand the additional componentsinvolved in root nodulation in legumes, re-searchers have been unraveling the regulatorynetwork at the later phase of the soybean-rhizobial interaction. Cloning and sequencingof miRNAs from functional nodules of soybeanidentified small RNAs belonging to 11 miRNAfamilies (101). Four of these belonged toconserved miR167, miR172, miR396, andmiR399 families, whereas another four familieshad sequences homologous to gma-miR1507,gma-miR1508, gma-miR1509, and gma-miR1510 that were also reported previously bySubramanian et al. (90). Three novel miRNAfamilies (gma-miR222, gma-miR383, andgma-miR235) that possibly play a role in ni-trogen fixation were reported. These miRNAsexhibited differential expression in rootnodules. High levels of gma-miR172 andgma-miR222 but low levels of gma-miR1508and gma-miR1510 were detected in rootnodules, prompting the authors to speculatethat these miRNAs play critical roles innodulation maturation and nitrogen fixation(Figure 2). Further investigations on functionsof these miRNAs and their putative targetswould provide insights on legume-rhizobiuminteractions during symbiosis and ultimatelyabout the mechanism of nitrogen fixation.

The levels of MtHAP2-1, a transcrip-tion factor of Medicago truncatula stronglyup-regulated during nodule developmentare controlled by miR169 (16). MtHAP2-1encodes a HAP2-type transcription factor(29) and is abundant in and limited to cellsof the nodule meristematic zone (16), whichfurther suggests its role in nitrogen fixation.MtHAP2-1 RNAi transgenic lines exhibiteddelayed nodule maturation because of arrestedgrowth and internment of the bacteriumSinorhizobium melitoti within the developingnodule. miR169 limits the expression ofMtHAP2-1 to the nodule meristematic zone,and the root growth of miR169-resistantMtHAP2-1 transgenic plants was reduced.All of these results strongly suggest that,during rhizobial infection of M. truncatula,MtHAP2-1 is critical in differentiation of

nodule cells and that its expression levels aretemporally and spatially tuned by miR169.The involvement of miR169 in regulatingMtHAP2-1 levels further emphasizes therole of small RNAs in symbiotic interactionbetween bacteria and plants.

Genome-wide small RNA profiling of rootapices and nodules of M. truncatula identified100 novel candidate miRNAs in addition tothe miRNAs that are homologous to knownmiRNAs from other species (59). Northernblot analysis and in situ localization studies re-vealed several miRNAs that were differentiallyexpressed between roots and nodules. miR167is highly expressed in the nodule peripheralvascular tissues, whereas miR172 and miR398are specifically localized in the infection zone,which suggests that they have roles in cell differ-entiation or infection by the symbiotic bacteria.These studies in soybean and Medicago indicatethat miRNAs indeed contribute to gene regu-lation of nodulation.

siRNAs

Although plants contain only several hundredmiRNAs, they contain huge numbers of en-dogenous siRNAs. As discussed earlier, thesesiRNAs have been classified into groups basedmainly on their biogenesis: the ta-siRNAs, ra-siRNAs, nat-siRNAs, and lsiRNAs. Their bio-logical roles, however, are not well understood.Our laboratory has identified a nat-siRNA anda lsiRNA specifically induced by the bacterialpathogen Pst (avrRpt2), and these siRNAs con-tribute to plant antibacterial immunity (54, 55).

nat-siRNAs. The first plant-endogenous nat-siRNA identified to be involved in plantimmunity is nat-siRNAATGB2, which reg-ulates R-gene mediated ETI (55). Thissmall RNA is generated from the overlap-ping region of a natural antisense transcript(NAT) pair, which encodes a Rab2-like GTP-binding protein gene ATGB2 and a penta-tricopepetide protein (PPR)-like gene PPRL.The endogenous RNA is specifically andstrongly induced by Pst (avrRpt2) and cleavesantisense PPRL mRNA for silencing. To


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R proteins: resistanceproteins

define the components involved in its biogene-sis, we examined the accumulation of ATGB2nat-siRNAs in Pst (avrRpt2)-challenged smallRNA-biogenesis mutants. This small RNA isprocessed by the DCL1-HYL1 complex, stabi-lized by HEN1-mediated methylation, and am-plified by RDR6, SGS3, and RNA Pol IV. Wefurther demonstrated that the induction of nat-siRNAATGB2 by Pst (avrRpt2) also requiresthe cognate host R gene RPS2 and its resis-tance signaling components, including NDR1.Constitutive overexpression of the target genePPRL without the nat-siRNA target site intransgenic Arabidopsis plants resulted in a de-layed hypersensitive response (HR) and atten-uated RPS2-mediated disease resistance. Theseresults strongly suggest that PPRL acts as a neg-ative regulator of RPS2-mediated resistance.PPRL is an atypical tandem PPR and is likelyto be localized in mitochondria (43, 62), whichmay contribute to Pst (avrRpt2)-triggered ox-idative burst, HR, and programmed cell death(PCD). Proteins containing the PPR domainare believed to be involved mainly in posttran-scriptional processes in organelles, includingRNA editing (87), mRNA silencing by cleav-age (103), and translational regulation (86).

lsiRNAs. During the search for pathogen-induced small RNAs by Northern blot analy-sis, we identified a novel class of endogenoussiRNA, the lsiRNAs (54). As the name sug-gests, lsiRNAs are longer than the normal 21to 24 nt siRNAs and are in the size range of30 to 40 nt. We found several lsiRNAs thatare mainly induced by bacterial infection orspecific growth conditions. The biogenesis ofAtlsiRNA-1 involves components of distinctsmall RNA pathways, including DCL1, HYL1,HEN1, HST, AGO7, RDR6, NRPD1a, andNRPD1b. Pst (avrRpt2)-mediated inductionof AtlsiRNA-1 specifically targets the AtRAPgene, which encodes a RNA-binding proteincontaining a putative RNA-binding RAP do-main (RNA binding domain abundant in Api-complexans). AtlsiRNA-1 employs a uniquemechanism to degrade target mRNA by DCP2-VCS (Decapping 2 and Varicose) mediated

decapping followed by a exoribonucleaseXRN4-mediated 5′-3′ decay. AtRAP is a neg-ative regulator of PTI and ETI becausethe knock-out mutant of this gene resultedin enhanced resistance to both avirulent Pst(avrRpt2) and a virulent strain Pst DC3000.

To be cost effective, plants have devel-oped a sophisticated regulatory mechanismto suppress the defense response systems un-der normal conditions, possibly by employ-ing negative regulators of plant defense, suchas PPRL and AtRAP. As the defense systemsmust be switched on rapidly upon pathogenattack, small RNAs such as miRNA393, nat-siRNAATGB2, and AtlsiRNA-1, are inducedin the early phases of infection and cleave ordegrade the mRNA of these negative regula-tors of plant immunity, thereby mounting rapidcounter defense mechanisms.

siRNAs generated from R gene loci (RPP5Locus and N gene MITEs). Plant resistance(R) proteins recognize specific pathogens bydirectly and indirectly interacting with corre-sponding avirulence (avr) effectors and trigger-ing a cascade of events leading to disease re-sistance (21, 64). These R genes are generallyclustered in the genome and encode proteinswith common motifs. To keep pace with thecontinuous and rapid evolution of pathogens, Rgenes undergo coevolution resulting in variablegene clusters. The Arabidopsis thaliana ecotypeColumbia RPP4 locus (known as RPP5 in Lands-berg erecta for recognition of the oomycetesHyaloperonospora parasitica 5) comprises sevenTIR-NBS-LRR class-R genes along with threerelated and two unrelated genes (73, 109). TwoR genes in this locus, named RPP4 and SNC1(for suppressor of npr1-1, constitutive 1), im-part resistance to both P. syringe maculicola andH. parasitica (89, 97, 108, 114). These two Rgenes are coordinately regulated by transcrip-tional activation and siRNA-mediated RNA si-lencing. Endogenous siRNAs generated at theRPP4 locus apparently target SNC1 because en-hanced transcript levels of SNC1 were observedin small RNA biogenesis deficient mutants suchas dcl4, upf1, and ago1 as well as in transgenic


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plants expressing P1/HC-Pro suppressor. Thisfine-tuning of R-gene expression is importantbecause it substantially reduces the fitness costfor constitutive activation of defense pathways.In other words, the siRNAs generated fromthe RPP4 locus may control the resistance re-sponses, thereby enhancing plant health andfitness.

MITEs (miniature inverted repeat transpos-able elements) are truncated DNA transposonsthat are generally less than 600 base pairs(bp), lack open reading frames, and depend onthe activity of transposons for their mobility(49, 50). MITEs are present in high copynumbers in several plant genomes and arepredicted as regulatory elements in plant geneexpression (33, 50). They are also thoughtto serve as a major evolutionary element intransposon-mediated gene regulation in plantsby generating small RNAs. The complexity ofthe tobacco mosaic virus (TMV) R gene N ismaintained by MITEs-mediated creation ofnew gene structures (56). Virus infection maylead to temporary inhibition of PTGS for N ex-pression and siRNA-guided cleavage. This mayinduce premature translation termination orprovide a polyadenylation signal or introducedeletions/insertions leading to gene diversity.Kuang et al. (56) suggested that biogenesis ofmost MITEs-derived small RNAs (MiS) de-pends on the NRPDla/RDR2/DCL3/AGO4pathway. DCL4 is also implicated in theaccumulation of MiS small RNAs (56). It willhelp our understanding of R gene evolution toelucidate the mechanisms of generating smallRNAs from MiS and their functionality ingene regulation in plants.

COMPONENTS OF THE SMALLRNA BIOGENESIS PATHWAYPLAY AN IMPORTANT ROLEIN PLANT DEFENSE

Many plant genomes encode multiple DCLs,RDRs, and AGOs in the RNAi silencingmachinery. The components within the samefamily have distinct or sometimes partiallyoverlapping functions in different small RNA

pathways. Arabidopsis has four DCLs, six RDRs,and ten AGOs, many of which are involved inplant-defense signaling pathways.

Dicer-Like Proteins (DCLs) andTheir Associated Proteins

Arabidopsis consists of four DCLs that processdsRNA or fold-back RNA precursors to gener-ate siRNA and miRNAs, respectively. Geneticexperiments using single, double, or triple mu-tants of DCLs have dissected the roles of in-dividual DCLs and their compensatory func-tions in the production of virus-derived smallRNAs (viRNAs). Recent studies demonstratethat loss-of-function mutations in both DCL4and DCL2 are necessary and sufficient to makeplants highly susceptible to several (+)ssRNAviruses (9, 22, 24, 35).

Deleris et al. (22) employed virus-inducedgene silencing (VIGS) to knock out endoge-nous phytoene desaturase (PDS) by inoculatingArabidopsis plants with modified tobacco rattlevirus (TRV). The virus was modified suchthat the plant PDS gene replaced the RNA2-encoded 2b and 2c sequences in the viralgenome. After infection, the modified virusdid not cause disease because of the siRNA-mediated antiviral response. That is, thesiRNAs targeted TRV-PDS and generatedPDS-targeting siRNAs. These PDS siRNAsinitiated degradation of endogenous PDSmRNA, which results in extensive photobleaching of the plants. The inoculation ofthe modified TRV-PDS in wild-type and dclmutants showed that DCL4 is the primarysensor and produces 21 nt siRNAs thatprogram a RISC effector complex againstviruses. In the absence of DCL4, DCL2 actsas a subordinate antiviral defense protein byproducing 22 nt siRNAs. Moreover, doublemutants of dcl2/dcl4 exhibited hypersuscep-tibility to the virus infection, which suggeststhe combined action of these two specificproteins in antiviral defense. Unlike DCL2and DCL4, DCL1 and DCL3 were notfound to be involved in antiviral immunity inthis study. A similar study by Qu et al. (82),


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aimed to determine the contribution of keyRNA-silencing pathway components in antivi-ral silencing, found that all four DCL proteinsare involved in mounting an antiviral defensein plants. This study confirmed that DCL2 andDCL4 are primary proteins, whereas DCL3has a minor role in this process. Interestingly,DCL1 was also implicated in antiviral silencingbut apparently plays a negative role as it down-regulates the expression of DCL4 and DCL3.These studies corroborate that there is a func-tional hierarchy of different plant DCL proteins(DCL4>DCL2>DCL3>DCL1) in process-ing viral RNAs into viRNAs (22). DCL3 alsohas clear antiviral roles in natural DNA virus in-fections (7, 67). Arabidopsis plants infected withCaMV had an increased viral load in dcl2-dcl3-dcl4 mutants. However, viral load determinedby immunoblot analysis of coat protein was notsignificantly different between wild-type plantsand single mutants (dcl2, dcl3, dcl4) or doublemutants (dcl2-dcl3, dcl2-dcl4, dcl3-dcl4). DCL1plays a positive role in antiviral defense by facil-itating synthesis of viRNAs derived from the in-tramolecular hairpins formed in the 35S-leadersequence of CaMV. These hairpins resemblemiRNA precursors, and DCL1 probably iden-tifies this structure, excises it, and then presentsit to other Dicers for further processing.

Small dsRNA-binding proteins (DRBs) areessential cofactors of DCL proteins (45, 69).These DRBs, however, do not exhibit hierar-chical redundancy as DCLs (19). DCL1 andDCL4 interact with DRB1/HYL1 and DRB4,respectively (45, 69). DRB4 contributes to an-tiviral defense, possibly by interacting withDCL4 (82). In contrast, DCL2 and DCL3 donot require any DRB for production of viRNAs(19). Another protein that contains a dsRNAbinding domain is HEN1, which plays an im-portant role in small RNA metabolism (75).The Arabidopsis hen1 mutant exhibits hyper-susceptibility to CMV by a fivefold increasein the accumulation of the viral RNA whencompared with that in wild type, which suggestthat HEN1 contributes to resistance against thevirus (10).

DCL proteins are also involved in the gen-eration of small RNAs that contribute to an-tibacterial immunity in plants. The dcl1 mutantshowed enhanced susceptibility to Pst DC3000hrcC-, a nonpathogenic strain that can elicitPTI (71). DCL1 is required for generatingmiR393. The accumulation of the target tran-scripts of miR393 (TIR1, AFB2, and AFB3) isincreased in the dcl1-9 mutant (70) when treatedwith Flg22 peptide. DCL1 is also involved inthe generation of nat-siRNAATGB2 (55) andAtlsiRNA-1 (54).

HYL1, the dsRNA-binding protein asso-ciated with DCL1, is also involved in resis-tance against bacterial infection as the hyl1mutant was susceptible to Pst (avrRpt2) (55).Moreover, the hyl1 mutant exhibited compro-mised accumulation of nat-siRNAATGB2 andAtlsiRNA-1 (54, 55).

RNA-Dependent RNAPolymerases (RDRs)

In plants, RDRs are important for siRNAformation because they synthesize dsRNAsfor downstream processing by DCL. Initialstudies showed that virus infection enhancesRDR activity, which led to the hypothesisthat RDRs are one of the many host factorsthat assist virus replication (27). Subsequently,extensive studies have implicated RDRs inantiviral defense in plants (4, 106, 112). Xieet al. (106) found that RDR1 activity is inducednot only by virus infection but also by defensesignaling compounds such as salicylic acid (SA).Reducing the expression levels of RDR1 intransgenic antisense Arabidopsis plants resultedin enhanced accumulation of viral RNAs andincreased susceptibility to TMV and potatovirus X (PVX) infection. AtRDR1, an orthologof NtRDR1, is also known to impart defenseagainst tobamovirus and tobravirus becauseArabidopis rdr1 mutant plants had enhancedlevels of viral RNAs (112). NtRDR1 is alsoinvolved in combating potato virus Y (PVY)infection; knocking down expression of RDR1in transgenic tobacco plants resulted in reduced


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expression of other defense-related genes suchas AOX1 and ERF5 (84). These studies raisethe question about whether different RDRproteins (for instance, the six members in Ara-bidopsis thaliana) have distinct or overlappingfunctions.

Arabidopsis RDR6 (SDE1/SGS2) was ini-tially considered to be important for transgene-induced PTGS and apparently had no rolein plant antiviral response because a RDR6mutant allele sde1 did not exhibit enhancedviral susceptibility (20, 68). Extensive studiesby Qu et al. (81), however, demonstrated thatNbRDR6 (a functional homolog of AtRDR6)plays an important role in antiviral defense. Thetransgenic tobacco plants wherein NbRDR6was downregulated showed hypersusceptibilityto many different viruses, and this response wasmore pronounced at elevated temperatures.This was consistent with earlier reports, whichdemonstrated that there is increased siRNAgeneration at higher temperatures (93). Thisstudy provided strong evidence that NbRDR6contributes to general antiviral defense by RNAsilencing and that environmental conditionsinfluence the plant-virus interactions. In a re-cent elegant study, Wang et al. (102) examinedsingle, double, and triple mutants of RDR1,RDR2, and RDR6 using a mutated CMV withno viral suppressor (VSR) 2b, and demonstratedthe role of both RDR1 and RDR6 in secondaryviRNA formation. Small RNA profilingrevealed that RDR1 preferentially amplifiesviRNAs that mapped at the 5′-terminal viralRNAs, whereas RDR6-dependent viRNAsmapped to the 3′-terminal half of viral RNAs.RDR6 interacts with a coiled-coil protein,SGS3, to produce secondary viRNAs (57, 99).An sgs3 mutant shows enhanced susceptibilityto cucumovirus (68), which indicates thatSGS3 also contributes to antiviral resistance inplants. The generation of nat-siRNAATGB2(55) and AtlsiRNA-1 (54) requires RDR6. PstDC3000 (avrRpt2) displayed enhanced growthin the rdr6 mutant (55), which provided directevidence for the function of RDR6 in plantimmunity.

Argonautes (AGOs)

AGOs are associated with small RNAs andform RISC complexes to induce silencingof target genes (41). Arabidopsis contains 10AGOs, and their role in plant immunity is yetto be determined. There is emerging evidencethat the methylation status of plant genomesare altered in response to attack by pathogens,including viruses, bacteria, and fungi (34, 39,76, 88). hc-siRNAs trigger transcriptional genesilencing (TGS) by guiding RNA-directedDNA methylation (RdDM) and histonemodification in plants (47, 66, 96). AGO4 isa major nuclear RNAi effector associated withhc-siRNAs or ra-siRNAs that direct DNAmethylation (60, 80). Involvement of AGO4in the disease-resistance response links DNAmethylation and plant defense. When attackedby viruses, plants employ DNA methylation torepress viral transcription and/or replication.Upon infection by either of two geminiviruses,cabbage leaf curl virus (CaLCuV) or beet curlytop virus (BCTV), Arabidopsis plants silenceviral chromatin by both cytosine and histonemethyltransferases (83). This is evident bythe hypersusceptibility methylation-deficientmutants to geminiviruses, including mutants ofcytosine methyltransferases (drm1 drm2, cmt3,and met1), histone H3K9 methyltransferase(kyp2), RdDM pathway components (ago4,ddm1, and nrpd2A), or methyl cycle enzymes(adk1 and adk2) (83). Viral suppressors AL2and L2 inhibit the activity of adenosinekinase (ADK), a cellular enzyme involved inthe generation of S-adenosyl-methionine (amethyltransferase cofactor). Therefore, plantsinfected with virus lacking L2 had hypermethy-lation of viral DNA. Additionally, recovery ofthe viral-infected plants from the disease symp-toms required AGO4 (83). Chromatin methy-lation may be a generalized process adopted byplants to evade infection by DNA. VIGS of twoNicotiana benthamiana homologs of ArabidopsisAGO4 blocked the R gene-mediated antiviralresponses through translational suppression ofviral RNAs (6). This result suggests that AGO4may have additional functions besides its role


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in the RdDM pathway. It is also possible thatthe NbAGO4 may not function the same wayas Arabidopsis AGO4, or there might be anotherunidentified N. benthamiana AGO that is moreclosely related to Arabidopsis AGO4.

AGO4 is also involved in antibacterial de-fenses. Mutant ago4-2 was identified from a ge-netic screening using a H2O2-responsive Ep5Cpromoter-driven GUS reporter (2). Assessmentof disease susceptibility revealed that ago4-2 ex-hibits reduced resistance to virulent Pst DC3000as well as to avirulent Pst (avrRpm1). How-ever, mutants of DCL3 and RDR2, the up-stream components of AGO4, and mutants ofchromomethylase 3 (CMT3) and defective inRNA-directed DNA methylation (DRD1) anddomains rearranged methyltransferase 1 and 2(DRM1 and DRM2), the downstream compo-nents of AGO4 in the RdDM pathway, showed

no change in Pst growth. These results suggestthat either these components are functionallyredundant with their close homologous genesor AGO4 simply has an unidentified RdDM-unrelated function in plant defense. These pos-sibilities should be investigated by testing thedisease resistance responses of double and triplemutants of these components in the RNAi andRdDM pathways.

In addition to AGO4, Qu et al. (82) con-clusively showed that AGO1 and AGO7 havean important role in slicing viral RNAs. AGO1is the primary slicer because it targets viralRNAs with more compact structures but AGO7is a surrogate slicer whose targets are lessstructured (82). The biogenesis of AtlsiRNA-1 is known to involve AGO7 as ago7 mutantdo not accumulate AtlsiRNA-1 (54). However,other ago mutant plants, including ago3, ago4,

Table 1 Mode of action of viral silencing suppressors in plants

Suppressor Source Mode of action ReferenceAC4 Geminivirus Competes with AGOs by binding to single-stranded siRNA and thereby preventing

RISC assembly.12

AC2 Begomovirus Transcriptional activator. Induces expression of any gene, one of which might be asilencing suppressor.

95

HcPro Potyvirus Mimics hen1 mutations. viRNAs are oligo-uridylated and partially degraded due tolack of 2′-O methylation.

111, 3, 105

Interacts with a calmodulin-related protein, overexpression of which suppressessilencing.

Amino acids 180, 205, and 396 of HcPro are critical for suppression of miRNA,tasiRNA, and VIGS pathway but not for sense PTGS.

P6 Cauliflowermosaic virus

Is imported in the nucleus and binds to DRB4 protein. Suppresses RNA silencingpathway, possibly by inactivating DRB4, which is an essential component requiredfor DCL4 action.

40

2b Cucumbermosaic virus

Interacts physically with siRNA-loaded RISC and inhibits its slicing action.In vitro assays suggest that 2b binds to siRNAs and to a lesser extent than to long

dsRNAs.2b inhibits the production of RDR1-dependent viral siRNAs.

113, 38, 24

P0 Polerovirus Promotes ubiquitin-dependent proteolysis of AGO1. 77P69 Tymovirus Inhibits viRNA amplification. 14AL2 Curtovirus Interacts with adenosine kinase, whose inhibition possibly prevents methylation of

viral DNA.100

p126 TMV Encodes methyltransferase and helicase. Binds duplex siRNA and inhibitsHEN1-dependent methylation and degradation.

7

RNAse III Closterviridae In vitro assays suggest that RNAse III suppresses siRNA silencing by cleaving 21,22, 24 bp siRNAs into 14 bp fragments.

18


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and ago9, showed no significant change in thelevel of AtlsiRNA-1 as compared with wild type.AGO7 is also associated with TAS3 tasiRNA (1,32, 36). The accumulation of bacteria-inducedAtlsiRNA-1 is dependent on AGO7, suggestinga role of AGO7 in antibacterial defense.

RNA SILENCING SUPPRESSORSOF PATHOGENS

Viral Suppressors ofRNA Silencing (VSRs)

Many viruses encode specific proteins that sup-press the host antiviral silencing response andthereby benefit viral infection. These viral sup-pressors of RNA silencing (VSRs) can act atthree levels, i.e., they can (a) inhibit genera-tion of viRNAs; (b) inhibit loading of viRNAsin RISC by binding to the viRNA, and(c) inhibit components of RISC. Ectopically ex-pressed VSRs, in conjugation with a sensor,have been used to decipher functions of manyVSRs. The use of viruses with disabled or mod-ified VSRs, however, has recently proven to bea very effective approach for determining VSRfunction. Two recent reviews provide compre-hensive coverage on this topic (23, 26). Table 1summarizes what is known about the mode ofaction of VSRs in plants.

Bacteria-Encoded Suppressorsof RNA Silencing (BSRs)

As noted in the previous paragraph, virusesencode VSRs that suppress host antiviralsilencing machinery and thereby promotetheir pathogenesis. Because small RNA andRNA-silencing machinery are also important

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Mechanism of action of bacterial suppressors ofRNA silencing (BSRs) in plants. Different steps insmall RNA pathways are suppressed by differenteffectors encoded by Pseudomonas syringae.FLS: Flagellin receptor; TTSS: Type III secretionsystem of bacterial pathogen; Pst: Pseudomonassyringae pv. tomato DC3000.


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for antibacterial defense, the question arisesas to whether bacterial pathogens also devel-oped similar silencing suppressors to counterantibacterial defense responses in plants.Recently, Navarro et al. (71) identified severalPst type III secretion effectors that suppresshost RNA silencing machinery and thereforeincrease disease susceptibility (Figure 3).AvrPtoB represses transcription of miRNAgenes and results in a low level of pri-miR393.Some pri-miRNAs were unaffected, however,and it is therefore unlikely that AvrPtoB isa general transcriptional suppressor of themiRNA pathway. AvrPtoB might suppress aspecific component involved in plant defensethat is required for transcription of miR393genes. Another effector, AvrPto, interfereswith processing of some miRNA precursorsand downregulates the level of mature miR393.It remains to be determined whether AvrPtodirectly suppresses miRNA-processing com-ponents, such as DCL1 and HYL1, or thecomponents required for miRNA stability, suchas HEN1. In addition, HopT1 inhibits the ac-tion of the AGO1 protein in the RISC complex.Thus, as with viruses, bacteria have evolved an

array of effectors that target different steps ofthe miRNA pathway. We speculate that otherpathogens, such as fungi and oomycetes, havealso developed RNAi suppressors to counteracthost antipathogen RNA-silencing mechanisms.

CONCLUDING REMARKS

More and more studies have shown that manyhost miRNAs and siRNAs are induced or sup-pressed by various pathogen challenges andthat modulation of miRNA and siRNA lev-els plays an important role in gene expres-sion reprogramming and fine-tuning plant re-sponses against a wide range of pathogens(Figure 4). These pathogen-responsive smallRNAs induce posttranscriptional gene silenc-ing by guiding mRNA cleavage/degradationor translational repression, or may guide tran-scriptional gene silencing by direct DNAmethylation or chromatin modification. Thisidea is supported by observations that manycomponents in the small RNA pathways are re-quired for plant defense responses and immu-nity. As a countermeasure, viruses and bacte-ria have developed VSRs and BSRs to suppress

Supressors ofRNA silencing

(VSRs and BSRs)

PTI

ETIPlants

Plants

Pathogens

miRNAsiRNA

miRNAsiRNA

Negativeregulators

Positiveregulators

PAMP/PRRs

Effector/R protein

miRNAsiRNA

miRNAsiRNA

Negativeregulators

Positiveregulators

Figure 4Regulation of immunity against pathogens is regulated by small RNAs in plants. An overview of PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) regulated by small RNAs, RNA silencingsuppressors (VSRs and BSRs) repress the small RNA silencing pathway. PAMPs: Pathogen-associated molecular patterns; PRRs: Pattern recognition receptors; VSRs: Viral suppressors of RNAsilencing; BSRs: Bacteria-encoded suppressors of RNA silencing.


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host RNAi machinery and compromise diseaseresistance in plants. To combat continuouslyevolving pathogens, plants have also evolvedcomponents, such as R proteins, that can rec-ognize VSRs and BSRs and trigger robust andrapid resistant responses, which are referred toas ETI.

The study of small RNA–mediated regu-latory mechanisms in plant immunity is anemerging field, and we expect that many morepathogen-responsive small RNAs will be iden-tified using new technologies, such as high-throughput deep sequencing. Characterization

of these small RNAs and their target geneswill reveal new components in plant resistancesignaling pathways and help us understand themolecular mechanisms of plant immunity. Wealso expect that more silencing suppressors willbe identified from viruses, bacteria, fungi, andoomycetes, and that such identification willincrease our understanding of the interactionand coevolution between pathogens and planthosts. These studies will elucidate the molecu-lar mechanisms of plant defense responses andwill ultimately lead to the development of ef-fective tools for controlling disease in the field.

SUMMARY POINTS

1. Endogenous small RNAs play pivotal roles in reprogramming of host gene expression inresponse to infection by a wide range of microbes, including viruses, bacteria, and fungi.

2. Small RNAs contribute to PTI or basal defense, as well as ETI or race-specific resistance.

3. Small RNAs are generated from pathogen-derived nucleic acids as seen in viruses andAgrobacterium. These pathogen-derived small RNAs may regulate the interaction ofpathogens with plants.

4. Endogenous small RNAs are induced or suppressed during legume-rhizobia symbioses.These small RNAs are thought to facilitate the plant-bacterium interaction by regulatingdifferent steps of development of nitrogen-fixing nodules.

5. Different components of small RNA pathways directly play important roles in mediatinghost immune responses against pathogens.

6. RNA silencing is activated in plants to counteract pathogen infection. To counter-counteract effects of silencing and to establish infection, rapidly evolving pathogenssecrete suppressors of RNA silencing (VSRs and BSRs) that inhibit different steps ofsmall RNA pathways.

7. To combat continuously evolving pathogens, plants have evolved components such asR proteins that can recognize pathogen-derived suppressors to mount robust and rapidresistant responses.

FUTURE ISSUES

1. Deep sequencing technologies will allow small RNA profiling of plants infectedwith various pathogens and identification of small RNAs involved in plant-microbeinteractions.

2. It is a challenge to identify and characterize the target genes of newly discovered smallRNAs that exhibit alteration in their expression level on pathogen infection. Overcomingthis challenge will allow rapid deciphering of new components in plant-pathogen interac-tions and lead to elucidation of the complex regulatory network of host immune systems.


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3. Identification of pathogen-derived small RNAs and their potential host targets will helpus understand how pathogens cause disease. Whether there is any functional interac-tion between pathogen-derived small RNAs and host mRNAs is an interesting area ofresearch.

4. Identification of different components of small RNA machinery during disease suscep-tibility and resistance responses remains an active area of research. It is still not knownwhether components of the disease resistance pathway are also regulating generation ofsmall RNAs.

5. Studying the changes in DNA methylation profiles of host plants after infection withvarious pathogens may help us understand the evolution of new components in plantdefense for combating rapidly evolving pathogens.

6. Microbes such as virus and bacteria encode suppressors that counteract the plant defensesystem. The identification of additional silencing suppressors from different pathogensand the elucidation of their functions and how they act as suppressors will shed light onthe generality of suppression of disease resistance. It is interesting and useful to identifythe targets of these suppressors and see whether these suppressors affect other pathwaysin plants besides small RNA pathways. Another tempting projection is that the pathogen-encoded suppressors could affect transposon activation and affect plant gene expression.Understanding the action of these suppressors will unravel the diversity and evolutionof pathogens as well as RNA silencing pathways.

7. Information generated from characterization of new small RNAs and the regulatorynetwork of host immune systems needs to be scrutinized for development of tools toenhance plant resistance against pathogens.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We are grateful to Manu Agarwal, Thomas Eulgem, Isgouhi Kaloshian, Shou-Wei Ding, andPadmanabhan Chellappan for critical reading and invaluable comments and Somya Sinha forhelping with the preparation of the manuscript. Katiyar-Agarwal Laboratory receives fundingfrom Department of Biotechnology, Ministry of Science and Technology, India and UniversityGrants Commission, India. Research in Jin Laboratory is supported by National Science Founda-tion Career Award MCB-0642843, University of California Discovery Grant Bio06-10566, andCalifornia Citrus Board grants 5210-131 and 5210-132. We apologize for not citing some publi-cations owing to space limitations.

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Role of Small RNAs in Host-Microbe Interactions · PY48CH11-Jin ARI 29 April 2010 19:41 R E V I E W...

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Transcript of Role of Small RNAs in Host-Microbe Interactions · PY48CH11-Jin ARI 29 April 2010 19:41 R E V I E W...