Poty Virus

PY48CH11-Jin ARI 5 July 2010 19:21

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. 48:225–46

First published online as a Review in Advance onMay 6, 2010

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

0066-4286/10/0908/0225$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 on 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.

225

Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


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, 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 by either mRNA degradation ortranslational repression. (b) trans-acting 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 the DCL4-DRB4 complex and cut into phased 21-nt small RNAs that undergo further methylation by HEN1. Theta-siRNAs are incorporated into a RISC containing AGO7 (in the case of TAS3) or AGO1 (in the case of TAS1 and 2), which results intarget 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 RISC complex. RDR6-SGS3, together with Pol IV, forms anamplification loop to generate 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 theformation of single-stranded RNA (ssRNA), which is converted into dsRNA by the action of RDR2. This dsRNA is processed intopredominantly 24-nt long hc-siRNAs by DCL3. These 24-nt siRNAs associate with AGO4 (or AGO6, or AGO9) through an adaptorprotein, KTF1, to form an RNA-directed DNA methylation (RdDM) effector complex that directly or indirectly recruits proteinsinvolved in heterochromatin 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, ordsRNAs from the action of Pol IV and RDRs. lsiRNAs are methylated by HEN1 and repress the expression of target genes by guidingmRNA decapping 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

226 Katiyar-Agarwal · Jin

Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


Pri

ma

ry m

iRN

A

Po

l II

7m

G

An

An

a m

iRN

A p

ath

way

b t

a-s

iRN

A p

ath

way

c n

at-

siR

NA

pa

thw

ayd

hc-

siR

NA

pa

thw

aye

Isi

RN

A p

ath

way

Tra

nsl

ati

on

al

rep

ress

ion

mR

NA

de

gra

da

tio

n

7m

G

An

AG

O1

Po

l II

TAS

pre

curs

or

7m

G

An

AG

O1

7m

GA

n

AG

O1

40

S

60

S

7m

G

An

7m

G

An

7m

G

An

AG

O7

TAS

3

miR

390

7m

G

An

AG

O1

TAS

1,2

TAS

1,2

TAS

3

HE

N1 ta-s

iRN

A t

arg

et

mR

NA

de

gra

da

tio

n

7m

G

An

AG

O1

+?

RD

R6

RD

R6

SG

S3

DC

L4D

RB

4

HE

N1

Po

l II

An

an

d/o

rD

CL2

RD

R2

dsR

NA

DC

L3

Po

l IV

HE

N1

AG

04

+ K

TF

1o

r A

G0

6o

r A

G0

9

Po

l V +

RD

M4

DR

M2

/DR

D1

/DM

S3

or

RD

R6

dsR

NA

dsR

NA

Pol IV and RDR6 - SGS 3

Pol IV and RDR6

De

cap

pin

g

An

An

AG

O7

7m

G

An

AG

O7

mR

NA

de

gra

da

tio

n

mR

NA

de

gra

da

tio

n

DC

P2

VC

S

7m

GA

n

AG

O?

Po

l II

Pre

curs

or

miR

NA

miR

NA

/miR

NA

*

HE

N1

DC

L1D

DL

SE

HY

L1 DC

L1

HY

L1

HY

L1

DC

L1

HY

L1

DC

L1

Targ

et

ge

ne

Targ

et

ge

ne

Targ

et

ge

ne

Targ

et

ge

ne

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

me

HST

HST

XR

N4

Cy

top

lasm

Nu

cle

us

miR

173

SG

S3

www.annualreviews.org • Small RNAs in Plant Immunity 227

Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


RISC: RNA-inducedsilencing complex

PAMP: pathogen-associated 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 act asa 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 nt 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 addition to21 to 24 nt siRNAs, a class of lsiRNAs in thesize range of 30 to 40 nt was discovered (54).The biogenesis of lsiRNAs is dependent onDCL1, HYL1, HEN1, AGO7, and 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 examplesof how pathogens are encountered at variouslevels. The preliminary interaction betweenthe pathogen and its host is responsiblefor pathogen-associated molecular pattern(PAMP)-triggered immunity (PTI) in plants(15, 52). Bacteria counteract PTI by secretingand injecting effector proteins into plant cells,which leads to suppression of PTI. Host plants,in turn, have evolved resistance componentssuch as resistance (R) proteins that can rec-ognize effectors and elicit effector-triggeredimmunity (ETI) (15, 52).

An important role of 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 between


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


TTSS: type IIIsecretion system

plant and pathogen that is mediated by smallRNAs. 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, viruses, and fungi. Moreover,it is 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,Flg22. miR393 negatively regulates auxin sig-naling 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 cleavagebecause of extra mismatches in the miRNA tar-get site. Transgenic lines expressing Myc-AFB1in tir1-1 background were more susceptible tovirulent Pseudomonas syringae pv. tomato strainDC3000 (Pst DC3000) and displayed enhanceddisease symptoms. To determine whethermiR393 is involved in race-specific resistance,these transgenic plants were inoculated withavirulent Pst DC3000 carrying an effector gene,avrRpt2. Bacterial growth in transgenic lines ex-pressing Myc-AFB1 did not differ from non-transformed plants even at 4 days postinocula-

tion (dpi). These data suggest that miR393 hasa role in imparting basal resistance but not race-specific resistance. Induction of miR393 wasfurther confirmed by Fahlgren et al. (31) whenthey carried out deep sequencing of Arabidopsisleaves at 1 h and 3 h postinoculation (hpi) witha nonpathogenic strain, Pst DC3000 hrcC−,which has a mutation in the type III secretionsystem (TTSS). Relative to uninfected leaves,miR393 was induced tenfold in the infectedleaves at 3 hpi. Overexpression of miR393afrom a strong constitutive promoter resultedin lower levels of TIR1 mRNA in transgeniclines, and these lines exhibited restricted bacte-rial growth. Besides miR393, two other miRNAfamilies, miR160 and miR167, were also upreg-ulated at 3 hpi. These miRNAs target mem-bers of the auxin-response factor (ARF) familyof transcription factors that are also involved inauxin signaling (85). Thus, in response to bacte-rial infection, plants suppress multiple compo-nents of the auxin signaling pathways. AnothermiRNA, miR825, which is predicted to targetremorin, zinc finger homeobox family protein,and frataxin-related protein, was also elevatedduring Pst hrcC − infection (31).

The interaction between the plant andanother 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, whereas thedisarmed strain (i.e., a strain lacking tumor-inducing properties) did not induce miR393.Interestingly, the levels of miR393 and miR167,which repress the auxin signaling pathway,were greatly reduced in tumors induced by A.tumefaciens infection. Derepression of the auxinsignaling pathway promotes tumor growth.Moreover, roots and stems of miRNA-deficientmutants, dcl1 and hen1, were immune to A.tumefaciens infection (28). All of these resultsindicate a possible role of miR393 and miR167in regulating auxin responses in tumor tissue.However, the levels of all other miRNAs stud-ied were moderately altered in tumor tissue.All these results demonstrate the importance


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


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 upregulation 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 galled stem. These re-duced levels of miRNAs in galled stems rela-tive 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 inthe stem above the gall, their target transcriptswere significantly upregulated in stem abovethe gall as compared with healthy stems. Thisresult suggests that fungal infection at the gallprobably immunizes the uninfected stem andmay provide protection ahead of the spreadinginfection. 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 pathogenic fungiand bacteria. This prompts the question ofwhether host miRNAs respond to viral in-fection. Two miRNAs, bra-miR158 and bra-miR1885, were greatly upregulated when Bras-sica rapa was infected by Turnip mosaic virus(TuMV) (42). The induction of bra-miR158and bra-miR1885 is highly specific to TuMVinfection because infection of B. rapa and B.napus with Cucumber mosaic virus, Tobaccomosaic virus (TMV), or the fungal pathogenSclerotinia sclerotiorum had 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.


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


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 of

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 miR159and miR393 exhibited significant upregula-tion at 3 hpi, miR160 and miR169 showeddownregulation. Rhizobial infection changesthe levels of miR160, miR393, miR164, andmiR168, which target ARFs (ARF10, ARF16,and ARF17), 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 nodule development for nitrogen fixation.Different steps in nodule formation are shown along with the miRNAs predicted to be involved at specificsteps. (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 equilibriumlead 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.


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


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 to con-served miR167, miR172, miR396, and miR399families, whereas another four families hadsequences homologous to gma-miR1507, gma-miR1508, gma-miR1509, and gma-miR1510,which 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 in nodulematuration and nitrogen fixation (Figure2). Further investigations on functions ofthese 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 stronglyupregulated during nodule development,are 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-endogenousnat-siRNA identified as involved in plant im-munity is nat-siRNAATGB2, which regulatesR-gene mediated ETI (55). This small RNAis generated from the overlapping regionof a NAT pair, which encodes a Rab2-likeGTP-binding protein gene ATGB2 and apentatricopepetide protein (PPR)-like genePPRL. The endogenous RNA is specifically andstrongly induced by Pst (avrRpt2) and cleavesantisense PPRL mRNA for silencing. Todefine the components involved in its


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


R proteins: resistanceproteins

biogenesis, we examined the accumulationof ATGB2 nat-siRNAs in Pst (avrRpt2)-challenged small RNA-biogenesis mutants.This small RNA is processed by the DCL1-HYL1 complex, stabilized by HEN1-mediatedmethylation, and amplified by RDR6, SGS3,and RNA Pol IV. We further demonstratedthat the induction of nat-siRNAATGB2 by Pst(avrRpt2) also requires the cognate host R geneRPS2 and its resistance signaling components,including NDR1. Constitutive overexpressionof the target gene PPRL without the nat-siRNAtarget site in transgenic Arabidopsis plants re-sulted in a delayed hypersensitive response(HR) and attenuated RPS2-mediated diseaseresistance. These results strongly suggestthat PPRL acts as a negative regulator ofRPS2-mediated resistance. PPRL is an atypicaltandem PPR and is likely to be localized inmitochondria (43, 62), which may contributeto Pst (avrRpt2)-triggered oxidative burst, HR,and programmed cell death (PCD). Proteinscontaining the PPR domain are believed tobe involved mainly in posttranscriptionalprocesses in organelles, including RNA editing(87), mRNA silencing by cleavage (103), andtranslational 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 21-to 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 an 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 rapidcounterdefense 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 trig-gering a cascade of events leading to diseaseresistance (21, 64). These R genes are gen-erally clustered in the genome and encodeproteins with common motifs. To keep pacewith the continuous and rapid evolution ofpathogens, R genes undergo coevolution result-ing in variable gene clusters. The Arabidopsisthaliana ecotype Columbia RPP4 locus (knownas RPP5 in Landsberg erecta for recognitionof the oomycetes Hyaloperonospora parasitica 5)is composed of seven TIR-NBS-LRR class-Rgenes along with three related and two unre-lated genes (73, 109). Two R genes in this lo-cus, named RPP4 and SNC1 (for suppressorof npr1-1, constitutive 1), impart resistance toboth P. syringe pv. maculicola and H. parasitica(89, 97, 108, 114). These two R genes are coor-dinately regulated by transcriptional activationand siRNA-mediated RNA silencing. Endoge-nous siRNAs generated at the RPP4 locus ap-parently target SNC1 because enhanced tran-script levels of SNC1 were observed in smallRNA biogenesis-deficient mutants such as dcl4,


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


upf1, and ago1 as well as in transgenic plantsexpressing P1/HC-Pro suppressor. This fine-tuning of R-gene expression is important be-cause 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 fewer than 600 base pairs(bp) long, lack open reading frames, anddepend on the activity of transposons for theirmobility (49, 50). MITEs are present in highcopy numbers in several plant genomes and arepredicted to be regulatory elements in plantgene expression (33, 50). They are also thoughtto serve as a major evolutionary element intransposon-mediated gene regulation in plantsby generating small RNAs. The complexity ofthe TMV R gene N is maintained by MITE-mediated creation of new gene structures (56).Virus infection may lead to temporary inhi-bition of PTGS for N expression and siRNA-guided cleavage. This may induce prematuretranslation termination, provide a polyadeny-lation signal, or introduce deletions/insertionsleading to gene diversity. Kuang et al. (56) sug-gested that biogenesis of most MITE-derivedsmall RNAs (MiS) depends on the NR-PDla/RDR2/DCL3/AGO4 pathway. DCL4is also implicated in the accumulation of MiSsmall RNAs (56). It will help our understandingof R gene evolution to elucidate the mech-anisms of generating small RNAs from MiSand their functionality in gene regulation inplants.

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 partially

overlapping functions in different small RNApathways. Arabidopsis has four DCLs, six RDRs,and ten AGOs, many of which are involved inplant-defense signaling pathways.

Dicer-Like Proteins andTheir Associated Proteins

Arabidopsis contains four DCLs that processdsRNA or fold-back RNA precursors to gener-ate siRNAs 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 photo-bleaching 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 and dcl4 exhibited hypersuscep-tibility to the viral infection, which suggeststhe combined action of these two specificproteins in antiviral defense. Unlike DCL2and DCL4, DCL1 and DCL3 were not foundto be involved in antiviral immunity in this


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


study. A similar study by Qu et al. (82), whichaimed 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 wasnot significantly different between wild-typeplants and single mutants (dcl2, dcl3, dcl4) ordouble mutants (dcl2-dcl3, dcl2-dcl4, dcl3-dcl4).DCL1 plays a positive role in antiviral defenseby facilitating synthesis of viRNAs derivedfrom the intramolecular hairpins formed in the35S-leader sequence of CaMV. These hairpinsresemble miRNA precursors, and DCL1probably identifies this structure, excises it,and then presents it to other Dicers for furtherprocessing.

Small dsRNA-binding proteins (DRBs) areessential cofactors of DCL proteins (45, 69).These DRBs, however, do not exhibit hier-archical redundancy as do DCLs (19). DCL1and DCL4 interact with DRB1/HYL1 andDRB4, respectively (45, 69). DRB4 contributesto antiviral 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 increase inthe accumulation of the viral RNA when com-pared with that in wild type, which suggests

that 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 RNA Polymerases

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 becauseArabidopsis rdr1 mutant plants had enhancedlevels of viral RNAs (112). NtRDR1 is alsoinvolved in combating potato virus Y (PVY) in-fection; knocking down expression of RDR1 in


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


transgenic tobacco plants resulted in reducedexpression of other defense-related genessuch as AOX1 and ERF5 (84). These studiesraise the question of 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 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 genomes isaltered in response to attack by pathogens, in-cluding viruses, bacteria, and fungi (34, 39, 76,88). hc-siRNAs trigger transcriptional gene si-lencing (TGS) by guiding RNA-directed DNAmethylation (RdDM) and histone modificationin plants (47, 66, 96). AGO4 is a major nuclearRNAi effector associated with hc-siRNAs or ra-siRNAs that direct DNA methylation (60, 80).Involvement of AGO4 in the disease-resistanceresponse links DNA methylation and plant de-fense. When attacked by viruses, plants employDNA methylation to repress viral transcriptionand/or replication. Upon infection by eitherof two geminiviruses, cabbage leaf curl virus(CaLCuV) or beet curly top virus (BCTV),Arabidopsis plants silence viral chromatin byboth cytosine and histone methyltransferases(83). This is evident from the hypersuscepti-bility of the methylation-deficient mutants togeminiviruses, including mutants of cytosinemethyltransferases (drm1, drm2, cmt3, andmet1), histone H3K9 methyltransferase (kyp2),RdDM pathway components (ago4, ddm1,and nrpd2A), or methyl cycle enzymes (adk1and adk2) (83). Viral suppressors AL2 and L2inhibit the activity of adenosine kinase (ADK),a cellular enzyme involved in the generationof S-adenosylmethionine (a methyltransferasecofactor). Therefore, plants infected withvirus lacking L2 had hypermethylation of viralDNA. Additionally, recovery of the virus-infected plants from the disease symptomsrequired AGO4 (83). Chromatin methylationmay be a generalized process adopted by plantsto 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


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


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 exhibits reduced resistance to virulent PstDC3000 as well as to avirulent Pst (avrRpm1).However, 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 vi-ral RNAs with more compact structures, butAGO7 is a surrogate slicer whose targets are lessstructured (82). The biogenesis of AtlsiRNA-1 is known to involve AGO7, as ago7 mutantdoes not accumulate AtlsiRNA-1 (54). How-ever, other ago mutant plants, including ago3,

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, 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,ta-siRNA, 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 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 prevent 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, and 24 bp siRNAs into 14-bp fragments.

18


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


7m

G

An

AG

O

miR

NA

gen

esPr

e-m

iRN

A

ssRN

AG

enes

and

inte

rgen

icre

gion

s

Po

l II

Po

l II

Pri-m

iRN

A

dsRN

A

AG

O

7m

G

An

AG

O

mR

NA

de

gra

dat

ion

Tran

slat

ion

alre

pre

ssio

n

Imm

unit

yag

ains

tpa

thog

enm

iRN

A

siRN

A

AG

O p

rote

inac

tio

n

miR

NA

pro

cess

ing

TT

SS

Tran

scri

pti

on

of

miR

NA

ge

ne

Pst

FLS Po

l IV

HE

N1

RD

Rs

DC

L +

DR

B

HE

N1

DC

L1-H

YL1

DC

L1-H

YL1

SE

DD

L

avrP

toav

rPto

BH

op

T1

me

me

me

me

me

me

me

me

me

me

ago4, and ago9, showed no significant changein the level of AtlsiRNA-1 as compared withwild type. AGO7 is also associated with TAS3ta-siRNA (1, 32, 36). The accumulation ofbacteria-induced AtlsiRNA-1 is dependent onAGO7, suggesting a role of AGO7 in antibac-terial defense.

RNA SILENCING SUPPRESSORSOF PATHOGENS

Viral Suppressors of RNA Silencing

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

As noted in the previous paragraph, virusesencode VSRs that suppress host antiviralsilencing machinery and thereby promotetheir pathogenesis. Because small RNAs 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.


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


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

PAMPs/PRRs

Effectors/R proteins

miRNAsiRNA

miRNAsiRNA

Negativeregulators

Positiveregulators

Figure 4Immunity against pathogens is regulated by small RNAs in plants. In PAMP-triggered immunity (PTI) andeffector-triggered immunity (ETI) regulated by small RNAs, RNA silencing suppressors (VSRs and BSRs)repress the small RNA silencing pathway. PAMPs, pathogen-associated molecular patterns; PRRs, patternrecognition receptors; VSRs, viral suppressors of RNA silencing; BSRs, bacteria-encoded suppressors ofRNA silencing.


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


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 in the 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 rapidresistance 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 discoveredsmall RNAs that exhibit alteration in their expression level upon pathogen infection.Overcoming this challenge will allow rapid deciphering of new components in plant-pathogen interactions and lead to elucidation of the complex regulatory network of hostimmune systems.


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


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 also regulate generation of smallRNAs.

5. Studying the changes in the 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 viruses and bacteria encode suppressors that counteract the plantdefense system. The identification of additional silencing suppressors from differentpathogens and the elucidation of their functions and how they act as suppressors willshed light on the generality of suppression of disease resistance. It is interesting and use-ful to identify the targets of these suppressors and see whether these suppressors affectother pathways in plants besides small RNA pathways. Another tempting projection isthat the pathogen-encoded suppressors could affect transposon activation and affect plantgene expression. Understanding the action of these suppressors will unravel the diversityand evolution of 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 to Somya Sinha forhelping with the preparation of the manuscript. The Katiyar-Agarwal laboratory receives fundingfrom Department of Biotechnology, Ministry of Science and Technology, India and UniversityGrants Commission, India. Research in the Jin laboratory is supported by National Science Foun-dation 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.

LITERATURE CITED

1. Adenot X, Elmayan T, Lauressergues D, Boutet S, Bouche N, et al. 2006. DRB4-dependent TAS3trans-acting siRNAs control leaf morphology through AGO7. Curr. Biol. 16(9):927–32

2. Agorio A, Vera P. 2007. ARGONAUTE4 is required for resistance to Pseudomonas syringae in Arabidopsis.Plant Cell 19:3778–90


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


3. Anandalakshmi R, Marathe R, Ge X, Herr JM Jr, Mau C, et al. 2000. A calmodulin-related protein thatsuppresses posttranscriptional gene silencing in plants. Science 290:142–44

4. Baulcombe D. 1999. Viruses and gene silencing in plants. Arch. Virol. Suppl. 15:189–2015. Baulcombe D. 2004. RNA silencing in plants. Nature 431:356–636. Bhattacharjee S, Zamora A, Azhar MT, Sacco MA, Lambert LH, Moffett P. 2009. Virus resistance

induced by NB-LRR proteins involves Argonaute4-dependent translational control. Plant J. 58(6):940–51

7. Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, et al. 2006. Four plant Dicersmediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res. 34:6233–46

8. Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK. 2005. Endogenous siRNAs derived from a pair ofnatural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123:1279–91

9. Bouche N, Lauressergues D, Gasciolli V, Vaucheret H. 2006. An antagonistic function for ArabidopsisDCL2 in development and a new function for DCL4 in generating viral siRNAs. EMBO J. 25:3347–56

10. Boutet S, Vazquez F, Liu J, Beclin C, Fagard M, et al. 2003. Arabidopsis HEN1: a genetic link betweenendogenous miRNA controlling development and siRNA controlling transgene silencing and virus re-sistance. Curr. Biol. 13:843–48

11. Chapman EJ, Carrington JC. 2007. Specialization and evolution of endogenous small RNA pathways.Nat. Rev. Genet. 8:884–96

12. Chellappan P, Vanitharani R, Fauquet CM. 2005. MicroRNA-binding viral protein interferes withArabidopsis development. PNAS 102:10381–86

13. Chellappan P, Zhang X, Jin H. 2009. Host small RNAs are big contributors to plant innate immunity.Curr. Opin. Plant Biol. 12:465–72

14. Chen J, Li WX, Xie D, Peng JR, Ding SW. 2004. Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microRNA in host gene expression. Plant Cell 16:1302–13

15. Chisholm ST, Coaker G, Day B, Staskawicz BJ. 2006. Host-microbe interactions: shaping the evolutionof the plant immune response. Cell 124(4):803–14

16. Combier JP, Frugier F, de Billy F, Boualem A, El-Yahyaoui F, et al. 2006. MtHAP2–1 is a key transcrip-tional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula.Genes Dev. 20:3084–88

17. Cooper JE. 2007. Early interactions between legumes and rhizobia: disclosing complexity in a moleculardialogue. J. Appl. Microbiol. 103:1355–65

18. Cuellar WJ, Kreuze JF, Rajamaki ML, Cruzado KR, Untiveros M, Valkonen JP. 2009. Elimination ofantiviral defense by viral RNase III. Proc. Natl. Acad. Sci. USA 106(25):10354–58

19. Curtin SJ, Watson JM, Smith NA, Eamens AL, Blanchard CL, Waterhouse PM. 2008. The roles ofplant dsRNA-binding proteins in RNAi-like pathways. FEBS Lett. 582:2753–60

20. Dalmay T, Hamilton A, Rudd S, Angell S, Baulcombe DC. 2000. An RNA-dependent RNA polymerasegene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not bya virus. Cell 101:543–53

21. Dangl JL, Jones JD. 2001. Plant pathogens and integrated defence responses to infection. Nature411:826–33

22. Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O. 2006. Hierarchicalaction and inhibition of plant Dicer-like proteins in antiviral defense. Science 313:68–71

23. Diaz-Pendon JA, Ding SW. 2008. Direct and indirect roles of viral suppressors of RNA silencing inpathogenesis. Annu. Rev. Phytopathol. 46:303–26

24. Diaz-Pendon JA, Li F, Li WX, Ding SW. 2007. Suppression of antiviral silencing by cucumber mosaicvirus 2b protein in Arabidopsis is associated with drastically reduced accumulation of three classes of viralsmall interfering RNAs. Plant Cell 19:2053–63

25. Dietrich RA, Richberg MH, Schmidt R, Dean C, Dangl JL. 1997. A novel zinc finger protein is encodedby the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death. Cell 88(5):685–94

26. Ding SW, Voinnet O. 2007. Antiviral immunity directed by small RNAs. Cell 130:413–2627. Dorssers L, Zabel P, Van Der Meer J, van Kammen A. 1982. Purification of a host-encoded RNA-

dependent RNA polymerase from cowpea mosaic virus–infected cowpea leaves. Virology 116:236–49


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


28. Dunoyer P, Himber C, Voinnet O. 2006. Induction, suppression and requirement of RNA silencingpathways in virulent Agrobacterium tumefaciens infections. Nat. Genet. 38(2):258–63

29. El Yahyaoui F, Kuster H, Ben Amor B, Hohnjec N, Puhler A, et al. 2004. Expression profiling inMedicago truncatula identifies more than 750 genes differentially expressed during nodulation, includingmany potential regulators of the symbiotic program. Plant Physiol. 136:3159–76

30. Ellendorff U, Fradin EF, de Jonge R, Thomma BP. 2009. RNA silencing is required for Arabidopsisdefence against Verticillium wilt disease. J. Exp. Bot. 60:591–602

31. Fahlgren N, Howell MD, Kasschau KD, Chapman EJ, Sullivan CM, et al. 2007. High-throughputsequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes. PLoSOne 2:e219

32. Fahlgren N, Montgomery TA, Howell MD, Allen E, Dvorak SK, et al. 2006. Regulation of AUXINRESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis.Curr. Biol. 16:939–44

33. Feschotte C. 2004. Merlin, a new superfamily of DNA transposons identified in diverse animal genomesand related to bacterial IS1016 insertion sequences. Mol. Biol. Evol. 21:1769–80

34. Finnegan EJ, Genger RK, Peacock WJ, Dennis ES. 1998. DNA methylation in plants. Annu. Rev. PlantPhysiol. Plant Mol. Biol. 49:223–47

35. Fusaro AF, Matthew L, Smith NA, Curtin SJ, Dedic-Hagan J, et al. 2006. RNA interference-inducinghairpin RNAs in plants act through the viral defence pathway. EMBO Rep. 7:1168–75

36. Garcia D, Collier SA, Byrne ME, Martienssen RA. 2006. Specification of leaf polarity in Arabidopsis viathe trans-acting siRNA pathway. Curr. Biol. 16:933–38

37. Gasciolli V, Mallory AC, Bartel DP, Vaucheret H. 2005. Partially redundant functions of ArabidopsisDICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr. Biol. 15:1494–500

38. Goto K, Kobori T, Kosaka Y, Natsuaki T, Masuta C. 2007. Characterization of silencing suppressor 2bof cucumber mosaic virus based on examination of its small RNA-binding abilities. Plant Cell Physiol.48(7):1050–60

39. Guseinov VA, Kiryanov GI, Vanyushin BF. 1975. Intragenome distribution of 5-methylcytosine in DNAof healthy and wilt-infected cotton plants (Gossypium hirsutum L.). Mol. Biol. Rep. 2:59–63

40. Haas G, Azevedo J, Moissiard G, Geldreich A, Himber C, et al. 2008. Nuclear import of CaMV P6 isrequired for infection and suppression of the RNA silencing factor DRB4. EMBO J. 27(15):2102–12

41. Hannon GJ. 2002. RNA interference. Nature 418(6894):244–5142. He X-F, Fang Y-Y, Feng L, Guo H-S. 2008. Characterization of conserved and novel microRNAs and

their targets, including a TuMV-induced TIR-NBS-LRR class R gene-derived novel miRNA in Brassica.FEBS Lett. 582:2445–52

43. Heazlewood JL, Tonti-Filippini JS, Gout AM, Day DA, Whelan J, Millar AH. 2004. Experimental anal-ysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components providesassessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins. PlantCell 16:241–56

44. Herr AJ. 2005. Pathways through the small RNA world of plants. FEBS Lett. 579(26):5879–8845. Hiraguri A, Itoh R, Kondo N, Nomura Y, Aizawa D, et al. 2005. Specific interactions between Dicer-

like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol. Biol.57:173–88

46. Howell MD, Fahlgren N, Chapman EJ, Cumbie JS, Sullivan CM, et al. 2007. Genome-wide analysisof the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidopsis revealsdependency on miRNA- and tasiRNA-directed targeting. Plant Cell 19(3):926–42

47. Huettel B, Kanno T, Daxinger L, Bucher E, Van Der Winden J, et al. 2007. RNA-directed DNAmethylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing inplants. Biochim. Biophys. Acta 1769(5–6):358–74

48. Jagadeeswaran G, Saini A, Sunkar R. 2009. Biotic and abiotic stress down-regulate miR398 expressionin Arabidopsis. Planta 229:1009–14

49. Jiang N, Bao Z, Zhang X, Hirochika H, Eddy SR, et al. 2003. An active DNA transposon family in rice.Nature 421:163–67


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


50. Jiang N, Feschotte C, Zhang X, Wessler SR. 2004. Using rice to understand the origin and amplificationof miniature inverted repeat transposable elements (MITEs). Curr. Opin. Plant Biol. 7:115–19

51. Jin H. 2008. Endogenous small RNAs and antibacterial immunity in plants. FEBS Lett. 582:2679–8452. Jones JD, Dangl JL. 2006. The plant immune system. Nature 444(7117):323–2953. Jones-Rhoades MW, Bartel DP, Bartel B. 2006. MicroRNAs and their regulatory roles in plants. Annu.

Rev. Plant. Biol. 57:19–5354. Katiyar-Agarwal S, Gao S, Vivian-Smith A, Jin H. 2007. A novel class of bacteria-induced small RNAs

in Arabidopsis. Genes Dev. 21:3123–3455. Katiyar-Agarwal S, Morgan R, Dahlbeck D, Borsani O, Villegas A Jr, et al. 2006. A pathogen-inducible

endogenous siRNA in plant immunity. Proc. Natl. Acad. Sci. USA 103:18002–756. Kuang H, Padmanabhan C, Li F, Kamei A, Bhaskar PB, et al. 2009. Identification of miniature inverted-

repeat transposable elements (MITEs) and biogenesis of their siRNAs in the Solanaceae: new functionalimplications for MITEs. Genome Res. 19:42–56

57. Kumakura N, Takeda A, Fujioka Y, Motose H, Takano R, Watanabe Y. 2009. SGS3 and RDR6 interactand colocalize in cytoplasmic SGS3/RDR6-bodies. FEBS Lett. 583:1261–66

58. Lamb C, Dixon RA. 1997. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. PlantMol. Biol. 48:251–75

59. Lelandais-Briere C, Naya L, Sallet E, Calenge F, Frugier F, et al. 2009. Genome-wide Medicago truncatulasmall RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules.Plant Cell 21(9):2780–96

60. Li CF, Henderson IR, Song L, Fedoroff N, Lagrange T, Jacobsen SE. 2008. Dynamic regulation ofARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana. PLoS Genet. 4:e27

61. Lu S, Sun YH, Amerson H, Chiang VL. 2007. MicroRNAs in loblolly pine (Pinus taeda L.) and theirassociation with fusiform rust gall development. Plant J. 51:1077–98

62. Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, et al. 2004. Genome-wide analysis of Arabidopsispentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16:2089–103

63. Mallory AC, Vaucheret H. 2006. Functions of microRNAs and related small RNAs in plants. Nat. Genet.38(Suppl):S31–36

64. Martin GB, Bogdanove AJ, Sessa G. 2003. Understanding the functions of plant disease resistanceproteins. Annu. Rev. Plant Biol. 54:23–61

65. Matzke MA, Birchler JA. 2005. RNAi-mediated pathways in the nucleus. Nat. Rev. Genet. 6(1):24–3566. Matzke M, Kanno T, Daxinger L, Huettel B, Matzke AJ. 2009. RNA-mediated chromatin-based silencing

in plants. Curr. Opin. Cell Biol. 21(3):367–7667. Moissiard G, Voinnet O. 2006. RNA silencing of host transcripts by cauliflower mosaic virus requires

coordinated action of the four Arabidopsis Dicer-like proteins. Proc. Natl. Acad. Sci. USA 103:19593–9868. Mourrain P, Beclin C, Elmayan T, Feuerbach F, Godon C, et al. 2000. Arabidopsis SGS2 and SGS3 genes

are required for posttranscriptional gene silencing and natural virus resistance. Cell 101:533–4269. Nakazawa Y, Hiraguri A, Moriyama H, Fukuhara T. 2007. The dsRNA-binding protein DRB4 interacts

with the Dicer-like protein DCL4 in vivo and functions in the trans-acting siRNA pathway. Plant Mol.Biol. 63:777–85

70. Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, et al. 2006. A plant miRNA contributes toantibacterial resistance by repressing auxin signaling. Science 312:436–39

71. Navarro L, Jay F, Nomura K, He SY, Voinnet O. 2008. Suppression of the microRNA pathway bybacterial effector proteins. Science 321:964–67

72. Neill SJ, Desikan R, Clarke A, Hurst RD, Hancock JT. 2002. Hydrogen peroxide and nitric oxide assignalling molecules in plants. J. Exp. Bot. 53:1237–47

73. Noel L, Moores TL, Van Der Biezen EA, Parniske M, Daniels MJ, et al. 1999. Pronounced intraspecifichaplotype divergence at the RPP5 complex disease resistance locus of Arabidopsis. Plant Cell 11:2099–112

74. Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, et al. 2003. Control of leaf morphogenesis bymicroRNAs. Nature 425:257–63

75. Park W, Li J, Song R, Messing J, Chen X. 2002. CARPEL FACTORY, a Dicer homolog, and HEN1,a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol. 12:1484–95


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


76. Pavet V, Quintero C, Cecchini NM, Rosa AL, Alvarez ME. 2006. Arabidopsis displays centromeric DNAhypomethylation and cytological alterations of heterochromatin upon attack by Pseudomonas syringae.Mol. Plant-Microbe Interact. 19:577–87

77. Pazhouhandeh M, Dieterle M, Marrocco K, Lechner E, Berry B, et al. 2006. F-box-like domain in thepolerovirus protein P0 is required for silencing suppressor function. Proc. Natl. Acad. Sci. USA 103:1994–99

78. Pikaard CS, Haag JR, Ream T, Wierzbicki AT. 2008. Roles of RNA polymerase IV in gene silencing.Trends Plant Sci. 13(7):390–97

79. Pruss GJ, Nester EW, Vance V. 2008. Infiltration with Agrobacterium tumefaciens induces host defenseand development-dependent responses in the infiltrated zone. Mol. Plant-Microbe Interact. 21:1528–38

80. Qi Y, He X, Wang XJ, Kohany O, Jurka J, Hannon GJ. 2006. Distinct catalytic and non-catalytic rolesof ARGONAUTE4 in RNA-directed DNA methylation. Nature 4437:1008–12

81. Qu F, Ye X, Hou G, Sato S, Clemente TE, Morris TJ. 2005. RDR6 has a broad-spectrum buttemperature-dependent antiviral defense role in Nicotiana benthamiana. J. Virol. 79:15209–17

82. Qu F, Ye X, Morris TJ. 2008. Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proc. Natl. Acad. Sci. USA105:14732–37

83. Raja P, Sanville BC, Buchmann RC, Bisaro DM. 2008. Viral genome methylation as an epigenetic defenseagainst geminiviruses. J. Virol. 82:8997–9007

84. Rakhshandehroo F, Takeshita M, Squires J, Palukaitis P. 2009. The influence of RNA-dependent RNApolymerase 1 on potato virus Y infection and on other antiviral response genes. Mol. Plant-MicrobeInteract. 22:1312–18

85. Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP. 2002. Prediction of plant microRNAtargets. Cell 110:513–20

86. Schmitz-Linneweber C, Williams-Carrier R, Barkan A. 2005. RNA immunoprecipitation and microarrayanalysis show a chloroplast pentatricopeptide repeat protein to be associated with the 5′ region of mRNAswhose translation it activates. Plant Cell 17:2791–804

87. Shikanai T. 2006. RNA editing in plant organelles: machinery, physiological function and evolution.Cell Mol. Life Sci. 63:698–708

88. Steward N, Ito M, Yamaguchi Y, Koizumi N, Sano H. 2002. Periodic DNA methylation in maizenucleosomes and demethylation by environmental stress. J. Biol. Chem. 277:37741–46

89. Stokes TL, Richards EJ. 2002. Induced instability of two Arabidopsis constitutive pathogen-responsealleles. Proc. Natl. Acad. Sci. USA 99:7792–96

90. Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O. 2008. Novel and nodulation-regulatedmicroRNAs in soybean roots. BMC Genomics 9:160

91. Sunkar R, Kapoor A, Zhu JK. 2006. Posttranscriptional induction of two Cu/Zn superoxide dismutasegenes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance.Plant Cell 18:2051–65

92. Sunkar R, Zhu JK. 2007. Micro RNAs and short-interfering RNAs in plants. J. Integr. Plant Biol. 49:817–826

93. Szittya G, Silhavy D, Molnar A, Havelda Z, Lovas A, et al. 2003. Low temperature inhibits RNAsilencing-mediated defence by the control of siRNA generation. EMBO J. 22:633–40

94. Torres MA, Jones JD, Dangl JL. 2006. Reactive oxygen species signaling in response to pathogens. PlantPhysiol. 141(2):373–78

95. Trinks D, Rajeswaran R, Shivaprasad PV, Akbergenov R, Oakeley EJ, et al. 2005. Suppression of RNAsilencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes. J. Virol.79:2517–27

96. Vaistij FE, Jones L, Baulcombe DC. 2002. Spreading of RNA targeting and DNA methylation in RNAsilencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase.Plant Cell 14:857–67

97. Van Der Biezen EA, Freddie CT, Kahn K, Parker JE, Jones JD. 2002. Arabidopsis RPP4 is a memberof the RPP5 multigene family of TIR-NB-LRR genes and confers downy mildew resistance throughmultiple signalling components. Plant J. 29:439–51


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.


98. Vaucheret H. 2006. Post-transcriptional small RNA pathways in plants: mechanisms and regulations.Genes Dev. 20:759–71

99. Vazquez F. 2006. Arabidopsis endogenous small RNAs: highways and byways. Trends Plant Sci. 11(9):460–68

100. Wang H, Buckley KJ, Yang X, Buchmann RC, Bisaro DM. 2005. Adenosine kinase inhibition andsuppression of RNA silencing by geminivirus AL2 and L2 proteins. J. Virol. 79:7410–18

101. Wang Y, Li P, Cao X, Wang X, Zhang A, Li X. 2009. Identification and expression analysis of miRNAsfrom nitrogen-fixing soybean nodules. Biochem. Biophys. Res. Commun. 378:799–803

102. Wang X-B, Wu Q, Ito T, Cillo F, Li W-X, et al. 2010. RNAi-mediated viral immunity requires ampli-fication of virus-derived siRNAs in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 107:484–89

103. Wang Z, Zou Y, Li X, Zhang Q, Chen L, et al. 2006. Cytoplasmic male sterility of rice with Boro IIcytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinctmodes of mRNA silencing. Plant Cell 18:676–87

104. Wendehenne D, Durner J, Klessig DF. 2004. Nitric oxide: a new player in plant signalling and defenceresponses. Curr. Opin. Plant Biol. 7:449–55

105. Wu HW, Lin SS, Chen KC, Yeh SD, Chua NH. 2010. Discriminating mutations of HC-pro of zucchiniyellow mosaic virus with differential effects on small RNA pathways involved in viral pathogenicity andsymptom development. Mol. Plant-Microbe Interact. 23(1):17–28

106. Xie Z, Fan B, Chen C, Chen Z. 2001. An important role of an inducible RNA-dependent RNA polymerasein plant antiviral defense. Proc. Natl. Acad. Sci. USA 98:6516–21

107. Xie Z, Qi X. 2008. Diverse small RNA-directed silencing pathways in plants. Biochim. Biophys. Acta1779(11):720–24

108. Yang S, Hua J. 2004. A haplotype-specific resistance gene regulated by BONZAI1 mediates temperature-dependent growth control in Arabidopsis. Plant Cell 16:1060–71

109. Yi H, Richards EJ. 2007. A cluster of disease resistance genes in Arabidopsis is coordinately regulated bytranscriptional activation and RNA silencing. Plant Cell 19:2929–39

110. Yu B, Bi L, Zheng B, Ji L, Chevalier D, et al. 2008. The FHA domain proteins DAWDLE in Arabidopsisand SNIP1 in humans act in small RNA biogenesis. Proc. Natl. Acad. Sci. USA 105:10073–78

111. Yu B, Chapman EJ, Yang Z, Carrington JC, Chen X. 2006. Transgenically expressed viral RNA silencingsuppressors interfere with microRNA methylation in Arabidopsis. FEBS Lett. 580:3117–20

112. Yu D, Fan B, MacFarlane SA, Chen Z. 2003. Analysis of the involvement of an inducible ArabidopsisRNA-dependent RNA polymerase in antiviral defense. Mol. Plant-Microbe Interact. 16:206–16

113. Zhang X, Yuan YR, Pei Y, Lin SS, Tuschl T, et al. 2006. Cucumber mosaic virus-encoded 2b suppressorinhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes Dev. 20:3255–68

114. Zhang Y, Goritschnig S, Dong X, Li X. 2003. A gain-of-function mutation in a plant disease resistancegene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1–1,constitutive 1. Plant Cell 15:2636–46

115. Zhu JK. 2009. Active DNA demethylation mediated by DNA glycosylases. Annu. Rev. Genet. 43:143–66


Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.

PY48-FM ARI 7 July 2010 17:52

Annual Review ofPhytopathology

Volume 48, 2010Contents

Go Where the Science Leads YouRichard S. Hussey � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Induced Systemic Resistance and Plant Responses to FungalBiocontrol AgentsMichal Shoresh, Gary E. Harman, and Fatemeh Mastouri � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �21

Plant Proteins Involved in Agrobacterium-Mediated GeneticTransformationStanton B. Gelvin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �45

Cellular Remodeling During Plant Virus InfectionJean-Francois Laliberte and Helene Sanfacon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �69

The Strigolactone StoryXiaonan Xie, Kaori Yoneyama, and Koichi Yoneyama � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �93

Current Epidemiological Understanding of Citrus HuanglongbingTim R. Gottwald � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 119

Pathogen Refuge: A Key to Understanding Biological ControlKenneth B. Johnson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 141

Companion Cropping to Manage Parasitic PlantsJohn A. Pickett, Mary L. Hamilton, Antony M. Hooper, Zeyaur R. Khan,and Charles A.O. Midega � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

Principles of Predicting Plant Virus Disease EpidemicsRoger A.C. Jones, Moin U. Salam, Timothy J. Maling, Arthur J. Diggle,and Deborah J. Thackray � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 179

Potyviruses and the Digital RevolutionAdrian Gibbs and Kazusato Ohshima � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 205

Role of Small RNAs in Host-Microbe InteractionsSurekha Katiyar-Agarwal and Hailing Jin � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 225

v

Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.

PY48-FM ARI 7 July 2010 17:52

Quantitative Disease Resistance and Quantitative Resistance Loci inBreedingDina A. St.Clair � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 247

Engineering Pathogen Resistance in Crop Plants: Current Trends andFuture ProspectsDavid B. Collinge, Hans J.L. Jørgensen, Ole S. Lund, and Michael F. Lyngkjær � � � � � � 269

Plant Pathology: A Story About BiologyThomas R. Gordon and Johan H.J. Leveau � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 293

Managing Nematodes Without Methyl BromideInga A. Zasada, John M. Halbrendt, Nancy Kokalis-Burelle, James LaMondia,Michael V. McKenry, and Joe W. Noling � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 311

Hyaloperonospora arabidopsidis as a Pathogen ModelMary E. Coates and Jim L. Beynon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 329

Playing the “Harp”: Evolution of Our Understanding of hrp/hrc GenesAnastasia P. Tampakaki, Nicholas Skandalis, Anastasia D. Gazi,Marina N. Bastaki, Panagiotis F. Sarris, Spyridoula N. Charova,Michael Kokkinidis, and Nickolas J. Panopoulos � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 347

Ecology of Plant and Free-Living Nematodes in Natural andAgricultural SoilDeborah A. Neher � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 371

Translational Research on Trichoderma: From ’Omics to the FieldMatteo Lorito, Sheridan L. Woo, Gary E. Harman, and Enrique Monte � � � � � � � � � � � � � � � 395

Xanthomonas AvrBs3 Family-Type III Effectors: Discoveryand FunctionJens Boch and Ulla Bonas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 419

Cowpea mosaic Virus: The Plant Virus–Based Biotechnology WorkhorseFrank Sainsbury, M. Carmen Canizares, and George P. Lomonossoff � � � � � � � � � � � � � � � � � � � 437

Studying Plant-Pathogen Interactions in the Genomics Era: BeyondMolecular Koch’s Postulates to Systems BiologyDavid J. Schneider and Alan Collmer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 457

Errata

An online log of corrections to Annual Review of Phytopathology articles may be found athttp://phyto.annualreviews.org/

vi Contents

Ann

u. R

ev. P

hyto

path

ol. 2

010.

48:2

25-2

46. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

by S

ung

Kyu

n K

wan

Uni

vers

ity -

Jon

gro

Gu

on 1

1/27

/12.

For

per

sona

l use

onl

y.

Poty Virus

Documents

Transcript of Poty Virus