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Roles of Plant Small RNAs
in Biotic Stress Responses
Virginia Ruiz-Ferrer and Olivier Voinnet
Institut de Biologie Moléculaire des Plantes du CNRS, UPR2357, 67084 Strasbourg Cedex,
France; email: [email protected]
Annu. Rev. Plant Biol. 2009. 60:485–510
Key Words
The Annual Review of Plant Biology is online at
plant.annualreviews.org
RNA silencing, innate immunity, virus, bacteria, suppressors
This article’s doi:
10.1146/annurev.arplant.043008.092111
Abstract
c 2009 by Annual Reviews.
Copyright All rights reserved
1543-5008/09/0602-0485$20.00
A multitude of small RNAs (sRNAs, 18–25 nt in length) accumulate
in plant tissues. Although heterogeneous in size, sequence, genomic
distribution, biogenesis, and action, most of these molecules mediate
repressive gene regulation through RNA silencing. Besides their roles
in developmental patterning and maintenance of genome integrity,
sRNAs are also integral components of plant responses to adverse
environmental conditions, including biotic stress. Until recently, antiviral RNA silencing was considered a paradigm of the interactions
linking RNA silencing to pathogens: Virus-derived sRNAs silence viral gene expression and, accordingly, viruses produce suppressor proteins that target the silencing mechanism. However, increasing evidence
shows that endogenous, rather than pathogen-derived, sRNAs also have
broad functions in regulating plant responses to various microbes. In
turn, microbes have evolved ways to inhibit, avoid, or usurp cellular
silencing pathways, thereby prompting the deployment of countercounterdefensive measures by plants, a compelling illustration of the
neverending molecular arms race between hosts and parasites.
*This PDF amended on May 15, 2009.
See explanation at http://arjournals.annualreviews.org/doi/full/10.1146/annurev.pp.60.090515.200001
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Contents
Annu. Rev. Plant Biol. 2009.60:485-510. Downloaded from arjournals.annualreviews.org
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BUILDING BLOCKS OF THE
MAIN RNA-SILENCING
PATHWAYS IN PLANTS . . . . . . . . .
Processors and Operators of RNA
Silencing in Arabidopsis . . . . . . . . . .
Major Cellular RNA-Silencing
Pathways of Arabidopsis . . . . . . . . . .
INDUCTION OF
RNA SILENCING DURING
DEFENSE RESPONSES. . . . . . . . . .
Small RNAs Produced from
Pathogen-Derived Nucleic
Acids . . . . . . . . . . . . . . . . . . . . . . . . . . .
Endogenous Small RNAs Whose
Expression Is Altered by
Pathogens . . . . . . . . . . . . . . . . . . . . . .
OPERATING RNA SILENCING
IN PLANT-PATHOGEN
INTERACTIONS . . . . . . . . . . . . . . . .
Antiviral RNA-Induced Silencing
Complexes and Their Possible
Activities . . . . . . . . . . . . . . . . . . . . . . .
Effecting miRNA, nat-siRNA, and
lsiRNA Defensive Functions . . . . .
486
486
488
488
489
491
496
497
Processors and Operators of RNA
Silencing in Arabidopsis
486
Plant RNA-silencing phenomena share four
consensus biochemical steps: (a) induction by
double-stranded RNA (dsRNA), (b) dsRNA
processing into 18–25-nt small RNA (sRNA),
(c) 2 -O-methylation of sRNA, and (d ) sRNA
incorporation into effector complexes that
associate with partially or fully complementary
target RNA or DNA. dsRNA might derive directly from virus replication, inverted repeats,
or convergent transcription of transgenes or
transposons. dsRNA formation may also be
genetically programmed at endogenous loci
Ruiz-Ferrer
·
Voinnet
500
500
500
503
505
505
505
505
499
BUILDING BLOCKS OF THE
MAIN RNA-SILENCING
PATHWAYS IN PLANTS
Effectors: virulence
factors injected or
secreted into host cells
by pathogens such us
bacteria, fungi,
nematodes, or insects
Role of AGO4 in Antibacterial
Defense: A Link to
RNA-Directed DNA
Methylation? . . . . . . . . . . . . . . . . . . .
SUPPRESSION, AVOIDANCE,
AND USURPATION OF RNA
SILENCING BY
PATHOGENS . . . . . . . . . . . . . . . . . . . .
Plant Viruses . . . . . . . . . . . . . . . . . . . . . .
RNA Silencing Suppression
by Other Pathogens . . . . . . . . . . . . .
HOST RESPONSES TO
SILENCING SUPPRESSION
BY PATHOGENS . . . . . . . . . . . . . . . . .
R Proteins and Direct Targeting
of VSR and BSR
Integrity/Function . . . . . . . . . . . . . .
Altering the Levels of Endogenous
Small RNAs That Regulate
Antiviral or Antibacterial
Components . . . . . . . . . . . . . . . . . . . .
Sentinel Small RNAs Generated
at Complex R Gene Loci . . . . . . . .
that produce transcripts with internal stemloop structures. Alternatively, dsRNA may
be synthesized by one of six RNA-dependent
RNA polymerases (RDR1–6) that copy singlestranded RNA (ssRNA). RDR templates
include mRNAs with aberrant features or
transcripts produced by a putative plantspecific RNA polIV, whose subunit NRPD1a
is found at certain methylated loci (reviewed in
References 11 and 14) (Figure 1a).
In Arabidopsis, the dsRNA is processed into
specifically sized sRNA duplexes by one of
four Dicer-like (DCL1–4) proteins. DCL1
synthesizes 18–21-nt-long sRNA, whereas
the products of DCL2, DCL3, and DCL4
are 22 nt, 24 nt, and 21 nt long, respectively
(78). dsRNA processing, called dicing, is facilitated by one of five dsRNA-binding proteins
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d
dsRNA production
sRNA function
NRPD1a and/or RNA Pol II
1
2
RNA Pol II
1
(A)n
or
aberrant RNA
RDR
(A)n
(A)n
AGO
RNA cleavage
RNA-dependent RNA polymerases: RDR1–6
MIRNA gene
3
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40S
4
RNA Pol II
(A)n
(A)n
60S
(A)n
AGO
RNA Pol II
NAT pair
b
2
Repression of translation
Inverted repeat
c
sRNA processing
sRNA stabilization & export
3
CH3
DCL
DRB
Nucleus
19–25 nt
AGO
HST
Cytosol
CH3
Dicer-like proteins: DCL1–4
dsRNA-binding proteins: HYL1, DRB2–5
CH3
CH3
HEN1
NRPD1b
DNA cytosine and/or
histone methylation
ARGONAUTE proteins:
AGO1–10
Figure 1
Consensus steps of Arabidopsis RNA silencing pathways. (a) Various sources of double-stranded RNA (dsRNA) and (b) its processing
into small RNAs (sRNAs) by one of four Dicer-like proteins (DCLs) assisted by dsRNA-binding proteins. (c) HUA ENHANCER 1
(HEN1)-mediated sRNA stabilization and export by HASTY (HST). (d ) sRNA operation. Selected strands of sRNA duplexes guide
ARGONAUTE (AGO)-containing RNA-induced silencing complex (RISC) to target RNAs (1) for endonucleolytic cleavage,
(2) for translation repression, or (3) to target chromatin for cytosine and/or histone methylation together with NRPD1b.
Figure 1 amended May 15, 2009.
(HYPONASTIC 1 or HYL1 and DRB2–5) that
interact with specific DCLs (Figure 1b). Upon
dicing, the sRNA 3 overhanging ends are 2 O-methylated by the methyltransferase HUA
ENHANCER 1 (HEN1) (83), which protects
them from oligouridylation and degradation
(Figure 1c). Stabilized sRNA duplexes are then
retained nuclearly for chromatin-level activities
or exported cytoplasmically, possibly via the
exportin-5 homolog HASTY (HST), for posttranscriptional gene silencing (PTGS). One selected sRNA strand incorporates one or several
RNA-induced silencing complexes (RISC) that
scan the cell for complementary nucleic acids
to execute their function. sRNA-directed RISC
activities include (a) RNA endonucleolytic
cleavage (slicing) at the center of sRNA-target
hybrids, (b) translational repression through
unknown mechanisms, and (c) DNA cytosine
and/or histone methylation (Figure 1d ) with
the assistance of Pol IV subunit b (NRPD1b).
Eukaryotic RISCs invariably include an
ARGONAUTE (AGO) protein. AGOs contain
a sRNA-binding PAZ domain and a PIWI domain with catalytic residues conferring endonucleolytic activity to those RISCs programmed
to slice RNA. Among the ten predicted Arabidopsis family members (AGO1–10), roles for
AGO1, AGO4, AGO6, and AGO7 in sRNAdirected silencing have been established, and a
slicer activity has been demonstrated for AGO1
and AGO4 (reviewed in References 11 and 14).
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Major Cellular RNA-Silencing
Pathways of Arabidopsis
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RNA interference
(RNAi): RNA
silencing process by
which exogenous
dsRNA directs the
posttranscriptional
silencing of
homologous genes
Transfer DNA
(T-DNA): DNA of
tumor-inducing (Ti)
plasmids of some
species of plant
bacteria such as
A. tumefaciens and
A. rhizogenes
488
High-throughput cloning and sequencing
show that the Arabidopsis sRNA repertoire is
largely dominated by short-interfering RNAs
(siRNAs) that act mostly at the chromatin level
and by microRNAs (miRNAs) (29, 63). Synthesis of other classes of cellular sRNA, some
of which are covered in specific sections of
this review, occurs through combinations of the
miRNA and siRNA pathways detailed below.
microRNA pathway. Most MIRNA genes
are intronic or intergenic RNA pol II transcription units, whose expression frequently
exhibits high tissue specificity and/or sensitivity to external stimuli including microbial
challenges. MIRNA transcription yields a
primary miRNA transcript (pri-miRNA) that
forms an imperfect fold-back structure. The
pri-miRNA is processed into a stem-loop precursor (premiRNA) and then diced as a duplex
containing the mature miRNA and a labile
passenger strand called miRNA∗ (reviewed in
Reference 36). The forkhead-associated domain protein DAWDLE (DDL) is required
for pri-RNA accumulation (82), whereas
both HYL1 and the zinc-finger protein
SERRATE (SE) are required for pri-miRNAto-premiRNA processing. Most Arabidopsis
miRNAs are matured in subnuclear bodies by
DCL1, although a few appear to be DCL4
dependent (29, 63). Upon HEN1-mediated
2 O-methylation, the mature miRNA strand
is selectively incorporated into AGO1containing or AGO10-containing RISCs to
promote slicing or translational repression
of target transcripts (10) (Figure 5a, right).
miRNA function is believed to be cytoplasmic,
following HST-mediated transport of miRNARISCs or miRNA duplexes from the nucleus.
More than 180 Arabidopsis MIRNA loci have
been identified, representing nearly 80 miRNA
families, many of which are important for plant
development. Some miRNA are also induced
or repressed by abiotic and biotic stresses (36),
as detailed in this review.
Ruiz-Ferrer
·
Voinnet
Short-interfering RNA pathway. Among the
endogenous siRNA pathways of Arabidopsis,
the heterochromatic pathway largely dominates by the sheer amount and sequence diversity of sRNA it produces at transposon
loci and DNA repeats. RDR2, RNA pol IV,
and DCL3 cooperatively generate the largest
bulk of heterochromatin-associated 24-nt
siRNAs. These siRNAs incorporate into AGO4
or AGO6 and guide cytosine methylation in
all sequence contexts, a landmark of RNAdirected DNA methylation (RdDM). RdDM is
also accompanied by histone modifications, including deacetylation and methylation. Heterochromatic siRNAs are often referred to as cisacting siRNAs because they affect the genomic
loci that produce them, which often results in
their transcriptional gene silencing (TGS; reviewed in Reference 13). Other siRNAs produced at discrete endogenous loci act in trans
to direct PTGS of mRNAs notably involved
in developmental phase changes and organ polarity. These trans-acting siRNAs (ta-siRNAs)
are produced upon miRNA-guided cleavage of
noncoding primary transcripts that are then
converted into dsRNA by RDR6. The dsRNA
is sequentially diced by DCL4 in a phased reaction that can be carried out by DCL2 when
DCL4 is genetically inactivated (reviewed in
Reference 14). DCL4 and DCL2 also redundantly mediate dsRNA-mediated RNA interference (RNAi) used for experimental gene
knockdown in plants (25).
INDUCTION OF
RNA SILENCING DURING
DEFENSE RESPONSES
This section discusses the genetic pathways
underlying RNA silencing activation by plant
parasites. Some sRNAs are produced directly
from pathogen-derived nucleic acids, including viral RNA and Agrobacterium tumefaciens
transfer DNAs (T-DNAs). Other pathogens
and pests induce modification of cellular
sRNA profiles, which in turn impact expression of regulators or effectors of host defense
pathways.
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Small RNAs Produced from
Pathogen-Derived Nucleic Acids
Viruses. sRNA cloning and high-throughput
sequencing in virus-infected plants identified
fold-back structures within single-stranded
viral transcripts as major dsRNA sources
from DNA viruses, RNA viruses, satellites, and
viroids. Replication intermediates (RIs) synthesized by viral-encoded replicases also account
for dsRNA production by some RNA viruses
(32, 33, 47), whereas converging transcription
of nuclear transcripts is a major dsRNA source
from DNA geminiviruses (16). dsRNA arising
directly from structural features or replication
of viral genomes generates primary viral small
RNA (vsRNA), in contrast to secondary vsRNA
produced by host-encoded RDRs that convert
viral ssRNA into dsRNA. Arabidopsis RDR6
and RDR1 [the latter is induced by salycilic
acid (SA), a defense-signaling compound commonly produced during pathogen infections]
play major and probably redundant antiviral
roles (21, 23, 61*). These functions require
cofactors, including the putative RNA helicase
SILENCING DEFECTIVE 3 (SDE3) and
the coiled-coil domain protein SUPPRESSOR
OF GENE SILENCING 3 (SGS3), necessary
for RDR6-mediated suppression of RNA and
DNA virus accumulation (7, 19, 50, 51). The
heterochromatic RDR2 is additionally required for silencing DNA viruses and the RNA
virus Tobacco rattle virus (TRV) (23, 62). Host
RDR activities are stimulated by RNAs lacking
quality control marks, such as a 5 -cap or a 3 polyA tail, features frequently exhibited by viral
RNAs (reviewed in Reference 75). Additionally,
primary vsRNAs may promote RDR activities
by generating uncapped or polyA− RNA
fragments upon RISC-mediated slicing of viral
transcripts or by acting as primers for RDRdirected dsRNA synthesis (45) (Figure 2). RDR
functions are also required for amplifying a systemic silencing response that contributes immunizing tissues that are yet to be infected (see
Non-Cell-Autonomous Silencing in Plants).
These intricate dsRNA-generating pathways make it difficult to distinguish the rel-
NON-CELL-AUTONOMOUS SILENCING
IN PLANTS
Unlike the miRNA pathway, the DCL4-dependent siRNA pathway is not cell autonomous in plants: 21-nt siRNAs or their long
dsRNA precursors move between cells through plasmodesmata
and over long distances through the phloem. RDRs involved in
PTGS, most notably RDR6, play a crucial role in this movement
process by amplifying signal molecules or their precursors, thus
ensuring a potent and sustained response throughout plants. Although silencing spread has been studied mostly in the context of
transgene silencing, it has also been observed indirectly during
virus infections, where it likely constitutes the systemic component of antiviral RNA silencing. For instance, viral suppressors of
RNA silencing (VSR)-deficient viruses accumulating in vascular
bundles fail to unload into neighboring cells. Although virus-free,
these cells exhibit sequence-specific resistance to secondary infection, a phenomenon alleviated in the Arabidopsis loss-of-function
dcl4 mutant. Thus, cell-to-cell spread and amplification of
DCL4-dependent silencing signals likely immunize tissues just
ahead of the infection. Phloem-mediated silencing spread between distant organs has also been demonstrated with movementdeficient and replication-proficient recombinant viruses, which
promote systemic silencing responses in noninoculated tissues.
As in the case of cell-to-cell movement, phloem-mediated transport of RNA silencing also likely has antiviral roles because it
is precluded by the CMV-encoded VSR 2b. Moreover, silencing amplification upon vascular transport probably helps immunize recipient tissues, because RDR6 activities that enable
detection/amplification of long-distance transgene silencing exclude many plant viruses from meristems in apical growing points.
ative importance of primary versus secondary
vsRNA synthesis in antiviral defense, which
moreover might vary from one virus to another. Thus, TRV-derived vsRNA are nearly
eliminated in triple rdr1 rdr2 rdr6 mutants, indicating that these molecules originate mostly
from dicing of RDR products rather than from
intrinsic dsRNA features of the TRV genome
(23). Another difficulty in distinguishing primary and secondary vsRNA contribution to antiviral silencing is that both derive from similar DCL activities. Hence, DCL4 and DCL2
act redundantly upstream (primary vsRNAs)
and downstream (secondary siRNAs) of RDR6
Satellite: a subviral
agent composed of
nucleic acid that
depends on the
coinfection of a host
cell with a helper or
master virus for its
multiplication
*Reference 61 added to text, May 15, 2009.
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489
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or
15:37
CH3
(A)n
CH3
RNA virus
4/6
AGO
24nt
RDR2
?
?
Episome
DNA
DCL3
DRBX
DCL4 DRB4
Geminivirus
DRB4 DCL4
(A)n
Nucleus
Cytosol
(A)n
(A)n
DRBX
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RI
Replicase
CH3
DCL2
DRBX
DCL2
21nt 22nt
or
DRB4
DCL2
SA
40S
&
(A)n
60S
RDR6
SGS3
+
?
AGO1
RDR1
?
?
SDE3
AGO1
AGO1
VAMP?
DRBX
21nt 22nt
(A)n
(A)n
(A)n
(A)n
DCL4
Silencing
signal
Amplification
Systemic
defense
responses
SA
Figure 2
Antiviral silencing in Arabidopsis. Dicer-like4 (DCL4) primarily processes viral double-stranded RNA (dsRNA) into 21-nt viral small
RNAs (vsRNAs). If suppressed by viruses, DCL4 function is substituted by DCL2, which generates 22-nt vsRNAs. ARGONAUTE1
(AGO1) is presented as the major antiviral AGO that mediates slicing and possibly translational repression of viral RNA. However,
other AGO paralogs, including AGO7, are likely to be involved. DNA virus genomes (here shown as geminivirus) undergo
DNA/histone methylation in the nucleus by DCL3-dependent vsRNAs. Primary vsRNAs are amplified into secondary vsRNAs by
RNA-dependent RNA polymerase 6 (RDR6) and the salycilic acid (SA)-induced RDR1. Cellular and systemic SA production might be
induced upon possible recognition of an as yet uncharacterized viral-associated molecular pattern (VAMP). Aberrant viral mRNAs can
enter RNA-dependent RNA polymerase (RDR1, RDR2, RDR6) pathways independently of primary vsRNA synthesis. Alternatively,
vsRNA might act as primers for RDRs. An amplified, DCL4-dependent silencing signal (possibly the 21-nt product of DCL4) moves
through the plasmodesmata to immunize neighboring cells. DRB, dsRNA binding; SGS, SUPPRESSOR OF GENE SILENCING;
SDE, SILENCING DEFECTIVE; RI, replication intermediate.
Figure 2 amended May 15, 2009.
Viroid: an
autonomously
replicating
plant-specific subviral
pathogen that consists
of a short stretch of
highly complementary,
circular, and ssRNA
without an ORF
490
and RDR1 action during RNA virus infections (23, 45) (Figure 2). DCL2 can be considered a DCL4 surrogate because its antiviral role is evident only if the action of DCL4
is genetically compromised or suppressed by
dedicated proteins called viral suppressors of
RNA-silencing (VSRs) (20, 21). The miRNAspecific enzyme DCL1, although not implicated in a major way in processing dsRNA from
Ruiz-Ferrer
·
Voinnet
RNA viruses, modulates DCL4 expression negatively such that antiviral silencing is exacerbated in dcl1 hypomorphic mutants (61). Thus,
as yet unidentified DCL1-dependent miRNAs
could target DCL4 transcripts for degradation or negatively control transcription factors
required for DCL4 expression. Consistently,
DCL4 transcript levels are enhanced in Arabidopsis deprived of the miRNA effector protein
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AGO1 (61). All four Arabidopsis DCLs are
critically involved in vsRNA production from
DNA viruses (gemini- and pararetroviruses;
7, 46). Notably, DCL3-dependent, 24-nt-long
vsRNA may dampen viral transcription by
inducing chromatin condensation of nuclear viral episomes and minichromosomes (62). Optimal vsRNA production requires DRBs, among
which DRB4 facilitates synthesis of DCL4dependent vsRNA from RNA and DNA viruses
(31, 61). vsRNA duplexes are then stabilized by
HEN1 (7, 42).
In vertebrates, viral dsRNA is perceived
as a pathogen-associated molecular pattern
(PAMP) through dedicated Toll-like receptors (TLRs). Activated TLRs signal downstream innate immune responses, which,
unlike RNA silencing, are not sequence specific
(40). Although the existence of plant dsRNAstimulated immune receptors remains enigmatic, the expression of several antiviral silencing components is induced by virus-triggered
plant hormones, notably SA (2), which is also
typically produced upon recognition of microbial PAMPs and virulence factors (71). Thus,
detection of viral signatures other than dsRNA
could potentially exacerbate antiviral silencing
by promoting a generalized, hormone-based
immune reaction. The catalytic triad GDD
found in all RNA virus replicases (54) might
constitute one of these putative viral-associated
molecular patterns (VAMPs) (Figure 2). We
finally note that global changes incurred by
viruses to cellular sRNAs, including miRNAs
that might control nonsilencing-based antiviral
defense pathways, have so far eluded characterization in plants.
Agrobacterium tumefaciens. In crown gall
disease, A. tumefaciens transferred T-DNAs
integrate into plant genomes to express oncogenes. Bacteria then thrive on the resulting
tumors by metabolizing nutrients produced
by dedicated T-DNA-encoded enzymes (72).
Although oncogene-free T-DNAs are widely
exploited for plant transformation, transgenes
are often poorly expressed owing to an RNAsilencing phenomenon that recapitulates a
host defense reaction preventing expression
of tumor-inducing T-DNAs of virulent bacteria (26). Hence, siRNAs with sequences
of T-DNA-encoded oncogenes accumulate
in tobacco leaves infected with virulent
A. tumefaciens. Production of these 21-nt-long
siRNAs likely involves DCL4 and the upstream
action of (at least) RDR6, because rdr6 lossof-function mutants are more susceptible to
A. tumefaciens than WT Arabidopsis (Figure 3).
Presumably, RDR6 converts uncapped or
polyA− RNAs produced from integrated or
episomal T-DNA into dsRNA substrates
for DCL4. T-DNA arrays with head-to-tail
configuration, frequently found at genomic
insertion sites, might also contribute to siRNA
production by generating dsRNA directly
(26).
Endogenous Small RNAs Whose
Expression Is Altered by Pathogens
Natural antisense and long siRNAs.
Defense responses to bacteria, fungi, and
oomycetes can be conceptually separated
into PAMP-triggered immunity (PTI) and
effector-triggered immunity (ETI) (see Nonhost Resistance, PAMP-Triggered Immunity,
and Effector-Triggered Immunity). The link
between ETI and RNA silencing was discovered by comparing global sRNA profiles with
the expression of known pathogen-regulated
genes in Arabidopsis. A natural antisense
RNA (nat-siRNA) called nat-siRNAATGB2
is specifically induced upon recognition of
Pseudomonas syringae effector AvrPt2 by the
cognate Arabidopsis disease resistance (R)
protein RPS2 (39). nat-siRNAATGB2 derives from the overlapping region of a pair
of natural antisense (NAT) transcripts: a
Rab2-like small GTP-binding protein gene,
ATGB2, and a constitutively expressed PPR
(pentatricopeptide repeats) protein–like gene,
PPRL. nat-siRNAATGB2 synthesis presumably requires ATGB2 transcriptional induction
through a mechanism involving the plasma
membrane protein NDR1, which is required
for RPS2-mediated resistance (Figure 4a,
www.annualreviews.org • Plant Small RNAs in Biotic Stress Responses
Replication
intermediate (RI):
long dsRNA
intermediate thought
to be produced by viral
replicases of
positive-strand RNA
viruses
Salycilic acid (SA): a
phytohormone
involved in plant
defense against insects
and pathogens; has
intrinsic antimicrobial
properties; is the active
principle of aspirin
Pathogen-associated
molecular pattern
(PAMP): signature
molecules that are
indispensable to
microbial growth;
include
lipopolisaccharides,
flagellin, elongation
factor Tu, cold-shock
proteins,
peptidoglycans, and
dsRNA
491
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a
15:37
b
HEN1
c
3'
siRNA
ND
5'
QR
122
P19
UUU
PIWI
P19
UUU
3' uridylation
5'
siRNA
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PAZ 3'
Mid
CH3
CH3
5'
2b
ANRV375-PP60-22
Degradation
2b
P0 ND
5'
Mid
2b
d
PAZ 3'
QR
PIWI
e
U
Ler
Col-0
dcl1
rdr6
AGO1
AGO1
AGO1
AGO1
AGO1
Saturation
Inaccessibility
Figure 3
Viral suppressors of RNA silencing (VSRs) from RNA viruses and silencing usurpation strategies. (a) P122-mediated block of HUA
ENHANCER 1 (HEN1) function resulting in viral small RNA (vsRNA) uridylation and degradation. (b) Upper: P19 acts as a vsRNA
caliper that binds specifically to 21-bp duplexes. Lower: The hook-like structures of 2b can interact with long and short double-stranded
RNA (dsRNA). (c) 2b and P0 inhibit ARGONAUTE1 (AGO1) activity through direct interaction with PAZ, PIWI, and ND domains
(2b) or just PAZ and ND domains (P0). (d ) Efficient dicing of viroid genomes might generate large amounts of vdsRNAs with 5 -U
termini. These may saturate AGO1, preventing its productive use of other vdRNAs, which may, in any case, be largely inert owing to
the strong secondary structures of viroid target RNA that prevent RNA-induced silencing complex (RISC) action. (e) Left:
Agrobacterium tumefaciens stab-inoculated stems of Dicer-like1 (dcl1) mutants are immune to tumor formation compared with WT
Arabidopsis (Ler). Middle: Root inoculation assays unravel the hypersusceptibility of the Arabidopsis RNA-dependent RNA polymerase6
(rdr6) mutant to A. tumefaciens compared with WT Col0 plants (Col-0). Right: Tumors developing on GFP-silenced Nicotiana
benthamiana plants appear bright green under UV illumination, demonstrating RNA-silencing suppression in proliferating cells.
left). nat-siRNAATGB2 biogenesis entails a
complex series of reactions whose precise order
is unspecified, involving DCL1, HYL1, and
HEN1 (miRNA pathway); RDR6 and SGS3
(siRNA amplification); and the RNA pol IV
492
Ruiz-Ferrer
·
Voinnet
subunit NRPDla (heterochromatic silencing)
(39) (Figure 4a, right).
A class of siRNAs that is atypical in size (39–
41-nt long), dubbed long siRNAs (lsiRNAs),
also accumulates in biotically stressed
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Arabidopsis. Among these, lsiRNA-1 is specific
to RPS2-mediated resistance. Similarly to natsiRNAATGB2, lsiRNA-1 production depends
on a NAT pair in which induction of a receptorlike kinase (RLK) transcript requires RPS2
activation by bacterial AvrPt2. lsiRNA-1 is
produced at the overlap between RLK and the
3 -UTR of the antisense transcript RAP (37).
The biogenesis of lsiRNA-1 is similar, albeit
not identical, to that of siRNAATGB2 because
it involves the additional action of DCL4,
ARGONAUTE 7 (AGO7), and NRPD1b, but
not of RDR6 and SGS3, again through an as
yet unspecified order of events (Figure 4b,
left). Mutations in RDR6 and HYL1, but not
in silencing components not required for their
biogenesis, compromise RPS2-mediated resistance, suggesting a role for nat-siRNAATGB2
and lsiRNA-1 in AvrPt2-specified ETI (37).
An outstanding issue pertains to the nature
of signaling cascades that specifically induce
ATGB2 or RLK to act as major on/off switches
to control nat-siRNA and lsiRNA production,
respectively (Figure 4b, right). The AvrPt2RPS2 interaction likely involves additional
nat-siRNA or lsiRNAs, and more generally,
many other nat-si or lsiRNAs might orchestrate ETI and PTI (see below) against a wide
range of pathogens. More than 3000 Arabidopsis
sRNAs match >60% of protein-coding NAT
pairs (77). Furthermore, more than 30% of
the Arabidopsis genome can produce transcripts
from both sense and antisense strands (80),
some of which might be transcriptionally
induced by biotic stresses to form dsRNA
substrates for DCLs.
microRNAs. Arabidopsis miR393 was the first
sRNA implicated in bacterial PTI (52). MIR393
transcription is induced by the flagellin-derived
PAMP peptide, flg22, to target mRNAs encoding the F-box auxin receptor transport inhibitor response 1 (TIR1) and related proteins
(Figure 5a). Enhanced miR393 accumulation
was similarly found during sRNA profiling
in Arabidopsis challenged with P. syringae pv.
tomato (Pst) DC3000 hrcC, which lacks a functional type-III secretion system required for vir-
NONHOST RESISTANCE, PAMP-TRIGGERED
IMMUNITY, AND EFFECTOR-TRIGGERED
IMMUNITY
Plants have evolved multiple obstacles to protect themselves
from pathogen attacks, including (a) nonhost resistance via
physical barriers, (b) PAMP-triggered immunity (PTI), and
(c) effector-triggered immunity (ETI). The first obstacle accounts
for plant resistance to a majority of pathogens: deployment of
waxy cuticles and thickened cell walls or deprivation of factors required for pathogen growth. Successful pathogens then
encounter the PTI defense layer, orchestrated by transmembrane pattern-recognition receptors (PRRs) that sense pathogenassociated molecular patterns (PAMPs). The best-characterized
plant PAMP-PRR interaction involves recognition of a highly
conserved 22-amino-acid epitope (flg 22) in the N terminus of
bacterial flagellin by the leucine-rich-repeat receptor-like kinase
FLAGELLIN-SENSING 2 (FLS2). PAMP recognition triggers
a cascade of reactions known as basal defense, which involves activation of mitogen-activated protein (MAP) kinases, production of
reactive oxygen species and nitric oxide, cell wall reinforcement,
and salycilic acid (SA) synthesis and signaling. On occasion, potent basal defense might stop pathogen attack, resulting in nonhost resistance. However, many pathogens can overcome PTI
by delivering virulence factors (effectors), which suppress basal
defense signaling, into plant cells. As a counter-counterdefensive
response to PTI suppression, plants deploy resistance (R) proteins
that recognize specific pathogen effectors (avirulence or Avr proteins) or modifications incurred by pathogen effectors to host defense components. Specific R-mediated recognition of Avrs then
triggers ETI signaling, thought to be quantitatively stronger than
PTI signaling. ETI often culminates in a form of programmed
cell death called the hypersensitive response (HR) and is accompanied by a potent SA-mediated systemic defense response.
ulence (29) (see Nonhost Resistance, PAMPTriggered Immunity, and Effector-Triggered
Immunity). Accordingly, constitutive overexpression in a tirl-1 mutant background of a
miR393-resistant TIR1 paralog enhanced susceptibility to Pst DC3000, whereas bacterial
growth was reduced in miR393-overexpressing
lines (52). Pst DC3000 hrcC infection upregulated many additional miRNAs unrelated to auxin signaling, suggesting a global
contribution of the miRNA pathway to PTI
www.annualreviews.org • Plant Small RNAs in Biotic Stress Responses
Type-III secretion
system: a protein
secretion apparatus
used by some
gram-negative bacteria
to inject virulence
factors (or effectors)
into the cytoplasm of
hosts
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b
FLS2
FLS2
BAK1
BAK1
Pst
Pst
nat-siRNA
lsiRNA
HYL1
HYL1
DCL1
pt2
avrR
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NDR1
HEN1
RPS2
DCL1
pt2
avrR
NDR1
HEN1
RPS2
AGO7
NRPD1a
ATGB2
RDR6
RLK
NRPD1a
SGS3
RDR6
RAP
PPRL
SDE3
?
DCL4
(A)n
(A)n
(A)n
(A)n
NRPD1b
mRNA decay?
nat-siRNA ATGB2
lsiRNA
(A)n
(A)n
VCS
TDT
PPRL
PPRL
Figure 4
Contribution of Arabidopsis small RNA (sRNA) to race-specific resistance against Pseudomonas syringae carrying avRpt2 mediated by
RPS2. (a) The 22-nt-long natural antisense RNA (nat-siRNA) ATGB2 produced at the ATGB2-PPRL overlap specifically
downregulates the expression of PPRL, a possible negative regulator of defense. (b) In a related scheme, the 40-nt long siRNA1
(lsiRNA1) produced at the receptor-like kinase (RLK)/RAP overlap contributes to PPRL mRNA decay through VARICOSE (VCS) and
TRIDENT (TDT). The precise order of action of the RNA silencing components required for nat-siRNA and lsiRNA generation
[right side of (a) and (b), respectively] remains to be formally established. AGO7, ARGONAUTE7; BAK1, BRI1-associated
receptor-kinase 1; DCL1, Dicer-like1; FLS2, FLAGELLIN-SENSING 2; HEN1, HUA ENHANCER 1; HYL1, HYPONASTIC 1;
NDR, non–race specific disease resistance; NRPD1a or b, Pol IV subunit a or b; Pst, P. syringae pv. tomato; RDR6, RNA-dependent
RNA polymerase6; SDE3, SILENCING DEFECTIVE 3; SGS3, SUPPRESSOR OF GENE SILENCING 3.
(29) (Figure 5a). Indeed, growth and symptoms
from normally nonvirulent Pst DC3000 hrcC
were appreciably, albeit not completely, rescued in miRNA-deficient dcl1, but not in siRNA
494
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pathway mutants of Arabidopsis (53). Moreover, dcl1 also sustained growth from bacteria not infecting Arabidopsis, unraveling strong
miRNA contributions to nonhost resistance
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Figure 5
microRNAs (miRNAs) in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). (a) Left panel: Under low
miR393 levels, the transport inhibitor response 1 (TIR1) and related F-box proteins signal to increase the ubiquitin-mediated
degradation of auxin/indole-3-acetic acid (Aux/IAA) factors, promoting Aux-responsive gene expression and suppression of defense.
Middle panel: Upon flagellin elicitation of FLAGELLIN-SENSING 2 (FLS2), MIR393 is transcriptionally activated, leading to
suppression of TIR1 mRNA and protein production. The ensuing Aux/IAA accumulation reduces Aux-responsive gene expression,
enhancing PTI. Right panel: Factors required for miR393 biogenesis and action. (b) A balanced output of miRNA action during PTI.
AGO1, ARGONAUTE1; ARF, auxin response factor; BAK1, BRI1-associated receptor-kinase 1; DCL1, Dicer-like1; DDL,
DAWDLE; HEN1, HUA ENHANCER 1; HYL1, HYPONASTIC 1; Pst, P. syringae pv. tomato; R, resistance; RLK, receptor-like
kinase; SCF, Skp, Cullin, F-box containing; SE, SERRATE.
Figure 5 amended May 15, 2009.
(see Nonhost Resistance, PAMP-Triggered Immunity, and Effector-Triggered Immunity).
Nonetheless, Pst DC3000 hrcC growth was consistently higher in hen1 than in dcl1 mutants
(53). Because HEN1 affects all known Arabidopsis silencing pathways, other cellular sRNA
classes might thus orchestrate PTI in addition to miRNAs, including as yet unidentified lsiRNAs and nat-siRNAs, whose biogenesis
also relies heavily on DCL1 and HEN1 (37, 39).
High-throughput sequencing identified several DC3000 hrcC-downregulated, rather thanupregulated, miRNAs, among which miR825
targets three potential positive PTI regulators
(29, 38). This result could explain why rescue
of Pst DC3000 hrcC growth was only partial in
dcl1 and hen1 mutants, because reduced miRNA
accumulation in both backgrounds would
www.annualreviews.org • Plant Small RNAs in Biotic Stress Responses
Nonhost resistance:
the resistance observed
when all members of a
plant species exhibit
resistance to all
members of a given
pathogen species
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Jasmonic acid ( JA):
a phytohormone
involved in plant
defense against some
plant pathogens, as
well as wounding,
growth inhibition,
senescence, and leaf
abscission
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15:37
elevate the levels of miR825 (and possibly of
other miRNA) targets to promote defense.
Downregulation of 10 out of 11 large
miRNA families of loblolly pine (Pinus taeda)—
including seven pine-specific families—was also
observed in galls induced by the rust fungus
Cronartium quercuum (43). Remarkably, most
validated targets of the pine-specific families
encode defense factors, including R proteins
and RLKs; others include orthologs of highly
conserved miRNAs that repress organ development. Nearly all mRNA targets accumulated highly in healthy stem tissues surrounding galls, suggesting that miRNA suppression
activates growth- and resistance-related genes
to restrict fungal growth (43). Therefore, the
miRNA involvement in PTI and basal defense
during compatible interactions probably entails
a balanced output of (a) induction of miRNAmediated repression of negative defense regulators and (b) repression of miRNA-mediated
repression of positive effectors of defense
(Figure 5b).
Some miRNAs facilitate symbiotic and parasitic plant-bacterium interactions: Restriction
by miR169 of the MtHAP2-1 transcription
factor to nodule meristematic zones facilitates differentiation of nitrogen-fixing cells
in Rhizobium-inoculated Medicago roots (18).
Likewise, miR166 expression in root vascular
and apical regions promotes nodule differentiation (9). Whereas the RDR6-DCL4-dependent
pathway restricts Agrobacterium growth, successful tumor development requires miRNA
pathway integrity, because Arabidopsis hen1 and
hypomorphic dcl1 mutants are immune to
crown gall formation (26) (Figure 3e). Specific host miRNAs might be indispensable for
differentiation of cells required for tumor vascularization or for elimination of putative tumor suppressors, for example. Alternatively,
T-DNA-encoded miRNAs may act as virulence factors, similar to miRNAs produced
by some mammalian-infecting DNA viruses
(reviewed in Reference 60). These examples
unravel highly complex interactions that link
plant microbes to miRNAs and the extraor-
Ruiz-Ferrer
·
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dinary variety of their possible outcomes. As
for lsiRNAs and nat-siRNAs, a major challenge ahead will be to determine which signaling pathways and regulatory DNA elements
modulate, positively or negatively, specific arrays of MIRNA genes during various biotic
stresses.
NaRDR1-dependent sRNAs in herbivore
attacks. A central role in herbivore resistance was identified for the Nicotinana attenuata RDR1 (NaRDR1) ortholog: Insect oral secretions and SA or jasmonate ( JA)—hormones
commonly produced in response to herbivore attack—strongly enhance NaRDR1 expression (56). Comparative sRNA profiling in
WT versus NaRDR1-knockdown plants before and after herbivore elicitation identified
NaRDR1-specific siRNAs with predicted targets involved in SA and JA signaling (57). Moreover, NaRDR1 knockdown severely altered induced transcript accumulation of most targets
tested, and exogenous JA application restored
insect resistance. Thus, NaRDR1-dependent
siRNAs seem key to JA signaling and probably
contribute to NaRDR1 homeostasis through
positive feedback. NaRDR1 might control a
vast array of additional traits because only 6%
overlap was found between global sRNA profiles of WT versus NaRDR1-knockdown plants
(57). Although lack of genomic information in
N. attenuata precludes identification of the
NaRDR1 templates involved, these results
clearly implicate RDR1 beyond mere antiviral
defense in Nicotianae.
OPERATING RNA SILENCING
IN PLANT-PATHOGEN
INTERACTIONS
This section illustrates how host proteins operate RNA silencing, notably as part of RISCs.
This section also highlights original gene expression regulatory schemes in which RNA
silencing provides safeguards against constitutive defense activation, thereby reducing fitness
costs to plants.
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Antiviral RNA-Induced Silencing
Complexes and Their Possible
Activities
Plant recombinant viruses whose genomes incorporate fragments of host transcripts induce
symptoms that mimic those of knockdown mutations in corresponding mRNAs (65). Therefore, vsRNAs can inhibit expression of complementary RNA in trans upon loading into
AGO(s). Consistently, Arabidopsis plants with
knockdown mutations in the miRNA effector
AGO1 are hypersusceptible to RNA virus infections (49). Although generally interpreted
as evidence for an AGO1-associated antiviral RISC, this hypersusceptibility could also
be partially contributed to by reduced activities of cellular miRNAs that normally control negative regulators of antiviral defense.
Studies of Cymbidium ringspot virus (CyRSV)
provide perhaps the most compelling support
for an AGO1-associated antiviral RISC. Thus,
CyRSV-derived vsRNAs and cellular miRNAs
cofractionate into two protein complexes likely
corresponding to free AGO1 and partially or
fully assembled RISC (58). Moreover, a similarly sized complex containing vsRNAs from a
CyRSV-related virus exhibits virus sequence–
preferential and ssRNA-specific nuclease activity (55), and AGO1 immunoprecipitates
isolated from Cucumber mosaic virus (CMV)infected Arabidopsis contain CMV-derived
vsRNAs (84).
Nevertheless, immunoprecipitations with
AGO2 and AGO5 yield similar results (68), yet
their role in antiviral defense has never been
established; AGO2 even lacks catalytic residues
potentially required for viral RNA slicing. Selective sRNAs loading into specific AGOs seem
strongly (albeit not entirely) influenced by their
5 terminal nucleotide (44, 48, 68). Thus, miRNAs with predominant 5 -Us are frequently
loaded into AGO1, whereas most AGO2- and
AGO5-associated sRNAs have 5 -G and 5 -U
termini, respectively. Assuming similar rules
apply to vsRNAs and viroid-derived sRNAs
(vdsRNAs), many vdsRNAs might thus incorporate AGOs with little or no intrinsic an-
tiviral activity. Second, vs/vdsRNA allocation
to plant AGOs might vary extensively from
one virus to another, or even between viral
strains, owing to differences in vsRNA populations and 5 -nucleotide polymorphisms. By
extension, some plant AGOs might strongly
inhibit specific virus subsets, but not others.
This hypothesis might partly explain the recurrent difficulties in identifying single ago
mutations with broad antiviral silencing defects in Arabidopsis. Moreover, susceptibility assays usually involve viruses that produce VSRs
whose effects are redundant with those of mutations in DCLs, RDRs, and AGOs. Hence, only
with P38-deficient Turnip crinkle virus (TCV )
(P38 is the TCV-encoded VSR) was the role
of DCL2 and DCL4 and AGO1 in restricting TCV accumulation clearly appreciated
(Figure 6), as was the milder AGO7 contribution to this process (20, 61). Interestingly, P38-deficient TCV with a foreign GFP
RNA insert becomes far more sensitive to
RDR6 and AGO7 antiviral activities, suggesting that AGO7 might specifically incorporate
secondary, rather than primary, vsRNAs (61).
Therefore, distinct RISCs might simultaneously operate in virus-infected cells.
Antiviral AGO-mediated silencing is
thought to occur mainly via vsRNA-directed
slicing. However, global viral RNA levels
rather than site-specific endonucleolytic
cleavage events are usually measured experimentally. Yet, viral RNA accumulation
depends ultimately upon the rate of viral
protein synthesis (e.g., replicase), of viral
RNA decay and, possibly, of slicing, layers
of gene regulation that are all influenced by
sRNA activities in plants and animals (10,
28). Repression of viral protein synthesis
certainly deserves careful attention, especially
with vsRNAs derived from imperfect hairpins
within mRNAs (47), because they would be
only partially complementary to the other
arm of the stem and could, therefore, favor
translational inhibition of targeted transcripts,
as with imperfectly matched plant and animal
miRNAs (4, 24). Moreover, even sRNAs with
100% target complementary (for example,
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a
Replicase
p9
P38
WT TCV
GFP
TCV-ΔP38
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p8
b
Low
WT
Infectivity
dcl4
dcl2 dcl4
High
Figure 6
Redundancy between viral suppressor of RNA silencing (VSR) function and RNA silencing mutations.
(a) Schematics of WT Turnip crinkle virus (TCV) (expressing the P38 VSR) and TCV-P38, in which the
P38 open reading frame is replaced by that of the GFP, used to image viral accumulation. (b) TCV-P38
primary lesions remain small in inoculated leaves of WT plants. They expand, however, in leaves of dcl4
mutants and become confluent in leaves of dcl4 dcl2 double mutant plants, in which viral systemic movement
is also specifically restored. This simple rescue experiment identifies Dicer-like2 (DCL2) and DCL4 as
genetic targets of P38.
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those directing experimental RNAi) can have
extensive translational inhibitory effects under
appropriate circumstances, possibly including
viral infections (10). Nonetheless, vsRNA
loading into AGOs does not guarantee their
functionality, because sRNA efficacy is largely
influenced by RISC target site accessibility,
which is potentially inhibited by viral or host
protein binding or local secondary RNA
structures (3). The latter is well illustrated
by viroids, whose tight, rod-like, and circular
RNA genome is amenable to dicing but largely
resistant to RISC (34) (Figure 3d ).
AGOs also exert chromatin-level antiviral effects against nuclear genomes of DNA
viruses. Hence, the RdDM pathway likely accounts for methylation of geminivirus DNA
and histone H3 lysine 9, because Arabidopsis
RdDM mutants are hypersusceptible to geminivirus infection (62). Moreover, accumulation
of VSR-deficient geminivirus is specifically rescued in ago4 mutants. Geminivirus infection
produces large amounts of DCL3-dependent,
24-nt-long vsRNA, and similarly sized endogenous siRNAs direct nuclear RdDM through
AGO4 (62). Therefore, AGO4 contributes
to heterochromatinization of geminiviral episomes, probably as does AGO6, because it
acts redundantly in endogenous RdDM (85)
(Figure 2).
Effecting miRNA, nat-siRNA, and
lsiRNA Defensive Functions
Bacterial- and fungal-induced miRNAs likely
operate through AGO1. This is the case
for miR393, which prevails in PTI against
P. syringae by targeting the auxin receptor TIR1
and paralogs (29, 52). Because TIR1 interacts
with and degrades Aux/IAA proteins, miR393
induction increases the cellular Aux/IAA availability to repress auxin response factors (ARFs)
through heterodimerization and thereby
inhibit auxin-responsive gene expression
(Figure 5a). AGO1-loaded miR160 and
miR167, both induced by Pst DC3000 HrcC,
target mRNAs of additional ARF family
members (29). Therefore, AGO1-mediated
suppression of auxin signaling seems integral to bacterial PTI, suggesting that auxin
promotes disease susceptibility through mechanisms awaiting characterization. During
ETI through AvRpt2-elicitation of RPS2,
nat-siRNAATGB2 suppresses in cis accumulation of a constitutive PPRL transcript
(39), but the AGO involved, if any, is currently unknown (Figure 4a). Because PPRL
overexpression attenuates disease resistance,
PPRL may normally regulate negatively the
RPS2-mediated response. Similarly, RPS2dependent elicitation of lsiRNA1 production
induces degradation in cis of constitutively
expressed RAP transcripts produced at the
RLK-RAP NAT pair (Figure 4b). rap mutants
display enhanced resistance to virulent and
avirulent P. syringae, suggesting that the RAPencoded RNA-domain protein negatively
regulates RPS2-mediated resistance through
unspecified mechanisms (37). The lsiRNA1
pathway probably involves the biogenesis
rather than the action of AGO7. Indeed, RAP
turnover appears to be slicing independent but
requires the decapping factors VARICOSE
(VCS) and TRIDENT (TDT) and the presence of the lsiRNA1-complementary site in the
RAP 3 -UTR, indicating sequence specificity
(37) (Figure 4b).
Analyses of miRNAs in PTI and of NATderived siRNAs in ETI reveal a consensual gene
regulation pattern, whereby pathogen-induced
sRNAs repress constitutively expressed negative regulators of defense. This modus operandus likely reflects an adaptation against prolonged defense activation, which considerably
reduces plant fitness (70). In this context, one
obvious advantage of using sRNAs lies in the
necessity to deplete cells of mRNA and protein
pools of negative regulators already present at
the time of pathogen elicitation. A second anticipated advantage is reversibility. Indeed, plant
sRNA action has a widespread translational repression component (10), which can be reversed
within minutes, at least in mammalian cells (6).
Similar reversibility in silencing mediated by
microbe-induced sRNAs in plants would ensure
that translation of negative regulators resumes
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rapidly once the burst of defense responses has
elapsed. The compatible interaction between
pine and C. quercuum illustrates an alternative
yet related regulation mode (43), where defense
pathway components might be repressed constitutively by specific miRNAs in healthy tissues
but activated upon stress-induced inhibition of
those miRNAs (Figure 5b).
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Role of AGO4 in Antibacterial
Defense: A Link to RNA-Directed
DNA Methylation?
Pst DC3000 induces DNA hypomethylation
of genomic loci, including pericentromeric
repeats and retrotransposons, as well as decondensation of chromocenters in infected Arabidopsis tissues (59). The ago4-2 mutant, isolated
in a forward genetic screen, also displays enhanced susceptibility to virulent Pst DC3000,
to avirulent Pst DC3000 carrying the effector avrRpml, and to the nonhost P. syringae
pv. phaseolicola. In fact, all ago4 alleles tested—
including loss-of-function alleles—exhibit this
phenotype, suggesting the involvement of heterochromatic siRNAs (1). However, none of
the factors normally associated with AGO4
function in RdDM, most prominently DCL3,
RDR2, and chromomethylase 3 (CMT3), could
be linked to the ago4-enhanced susceptibility phenotype (1). Although this could reflect
functional redundancy among RdDM factors,
a novel AGO4 function might also contribute
to disease resistance independently of RdDM
or, indeed, of sRNAs.
SUPPRESSION, AVOIDANCE,
AND USURPATION OF RNA
SILENCING BY PATHOGENS
The neverending molecular arms race between
parasites and their hosts is also a feature of antimicrobial silencing. This section highlights
some of the sophisticated mechanisms by which
viruses or pathogenic bacteria may actively suppress, evade, or sometimes usurp host RNA silencing pathways to cause disease.
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Plant Viruses
Viral suppressors of RNA silencing. A
widespread viral counterdefensive strategy
against RNA silencing is the deployment of
VSRs, which are highly diverse in sequence,
structure, and activity (reviewed in Reference
22). Single VSRs may target multiple points
in RNA silencing pathways; viruses with large
genomes may encode several functionally distinct proteins to achieve this effect. Whereas
illustrations of VSR action abound, we provide molecularly well-characterized examples
of proteins that each inhibit at least one step in
the antiviral silencing pathway. We start with
inhibition of host-directed dsRNA production
by the cytoplasmic V2 protein of Tomato yellow leaf curl virus (TYLCV, DNA virus). V2
binds to and colocalizes with the tomato ortholog of SGS3, which is required for RDR6
function (30). Disruption of binding prevents
V2’s VSR activity, suggesting that V2 interaction with SGS3 compromises its function
(Figure 7a). Studies of the Cauliflower mosaic
virus (CaMV, DNA genome) P6 protein show
how viruses impinge upon dsRNA processing
into vsRNAs (31). Although most P6 molecules
aggregate into cytoplasmic inclusion bodies (viroplasms), a small, nuclear fraction is essential
to CaMV infectivity. Transgenic P6 expression
in Arabidopsis is genetically equivalent to inactivating the nuclear protein DRB4, which facilitates synthesis by DCL4 of 21-nt-long vsRNAs.
Moreover, P6 is detected in DRB4 immunoprecipitates isolated from CaMV-infected cells,
suggesting not only genetic, but also physical
interactions between the two factors in the nucleus (31) (Figure 7b). Several VSRs inhibit
HEN1-mediated 2 O-methylation of vsRNA
duplexes, including the P122 kDa replicase
(Figure 3a) of Tobacco mosaic virus (TMV, RNA
virus) (42).
Direct sequestration of vsRNA duplexes
is one mode of action of the P19 protein of
tombusviruses and 2b protein of cucumoviruses
(RNA viruses). P19 uses an extended β-sheet
surface and a small α-helix to form a caliperlike structure for binding and measuring
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a
b
Geminivirus
CH3
Episome
24nt
CaMV
DCL4 DRB4
4/6
AGO
Reduced
methylation
24nt
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DCL3 DRB3
CH3
CH3 CH3
CH3
CH3
CH3
CH3
CH3
CH3
DNA
CH3
CH3
CH3
CH3
CH3 CH3
Nucleus
(A) n
CaMV 35S RNA
21nt
P6
α
β
Nucleus
Cytosol
Cytosol
CH3
Reduced
methyl levels
P6
or
(A)n
SAM
Met
Free P6
P6
P6
P6
α
β
SAH
Adenosine
Hcy
V2
RDR6
P6
SGS3
Viroplasm
SDE3
L2
ADK
DCL4 DRB4
P6
P6
P6
P6
P6
P6
P6
P6
P6
P6
P6
AMP
21nt
Figure 7
Modes of action of DNA virus–encoded viral suppressors of RNA silencing (VSRs). (a) Two strategies deployed by geminiviruses
against RNA silencing: (i ) inhibition of RNA-dependent RNA polymerase6 (RDR6) function through SUPPRESSOR OF GENE
SILENCING 3 (SGS3) interference by cytoplasmic V2 and (ii ) indirect suppression of RNA-directed DNA methylation (RdDM)
through inhibition of the methyl cycle by nuclear L2. (b) Free cytoplasmic P6 molecules produced by Cauliflower mosaic virus (CaMV)
interact with importin (αβ) and are translocated to the nucleus to inhibit dsRNA binding 4 (DRB4) function through physical
interactions.
Figure 7 amended May 15, 2009.
the characteristic length of vsRNAs to preferentially sequester DCL4-dependent, 21-bp
duplexes (73) (Figure 3b). By contrast, 2b uses
a pair of hook-like structures that interact more
promiscuously with long and short dsRNA
(73). Additionally, CMV 2b binds AGO1 and
blocks slicing without interfering with sRNA
loading in vitro (84). Although seemingly
contradictory, these two antisilencing 2b
activities are reconcilable, because 2b’s affinity
for dsRNA is weak compared with that of
P19. Thus, its interaction with AGO1 could
increase 2b local concentrations and enhance
specific binding to vsRNAs (Figure 3b). The
polerovirus (RNA viruses) VSR, P0, contains an
F-box-like domain that interacts with components of the SKP1-Cullin-F box (SCF) family
of ubiquitin ligases. Because P0 binds AGO1
and promotes its decay, direct P0-mediated
ubiquitination of AGO1 might induce its 26S
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proteasome–dependent degradation (8). However, the P0-AGO1 interaction maps outside
the F-box domain, and the 26S proteasome
inhibitor MG132 does not affect P0-mediated
AGO1 turnover (5). P0 is possibly a dominantnegative inhibitor of a host F-box protein that
regulates AGO1 homeostasis, or it may direct
erroneous AGO1 ubiquitination patterns
that interfere with RISC function/assembly
(Figure 3c). The geminivirus-encoded L2
protein illustrates indirect inhibition of RdDM
of viral DNA. L2 interacts with and inhibits
adenosine kinase (ADK), which is required
for production of the methyl group donor
S-adenosyl methionine (SAM), such that ADKdeficient plants display global methylation
defects, consistent with their hypersusceptibility to geminivirus infection (62, 76)
(Figure 7a).
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Other viral strategies to suppress, avoid, or
usurp RNA silencing. Because HEN1 and
AGO1 are shared components of multiple endogenous silencing pathways, VSRs that affect those factors are likely to broadly interfere
with cellular sRNA functions. Indeed, VSRoverexpressing plants often exhibit developmental anomalies typical of miRNA pathway
mutants, which are usually considered to be
side effects of primary inhibition of vsRNAdirected antiviral silencing (15, 27). However,
VSR expression in these plants occurs in a
much broader tissue range than in natural infections. Second, miRNAs and other cellular
sRNAs may regulate innate antiviral responses
unrelated to RNA silencing (akin to fungal and
bacterial PTI); their inhibition may thus reflect
deliberate viral strategies. For instance, DCL1,
which represses transcript accumulation of antiviral DCL4 (61), is itself negatively regulated
by miR162 (79). Viral inhibition of miRNA
functions could thus augment DCL1 levels and
activity, resulting in reduced DCL4 accumulation and, hence, attenuated antiviral silencing. Assuming the 5 nt bias for sRNA sorting into specific AGOs applies to vsRNAs (44,
48, 68), viral production of large amounts of
vsRNA with 5 -terminal U could possibly satu502
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rate AGO1 to impair its action in antiviral defense and possibly in innate immunity mediated
by endogenous sRNAs. This might be particularly true of viroids, whose genomes are efficiently diced but largely immune to RISC (34)
(Figure 3d ). Titration of DCL activities likely
explains how Red clover necrotic mosaic virus
(RCNMV, RNA virus) might use replicating
RNA rather than dedicated proteins to inhibit silencing (69). Because many plant viruses
replicate into cytoplasmic membranous compartments, this raises the question as to how
their genome is accessed by DCLs, which
seem nearly exclusively nuclear in Arabidopsis,
DCL2 aside (78). Membrane-associated replication might thus allow viral evasion rather than
suppression of silencing. Evasion might also
occur through acquisition of mutations within
vsRNA target sites owing to error-prone viral
replication. Hence, recombinant Plum pox virus
(PPV, RNA virus) containing target sites of cellular miRNAs rapidly evaded their inhibitory
effects by mutating the seed region essential for
miRNA-target interactions (67).
Plant viruses might also usurp RNA silencing, as do mammalian DNA viruses, which
encode their own miRNAs to regulate viral
gene expression and/or target transcripts of antiviral, proapoptotic, or antiproliferating host
factors (reviewed in Reference 60). A parallel in plants is provided by vsRNAs produced
from an extensive stem-loop structure within
the CaMV-encoded 35S RNA (Figure 7a), of
which several exhibit near-perfect complementarity to Arabidopsis mRNAs that are effectively
downregulated during infection (46). vsRNAmediated downregulation of cellular mRNAs
is likely often fortuitous, but some targeting
events might be positively selected, for instance
if they affect host defense factors. Although
seemingly contradictory to the deployment of
VSR (to inhibit RNA silencing), studies of
the CaMV-encoded P6 protein illustrate how
the two processes might coexist within cells
(31). Indeed, by inhibiting the DCL4 cofactor DRB4, P6 reduces but does not eliminate
DCL4 function, resulting in 21-nt vsRNA levels low enough to dampen antiviral defense
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but high enough to target host gene expression
(Figure 7a).
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RNA Silencing Suppression
by Other Pathogens
Agrobacterium infections illustrate an indirect
form of bacterial-mediated silencing suppression. Although the RDR6/DCL4-dependent
siRNA pathway initially limits T-DNA oncogene expression, primary tumors become progressively resistant to this process owing to
compromised DCL4-mediated siRNA processing (Figure 3e). Silencing suppression seems
independent of T-DNA-encoded proteins or
bacterial virulence factors, because it is recapitulated in calli derived from A. tumefaciens–free
silenced tissues (26). Therefore, Agrobacteriuminduced cell proliferation and/or dedifferentiation likely promotes a general block on
siRNA production. Although as yet uncharacterized, the underlying molecular processes
must be highly specific, because miRNA
accumulation/activity is not affected in primary
tumors, consistent with the strict requirement
for the miRNA pathway in crown gall disease
(26).
The partial rescue of P. syringae type-III secretion mutants in miRNA- but not siRNAdeficient Arabidopsis demonstrates a key role
for host miRNAs in PTI. A corollary, therefore, is that some bacterial virulence factors
injected into host cells through the type-III
apparatus to suppress PTI should also suppress the miRNA pathway. This idea was validated in a study providing examples of DC3000
effector proteins (named bacterial suppressors of RNA-silencing, BSRs) that inhibit
miRNA transcription, biogenesis/stability, or
activity (53) (Figure 8a). Transcriptional suppression of PAMP-responsive MIRNA genes,
such as flg22-induced MIR393, was demonstrated with AvrPtoB. Suppression was independent of AvrPtoB’s E3-ligase activity, as is
the case for AvrPtoB-mediated suppression of
PAMP-responsive protein-coding genes that
mediate PTI. This result indicates a general interference with PTI signaling at or downstream
of the FLS22 receptor-like kinase (53).
A somewhat less trivial scenario is required to explain how the DC3000 effector AvrPto broadly suppresses accumulation of
PTI-related and PTI-unrelated miRNAs, presumably by inhibiting DCL1-mediated processing (53). The fact that mutations preventing AvrPto plasma membrane localization alter
its antimiRNA activity is indeed intriguing because AvrPto’s membrane anchoring is required
for its virulence function through inhibition of
the kinase activities of the PAMP receptors, including FLS2. Although this result could suggest the existence of an as yet uncharacterized
membranous pool of miRNA-processing factors, an alternative explanation lies in the interaction of AvrPto with the plasma membrane
protein BAK1 (BRI1-associated receptor kinase
1) (66). BAK1 is a shared signaling partner of
FLS2 and of BRI1 (brassinosteroid-insensitive
1), the receptor of the phytohormone brassinosteroid, which is required for development (17).
Constitutive AvrPto expression indeed generates developmental phenotypes indistinguishable from those of Arabidopsis bri1 mutants, indicating inhibition of brassinosteroid signaling
(66). One possible explanation for the AvrPto
effects on miRNA biogenesis is therefore that
brassinosteroids are constitutively required for
optimal expression of some miRNA processing
factors*.
Suppression of miRNA activity was documented with the bacterial effector HOPT11, which inhibits cleavage or translational repression of several endogenous miRNA targets,
mimicking the effects of Arabidopsis ago1 mutant
alleles. Thus, HOPT1-1 might directly interact
with AGO1 or with a component of AGO1RISC (53). Bacterial suppression of RNA silencing in ETI was also suggested to explain
the unusual size of At-lsiRNA1, produced upon
RPS2 elicitation by Pst DC3000 AvRpt2 (37).
*Unpublished data removed, May 15, 2009.
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a
Systemic
defense SA
responses
FLS2
BAK1
Pst
b
AS
R1
SNC1
AGO1
R2
(A)n
SNC1
AGO1
DCL4
DRB4
MIR
BSR
DCL4
DRB4
21nt
Sentinel sRNA
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VSR
miRNA
Cosuppression
of R locus
vsRNA
c
(A)n
(A)n
AGO1
AGO1
AGO1
AS
R1
SNC1
R2
(A)n
R
SNC1
DCL4
DRB4
SA
R
R
RDR1
HR
+
Other silencing
components
21nt
BSR
AGO1
R locus activation
VSR
Figure 8
Host counter-counterdefense strategies against silencing suppression. (a) The effects of viral suppressors of RNA silencing (VSRs) or
bacterial suppressors of RNA-silencing (BSRs) [here P. syringae pv. tomato (Pst) effectors] might be detected by dedicated resistance (R)
proteins guarding key effectors of RNA-silencing pathway components. R protein activation might induce a hypersensitive response
(HR) and salycilic acid (SA) production, leading to exacerbated RNA silencing and systemic defense responses. (b) SNC1 is one of
several R genes present at the RPP4 locus that share extensive sequence homology (orange). Endogenous 21-nt siRNAs or sentinel
siRNAs produced by antisense transcription at Suppressor of nprl-1, constitutive 1 (SNC1) and operating through ARGONAUTE1
(AGO1) coordinately cosuppress the R genes in the locus. (c) Inhibition of AGO1 function by VSR or BSR prevents the action of
sentinel short-interfering RNAs (siRNAs) and releases inhibition of RPP4-like gene expression, potentially promoting defense against
the VSR- or BSR-producing pathogens. BAK1, BRI1-associated receptor-kinase 1; DCL, Dicer-like; DRB4, dsRNA binding 4; FLS2,
FLAGELLIN-SENSING 2; RDR1, RNA-dependent RNA polymerase1; vsRNA, viral small RNA.
Although likely synthesized by DCL1 (which
typically produces miRNAs in the 19–24-nt
size range) the 30-nt-long at-lsiRNA1 may
arise from AvRpt2-mediated interference with
DCL1 or interacting partners in the miRNA
processing machinery, including the dsRNAbinding protein HYL1.
RNA silencing suppression by pathogens
other than viruses, P. syringae, and Agrobacterium awaits demonstration. However, given
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the broad involvement of endogenous sRNAs in
biotic stress responses and the availability of robust silencing reporter systems (25, 53), effector
proteins injected or secreted into host cells by
fungi, nematodes, or insects will probably soon
be trivially identified as silencing inhibitors.
Key issues will be to identify the mode of action
and targets of these suppressors and, perhaps
more importantly, to understand their spatial
and temporal dynamics of expression/action in
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authentic infections rather than nonbiological
expression systems.
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HOST RESPONSES TO
SILENCING SUPPRESSION
BY PATHOGENS
This final section illustrates how silencing suppression by pathogens might be perceived and
reacted against by plants. Although less well
documented or understood, the deployment of
counter-counterdefensive measures to accommodate VSR or BSR detrimental effects probably underlies the proliferation of plant antimicrobial silencing components and also explains,
in turn, the remarkable fluidity of genes encoding VSR (and possibly BSR), presumably
required for their constant adaptation to the
various host responses to silencing suppression.
R Proteins and Direct Targeting of
VSR and BSR Integrity/Function
Plants exploit rapidly evolving R proteins to
monitor or guard the integrity of specific host
defense components called guardees, which are
the primary targets of a pathogen’s virulence
factors (35). During ETI, changes in the status
of guardees usually result in R proteins triggering host defense reactions. These reactions
sometimes culminate in a form of programmed
cell death called the hypersensitive response
(HR) and are accompanied by phytohormone release (see Nonhost Resistance, PAMPTriggered Immunity, and Effector-Triggered
Immunity). VSR and BSR exert their virulence
functions at least partly through suppression
of RNA silencing. Therefore, some R genes
may have evolved to specifically sense the modifications incurred by pathogens to antiviral
and antimicrobial silencing components, particularly if these components are shared between multiple defense pathways (e.g., AGO1).
An R-based system to counteract VSR or BSR
effects could also be advantageous in further
promoting phytohormone-mediated stimulation of RNA-silencing components, as seen
with SA- or JA-mediated induction of RDR1
during defense (21, 56) (Figure 8a). Although
no experimental proof supports these ideas as
yet, strikingly, several VSR are known to trigger
R-gene-dependent HR in specific hosts and, in
one case, VSR mutations that compromised silencing suppression also compromised HR (41,
64). Whether the same applies to bacterial effector alleles known to escape R protein recognition during ETI awaits characterization.
Hosts could also directly neutralize silencing suppressors through activities that degrade
or relocate them into inappropriate subcellular
compartments. The former probably explains
the failure of specific alleles of the CMVencoded 2b protein to accumulate in Arabidopsis, possibly because of allele-dependent
proteolysis (84). The latter is suggested by the
nuclear relocation of the tombusviral P19 protein caused by host-encoded P19-interacting
ALY proteins (12). The varying efficacy of
host-directed degradation/relocation of microbial silencing suppressors and polymorphism
among these factors could thus contribute to
differences in viral or microbial susceptibility
between plant ecotypes or species.
Altering the Levels of Endogenous
Small RNAs That Regulate Antiviral
or Antibacterial Components
Together with miR825, miR162 and miR168
are downregulated upon elicitation of PTI by
Pst DC3000 HrcC (29). This phenomenon is
highly significant, because miR162 and miR168
normally suppress DCL1 and AGO1, the major processor and effector of the miRNA
pathway, respectively (74, 79). Thus, bacterial
PAMP recognition might elevate DCL1 and
AGO1 cellular levels, potentially resulting in
enhanced PTI. Suppression of miRNA processing or AGO1 function by bacterial effectors or
VSR is similarly predicted to enhance DCL1
and AGO1 levels during ETI and antiviral
defense.
Sentinel Small RNAs Generated
at Complex R Gene Loci
R genes are constantly acquiring new specificity through high recombination rates,
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*Text changed to note Figure 8c, May 15, 2009.
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which typically results in complex arrays of
silencing-prone gene clusters. For instance,
the Arabidopsis Columbia RPP4 locus (named
RPP5 in the Landsberg ecotype) contains
seven TIR-nucleotide binding site (NBS)leucine-rich repeat (LRR) class R genes, of
which two, RPP4 and SNC1 (for Suppressor
of nprl-1, constitutive 1), confer resistance to
the bacterium P. syringae and the oomycete
Hyaloperonospora parasitica, respectively (81).
These R genes are interspersed with two
non-R genes and three related sequences that
are coordinately regulated by transcriptional
activation and RNA silencing. Endogenous
siRNAs indeed accumulate at the RPP4 locus,
possibly originating through annealing of
sense and antisense transcripts detected at this
region. SNC1 mRNA levels are elevated in dcl4
and ago1 mutants (defective in processing and
activity of 21-nt-long siRNAs, respectively)
suggesting that SNC1 and the other highly
related RPP4 genes are coordinately cosuppressed by siRNAs (81) (Figure 8c*, upper). As
explained earlier, reversible posttranscriptional
silencing of resistance-related genes might be
required to reduce the fitness costs of constitutive defense activation. However, an added
advantage is anticipated from the observation
that expression of helper component proteinase
(HcPro), a potyviral VSR, enhances steadystate expression of SNC1, presumably because
HcPro inhibits the action of DCL4-dependent
siRNAs, notably found at the RPP4 locus
(81) (Figure 8c). Put into a broader context
of plant-microbe interactions, these results
suggest that sRNA involved in dampening R
gene expression may directly sense silencing
suppression caused by pathogens and stimulate
an immediate and global enhancement of
defense.
SUMMARY POINTS
1. Plant parasites activate RNA silencing through at least two different mechanisms: (a) by
producing their own small RNAs (sRNAs) (viruses and A. tumefaciens) or (b) by altering
endogenous sRNA levels in plant hosts.
2. sRNAs regulate gene expression by mediating mRNA degradation, translational inhibition, or promoting chromatin modifications.
3. Many sRNAs are induced or repressed in response to pathogen attack. These sRNAs
contribute to basal and race-specific defense responses upon their incorporation into
effector complexes containing ARGONAUTE proteins.
4. Healthy plants may use cellular sRNAs to reversibly attenuate expression of many
resistance-related genes, thereby reducing the fitness costs of constitutive defense activation.
5. Classical hormonal responses to pathogens, notably based on salycilate and jasmonate,
appear to be intimately linked to the operation of antimicrobial RNA silencing.
6. To counteract the effects of sRNA, pathogens produce virulence factors, called suppressors of RNA silencing, that inhibit various steps of the RNA silencing machinery. The
mode of action and cellular targets of viral and bacterial suppressors of RNA silencing
(VSR and BSR) are being progressively elucidated.
7. Viruses and pathogenic bacteria may also evade or sometimes usurp host RNA silencing
pathways to cause disease.
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8. Plants can sense the modifications incurred by pathogens to RNA silencing components
and respond through different strategies. These strategies possibly include resistance
protein-mediated recognition of VSR/BSR and ensuing enhancement of innate immune
responses, transcriptional or posttranscriptional stimulation of RNA silencing components, or the direct use of cellular sRNA as sensors or sentinels against aggressions.
DISCLOSURE STATEMENT
Annu. Rev. Plant Biol. 2009.60:485-510. Downloaded from arjournals.annualreviews.org
by University of Crete on 04/16/10. For personal use only.
The authors are not aware of any biases that might be perceived as affecting the objectivity of this
review.
ACKNOWLEDGMENTS
The authors are supported by a grant from the Bettencourt Foundation for Life Sciences, a
grant from European Union–integrated project SIROCCO (Silencing RNAs: Organisers and
Coordinators of Complexity in Eukaryotic Organisms; LSHG-CT-2006-037900), a starting grant
from the European Research Council (ERC, Frontiers of RNAi, 210890) to O.V., and a European
Union Marie Curie fellowship (041419) to V.R.F.
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Volume 60, 2009
Annu. Rev. Plant Biol. 2009.60:485-510. Downloaded from arjournals.annualreviews.org
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My Journey From Horticulture to Plant Biology
Jan A.D. Zeevaart p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Roles of Proteolysis in Plant Self-Incompatibility
Yijing Zhang, Zhonghua Zhao, and Yongbiao Xue p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p21
Epigenetic Regulation of Transposable Elements in Plants
Damon Lisch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p43
14-3-3 and FHA Domains Mediate Phosphoprotein Interactions
David Chevalier, Erin R. Morris, and John C. Walker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p67
Quantitative Genomics: Analyzing Intraspecific Variation Using
Global Gene Expression Polymorphisms or eQTLs
Dan Kliebenstein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p93
DNA Transfer from Organelles to the Nucleus: The Idiosyncratic
Genetics of Endosymbiosis
Tatjana Kleine, Uwe G. Maier, and Dario Leister p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 115
The HSP90-SGT1 Chaperone Complex for NLR Immune Sensors
Ken Shirasu p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 139
Cellulosic Biofuels
Andrew Carroll and Chris Somerville p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 165
Jasmonate Passes Muster: A Receptor and Targets
for the Defense Hormone
John Browse p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 183
Phloem Transport: Cellular Pathways and Molecular Trafficking
Robert Turgeon and Shmuel Wolf p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 207
Selaginella and 400 Million Years of Separation
Jo Ann Banks p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 223
Sensing and Responding to Excess Light
Zhirong Li, Setsuko Wakao, Beat B. Fischer, and Krishna K. Niyogi p p p p p p p p p p p p p p p p p p p p 239
Aquilegia: A New Model for Plant Development, Ecology, and
Evolution
Elena M. Kramer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 261
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Environmental Effects on Spatial and Temporal Patterns of Leaf
and Root Growth
Achim Walter, Wendy K. Silk, and Ulrich Schurr p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 279
Short-Read Sequencing Technologies for Transcriptional Analyses
Stacey A. Simon, Jixian Zhai, Raja Sekhar Nandety, Kevin P. McCormick,
Jia Zeng, Diego Mejia, and Blake C. Meyers p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 305
Biosynthesis of Plant Isoprenoids: Perspectives for Microbial
Engineering
James Kirby and Jay D. Keasling p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 335
Annu. Rev. Plant Biol. 2009.60:485-510. Downloaded from arjournals.annualreviews.org
by University of Crete on 04/16/10. For personal use only.
The Circadian System in Higher Plants
Stacey L. Harmer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 357
A Renaissance of Elicitors: Perception of Microbe-Associated
Molecular Patterns and Danger Signals by Pattern-Recognition
Receptors
Thomas Boller and Georg Felix p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 379
Signal Transduction in Responses to UV-B Radiation
Gareth I. Jenkins p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 407
Bias in Plant Gene Content Following Different Sorts of Duplication:
Tandem, Whole-Genome, Segmental, or by Transposition
Michael Freeling p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 433
Photorespiratory Metabolism: Genes, Mutants, Energetics,
and Redox Signaling
Christine H. Foyer, Arnold Bloom, Guillaume Queval, and Graham Noctor p p p p p p p p p p p 455
Roles of Plant Small RNAs in Biotic Stress Responses
Virginia Ruiz-Ferrer and Olivier Voinnet p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 485
Genetically Engineered Plants and Foods: A Scientist’s Analysis
of the Issues (Part II)
Peggy G. Lemaux p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 511
The Role of Hybridization in Plant Speciation
Pamela S. Soltis and Douglas E. Soltis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 561
Indexes
Cumulative Index of Contributing Authors, Volumes 50–60 p p p p p p p p p p p p p p p p p p p p p p p p p p p 589
Cumulative Index of Chapter Titles, Volumes 50–60 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 594
Errata
An online log of corrections to Annual Review of Plant Biology articles may be found at
http://plant.annualreviews.org/
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Contents