DOI: 10.1126/science.1126088
, 436 (2006);312Science
et al.Lionel Navarro
Repressing Auxin Signaling
A Plant miRNA Contributes to Antibacterial Resistance by
This copy is for your personal, non-commercial use only.
clicking here.colleagues, clients, or customers by
, you can order high-quality copies for yourIf you wish to distribute this article to others
here.following the guidelines
can be obtained byPermission to republish or repurpose articles or portions of articles
):October 31, 2014www.sciencemag.org (this information is current as of
The following resources related to this article are available online at
http://www.sciencemag.org/content/312/5772/436.full.html
version of this article at:
including high-resolution figures, can be found in the onlineUpdated information and services,
http://www.sciencemag.org/content/suppl/2006/04/18/312.5772.436.DC1.html
can be found at:Supporting Online Material
http://www.sciencemag.org/content/312/5772/436.full.html#related
found at:
can berelated to this articleA list of selected additional articles on the Science Web sites
http://www.sciencemag.org/content/312/5772/436.full.html#ref-list-1
, 9 of which can be accessed free:cites 25 articlesThis article
203 article(s) on the ISI Web of Sciencecited byThis article has been
http://www.sciencemag.org/content/312/5772/436.full.html#related-urls
100 articles hosted by HighWire Press; see:cited byThis article has been
http://www.sciencemag.org/cgi/collection/botany
Botany
subject collections:This article appears in the following
registered trademark of AAAS.
is aScience2006 by the American Association for the Advancement of Science; all rights reserved. The title
CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005.
(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience
onOctober31,2014www.sciencemag.orgDownloadedfromonOctober31,2014www.sciencemag.orgDownloadedfromonOctober31,2014www.sciencemag.orgDownloadedfromonOctober31,2014www.sciencemag.orgDownloadedfromonOctober31,2014www.sciencemag.orgDownloadedfrom
in the long run. However, migration rate in our
experiment was density independent and migration
was confined to the two nearest neighbors,
whereas it is known that the dynamics of a
metapopulation can vary depending on the exact
form of density dependence (31) and scheme of
migration (11). Moreover, growth rates of
Drosophila (and most insects, microbes, and
fishes) are higher than those of mammals and
birds, which are generally of greater concern
for conservation. The intrinsic growth rates of
subpopulations are also known to interact
strongly with migration rate in producing
observed metapopulation dynamics (12). Therefore,
due caution should be exercised when
extrapolating our results to natural populations.
References and Notes
1. I. Hanski, Metapopulation Ecology (Oxford Univ. Press,
New York, 1999).
2. M. Gyllenberg, G. So¨derbacka, S. Ericsson, Math. Biosci.
118, 25 (1993).
3. B. E. Kendall, G. A. Fox, Theor. Popul. Biol. 54, 11 (1998).
4. A. Hastings, Ecology 74, 1362 (1993).
5. J. C. Allen, W. M. Schaffer, D. Rosko, Nature 364, 229
(1993).
6. G. D. Ruxton, Proc. R. Soc. London Ser. B 256, 189
(1994).
7. E. Ranta, V. Kaitala, P. Lundberg, Oikos 83, 376
(1998).
8. J. Ripa, Oikos 89, 175 (2000).
9. G. Nachman, J. Anim. Ecol. 56, 267 (1987).
10. G. Nachman, Biol. J. Linn. Soc. 42, 285 (1991).
11. D. J. D. Earn, S. A. Levin, P. Rohani, Science 290, 1360
(2000).
12. S. Dey, S. Dabholkar, A. Joshi, J. Theor. Biol. 238, 78
(2006).
13. L. D. Mueller, A. Joshi, Stability in Model Populations
(Princeton Univ. Press, Princeton, NJ, 2000).
14. J. Lecomte, K. Boudjemadi, F. Sarrazin, K. Cally, J. Clobert,
J. Anim. Ecol. 73, 179 (2004).
15. R. Levins, Bull. Entomol. Soc. Am. 15, 237 (1969).
16. G. D. Ruxton, J. Anim. Ecol. 65, 161 (1996).
17. I. Hanski, D. Y. Zhang, J. Theor. Biol. 163, 491
(1993).
18. G. Nachman, Oikos 91, 51 (2000).
19. A. R. Ives, S. T. Woody, E. V. Nordheim, C. Nelson, J. H.
Andrews, Am. Nat. 163, 375 (2004).
20. In the experiment, the nine subpopulations (single-vial
Drosophila cultures) were arranged on the periphery of a
circle, with each vial exchanging migrants only with its
two nearest neighbors (i.e., a one-dimensional array with
periodic boundary conditions). In nature, such metapopulations
might exist, for example, on the shorelines of
lakes or along the edges of ecosystems. The simulations
mimicked the experimental system and focused on the
behavior of nine coupled Ricker maps, with periodic
boundary conditions. See supporting material on
Science Online.
21. V. Grimm, C. Wissel, Oecologia 109, 323 (1997).
22. We define a population whose size fluctuates with higher
amplitude across time to be less stable than one that has
lower amplitude of fluctuation [‘‘constancy’’ attribute of
stability (21)]. We introduce a measure of stability, the
fluctuation index (FI) of a series, given as
FI 0
1
TN
XTj1
t00
absðNtþ1 j NtÞ
where Nj is the mean population size over T generations.
FI thus reflects the mean one-step change in population
size, scaled by average population size, over the study
duration.
23. O. N. Bjørnstad, R. A. Ims, X. Lambin, Trends Ecol. Evol.
14, 427 (1999).
24. P. A. P. Moran, Aust. J. Zool. 1, 291 (1953).
25. In the experiment, a subpopulation was deemed extinct
when it did not contain at least one male and one female
fly. When a CM subpopulation became extinct, it was
restarted using four males and four females from a
backup vial. The backup vials were maintained in
parallel to the three treatments, under high larval
crowding and yeast supplement to the adults. There
were no restarts in LMMs or HMMs; extinct subpopulation
vials remained empty until recolonized by migrants
from a neighboring subpopulation.
26. A˚. Bra¨nnstro¨m, D. J. T. Sumpter, Proc. R. Soc. London
Ser. B 272, 2065 (2005).
27. R. M. May, G. F. Oster, Am. Nat. 110, 573 (1976).
28. For example, in simulations with r 0 2.8, comparable
to the estimated r in our CM subpopulations, the
mean extinction rate was 0.75 out of 9 subpopulations
per generation, versus 3.35 out of 9 for experimental
CMs.
29. R. A. Cheke, J. Holt, Ecol. Entomol. 18, 109
(1993).
30. V. Sheeba, A. Joshi, Curr. Sci. 75, 1406 (1998).
31. R. A. Ims, H. P. Andreassen, Proc. R. Soc. London Ser. B
272, 913 (2005).
32. We thank V. K. Sharma for useful discussions; three
anonymous reviewers for helpful comments on the
manuscript; M. Shakarad, M. Rajamani, and N.
Raghavendra for crucial help in fly handling; and
J. Mohan, S. Ghosh, K. M. Satish, N. Rajanna, and
M. Manjesh for help in diverse ways during the
experiment. Supported by a senior research fellowship
from the Council for Scientific and Industrial Research,
Government of India (S.D.) and by grants from
Department of Science and Technology, Government
of India.
Supporting Online Material
www.sciencemag.org/cgi/content/full/312/5772/434/DC1
Materials and Methods
SOM Text
Fig. S1
References
24 January 2006; accepted 16 March 2006
10.1126/science.1125317
A Plant miRNA Contributes to
Antibacterial Resistance by
Repressing Auxin Signaling
Lionel Navarro,1,2
Patrice Dunoyer,2
Florence Jay,2
Benedict Arnold,3
Nihal Dharmasiri,4
Mark Estelle,4
Olivier Voinnet,2
*† Jonathan D. G. Jones1
*†
Plants and animals activate defenses after perceiving pathogen-associated molecular
patterns (PAMPs) such as bacterial flagellin. In Arabidopsis, perception of flagellin increases
resistance to the bacterium Pseudomonas syringae, although the molecular mechanisms
involved remain elusive. Here, we show that a flagellin-derived peptide induces a plant
microRNA (miRNA) that negatively regulates messenger RNAs for the F-box auxin receptors
TIR1, AFB2, and AFB3. Repression of auxin signaling restricts P. syringae growth, implicating
auxin in disease susceptibility and miRNA-mediated suppression of auxin signaling
in resistance.
P
lants perceive a 22–amino acid peptide
(flg22) from the N terminus of eubacterial
flagellin (1). In Arabidopsis, flg22 triggers
rapid changes in transcript levels, including
down-regulation of a gene subset,
potentially by posttranscriptional mechanisms
(2). One posttranscriptional mechanism is RNA
silencing, a sequence-specific mRNA degradation
process mediated by 20- to 24-nucleotide
(nt) RNAs known as short interfering RNAs
(siRNAs) and microRNAs (miRNAs). Both are
made from double-stranded RNA (dsRNA) by
the ribonuclease III enzyme Dicer. Four
paralogs (Dicer-likes, or DCLs) are found in
Arabidopsis. DCL2 produces viral-derived
siRNAs (3) and siRNAs from antisense
overlapping transcripts (4). DCL3 generates
DNA repeat–associated siRNAs (3), whereas
DCL4 synthesizes trans-acting siRNAs and
mediates RNA interference (5–7). DCL1
excises miRNAs from intergenic stem-loop
transcripts to promote cleavage of cellular
transcripts carrying miRNA-complementary
sequences (8).
We examined whether small RNAs—
especially miRNAs—contribute to the rapid
changes elicited by flg22. We analyzed
transgenic Arabidopsis expressing the P1Hc-Pro,
P19, and P15 viral proteins that
suppress miRNA- and siRNA-guided functions
(9, 10), anticipating that transcripts
repressed by flg22-stimulated small RNAs
would be more abundant in these lines.
Comparative transcript profiling of untreated
1
The Sainsbury Laboratory, John Innes Centre, Colney Lane,
Norwich NR4 7UH, UK. 2
Institut de Biologie Mole´culaire des
Plantes du Centre National de la Recherche Scientifique,
67084 Strasbourg Cedex, France. 3
John Innes Centre, Colney
Lane, Norwich NR4 7UH, UK. 4
Department of Biology, Indiana
University, Bloomington, IN 47405, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
jonathan.jones@sainsbury-laboratory.ac.uk (J.D.G.J.); olivier.
voinnet@ibmp-ulp.u-strasbg.fr (O.V.)
REPORTS
21 APRIL 2006 VOL 312 SCIENCE www.sciencemag.org436
transgenic seedlings and flg22-elicited wildtype
seedlings identified a subset of mRNAs
that fulfilled this criterion, including TIR1
(Transport Inhibitor Response 1) and two of
its three functional paralogs, AFB2 and AFB3
(Auxin signaling F-Box proteins 2 and 3)
(fig. S1, A and B). However, accumulation of
AFB1, the third TIR1 paralog, was not
discernibly altered in the suppressor lines
(fig. S1B).
The F-box proteins TIR1, AFB1, AFB2,
and AFB3 are receptors for the plant hormone
auxin (11–13). Additionally, TIR1 and AFB
transcripts are targets of miR393, a conserved
miRNA (fig. S2) (14, 15). A modified rapid
amplification of cDNA ends (RACE) assay
confirmed that TIR1, AFB2, and AFB3
mRNAs are specifically cleaved by miR393 in
a DCL1-dependent manner (Fig. 1A and fig.
S3). However, polymerase chain reaction
(PCR)–amplified cleavage products derived
from AFB1 were hardly detectable and rarely
cloned (Fig. 1A and fig. S3), confirming that
AFB1 is partially resistant to miR393-directed
cleavage. Presumably, this is due to a single
mismatch in the miR393 complementary site
found specifically in AFB1 mRNA (fig. S3).
Quantitative reverse transcription–polymerase
chain reaction (RT-qPCR) analyses with primers
flanking the miR393 target sites revealed
a two- to threefold reduction in the levels
of TIR1, AFB1, AFB2, and AFB3 30 min after
flg22 elicitation of wild-type seedlings
(Fig. 1B). Repression of TIR1, AFB2, and
AFB3 was partially compromised in dcl1-9
seedlings (Fig. 1B), suggesting that a miRNAdirected
pathway, probably involving miR393,
contributes to TIR1, AFB2, and AFB3 downregulation.
However, flg22-triggered downregulation
of AFB1 was unaffected in dcl1-9,
in agreement with its reduced sensitivity to
miR393 (Fig. 1A and fig. S3). Therefore, repression
of AFB1 is miRNA-independent and
may exclusively occur at the transcriptional
level.
We analyzed miR393 levels in flg22elicited
Arabidopsis seedlings. Northern analysis
showed a twofold increase in miR393
accumulation after 20 and 60 min, whereas
levels of the unrelated miR171 remained
unaltered (Fig. 2A). Similarly, miR393 levels
were unaltered in seedlings treated with
control flg22A.tum inactive peptide (Fig. 2A)
(1). The Arabidopsis genome contains two
miR393 precursors, on chromosomes 2 (AtmiR393a)
and 3 (At-miR393b) (16). We fused
1.5-kb DNA fragments upstream of AtmiR393a
and At-miR393b to the enhanced
Green Fluorescence Protein (eGFP) and
transformed them into Arabidopsis, producing
transgenic lines AtmiR393a-p::eGFP
and AtmiR393b-p::eGFP. Using RT-qPCR,
we observed a twofold increase in eGFP
mRNA level in three independent flg22treated
AtmiR393a-p::eGFP lines (Fig. 2B),
consistent with the miR393 Northern analysis
(Fig. 2A). However, eGFP levels were
unaltered in three independent At-miR393bp::eGFP–elicited
lines (Fig. 2B). These data
suggest that up-regulation of miR393 by
flg22 results from enhanced transcription of
At-miR393a.
To assay TIR1 protein levels after flg22
treatment, we used Arabidopsis transformants
expressing a Myc epitope–tagged TIR1 under
the dexamethasone (Dex)-inducible promoter
(Dex::TIR1-Myc) (12). Western blotting revealed
a rapid reduction in TIR1-Myc levels
upon flg22, but not flg22A.tum, treatment (Fig.
3A). Because TIR1 is part of the ubiquitinligase
complex SCFTIR1 that interacts with
Aux/IAA proteins to promote their degradation
(17), we investigated whether Aux/IAA
proteins became stabilized upon flg22 treatment.
We used transgenic lines expressing a
Fig. 2. Flg22 triggers miR393 induction mainly
through transcriptional activation of At-miR393a.
(A) Northern analysis of miR393 (left panels) and
miR171 (right panels) upon treatment with flg22
(upper panels) or flg22A.tum (bottom panels). Col-0
seedlings were treated with either 10 mM flg22 or
10 mM flg22A.tum. rRNA, ethidium bromide
staining of ribosomal RNA; R, miRNA signal ratio
between flg22-treated versus flg22A.tum-treated
samples at each time point. (B) Transcriptional
activation of At-miR393a in response to flg22. T2
transgenic lines were treated for 60 min with
either 10 mM flg22 (þ) or 10 mM flg22A.tum (j).
Bar graph representing the relative mRNA level of
eGFP upon treatment with flg22 (þ) versus
flg22A.tum (j) as assayed by qRT-PCR. Error bars
represent the standard deviation from three PCR
results, and similar results were obtained in two
independent experiments. At-miR393a-p::eGFP
and At-miR393b-p::eGFP represent transgenic lines
expressing miR393a and miR393b promoter-eGFP
fusions, respectively. The dashed line indicates the
twofold threshold induction observed in three
independent flg22-treated At-miR393a-p::eGFP
lines.
Fig. 1. Flg22 triggers a reduction
in TIR1 and AFB mRNAs
through miRNA-dependent and
miRNA-independent regulatory
mechanisms. (A) PCR-amplified
cleavage products from TIR1
and AFB mRNAs. Col-0 samples
are not shown (27). (B) Repression
of TIR1, AFB1, AFB2, and
AFB3 mRNAs in response to
flg22. Seedlings were treated for
30 min with either 10 mM flg22
(þ) or 10 mM flg22A.tum (j).
qRT-PCRs were done to assess the relative
mRNA levels of TIR1 (At3g62980), AFB3
(At1g12820), AFB2 (At3g26810), and AFB1
(At4g03190) upon treatment with flg22 or
flg22A.tum. Error bars represent the standard
deviation from four PCR results, and
similar results were obtained in three
independent experiments.
REPORTS
www.sciencemag.org SCIENCE VOL 312 21 APRIL 2006 437
heat shock–inducible AXR3/IAA17 protein
fused to the b-glucuronidase (GUS) reporter
(HS::AXR3NT-GUS) (17). Seedlings were
treated with either flg22 or flg22A.tum for 2
hours at 37-C and then stained for GUS.
Flg22, but not flg22A.tum, triggered stabilization
of AXR3NT-GUS (Fig. 3B), starting
from 1.5 hours after flg22 elicitation (Fig.
3C), which coincided with the TIR1-Myc
repression (Fig. 3A).
Aux/IAA proteins repress auxin signaling
through heterodimerization with Auxin Response
Factors (ARFs) (18). Those transcription
factors ARFs bind to auxin-responsive
elements (AuxREs) in promoters of primary
auxin-response genes and activate (or repress)
transcription (19). The flg22-induced stabilization
of AXR3/IAA17 prompted us to determine
whether flg22 inhibits auxin-response
gene activation. Seedlings were treated for
1.5 hours with either flg22 or flg22A.tum, and
transcripts of the primary auxin-response
genes GH3-like, BDL/IAA12, and AXR3/IAA17
were monitored by RT-qPCR. This time point
was chosen on the basis of the flg22-induced
stabilization profile of AXR3/IAA17 (Fig.
3C). All three auxin-response genes were
repressed at this time point (Fig. 3D). Thus,
flg22 triggers events that contribute to rapid
down-regulation of primary auxin-response
genes.
Does repression of auxin signaling enhance
bacterial disease resistance? We tested,
in a tir1-1 mutant background, resistance in
Arabidopsis transgenic lines that constitutively
overexpress Myc epitope–tagged AFB1
(20). These lines contain higher levels of
AFB1 mRNA as compared with tir1-1, and
exhibit high AFB1-Myc protein accumulation
(Fig. 4A). The partial resistance of AFB1
mRNA to miR393, together with its constitutive
overexpression, suggested that both
AFB1 mRNA and AFB1-Myc would remain
unaffected by flg22 treatment, which was
confirmed in semiquantitative RT-PCR and
Western analysis (Fig. 4, B and C). We anticipated
that constitutive overexpression of
AFB1 would prevent the miR393-mediated
suppression of auxin signaling, perhaps
resulting in enhanced disease sensitivity.
When AFB1-Myc–overexpressing plants
were inoculated with virulent P. syringae
pv. tomato (Pto) DC3000, they had bacterial
titers that were about 20-fold higher than
those of nontransformed or tir1-1 plants and
they displayed enhanced disease symptoms
(Fig. 4D, fig. S4A). Furthermore, no difference
was observed with avirulent Pto
DC3000 carrying AvrRpt2, which triggers
race-specific resistance via resistance gene
RPS2 (Fig. 4E) (21). Thus, suppression of
auxin signaling, mediated at least partly by
miR393, might specifically promote resistance
to virulent Pto DC3000 but is not
implicated in race-specific resistance.
We also transformed Arabidopsis with a
construct with At-miR393a constitutively
transcribed from the strong 35S promoter
(Fig. 4F). Three independent T2 transgenic
lines were selected for high miR393 accumulation
(Fig. 4F). These lines showed
lower TIR1 mRNA levels than controls
(Fig. 4F), and in older plants, they displayed
reduced apical dominance with multiple
shoots, reminiscent of auxin signaling mutants.
However, these developmental alterations
were minor compared with those
observed in tir1/afb multiple mutants (20).
Four days after inoculation with virulent Pto
DC3000, all three miR393-overexpressing
lines, but not the controls, displayed fivefold
lower bacterial titer, confirming that
miR393 restricts Pto DC3000 growth (Fig.
4G). There was no difference in bacterial
growth on transgenic lines overexpressing an
artificial miRNA directed against GFP (22)
(Fig. 4G).
We show here that a bacterial PAMP
down-regulates auxin signaling in Arabidopsis
by targeting auxin receptor transcripts.
Augmenting auxin signaling through overexpressing
a TIR1 paralog that is partially
refractory to miR393 enhances susceptibility
to virulent Pto DC3000, and, conversely,
repressing auxin signaling through miR393
overexpression increases bacterial resistance.
These results indicate that down-regulation of
auxin signaling, resulting in ARF inactivation,
is part of a plant-induced immune response
(fig. S5). They also suggest that auxin
promotes susceptibility to bacterial disease.
This is consistent with other published
findings. The tumorigenic P. syringae pv.
savastonoi synthesizes high levels of indole-
3-acetic acid (IAA) (23). Also, most P.
syringae strains can produce IAA, and Pto
DC3000 infection triggers increased IAA
levels in Arabidopsis (24, 25). Consistent with
these observations, exogenous application of
the auxin analog 2,4-dichlorophenoxyacetic
acid enhances Pto DC3000 disease symptoms
(fig. S4B).
In addition to the contribution of miR393
in flg22-triggered repression of auxin signaling,
our analysis also reveals a miR393Fig.
3. Flg22 triggers repression of
TIR1 protein, stabilization of AXR3/
IAA17, and repression of three
primary auxin-response transcripts.
(A) TIR1-Myc repression in response
to flg22. (Upper panel) Western
analysis using a Myc-specific antibody
(anti-Myc). (Bottom panel)
Ponceau staining of total proteins.
Similar results were obtained in two
independent experiments. (B)
AXR3/IAA17 stabilization in leaves.
HS::AXR3NT-GUS seedlings were
transferred for 2 hours at 37-C
with either 10 mM flg22 (right leaf)
or 10 mM flg22A.tum (left leaf).
Similar results were obtained in five
independent experiments. (C) Kinetics
of AXR3/IAA17 stabilization.
HS::AXR3NT-GUS seedlings were
treated as in (B). (Upper panel)
Western analysis using anti-GUS.
(Bottom panel) Ponceau staining
of total proteins. Similar results
were obtained in two independent
experiments. (D) Flg22 reduces accumulation
of three primary auxinresponse
genes. Col-0 seedlings
were treated for 1.5 hours with
either 10 mM flg22 (þ) or 10 mM
flg22A.tum (j). qRT-PCRs were done
to assess the relative mRNA level of
GH3-like (At4g03400), BDL/IAA12
(At1g04550), and AXR3/IAA17
(At1g04250) upon flg22 as compared
with flg22A.tum treatment.
Error bars represent the standard
deviation from four PCR results, and
similar results were obtained in
three independent experiments.
REPORTS
21 APRIL 2006 VOL 312 SCIENCE www.sciencemag.org438
independent pathway that probably involves
transcriptional repression of TIR1 and its
paralogs. This is consistent with Bmutual
exclusion[ in Drosophila, whereby miRNAs
prevent unwanted expression of genes that
become transcriptionally repressed in the
miRNA-producing cells (26). Rapid induction
of miR393 by flg22 might deplete
TIR1 mRNAs present at the time of elicitation
and thus confer robustness to the
transcriptional repression provoked during
elicitation. This regulatory feature has not
previously been reported for plant miRNAs
involved in development, and it will be
interesting to establish if these observations
on miR393 hold for other stress-induced
miRNAs.
References and Notes
1. G. Felix, J. D. Duran, S. Volko, T. Boller, Plant J. 18, 265
(1999).
2. L. Navarro et al., Plant Physiol. 135, 1113 (2004).
3. Z. Xie et al., PLoS Biol. 2, E104 (2004).
4. O. Borsani, J. Zhu, P. E. Verslues, R. Sunkar, J. K. Zhu, Cell
123, 1279 (2005).
5. Z. Xie, E. Allen, A. Wilken, J. C. Carrington, Proc. Natl.
Acad. Sci. U.S.A. 102, 12984 (2005).
6. V. Gasciolli, A. C. Mallory, D. P. Bartel, H. Vaucheret,
Curr. Biol. 15, 1494 (2005).
7. P. Dunoyer, C. Himber, O. Voinnet, Nat. Genet. 37, 1356
(2005).
8. D. P. Bartel, Cell 116, 281 (2004).
9. P. Dunoyer, C. H. Lecellier, E. A. Parizotto, C. Himber, O.
Voinnet, Plant Cell 16, 1235 (2004).
10. E. J. Chapman, A. I. Prokhnevsky, K. Gopinath, V. V. Dolja,
J. C. Carrington, Genes Dev. 18, 1179 (2004).
11. N. Dharmasiri, S. Dharmasiri, M. Estelle, Nature 435,
441 (2005).
12. W. M. Gray et al., Genes Dev. 13, 1678 (1999).
13. S. Kepinski, O. Leyser, Nature 435, 446 (2005).
14. M. W. Jones-Rhoades, D. P. Bartel, Mol. Cell 14, 787
(2004).
15. R. Sunkar, J. K. Zhu, Plant Cell 16, 2001 (2004).
16. A. M. Gustafson et al., Nucleic Acids Res. 33, D637
(2005).
17. W. M. Gray, S. Kepinski, D. Rouse, O. Leyser, M. Estelle,
Nature 414, 271 (2001).
18. E. Liscum, J. W. Reed, Plant Mol. Biol. 49, 387
(2002).
19. G. Hagen, T. Guilfoyle, Plant Mol. Biol. 49, 373 (2002).
20. N. Dharmasiri et al., Dev. Cell 9, 109 (2005).
21. M. C. Whalen, R. W. Innes, A. F. Bent, B. J. Staskawicz,
Plant Cell 3, 49 (1991).
22. E. A. Parizotto, P. Dunoyer, N. Rahm, C. Himber, O.
Voinnet, Genes Dev. 18, 2237 (2004).
23. T. Yamada et al., in Molecular Strategies of Pathogens and
Host Plants, D. L. Mills, C. Vance, S. Ouchi, S. S. Patil, Eds.
(Springer, New York, 1991), pp. 83–94.
24. E. Glickmann et al., Mol. Plant Microbe Interact. 11, 156
(1998).
25. P. J. O’Donnell et al., Plant J. 33, 245 (2003).
26. A. Stark, J. Brennecke, N. Bushati, R. B. Russell, S. M.
Cohen, Cell 123, 1133 (2005).
27. Materials and methods are available as supporting
material on Science Online.
28. We thank B. Kunkel and P. Brodersen for helpful
comments and L. Buhot for technical advice. Supported
by the Gatsby Foundation (J.D.G.J); a long-term Fellowship
from the Federation of European Biochemical
Societies and by the Gatsby Foundation (L.N.); an Action
The´matique Incitative sur Programme grant from the
CNRS and a grant from the trilateral ge´noplante–German
Plant Genome Research Program–Spanish ministry of
Research (P.D, F.J, and O.V); and by grants from NIH, NSF,
and the U.S. Department of Energy (N.D and M.E.)
Supporting Online Material
www.sciencemag.org/cgi/content/full/312/5772/436/DC1
Materials and Methods
SOM Text
Figs. S1 to S5
References
10 February 2006; accepted 23 March 2006
10.1126/science.1126088
Fig. 4. Down-regulation of auxin signaling is required for Pto
DC3000 disease resistance. (A) Molecular characterization of
AFB1-overexpressing lines. (Upper panel) RT-PCR analysis of
AFB1 transcript. (Bottom panel) Western analysis using antiMyc.
(B) AFB1 mRNA levels are not altered in AFB1overexpressing
lines treated with flg22. The 35S::AFB1-Myc
seedlings were treated with either 10 mM flg22 or 10 mM
flg22A.tum. RT-PCR analysis was done as in (A). (C) AFB1-Myc
protein levels are not altered in AFB1-overexpressing challenged
lines. The 35S::AFB1-Myc seedlings were treated as in
(B). (Upper panel) Western analysis using anti-Myc. (Bottom
panel) Ponceau staining of total proteins. (D) Growth of Pto
DC3000 on AFB1-overexpressing lines. Six-week-old plants
were inoculated with 105 colony-forming units (cfu/ml) of
bacteria. Error bars represent the standard error of logtransformed
data from five independent samples, and similar
results were obtained in three independent experiments. (E)
Growth of Pto DC3000 (AvrRpt2) on AFB1-overexpressing lines.
Inoculation was performed as in (D), and results are presented
as in (D). Similar results were obtained in two independent
experiments. (F) Molecular characterization of miR393-overexpressing
lines. (Top panel) Schematic representation of the
35S::At-miR393a construct. (G) Growth of Pto DC3000 on
miR393-overexpressing lines. Inoculation was performed as in
(D). Similar results were obtained in two independent
experiments. (Middle panel) Northern analysis of miR393
overexpression in independent T2 transgenic lines; EV, empty
vector; rRNA, ethidium bromide staining of ribosomal RNA.
(Bottom panel) RT-PCR of the TIR1 transcript.
REPORTS
www.sciencemag.org SCIENCE VOL 312 21 APRIL 2006 439