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Proc Natl Acad Sci U S A. May 12, 2009; 106(19): 8067–8072.
Published online Apr 29, 2009. doi:  10.1073/pnas.0810206106
PMCID: PMC2683104
Plant Biology

Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling


Mitogen-activated protein kinase (MAPK)–mediated responses are in part regulated by the repertoire of MAPK substrates, which is still poorly elucidated in plants. Here, the in vivo enzyme–substrate interaction of the Arabidopsis thaliana MAP kinase, MPK6, with an ethylene response factor (ERF104) is shown by fluorescence resonance energy transfer. The interaction was rapidly lost in response to flagellin-derived flg22 peptide. This complex disruption requires not only MPK6 activity, which also affects ERF104 stability via phosphorylation, but also ethylene signaling. The latter points to a novel role of ethylene in substrate release, presumably allowing the liberated ERF104 to access target genes. Microarray data show enrichment of GCC motifs in the promoters of ERF104–up-regulated genes, many of which are stress related. ERF104 is a vital regulator of basal immunity, as altered expression in both erf104 and overexpressors led to more growth inhibition by flg22 and enhanced susceptibility to a non-adapted bacterial pathogen.

Keywords: defense, elicitor, FRET, signal transduction

Mitogen-activated protein kinase (MAPK) cascades transduce external signals into cellular responses in eukaryotes (1). In plants, MAPKs orthologous to the Arabidopsis MPK3, MPK4, and MPK6 are activated by various stimuli including flg22, a bacterial flagellin–derived peptide that acts as a pathogen-associated molecular pattern (PAMP) (25). These three MAPKs control defense positively (MPK3/MPK6) (3, 6) or negatively (MPK4) (7).

Many phytohormones have been shown to affect defense responses; but most progress has been made in regard to salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) (8). The tobacco MPK6 ortholog is activated by SA (9) and the Arabidopsis mpk4 mutant has elevated SA levels and enhanced pathogen resistance (7). Genetic evidence linking ET to MAPK signaling is also suggested by the negative regulator of the ET response, Constitutive Triple Response 1 (CTR1), a Raf-like kinase that was recently shown to control MPK3/6 activation via MKK9 (MAPK kinase 9) (10). Both JA and the ET precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), activate MPK6 in Arabidopsis (11, 12) but not in tobacco (13). Although responses may differ between plant species, the activation of MPK6 by ET/ACC is highly debated (14). In another report, ACC did not activate MPK6, but ET biosynthesis was positively regulated by MPK6 through posttranslational stabilization of the rate-limiting ACC synthase (ACS) isoforms, ACS2 and ACS6 (14, 15).

In addition to the cytoplasmic ACSs, MAPKs also target nuclear proteins (10, 16, 17); this may occur either after MAPK nuclear translocation following activation (18, 19) or as preformed nuclear protein complexes (20). The latter would imply movement of the upstream MKKs into the nucleus to modify the MAPKs or, alternatively, that the activated MAPKs enter the nucleus to displace the inactive MAPK from preformed complexes. Examples of nuclear targets include the MPK4 substrates, MKS1 and two MKS1-interactors of the WRKY transcription factor family, WRKY25 and WRKY33 (17). A MPK4/MKS1/WRKY33 complex is thought to control camalexin biosynthesis via the action of WRKY33 (20). MKS1 acts downstream of MPK4 to regulate the SA-dependent pathway but is not involved in the ET/JA pathway. However, as mpk4 mutants are affected in JA/ET-mediated defense gene expression, additional unknown MPK4 substrates must be involved. Thus, the understanding of defense regulation via MAPKs is limited by the current knowledge of plant MAPK substrates. Using a yeast-2-hybrid screen, we identified a transcription factor of the Ethylene Response Factor family, ERF104, which interacted with MPK6. We validated this interaction in vivo and showed ERF104 to be a nuclear substrate involved in plant defense. The release of ERF104 from MPK6 in the nucleus required rapid ET signaling, which could indicate novel roles of hormone signaling in mediating substrate release.


MPK6 and ERF104 Show Dynamic Interaction in Fluorescence Resonance Energy Transfer (FRET) Assays.

The Ethylene Response Factor, ERF104 (At5g61600), interacted with MPK6 in a yeast-2-hybrid (Y2H) screen. A further hint of bona fide interaction is that MPK6-YFP, normally of nucleocytoplasmic distribution, became nuclear-localized when cotransfected with the nuclear-localized ERF104-CFP (Fig. 1A). These nuclear signals are not caused by cleavage products of fluorescent proteins, as Western blot showed intact fusion proteins [supporting information (SI) Fig. S1A]. The tagged proteins can also be activated by flg22 and hence are functional (Fig. S1B). Acceptor photobleaching-based FRET (21), initially tested with various positive and negative controls (Fig. 1, Fig. S1C), was used to validate the interaction. Positive FRET indicating in vivo protein–protein interaction occurred between ERF104 and MPK6 but not with MPK3 or MPK4 (Fig. 1B).

Fig. 1.
In vivo protein-protein interaction based on FRET analysis. (A) Localization of the indicated tagged proteins in transfected Arabidopsis protoplasts. Scale bar, 5 μm. (B) FRET analysis of the indicated components. A CFP-YFP fusion (positive control) ...

Loss of the FRET signal upon flg22 elicitation suggests MPK6/ERF104 complex disruption within 5–15 minutes (Fig. 1B), which could be validated to some extent by co-immunoprecipitation (Fig. S1D). By contrast, flg22 did not abrogate interaction between MPK6 and its upstream kinase MKK4, indicating that the flg22 effect is no FRET artifact, although not ruling out the existence of MKK4/MPK6 complexes in non-flg22-regulated pathways. No flg22-mediated disruption was seen by co-treatment with an excess of flg22 antagonist, flg15d5 (22) (Fig. 1C) or in fls2-derived protoplasts (Fig. S1E). Hence, flg22 perception via the FLS2 receptor is needed. In addition, MPK6 activity is required for the flg22-mediated disruption, as inactive MPK6 variants (mutations in ATP binding pocket, MPK6KR, or the activation loop, MPK6AEF) interacted with ERF104 despite flg22 treatment (Fig. 1D).

ET Signaling Affects ERF104/MPK6 Interaction.

The ET precursor, ACC, had no effect on the positive control (Fig. S1F) but reduced the ERF104/MPK6 FRET signal (Fig. 2A). The ACC effect is lost in ET-insensitive mutants (Fig. 2B) and hence is caused by ET signaling. To determine the role of ET signaling, protoplasts were pretreated with aminoethoxyvinylglycine (AVG, an ACS inhibitor) or silver ions (ET perception inhibitor) for 10 minutes before flg22 stimulation. Both treatments blocked the flg22-induced loss of FRET (Fig. 2A). Flg22 disruption of the ERF104/MPK6 complex was abrogated in strong ET-insensitive mutants (ein2 or ein3/eil1) (Fig. 2B) but not in weaker mutants (ein3 or etr1, Fig. S2A). Hence, the disruption of MPK6/ERF104 interaction by flg22 requires ET biosynthesis and signaling.

Fig. 2.
Role of ET and MPK6 activity in the ERF104/MPK6 interaction. (A) To study the role of ET signaling in ET-insensitive mutants required the use of plant-derived protoplast. FRET was first tested in protoplasts of wild-type Arabidopsis to show that leaf- ...

The requirement of MPK6 activity for ERF104 release (Fig. 1D) may suggest a “simple” enzyme-substrate relationship or that the inactive MPK6 interfered dominant-negatively with ET biosynthesis induced by flg22 (12). The latter can be excluded because ACC did not disrupt the MPK6KR/ERF104 complex (Fig. 2C). Alternatively, the inactive MPK6 may act as a “substrate trap” for ERF104 (but unfortunately unspecific binding of MPK6 to the matrices used deterred binding affinity measurements by Biacore). Taken together, both kinase activity and ET signaling are required for complex disruption. Although flg22-induced ET production in tomato and Arabidopsis is known (14, 23), our data suggest ET signaling within minutes after flg22 addition, which is as quick as that of MAPK activation and raises the possibility of ET signaling being upstream of MAPK activation.

Is ET Upstream or Downstream of MAPK Activation?

There are conflicting viewpoints in regard to this issue (10, 12, 14, 24, 25), wherein ACC activation of MAPKs including MPK6 (10, 12) is disputed (14). We re-evaluated the ability of ACC to activate MAPK but no rapid MPK6 activation, based on the sensitive immune-complex kinase assay, was detected (not shown). Enhanced MPK6 activity in the ctr1 mutant (10, 12) was also not seen (Fig. 2D), which may be caused by different experimental procedures such as the use of transfected ctr1 protoplasts (10) compared with intact unstressed seedlings in our system. Reduction of ET production by preincubation with AVG before flg22 stimulation had no or only marginal effects on MPK6 activation (Fig. S2B). Although minor delay/reduction of MPK6 activation by flg22 is seen in the various ET signaling mutants (Fig. 2D), this varied between experiments and, more importantly, it is not completely blocked. Thus, our data do not support ET being upstream of MAPK6 activation, but in agreement with Liu and Zhang (14), imply that the rapid flg22-induced ET signal lies downstream of MPK6 activation.

ERF104 Is an MPK6 Substrate.

ERF104 possesses two potential MAPK phosphorylation sites (26) (Fig. 3D). When ERF104-HA was co-expressed with a constitutive active (CA) MKK5 (19) (which activated MPK3 and MPK6 but not MPK4, Fig. 3A), an additional ERF104-HA band of reduced mobility appeared. This was not seen with the corresponding inactive MKK5-LF or when performed in mpk6 background (Fig. 3B). The inclusion of a protein phosphatase (At2g40180), as a third transfection component, eliminated this upper band. In “Phos-tag” gels, a method for retarding mobility of phosphoproteins in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (27), the mobility shift was enhanced (Fig. 3B). Moreover, MPK6, but not MPK3 or MPK4, immunoprecipitated from flg22-treated cells, accepted recombinant GST-ERF104 as substrate (Fig. 3C). Hence, ERF104 is specifically phosphorylated by MPK6.

Fig. 3.
MPK6 phosphorylates ERF104. (A) MBP kinase assays with immunoprecipitated MAPKs showing that transfection with a “constitutive-active, CA,” but not an inactive “loss-of-function, LF,” MKK5 led to the activation of MPK3/MPK6 ...

Two synthetic peptides spanning the putative phosphorylation sites (26) were tested as substrates (Fig. 3D). Pep2 was marginally, whereas pep1 was strongly, phosphorylated (Fig. 3E). When both serines within the (S/T)P motifs in pep1 were exchanged by alanine, no phosphorylation was seen; if only one was mutated, the first motif appeared to be the major phospho-site (Fig. 3 D and E). When the two corresponding phosphorylation sites (encompassed by pep1) were altered in the full-length ERF104, the mutated ERF104 (ERF104m) was no longer phosphorylated by MPK6 (Fig. 3F). Although this suggests that one or both of these two sites is/are the major phospho-sites, the issue is complicated by the fact that no FRET was seen with ERF104m and MPK6 (Fig. S2D). However, MPK6-YFP is still remobilized to the nucleus when cotransfected with ERF104m (Fig. S2E), suggesting that interaction may still occur but is not detectable, perhaps because conditions required for FRET (e.g., transition dipole orientation for optimal energy transfer) are not met.

Mutated ERF104 Is a Functional Transcription Factor but Less Stable.

Treatment of plants with cycloheximide to block protein synthesis revealed reduced stability of ERF104m compared with ERF104, especially after flg22 treatment (Fig. 4A). To appraise the effect of the mutations on the protein function, we first determined the ERF104 activity. It binds GCC-containing DNA probes in electrophoretic mobility shift assays (EMSA) with high specificity, as is shown by competition with an excess of unlabeled DNA, the failure of an unrelated WRKY binding element to compete this binding and the lack of binding to a mutated GCC element (Fig. S3A), or to S, DRE, G, JERE, and WRKY elements often found in promoters of various pathogen- and stress-responsive genes (28) (data not shown). In particular, the single nucleotide difference between S and GCC elements (Fig. 4B) highlights this specificity.

Fig. 4.
ERF104 is a transcriptional activator, and “phospho-site” mutation reduces its stability. (A) ERF104m is less stable than ERF104. Transgenic plants of Strep-tagged ERF104 variants were treated with cycloheximide (to block protein synthesis) ...

ERF104 was transiently expressed in protoplasts, which raised the activity of co-transfected synthetic promoter with GCC elements but not those with mutated GCC or the closely related S-Box element (Fig. 4B). Similarly, stably transformed 35S::ERF104 (ERF104OE) plants show GUS expression in reporter lines with promoters containing GCC, but not other elements (28) (Fig. 4C). A weak GUS staining in plants with the JERE-promoter element is likely an indirect effect as ERF104 did not bind JERE elements. Thus, ERF104 activates promoters with GCC cis-acting elements.

In contrast to native ERF104, promoter activity is highly variable after transient expression of the mutated ERF104m and also slightly more variable in stable transgenic 35S::ERF104m (ERF104mOE) plants (Fig. S3B and C). This higher variation seen in protoplasts may be accounted by stress-induced instability of ERF104m (Fig. 4A). In summary, the transcription factor function in ERF104m is intact, but its stability is reduced upon stress (flg22) treatment. In line with this, the necrotic-like specks in older leaves, which are stainable by trypan blue (dead cells) and DAB (H2O2), in the ERF104OE lines were also seen in ERF104mOE lines but less frequently (Fig. S3D) and several tested ERF104-up-regulated genes were similarly expressed in the ERF104m lines (not shown).

Expression Profiling to Find Putative ERF104 Targets.

To dissect the role of the MPK6/ERF104 protein complex in flg22 signaling, we profiled gene expression in mpk6 and erf104 mutants by microarray analysis. Most flg22-responsive genes were similarly regulated (Fig. S4D) and there was no clear trend of specific pathways being affected, suggesting that redundancies likely mask most of the effects in single mutants. In contrast, ERF104OE plants showed 534 up- and 17 down-regulated genes (at least 3-fold changes; False Discovery Rate, FDR, P < 0.05) (Table S1), with the strongest induction (≈1000 fold) for two PDF1.2 genes (Table 1, Fig. S4C). The 1-kb upstream regions of genes up-regulated >10-fold by ERF104 are enriched for GCC elements (based on “Motiffinder”), suggesting these to be direct targets of ERF104. Indeed, chromatin immunoprecipitation (ChIP) showed occupation of the PDF1.2 promoter by ERF104 (Fig. 5A). When compared with other microarray data for signals that also target GCC elements, only a partial overlap with ERF1–up-regulated (29) or JA- or ET-responsive genes was found (Fig. S4A, Table S6), implying that pathways controlled by ERF104, ERF1, ET, or JA are not identical.

Table 1.
Selected genes upregulated in ERF104OE plants
Fig. 5.
Effects of modulating ERF104 expression. (A) Chromatin immunoprecipitation (ChIP) shows higher levels of the PDF1.2 promoter in immunoprecipitates of Strep-tagged ERF104 from ERF104OE compared with Col-0 plants. (B) Botrytis cinerea disease progression, ...

Many of the ERF104–up-regulated genes are pathogenesis related or may be involved in further signal amplification of defense signaling, such as MKK4, RBOHD, ERF4, WRKY33, TGA1.3 (Table 1). Functional grouping revealed a high percentage (20%) of stress-responsive genes in the up-regulated genes, which represent only ≈8% in the repressed genes set, as is normally seen at the global genome level. Reciprocally, genes involved in signal transduction (& transcription regulation), normally of 4–5%, cover ≈20% among repressed genes (Fig. S4B). Thus, ERF104 targets several stress-related genes either directly and indirectly to coordinate stress responses.

Responses Affected in Plants with Altered ERF104 Expression.

No difference in the ET-induced triple response was seen in the ERF104OE plants (not shown). Although stronger symptoms developed, there was no enhanced bacterial growth after virulent Pseudomonas syringae infection (Fig. S5A). ERF overexpression often increases resistance to necrotrophic fungi (2931). However, both disease symptom development and biomass quantification of Botrytis cinerea in the ERF104OE plants showed a tendency toward greater susceptibility. Surprisingly, the erf104 mutant showed the same trend (Fig. 5B).

To assess basal resistance, the non-adapted bacterial pathogen, P. syringae pv. phaseolicola (Psp) 1448A, which normally infects beans but not Arabidopsis, was tested. The erf104 mutant and an RNAi line showed enhanced symptom development and more bacterial growth (Fig. 5C). The ERF104OE plants also had higher Psp growth and a soaked lesion phenotype that resembles that seen during compatible interactions. Similarly, root growth inhibition by flg22 is enhanced in both the erf104 mutant and ERF104OE/ERF104mOE plants (Fig. 5D). These results suggest that (i) the PAMP response that mediates resistance to non-adapted bacteria may be coordinated through flg22-mediated MPK6 activation and downstream components such as ERF104, and (ii) ERF104 is likely a vital component, as altering ERF104 expression in either direction changes the response.


Mis-expression of key signal components often has severe phenotypic effects, and thus they are typically under tight control. Accordingly, ERF104 levels are regulated by mRNA stability (32) and phosphorylation-regulated protein stability (Figs. 4A and and6).6). ERF104 is exclusively phosphorylated by MPK6 but not MPK3 or MPK4 (Fig. 3) and, in analogy to the unique yeast MAPK substrates in mating/starvation pathways (33), may confer signal specificity. Thus, despite apparent functional redundancies, MPK3 and MPK6 must have separate non-redundant signaling roles.

Fig. 6.
Model of flg22 effect on MPK6/ERF104 interaction. The ERF104/MPK6 complex disruption requires flg22 stimulation of MPK6 activity (1) that also positively affects ERF104 stability, as well as ET signaling (2). The rapid ET signal may be upstream (A) or ...

Overexpression of ERF104 did not enhance disease resistance. In fact, both erf104 and ERF104OE showed reduced immunity against B. cinerea and the non-adapted Psp and enhanced the growth inhibition response to flg22 (Fig. 5 B–D). These results are difficult to explain but can only mean that ERF104 must be maintained at an optimal level and any alterations of this crucial threshold can tip the “signaling balance.” For instance, it is possible that the ERF104OE may sequestrate some ERF104-interactors into the nucleus (cf. MPK6, Fig. 1). In support of this, the root growth inhibition by flg22 (Fig. 5D) and the Psp response of the ERF104OE (Fig. S5B) are reversed in the mpk6 background, and thus dependent on MPK6. Taken together, the data are in agreement with a signal cascade involving flg22-mediated activation of MPK6 and downstream targets such as ERF104 to control defense responses.

An important finding in this report is the rapid disruption of ERF104/MPK6 complex, which implied that the flg22-triggered ET biosynthesis is faster (i.e., minutes, not hours) than previously reported (14). The discrepancy may lie in lower sensitivities of the “headspace capture” ET measurement method or in the differential accessibility of flg22 to protoplasts and leaf tissue. The speed of ET production may hint at it being upstream, where MPK6 is activated by ACC (12), but our data are compatible with it being downstream of MPK6 (see model, Fig. 6). Recently, ACC (200 μM for 1 hour) was shown to activate MPK6 in detached leaves, and a model is proposed wherein bifurcate pathways downstream of CTR1 antagonistically control EIN3 stability via two different phosphorylation sites (10) (Fig. 6). One of these pathways is thought to contain an MKK9 complex with MPK3/6. It should be noted that direct proof of a complex was not shown but deduced from the ability of MKK9 to activate MPK3/6 and that MPK3/6 activities are higher in a ctr1 background, which can be suppressed by reintroducing active CTR1 (albeit a truncated CTR1). Another recent work showed that constitutively active MKK9 led to enhanced ET levels (25), which may explain some of the findings in (10). The conflicting data of the two groups on whether downstream responses are reduced by blocking ET signaling (e.g., by Ag+ and AVG treatment) would need to be clarified (24). Nevertheless, this proposed CTR1/MKK9/MPKs pathway cannot account for the quick reaction we observe and accordingly, the ERF104/MPK6 complex disruption still occurred in mkk9-derived protoplasts (not shown).

Our current model is that the flg22 signal network includes one pathway for MPK6 to target ERF104 directly through phosphorylation and on a separate branch, to stimulate ET production, which triggers a yet unknown mechanism (that is dependent on EIN2 and the EIN3/EIL members) for the release of MPK6 from ERF104 in the nucleus (Fig. 6). It is conceivable that the continued binding of MPK6 to ERF104 might constrain physical interactions with subsequent ERF104 targets and impinge on its role in transcription activation. Along this line, Qiu et al. showed that flg22 and pathogen treatment caused the release of the MKS1/WRKY33 complex from MPK4; thus allowing WRKY33 to evoke camalexin production by targeting the PAD3 promoter (20). However, unlike our data, the release of MKS1/WRKY33 is independent of substrate (MKS1) phosphorylation and the timing of the complex disruption was not provided, so that it is not clear whether it is as rapid as reported here. Moreover, additional factors are probably involved, because flg22 alone does not induce camalexin production and “altered PAD3 expression in mks1 is insufficient to affect camalexin production” (20). WRKY33 expression is enhanced in ERF104OE (Table 1), so that components downstream of MPK6 signaling may feed into the MPK4 pathway, thus linking the two opposing branches of flg22-regulated MAPK pathways that control defense responses.

In summary, transcription factor release may be a common theme after MAPK activation to control downstream gene expression. We further showed that ET signaling is required to mediate such substrate release, and it is tempting to speculate that other phytohormones may similarly control protein–protein interactions to coordinate downstream responses.

Materials and Methods

Additional methods are given in the SI Text.

FRET Analysis.

Protoplast isolation and transfection were performed as described in (3, 19). FRET measurements were done with YFP/CFP fusion proteins, using a LSM 510 Meta confocal microscope (Zeiss) in the channel mode (setup: CFP filter, 473.3–505.4 nm; YFP emission filter, 516.1–548.2 nm). The acceptor photo-bleaching method (21) was used. For every analysis, 12 or more protoplasts (i.e., the minimal number for performing Kolmogorov-Smirnov test; see below) were analyzed and scanned at 458 nm twice before and twice after bleaching of the YFP (full laser power, 514 nm, 80 times). The relative fluorescence intensity (I) in a certain region of interest (ROI) was measured using the ROI mean function of the Zeiss software. FRET efficiency (EF) was then calculated with the following equation:

equation image

The following statistical analysis was performed to evaluate the considerable variation in EF within each data series. First, Gaussian distribution of the data points was established using the Kolmogorov-Smirnov test, followed by removal of outliers via Grubb's test (GraphPad Prism software package), followed by a Mann-Whitney significance analysis (P < 0.05) against both positive and negative controls.

Protein Work.

Protein extraction, immunoprecipitation (α-MAPK or α-GFP [BD Living Colors]), in vitro phosphorylation reactions with the indicated substrates were performed as described (18). ERF104 stability was tested after incubating leaves with 100 μM cycloheximide (±10 μM flg22), and visualized with α-Strep-Tactin HRP conjugate (IBA-BioTAGnology, Germany) in Western blots.

Microarray Hybridization and Analysis.

Six-week old Col-0, erf104, mpk6 and ERF104OE plants were infiltrated with 1 μM flg22 or H20 and harvested 4 hours later. Total RNA was processed according to the Affymetrix protocol for biotin-labeled cRNA and hybridized to the Affymetrix ATH1 chip. The data were analyzed with Genespring GX 7.3.1 software (Agilent) with the following parameters: Filter on Flags for present or marginal in 50% of all considered experiments, Filter for reliable differentially expressed genes based on volcano plot (P <0.05 and >3-fold change in expression). Moreover, a Benjamini-Hochberg false discovery rate (FDR) of 5% was implemented. For the flg22 experiments, analysis for each genotype was separately performed and a composite list of flg22-regulated genes compiled, with the aim of including genes that may be differentially regulated in the genotype. Global expression profile was visualized by k-means clustering and condition tree (Genespring). Microarray data for JA and ET were obtained from public databases (GARNET, Genevestigator).

Supplementary Material

Supporting Information:


We thank Simone Altmann, Joseph Ecker, Bettina Hause, Kai Naumann, Thorsten Nürnberger, Ralph Panstruga, Stefan Posch, Sabine Rosahl, Ralf Reski, Jen Sheen, Imre Somssich, and Bernhard Westermann for sharing advice, materials, and protocols. This work was financed by the German Research Foundation within the SFB648 program.


The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The microarray CEL files in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE11807).

This article contains supporting information online at www.pnas.org/cgi/content/full/0810206106/DCSupplemental.


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