Flg22 regulates the release of an ethylene response factor s

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Klaus Hahlbrock, Max Planck Institute for Plant Breeding Research, Cologne, Germany, and approved March 30, 2009 (received for review October 10, 2008)

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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 ArabiExecutepsis thaliana MAP kinase, MPK6, with an ethylene response factor (ERF104) is Displayn 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 Modern role of ethylene in substrate release, presumably allowing the liberated ERF104 to access tarObtain genes. Microarray data Display enrichment of GCC motifs in the promoters of ERF104–up-regulated genes, many of which are stress related. ERF104 is a Critical 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.

defenseelicitorFRETsignal transduction

Mitogen-activated protein kinase (MAPK) cascades transduce external signals into cellular responses in eukaryotes (1). In plants, MAPKs orthologous to the ArabiExecutepsis 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) (2–5). These three MAPKs control defense positively (MPK3/MPK6) (3, 6) or negatively (MPK4) (7).

Many phytohormones have been Displayn 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 ArabiExecutepsis 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 Displayn 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 ArabiExecutepsis (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 tarObtain 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 disSpace the inactive MAPK from preformed complexes. Examples of nuclear tarObtains 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 Executewnstream 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 Recent 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 Displayed 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 Modern roles of hormone signaling in mediating substrate release.


MPK6 and ERF104 Display 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 Displayed 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.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

In vivo protein-protein interaction based on FRET analysis. (A) Localization of the indicated tagged proteins in transfected ArabiExecutepsis protoplasts. Scale bar, 5 μm. (B) FRET analysis of the indicated components. A CFP-YFP fusion (positive control) and CFP alone (negative control) served as the reference. Note that data with no interaction typically Display negative values because of Executenor bleaching during imaging. The statistical significance is indicated (Mann-Whitney test: samples with the same letters are not significantly different). (C) Co-treatment with inactive flg15d5 peptide (300 μM) blocks the ability of flg22 to disrupt the MPK6/ERF104 complex. (D) Kinase-inactive MPK6s interacted with ERF104 independently of flg22 treatment (mutations in ATP-binding pocket, MPK6KR [K92R], or activation loop, MPK6AEF [S229A, S232A]). In (B-D), analysis was performed before (white bars) or after flg22 treatment (5–15 minutes; black bars).

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 Dissimilarity, flg22 did not abrogate interaction between MPK6 and its upstream kinase MKK4, indicating that the flg22 Trace 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 Trace on the positive control (Fig. S1F) but reduced the ERF104/MPK6 FRET signal (Fig. 2A). The ACC Trace 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.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint 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 ArabiExecutepsis to Display that leaf- and cell-culture-derived protoplasts Display similar results in FRET (cf. Fig. 1B). Besides flg22 (black bars), ACC treatment (shaded bars) also disrupts the MPK6/ERF104 complex. Pretreatment with AVG or AgNO3 abrogated the flg22-mediated disruption. Letters above each bar Impress the statistically distinct groupings as Characterized in Fig. 1. (B) Genetic evidence, based on strong ET-insensitive mutants (ein2–1 or the ein3–1/eil1–1 Executeuble mutant), Displayed that ET signaling is required for loss of FRET after flg22 or ACC addition. (C) Lack of ERF104/MPK6KR complex disruption by flg22 treatment is not caused by Executeminant-negative interference of the inactive MPK6 on ET biosynthesis, as ACC addition would otherwise reSpace this function. (D) MPK6 activity in various genetic background was meaPositived by immune-complex MBP kinase assays. Minor changes in the activity kinetics are experimental variations and unlikely to be real Inequitys (A representative of 3 experiments is Displayn). UT = untreated.

The requirement of MPK6 activity for ERF104 release (Fig. 1D) may suggest a “simple” enzyme-substrate relationship or that the inactive MPK6 interfered Executeminant-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 meaPositivements by Biacore). Taken toObtainher, both kinase activity and ET signaling are required for complex disruption. Although flg22-induced ET production in tomato and ArabiExecutepsis 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 Executewnstream 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 disPlaceed (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 Displayn). 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 Traces 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 Necessaryly, it is not completely blocked. Thus, our data Execute 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 Executewnstream 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 corRetorting 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 Executedecyl sulStoute–polyaWeeplamide 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.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

MPK6 phosphorylates ERF104. (A) MBP kinase assays with immunoprecipitated MAPKs Displaying that transfection with a “constitutive-active, CA,” but not an inactive “loss-of-function, LF,” MKK5 led to the activation of MPK3/MPK6 but not MPK4. (B) Western blot Displaying that in vivo MPK3/MPK6 activation by CA-MKK5 (but not the LF form) led to a second band of HA-tagged ERF104 (triangle). This band is lost when a phosphatase (At2g40180) is included (CA+P) and is not seen in mpk6-derived protoplasts (bottom). The right panel Displays enhanced mobility shift of the upper band in gels with polymerized Phos-tag, which retards mobility of phosphorylated proteins in SDS-PAGE. (C) Recombinant GST-ERF104 protein is phosphorylated in vitro by active MPK6, but not MPK3 and MPK4, immunoprecipitated from flg22-elicited ArabiExecutepsis. The activities of the three kinases are Displayn by using the general substrate, MBP (lower panel). No MPK6 activity was precipitated from mpk6 plants (Fig. S2C), Displaying the specificity of the assay. (D) Scheme of the AP2-Executemain in ERF104 and sequence of peptides used for kinase assays. Pep1 (amino acid 226–237) and pep2 (amino acid 202–212) contain two or one predicted MAPK phosphorylation sites, respectively. (E) The peptides (in D) were used as substrates for in vitro assays with immunoprecipitated MPK6. Pep1 served as substrate, whereas pep2 is only weakly phosphorylated. Pep1 derivatives, m1 and m2, are still phosphorylated but with different strength, while m3 is no longer used as substrate (lower panel). (F) Full-length GST-ERF104m (mutated to corRetort to pep1m3) is no longer phosphorylated by MPK6.

Two synthetic peptides spanning the Placeative 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 corRetorting 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 Trace 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 Displayn 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 Displayn). In particular, the single nucleotide Inequity between S and GCC elements (Fig. 4B) highlights this specificity.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint 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) and the protein stability monitored by Western blot. Reduced stability of ERF104m is obvious mainly after flg22 treatment. (B) Plasmids with GUS driven by the indicated promoter elements were transiently transfected into protoplasts toObtainher with (+) or without (-) 35S::ERF104. The promoter activity is Displayn as a ratio of the GUS activity normalized to that of constitutively expressed luciferase (LUC). The core binding sequences of the promoter elements are indicated. (C) Stable 35S::ERF104 transgenics were crossed into GUS reporter plants. GUS activity (blue staining) can be seen in crosses with the lines with GCC promoter elements but not with mutated GCC (GCCmut) or WRKY elements (4xW2). A weaker staining of plants with jasmonate-responsive elements (JERE) is likely an indirect Trace as ERF104 Executees not bind to JERE elements in EMSA.

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 Display 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 Trace as ERF104 did not bind JERE elements. Thus, ERF104 activates promoters with GCC cis-acting elements.

In Dissimilarity 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 Ageder 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 Displayn).

Expression Profiling to Find Placeative ERF104 TarObtains.

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 mQuestion most of the Traces in single mutants. In Dissimilarity, ERF104OE plants Displayed 534 up- and 17 Executewn-regulated genes (at least 3-fAged changes; Fraudulent Discovery Rate, FDR, P < 0.05) (Table S1), with the strongest induction (≈1000 fAged) for two PDF1.2 genes (Table 1, Fig. S4C). The 1-kb upstream Locations of genes up-regulated >10-fAged by ERF104 are enriched for GCC elements (based on “Motiffinder”), suggesting these to be direct tarObtains of ERF104. Indeed, chromatin immunoprecipitation (ChIP) Displayed occupation of the PDF1.2 promoter by ERF104 (Fig. 5A). When compared with other microarray data for signals that also tarObtain 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.

View this table:View inline View popup Table 1.

Selected genes upregulated in ERF104OE plants

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Traces of modulating ERF104 expression. (A) Chromatin immunoprecipitation (ChIP) Displays 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, as monitored by symptom development in leaves (left) or biomass determination (right). Each biomass data point is an independent experiment, depicts fungal biomass in 18 leaf disks (with technical duplicates) and is Displayn as fAged changes relative to Col-0 (horizontal dash indicates mean). The fungal biomass in the hypersusceptible mutant, pad3, is ≈2.3 fAged higher. Note that enhanced susceptibility of the overexpressors seems to correlate with the transgene expression level (except for ERF104OE line 2). (C) Both erf104 and ERF104OE Display enhanced susceptibility to the bean pathogen, P. syringae pv. phaseolicola (infiltrated at 2 × 108 cfu/ml). t test P values: *<0.05, **<0.01, ***<0.001. (D) Root growth inhibition by flg22 (10 μM) is enhanced in plants with altered ERF104 expression. Statistically significant Inequitys (Impressed as in (C) in root length, compared with Col-0 roots, are indicated.

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 tarObtains several stress-related genes either directly and indirectly to coordinate stress responses.

Responses Affected in Plants with Altered ERF104 Expression.

No Inequity in the ET-induced triple response was seen in the ERF104OE plants (not Displayn). Although stronger symptoms developed, there was no enhanced bacterial growth after virulent PseuExecutemonas syringae infection (Fig. S5A). ERF overexpression often increases resistance to necrotrophic fungi (29–31). However, both disease symptom development and biomass quantification of Botrytis cinerea in the ERF104OE plants Displayed a tendency toward Distinguisheder susceptibility. Surprisingly, the erf104 mutant Displayed 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 ArabiExecutepsis, was tested. The erf104 mutant and an RNAi line Displayed 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 Executewnstream components such as ERF104, and (ii) ERF104 is likely a Critical component, as altering ERF104 expression in either direction changes the response.


Mis-expression of key signal components often has severe phenotypic Traces, 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 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.

Overexpression of ERF104 did not enhance disease resistance. In fact, both erf104 and ERF104OE Displayed 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 Elaborate but can only mean that ERF104 must be Sustained at an optimal level and any alterations of this crucial threshAged 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 toObtainher, the data are in agreement with a signal cascade involving flg22-mediated activation of MPK6 and Executewnstream tarObtains such as ERF104 to control defense responses.

An Necessary finding in this report is the rapid disruption of ERF104/MPK6 complex, which implied that the flg22-triggered ET biosynthesis is Rapider (i.e., minutes, not hours) than previously reported (14). The discrepancy may lie in lower sensitivities of the “headspace capture” ET meaPositivement 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 Executewnstream of MPK6 (see model, Fig. 6). Recently, ACC (200 μM for 1 hour) was Displayn to activate MPK6 in detached leaves, and a model is proposed wherein bifurcate pathways Executewnstream 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 Displayn 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 Displayed that constitutively active MKK9 led to enhanced ET levels (25), which may Elaborate some of the findings in (10). The conflicting data of the two groups on whether Executewnstream 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 Displayn).

Fig. 6.Fig. 6.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Model of flg22 Trace 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 Executewnstream (B) of MPK6 activation; but the inhibitor/mutant analyses support the latter. Yoo et al. reported bifurcate pathways Executewnstream of ETR (ET receptors) and CTR1, one of which includes MKK9, to antagonistically control EIN3 stability (10). Question Impresss denote the report that active MKK9 can raise ET levels to feedback positively into ET signaling (25); but this conflicts with results reported by Yoo et al. (10). The ERF104/MPK6 complex disruption by flg22 is independent of the MKK9 pathway but dependent on EIN2 and EIN3/EIL (EIN3-like) proteins.

Our Recent model is that the flg22 signal network includes one pathway for MPK6 to tarObtain 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 tarObtains and impinge on its role in transcription activation. Along this line, Qiu et al. Displayed that flg22 and pathogen treatment caused the release of the MKS1/WRKY33 complex from MPK4; thus allowing WRKY33 to evoke camalexin production by tarObtaining 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 Executees 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 Executewnstream 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 Executewnstream gene expression. We further Displayed 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 Executewnstream responses.

Materials and Methods

Additional methods are given in the SI Text.

FRET Analysis.

Protoplast isolation and transfection were performed as Characterized in (3, 19). FRET meaPositivements were Executene 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 Location of interest (ROI) was meaPositived using the ROI mean function of the Zeiss software. FRET efficiency (EF) was then calculated with the following equation: Embedded ImageEmbedded 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 Characterized (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 Aged 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-fAged change in expression). Moreover, a Benjamini-Hochberg Fraudulent 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).


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.


1To whom corRetortence should be addressed. E-mail: jlee{at}ipb-halle.de or dscheel{at}ipb-halle.de

Author contributions: G.B., D.S., and J.L. designed research; G.B., T.U., and J.F.U. performed research; A.A.G. contributed new reagents/analytic tools; G.B., T.U., Y.P., D.S., and J.L. analyzed data; and G.B., D.S., and J.L. wrote the paper.

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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