Repression of Sucrose/Ultraviolet B Light-InducedFlavonoid Accumulation in Microbe-AssociatedMolecular Pattern-Triggered Immunity in Arabidopsis1[W]
Mario Serrano2, Kazue Kanehara3, Martha Torres, Kohji Yamada, Nico Tintor, Erich Kombrink,Paul Schulze-Lefert4, and Yusuke Saijo4*
Department of Plant-Microbe Interactions, Max-Planck-Institute for Plant Breeding Research, Cologne 50829,Germany
Recognition of microbe-associated molecular patterns (MAMPs) leads to the generation of MAMP-triggered immunity (MTI),which restricts the invasion and propagation of potentially infectious microbes. It has been described that the perception ofdifferent bacterial and fungal MAMPs causes the repression of flavonoid induction upon light stress or sucrose application.However, the functional significance of this MTI-associated signaling output remains unknown. In Arabidopsis (Arabidopsisthaliana), FLAGELLIN-SENSING2 (FLS2) and EF-TU RECEPTOR act as the pattern recognition receptors for the bacterialMAMP epitopes flg22 (of flagellin) and elf18 (of elongation factor [EF]-Tu), respectively. Here, we reveal that reactive oxygenspecies spiking and callose deposition are dispensable for the repression of flavonoid accumulation by both pattern recognitionreceptors. Importantly, FLS2-triggered activation of PATHOGENESIS-RELATED (PR) genes and bacterial basal defenses areenhanced in transparent testa4 plants that are devoid of flavonoids, providing evidence for a functional contribution offlavonoid repression to MTI. Moreover, we identify nine small molecules, of which eight are structurally unrelated, thatderepress flavonoid accumulation in the presence of flg22. These compounds allowed us to dissect the FLS2 pathway.Remarkably, one of the identified compounds uncouples flavonoid repression and PR gene activation from the activation ofreactive oxygen species, mitogen-activated protein kinases, and callose deposition, corroborating a close link between theformer two outputs. Together, our data imply a model in which MAMP-induced repression of flavonoid accumulation serves arole in removing the inherent inhibitory action of flavonoids on an MTI signaling branch.
The detection of molecular structures typical of amicrobial class, termed microbe-associated molecularpatterns (MAMPs), is central to the generation of anenhanced cellular state of immunity in plants (Bollerand Felix, 2009). The so-called MAMP-triggered im-munity (MTI) represents a first layer of inducibledefenses, thereby restricting the invasion and propa-gation of pathogenic microbes. MAMPs described todate include bacterial flagellin, the elongation factor(EF)-Tu, lipopolysaccharides, and peptidoglycans butalso fungal cellulysin and cell wall components such
as chitin (GlcNAc polymer/oligomers; Boller andFelix, 2009). MTI provides functional links to otherimportant branches of plant immunity, such as effec-tor-triggered immunity and systemic acquired resis-tance, and thus serves as a basis for inducible defensesin plants (Jones and Dangl, 2006; Mishina and Zeier,2007; Tsuda and Katagiri, 2010). Salicylic acid (SA) andjasmonic acid, as well as their derivatives, act as keyphytohormones that often antagonize or complementeach other in defense signaling in the coordination ofthe above branches (Grant and Jones, 2009; Pieterseet al., 2009).
Pattern recognition receptors (PRRs) identified todate are limited to membrane-resident proteins inplants (Zipfel, 2008). Among the best characterizedmembers are the leucine-rich repeat (LRR)-receptorprotein kinases (RKs) FLAGELLIN-SENSING2 (FLS2)and EF-TU RECEPTOR (EFR) that recognize bacterialflagellin and EF-Tu (as well as their bioactive epitopesflg22 and elf18), respectively (Gomez-Gomez andBoller, 2000; Zipfel et al., 2006). In the reference plantArabidopsis (Arabidopsis thaliana), known flg22- andelf18-induced responses are entirely dependent onFLS2 or EFR, respectively. Loss of either FLS2 or EFRallows increased growth of both adapted and non-adapted bacterial strains in plants (Zipfel et al., 2004,2006; Nekrasov et al., 2009; Saijo et al., 2009), providingevidence for a key role of both PRRs in the overall host
1 This work was supported by the Max Planck Society, by theDeutsche Forschungsgemeinschaft (grant no. SFB670 to P.S.-L. and Y.S.), and by a Ph.D. fellowship from the International Max PlanckResearch School Program (to N.T.).
2 Present address: Department of Biology, University of Fribourg,Chemin du Musee 10, 1700 Fribourg, Switzerland.
3 Present address: Institute of Plant and Microbial Biology, Aca-demia Sinica 128 Section 2, Nankang, Taipei 11529, Taiwan, Republicof China.
4 These authors contributed equally to the article.* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Yusuke Saijo ([email protected]).
[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.111.183459
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immunity. The ligand-binding extracellular LRR do-mains of FLS2 and EFR undergo Asn-linked glycosy-lation and quality control (QC) in the endoplasmicreticulum (ER). Genetic studies on Arabidopsis mu-tants that are insensitive to elf18 have revealed acritical function of evolutionarily conserved ERQCcomponents in an N-glycosylation pathway for thestable accumulation of EFR (Saijo, 2010).The perception of distinct MAMPs by cognate PRRs
converges on serial activation of stereotypic cellularresponses that are detectable from minutes to days afterelicitation. Immediately upon ligand perception, FLS2and EFR recruit the LRR-receptor protein kinase BRI1-ASSOCIATED KINASE1 (BAK1; and/or its relatedSERK members), thereby leading to the formation of amolecular platform that is thought to trigger down-stream signaling (Chinchilla et al., 2007; Heese et al.,2007; Roux et al., 2011). This is followed by reactiveoxygen species (ROS) spiking, mitogen-activated pro-tein kinase (MAPK) activation, ethylene generation,extensive reprogramming of the transcriptome andmetabolome, and PMR4/GSL5-dependent callose depo-sition (Boller and Felix, 2009). Although these MAMP-induced responses have been regarded as hallmarksof MTI activation, it remains elusive or controversialwhether and how these MTI proxies contribute to theoverall host immunity and influence each other. More-over, themolecular mechanisms that link PRR activationto these signaling outputs remain poorly understood.In nature, plants mount MTI while coping with a
combination of different stresses in an environment.However, as the majority of research to date has focusedon immune responses under optimal growth conditions,our knowledge is still limited regarding the strategiesand mechanisms by which plants accomplish this taskduring simultaneous exposure to different stress cues. Ithas been documented in several plant-pathogen inter-actions that plants trigger immune responses at theexpense of abiotic stress-induced flavonoid accumula-tion (Lozoya et al., 1991; Lo and Nicholson, 1998;McLusky et al., 1999; Logemann and Hahlbrock, 2002;Schenke et al., 2011). The flavonoids define a family ofsecondary metabolites with more than 9,000 individualcompounds that are largely classified into six majorsubclasses present in most higher plants: the chalcones,flavones, flavonols, flavandiols, anthocyanins, andcondensed tannins (Winkel-Shirley, 2001). These com-pounds have a diverse array of physiological func-tions, not only acting as antioxidants and/orsunscreen pigments to protect plants from oxidativeand UV light damage but also as modulators of auxintransport (Winkel-Shirley, 2001; Buer et al., 2010).Flavonoids are induced in response to various abioticstresses, such as UV-B irradiation, high concentrationsof Suc, drought, or nutrient deprivation (Winkel-Shirley, 2001; Buer et al., 2010). Flavonoid inductionalso occurs in interactions with pathogens (Pasoldet al., 2010); conversely, it has been demonstrated thatUV-B irradiation induces pathogen resistance (Kunzet al., 2008). The apparent contradiction between these
observations and the aforementioned flavonoid re-pression upon MTI activation might reflect the possi-bility that the role of flavonoid repression in plantimmunity differs in a context-dependent manner. Thisalso predicts the importance of fine-tuning flavonoidmetabolism as a critical step in the adaptation todifferent biotic and abiotic stresses.
In Arabidopsis, single-copy genes encode a series ofenzymes engaged in a central flavonoid metabolicpathway leading to the biosynthesis of flavonols andanthocyanins, allowing the recovery of mutant plantsdisrupted at different steps in the flavonoid pathway(Winkel-Shirley, 2001). Genetic evidence demonstrates acritical role of flavonoids for the tolerance of plantsunder the flavonoid-inducible abiotic stress conditionsdescribed above (Buer et al., 2010). However, geneticstudies examining links between flavonoid metabolismand plant immunity are limited, making it difficult toassess a potential functional relationship(s).
We have recently shown that both flg22 and elf18repress the Suc-dependent induction of anthocyaninsin Arabidopsis (Saijo et al., 2009). The use of thisreadout in a genetic screen led to the isolation of anumber of priority in sweet life mutants that derepressanthocyanins and fail to trigger MTI-characteristicoutputs in the presence of elf18. Studies on thesemutants have revealed that postrecognition signalingof EFR is impaired in the presence of weakly dysfunc-tional ERQC, despite the stable accumulation of thereceptor (Lu et al., 2009; Saijo et al., 2009). In anER-resident GLUCOSIDASE II a-subunit allele, des-ignated radially swollen root3 (rsw3), EFR-triggeredimmunity is largely collapsed despite wild-type-likecoactivation of ROS spiking, MAPKs, ethylene gener-ation, and callose deposition in response to elf18.However, EFR no longer sustains the activation ofdefense-related genes, including antimicrobial PATH-OGENESIS-RELATED (PR) genes, which is tightlylinked to host immune states (van Loon et al., 2006),in a late phase of MTI in rsw3 plants (Lu et al., 2009).This points to a close correlation between flavonoidrepression and sustained transcriptional reprogram-ming and further implies a role of the two outputs forrobust MTI activation. However, to date, direct evi-dence is still missing for how the two key MTI outputsare linked to and influence each other.
Here, we verify that different MAMPs derivedfrom bacteria or fungi repress abiotic stress-inducedflavonoid accumulation in Arabidopsis seedlings.The ease and robustness of flavonoid visualizationand measurement also facilitate the dissection of PRRpathways leading to flavonoid repression duringMTIactivation. By screening a natural small moleculelibrary, we identified nine (including eight structur-ally unrelated) compounds that not only compromiseflavonoid repression but also differentially influenceother characteristic FLS2-triggered outputs. In theFLS2 pathway, our results point to (1) an uncouplingbetween early outputs and sustained activation of PRgenes and (2) a close association between the latter and
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flavonoid repression, as described earlier in the EFRpathway. We further provide evidence that the removalof flavonoids enhances MAMP-triggered activation ofSA-inducible PR genes and host bacterial defenses,suggesting that MAMP-triggered flavonoid repressionfacilitates SA-mediated responses in MTI.
RESULTS
Perception of Different MAMPs Leads to the
Repression of Suc-Induced AnthocyaninAccumulation in Arabidopsis Seedlings
Our earlier studies have shown that application ofthe bacterial MAMPs flg22 and elf18 represses Suc-induced anthocyanin accumulation in Arabidopsisseedlings (Saijo et al., 2009). To find out whether thisflavonoid repression is conserved in response to fun-gus-derived elicitors, we tested the effects of the fungalMAMPs chitin and cellulysin. We subjected 3-d-oldseedlings grown in Suc-free submerged culture to 100mM Suc. Anthocyanins represent a major subclass offlavonoids that are induced under Suc stress, and theiraccumulation reach saturation in Arabidopsis seedlingsat the concentrations used (Solfanelli et al., 2006). Underour conditions, anthocyanin accumulation becomesapparent within 2 to 3 d, as represented by red anddark green pigmentation on cotyledons and hypocotyls(Fig. 1, A and B). By contrast, anthocyanin inductionis diminished, albeit to a varied extent, when exogenousSuc is simultaneously applied with the tested MAMPs(Fig. 1, A and B). At the concentrations used, thefungal MAMPs have been described to effectivelyactivate defense response genes in Arabidopsis(Ramonell et al., 2002; Serrano et al., 2007). The ob-
served variations in the repression of anthocyaninsbetween individual MAMPs might thus reflect possi-ble differences in their threshold concentrations re-quired for anthocyanin repression that might behigher than for the activation of defense-related genes.Nonetheless, the lack of flg22 or elf18 responsivenessin fls2 or efr plants, respectively, verifies that anthocy-anin repression is a consequence of the activation ofthe earlier defined, authentic PRR-initiated pathways(Fig. 1, C and D). In sum, our data point to therepression of anthocyanin accumulation as a commonsignaling output in response to different MAMPs thatare derived from bacteria or fungi.
We note that maximum anthocyanin accumulationsaturated at 100 mM Suc (Solfanelli et al., 2006) issubstantially repressed by both flg22 and elf18 at sub-optimal concentrations (Supplemental Fig. S1). In ad-dition, wild-type seedlings treated withMAMPs for 3 dcan be rescued and resume normal growth after theremoval of the MAMPs. Thus, it is unlikely that antho-cyanin accumulation is blocked by a possible toxic effectof extremely high doses of these MAMPs. In addition,the results disfavor the possibility that either theMAMP- or Suc-induced response is prioritized accord-ing to relative input levels of the two stimuli; rather,they suggest that MAMP-triggered anthocyanin repres-sion inherently overrides Suc-induced anthocyanin ac-cumulation in Arabidopsis.
MAMP Signaling Activation Also Blocks
UV-B-Induced Flavonoid Accumulation
We next tested whether MAMP signaling activationrepresses the induction of flavonoid accumulationunder abiotic stress conditions other than Suc stress.
Figure 1. Different MAMPs from bac-teria and fungi repress Suc-inducedanthocyanin accumulation. A, Cotyle-dons of 6-d-old seedlings exposed to100 mM Suc with or without (2) theindicated MAMPs for 3 d under con-tinuous light. MAMP concentrationsused are 1 mM for flg22 and elf18 and200 mg mL21 for cellulysin and chitin.B, Anthocyanin content of the seed-lings described in A. C, Six-day-oldseedlings of fls2 and efrmutants grownin submerged culture with (+) or with-out (2) 100 mM Suc and 0.5 mM
MAMPs. D, Anthocyanin content ofthe seedlings described in C. Errorbars represent SD of six replicates (n =6–8 each), and asterisks denote statis-tical significance from the values in thepresence of 100 mM Suc withoutMAMP (* P , 0.01, t test). FW, Freshweight.
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To this end, we examined whether UV-B-inducedaccumulation of flavonols is also affected by flg22 orelf18. To visualize colorless flavonols that accumulatein plant tissues in the absence or presence of UV-Birradiation, we stained whole seedlings with diphe-nylboric acid-2-aminoethyl ester (DPBA) and thenmonitored detached cotyledons for flavonol-derivedfluorescence with an epifluorescence microscope.Twelve hours after UV-B irradiation, histochemicalanalysis detected the fluorescence that represents theaccumulation of quercetin (orange) and/or naringenin-chalcone (bright yellow; Peer et al., 2001; Solfanelliet al., 2006; Fig. 2A). By contrast, in the presence offlg22 or elf18, no significant fluorescence was detect-able, indicating that the UV-B-induced flavonol accu-mulation is abolished. Flg22- and elf18-mediatedrepression again requires the cognate PRRs FLS2 andEFR, respectively (Fig. 2B). These results are consistentwith recent data obtained in Arabidopsis suspension-cultured cells exposed to flg22 and UV-B irradiation(Schenke et al., 2011). We conclude that PRR signalingactivation represses flavonoid accumulation, com-mencing with the exposure to Suc or UV-B stressconditions.The transcription factor HY5 is up-regulated in re-
sponse to UV-B irradiation and plays a critical role inthe acquisition of UV-B stress tolerance in Arabidopsis(Ulm et al., 2004; Brown and Jenkins, 2008). We exam-ined possible effects of flg22 onUV-B-induced elevationof HY5 accumulation. Immunoblot analysis of proteinextracts derived from seedlings shows that the steady-state levels of HY5 are increased uponUV-B irradiation,irrespective of the presence or absence of flg22 (Fig. 2C).This demonstrates that at least UVB-dependent pro-cesses from UV-B perception to HY5 elevation remainunaffected upon flg22 elicitation, indicating that MTIactivation represses only a portion and/or subset ofUV-B-triggered responses.
Down-Regulation of Chalcone Synthase Steady-StateLevels upon MAMP Signaling Activation
As a first step to explore a possible link betweenMAMP signaling and flavonoid metabolism, weexamined the steady-state levels of chalcone syn-thase (CHS) that define the first committed andessential enzyme in the flavonoid biosynthesis path-way. CHS expression has been described to be up-regulated in response to exogenous Suc applicationand UV-B irradiation (Tsukaya et al., 1991; Jenkinset al., 2001). Immunoblot analysis shows that thesteady-state levels of the enzyme increase upon Sucstress in wild-type, fls2, and efr plants (Fig. 3A). Thisalso excludes constitutive CHS accumulation inboth mutants (Fig. 3A). However, simultaneous ap-plication of flg22 or elf18 with Suc reduces theSuc-induced elevation of CHS in a cognate PRR-dependent manner (Fig. 3A). Thus, it appears thatPRR activation prevents the full induction of CHS,which would at least in part account for the ob-
served low flavonoid accumulation in the presenceof these MAMPs (Fig. 1). We then determinedwhether, and if so how fast, PRR-triggered signalinginterferes with CHS up-regulation at the protein andtranscript levels. To this end, we initially subjectedseedlings to Suc stress for 24 h, allowing high-levelCHS accumulation, and subsequently applied flg22peptide. Consistent with the above results, the flg22application substantially reduces the protein levelsin 24 h despite the persistence of high concentrationsof Suc (Fig. 3B). We also traced CHS transcriptaccumulation under a similar experimental setting.As described previously (Tsukaya et al., 1991; Jenkinset al., 2001), CHS transcript levels are increased inseedlings following the exposure to high concentrationsof Suc for 24 h (Fig. 3C). Remarkably, a steep decrease(by greater than 2-fold within 1–2 h) of the elevatedCHS
Figure 2. The MAMPs flg22 and elf18 repress UV-B-induced flavonoidaccumulation. A, Cotyledons of 1-week-old seedlings were stainedwith DPBA after exposure to UV-B irradiation for 16 h without or withthe indicated MAMPs. The orange/yellow fluorescence of DPBA-stained leaves represents flavonol accumulation. B, Cotyledons of fls2and efr plants prepared as described in A. C, Immunoblot analysis withanti-HY5 antibodies of protein extracts from wild-type (WT) seedlingsexposed to UV-B irradiation for the indicated time with (+) or without(2) 0.5 mM flg22. hy5 seedlings were used as a control to verify thesignal identity. A blot probed with anti-Rpt5 antibodies is shown as aloading control.
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levels occurs in response to subsequent flg22 application(Fig. 3C). Essentially the same conclusion was obtainedwith seedlings exposed to UV-B irradiation prior toflg22 application, albeit at a slightly lower rate of CHStranscript decrease (Supplemental Fig. S2). These resultsstrongly suggest that an early output of FLS2 signalinginvolves the down-regulation of CHS transcript accu-mulation despite the presence of otherwise stimulatoryinputs, which would eventually contribute to the ob-served decrease in enzyme accumulation (Fig. 3A).Flg22-triggered rapid down-regulation of CHS, evenafter its high induction by the two tested abiotic stresses,points to robust in planta prioritization for MTI activa-tion over an adaptive program to the two abioticstresses. However, of note, in the presence of theMAMPs, Suc-mediated anthocyanin induction is barelydetectable (Fig. 1), despite lower but evident elevation ofthe enzyme steady-state levels (Fig. 3A). This impliesthe existence of an additional mechanism(s) by whichPRR signaling represses flavonoid accumulation.
Alterations in FLS2-Triggered Reprogramming ofDefense-Related Gene Expression in transparenttesta4 Plants That Are Devoid of Flavonoids
The robust in planta prioritization of MTI activationover Suc/UV-B stress-induced flavonoid accumula-tion prompts the question of whether flavonoidbiosynthesis has an as-yet-undefined inhibitory rolein one or several MTI signaling outputs. The genetictractability of Arabidopsis allows us to directly assesspossible alterations of MTI-associated responses in theabsence of a central flavonoid metabolic pathwayleading to the biosynthesis of flavonols and anthocy-
anins (Winkel-Shirley, 2001). We analyzed flg22 re-sponses of transparent testa4 (tt4) plants that carry amutation in the CHS locus and thus are devoid offlavonoids (Shirley et al., 1995). Immunoblot analysisof total protein extracts derived from nonelicitedseedlings grown in the presence of exogenous Sucrevealed that FLS2 steady-state levels remain essen-tially unaltered in tt4 plants (Fig. 4A). In addition,characteristic outputs of FLS2 signaling, ROS spiking,MAPK activation, and callose deposition are not sig-nificantly enhanced in tt4 plants (Supplemental Fig.S3). Thus, the absence of flavonoid biosynthesis ap-pears to have little impact on these PRR signalingoutputs. In light of the earlier described uncoupling ofthese outputs from sustained transcriptional reprog-ramming in the EFR pathway (Lu et al., 2009), we alsoexamined potential alterations in flg22-elicited defensegene expression in tt4 seedlings. Among the earlyMAMP-inducible genes is WRKY22, encoding a mem-ber of the WRKY transcription factor family that isthought to promote MTI (Asai et al., 2002; Zhang et al.,2007). Our quantitative reverse transcription (qRT)-PCR data suggest that WRKY22 activation might beslightly increased in tt4 plants in response to flg22 (Fig.4B). To assess the significance of the tt4 mutations ondefense gene expression, we extended our analysis toinclude late-responsive PR genes such as PR-1 and PR-2, which encode a putative antimicrobial peptide andb-1,3-glucanase, respectively (van Loon et al., 2006). Intt4 plants, the levels of both PR-1 and PR-2 transcriptsare much higher compared with wild-type plants inthe presence of flg22 (Fig. 4C). We noticed that, unlikeWRKY22 and PR-1, significant PR-2 activation occursin tt4 plants in the absence of exogenous MAMP
Figure 3. Suc-induced CHS expression is reduceduponMAMPapplication. A, Immunoblot analysis withanti-CHS antibodies of protein extracts from 6-d-oldseedlings exposed to 100 mM Suc for 3 d with (+) orwithout (2) the indicated MAMPs at 0.5 mM undercontinuous light. B and C, Flg22 down-regulates CHSexpression despite preceding and persistent Suc stress.Three-day-old seedlings were exposed to 100 mM Sucfor 24 h, allowing high CHS induction prior to flg22application at 0.5 mM for the indicated times undercontinuous light. Immunoblot (B) and qRT-PCR (C)results are shown. Numbers under the lanes in A and Bindicate relative band intensities that were quantifiedand normalized, with the value in non-flg22-elicitedwild-type (WT) seedlings at 100 mM Suc as 1. Coo-massie blue (CB)-stained blots are shown to verifyequal loading (A and B). A representative data set isshown with SD of experimental replicates (C).
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application (Fig. 4, B and C). Moreover, by chemicalcomplementation of tt4 plants, we verified that theobserved mutant phenotype is a consequence of theloss of flavonoid biosynthesis. Naringenin is produceddownstream of the CHS-catalyzed step and acts as aprecursor for the biosynthesis of flavonols and antho-cyanins (Shirley et al., 1995). The application of nar-ingenin allows tt4 seedlings to exhibit wild-type-likered and/or dark green pigmentation in the presenceof Suc (Supplemental Fig. S4), as described earlier(Buer et al., 2010), indicating that anthocyanin biosyn-thesis is restored in the mutant. We further found thatsimultaneous flg22 application with Suc and naringe-nin represses the anthocyanin accumulation otherwiserestored in tt4 plants in the presence of exogenouslyapplied naringenin (Supplemental Fig. S4), pointing tothe existence of another step than CHS expression inthe flavonoid biosynthesis pathway that is blocked byFLS2 signaling. Importantly, the addition of naringe-nin to the medium prior to flg22 application, thereby
allowing flavonoid induction in tt4 plants, leads to adecrease in flg22-triggered activation of PR-1 and PR-2(Fig. 4D). Together, our data strongly suggest a repres-sive role of flavonoids (and/or their derivatives) inMAMP-triggered activation of these PR genes.
We then assessed whether host immunity is en-hanced in the absence of flavonoid biosynthesis. Tothis end, we challenged wild-type and tt4 plants withthe phytopathogenic bacterium Pseudomonas syringaepv tomato (Pst) DC3000 and then compared bacterialgrowth in leaves. In tt4 plants, a less virulent DavrPto/DavrPtoBmutant strain of Pst DC3000 (Lin andMartin,2005) did not grow as well as in wild-type plants,indicating that the loss of flavonoids indeed leads to anincrease in host basal immunity (Fig. 4E).
Earlier studies have shown that PR-1 and PR-2genes are not only activated upon MAMPs but alsoupon Suc stress or UV-B irradiation in Arabidopsis,representing common signaling outputs between thebiotic and abiotic stress responses (Thibaud et al.,
Figure 4. Enhanced PR gene activation in tt4 plantsthat are devoid of flavonoids. A, Immunoblot analysisof protein extracts derived from 2-week-old noneli-cited seedlings. The positions of molecular massmarkers are shown on the left (kD). A Coomassieblue (CB)-stained blot is shown at the bottom to verifyequal loading. B to D, qRT-PCR analysis of 2-week-old seedlings exposed to 0.5 mM flg22 for the indi-cated times. In D, tt4 GK02 plants were supplied withnaringenin at 100 mM (+) for 24 h before flg22application for the indicated times. The relativeinduction is shown in fold, with the gene/ACTINexpression value at 0 h in wild-type (WT) plants as 1.Error bars represent SD of experimental replicates. E,Growth of a DavrPto/DavrPtoB mutant strain of PstDC3000 in 4-week-old plant leaves 3 d after sprayinoculation with bacteria at 1 3 109 colony-formingunits (cfu) mL21. Error bars and the asterisk representSD of experimental replicates and statistical signifi-cance (* P , 0.01, t test), respectively.
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2004). We assessed how flg22- and Suc stress-triggeredsignaling influence each other for the activation ofthese PR genes. Under our conditions, the Suc stressalone merely allows a slight increase in PR-1 transcriptlevels (approximately 3-fold), which is much less thanthe flg22 induction fold of the gene (typically 30- to100-fold; Supplemental Fig. S5). However, remarkably,PR-1 is vastly activated (up to 1,500- to 2,500-fold) inthe presence of both Suc stress and flg22 application,pointing to a synergistic effect between the two stimuli(Supplemental Fig. S5). This synergisticPR-1 activationis largely unaffected in tt4 plants (Supplemental Fig.S5). This indicates that the observed Suc-flg22 syner-gies occur independently of flavonoids and is in goodagreement with our data that flavonoid induction isessentially abolished upon simultaneous applicationof flg22 and Suc (Fig. 1; Supplemental Fig. S1). Bycontrast, the Suc stress alone is sufficient to activatePR-2 by typically approximately 10-fold, which iscomparable to the fold induction (typically 10- to 20-fold) upon flg22 application under normal Suc condi-tions (25 mM). However, unlike for PR-1, there is nosynergistic effect for PR-2 activation between the twostimuli (Supplemental Fig. S5). In tt4 plants, PR-2 isconstitutively activated irrespective of the Suc condi-tions tested (Supplemental Fig. S5). This suggests thatflavonoid repression greatly contributes to and islargely sufficient to confer PR-2 activation duringflg22-triggered immunity. In sum, our results forboth PR genes indicate that a subset of Suc-dependentresponses, in particular those closely associated withdefense activation, are not repressed but rather enhancedor possibly coopted by FLS2-triggered signaling.
Genetic Requirements for MTI-Related Elements inFLS2- and EFR-Triggered Anthocyanin Repression
To identify the genetic requirements for MAMP-triggered flavonoid repression, we assessed Suc-induced anthocyanin accumulation in previouslydefined MTI-related mutant plants in the presence offlg22 or elf18. In Arabidopsis, earlier studies haveidentified that flg22-induced ROS spiking and callosedeposition are abolished in the absence of the NADPHoxidase subunit AtRbohD and of the callose synthasePMR4/GSL5, respectively (Kim et al., 2005; Nuhseet al., 2007; Zhang et al., 2007). As shown in Figure 5A,PMR4 is also required for elf18-induced callose depo-sition. Both rbohD rbohF and pmr4 mutant plants showno discernible alterations in seedling developmentunder our conditions and also retain wild-type-likeanthocyanin repression in the presence of flg22 orelf18 at the range of concentrations used (Fig. 5B).Thus, transient ROS spiking and callose depositionare dispensable for anthocyanin repression in re-sponse to the two MAMPs. On the other hand, ROSspiking and callose deposition occur without signifi-cant anthocyanin repression in response to elf18 inrsw3 plants (Lu et al., 2009). Together, these resultssupport a nonlinear model in which these signaling
outputs are under the control of separate pathwaysemanating from the PRRs.
Accumulating evidence points to a role of the LRR-RLK BAK1 that is rapidly recruited to FLS2 and EFRupon the elicitation of these PRRs by cognate MAMPs,which is thought to provide a platform for postrecog-nition signaling (Chinchilla et al., 2007; Heese et al.,2007; Roux et al., 2011). Consistent with this idea, thetwo bak1 alleles tested allow the derepression of antho-cyanin accumulation in the presence of flg22 at 0.05 mM
(Fig. 5C). However, elf18-triggered anthocyanin repres-sion is less affected in these bak1 plants (Fig. 5C). Thus,as earlier described for MAPK activation (Chinchillaet al., 2007), EFR is more tolerant than FLS2 against lossof BAK1 for anthocyanin repression as well.
Chemical Screens for Natural Compounds ThatPerturb FLS2-Triggered Anthocyanin Repression
To further dissect the FLS2 pathway, we have screeneda chemical library of 6,800 natural small molecules forcompounds that affect the dominant effect of flg22 overSuc stress-induced anthocyanin accumulation. We havemodified and applied a previously established chemicalscreening procedure (Serrano et al., 2007) to the afore-mentioned Suc-anthocyanin assays in the presence offlg22. Three-day-old seedlings grown in submerged Suc-free culture were exposed to 100mM Sucwith or withoutflg22 in the presence of each single compound at 10 mM
for a further 3 d. Our screens have revealed ninecompounds that allow the derepression of anthocyaninaccumulation in the presence of flg22 (Fig. 6). In theabsence of flg22, none of these compounds significantlyalters anthocyanin content at 100mM Suc comparedwitha mock control (Fig. 6A). Although AN8-A10 slightlyelevates anthocyanin levels in the absence of exoge-nous Suc, the rest of these compounds do not showany discernible effects (Supplemental Fig. S6A).Thus, it can be essentially ruled out that thesecompounds constitutively activate anthocyanin ac-cumulation. Taken together, it appears that thesecompounds specifically interfere with flg22 respon-siveness rather than Suc responsiveness or anthocya-nin biosynthesis per se.
All identified compounds, except AN14-F3 andAN14-G3, which share a similar backbone, are unre-lated in structure (Fig. 6B). Despite a certain degree ofvariation in their dose dependence, these compoundseffectively block flg22-dependent anthocyanin repres-sion (Supplemental Fig. S6B). At present, relevantcellular targets of these compounds remain elusiveon the basis of their structures (http://pubchem.ncbi.nlm.nih.gov and http://www.drugbank.ca). Never-theless, these compounds provide a valuable tool forfurther studies, as they are expected to perturb acritical step in the FLS2 pathway leading to therepression of flavonoid accumulation.
We investigated possible alterations in severalcharacteristic FLS2 signaling outputs in the presenceof the identified compounds other than the derepres-
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sion of anthocyanin accumulation. Our results defineflg22-dependent down-regulation of CHS expressionas a new output of PRR signaling (Fig. 3). Thus, wefirst examined whether the identified compoundspermit Suc-induced accumulation of the enzyme inthe presence of flg22. Unexpectedly, none of thesecompounds restores CHS steady-state levels as highas in the absence of flg22 (Fig. 7), despite evidentderepression of anthocyanin accumulation (Fig. 6).This indicates that a recovery of anthocyanin accu-mulation occurs without the full restoration of CHSaccumulation in the presence of these compounds.Together with the aforementioned flg22-triggeredrepression of anthocyanin accumulation restored intt4 plants upon naringenin application (Supplemen-tal Fig. S4), these results suggest that FLS2 signalingrepresses another key step than CHS accumulation inSuc-induced anthocyanin accumulation. It seemslikely that this unknown output(s) is impaired uponchemical perturbation by these compounds. Thus,future identification of cellular target(s) of thesecompounds should reveal a presumed convergencepoint(s) between FLS2 signaling and the flavonoidmetabolic pathway.
FLS2-Triggered Activation of ROS Spikingand Callose Deposition in the Presence of the
Identified Compounds
We next tested flg22-induced ROS spiking in leafdiscs derived from mature rosette leaves followingestablished procedures (Saijo et al., 2009) in the presenceof the identified nine compounds. Preincubation with
these compounds did not stimulate ROS generationat detectable levels in the absence of flg22 elicitation(at 0 min in Fig. 8). Whereas flg22 induces a transientoxidative burst in amock control, the compounds testedinhibit this early output of FLS2 signaling to a varieddegree (Fig. 8). Based on the severity of the defects, weclassify the nine compounds into three groups: stronginhibition is apparent in the presence of AN3-E2, AN8-D9, and AN14-G3; weak but substantial inhibition isdetectable in the presence of AN3-H7, A21-A1, A44-B6,AN14-F3, andAN8-A10; no significant inhibition occursin the presence of A7-G7 (Fig. 8). These results stronglysuggest that at least the former two classes (containingeight of the nine compounds) influence a very early stepupstream of FLS2-triggered ROS spiking.
We also examined whether flg22-induced callosedeposition is affected in the presence of these ninecompounds. We verified that none of these com-pounds alone stimulates callose deposition in theabsence of MAMP elicitation (data not shown).Whereas eight of the nine compounds significantlyreduce the peak level of ROS spiking, as describedabove, only AN8-A10 exhibits the significant repres-sion of callose deposition under our conditions (Fig.9). Since both ROS spiking and callose deposition aredispensable for anthocyanin repression (Fig. 5B), itseems unlikely that the decrease in ROS spiking and/or callose deposition results in the interference withFLS2-mediated anthocyanin repression in the pres-ence of these compounds (Fig. 6). Rather, our datasuggest that their cellular targets do not belong to themachineries executing ROS spiking or callose depo-sition per se.
Figure 5. Genetic requirements for FLS2- andEFR-triggered repression of Suc-induced antho-cyanin accumulation. A, PMR4-dependent cal-lose deposition. Callose deposits were stainedwith aniline blue in the cotyledons of wild-type(WT) and pmr4 plants exposed to 2 mM flg22 orelf18 for 24 h. B and C, Anthocyanin content of6-d-old seedlings exposed to water (2 suc) or 100mM Suc (+ suc) with (+) or without (2) flg22 orelf18 at the indicated concentrations for 3 d undercontinuous light. Error bars represent SD of four ormore experimental replicates (n = 6–8 each), andasterisks denote statistical significance from thevalues of the corresponding wild-type controls(* P, 0.01, ** P, 0.05, t test). FW, Fresh weight.
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Uncoupling between FLS2-Triggered EarlySignaling Outputs and PR-1 Activation
upon Pharmacological Perturbation
In the EFR pathway, sustained activation of defensegenes is impaired in the presence of an impairedERQC of the PRR, despite nearly full coactivation ofseveral early outputs (Lu et al., 2009). We wondered ifsuch an uncoupling of signaling outputs also occurs inthe FLS2 pathway in the presence of A7-G7, whichblocks anthocyanin repression but does not signifi-cantly affect FLS2 accumulation, ROS spiking, or cal-lose deposition (Figs. 6, 8, and 9; Supplemental Figs. S6and S7). To test this possibility, we assessed the flg22-induced activation of the early-responsive WRKY22gene, which encodes a defense-promoting member ofthe WRKY transcription factor family (Asai et al.,2002), and of the late-responsive PR-1 gene, whichencodes a putative antimicrobial peptide (van Loonet al., 2006). We also tested the effects of AN14-G3 andAN14-F3, which strongly and weakly reduce ROSspiking, respectively (Fig. 8). Consistent with the reten-tion of wild-type-like callose deposition (Fig. 9), FLS2
accumulation is largely unaffected in the presence ofAN14-G3, AN14-F3, or A7-G7 (Supplemental Fig. S7).
Our qRT-PCR data indicate that FLS2-triggered PR-1 activation is significantly reduced in the presence ofA7-G7, albeit to a lesser extent than in the presence ofAN14-G3 and AN14-F3 (Fig. 10). By contrast, earlyactivation of WRKY22 is largely retained in the pres-ence of either of these compounds (Fig. 10). This pointsto a separation of early WRKY22 activation and latePR-1 activation in the FLS2 pathway upon pharmaco-logical disturbance with these compounds. Moreover,FLS2-mediated full PR-1 activation and anthocyaninrepression are impaired in the presence of A7-G7,despite nearly intact coactivation of ROS spiking,callose deposition, and early WRKY22 activation.These results strongly suggest that the latter MTI-characteristic outputs are insufficient to drive strongPR-1 activation at a late phase in FLS2-tiggeredimmunity. This corroborates our recent finding on theEFR pathway that sustained activation of defensegenes and flavonoid repression can be geneticallyuncoupled from early signaling outputs (Lu et al.,
Figure 6. Chemical compounds thatderepress Suc-induced anthocyaninaccumulation despite the presence offlg22. A, Anthocyanin content of 6-d-old seedlings exposed to 100 mM Sucwith (+) or without (2) 1 mM flg22 inthe presence of the indicated com-pounds at 10 mM. Error bars representSD of six replicates (n = 6 each), andasterisks denote statistical significancefrom the value of the mock control inthe presence of Suc and flg22 (* P ,0.01, t test). B, Chemical structures ofthe compounds used. FW, Freshweight.
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2009) and extend such uncoupling of MTI outputs tothe FLS2 pathway by pharmacological intervention.
DISCUSSION
Here, we show in Arabidopsis that MTI activationupon application of different MAMPs derived from
bacteria or fungi leads to the repression of flavonoidaccumulation, even in the presence of Suc stress or UV-B irradiation. Following our earlier findings that flg22and elf18 repress Suc-induced anthocyanin accumula-tion (Saijo et al., 2009), this study highlights a domi-nance of MTI activation over flavonoid induction inresponse to two different abiotic stresses. It has beendocumented that simultaneous application of fungalelicitor represses UV-B-induced flavonoid accumula-tion and the expression of light-responsive genes inparsley (Petroselinum crispum) cell culture (Lozoyaet al., 1991; Logemann and Hahlbrock, 2002). Com-plete suppression of anthocyanin accumulation atincipient Botrytis allii infection sites has been describedin epidermal cells of onion (Allium cepa; McLuskyet al., 1999). Likewise, inoculation of monocotyledon-ous sorghum (Sorghum bicolor) with the nonadaptedpathogenic fungus Cochliobolus heterostrophus drasti-cally reduced the light-induced accumulation of an-thocyanin by repressing the transcription of theanthocyanin biosynthesis genes encoding flavanone
Figure 8. Effects of the identified chemical compounds on ROS spiking in response to flg22. ROS spiking triggered from leafdiscs derived from expanded rosette leaves of 4-week-old plants is traced for the indicated times in the presence of thecompounds shown in each panel, with the addition of 1 mM flg22 at 0 min. Error bars indicate SD of six experimental replicates.Luminescence emission is given as relative light units (RLU).
Figure 7. Immunoblot analysis of protein extracts derived from 6-d-oldseedlings exposed towater (2) or 100mM Suc (+)with (+) orwithout (2) 0.5mMflg22 in the presence of the indicated compounds at 10mM.ACoomassieblue (CB)-stained blot is shown at the bottom to verify equal loading.
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3-hydroxylase, dihydroflavonol 4-reductase, andanthocyanidin synthase (Lo and Nicholson, 1998).Thus, prioritization of immune responses over abioticstress-inducible flavonoid/anthocyanin accumulationis a widespread phenomenon in flowering plantsduring their interactions with various, if not all, mi-crobes and might be mediated by an evolutionarilyancient regulatory cross talk mechanism.
In nature, plants are often exposed to and must copewith a combination of different stresses. Of note, anadaptive program optimized under a particular stresscondition might be suboptimal or even detrimental toadaptations under different stress conditions. On the
other hand, prompt activation of an appropriate adap-tive response is crucial for the timely acquisition ofeffective stress tolerance. Thus, upon sensing a com-bination of different stress cues, it would in principlebe preferable if an adaptive program is flexibly butquickly chosen according to their relative input levels,as earlier described with coapplication of SA andjasmonic acid (Mur et al., 2006). However, our resultsdemonstrate that MTI activation, even at suboptimallevels, represses saturated flavonoid accumulationinduced by Suc stress or UV-B irradiation. This ratherindicates a hierarchical structure in the signaling crosstalk between MTI and the abiotic stress-triggered
Figure 9. Effects of the identified chemical com-pounds on callose deposition in response to flg22.Callose deposits were stained with aniline blue in thecotyledons of wild-type plants exposed to 1 mM flg22for 24 h in the presence of the indicated compounds.Callose deposition was quantified and is shown in thebottom panel with SD (n = 5). The asterisks denotestatistical significance from the value of the mockcontrol (* P , 0.01, t test). Experiments were per-formed three times with the same conclusion.
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flavonoid induction. This might be explained by costsincurred from the aforementioned flexible coordina-tion between the two stress responses and/or by thebenefit of MTI, which might exceed that of the abioticstress tolerance, for plant fitness in a microbe-richenvironment.The characterization of tt4 plants that are depleted
of flavonoids revealed that FLS2-triggered activationof late-responsive PR genes is enhanced without adiscernible increase in the activation of ROS, MAPKs,and callose accumulation by the receptor. Togetherwith our data that MAMP-induced ROS spiking andcallose deposition are dispensable for anthocyaninrepression (Fig. 5), this points to a close associationbetween flavonoid repression and a sustained activa-tion of late-phase defense-related genes in MTI, asdescribed earlier in the EFR pathway (Lu et al., 2009).More importantly, this provides evidence for a repres-sive role of flavonoids in the establishment and/ormaintenance of the defense-related transcriptome. Ofnote, PR2 activation occurs at high levels in nonelicitedtt4 plants, the extent of which is comparable with oroccasionally even higher than that in flg22-elicitedwild-type plants (Fig. 4). Our results demonstrate thata loss of flavonoids is sufficient to confer activation of
the PR2 gene without PRR elicitation. Thus, flavonoid-dependent inhibition of late-responsive defense-relatedgenes and MAMP-induced flavonoid down-regulationstrongly suggest the existence of a mutually inhibitoryconnectivity between these two adaptive responses(Fig. 11), which, to our knowledge, has not been de-scribed before. Together, we infer from our results thatMAMP-induced flavonoid down-regulation increasesthe amplitude of late-phase defense gene expression byremoving an intrinsic negative feedback mediated byflavonoids on this MTI output. Thus, it might be thatactive avoidance of flavonoid induction contributes tothe effectiveness of MTI and/or other branches ofimmunity that are linked to MTI.
We also revealed that Suc-dependent activation ofthese PR genes is not repressed but rather enhancedby flg22 application (Supplemental Fig. S5). Thisstrengthens the notion that MAMP signaling activationdoes not uniformly repress entire Suc stress-inducedresponses but selectively represses the flavonoid bio-synthesis pathway. Moreover, in tt4 plants, the syner-gies between flg22 and Suc stress remain largelyunaffected for PR-1 activation, while constitutive PR-2activation occurs irrespective of the Suc conditionstested (Supplemental Fig. S5). This suggests that therepressive role and mode of action of flavonoids differeven between the two PR genes that otherwise arehighly coexpressed in Arabidopsis under differentgrowth conditions (http://atted.jp/). Thus, a subsetof defense-related pathway(s) might be highly sensitiveto changes in cellular flavonoid levels and vulnerable toflavonoid-mediated repression. It will be interesting toexplore a possible correlation between the responsive-ness of MTI-associated genes to high amounts of Sucand their enhanced activation upon the depletionof flavonoids in future genome-wide transcriptomeanalyses.
Remarkably, our natural compound survey hasagain revealed an uncoupling of PR1 gene activationfrom ROS spiking and callose deposition in the FLS2pathway. With a focus on the MAMP-Suc signaling
Figure 10. Chemical perturbation of flg22-induced activation of de-fense-related genes. qRT-PCR analysis is shown for flg22-induced PR-1 and WRKY22 activation in 2-week-old seedlings exposed to 0.5 mM
flg22 for 24 h in the presence of the indicated compounds at 10 mM. Therelative induction is shown in fold, with the gene/ACTIN expressionvalue at 0 h in fls2 plants as 1. A representative data set is shown with SD
of experimental replicates. WT, Wild type.
Figure 11. Model for signaling cross talk between MAMP-triggeredimmunity and abiotic stress-triggered flavonoid accumulation.
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cross talk, we have revealed nine compounds thatderepress anthocyanins in the presence of flg22 anddifferentially impair previously defined characteristicoutputs of FLS2 signaling. Of note, one of the com-pounds, A7-G7, compromises both PR1 activation andflavonoid repression, despite almost full retentionof ROS spiking, callose deposition, and WRKY22 acti-vation in response to flg22. This corroborates theexistence of two major phases or branches in PRRsignaling that can be uncoupled by chemical interfer-ence (this study) and by genetic means (Lu et al., 2009).Whether the observed uncoupling of FLS2 signalingoutputs occurs at the level or downstream of PRR-triggered signal initiation remains unknown. Futureidentification of a cellular target(s) of A7-G7 willprovide insight into the mechanisms that separatethe two phases (or branches) in the FLS2 pathway.
It appears that the existence of two separate phases orbranches represents a common principle in MTI. Thetwo phases (or branches) might correspond to an initialtransient elicitation and subsequent robust activation/maintenance of immunity. Our data suggest that flavo-noid down-regulation is closely associatedwith the latterphase (branch). Considering the energy- and nutrient-intensive nature of immune responses, in particular ofthe latter phase of the above two, it is conceivable thatshutting down of flavonoid biosynthesis facilitates therelocation of limited resources to costly defense re-sponses. The flavonoid biosynthesis pathway representsone of the branches derived from the phenylpropanoidmetabolic pathway and shares precursors with meta-bolic pathways for the biosynthesis, for example, of SA,lignins, and isoflavonoid-phytoalexins (Winkel-Shirley,2001). Although the isoflavonoid pathway does not seemto exist in Arabidopsis (Winkel-Shirley, 2001), the othertwo pathways are closely linked and plausibly influ-enced by the activity of the flavonoid pathway. Ourevidence that flavonoids reduce flg22-triggered activa-tion of the SA-induced defense markers, PR-1 and PR-2,is compatible with the idea that flavonoid repressionserves to promote SA-mediated immunity during MTI.
Lignin deposition defines an important componentin plant immunity, by the reinforcement of cell walls toprovide a physical barrier against invasive microbes. InArabidopsis suspension-cultured cells, flg22 applica-tion leads to an increase in lignin formation (Schenkeet al., 2011). However, no detectable alterations havebeen described in total lignin content or compositionbetween nonelicited wild-type and null tt4 plants (Liet al., 2010). This indicates that the repression of flavo-noid biosynthesis alone is insufficient to increase ligninbiosynthesis by the relocation of a shared precursor,p-coumarate, to the latter pathway.
Unlike Arabidopsis, for example, legume plants havemultiple members that encode enzymes engaged in aflavonoid metabolic pathway and the isoflavonoidbranch, which leads to the biosynthesis of antimicrobialphytoalexins. In good agreement with these differences,legume and Arabidopsis plants seem to differ in themode of regulation of flavonoid pathway genes during
defense responses. For example, soybean has eight CHSgenes, of which all but one are up-regulated uponeffector-triggered immunity activation with an incom-patible strain of Pseudomonas syringae pv glycinea (Zabalaet al., 2006). Further studies will be required to elucidatethe presumably species-, genus-, and/or class-specificfunctions and regulations of flavonoids, as well as theirpotential roles as signaling molecules that modulateplant immunity, in diverse plant-microbe interactions.
MATERIALS AND METHODS
Plant Materials and Growth Conditions
The wild-type control used was Arabidopsis (Arabidopsis thaliana) ecotype
