A MPK3/6-WRKY33-ALD1-Pipecolic acid Regulatory Loop Contributes to Systemic Acquired Resistance

Transcription

1 Plnt Cell Avne Pulition. Pulishe on Septemer 18, 218, oi:1.115/tp RESEARCH ARTICLE A MPK3/6-WRKY33-ALD1-Pipeoli i Regultory Loop Contriutes to Systemi Aquire Resistne Yiming Wng, Stefn Shuk,, Jingni Wu,1, Ping Yng,2, Anne-Christin Döring, Jürgen Zeier,,*, n Kenihi Tsu,* Deprtment of Plnt Miroe Intertions, Mx Plnk Institute for Plnt Breeing Reserh, Crl-von-Linné Weg 1, 5829, Cologne, Germny. Deprtment of Moleulr Eophysiology of Plnts, Heinrih Heine University Düsselorf, Universitätsstrße 1, 225 Düsselorf, Germny Cluster of Exellene on Plnt Sienes (CEPLAS), Heinrih Heine University, Universitätsstrße 1, 225 Düsselorf, Germny Present ress: 1 Shnghi Institute of Plnt Physiology n Eology, Chinese Aemy of Sienes, Shnghi 232, Chin 2 Institute of Crop Sienes, Chinese Aemy of Agriulturl Sienes, Beijing 181, Chin. * To whom orresponene shoul e resse: Kenihi Tsu (tsu@mpipz.mpg.e) n Jürgen Zeier (Juergen.Zeier@uni-usselorf.e) Short title: MPK3/6-WRKY33-ALD1 loop for Pip-meite SAR One sentene summry: A positive feek loop onsisting of MPK3/6, WRKY33, ALD1, n pipeoli i regultes lol immune mplifition ontriuting to systemi quire resistne in Ariopsis thlin. The uthors responsile for istriution of mterils integrl to the finings presente in this rtile in orne with the poliy esrie in the Instrutions for Authors ( re: Kenihi Tsu (tsu@mpipz.mpg.e) n Jürgen Zeier (Juergen.Zeier@uni-usselorf.e). ABSTRACT Plnts inue systemi quire resistne (SAR) upon lolize exposure to pthogens. Pipeoli i (Pip) proution vi AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1) is key for SAR estlishment. Here, we report positive feek loop importnt for SAR inution in Ariopsis thlin. We showe tht lol tivtion of the MAP kinses MPK3 n MPK6 is suffiient to trigger Pip proution n mount SAR. Consistent with this, muttions in MPK3 or MPK6 le to ompromise Pip umultion upon inoultion with the teril pthogen Pseuomons syringe pv. tomto DC3 (Pto) AvrRpt2, whih triggers strong sustine MAPK tivtion. By ontrst, P. syringe pv. muliol (Pm) n Pto, whih inue trnsient MAPK tivtion, trigger Pip iosynthesis n SAR inepenently of MPK3/6. ALD1 expression, Pip umultion, n SAR were ompromise in mutnts efetive in the Amerin Soiety of Plnt Biologists. All Rights Reserve

2 MPK3/6-regulte trnsription ftor WRKY33. Chromtin immunopreipittion showe tht WRKY33 ins to the ALD1 promoter. We foun tht Pip triggers tivtion of MPK3 n MPK6 n tht MAPK tivtion fter Pto AvrRpt2 inoultion is ompromise in wrky33 n l1 mutnts. Colletively, our results revel positive regultory loop onsisting of MPK3/MPK6, WRKY33, ALD1, n Pip in SAR inution n suggest the existene of istint SAR tivtion pthwys tht onverge t the level of Pip iosynthesis INTRODUCTION Plnts hve evolve two types of innte immune system to el with ttks y miroil pthogens: ell surfe reeptor-meite immunity (pttern-triggere immunity or PTI) n intrellulr reeptor-meite immunity (effetor-triggere immunity or ETI) (Jones n Dngl, 26; Tsu n Ktgiri, 21). PTI is inue y the reognition of miroe-ssoite moleulr ptterns (MAMPs) y pttern reognition reeptors (PRRs) on the plsm memrne, whih re reeptor-like kinses (RLKs) or reeptor-like proteins (RLPs) (Jones n Dngl, 26; Boutrot n Zipfel, 217; Yu et l., 217). For instne, the teril MAMP flg22, prt of teril flgellin, is reognize y FLAGELLIN-SENSITIVE2 (FLS2) n the o-reeptors BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) n BAK1-LIKE1 (BKK1) in Ariopsis thlin (Gomez-Gomez n Boller, 2; Zipfel et l., 2; Chinhill et l., 27; Roux et l., 211). ETI is triggere y reognition of virulene ftors suh s teril type III effetors (T3Es) with whih pthogens suvert plnt immunity in suseptile plnts y mostly nuleotie-ining/leuine-rih repet (NLR) reeptors (Jones n Dngl, 26; Cui et l., 215; Trn et l., 217; Zhng et l., 217). For instne, AvrRpt2 n AvrRpm1 re T3Es of the teril pthogen Pseuomons syringe whose virulene tions re reognize y the NLR reeptors RESISTANCE TO P. SYRINGAE (RPS2) n RESISTANCE TO P. SYRINGAE PV MACULICOLA 1 (RPM1), respetively, in A. thlin (Mkey et l., 22; Axtell n Stskwiz, 23; Mkey et l., 23). PTI n ETI shre signling omponents suh s the phytohormone sliyli i (SA) n mitogen-tivte protein kinses (MAPKs) (Tsu n Ktgiri, 21). SA regultes mjor portion of plnt immunity ginst iotrophi n hemiiotrophi pthogens suh s P. syringe vi the entrl regultor/reeptor of SA signling 2

3 NONEXPRESSER OF PR GENES 1 (NPR1) (Delney et l., 199; Co et l., 1997; Wu et l., 212; Pjerowsk-Mukhtr et l., 213; Ding et l., 218). A. thlin MAPKs, MPK3 n MPK6, positively ontriute to immunity ginst wie rnge of pthogens vi phosphoryltion of sustrtes in prtilly reunnt mnner (Bekers et l., 29; Meng n Zhng, 213; Xu et l., 216; Ding et l., 218). For instne, the WRKY fmily trnsription ftor WRKY33, iret phosphoryltion trget of MPK3 n MPK6, is neessry for MPK3 n MPK6-meite proution of the phytolexin mlexin n the phytohormone ethylene (Mo et l., 211; Li et l., 212). Plnts systemilly inue ro spetrum resistne lle systemi quire resistne (SAR) upon lolize exposure to pthogens (Fu n Dong, 213). Although the ientity of the moile signl tht relys lol immune tivtion for SAR tivtion in systemi tissues is still uner ete, severl moleules hve een implite in the estlishment of SAR suh s methyl sliylte (Prk et l., 27), ehyroietinl (Chturvei et l., 212), glyerol-3-phosphte (Chn et l., 211), zeli i (Jung et l., 29), n pipeoli i (Pip) (Nvrov et l., 212). Pip is Lys tolite tht is present uiquitously in the plnt kingom n umultes to high levels in P. syringe-inoulte leves n in istnt, uninfete leves t the onset of SAR (Nvrov et l., 212; Zeier, 213). Pip is synthesize y AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1) n SAR-DEFICIENT (SARD) (Nvrov et l., 212; Ding et l., 216; Hrtmnn et l., 217). The iosynthesis of Pip is fully epenent on ALD1 whih funtions s n -L-Lys minotrnsferse n genertes the iosyntheti intermeite 2,3-ehyropipeoli i (2,3-DP). 2,3-DP is susequently reue to Pip y SARD n nother reutse tivity (Nvrov et l., 212; Ding et l., 216; Hrtmnn et l., 217). Pip is further onverte y Flvinepenent monooxygense1 (FMO1) to N-hyroxypipeoli i (NHP), whih is ritil omponent for SAR tivtion (Chen et l., 218; Hrtmnn et l., 218). The umultion of Pip n NHP in pthogen-inoulte plnts is require for SAR, n exogenous pplition of Pip or NHP is suffiient to systemilly trigger immunity (Nvrov et l., 212; Vogel-Aghough et l., 213; Chen et l., 218; Hrtmnn et l., 218). The expression of ALD1 n SARD is positively regulte y the trnsription ftors SAR-DEFICIENT 1 (SARD1) n CALMODULIN BINDING PROTEIN 6g (CBP6g) (Sun et l., 215; Sun et l., 217), whih lso regulte expression of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), enoing n SA iosynthesis enzyme tht is require for SA proution upon pthogen infetion in A. 3

4 thlin (Wilermuth et l., 21; Zhng et l., 21; Wng et l., 211). It ws reently foun tht expression of SARD1 n CBP6g is regulte y the trnsription ftors TGA1, TGA, n WRKY7 (Sun et l., 217; Zhou et l., 217). Although SA is not the moile signl for SAR, it ontriutes to SAR (Vernooij et l., 199; Lwton et l., 1995; Prk et l., 27). SA is require for SAR in systemi leves ut not lol infete leves of too (Niotin tum) plnts (Vernooij et l., 199). Furthermore, SA ontriutes to SAR signl mplifition together with ALD1 n FMO1 in A. thlin systemi leves, exemplifying the importnt role of SA in systemi tissues for SAR (Bernsorff et l., 216). Previous reserh showe tht MPK3 n MPK6 n regulte immune responses reunntly with SA signling when they re tivte in sustine mnner ut not in trnsient mnner (Tsu et l., 213). Artifiil sustine tivtion of MPK3 n MPK6 triggere y exmethsone (DEX)-inue expression of MKK DD, onstitutively tive form of MAPK kinse tht n phosphorylte the ownstrem MPK3 n MPK6 (Ren et l., 22; Tsu et l., 213), ws suffiient to inue expression of SA-responsive genes without SID2 (Nwrth n Metrux, 1999; Wilermuth et l., 21; Tsu et l., 213). These results suggest tht MPK3 n MPK6 ontriute to SAR. Inee, it hs een shown tht MPK3 is require for SAR triggere y lol infetion with Pto AvrRpt2 (Bekers et l 29). However, the moleulr mehnism y whih the MAPK signling regulte the estlishment of SAR is yet unknown. Here, we show tht positive regultory loop for lol Pip umultion ontriutes to SAR in A. thlin. Sustine MAPK tivtion inues ALD1 expression vi WRKY33 to inrese lol Pip umultion. Pip pplition triggers tivtion of MPK3 n MPK6. MAPK tivtion uring Pto AvrRpt2 infetion is ompromise in wrky33, l1, n fmo1 mutnt plnts. These results suggest tht the regultory loop onsisting of MPK3/MPK6, WRKY33, ALD1, n Pip in lol leves plys ritil role in the estlishment of SAR when the MAPKs re lolly tivte in sustine mnner RESULTS Lol MAPK tivtion triggers SAR

5 SA pplition triggers SAR (Lwton et l., 1995), n MPK3 n MPK6 regulte immune responses reunntly with SA, when they re tivte in sustine mnner (Tsu et l., 213). Therefore, we hypothesize tht sustine MPK3/MPK6 tivtion in lol leves triggers SAR in systemi leves of A. thlin. Trnsgeni plnts expressing MKK DD (MKK DD ) uner the ontrol of DEX-inuile promoter (Ren et l., 22; Tsu et l., 213) were employe to investigte the effet of lolize MAPK tivtion. DEX tretment inue the expression of efense mrker genes PATHOGENESIS-RELATED 1 (PR1) n FLG22-INDUCED RECEPTOR-LIKE KINASE 1 (FRK1) in MKK DD plnts s well s in MKK DD si2 (Figure 1A). Interestingly, expression of ALD1 ws lso inue y tivtion of MPK3 n MPK6 in oth MKK DD n MKK DD si2 plnts (Figure 1A), pointing to role of MPK3/MPK6 in SAR estlishment without SA. Inee, we oserve tht SAR is triggere in MKK DD n to lesser extent, MKK DD si2 plnts fter DEX tretment in lol leves (Figure 1B), wheres no SAR ws oserve fter DEX tretment in, si2, n trnsgeni plnts hroring DEX-inuile GUS (GVG:GUS) (Figure 1B). We i not etet expression of the MKK DD or GUS trnsgene in systemi leves of MKK DD or GVG:GUS plnts, respetively, fter lol DEX pplition, suggesting tht DEX itself i not trnslote from lol leves to systemi leves (Supplementl Figure 1A n 1B). Thus, lol MAPK tivtion ppere to trigger SAR. Consistent with this, expression of PR1, FRK1, n ALD1, n SAR, were inue in oth n si2 plnts upon infetion with Pto AvrRpt2 (Figure 1C n 1D), whih triggers strong sustine MAPK tivtion (Tsu et l., 213) MAPK-meite SAR requires ALD1 Next, we teste whether known SAR omponents re require for the MAPK tivtion-triggere SAR. MKK DD plnts were rosse with fmo1, l1, n npr1 mutnts, in whih SAR ws shown to e roustly ompromise in vrious onitions (Co et l., 1997; Mishin n Zeier, 26; Bernsorff et l., 216; Hrtmnn et l., 218). SAR ssy fter lol DEX pplition showe tht FMO1, ALD1, n NPR1, ut not SID2 re require for the MAPK-meite SAR (Figure 2A). Immunolotting of MKK DD -flg, MPK3, n MPK6 showe tht the MKK DD inuile system is intt n MPK3 n MPK6 protein umultion remin unltere in these geneti kgrouns (Figure 2B). Notly, MAPK tivtion ws ompromise in l1 n 5

6 fmo1 kgrouns (Figure 2B), suggesting tht MAPK tivtion triggere y MKK DD requires the Pip pthwy. Consistent with this, we foun tht FMO1, ALD1, n NPR1 re require for SAR triggere y lol Pto AvrRpt2 infetion (Supplementl Figure 2A). MAPK-meite ALD1 (Figure 1A) n FMO1 inution (Figure A) prompte us to test whether MAPK tivtion triggers inrese Pip n NHP umultion in lol leves. Inee, Pip n NHP umultion ws inrese in lol leves of MKK DD plnts fter DEX pplition (Figure 2C). These results suggest tht MAPK tivtion inues ALD1 n FMO1 expression to inrese lol Pip n NHP umultion, therey ontriuting to SAR. Consiering tht Pip is metolize to NHP y FMO1 n tht Pip-inue responses require FMO1 (Chen et l., 218; Hrtmnn et l., 218), this inites tht NHP is the key signling moleule in MAPK-meite SAR. Consistent with previous reports (Ren et l., 28), umultion of the phytolexin mlexin lso inrese (Supplementl Figure 1C) MPK3 n MPK6 positively regulte Pip umultion upon infetion with Pto AvrRpt2 We investigte the geneti requirement of MPK3 or MPK6 for the estlishment of SAR. We first employe two systems to trigger SAR, lol infetion with Pto or Pto AvrRpt2, whih tivtes MPK3 n MPK6 in trnsient or sustine mnner, respetively (Tsu et l., 213). Upon lol infetion with Pto, SAR ws etete in, mpk3, n mpk6 ut not si2, mpk3 si2, n mpk6 si2 (Supplementl Figure 3A), suggesting tht SA is require for Pto-triggere SAR. In ontrst, upon lol infetion with Pto AvrRpt2, SAR ws oserve in, mpk3, mpk6, n si2 ut not in mpk3 si2 n mpk6 si2 (Figure 3A). Pto AvrRpt2-triggere inution of ALD1 n FMO1 in lol leves ws ompromise in mpk3, mpk6, mpk3 si2, n mpk6 si2, ut not in si2 (Figure 3B), pointing to the positive roles of MPK3 n MPK6 for lol ALD1 n FMO1 expression. As previously reporte (Tsu et l., 213), PR1 expression ws reunntly regulte y the MAPKs n SA (Figure 3B). Lol Pip umultion ws erese in mpk3 n mpk6 ompre to n in mpk3 si2 n mpk6 si2 ompre to si2 (Figure 3C), initing positive roles of MPK3 n MPK6 in Pip umultion. Pip umultion ws elevte in si2 ompre with Col- n in mpk3 si2 n mpk6 si2 ompre to mpk3 n mpk6, suggesting tht SA negtively regultes Pip umultion (Figure 3C). Curiously, mpk3 si2 n mpk6 6

7 si2 showe -like Pip umultion yet ompromise SAR (Figure 3A n 3C). Thus, the mount of lolly umulting Pip lone oes not explin the oserve SAR phenotypes MAPK-meite Pip umultion n SAR re ompromise in wrky33 The trnsription ftor WRKY33 regultes efense responses ginst wie rnge of pthogens (Zheng et l., 26; Liu et l., 215; Lio et l., 216; Liu et l., 217) n is tivte y MPK3 n MPK6 vi phosphoryltion (Mo et l., 211). Therefore, we hypothesize tht WRKY33 regultes MAPK-meite ALD1 expression n Pip umultion. Consistent with our hypothesis, levels of lol ALD1 expression s well s PR1 n FMO1 n Pip umultion fter MAPK tivtion were reue in wrky33 (Figure A n B) while MKK DD protein ws inue fter DEX tretment similrly to wil type kgroun (Figure 2B). MAPK-meite SAR ws lso prtilly ut signifintly reue in MKK DD wrky33 ompre to MKK DD plnts (Figure C), suggesting tht WRKY33 meites MAPK-regulte SAR vi ALD1 inution. Consistent with this, the levels of ALD1 expression s well s PR1 n FMO1, Pip umultion, n SAR were signifintly reue in wrky33 upon lol Pto AvrRpt2 infetion (Figure D n E). Similr to Figure 3, wrky33 si2 showe -like Pip umultion ut ompromise SAR n Pip umultion ws elevte in si2 ompre with n in wrky33 si2 ompre to wrky33 (Figure E n F). Moreover, the Pto-inue SAR fully epene on funtionl WRKY33 s well (Supplementl Figure 3B) SAR triggere y multiple stimuli onverges t WRKY33, ALD1, n FMO1 To etter unerstn the roles of the MAPK-WRKY33 n SA pthwys in SAR inue y ifferent stimuli, we lso investigte lol Pip umultion n SAR triggere y lol infetion with the teril strin Pseuomons syringe pv. muliol ES326 (Pm). Pm inoultion inues roust SAR in A. thlin n hs een use extensively to investigte the unerlying moleulr mehnisms (Mishin n Zeier, 26; Liu et l., 211; Bernsorff et l., 216; Hrtmnn et l., 218). Similr to Pto ut in ontrst to Pto vrrpt2 or Pm expressing vrrpm1, Pm i not trigger sustine MAPK tivtion in inoulte leves (Supplementl Figure A). Consistent 7

8 with previous finings (Bernsorff et l., 216), wek ut signifint SAR response ws oserve in si2 fter Pm inoultion, s well s in mpk3 si2 (Figure 5A). Thus, similr to SAR triggere y lol Pto infetion, the Pm-triggere SAR estlishment ws preominntly epenent on SA (Figure 5A). In ition, Pm inoultion triggere SAR in oth mpk3 n mpk6 to nerly sme levels with wil type (Figure 5A). This ws ompnie with wil type-like umultion of Pip in the lolly inoulte n systemi leves of oth mpk3 n mpk6 mutnts (Figure 5C). The Pminue iosynthesis of NHP n mlexin, however, ws speifilly reue in mpk3 (Figure 5C, Supplementl Figure B). Moreover, SAR triggere y Pm ws ttenute in wrky33 plnts (Figure 5B), n lol Pip n NHP, s well s systemi Pip umultion ws reue in wrky33 upon lol Pm infetion (Figure 5C). Thus, the sene of just MPK3 or MPK6 ppers to hve only minor effets on Pip umultion n SAR when lol MAPK tivtion is not sustine, s is the se for Pm- n Pto-inoultion. Nevertheless, WRKY33 plys ommon roles in SAR triggere y multiple pthogen stimuli irrespetive of the MAPK tivtion kinetis in lol leves (Figure F n 5B; Supplementl Figure B), suggesting tht WRKY33 tivity my lso e regulte y other ftors thn the MAPKs. The growth ssys with the ifferent teril strins lso inite WRKY33-inepenent signling rnh to SAR tht is tivte fter Pm inoultion n inues prtil SAR. Consistent with previous reports (Návrová et l., 212, Hrtmnn et l., 218), these ifferent signling pthwys leing to SAR inution onverge t ALD1 (Figure 2A, Supplementl Figure 2) WRKY33 ins to the ALD1 promoter Compromise ALD1 expression n Pip umultion triggere y MAPK tivtion in wrky33 (Figure A n B), suggests WRKY33 iretly regultes ALD1 expression. Inee, three W-oxes, the ining motif of WRKYs, were foun in the ALD1 promoter (Figure 6A). Therefore, we investigte WRKY33 ining to these W-oxes y hromtin immunopreipittion followe y quntittive PCR (ChIP-qPCR) in Pwrky33:WRKY33-HA plnts (Liu et l., 215). Aumultion of WRKY33 ws etete from 6 h post infiltrtion with Pto AvrRpt2 (Figure 6B). We etete strong enrihment of Pwrky33:WRKY33-HA for W-ox 2/3 ompre to ontrol ut not for W-ox 1 (Figure 6C). This result is onsistent with the lrge sle ChIP sequening 8

9 stuy showing tht WRKY33 ins to W-ox 2/3 ut not W-ox 1 in the ALD1 promoter (Birkenihl et l., 217). Thus, WRKY33 ppers to regulte ALD1 expression vi iret ining to the W-ox 2/3 in the ALD1 promoter Pip triggers MAPK tivtion n ALD1 is require for sustine MAPK tivtion upon Pto AvrRpt2 infetion Sine it is known tht efense tivtion often results in growth retrtion (Huot et l., 21), we investigte whether Pip ffets root growth. We foun tht Pip triggers root growth retrtion lthough its effet ws weker thn tht of flg22, MAMP n known inuer of root growth retrtion (Chinhill et l., 27) (Figure 7A). Root growth retrtion triggere y Pip s well s flg22 ws olishe in k1 kk1, whih is efiient in the o-reeptors of some memrne-lolize MAMP reeptors (Figure 7A). Moreover, Pip-triggere root growth retrtion ws not oserve with the isomeri n non-tive form of Pip (D-Pip) ut the tive form of Pip (L-Pip) (Nvrov et l., 212) (Figure 7A). We then explore the possiility tht Pip triggers MAPK tivtion. Strikingly, L-Pip ut not D-Pip triggere trnsient tivtion of MPK3 n MPK6 epenently on BAK1 BKK1 (Figure 7B; Supplementl Figure 5). These results my suggest tht Pip is sense y plnt memrne-lolize reeptor(s) to trigger MAPK tivtion. Sine the MAPK-WRKY33-ALD1 pthwy positively regulte Pip umultion uner sustine MAPK tivtion onitions (Figure 2B n B), these results lso suggest the existene of regultory loop for efense mplifition. If this hols true, MAPK tivtion shoul e ompromise in mutnt plnts efiient in this regultory loop. Inee, we oserve tht sustine lol MAPK tivtion triggere y Pto AvrRpt2 infetion ws ompromise in wrky33, l1, n fmo1 (Figure 7C n 7D), supporting the regultory loop onsisting of MPK3/MPK6, WRKY33, ALD1, FMO1, n Pip/NHP for SAR estlishment uner inution onitions involving lol sustine MAPK tivtion. This mplifition loop might e irumvente if Pip levels were elevte in the plnt to high levels y exogenous tretment. To test this hypothesis, we supplemente plnts with ose of 1 µmol Pip, tretment known to result in Pip ugmenttion in leves to SAR-like levels n in the inution of systemi immunity (Nvrov et l., 212; Vogel-Aghough et l., 213; Bernsorff et l., 216), n performe Pm growth ssy in, mpk3, mpk6, n wrky33 plnts. We oserve 9

10 signifint Pip-inue resistne ginst Pm in, s well s mpk3, mpk6, n wrky33 (Figure 7E), initing tht the MPK3/MPK6- n WRKY33-se regultory loop n e ypsse y high mounts of Pip. However, onsistent with our oservtion tht Pip-inue root growth inhiition n MAPK tivtion were ompromise in k1 kk1 mutnt plnts (Figure 7A n 7B), Pip-inue immunity ginst Pm require BAK1 BKK1 (Figure 7E) DISCUSSION In this stuy, we ientifie regultory loop for Pip umultion upon lol pthogen exposure whih ontriutes to SAR in A. thlin (Figure 7F). Our results n previous pulitions emonstrte tht (1) Lol MAPK tivtion triggers SAR; (2) Sustine MAPK tivtion inues ALD1 n FMO1 expression s well s Pip n NHP umultion; (3) Diret tivtion of WRKY33 y MPK3 n MPK6 ws previously shown (Mo et l., 211); () WRKY33 iretly regultes ALD1 expression; (5) MAPKmeite SAR is ompromise in wrky33, l1, n fmo1 mutnt plnts; (6) Pip triggers MAPK tivtion; (7) Sustine MAPK tivtion triggere y Pto AvrRpt2 is ompromise in wrky33, l1, n fmo1 mutnts. Thus, the positive regultory loop onsisting of MPK3/MPK6, WRKY33, ALD1, FMO1, n Pip/NHP ontriutes to the estlishment of SAR triggere y lol sustine MAPK tivtion. The SAR proesses n e ivie into three steps; lol immune tivtion, informtion rely from lol to systemi tissues y moile signl(s), n efense tivtion n priming in systemi tissues (Jung et l., 29; Shh n Zeier, 213). In this stuy, we fouse on lol immune tivtion importnt for SAR estlishment. We showe tht rtifiil lol tivtion of MPK3 n MPK6 y the MKK DD system is suffiient to trigger SAR (Figure 1). Geneti requirement of MPK3 n MPK6 for SAR ws lso etete when SAR is tivte y lol Pto AvrRpt2 infetion, whih triggers sustine MAPK tivtion (Figure 3). In ontrst, SAR preominntly epens on SA when it is tivte y lol infetion with Pto n Pm, oth of whih o not trigger sustine MAPK tivtion [Figure 5A; Supplementl Figure 3A; Supplementl Figure A; (Tsu et l., 213)]. Thus, the regultory loop for SAR ientifie in this stuy my kik in n ply ritil roles in SAR when lol MAPK tivtion is sustine. 1

11 ALD1 is ommonly require for SAR triggere y lol infetion with Pm n Pto AvrRpt2 n y lol MAPK tivtion s well s for systemi immunity inue y β-minoutyri i n zeli i (Figure 2A; Supplementl Figure 2) (Zimmerli et l., 2; Jung et l., 29; Nvrov et l., 212). ALD1- n FMO1-meite Pip n NHP proution, respetively, re highly inue uring immunity (Nvrov et l., 212; Hrtmnn et l., 217; Hrtmnn et l., 218). Thus, the regultion of pthogeninue ALD1 n FMO1 expression is ruil for SAR. We showe tht WRKY33 positively regultes ALD1 vi its iret ining to the ALD1 promoter to inrese Pip umultion (Figure 6). Inution of ALD1 expression n Pip umultion ws not totlly ompromise in wrky33 (Figure A n B), suggesting tht other trnsription ftors lso ontriute to ALD1 expression. Inee, the trnsription ftors SARD1 n CBP6g iretly regulte expression of ALD1 s well s SARD (Sun et l., 215; Sun et l., 217). SARD enoes the ehyropipeolte reutse enzyme tht reues ALD1-proue 2,3-ehyropipeoli i to Pip (Ding et l., 216; Hrtmnn et l., 217). More reently, it ws shown tht trnsription ftors TGA1 n TGA iretly regulte the expression of SARD1 n CBP6g n tht Pip umultion upon Pm infetion is signifintly reue ut not olishe in tg1 tg n sr1 p6g mutnts (Sun et l., 217). Furthermore, SAR ws olishe in wrky33 when it is tivte y lol Pto infetion irrespetive to moes of MAPK tivtion (Figure n Supplementl Figure 3B), wheres SAR tivte y lol Pm infetion ws ttenute ut not fully olishe in wrky33 (Figure 5B). These results suggest tht oth TAG1/TAG-SARD1/CBP6g n WRKY33 regulte Pip umultion n SAR triggere y lol Pm infetion while WRKY33 is the mjor regultor of SAR triggere y lol Pto infetion. Our stuy thus inites the existene of istint rnhes of SAR signling tht re ifferentilly tivte y ifferent pthogen types. These signling rnhes onverge t ALD1 n Pip proution (Figure 7F). SAR inution lso epens on FMO1 for oth MAPK tivting Pto AvrRpt2 n non-tivting Pm (Supplementl Figure 2) (Nvrov et l., 212), supporting the fining tht Pip to NHP onversion y FMO1 is ritil step for SAR tivtion (Hrtmnn et l., 218). SA is require for SAR in systemi leves ut not lol infete leves of too plnts (Vernooij et l., 199). Furthermore, SA ontriutes to SAR signl mplifition together with ALD1 n FMO1 in A. thlin systemi leves (Bernsorff et l., 216). These results suggest tht SA is n importnt omponent for SAR in systemi tissues ut not lol infete tissues. Previous results inite tht the SA- 11

12 efiient si2 mutnt strongly overproues NHP upon Pm infetion, suggesting tht SA negtively moultes levels of NHP (Hrtmnn et l., 218). Similrly, we foun here tht SA ts s negtive regultor of Pto AvrRpt2-triggere lol Pip umultion (Figure 3C n E). This elevte Pip umultion ppers to e suffiient for Pto AvrRpt2-triggere SAR in the sene of SID2-proue SA (Figure 3A n 3C). However, mpk3 si2 n mpk6 si2, whih umultes wil type levels of Pip in lol leves, i not trigger SAR fter Pto AvrRpt2 infetion (Figure 3A n 3C). One explntion for these oservtions is tht elevte Pip/NHP levels ut not wil type levels of Pip in lol leves re suffiient for SAR signl mplifition in systemi leves without SA. Thus, the strength of SAR ppers to e etermine y tivities of lol Pip/NHP pthwy n systemi SA pthwy. Interestingly, WRKY33 negtively regultes SA umultion n signling (Birkenihl et l., 212; Liu et l., 215; Birkenihl et l., 217). Consistent with this, we foun tht ompre with wil type plnts, wrky33 plnts re more resistnt ginst Pm, whih is sensitive to SAmeite immunity (Figure 5B). Thus, WRKY33 is negtive regultor of lol efense vi SA suppression n positive regultor of SAR vi Pip umultion, exemplifying tht regultions of lol immunity n SAR re tightly linke. Pip-triggere MAPK tivtion, root-growth inhiition, n Pip-inue SAR require BAK1 n BKK1 (Figure 7A, 7B, n 7E). BAK1 n BKK1 elong to the SOMATIC EMBRYOGENESIS RECEPTOR KINASEs (SERKs) tht funtion s oreeptors for the reognition of multiple MAMPs s well s plnt-erive mgessoite moleulr ptterns (DAMPs) on the plsm memrne (Chinhill et l., 27; Heese et l., 27; Shn et l., 28; Krol et l., 21). The o-reeptors BAK1 n BKK1 funtion with reeptors elonging to RLKs or RLPs. Therefore, it is tempting to speulte tht Pip or perhps Pip-erive prout is sense y RLKs or RLPs together with BAK1/BKK1, therey triggering MAPK tivtion s esrie for flg22 reognition y FLS2 (Asi et l., 22; Bekers et l., 29). Reently, n NHP-hexose onjugte tht umultes epenently on ALD1 n FMO1 in Pm-inoulte leves ws etete in A. thlin (Hrtmnn n Zeier, 218). Forwr/reverse geneti sreens n genome-wie ssoition nlysis using iverse A. thlin essions might help to ientify reeptor(s) for Pip, NHP, or further NHP erivtive(s) suh s NHP-hexose, whih my funtion s moile metolites involve in SAR longistne signling (Chen et l., 218; Hrtmnn n Zeier, 218). In A. thlin, the DAMP reeptors PEPR1 n PEPR2 reognize enogenous PROPEP-erive Pep 12

13 epitopes tht tivte immunity n funtion together with BAK1/BKK1 (Boller n Felix, 29; Ymguhi et l., 21; Ym et l., 216). Interestingly, mutnt plnts efiient in PEPR1 n PEPR2 showe ompromise SAR phenotypes triggere y lol infetion with Pto AvrRpm1 tht triggers strong sustine MAPK tivtion (Ross et l., 21). Likewise, pplition of Pep epitopes tivtes immune responses suh s MAPK tivtion (Ym et l., 216). Thus, the SAR inuer Pip n the DAMPs Pep epitopes re oth enogenously proue in plnts n tivte immunity n SAR, whih reners the ifferene of DAMPs n SAR-relte moleules miguous. Further reserh will e require to fully estlish the ifferene n similrity etween DAMPs n SAR inuers. Pip triggers trnsient tivtion of MPK3 n MPK6 (Figure 7B n Supplementl Figure 5) yet ALD1 n FMO1 ontriute to sustine MAPK tivtion fter Pto AvrRpt2 infetion (Figure 7C). One explntion for this is tht sustine MAPK tivtion is hieve y multiple signl inputs inluing Pip/NHP n others. Alterntive ut not exlusive explntion is tht Pip/NHP triggers trnsient tivtion of the MAPKs in ifferent ells t ifferent time points, resulting in sustine MAPK tivtion in lol infete leves. Mesuring the temporl ynmis of the MAPK tivities t the single ell resolution woul help solve this issue. The oservtion tht MAPK tivtion triggere y MKK DD requires ALD1 n FMO1 (Figure 2B) ws rther surprising to us euse MKK DD woul e le to iretly phosphorylte MPK3 n MPK6 without other omponents. However, this suggests tht MKK DD requires itionl omponents whose tivity epens on Pip/NHP to hieve sustine tivtion of MPK3 n MPK6 in plnts. We speulte tht Pip/NHP my onition the proper formtion of MKKDD-MPK3/MPK6 omplex through, for instne, ffeting the suellulr loliztion of MKK DD, MPK3, n MPK6. Alterntively, MKK DD triggers initil phosphoryltion of MPK3 n MPK6, whih then triggers sustine tivtion of MPK3 n MPK6 epenently on Pip/NHP. Reent work showe tht MPK6 phosphoryltes the upstrem MAPK kinse kinse MAPKKK5 to enhne tivtion of MPK3 n MPK6 (Bi et l., 218). Thus, Pip/NHP signling my ensure, for instne, expression of MAPKKK5 n this positive feek mehnism my e require for MKK DD -triggere sustine tivtion of MPK3 n MPK6. Nevertheless, this speultion nees to e experimentlly teste. 2 13

14 MATERIALS AND METHODS Plnt mterils n growth onitions Ariopsis thlin plnts were grown in hmer t 22ºC with 1 h light (white fluoresene lmps) perio n 6% reltive humiity. The A. thlin ession Col- ws use s the wil-type. A. thlin mutnts n trnsgeni lines, si2-2 (Wilermuth et l., 21), mpk3-1 (Wng et l., 27), mpk6-2 (Liu n Zhng, 2), mpk3-1 si2-2, mpk6-2 si2-2 (Tsu et l., 213), l1-t2 (Mishin n Zeier, 26), fmo1-1 (Nvrov et l., 212), npr1-1 (Co et l., 1997), wrky33-2 (Zheng et l., 26), Pwrky33:WRKY33-HA (Liu et l., 215), MKK DD, MKK DD si2 (Ren et l., 22), MKK DD npr1 (Tsu et l., 213), n rpm1-3 rps2 11C (Belkhir et l., 2) were previously esrie. The MKK DD fmo1, MKK DD l1, n MKK DD wrky33 mutnts were generte y rossing MKK DD with fmo1, l1, n wrky33. The wrky33 si2 oule mutnt ws generte y rossing wrky33-2 with si2-2. The primers n methos use for mutnt genotyping re liste in Supplementl Tle Bteril ultivtion n inoultion Pseuomons syringe pv. tomto DC3 hroring empty vetor (Pto) or AvrRpt2 (Pto AvrRpt2) ws ultivte s esrie (Tsu et l., 213). Bteril ells were wshe with H2O, ilute to the pproprite ensity, n infiltrte into A. thlin leves using neeleless syringe. Similrly, Pseuomons syringe pv. muliol ES326 (now lssifie s Pseuomons nnin pv. lislensis ES326), rrying either none trnsgene (Pm), vrrpm1 (Pm AvrRpm1), or the luxcdabe operon of Photorhus luminesens (Pm lux) were grown in King s B meium ontining 5 µg/ml rifmpiin. For Pm AvrRpm1 15 µg/ml tetryline n for Pm lux 5 µg/ml knmyin were itionlly e to the meium. Bteri from overnight ultures were wshe three times with 1 mm MgCl2 efore justing to the pproprite ensity Bteril growth ssy To inue SAR with Pto or Pto AvrRpt2, teril suspension ws infiltrte into three lol leves of -week-ol A. thlin plnts. Sterilize wter ws infiltrte s mok ontrol. Systemi leves were inoulte with Pto 2 h post lol infiltrtion. The teril titer in systemi leves ws etermine 2 ys post systemi infiltrtion. For 1

15 Pm-inue SAR, three lol leves of -5 week ol Ariopsis plnts were infiltrte with either mok (1 mm MgCl2) or Pm. Two ys fter tretment, three systemi leves were infiltrte with Pm lux. Bteril growth in systemi leves ws ssesse two ys post systemi infiltrtion vi luminesene s esrie in Hrtmnn et l. (217). To ssess Pip-inue resistne to Pm, plnts were wtere with 1 ml of 1 mm Pip or wter one y prior to infiltrtion of three rosette leves with Pm lux s esrie in (Nvrov et l., 212). Log1-trnsforme olony-forming units (fu) per m 2 lef surfe re or reltive luminesene light units (rlu) per m 2 were lulte n the following moel ws fit to the t; CFUgyr = GYgy+Rr+egyr, where GY, genotype:tretment intertion, n rnom ftors; R, iologil replite; e, resiul. The men estimtes of the fixe effets were use s the moele teril titers n ompre y two-tile t-test RNA isoltion n quntittive RT-PCR nlysis Totl RNA ws isolte from plnt smples using TRIzol regent (Thermo Fisher Sientifi) following the mnufturer s instrutions. Five mirogrm of totl RNA were reverse trnsrie using SuperSript II first-strn synthesis system (Thermo Fisher Sientifi) with n oligo(t) primer. Rel-time DNA mplifition ws monitore using Bio-R iq5 optil system softwre (Bio-R). The expression level of genes of interest ws normlize to tht of the enogenous referene gene ACTIN2. Primers use re liste in Supplementl Tle 1. The following moels were fit to the reltive Ct vlue t ompre to ACTIN2: Ctgyr = GYgy+Rr+egyr, where GY, genotype:tretment intertion, n rnom ftors; R, iologil replite; e, resiul; Ctytr = YTyt+Rr+eytr, where YT, tretment:time intertion; Ctgytr = GYTgyt+Rr+egytr, where GYT, genotype:tretment:time intertion. The men estimtes of the fixe effets were use s the moele reltive Ct vlues, visulize s the reltive log2 expression vlues n ompre y two-tile t-test Metolite quntifition Determintion of pipeoli i levels in leves ws performe using protool etile in (Nvrov et l., 212) using GC/MS-se nlysis following propyl hloroformte erivtistion. Cmlexin ws etermine y metho se on vpor- 15

16 phse extrtion n GC/MS nlysis of metolites s esrie previously (Attrn et l., 29; Nvrov et l., 212). Determintion of N-hyroxypipeoli i levels in leves ws performe using protool etile in (Hrtmnn et l., 218) using GC/MS-nlysis of lef extrts fter trimethylsilyltion of nlytes with N-methyl-Ntrimethylsilyltrifluoroetmie Growth suppression n MAP kinse ssy A. thlin sees were germinte on ½ MS pltes (.5 x MS slt, 1% w/v surose,.8% gr), n the 3-y-ol seelings with similr size were trnsferre on ½ MS plte with or without 1 µm L-pipeoli i (Sigm), D-pipeoli i (Sigm), or 1 µm flg22. Primry root length ws mesure seven ys fter the trnsfer. MAP kinse ssys were performe s esrie previously (Lee n Ellis, 27). Briefly, ten-yol seelings were trnsferre to 12-well pltes (3 seelings per well) ontining 2 ml of liqui MS meium with wter (mok), 1 µm L-pipeoli i, 1 µm D-pipeoli i, n 1 µm flg22. Seelings were frozen in liqui nitrogen t the inite time points. The frozen seelings were groun in liqui nitrogen n homogenize in MPK extrtion uffer (1 mm HEPES, ph 7.5, 5 mm EDTA, 5 mm EGTA, 2 mm DTT, protese inhiitor oktil (Rohe Applie Siene, Ininpolis, IN, USA) n phosphtse inhiitor oktil (Rohe Applie Siene)). The superntnt ws ollete fter entrifugtion t 12 rpm for 3 min t C. The protein onentrtion ws etermine using Brfor ssy (BIO-RAD, Herules, CA, USA) with ovine serum lumin s stnr. Five mirogrms of protein ws seprte in 12% SDS-PAGE. Immunolot nlysis ws performe using nti-phospho-p/2 MAPK (α-ptepy, 1:5, Cell Signling Tehnology) s the primry ntioy, n peroxise-onjugte got nti-rit IgG (1:2,, Sigm) s the seonry ntioy Chromtin Immunopreipittion (ChIP)-qPCR Four-week-ol n Pwrky33:WRKY33-HA plnts were infiltrte with Pto AvrRpt2 (OD=.1) or mok n the smples were ollete t 2 h post infiltrtion. ChIP ssy ws performe s esrie previously (Ymguhi et l., 21) using 16

17 rit polylonl nti-ha ntioy. ALD1 speifi primers esrie in Supplementl Tle 1 were use for qpcr nlysis s esrie ove Sttistil nlyses Sttistil nlysis ws performe using the mixe liner moel funtion (lmer) implemente in the pkge lme in the R environment. When pproprite, rw t were log trnsforme to meet the ssumptions of the mixe liner moel. For the t- tests, the stnr errors were lulte using the vrine n ovrine vlues otine from the moel fitting. The Benjmini-Hoherg metho ws pplie to orret for multiple hypothesis testing when ll pirwise omprisons of the men estimtes were me Aession Numers The ession numers for the genes isusse in this rtile re s follows: At2g161 (PR1), AT2G1919 (FRK1), AT1G771 (SID2), AT1G1925 (FMO1), AT2G1381 (ALD1), AT2G387 (WRKY33), AT1G628 (NPR1), AT1G5166 (MKK), AT3G565 (MPK3), AT2G379 (MPK6), AT3G1878 (ACTIN2) Supplementl Dt Supplementl Figure 1. Lol DEX pplition oes not tivte systemi tivtion of GVG system. Supplementl Figure 2. Pto AvrRpt2-triggere SAR requires ALD1. Supplementl Figure 3. SA n WRKY33 ontriute to SAR triggere y Pto infetion. Supplementl Figure. MAPK tivtion n mlexin umultion y Pm infetion. Supplementl Figure 5. Pipeoli i triggers trnsient MAPK tivtion. Supplementl Tle 1. Primers use in this stuy

18 Aknowlegements We thnk Dr. Imre Somssih for wrky33, Pwrky33:WRKY33-HA mutnts. This work ws supporte y the Mx Plnk Soiety n Deutshe Forshungsgemeinshft grnt SFB67 for K.T., n grnt to J.Z. y the Germn Reserh Fountion (ZE67/6-1). Y.W. ws supporte y Mx Plnk Soiety n Alexner von Humolt-Fountion Author ontriutions YW, JZ, KT oneive n esigne the experiments; YW, SS, JW, PY, ACD performe experiments; YW, JZ, n KT nlyze the t; YW, SS, JZ n KT wrote the pper. All uthors ommente on the mnusript Conflit of interest The uthors elre no ompeting finnil interests. 18

19 Figure legens Figure 1. MAPK-meite SAR is lrgely inepenent of SA. (A) n (C) Expression levels of PR1, FRK1, n ALD1 reltive to ACTIN2 in -week-ol leves etermine y RT-qPCR. Leves of DEX-inuile GUS (GUS), MKK DD, n MKK DD si2 plnts were hrveste 2 h fter infiltrtion with 1 µm DEX or mok (A) n of n si2 2 h fter infiltrtion with Pto AvrRpt2 (OD6=.1) or mok (C). (B) n (D) Bteril titers in systemi leves. Primry leves of, si2, GUS, MKK DD, n MKK DD si2 were infiltrte with 1 µm DEX or mok (B) n of n si2 with Pto AvrRpt2 (OD6=.1) or mok (D). After 1 y, systemi leves were infiltrte with Pto (OD6=.1), n teril titers in the systemi leves were mesure t 2 ys post systemi infetion. (A) to (D) Brs represent mens n stnr errors lulte from three inepenent experiments eh with three iologil replites using mixe liner moel. (, P <.1; two-tile Stuent s t- tests. n.s., not signifint) Figure 2. MAPK-meite SAR requires ALD1. (A) Bteril titers in systemi leves of GUS, MKK DD, MKK DD si2, MKK DD fmo1, MKK DD l1, n MKK DD npr1. Primry leves were infiltrte with 1 µm DEX or mok. After 1 y, systemi leves were infiltrte with Pto (OD6=.1), n teril titers in the systemi leves were mesure t 2 ys post systemi infetion. Brs represent mens n stnr errors lulte from three inepenent experiments eh with three iologil replites using mixe liner moel. inites signifint ifferene from Mok (P <.1; two-tile Stuent s t-tests). n.s., not signifint (B) Phosphoryltion of MPK3 n MPK6, n protein level umultion of MKK DD, MPK3, n MPK6 in lol leves of MKK DD, MKK DD si2, MKK DD fmo1, MKK DD l1, MKK DD npr1, n MKK DD wrky33 plnts t the inite time points fter infiltrtion with 1 µm DEX. (C) Pipeoli i n N-hyroxypipeoli i levels in lol leves of MKK DD plnts infiltrte with 1 µm DEX or mok t the inite time points. N.D. inites uner the etetion limit. Brs represent mens n stnr errors lulte from three inepenent experiments. inites signifint ifferene from hpi (P <.1; two-tile Stuent s t-tests)

20 Figure 3. MPK3 n MPK6 positively regulte Pip umultion uring Pto AvrRpt2 infetion. (A) Bteril titers in systemi leves of, mpk3, mpk6, si2, mpk3 si2, n mpk6 si2. Primry leves were infiltrte with Pto AvrRpt2 (OD6=.1) or mok. After 1 y, systemi leves were infiltrte with Pto (OD6=.1), n teril titers were mesure t 2 ys post systemi infetion. Brs represent mens n stnr errors lulte from six inepenent experiments eh with four iologil replites using mixe liner moel. (B) Expression of PR1, ALD1, n FMO1 in, mpk3, mpk6, si2, mpk3 si2, n mpk6 si2 t 2 h post infiltrtion with Pto AvrRpt2 (OD6=.1) or mok etermine y RT-qPCR. Brs represent mens n stnr errors of the log2 expression levels reltive to ACTIN2 lulte from three inepenent experiments eh with three iologil replites using mixe liner moel. (C) Lol Pip umultion in leves of, mpk3, mpk6, si2, mpk3 si2, n mpk6 si2 t 2 h post infiltrtion with Pto AvrRpt2 (OD6=.1) or mok. Brs represent mens n stnr errors of four inepenent iologil replites. Sttistil ifferenes were lulte using mixe liner moel followe y two-tile Stuent s t-tests. (A) to (C) Different letters ove the rs enote sttistilly signifint ifferenes (juste P <.5). Upperse letters inite omprisons etween genotypes for SAR effets Figure. MAPK-meite Pip umultion n SAR re ompromise in wrky33. (A) n (D) PR1, ALD1, n FMO1 expression in leves of MKK DD n MKK DD wrky33 t 2 h fter infiltrtion with 1 µm DEX or mok etermine y RTqPCR (A) n of, wrky33, si2 n wrky33 si2 t 2 h fter infiltrtion with Pto AvrRpt2 (OD6=.1) or mok (D). Brs represent mens n stnr errors lulte from two inepenent experiments eh with three iologil replites using mixe liner moel. (B) n (E) Pip umultion in leves of MKK DD n MKK DD wrky33 t 2 h fter infiltrtion with 1 µm DEX (B) n of, wrky33, si2 n wrky33 si2 t 7 h, 12 h, n 2 h fter infiltrtion with Pto AvrRpt2 (OD6=.1) or mok (E). Brs represent mens n stnr errors lulte from five (B) or three (E) inepenent iologil replites. (E) Sttistil ifferenes were lulte using mixe liner moel followe y two-tile Stuent s t-tests. (C) n (F) Bteril titers in systemi leves of MKK DD n MKK DD wrky33 (C) n of, wrky33, si2 n wrky33 si2 (F). Primry leves were infiltrte with 1 µm DEX or 2

21 mok (C) n with Pto AvrRpt2 (OD6=.1) or mok (F). After 1 y, systemi leves were infiltrte with Pto (OD6=.1), n teril titers in the systemi leves were mesure t 2 ys post systemi infetion. Brs represent mens n stnr errors lulte from t lest four inepenent experiments eh with three iologil replites using mixe liner moel. (A) to (F) Different letters ove the rs enote sttistilly signifint ifferenes (juste P <.5)., P <.1; two-tile Stuent s t-tests. n.s., not signifint Figure 5. SA n WRKY33 ontriute to SAR triggere y Pm infetion. (A) n (B) Bteril titers in systemi leves of, mpk3, mpk6, si2, mpk3 si2, n mpk6 si2 (A) n of n wrky33 (B). Primry leves were infiltrte with Pm (OD6=.5) or mok. After 2 ys, systemi leves were infiltrte with Pm lux (OD6=.1), n the ioluminesene of Pm lux ws etermine t 6 h post systemi infetion. Brs represent mens n stnr errors lulte from t lest four inepenent experiments eh with three iologil replites using mixe liner moel. (C) Pip umultion in lol leves t 2 h n systemi leves t 8h, n N-hyroxypipeoli i umultion in lol leves of, mpk3, mpk6, n wrky33 t 2 h post infiltrtion with Pm (OD6=.1) or mok (1 mm MgCl2). Brs represent mens n stnr errors of three iologil replites. N.D. inites uner the etetion limit. Sttistil ifferenes were lulte using mixe liner moel followe y two-tile Stuent s t-tests. (A) to (C) Different letters ove the rs enote sttistilly signifint ifferenes (juste P <.5). Upperse letters inite omprisons etween genotypes for SAR effets., P <.1; two-tile Stuent s t-tests Figure 6. WRKY33 ins to ALD1 promoter. (A) Shemti igrm of ALD1 promoter. The vertil lk rs represent W-oxes. The horizontl lines show the regions mplifie y ifferent qpcr primers. (B) Protein umultion of WRKY33 in WRYK33-HA wrky33 plnts fter infiltrtion with Pto AvrRpt2 (OD6=.1) or mok t the inite time points visulize y immunolotting using nti-ha ntioy. Poneu S-stine RuBisCo is shown s loing ontrol. (C) ChIP-qPCR ws performe using n WRYK33-HA wrky33 t 1 y fter infiltrtion with Pto AvrRpt2 (OD6=.1). Brs represent mens n stnr errors of the fol 21

22 enrihment reltive to (set to 1), lulte from three inepenent iologil replites (, P-vlue <.1, two-tile Stuent s t-tests. n.s., not signifint) Figure 7. Pip triggers MAPK tivtion. (A) n k1 kk1 plnts were grown on ½ MS meium ontining 1 µm flg22, 1 µm L-Pip, 1 µm D-Pip, or mok, n primry root length ws mesure t 1 ys ol. Brs represent mens n stnr errors lulte from four inepenent iologil replites using mixe liner moel. (B) MAPK tivtion in 1-y-ol seelings of n k1 kk1. Seelings were ollete t 15 min fter the tretment with 1 µm flg22, 1 µm L-Pip, 1 µm D-Pip, or mok. (C) n (D) MAPK tivtion in leves of -week-ol, wrky33, l1, n fmo1 fter infiltrtion with Pto AvrRpt2 (OD6=.1), n smples were ollete t the inite time points. (B) to (D) Proteins were etete y immunolotting using the inite ntioies. Poneu S-stine RuBisCo is shown s loing ontrol. Similr results were oserve in three inepenent experiments. (E) Bteril titers in leves of, mpk3, mpk6, wrky33, n k1 kk1. Five-week-ol plnts were supplie with 1 ml of 1 mm Pip (osge of 1 µmol) or wter vi the root system. Three leves per plnt were infiltrte with Pm lux (OD6=.1) t 1 y post tretment, n reltive luminesene light units (rlu) per m 2 (log1) were mesure t 6 hours post systemi infetion. Brs represent mens n stnr errors of t lest three inepenent iologil replites using mixe liner moel. (A) n (E) Different letters ove the rs enote sttistilly signifint ifferenes (juste P <.1). (F) Moel for the immune-mplifition loop onsisting of MPK3/6, WRKY33, ALD1, n pipeoli i Referenes Asi, T., Ten, G., Plotnikov, J., Willmnn, M.R., Chiu, W.L., Gomez-Gomez, L., Boller, T., Ausuel, F.M., n Sheen, J. (22). MAP kinse signlling se in Ariopsis innte immunity. Nture 15, Attrn, E., Zeier, T.E., Grieel, T., n Zeier, J. (29). Methyl Sliylte Proution n Jsmonte Signling Are Not Essentil for Systemi Aquire Resistne in Ariopsis. Plnt Cell 21,

23 Axtell, M.J., n Stskwiz, B.J. (23). Initition of RPS2-speifie isese resistne in Ariopsis is ouple to the AvrRpt2-irete elimintion of RIN. Cell 112, Bekers, G.J.M., Jskiewiz, M., Liu, Y.D., Unerwoo, W.R., He, S.Y., Zhng, S.Q., n Conrth, U. (29). Mitogen-Ativte Protein Kinses 3 n 6 Are Require for Full Priming of Stress Responses in Ariopsis thlin. Plnt Cell 21, Belkhir, Y., Nimhuk, Z., Huert, D.A., Mkey, D., n Dngl, J.L. (2). Ariopsis RIN negtively regultes isese resistne meite y RPS2 n RPM1 ownstrem or inepenent of the NDR1 signl moultor n is not require for the virulene funtions of teril type III effetors AvrRpt2 or AvrRpm1. Plnt Cell 16, Bernsorff, F., Doring, A.C., Gruner, K., Shuk, S., Brutigm, A., n Zeier, J. (216). Pipeoli Ai Orhestrtes Plnt Systemi Aquire Resistne n Defense Priming vi Sliyli Ai-Depenent n -Inepenent Pthwys. Plnt Cell 28, Bi, G., Zhou, Z., Wng, W., Li, L., Ro, S., Wu, Y., Zhng, X., Menke, F.L.H., Chen, S., n Zhou, J.M. (218). Reeptor-Like Cytoplsmi Kinses Diretly Link Diverse Pttern Reognition Reeptors to the Ativtion of Mitogen- Ativte Protein Kinse Cses in Ariopsis. Plnt Cell 3, Birkenihl, R.P., Diezel, C., n Somssih, I.E. (212). Ariopsis WRKY33 is key trnsriptionl regultor of hormonl n metoli responses towr Botrytis inere infetion. Plnt Physiol 159, Birkenihl, R.P., Krher, B., Roro, M., n Somssih, I.E. (217). Inue genome-wie ining of three Ariopsis WRKY trnsription ftors uring erly MAMP-triggere immunity (vol 29, pg 2, 217). Plnt Cell 29, Boller, T., n Felix, G. (29). A Renissne of Eliitors: Pereption of Miroe- Assoite Moleulr Ptterns n Dnger Signls y Pttern-Reognition Reeptors. Annul Review of Plnt Biology 6, Boutrot, F., n Zipfel, C. (217). Funtion, Disovery, n Exploittion of Plnt Pttern Reognition Reeptors for Bro-Spetrum Disese Resistne. Annul Review of Phytopthology, Vol 55 55,

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26 Lee, J.S., n Ellis, B.E. (27). Ariopsis MAPK phosphtse 2 (MKP2) positively regultes oxitive stress tolerne n intivtes the MPK3 n MPK6 MAPKs. J Biol Chem 282, Li, G.J., Meng, X.Z., Wng, R.G., Mo, G.H., Hn, L., Liu, Y.D., n Zhng, S.Q. (212). Dul-Level Regultion of ACC Synthse Ativity y MPK3/MPK6 Cse n Its Downstrem WRKY Trnsription Ftor uring Ethylene Inution in Ariopsis. Plos Genet 8. Lio, C.J., Li, Z., Lee, S., Yun, D.J., n Mengiste, T. (216). Ariopsis HOOKLESS1 Regultes Responses to Pthogens n Asisi Ai through Intertion with MED18 n Aetyltion of WRKY33 n ABI5 Chromtin. Plnt Cell 28, Liu, P.P., von Dhl, C.C., n Klessig, D.F. (211). The Extent to Whih Methyl Sliylte Is Require for Signling Systemi Aquire Resistne Is Depenent on Exposure to Light fter Infetion. Plnt Physiol 157, Liu, S., Ziegler, J., Zeier, J., Birkenihl, R.P., n Somssih, I.E. (217). Botrytis inere B5.1 promotes isese evelopment in Ariopsis y suppressing WRKY33-meite host immunity. Plnt Cell Environ, Liu, S.A., Krher, B., Ziegler, J., Birkenihl, R.P., n Somssih, I.E. (215). Negtive regultion of ABA signling y WRKY33 is ritil for Ariopsis immunity towrs Botrytis inere 21. Elife. Liu, Y.D., n Zhng, S.Q. (2). Phosphoryltion of 1-minoylopropne-1- roxyli i synthse y MPK6, stress-responsive mitogen-tivte protein kinse, inues ethylene iosynthesis in Ariopsis. Plnt Cell 16, Mkey, D., Holt, B.F., Wiig, A., n Dngl, J.L. (22). RIN interts with Pseuomons syringe type III effetor moleules n is require for RPM1- meite resistne in Ariopsis. Cell 18, Mkey, D., Belkhir, Y., Alonso, J.M., Eker, J.R., n Dngl, J.L. (23). Ariopsis RIN is trget of the type III virulene effetor AvrRpt2 n moultes RPS2-meite resistne. Cell 112, Mo, G.H., Meng, X.Z., Liu, Y.D., Zheng, Z.Y., Chen, Z.X., n Zhng, S.Q. (211). Phosphoryltion of WRKY Trnsription Ftor y Two Pthogen- Responsive MAPKs Drives Phytolexin Biosynthesis in Ariopsis. Plnt Cell 23,

27 Meng, X.Z., n Zhng, S.Q. (213). MAPK Cses in Plnt Disese Resistne Signling. Annu Rev Phytopthol 51, Mishin, T.E., n Zeier, J. (26). The Ariopsis flvin-epenent monooxygense FMO1 is n essentil omponent of iologilly inue systemi quire resistne. Plnt Physiol 11, Nvrov, H., Bernsorff, F., Doring, A.C., n Zeier, J. (212). Pipeoli Ai, n Enogenous Meitor of Defense Amplifition n Priming, Is Critil Regultor of Inuile Plnt Immunity. Plnt Cell 2, Nwrth, C., n Metrux, J.P. (1999). Sliyli i inution-efiient mutnts of Ariopsis express PR-2 n PR-5 n umulte high levels of mlexin fter pthogen inoultion. Plnt Cell 11, Pjerowsk-Mukhtr, K.M., Emerine, D.K., n Mukhtr, M.S. (213). Tell me more: roles of NPRs in plnt immunity. Trens Plnt Si 18, Prk, S.W., Kimoyo, E., Kumr, D., Mosher, S., n Klessig, D.F. (27). Methyl sliylte is ritil moile signl for plnt systemi quire resistne. Siene 318, Ren, D., Liu, Y., Yng, K.Y., Hn, L., Mo, G., Glzerook, J., n Zhng, S. (28). A fungl-responsive MAPK se regultes phytolexin iosynthesis in Ariopsis. Pro Ntl A Si U S A 15, Ren, D.T., Yng, H.P., n Zhng, S.Q. (22). Cell eth meite y MAPK is ssoite with hyrogen peroxie proution in Ariopsis. J Biol Chem 277, Ross, A., Ym, K., Hirum, K., Ymshit-Ym, M., Lu, X.L., Tkno, Y., Tsu, K., n Sijo, Y. (21). The Ariopsis PEPR pthwy ouples lol n systemi plnt immunity. Emo Journl 33, Roux, M., Shwessinger, B., Alreht, C., Chinhill, D., Jones, A., Holton, N., Mlinovsky, F.G., Tor, M., e Vries, S., n Zipfel, C. (211). The Ariopsis Leuine-Rih Repet Reeptor-Like Kinses BAK1/SERK3 n BKK1/SERK Are Require for Innte Immunity to Hemiiotrophi n Biotrophi Pthogens. Plnt Cell 23, Shh, J., n Zeier, J. (213). Long-istne ommunition n signl mplifition in systemi quire resistne. Front Plnt Si, 3. Shn, L.B., He, P., Li, J.M., Heese, A., Pek, S.C., Nurnerger, T., Mrtin, G.B., n Sheen, J. (28). Bteril effetors trget the ommon signling prtner 27

28 BAK1 to isrupt multiple MAMP reeptor-signling omplexes n impee plnt immunity. Cell Host Miroe, Sun, T., Bust, L., Zhng, Q., Ding, P., Jetter, R., n Zhng, Y. (217). TGACG- BINDING FACTOR 1 (TGA1) n TGA regulte sliyli i n pipeoli i iosynthesis y moulting the expression of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) n CALMODULIN-BINDING PROTEIN 6g (CBP6g). New Phytol. Sun, T.J., Zhng, Y.X., Li, Y., Zhng, Q., Ding, Y.L., n Zhng, Y.L. (215). ChIPseq revels ro roles of SARD1 n CBP6g in regulting plnt immunity. Nt Commun 6. Trn, D.T.N., Chung, E.H., Hring-Muller, A., Demr, M., Shw, R., Dngl, J.L., Weigel, D., n Che, E. (217). Ativtion of Plnt NLR Complex through Heteromeri Assoition with n Autoimmune Risk Vrint of Another NLR. Curr Biol 27, Tsu, K., n Ktgiri, F. (21). Compring signling mehnisms engge in pttern-triggere n effetor-triggere immunity. Curr Opin Plnt Biol 13, Tsu, K., Mine, A., Bethke, G., Igrshi, D., Botng, C.J., Tsu, Y., Glzerook, J., Sto, M., n Ktgiri, F. (213). Dul Regultion of Gene Expression Meite y Extene MAPK Ativtion n Sliyli Ai Contriutes to Roust Innte Immunity in Ariopsis thlin. Plos Genet 9. Vernooij, B., Frierih, L., Morse, A., Reist, R., Kolitzjwhr, R., Wr, E., Uknes, S., Kessmnn, H., n Ryls, J. (199). Sliyli-Ai Is Not the Trnslote Signl Responsile for Inuing Systemi Aquire-Resistne ut Is Require in Signl Trnsution. Plnt Cell 6, Vogel-Aghough, D., Sthl, E., Nvrov, H., n Zeier, J. (213). Pipeoli i enhnes resistne to teril infetion n primes sliyli i n niotine umultion in too. Plnt signling & ehvior 8, e Wng, H.C., Ngwenym, N., Liu, Y.D., Wlker, J.C., n Zhng, S.Q. (27). Stomtl evelopment n ptterning re regulte y environmentlly responsive mitogen-tivte protein kinses in Ariopsis. Plnt Cell 19,

29 Wng, L., Tsu, K., Trumn, W., Sto, M., Nguyen, L.V., Ktgiri, F., n Glzerook, J. (211). CBP6g n SARD1 ply prtilly reunnt ritil roles in sliyli i signling. Plnt J 67, Wilermuth, M.C., Dewney, J., Wu, G., n Ausuel, F.M. (21). Isohorismte synthse is require to synthesize sliyli i for plnt efene. Nture 1, Wu, Y., Zhng, D., Chu, J.Y., Boyle, P., Wng, Y., Brinle, I.D., De Lu, V., n Despres, C. (212). The Ariopsis NPR1 Protein Is Reeptor for the Plnt Defense Hormone Sliyli Ai. Cell Reports 1, Xu, J., Meng, J., Meng, X.Z., Zho, Y.T., Liu, J.M., Sun, T.F., Liu, Y.D., Wng, Q.M., n Zhng, S.Q. (216). Pthogen-Responsive MPK3 n MPK6 Reprogrm the Biosynthesis of Inole Gluosinoltes n Their Derivtives in Ariopsis Immunity. Plnt Cell 28, Ym, K., Ymshit-Ym, M., Hirse, T., Fujiwr, T., Tsu, K., Hirum, K., n Sijo, Y. (216). Dnger peptie reeptor signling in plnts ensures sl immunity upon pthogen-inue epletion of BAK1. Emo Journl 35, Ymguhi, N., Winter, C.M., Wu, M.F., Kwon, C.S., Willim, D.A., n Wgner, D. (21). PROTOCOLS: Chromtin Immunopreipittion from Ariopsis Tissues. The Ariopsis ook 12, e17. Ymguhi, Y., Huffker, A., Bryn, A.C., Tx, F.E., n Ryn, C.A. (21). PEPR2 Is Seon Reeptor for the Pep1 n Pep2 Pepties n Contriutes to Defense Responses in Ariopsis. Plnt Cell 22, Yu, X., Feng, B.M., He, P., n Shn, L.B. (217). From Chos to Hrmony: Responses n Signling upon Miroil Pttern Reognition. Annul Review of Phytopthology, Vol 55 55, Zeier, J. (213). New insights into the regultion of plnt immunity y mino i metoli pthwys. Plnt Cell Environ 36, Zhng, X.X., Dos, P.N., n Bernoux, M. (217). Wht Do We Know Aout NOD-Like Reeptors in Plnt Immunity? Annul Review of Phytopthology, Vol 55 55, Zhng, Y.X., Xu, S.H., Ding, P.T., Wng, D.M., Cheng, Y.T., He, J., Go, M.H., Xu, F., Li, Y., Zhu, Z.H., Li, X., n Zhng, Y.L. (21). Control of sliyli i synthesis n systemi quire resistne y two memers of plnt- 29

30 speifi fmily of trnsription ftors. P Ntl A Si USA 17, Zheng, Z.Y., Au Qmr, S., Chen, Z.X., n Mengiste, T. (26). Ariopsis WRKY33 trnsription ftor is require for resistne to nerotrophi fungl pthogens. Plnt J 8, Zhou, M., Lu, Y., Bethke, G., Hrrison, B.T., Htsugi, N., Ktgiri, F., n Glzerook, J. (217). WRKY7 prevents xeni tivtion of plnt immunity y iret repression of SARD1. New Phytol. Zimmerli, L., Jk, C., Metrux, J.P., n Muh-Mni, B. (2). Potentition of pthogen-speifi efense mehnisms in Ariopsis y et-minoutyri i. P Ntl A Si USA 97, Zipfel, C., Rotzek, S., Nvrro, L., Okeley, E.J., Jones, J.D.G., Felix, G., n Boller, T. (2). Bteril isese resistne in Ariopsis through flgellin pereption. Nture 28,

31 Mok DEX -6 n.s MKKDD MKKDD si2 GUS FRK1-2 - Reltive expression (log2) -1 n.s MKKDD MKKDD si2 GUS ALD n.s GUS Mok DEX MKKDD MKKDD si2 n.s si2 GUS MKKDD MKKDD si2 si2 FRK1-2 Mok Pto AvrRpt Col si2 ALD D n.s. n.s Reltive expression (log2) -1 PR1 Bteril titer [log1(cfu/m2)] Bteril titer [log1(cfu/m2)] B C - Reltive expression (log2) Reltive expression (log2) Reltive expression (log2) PR1 Reltive expression (log2) A si2 7 Mok Pto AvrRpt si2 Figure 1. MAPK-meite SAR is lrgely inepenent of SA. (A) n (C) Expression levels of PR1, FRK1, n ALD1 reltive to ACTIN2 in -week-ol leves etermine y RT-qPCR. Leves of DEX-inuile GUS (GUS), MKKDD, n MKKDD si2 plnts were hrveste 2 h fter infiltrtion with 1 µm DEX or mok (A) n of n si2 2 h fter infiltrtion with Pto AvrRpt2 (OD6=.1) or mok (C). (B) n (D) Bteril titers in systemi leves. Primry leves of, si2, GUS, MKKDD, n MKKDD si2 were infiltrte with 1 µm DEX or mok (B) n of n si2 with Pto AvrRpt2 (OD6=.1) or mok (D). After 1 y, systemi leves were infiltrte with Pto (OD6=.1), n teril titers in the systemi leves were mesure t 2 ys post systemi infetion. (A) to (D) Brs represent mens n stnr errors lulte from three inepenent experiments eh with three iologil replites using mixe liner moel. (, P <.1; two-tile Stuent s t-tests. n.s., not signifint).

32 A Bteril titer [log1(cfu/m2)] n.s. GUS n.s. si2 fmo1 MKK MKKDD α-flg α-mpk3 Pipeoli i (µg/g FW) α-mpk6 Poneu S l1 npr1 DD MKKDD si2 MKKDD l1 MKKDD fmo1 MKKDD npr1 MKKDD wrky h MPK6 MPK3 MKKDD MPK3 MPK6 Ruiso time post tretment 72 h N-hyroxypipeoli i (µg/g FW) α-ptepy n.s. n.s. 5 B C Mok DEX N.D. 72 h time post tretment Figure 2. MAPK-meite SAR requires ALD1. (A) Bteril titers in systemi leves of GUS, MKKDD, MKKDD si2, MKKDD fmo1, MKKDD l1, n MKKDD npr1. Primry leves were infiltrte with 1 µm DEX or mok. After 1 y, systemi leves were infiltrte with Pto (OD6=.1), n teril titers in the systemi leves were mesure t 2 ys post systemi infetion. Brs represent mens n stnr errors lulte from three inepenent experiments eh with three iologil replites using mixe liner moel. inites signifint ifferene from Mok (P <.1; two-tile Stuent s t-tests). n.s., not signifint (B) Phosphoryltion of MPK3 n MPK6, n protein level umultion of MKKDD, MPK3, n MPK6 in lol leves of MKKDD, MKKDD si2, MKKDD fmo1, MKKDD l1, MKKDD npr1, n MKKDD wrky33 plnts t the inite time points fter infiltrtion with 1 µm DEX. (C) Pipeoli i n N-hyroxypipeoli i levels in lol leves of MKKDD plnts infiltrte with 1 µm DEX or mok t the inite time points. N.D. inites uner the etetion limit. Brs represent mens n stnr errors lulte from three inepenent experiments. inites signifint ifferene from hpi (P <.1; two-tile Stuent s t-tests).

33 Bteril titer [log1(cfu/m2)] A Reltive expression (log2) AB B e e e 3 Col mpk3 mpk3 - mpk6 mpk6 mpk3 mpk6 si2 ALD1 e mpk3 mpk mpk3 si2 mpk6 si2 f -12 e -1 si2 FMO1 ef -2 mpk3 si2 mpk6 si2 e Mok Pto AvrRpt2 - mpk3si2 mpk6si2 mpk3 si2 mpk6 si si2 si2 PR1-12 Reltive expression (log2) A e AC e AB 5 B Reltive expression (log2) BC Mok Pto AvrRpt mpk3 mpk6 si2 mpk3 si2 mpk6 si2 C Pipeoli i (µg/g FW) 35 Mok Pto AvrRpt mpk3 mpk6 si2 mpk3 si2 mpk6 si2 Figure 3. MPK3 n MPK6 positively regulte Pip umultion uring Pto AvrRpt2 infetion. (A) Bteril titers in systemi leves of, mpk3, mpk6, si2, mpk3 si2, n mpk6 si2. Primry leves were infiltrte with Pto AvrRpt2 (OD6=.1) or mok. After 1 y, systemi leves were infiltrte with Pto (OD6=.1), n teril titers were mesure t 2 ys post systemi infetion. Brs represent mens n stnr errors lulte from six inepenent experiments eh with four iologil replites using mixe liner moel. (B) Expression of PR1, ALD1, n FMO1 in, mpk3, mpk6, si2, mpk3 si2, n mpk6 si2 t 2 h post infiltrtion with Pto AvrRpt2 (OD6=.1) or mok etermine y RT-qPCR. Brs represent mens n stnr errors of the log2 expression levels reltive to ACTIN2 lulte from three inepenent experiments eh with three iologil replites using mixe liner moel. (C) Lol Pip umultion in leves of, mpk3, mpk6, si2, mpk3 si2, n mpk6 si2 t 2 h post infiltrtion with Pto AvrRpt2 (OD6=.1) or mok. Brs represent mens n stnr errors of four inepenent iologil replites. Sttistil ifferenes were lulte using mixe liner moel followe y two-tile Stuent s t-tests. (A) to (C) Different letters ove the rs enote sttistilly signifint ifferenes (juste P <.5). Upperse letters inite omprisons etween genotypes for SAR effets.

34 MKKDD MKKDD wrky33 ALD MKKDD 2 MKKDD wrky33 FMO MKKDD MKKDD wrky33 E 1.2 Reltive expression (log2) D Reltive expression (log2) Mok DEX Reltive expression (log2) PR1 2 B Pipeoli i (µg/g FW) Pipeoli i (µg/g FW) Reltive expression (log2) Reltive expression (log2) Reltive expression (log2) A MKKDD wrky33 Mok Pto AvrRpt wrky33 si2 ALD1-2 - wkry33 si wrky33 si2 wkry33 si2 FMO1 wrky33 si2 6 wkry33 si2 7h 12 h 2 h i h gh 2 ef MKKDD MKKDD wrky33 F Bteril titer [log1(cfu/m2)] Bteril titer [log1(cfu/m2)] 6. PR1 efg fgh e MKKDD C 2 wrky33 si2 7 6 wkry33 si2 n.s. n.s. 5 wrky33 si2 wkry33 si2 Figure. MAPK-meite Pip umultion n SAR re ompromise in wrky33. (A) n (D) PR1, ALD1, n FMO1 expression in leves of MKKDD n MKKDD wrky33 t 2 h fter infiltrtion with 1 µm DEX or mok etermine y RT-qPCR (A) n of, wrky33, si2 n wrky33 si2 t 2 h fter infiltrtion with Pto AvrRpt2 (OD6=.1) or mok (D). Brs represent mens n stnr errors lulte from two inepenent experiments eh with three iologil replites using mixe liner moel. (B) n (E) Pip umultion in leves of MKKDD n MKKDD wrky33 t 2 h fter infiltrtion with 1 µm DEX (B) n of, wrky33, si2 n wrky33 si2 t 7 h, 12 h, n 2 h fter infiltrtion with Pto AvrRpt2 (OD6=.1) or mok (E). Brs represent mens n stnr errors lulte from five (B) or three (E) inepenent iologil replites. (E) Sttistil ifferenes were lulte using mixe liner moel followe y two-tile Stuent s t-tests. (C) n (F) Bteril titers in systemi leves of MKKDD n MKKDD wrky33 (C) n of, wrky33, si2 n wrky33 si2 (F). Primry leves were infiltrte with 1 µm DEX or mok (C) n with Pto AvrRpt2 (OD6=.1) or mok (F). After 1 y, systemi leves were infiltrte with Pto (OD6=.1), n teril titers in the systemi leves were mesure t 2 ys post systemi infetion. Brs represent mens n stnr errors lulte from t lest four inepenent experiments eh with three iologil replites using mixe liner moel. (A) to (F) Different letters ove the rs enote sttistilly signifint ifferenes (juste P <.5)., P <.1; two-tile Stuent s t-tests. n.s., not signifint.

35 C f 6.5 AB B 5.5 ef si2 C 5 Pipeoli i (µg/g FW) e e A Mok Pm mpk3 lol 2 h 3 2 mpk3 mpk6 mpk mpk6 si2 wrky mpk3 mpk6 25 Mok Pm wrky33 lol 2 h Mok Pm 16 Mok Pm 6. systemi 8 h Mok Pm 1 mpk3 si2 6.5 N-hyroxypipeoli i (µg/g FW) 7. fg B C g Pipeoli i (µg/g FW) Bteril titer [log1(rlu/m2)] 7.5 C Bteril titer [log1(rlu/m2)] A wrky N.D. N.D. mpk3 N.D. N.D. mpk6 wrky33 1 Figure 5. SA n WRKY33 ontriute to SAR triggere y Pm infetion. (A) n (B) Bteril 2 titers in systemi leves of, mpk3, mpk6, si2, mpk3 si2, n mpk6 si2 (A) n of n 3 wrky33 (B). Primry leves were infiltrte with Pm (OD6=.5) or mok. After 2 ys, systemi leves were infiltrte with Pm lux (OD6=.1), n the ioluminesene of Pm lux ws etermine 5 t 6 h post systemi infetion. Brs represent mens n stnr errors lulte from t lest four 6 inepenent experiments eh with three iologil replites using mixe liner moel. (C) Pip 7 umultion in lol leves t 2 h n systemi leves t 8h, n N-hyroxypipeoli i 8 umultion in lol leves of, mpk3, mpk6, n wrky33 t 2 h post infiltrtion with Pm 9 (OD6=.1) or mok (1 mm MgCl2). Brs represent mens n stnr errors of three iologil 1 replites. N.D. inites uner the etetion limit. Sttistil ifferenes were lulte using mixe 11 liner moel followe y two-tile Stuent s t-tests. (A) to (C) Different letters ove the rs enote 12 sttistilly signifint ifferenes (juste P <.5). Upperse letters inite omprisons etween 13 genotypes for SAR effets., P <.1; two-tile Stuent s t-tests.

36 A W-ox 1 B ALD1 W-ox 2/3 Pto AvrRpt2 Mok α-ha WRKY33 Ruiso Fol enrihment C Poneu S 2 15 Pwrky33:WRKY33-HA 1 5 n.s. W-ox 1 W1 W-ox 2/3 W2/3 1 Figure 6. WRKY33 ins to ALD1 promoter. (A) Shemti igrm of ALD1 promoter. The vertil 2 lk rs represent W-oxes. The horizontl lines show the regions mplifie y ifferent qpcr 3 primers. (B) Protein umultion of WRKY33 in WRYK33-HA wrky33 plnts fter infiltrtion with Pto AvrRpt2 (OD6=.1) or mok t the inite time points visulize y immunolotting using nti-ha 5 ntioy. Poneu S-stine RuBisCo is shown s loing ontrol. (C) ChIP-qPCR ws performe 6 using n WRYK33-HA wrky33 t 1 y fter infiltrtion with Pto AvrRpt2 (OD6=.1). Brs 7 represent mens n stnr errors of the fol enrihment reltive to (set to 1), lulte from 8 three inepenent iologil replites (, P-vlue <.1, two-tile Stuent s t-tests. n.s., not 9 signifint).

37 Root length (m) 8 6 E k1 kk1 Bteril titer [log1(rlu/m2)] A 2 H2O B flg22 L-pip k1 kk1 5.5 H 2O Pip F mpk3 Pm mpk6 k1 kk1 wrky33 Pto AvrRpt2 Pto MPK6 MPK3 AvrRpt2 Ruiso wrky MPK3/6 l MPK6 MPK3 α-mpk3 MPK3 α-mpk6 MPK6 Poneu S Ruiso h? X WRKY33 α-ptepy MPK6 MPK3 α-mpk3 MPK3 α-mpk6 MPK6 BAK1 BKK1 other TFs SARD1/CBP6g SA Lys fmo1 7 P TGA1/TGA 7 1 h α-ptepy Poneu S 6. k1 kk1 H2O flg22 H2O flg22 D-Pip L-Pip D-Pip L-Pip Poneu S D D-pip α-ptepy C 6.5 ALD1 Pip NHP SARD FMO1 lol lef X moile signl systemi lef SAR SA NHP WRKY33 Pip X Ruiso Figure 7. Pip triggers MAPK tivtion. (A) n k1 kk1 plnts were grown on ½ MS meium ontining 1 µm flg22, 1 µm L-Pip, 1 µm D-Pip, or mok, n primry root length ws mesure t 1 ys ol. Brs represent mens n stnr errors lulte from four inepenent iologil replites using mixe liner moel. (B) MAPK tivtion in 1-y-ol seelings of n k1 kk1. Seelings were ollete t 15 min fter the tretment with 1 µm flg22, 1 µm L-Pip, 1 µm D-Pip, or mok. (C) n (D) MAPK tivtion in leves of -week-ol, wrky33, l1, n fmo1 fter infiltrtion with Pto AvrRpt2 (OD6=.1), n smples were ollete t the inite time points. (B) to (D) Proteins were etete y immunolotting using the inite ntioies. Poneu S-stine RuBisCo is shown s loing ontrol. Similr results were oserve in three inepenent experiments. (E) Bteril titers in leves of, mpk3, mpk6, wrky33, n k1 kk1. Five-week-ol plnts were supplie with 1 ml of 1 mm Pip (osge of 1 µmol) or wter vi the root system. Three leves per plnt were infiltrte with Pm lux (OD6=.1) t 1 y post tretment, n reltive luminesene light units (rlu) per m2 (log1) were mesure t 6 hours post systemi infetion. Brs represent mens n stnr errors of t lest three inepenent iologil replites using mixe liner moel. (A) n (E) Different letters ove the rs enote sttistilly signifint ifferenes (juste P <.1). (F) Moel for the immune-mplifition loop onsisting of MPK3/6, WRKY33, ALD1, n pipeoli i.