Arabidopsis phospholipase Db1 modulates defense responses to bacterial and fungal pathogens

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1 Reserh Aridopsis phospholipse D1 modultes defense responses to teril nd fungl pthogens Jin Zho 1,, Shivkumr P. Devih 1, Cunxi Wng 1, Moyin Li,5, Ruth Welti 3 nd Xuemin Wng 1,,5 1 Deprtment of Biohemistry, Knss Stte University, Mnhttn, KS 5, USA; Ntionl Key Lortory of Crop Geneti Improvement, College of Plnt Siene nd Tehnology, Huzhong Agriulturl University, Wuhn, Chin; 3 Division of Biology, Knss Stte University, Mnhttn, KS 5, USA; Deprtment of Biology, University of Missouri, St Louis, MO, 311, USA; 5 Donld Dnforth Plnt Siene Center, St Louis, MO 313, USA Authors for orrespondene: Xuemin Wng Tel: Emil: swng@dnforthenter.org Jin Zho Tel: Emil: jinzho@mil.hzu.edu.n Reeived: 1 Jnury 13 Aepted: Ferury 13 New Phytologist (13) 199: 8 doi: /nph.15 Key words: Aridopsis thlin, Botrytis inere, lipid signlling, lysophospholipids, pthogeneses, phosphtidi id, phospholipse D1, Pseudomons syringe. Summry Pthogen infetion of higher plnts often indues rpid prodution of phosphtidi id (PA) nd hnges in lipid profiles, ut the enzymti sis nd the funtion of the lipid hnge in pthogen plnt intertions re not well understood. Infetion of phospholipse D 1(PLD1)-defiient plnts y Pseudomons syringe tomto pv DC3 (Pst DC3) resulted in less teril growth thn in wild-type plnts, nd the effet ws more profound in virulent Pst DC3 thn virulent Pst DC3 (rrying the virulene gene vrrpt) infetion. The expression levels of sliyli id (SA)-induile genes were higher, ut those induile y jsmoni id (JA) showed lower expression in PLD1 mutnts thn in wild-type plnts. However, PLD1-defiient plnts were more suseptile thn wild-type plnts to the fungus Botrytis inere. The PLD1-defiient plnts hd lower levels of PA, JA nd JA-relted defense gene expression fter B. inere inoultion. PLD1 plys positive role in pthogen-indued JA prodution nd plnt resistne to the nerotrophi fungl pthogen B. inere, ut negtive role in the SA-dependent signling pthwy nd plnt tolerne to infetion with iotrophi Pst DC3. PLD1 is responsile for most of the inrese in PA prodution in response to nerotrophi B. inere nd virulent Pst DC3 infetion, ut ontriutes less to virulent Pst DC3 (vrrpt)-indued PA prodution. Introdution Plnts hve developed different ut interrelted defense systems to ttle ginst invsion y pthogeni miroorgnisms (Durrnt & Dong, ; Armovith et l., ; Jones & Dngl, ). One system reognizes nd responds to moleules ommon to mny lsses of miroes, inluding nonpthogens. This defense response is initited y reognition of miroe- or pthogen-ssoited moleulr pttern (MAMP or PAMP, respetively) y the orresponding pttern reognition reeptor, whih is typilly n integrl plsm memrne protein. Another system of defense responds to pthogen virulene effetors, either diretly or through their effets on host trgets. The effetortriggered defense response involves resistne (R) proteins, usully polymorphi NB-LRR proteins tht speifilly reognize prtiulr pthogen effetors nd lunh speifi defense response, suh s the Aridopsis R protein RPS, whih reognizes the Pseudomons syringe effetor AvrRpt, type III effetor from the Pseudomons syringe (Glzerook, 5; Jones & Dngl, ). PAMP-triggered immunity (PTI) nd effetor-triggered immunity (ETI) re losely relted nd intert in n rry of defense responses, suh s the hypersensitive response, the iosynthesis of the signling moleules sliyli id (SA), jsmonte (JA) nd ethylene, nd the prodution of pthogenesisrelted (PR) proteins nd phytolexins (Durrnt & Dong, ; Armovith et l., ). Severl prllel, yet ross-tlking signling pthwys nd defense strtegies exist, suh s SA-dependent systemi quired resistne in virulent teril infetions nd JA/ethylene-dependent induile systemi resistne during nerotrophi fungl pthogen infetions (Glzerook 5; Spoel et l., 3; Trumn et l., 7). However, the iohemil pthwys mediting the ross-tlk of different defense responses re not well understood (Glzerook, 5; Jones & Dngl, ). Different spets of lipid metolism nd signling ply importnt roles in disese resistne nd suseptiility. For exmple, suppression of -Dioxygense1 (-DOX1), 1- nd 18-C ftty id-dioxygense, onfers enhned suseptiility to P. syringe (De Leon et l., ). Muttion of DEFECTIVE in INDUCED RESISTANCE 1 (DIR1), puttive lipid trnsfer protein, renders plnts defiient in systemti quired resistne (SAR; Mldondo et l., ). Muttion of plstidi steroyl yl-rrier protein desturse, SUPPRESSOR OF SALICYLIC ACID INSENSITIVITY (SSI), results in n inresed 8 New Phytologist (13) 199: 8 Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust

2 New Phytologist Reserh 9 SA-medited SAR (Nndi et l., ). Muttion of phospholipse A (PLA) suppressor of AvrBst-eliited resistne1 (SOBER1) inreses the resistne of Aridopsis eotype Pi- to Pseudomons syringe tomto (Pst) DC3 (vrbst, type III effetor from Xnthomons mpestris pv vesitori). This study lso indites potentil ross-tlk etween the lysophospholipid-produing PLA nd the phosphtidi id (PA)-produing phospholipse D (PLD) pthwys in the plnt teril pthogen intertion (Kirik & Mudgett, 9). PLD, whih hydrolyzes memrne phospholipids to generte PA nd free-hed group, is involved in ellulr proesses inluding retive oxygen speies (ROS) genertion (Sng et l., 1; Prk et l., ), hormone signling (Zhng et l., ), nd disese resistne (den Hrtog et l., 3; Andersson et l., ; Brgmnn et l., ; Ymguhi et l., 9). Plnt PLD is omposed of fmily of heterogeneous enzymes with distinguishle tlyti nd regultory properties (Wng et l., ). PLD1 inaridopsisindstoc + nd phosphtidylinositol-,5-isphosphte (PIP ) nd hydrolyzes phosphtidylethnolmine (PE) preferentilly over phosphtidylholine (PC) (Pppn et l., 1997; Zheng et l., ). The expression of Aridopsis PLD1 ws indued y oth teril nd fungl pthogen infetions (Zel et l., ).Tomto(Lyopersion esulentum) LePLD1 ws indued y fungl eliitors, nd RNAi knokdown of LePLD1 resulted in n inresed defense response to fungl eliitors (Lxlt et l., 1; Brgmnn et l., ). Knokdown of PLD1 in rie (Oryz stiv) inresed resistne to Pyriulri grise nd Xnthomons oryze pv oryze. These results indite tht PLD1 plys n importnt role in plnt pthogen intertions nd lso rise further questions. As phospholipid-hydrolyzing enzyme, wht effet would PLD1 hve on memrne glyerolipid speies without nd with pthogen infetion? How would gene-knokout of PLD1 ffet SA- nd JA-medited defense responses? In this study, we used PLD1-knokout nd RNAi-suppressed Aridopsis plnts to investigte the effet of PLD1 on the intertion of Aridopsis with the teril pthogen P. syringe nd the fungl pthogen Botrytis inere. The results indite tht PLD1 plys role in promoting PA prodution fter pthogen infetion. PLD1 plys negtive role in plnt resistne to teril pthogens ut positive role in the plnt response to the nerotrophi fungl pthogen B. inere. Mterils nd Methods PLD1 T-DNA-insertion knokout isoltion nd omplementtion A T-DNA insertion mutnt in PLD1 (At g1), designted pld1-1, ws identified from the Slk Aridopsis thlin (L.) Heynh T-DNA knokout olletion (Slk_79133) nd seeds were otined from the Aridopsis Biologil Resoure Center (ABRC) t Ohio Stte University. A PLD1 homozygous T-DNA insert mutnt ws isolted y PCR sreening using PLD1 gene-speifi primers 5 -ATA CCC TCC ACC TGA AAC TAA ACC GCA-3 (forwrd primer) nd 5 -TGTG GCTTATTGTGGAATGCATTACGA-3 (reverse primer), nd the T-DNA left order primer 5 -GCG TGG ACCGCT TGC TGCAACT-3. Positive T-DNA insertion lines were onfirmed Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust y sequening. The F3 genertion shows homozygous muttion, nd defiieny of PLD1 trnsripts ws onfirmed y northern lotting. pld1-1 showed o-segregtion with knmyin resistne in 3:1 rtio, inditing tht the mutnt hd single T-DNA insertion. For omplementtion of the PLD1 knokout mutnt, the ntive PLD1 gene, inluding its own promoter region, ws mplified from 1 p upstrem of the strt odon nd 3 p fter the stop odon nd then ws loned into the pec91 vetor. The primers for PLD1 omplementtion were 5 -ATGGCGCGCCAGATTCTCGTC CACTGAGGA-3 (forwrd) nd 5 -ATGGCGCGCCTAG AGATGGGCTCTGGAGAT-3 (reverse). The onstrut, pec91-pldg3, ws trnsformed into Agroterium tumfiens strin GV311 vi eletroportion. Homozygous pld1-1 plnts were trnsformed (Clough & Bent, 1998), the seeds were olleted, nd trnsformnts were seleted on medium ontining hlf-strength Murshige nd Skoog (MS) medium, 5 lg 1 ml 1 knmyin, nd 1% gr. PLD1 RNAi onstrut nd genertion of RNAi suppression lines The sense DNA frgment (9 p; the lst exon plus prt of the 3 untrnslted region (UTR), from 78 to 37 p in PLD1 mrna; GenBnk ession numer U858) followed y n intron (11 p; the lst intron, from to p in Aridopsis BAC lone TD; GenBnk ession numer U9938) ws mplified using PCR with the forwrd primer BIR51, 5 -CCCAAGCTTATTTAGAGTGATAATATATC-3 (HindIII site underlined), nd the reverse primer BIR31, 5 -CCGGAATTCAGATCTATGGATACAG AAT-3 (EoRI site underlined). Antisense DNA (9 p; the lst exon plus the 3 -UTR from 78 to 37 p in PLD1 mrna; GenBnk ession numer U858) ws mplified with the forwrd primer BIR5, 5 -CCGGAATTCTGGTCAGGTAAATCCCG CAAAC-3 (EoR1 site underlined), nd the reverse primer BIR3, 5 -CCGCTCGAGATTTAGAGTGATAATATATC-3 (XhoI site underlined). Two PCR produts were digested with EoRI nd then ligted into frgment of 11 p ontining two inverse DNA repets seprted y n intron. This frgment ws further digested with the XhoI nd HndIII restrition enzymes, nd the resulting frgment ws purified nd then ligted into the pkylx71-35s vetor t XhoI ndhndiii sites. The resulting RNAi vetor ws onfirmed y omplete sequening nd trnsformed into Aridopsis y the florl dipping method. F1 nd F seeds were sreened using oth knmyin pltes nd PCR. Two homozygous lines, nd, were finlly otined with drmtilly deresed PLD1 trnsripts in leves, s onfirmed y northern lotting. Pthogen growth nd inoultion Aridopsis thlin (L.) Heynh eotype Col-, pld1-1, RNAi mutnts, nd pld1-1 omplemented with PLD1 (PLD1-omplemented) plnts were grown in soil in growth hmers t 3 : 1 C nd 8% reltive humidity under 8 : 1 h New Phytologist (13) 199: 8

3 3 Reserh New Phytologist dy : night photoperiods (85 lmol m s 1 ) for pthogen inoultion or 1 : 8 h dy : night photoperiods (1 lmol m s 1 ) for physiologil nd geneti nlysis. The virulent strin Pseudomons syringe tomto (Pst) DC3 nd n virulent strin rrying the virulene gene vrrpt, Pst DC3 (vrrpt), were grown in King s medium B t 8 C with pproprite ntiiotis (5 mg l 1 of rifmpiin for Pst DC3 nd 5 mg l 1 of rifmpiin plus 5 mg l 1 of knmyin for Pst DC3 (vrrpt)). Five-week-old, soil-grown plnts were inoulted with Pst DC3 nd Pst DC3 (vrrpt). Bteril inoultions were performed s previously desried (Nndi et l., ). The fully expnded leves of eh plnt were infiltrted with suspension ontining 1 7 olony-forming units (CFU) per milliliter of Pst DC3 or Pst DC3 (vrrpt). In prllel, plnts similrly infiltrted with 1 mm MgCl served s the ontrols. At, 3 nd 5 d post inoultion, six infeted leves were olleted per genotype to mesure the growth of the pthogen. Bteril ounts re expressed s olony-forming units per lef dis. The rte of inrese in the lesion re ws determined ording to methods desried previously (Nndi et l., ). Eh dt point represents three replites, with three lef diss per replite. Approximtely 5 plnts of eh genotype nd > 3 leves were treted. Botrytis inere IMI19558 ws ultivted in potto dextrose roth medium t C in growth hmer. Spores were hrvested nd infetions performed s desried previously (Nndi et l., ). Briefly, 1-ll drop of freshly hrvested B. inere onidil spore suspension ( spores ml 1 ) ws pled on three leves of -wk-old soil-grown plnts priked with 3- guge needle. The inoulum ws llowed to ir-dry; plnts were overed with trnsprent plsti dome nd ultivted t 18 C (8 1% humidity) in growth hmer progrmmed for 1 : 1 h light : drk yle. Control plnts were inoulted with potto dextrose roth medium lone. The numer of leves showing different levels of lesion ws sored t 5 d post inoultion (Nndi et l., ). Approximtely 5 plnts of eh genotype nd > 3 leves were tested. Extrtion nd quntifition of SA Aout.3 g of fresh leves ws hrvested nd quikly frozen in liquid N. SA extrtion nd determintion were performed ording to method desried y Nndi et l. (). Briefly, frozen smples were ground nd underwent extrtion one with 3 ml of 9% methnol nd one with 3 ml of 1% methnol. The omined extrts were dried under N gs nd suspended in.5 ml of 5% trihloroeti id. The smples were idhydrolyzed y dding ll of HCl nd inuting t 95 C for 3 min. SA ws extrted with 5 ml of mixture ontining ylohexne : ethylette : isopropnol (5 : 5 : 1). The smple ws dried under N gs nd dissolved in.5 ml of the moile phse (9 : 7 : mix of wter : methnol : glil eti id). Smples were filtered through.-lm filter, nd 7.5 to ll ws used for high-performne liquid hromtogrphy. Smples were pssed over. 9 5-mm C18 reverse-phse olumn, nd SA ws eluted with the moile phse t flow rte of New Phytologist (13) 199: 8 ml min 1. Asorne of the eluted smples ws reorded t 31 nm, nd SA onentrtions were determined y omprison with SA stndrds. RNA lotting nd immunolotting Five-week-old Aridopsis seedlings tht were treted with pthogens or ontrol solution (wter or 1 mm MgSO ) were used for gene expression nlysis. Whole plnts were tken for extrtion of RNA (Zho et l., 11). Twenty mirogrms of RNA ws loded nd seprted y formldehyde-grose gel eletrophoresis nd trnsferred to hyridiztion memrne. 3 P- leled DNA proes for the pthogenesis-relted genes PR-1 (At g11), plnt defensin 1. (PDF1.) (At5 g), PLD1 (At3 g1573), PLD1 (At g1), lipoxygense (LOX) (At3 g51), llene oxide synthse (AOS) (At5 g5) nd PR-5 (At1 g75) were generted using full-length DNA of these genes. The immunolotting for PLD1 ws performed s desried previously (Zheng et l., ). Proteins were extrted from Aridopsis leves nd equl mounts of proteins were sujeted to 8% SDS-PAGE, followed y immunolotting using ntiodies ginst PLD1 nd PLD1. The PLD ntiodies were rised ginst the C-terminl 13-mino id residue peptide (Zheng et l., ). Rel-time qrt-pcr Rosette leves of pproprite ge were inoulted with pthogens. Leves were hrvested efore infetion nd mok tretments (time-point ) nd t the indited time-points fter tretment. Totl RNA ws isolted with the RNesy Plnt Mini kit (Qigen, USA). RNA ws reverse-trnsried using n M-MLV Reverse Trnsriptse kit (Life Tehnologies, Invitrogen, Grnd Islnd, NY, USA) ording to the mnufturer s instrutions. Primer pirs listed in Supporting Informtion Tle S1 online were used for rel-time quntifition y n iq 5 multiolor rel-time PCR detetion system (Bio-Rd). Individul PCR retion mixtures ontined 1 ll of diluted DNA, 1 ll of SYBR Green Mstermix (Thermo Fisher Sientifi In., St Louis, MO, USA), nd 5 lm of eh primer in finl volume of 1 ll. In ll experiments, three iologil replites of eh smple nd two tehnil (PCR) replites were performed. Dt were nlyzed with iq 5 OPTICAL SYSTEM softwre y the omprtive DDCT method. The mount of trget genes ws normlized to the undne of the onstitutive uiquitin 5 (UBQ5) nd Tuulin8 genes. Three iologil replites were nlyzed, eh onsisting of eight individully infeted leves. Detetion of O nd mesurement of H O After inoultion with teril or fungl pthogens for the time indited in the figures, inoulted leves were dethed nd infiltrted with nitrolue tetrzolium (NBT) for h. The purple formzn preipitte indites the lotion nd extent of O umultion. For quntittive mesurement of H O, frozen Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust

4 New Phytologist Reserh 31 leves (.. g) were ground to powder under liquid nitrogen nd homogenized with.5 ml of. M HClO in pre-ooled mortr nd pestle. The extrt ws entrifuged t 1 g for 1 min t C. The superntnt ws olleted nd neutrlized to ph with.8 M NH OH, nd riefly entrifuged t 3 g for 5 min to sediment the insolule mteril. After eightfold dilution, H O onentrtion ws mesured with n Amplex Red hydrogen peroxide/peroxidse ssy kit (Ct. no. A188; Moleulr Proes, Eugene, OR, USA). Lipid profiling nd JA mesurement The proess of lipid extrtion, nlysis, nd quntifition ws performed s desried previously (Welti et l., ). Briefly, oth teril nd fungl inoulted leves were olleted t the smpling time nd immersed immeditely in 3 ml of isopropnol with.1% utylted hydroxytoluene (BHT) for 15 min t 75 C to inhiit lipolyti tivities. This ws followed y the ddition of 1.5 ml of hloroform nd. ml of wter. After shking for 1 h, the extrting solvent ws trnsferred to len tue. The tissues were extrted with hloroform-methnol five times with 3-min gittion eh time. The extrts were omined nd wshed with 1 M KCl, followed y nother wsh with wter. The solvent ws evported with strem of nitrogen. The remining plnt tissues were dried in n oven t 15 C overnight nd then weighed for dry weight, whih here refers to the dry weight minus the lipid ontent. Lipid smples were nlyzed with n eletrospry ioniztion triple qudruple mss spetrometer (API ; Applied Biosystems, Foster City, CA, USA). The moleulr speies of eh lipid lss were quntified in omprison to two internl stndrds, s previously desried (Welti et l., ; Devih et l., ). Five replites of eh tretment for eh phenotype were proessed nd nlyzed. The Q-test for disordnt dt ws performed on the replites of totl lipid dt or smples. Pired vlues were sujeted to t-test to determine sttistil signifine. JA nlysis in B. inere-inoulted nd ontrol plnts ws rried out ording to the method desried previously (Yng et l., 7; Pn et l., 8). Lef smples were hrvested t, 1 nd h post inoultion. Aout 1 mg of fresh Aridopsis leves ws seled in 1.5-ml snp-p vils nd quikly frozen in liquid N. The lef tissues were ground into powder nd 5 ll of 1-propnol/H O/onentrted HCl ( : 1 :., v/v) with internl stndrds (5 ng) ws dded, nd the smples were gitted for 3 min t C. One ml of CH Cl ws dded, nd the smples were gitted for nother 3 min nd then entrifuged t 13 g for 5 min. The lower phse (5 ll) ws diretly infused into hyrid triple qudrupole/liner ion trp mss spetrometer (ABI Q-TRAP ; Applied Biosystems) outfitted with n eletrospry (ESI) ion soure. Sttistil nlysis For most experimentl dt, inluding SA nd H O mesurements, three independent experiments were performed; lipid Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust profiling ws performed with five repets. The dt were nlyzed using Student s t-test. The differenes etween two tils of dt with the error rs represent 95% onfidene limits. Results PLD1 deletion inresed defense response nd resistne to Pst DC3 To hrterize the funtion of PLD1, we generted RNAi mutnts; two lines, nd, in whih PLD1 expression ws suppressed to different degrees, were isolted nd used in this study (Fig. 1). RNA lotting indited tht hd. 5% of the wild-type level of PLD1 trnsript, wheres hd < 1% of the wild-type level of PLD1 trnsript (Fig. 1). Immunolotting using PLD1 ntiody lso onfirmed tht oth nd hd deresed level of PLD1 protein (Fig. 1). We lso isolted one knokout line (pld1-1) with the T-DNA inserted in the first exon of PLD1, 877 p downstrem of the strt odon (Fig. 1d). Elimintion of the PLD1 trnsript in pld1-1 ws onfirmed y RNA lotting (Fig. 1d). The three PLD1 mutnt lines with vrying dereses in PLD1 trnsripts were used for further study. Under norml growth onditions, the PLD1-defiient plnts displyed no pprent morphologil ltertions. We exmined the expression of PLD1 in leves treted with severl ompounds ssoited with stresses to gin insights into its funtion. PLD1 trnsript level ws inresed in leves with.1 mm methyl jsmonte (MeJA), ut deresed with.1 mm SA or methyl sliylte (MeSA) (Fig. S1,). When leves were treted with mm H O, the expression of PLD1 ws suppressed in the erly phse (3 h) ut inresed lter ( h). In ontrst, sisi id (ABA) tretments indued trnsient inrese in PLD1 expression t h. When Aridopsis leves were inoulted with pthogens, the expression of PLD1 ws indued y oth virulent (Pst DC3) nd virulent (Pst DC3(vrRpt)) teril pthogens (Fig., left pnel). Infetion with the fungl pthogen B. inere lso strongly indued PLD1 expression (Fig., right pnel). In ontrst, PLD ws not indued y the teril or fugl pthogens, When Col-, nd plnts were infeted with the pthogen B. inere,thepld1 trnsript ws inresed in Col- plnts, ut only smll mounts of PLD1 trnsripts were deteted in the RNAi lines (Fig. S). To investigte the role of PLD1 in the plnt response to pthogens, we inoulted PLD1 mutnt nd wild-type Aridopsis plnts with virulent Pst DC3 or Pst DC3 (vrrpt). Most plnts displyed lef ell ollpse in the pthogen-inoulted leves within h (Fig. ). However, the lef ell ollpse ws more evident in the two RNAi lines nd pld1-1 plnts thn in wild-type plnts, with PLD1 mutnts displying oth erlier development of ell ollpse nd lrger ollpse re (Fig. ). The elerted ell ollpse in PLD1 mutnts ourred with oth Pst DC3 nd Pst DC3 (vrrpt) (Fig. ). Geneti omplementtion of pld1-1 with ntive PLD1 restored the mutnt s ollpses to the wild-type phenotype, suggesting tht ltion of PLD1 results in n enhned resistne to the New Phytologist (13) 199: 8

5 3 Reserh New Phytologist () HndIII () () (d) LB ATG T-DNA Exon RB EoR1 Intron TGA Exon x 35S NOS PLDβ1 trnsripts (% wild type) PLDβ1 proteins (% wild type) PLDβ1 PLDα1 EtBr Col- PLDβ1 PLDα1 Col- PLDβ1 PLDα1 Xho1 DNA 35 p EtBr Fig. 1 Prodution nd onfirmtion of phospholipse D 1(PLD1) RNAi knokdown nd T-DNA insertion knokout Aridopsis lines. () Constrution of the PLD RNAi vetor. Inverted repets of exons (gry oxes) nd n intron (empty oxes) with restrition sites were loned in tndem, nd their expression ws driven y doule CMV 35S promoters nd terminted y the nopline synthse (NOS) termintor. () RNA lotting of PLD1 nd PLD1 expression in RNAi lines. Equl mounts of totl RNA from Aridopsis leves were loded nd rrna deteted with ethidium romide (EtBr) ws used s loding ontrol. PLD1 protein levels were lso used s loding ontrols. The density of the PLD1 RNA nd ws quntified from three experiments. Dt rs represent the men ( SD) of three repets. () Immunolotting of PLD1 nd PLD1 protein levels in RNAi lines. Equl mounts of Aridopsis lef proteins were sujeted to SDS-PAGE, followed y immunolotting using ntiodies ginst PLD1 nd PLD1. The nd density of PLD1 ws quntified from three experiments. Dt rs represent the men ( SD) of three repets. (d) T-DNA insertionl knokout of PLD1. Left pnel, T- DNA insertion position in PLD1 with exons (gry oxes) nd introns (rs etween exons). Right pnel, northern lot showing the sene of the PLD1 trnsript in T-DNA insertion knokout. PLD1 expression nd 8S RNA (deteted with ethidium romide) were used s loding ontrols. EtBr, ethidium romide. iotrophi pthogen Pst DC3 (Fig. ). No symptoms ourred in ontrol plnts treted only with 1 mm MgSO.A teril growth ssy showed tht PLD1 mutnt plnts were New Phytologist (13) 199: 8 suppressed in teril pthogen growth, nd the differene in pthogen growth etween pld1-1 nd wild-type fter virulent Pst DC3 infetion ws greter thn tht fter virulent Pst DC3 (vrrpt) infetion (Fig. ). Both RNAi mutnts nd pld1-1 produed higher levels thn in wild-type of totl SA (free SA nd SA glyoside) in response to Pst DC3 infetions (Fig. 3) nd Pst DC3 (vrrpt) infetions (Fig. S3). These differenes were evident in oth inoulted (lol) leves nd neighoring (distl) leves, suggesting systemti indution of SA prodution (Fig. 3). Both quntittive RT-PCR nd RNA lotting were used to evlute defenserelted gene expression in treted/nontreted smples. Consistent with the higher level of SA, the SA-induile pthogenesis-relted (PR) gene PR1 ws up-regulted in PLD1 mutnts (Figs 3, S). However, PLD1 mutnts displyed lower levels of expression of the JA iosyntheti genes AOS nd LOX, nd the JA/ethyleneregulted defensive gene PDF1., thn the wild-type (Figs 3, S). PLD1 mutnts inresed suseptiility to Botrytis inere When plnts were inoulted with spores of B. inere, PLD1 mutnts displyed elerted nd lrger neroti lesions ompred with wild-type plnts, s indited y lesion res mesured t dy 5 post inoultion (Fig. ). In ddition, more infeted leves hd neroti lesions with hloroti hlos (Fig. ). qrt- PCR nd RNA lotting nlyses showed tht PLD1 mutnts hd lower levels of expression of LOX, AOS, nd PDF1., ut higher level of PR1 expression thn wild-type in response to B. inere (Figs, S5). We then mesured endogenous JA nd SA onentrtions in PLD1-knokout (KO), wild-type, nd PLD1-omplemented plnts with or without pthogen inoultion. JA onentrtions in these plnts inresed gretly h fter B. inere spore inoultion, ut the JA onentrtion of pld1-1 ws signifintly lower thn tht of wild-type nd PLD1-omplemented plnts (Fig. d). Menwhile, SA mesurement results suggest tht PLD1-KO nd knokdown mutnts ll hd slightly higher onentrtions of totl SA fter 3 d of spores-inoultion (Fig. e), whih is onsistent with higher PR1 expression. These results suggest tht olishing PLD1 impirs fungl pthogen-indued JA prodution, rendering plnts more sensitive to the pthogen. PLD1 muttion inresed ROS prodution in response to pthogens We exmined whether there ws differene etween PLD1 mutnts nd wild-type in ellulr ROS umultion during the defense response. When O rets with NBT, preipitte of purple formzn forms (Ro et l., ) (Fig. 5). Pthogentreted leves of the PLD1-defiient mutnts displyed more intense lue stining thn those of the wild-type. The intensity of lue stining ws inversely ssoited with the level of PLD1 trnsript in the two RNAi mutnts nd pld1-1 (Fig. 5,). To verify the ssoition of PLD1 defiieny with ROS prodution, we mesured H O onentrtions in infeted leves. PLD1 mutnts generted more H O thn wild-type plnts in response Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust

6 New Phytologist Reserh 33 () Reltive expression PLDβ1 PLDβ Reltive expression PLDβ1 PLDβ MgSO Pst DC3 Pst DC3 (vrrpt) Time fter inoultion (h) () Col- -1 Col- Pst DC3 Pst DC3 (vrrpt) MgSO () Bteril prolifertion (inrese in log CFU/lef) PLDβ1-1 Col- Pst DC Dys post inoultion Bteril prolifertion (inrese in log CFU/lef) Pst DC3 (vrrpt) PLDβ1-1 Col Dys post inoultion Fig. Responses of phospholipse D 1(PLD1) Aridopsis mutnts to teril pthogens. Fully expnded leves of Columi (Col-), pld1-1, RNAi mutnts, nd pld1-1 omplementtion () plnts were infiltrted with suspension ontining Pseudomons syringe pv tomto DC3 (Pst DC3) with or without vrrpt. () Expression of PLD1 nd PLD in Col- plnts infeted with Pst DC3 pthogens or Pst DC3 (vrrpt, type III effetor gene from the Pseudomons syringe) pthogens ( CFU ml 1 ) for 1 h. Leves treted with 1 mm MgSO were used s ontrol (mok). The reltive expression level of PLD1 nd PLD ws nlyzed y quntittive PCR. Expression levels were normlized with respet to the housekeeping genes UBQ (uiquitin 5) nd Tuulin. Dt rs represent the men ( SD) of three repets. () Defensive response of Aridopsis plnts with ltered PLD1 expression to virulent nd virulent Pst DC3 ( CFU ml 1 ). The photogrphs show representtive leves inoulted with teril pthogen for 1 h. () Prolifertion of the virulent Pst DC3 (left pnel) nd virulent Pst DC3 (vrrpt)(9 1 5 CFU ml 1 ) (right pnel) pthogens in Col-, pld1-1 nd RNAi plnts. Bteril growth in five lef diss ws mesured. Bteril numers re expressed s the inrese in olonyforming units (CFU) per lef dis fter teril inoultion (i.e. log CFU post inoultion log CFU t time zero). Dt represent the verge of three smples SD. Different letters indite smple groups with signifint differenes (P <.5) from eh other. to infetions with the teril pthogens Pst DC3 nd Pst DC3 (vrrpt) nd the fungl pthogen B. inere (Figs 5,d, S). The enhned ROS prodution in PLD1-defiient plnts ws orrelted with stronger defensive response nd more severe neroti responses thn oserved in wild-type plnts. PLD1 ltion deresed virulent pthogen-indued prodution of phosphtidi id To determine whether ltion of PLD1 hnged the ellulr onentrtion of PA, we quntittively profiled phospholipids nd gltolipids in Col-, pld1-1, nd PLD1-omplemented plnts with or without pthogen tretments. There were no differenes in PA onentrtion mong Col-, pld1-1, nd PLD1-omplemented plnts without tretment. Inoultion with B. inere spores indued signifint inreses in PA in ll three genotypes, ut the PA inrese in pld1-1 ws signifintly lower thn tht in Col- nd PLD1-omplemented plnts (Fig. ). Conentrtions of the mjor PA speies, 3:-, 3:3-, 3:-, 3:5-, nd 3:-PA, in pld1-1 were signifintly lower thn those in wild-type plnts fter the fungl pthogen tretment (Fig. S7). Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust New Phytologist (13) 199: 8

7 3 Reserh New Phytologist Totl SA (μg g 1 FW) () Col Time post inoultion (h) () Totl SA (μg g 1 FW) Col- -1 Mok Distl Lol () PR1 expression (reltive to UBQ5) Col- Mok Pst3 Pst3 (vrrpt) AOS expression (reltive to UBQ5) 1 8 Col- Mok Pst3 Pst3 (vrrpt) PDF1. expression (reltive to UBQ5) Col- Mok Pst3 Pst3 (vrrpt) LOX expression (reltive to UBQ5 ) Col- Mok Pst3 Pst3 (vrrpt) Fig. 3 Sliyli id (SA) in pthogen-inoulted phospholipse D 1(PLD1) Aridopsis mutnts. Totl SA (free SA + SA glyosides) in the wild type, pld1-1,, nd were determined y HPLC. Dt re presented s men SD (n = 3). Different letters indite smple groups with signifint differenes (P <.5) from eh other. () SA onentrtions in inoulted leves s funtion of time post Pseudomons syringe pv tomto DC3 (Pst DC3) inoultion ( CFU ml 1 ). () SA onentrtions in lol nd distl leves from plnts infeted with Pst DC3 for 1 h. () Expression of defense-relted genes in Col-, pld1-1,,, nd pld1-1 omplementtion () plnts infeted with Pst DC3 pthogens (pst) or Pst DC3 (vrrpt) (Rpt) pthogens ( CFU ml 1 ) for 1 h. Leves treted with 1 mm MgSO (m) were used s ontrol. The reltive expression level of PR-1 ws nlyzed y quntittive PCR. Expression levels were normlized with respet to the housekeeping genes. Dt rs represent the men ( SD) of three repets. AOS, oxide synthse; LOX, lipoxygense; PR, pthogenesis-relted; PDF, plnt defensin. The PA inrese ws lso oserved when the plnts were treted with the virulent pthogen Pst DC3 (Fig. ). Pst DC3 infetion signifintly inresed PA prodution h post inoultion, ut pld1-1 hd lower PA onentrtions thn wild-type nd PLD1-omplemented plnts fter Pst DC3 infetion (Fig. ). The mjor PA speies, suh s 3:-, 3:3-, 3:-, 3:5- nd 3:-PA, were signifintly lower in pld1-1 thn in wild-type nd PLD1-omplemented plnts treted with virulent teril pthogens (Fig. S7). Inoultion with the virulent pthogen Pst DC3 (vrrpt) indued more PA prodution thn did Pst DC3 nd B. inere infetions, ut pld1-1 did not show muh differene from wild-type or PLD1-omplemented plnts in PA onentrtions (Fig. ). These results suggest tht PLD1 plys different role in virulent nd virulent teril pthogen-indued PAPA prodution: wheres it ontriutes signifintly to the virulent pthogen-indued PA prodution, ut less to the virulent pthogen-indued PA under this ssy ondition. The onentrtions of totl phosphtidylholine (PC), phosphtidylethnolmine (PE), phosphtidylinositol (PI); phosphtidylglyerol (PG), nd digltosyldiylglyerol (DGDG) New Phytologist (13) 199: 8 were omprle in wild-type nd pld1-1 with or without pthogen infetion (Fig. ). However, infetions resulted in different hnges in wild-type nd pld1-1 when lipid moleulr speies were nlyzed (Fig. S8). Tretment with B. inere tended to result in higher onentrtions of 3:3, 3:5 nd 3: PC nd 3:3 PE, nd tretment with Pst DC3 resulted in higher onentrtions of 3:5 nd 3: PC in the PLD1 mutnt reltive to wild-type (Fig. S8). These higher onentrtions of PC nd PE moleules in PLD1 mutnts versus wild-type plnts fter pthogen tretments were ssoited with the lower levels of PA prodution in PLD1 thn in wild-type. These dt might suggest tht PC nd PE re potentil in vivo sustrtes of PLD1. PLD1-KO hd elevted lysophospholipid onentrtions in virulent pthogen-infeted leves In ontrst to the smller inrese in PA in pld1-1, higher onentrtions of totl lysophosphtidylholine (LPC), lysophosphtidylethnolmine (LPE), nd lysophosphtidylglyerol (LPG) were found in pld1-1 thn in wild-type nd Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust

8 New Phytologist Reserh 35 () B. inere spores Control Control B. inere spores Col- Col- -1 () 1 Nerosis rte (%) Pthogen Control Col- -1 () LOX expression (reltive to UBQ) Col- 1 3 Dys fter Inoultion AOS expression (reltive to UBQ) Col- 1 3 Dys fter inoultion PDF1. expression (reltive to UBQ) (d) JA (ng mg 1 FW) Col- 1 3 Dys fter inoultion Col- -1 PR1 expression (reltive to UBQ) (e) Totl SA (μg g 1 FW) Col- Col- 1 3 Dys fter inoultion h 1 1t t 1 3 Time (hpi) Dys fter inoultion Fig. Responses of phospholipse D 1(PLD1) Aridopsis mutnts to Botrytis inere. () Leves of 3-wk-old Columi (Col-), pld1-1, RNAi, nd pld1-1 omplementtion () plnts were inoulted with B. inere spores ( spores ml 1 ) or wter (ontrol). The photogrphs show representtive leves 5 d post inoultion. () Lesions sored t 5 d post inoultion. The lesion development rte ws the numer of leves with neroti re mong the inoulted leves in eh line. Dt re presented s men SD (n = 5). Different letters indite smple groups with signifint differenes (P <.5) from eh other. () Expression of defense-relted genes in Col- nd PLD1 mutnts infeted with fungl pthogens determined y quntittive PCR. Expression levels were normlized with respet to uiquitin 5 (UBQ5). Dt rs represent the men ( SD) of three repets. Leves treted with wter were used s ontrol. (d) JA onentrtions in wild-type (Col-), pld1-1, nd pld1 omplementtion () plnts t vrious hours post inoultion (hpi) with B. inere. C indites ontrol (i.e. mok-inoulted) nd T indites treted (i.e. pthogen-inoulted). Three-week-old soilgrown seedlings were treted with B. inere spores. Vlues re men SE of three independent replite experiments. (e) Sliyli id (SA) in B. inere spore-inoulted PLD1 mutnts. Smples were hrvested fter inoultion with B. inere spores ( CFU ml 1 ) for different times. Totl SA (free SA + SA glyosides) in the wild-type, pld1-1,,, nd pld1-1 omplementtion plnts ws determined y HPLC. Dt re presented s men SD (n = 3). AOS, oxide synthse; LOX, lipoxygense; PR, pthogenesis-relted; PDF, plnt defensin. dd omplemented plnts fter B. inere infetions, wheres without infetion, pld1-1 nd wild-type displyed no signifint differene (Fig. 7, top pnel). After virulent Pst DC3 infetion, pld1-1 plnts lso hd higher onentrtions of LPE nd LPG Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust thn wild type (Fig. 7, middle pnel). However, fter infetion with the virulent pthogen Pst DC3 (vrrpt), only LPG ws signifintly higher in pld1-1 thn in wild-type, nd the differene in LPG etween wild type nd pld1-1 ws lso smller New Phytologist (13) 199: 8

9 3 Reserh New Phytologist () Col- -1 () Col- -1 () 1 H O (nmol g 1 FW) 1 8 (d) H O (nmol g 1 FW) Col Time post inoultion (h) Col Dys post inoultion (dy) Fig. 5 Retive oxygen speies (ROS) genertion in the Aridopsis response to pthogen infetion. The wild-type (Columi (Col-)), phospholipse D 1-1 (pld1-1),, nd plnts were inoulted with Pseudomons syringe pv tomto DC3 (Pst DC3) ( CFU ml 1 ) or Botrytis inere spores. Dt re presented s men SD (n = 5). () Nitrolue tetrzolium stining of 5-wk-old Aridopsis leves 1 h post inoultion with Pst DC3 teril pthogen ( CFU ml 1 ). () H O quntifition in Pst DC3-infeted leves 1 h post inoultion. () Nitrolue tetrzolium stining of 5-wk-old Aridopsis leves 1 h post inoultion with B. inere. (d) H O quntifition in B. inere sporeinoulted leves. Different letters indite smple groups with signifint differenes (P <.5) from eh other. Lipid/dry weight (nmol mg 1 ) Col- C -1 C C Col- T -1 T T B. inere Pst DC3 Pst DC3 (vrrpt) PC PE PI PA PS PG MGDG DGDG Lipid speies (totl yl rons: totl doule onds) Fig. Phospholipid nd gltolipid ontents in leves of wild-type nd phospholipse D 1-1 (pld1-1) Aridopsis plnts s ffeted y Botrytis inere, Pseudomons syringe pv tomto DC3 (Pst DC3), or virulent Pst DC3 (vrrpt) infetion. Columi (Col-), pld1-1, nd pld1 omplementtion () plnts were inoulted with ontrol solutions (-C) or with pthogens (-T). Control solutions were wter for B. inere spores ( spores ml 1 ) nd 1 mm MgSO for the virulent strin Pst DC3 nd the virulent strin Pst DC3 (vrrpt)(9 1 7 CFU ml 1 ). Lef smples were olleted for lipid profiling h post inoultion. Vlues re men SE (n = 5). Different letters indite smple groups with signifint differenes (P <.5) from eh other. PA, phosphtidi id; PC, phosphtidylholine; PE, phosphtidylethnolmine, PI, phosphtidylinositol; PG, phosphtidylglyerol; PS, phosphtidylserine, MGDG, monogltosyldiylglyerol; DGDG, digltosyldiylgly. thn tht for virulent Pst DC3-infeted plnts (Fig. 7, ottom pnel). In ddition, the virulent Pst DC3 (vrrpt) infetion showed n inrese in lysophospholipid onentrtions in wildtype, pld1-1, nd omplemented plnts (Fig. 7, ottom pnel). However, no suh inrese ws oserved in wild-type nd omplemented plnts fter virulent Pst DC3 or B. inere infetion (Fig. 7, top nd middle pnels). New Phytologist (13) 199: 8 The mjor LPC nd LPE speies were 18:3, 18:, nd 1: speies (Fig. S9,), wheres 18:3, 1:1, nd 1: were the mjor LPG speies (Fig. 7d). At h post inoultion with Pst DC3, oth 18:3-LPC nd 18:-LPC were higher in pld1-1 thn in wild-type nd omplemented plnts (Fig. S9). In the pld1-1 plnts, the formtion of 1:1-LPG fter pthogen inoultion ws prtiulrly pprent (Fig. S9). Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust

10 New Phytologist Reserh 37 Disussion Our results show tht PLD1 is negtive regultor of SAdependent resistne to Pst DC3, ut positive regultor of the JA-dependent pthwy nd resistne to B. inere. The negtive role of PLD1 ws indited y the inresed prodution of SA, SA-induile genes, nd deresed dmge of virulent teril infetion. The positive effet of PLD1 on the JA-dependent signling pthwy ws indited y deresed expression of JAiosyntheti nd -responsive genes, deresed JA prodution, nd inresed dmge in response to B. inere inoultion. Botrytis inere inoultion hs een reported to indue sustntil inrese in JA (Trumn et l., 7; Yng et l., 7). However, the expression of PLD1 is indued y JA nd lso y wounding (Wng et l., 1). Of the four PLDs (1, 1,, nd 1) exmined, the gretest inrese for PLD1 mrna ourred 3 min fter wounding, wheres tht for PLD1 nd PLD trnsripts ourred t min fter wounding nd tht for PLD mrna ourred t 3 to hr fter wounding (Wng et l., 1). However, the wounding-indued JA prodution in Aridopsis peked 1 h fter wounding (Wng et l., 1). These results ould men tht PLD1 is involved in pthogen- nd woundingindued JA prodution. The SA-dependent nd JA/ethylenedependent signling pthwys in Aridopsis ontriute to resistne to distint miroil pthogens (Spoel et l., 3; Durrnt & Dong, ; Trumn et l., 7). Yet, lose interply exists etween the two pthwys. For exmple, the NON-EXPRESSOR OF PR GENES1 (NPR1) mutnt, whih is impired in SA signling, produes higher onentrtion of JA nd shows n inresed suseptiility to pthogens reltive to wild type (Spoel et l., 3). The effets of PLD1 on plnt pthogen intertions indite tht PLD1 nd ssoited lipid hnges re involved in the SA-dependent nd JA/ethylene-dependent plnt defenses ginst teril nd fungl pthogens. Tretments of plnts with P. syringe with or without AvrRpm1 or AvrRpt, rhizoium, fungl pthogens, or eliitors hve ll een reported to indue PA prodution (Vn der Luit et l., ; de Jong et l., ; Andersson et l., ; Brgmnn et l., ). However, the speifi PLD responsile for the pthogen-indued PA prodution ws unknown. The urrent study showed tht knokout of PLD1 deresed the PA prodution indued y B. inere nd virulent Pst DC3 infetion, ut the loss of PLD1 hd no mjor impt on the PA prodution indued y the virulent pthogen Pst DC3 (vrrpt). The results indite tht PLD1 is responsile for mjor portion of the PA generted during virulent teril nd fungl pthogen ttk, ut it ontriuted less to virulent pthogen-indued PA prodution under the present ssy onditions. It is possile tht other PLDs, suh s PLD1 nd PLDd, esides PLD1, re tivted in the plnt virulent DC3 (vrrpt) intertion, thus msking the differene in PA prodution etween pld1-1 nd wild-type plnts. Speifilly, PLDd hs een shown to e responsile for most H O -indued PA prodution (Zhng et l., 3). The virulent infetion indued more H O prodution thn the virulent infetion (Fig. S versus Fig. 5d), whih my result in inresed PLDd tivity during the DC3 (vrrpt) infetion. Thus, these dt indite tht other PLDs, esides PLD1, re involved in pthogen-indued PA prodution, nd during virulent infetion the other PLDs ontriute more, thus overwhelming the effet of PLD1..1 Col- C -1 C C Col- T -1 T T B. inere Fig. 7 Conentrtions of totl LPC, LPE, nd LPG in leves of wild-type nd phospholipse D 1-1 (pld1-1) Aridopsis plnts with nd without inoultion with Botrytis inere, Pseudomons syringe pv tomto DC3 (Pst DC3), or virulent Pst DC3 (vrrpt). Columi (Col-), pld1-1, nd pld1 omplementtion () plnts were inoulted with ontrol solutions (-C) or with pthogens (-T). Control solutions were wter for B. inere nd 1 mm MgSO for the virulent strin Pst DC3 nd the virulent strin Pst DC3 (vrrpt). Lef smples were olleted for lipid profiling h post inoultion. Vlues re men SE (n = 5). Different letters indite vlues with signifint differenes (P <.5) from eh other LPC, lysophosphtidylholine; LPE, lysophosphtidylethnolmine; LPG, lysophosphtidylglyerol. Lipid/dry weight (nmol mg 1 ) Pst DC3 Pst DC3 (vrrpt) LPC LPE LPG Lipid speies (totl yl rons: totl doule onds) Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust New Phytologist (13) 199: 8

11 38 Reserh New Phytologist rogtion. However, pld1-1 still displyed inresed resistne to virulent DC3 (vrrpt) infetion, even though no differene in PA onentrtion ws deteted etween pld1-1 nd wild-type plnts. One possile explntion for this is tht the PA produed y other PLDs my ply less importnt (or different) role in heightening disese sensitivity thn PLD1- derived PA. Different PLDs nd their derived PA hve een shown to ply distinguishle roles in the plnt response to different stresses (Zhng et l., 3, 9). Unlike the ttenuted inrese in PA, the onentrtions of the lysophospholipids LPC, LPE, nd LPG in pthogeninfeted PLD1-defiient plnts were higher thn those in wild-type plnts. This inverse reltionship etween PA nd lysophospholipids in teril infetion ws lso oserved with nother mutnt, SOBER1, whih ffeted Aridopsis Pst DC3 (vrbst) intertions (Kirik & Mudgett, 9). SOBER1 is PLA tht negtively regultes pthogen-indued PA umultion. Both SOBER1 nd PLD1 re negtive regultors in Pst DC3 (vrbst) nd virulent Pst DC3 defense, respetively. The dt ould indite ross-tlk etween the PLD nd PLA pthwys in plnt pthogen intertions. Suh n intertion hs lso een proposed to our in the plnt response to wounding nd senesene (Ryu & Wng, 199; Ryu et l., 1997). LPE ws reported to inhiit PLD to derese PA prodution nd dely senesene (Ryu et l., 1997). It is oneivle tht lysophospholipids my inhiit PLD nd other PLDs to suppress PA prodution (Kirik & Mudgett, 9). The present findings suggest tht the ltion of PLD1 results in n inrese in PLA tivity. It would e of interest in future studies to determine how PLD nd its derived PA suppress PLA nd the prodution of lysolipids. PLD nd PA re oth involved in ROS prodution nd response (Sng et l., 1; Prk et l., ; Andersson et l., ). PLD1-derived PA hs een shown to diretly intert with NADPH oxidse on the plsm memrne nd promote ROS prodution (Zhng et l., 9). However, the present study showed tht PLD1-defiient plnts produed lower onentrtion of PA, ut higher onentrtion of ROS in response to virulent teril nd fungl pthogen infetion. The sme trend hs lso een oserved in PLD1- defiient rie nd tomto (Brgmnn et l., ; Ymguhi et l., 9). These results suggest tht PLD1 nd its derived PA re unlikely to promote ROS prodution, nd insted they my ply role in mediting the ROS response, similr to tht of PLDd, where ltion of PLDd rendered plnts more sensitive to ROS-promoted ell deth (Zhng et l., 3). PLD1 mutnts showed n inresed hypersensitive response phenotype in response to virulent or virulent Pst DC3 infetions. The effets my result from inresed ROS dmge in PLD1 mutnts ompred with wild-type plnts. The inresed onentrtion of ROS in PLD1-defiient mutnts my inrese SA iosynthesis, whih in turn inreses the ROS onentrtion euse SA inhiits tlse tivity (Durner & Klessig, 1995; Mrtinez et l., ). Rpid iosynthesis of SA is importnt for New Phytologist (13) 199: 8 plnts to estlish systemti resistne to Pst DC3 (Durrnt & Dong, ). Conlusion The present dt indite tht PLD1 plys positive role in pthogen-indued PA prodution, JA umultion, JA-dependent defense gene expression, nd plnt resistne to pthogenesis of the nerotrophi fungl pthogen B. inere. In ontrst, PLD1 suppresses the SA-dependent signling pthwy nd defense gene expression, thus negtively ffeting plnt resistne to infetion with iotrophi Pst DC3. In ddition, the results suggest tht PLD1 my suppress the prodution of lysolipids, implying ross-tlk etween PLD1- nd PLA-medited signling in the plnt response to teril nd fungl pthogens. Further study is needed to determine whether the inverse reltionship etween PA nd lysophospholipid prodution my underlie hemil mehnism tht modultes plnt pthogen intertions. Aknowledgements We thnk Mry Roth for lipid nlysis. The work ws supported y grnts from the Ntionl Siene Foundtion (IOS-8187; MCB-9879) nd the US Deprtment of Agriulture to X.W. Equipment quisition nd method development t the Knss Lipidomis Reserh Center were funded y the Ntionl Siene Foundtion (EPS 3913, MCB nd 93, DBI 51587), Knss Tehnology Enterprise Corportion, K-IDeA Networks of Biomedil Reserh Exellene (INBRE) of Ntionl Institute of Helth (PRR175), nd Knss Stte University. Referenes Armovith RB, Anderson JC, Mrtin GB.. Bteril eliittion nd evsion of plnt innte immunity. Nture Review of Moleulr Cell Biology 7: Andersson MX, Kourthenko O, Dngl JL, Mkey D, Ellerstr om M.. Phospholipse-dependent signlling during the AvrRpm1- nd AvrRpt- indued disese resistne responses in Aridopsis thlin. Plnt Journl 7: Brgmnn BO, Lxlt AM, Riet BT, Shouten E, vn Leeuwen W, Dekker HL, de Koster CG, Hring MA, Munnik T.. LePLDet1 tivtion nd reloliztion in suspension-ultured tomto ells treted with xylnse. 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Phospholipse D1 nd phosphtidi id regulte NADPH oxidse tivity nd prodution of retive oxygen speies in ABAmedited stomtl losure in Aridopsis. Plnt Cell 1: Zho J, Wng C, Bedir M, Welti R, Sumner LW, Bxter I, Xuemin Wng X. 11. Suppression of phospholipse Ds onfers inresed luminum resistne in Aridopsis thlin. PLoS ONE : e88. Zheng L, Krishnmoorthi R, Zolkiewski M, Wng X.. Distint lium inding properties of novel C domins of plnt phospholipse D nd. Journl of Biologil Chemistry 75: Zheng L, Shn J, Krishnmoorthi R, Wng X.. Ativtion of plnt phospholipse D y phosphtidylinositol,5-isphosphte: hrteriztion of inding site nd mode of tion. Biohemistry 1: Supporting Informtion Additionl supporting informtion my e found in the online version of this rtile. Fig. S1 PLD1 expression pttern. Fig. S PLD1 expression in Aridopsis leves indued y Botrytis inere infetion in Col- nd PLD1 mutnts. Fig. S3 Sliyli id in pthogen-inoulted Aridopsis leves. Fig. S Bteril infetion-indued defense gene expression in wild-type nd pld1-1 Aridopsis leves. Fig. S5 Defense-relted gene expression indued y Botrytis inere infetion in Aridopsis Col- nd PLD1 mutnts. Fig. S H O quntifition in Pst DC3 (vrrpt)-infeted Aridopsis leves. Fig. S7 Profiling of phosphotidi id speies in Col-, pld1-1, nd pld1-1 omplemented Aridopsis plnts inoulted with ontrol solutions (-C) or with pthogens (-T). Ó 13 The Authors New Phytologist Ó 13 New Phytologist Trust New Phytologist (13) 199: 8