Phospholipase C2 Affects MAMP-Triggered Immunity byModulating ROS Production1

Juan Martín D’Ambrosio,a Daniel Couto,b Georgina Fabro,c Denise Scuffi,a Lorenzo Lamattina,a

Teun Munnik,d Mats X. Andersson,e María E. Álvarez,c Cyril Zipfel,b and Ana M. Laxalta,2

aInstituto de Investigaciones Biológicas IIB-Consejo Nacional de Investigaciones Científicas y Técnicas,Universidad Nacional de Mar del Plata, 7600 Mar del Plata, ArgentinabSainsbury Laboratory, Norwich NR4 7UH, United KingdomcCentro de Investigaciones en Química Biológica de Córdoba, UNC-Consejo Nacional de InvestigacionesCientíficas y Técnicas, Universidad Nacional de Córdoba, X5000HUA Cordoba, ArgentinadSwammerdam Institute for Life Sciences, Section Plant Cell Biology, University of Amsterdam, 1098 XHAmsterdam, The NetherlandseDepartment of Biological and Environmental Sciences, University of Gothenburg, SE-405 30 Gothenburg, Sweden

ORCID IDs: 0000-0002-0084-4006 (L.L.); 0000-0002-4919-4913 (T.M.); 0000-0003-4279-6572 (M.X.A.); 0000-0001-7001-5898 (M.E.Á.);0000-0003-4935-8583 (C.Z.); 0000-0002-8225-2441 (A.M.L.).

The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition ofseveral microbe-associatedmolecular patterns (MAMPs) in plants. The Arabidopsis (Arabidopsis thaliana) PI-PLC gene family is composedof nine members. Previous studies suggested a role for PLC2 inMAMP-triggered immunity, as it is rapidly phosphorylated in vivo upontreatment with the bacterial MAMP flg22. Here, we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silencePLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficientbacterial strain Pseudomonas syringae pv tomato (Pst) DC3000 hrcC2 and to the nonadapted pea (Pisum sativum) powdery mildew Erysiphepisi. However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent (Pst DC3000 AvrRPM1) P.syringae strains, conserving typical hypersensitive response features. In response to flg22, PLC2-silenced plants maintain wild-typemitogen-activated protein kinase activation and PHI1, WRKY33, and FRK1 immune marker gene expression but have reducedreactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation ofROS upon flg22 treatment is compromised in the PLC2-defficient plants, suggesting an effect of PLC2 in a branch of MAMP-triggeredimmunity and nonhost resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidaseRBOHD, suggesting its potential regulation by PLC2.

Plants are constantly challenged by microbial patho-gens, and to resist them, they exhibit various defensemechanisms. A first line of inducible defenses is triggered

by the recognition of microbe-associated molecular pat-terns (MAMPs) by cell surface pattern recognition recep-tors (Antolín-Llovera et al., 2014). This recognition inducesMAMP-triggered immunity (MTI), which confers resis-tance to multiple microbes (Couto and Zipfel, 2016).Adapted plant pathogens use secreted effector proteins to,among other things, interfere with MTI, resulting in theso-called effector-triggered susceptibility. Eventually, mi-crobial effectors can become detected by intracellularnucleotide-binding Leu-rich repeat (NLR) proteins, trig-gering a second line of defense called effector-triggeredimmunity (ETI; Jones and Dangl, 2006).

After the recognition of MAMPs, a series of rapid re-sponses are initiated, including an increase in cytosolicCa2+, generation of apoplastic reactive oxygen species(ROS), activation of mitogen-activated protein kinases(MAPKs) and Ca2+-dependent protein kinases (CDPKs),callose deposition, and stomatal closure (Boller and Felix,2009; Segonzac andZipfel, 2011). Among the best-studiedresponses to MAMPs are those triggered following therecognition of bacterial flagellin (or the derived peptideflg22) by the Arabidopsis (Arabidopsis thaliana) Leu-richrepeat receptor kinase (LRR-RK) FLAGELLIN SENSING2

1 This work was supported by UNMdP, Consejo Nacional de Inves-tigaciones Científicas y Técnicas (CONICET; PIP 1142010010 0219), byAgencia Nacional de Promoción Científica y Tecnológica (PICT 2010No574, PICT 2012 No 2117, PICT 2014 No 1621, and PICT 2014 No 3255),by EMBO Short Term Fellowship (ASTF 477 - 2015) and by the CarlTrygger Foundation for Scientific Research to M.X.A. C.Z. was fundedby the Gatsby Charitable Foundation and the European Research Coun-cil (PHOSPHinnATE). T.M. was funded by the Netherlands Organiza-tion for Scientific Research (867.15.020).

2 Address correspondence to amlaxalt@mdp.edu.ar.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Ana M. Laxalt (amlaxalt@mdp.edu.ar).

A.M.L. conceived the original research plans; A.M.L., M.X.A., M.E.Á.,and C.Z. designed and supervised the experiments and analyzed thedata; J.M.D. performed most of the experiments and analyzed thedata; D.C., G.F., and D.S. performed some of the experiments; A.M.L.conceived the project and wrote the article with contributions of all theauthors; L.L. and T.M. supervised and complemented the writing.

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(FLS2; Felix et al., 1999; Gómez-Gómez and Boller, 2000;Sun et al., 2013). Upon ligand recognition, FLS2 formsa complex with the LRR-RK BRASSINOSTEROIDRECEPTOR1-ASSOCIATED KINASE1 (BAK1), alsoknown as SOMATIC EMBRYOGENESIS-RELATEDKINASE3 (SERK3; Chinchilla et al., 2007; Heese et al.,2007; Roux et al., 2011; Sun et al., 2013). This complexinteracts with and phosphorylates the receptor-like cy-toplasmic kinase BOTRYTIS INDUCED KINASE1(BIK1; Veronese et al., 2006; Lu et al., 2010; Zhanget al., 2010). Upon activation, BIK1 phosphorylates theplasma membrane NADPH oxidase RBOHD, thuspriming apoplastic ROS production (Kadota et al.,2014; Li et al., 2014).Several lipids and lipid-derived metabolites have been

shown to function in signal transduction pathways lead-ing to the activation of plant defense responses (Laxalt andMunnik, 2002; Munnik and Vermeer, 2010; Hung et al.,2014; Hong et al., 2016). Specifically, phosphoinositide-specific phospholipase C (PI-PLC) is rapidly activated inplant cells after the recognition of different MAMPs, suchas xylanase, flg22, and chitosan (van der Luit et al., 2000;Laxalt et al., 2007; Raho et al., 2011), or of pathogen effectorproteins (de Jong et al., 2004; Andersson et al., 2006).PI-PLC catalyzes the hydrolysis of phosphatidylinositol4-phosphate and phosphatidylinositol (4,5) bisphosphate(PIP2) to generatewater-soluble inositol bisphosphate (IP2)or IP3 and diacylglycerol (DAG), which remains in themembrane. In plants,DAGproducedbyPI-PLCactivity isphosphorylated by DAG kinase (DGK) to produce phos-phatidic acid (PA), which regulates several protein targets(Arisz et al., 2009; Testerink and Munnik, 2011; Munnik,2014). PA has been implicated specifically in the modu-lation of immune signaling components, such as MAPKsand PHOSPHOINOSITIDE-DEPENDENT PROTEINKINASE1 (PDK1; Farmer and Choi, 1999; Lee et al.,2001; Szczegielniak et al., 2005; Anthony et al., 2006).In particular, PA binds to the NADPH oxidase isoformsRBOHD and RBOHF to induce ROS during abscisic acid(ABA)-mediated stomatal closure (Zhang et al., 2009).Additionally, it has been shown that PLC activity is re-quired for ROS production during ETI responses (deJong et al., 2004; Andersson et al., 2006).In animals, IP3 triggers the release of Ca2+ from in-

tracellular stores by activating a ligand-gated calciumchannel at the endoplasmic reticulum. In plants, no clearhomolog of the IP3-activated Ca2+ channel has beenidentified (Munnik and Testerink, 2009). Instead, IP2 andIP3 are further phosphorylated by inositolpolyphosphatekinase (Williams et al., 2015) to generate (1) IP6, whichstimulates the release of Ca2+ from intracellular storesin guard cells (Lemtiri-Chlieh et al., 2000), affects genetranscription andmRNA export, and regulates the auxinreceptor TIR1 (Tan et al., 2007; Lee et al., 2015); (2) IP5,which is part of the jasmonate receptor COI1 (Sheardet al., 2010); and (3) IP7 and IP8, which are involved inplant defense (Laha et al., 2015). In addition, PIP andPIP2, originally characterized as PLC substrates, do havesignaling properties themselves, since many proteinsinvolved inmembrane trafficking and signal transduction

have domains that bind to these lipids (Munnik andNielsen, 2011; Delage et al., 2013; Heilmann, 2016).

The Arabidopsis genome contains nine genes en-coding PI-PLCs (AtPLC1–AtPLC9; Mueller-Roeberand Pical, 2002). AtPLC2 (hereafter PLC2) is the mostabundant PLC isoform, which is strongly and consti-tutively expressed and localizes to the plasma mem-brane (Pokotylo et al., 2014). PLC2 also is rapidlyphosphorylated following flg22 recognition (Nühseet al., 2007). In this work, we analyzed the role of PLC2in resistance to Pseudomonas syringae and Erysiphe pisiand in responses triggered upon flg22 perception. Wefound that PLC2 plays an important role in stomatalpreinvasion immunity and nonhost resistance andthat it associates with RBOHD, suggesting a potentialregulation of the Arabidopsis NADPH oxidase and,consequently, of ROS-dependent processes by PLC2.

RESULTS

PLC2 Silencing by Artificial MicroRNA

To study the role of PLC2 in plant defense, we de-veloped PLC2-silenced Arabidopsis plants by constitu-tively expressing a specific artificial microRNA (amiR).Expression analysis using quantitative PCR (qPCR) ofPLC2 in leaves of T4 amiR-PLC2 homozygous plantsshowed that PLC2 was stably silenced (Fig. 1A). Theexpression of PLC7 (the closest homolog to PLC2), PLC4(coexpressed with PLC2), and PLC1 (the second mostabundant PLC; Pokotylo et al., 2014) was not altered inPLC2-silenced plants (Supplemental Fig. S1A). Western-blot analysis using a specific anti-PLC2 antibody (Otterhaget al., 2001) showed strongly reduced levels of PLC2 pro-tein in amiR-silenced lines (Fig. 1B). The PLC2-silencedplants show similar morphological features to wild-typeplants under standard growth conditions (SupplementalFig. S1B).

PLC2-Silenced Plants Are More Susceptible to theBacterial P. syringae pv tomatoDC3000 hrcC2 Strain and tothe Nonadapted Fungus E. pisi

To investigate the role of PLC2 in plant innate im-munity, we tested the interaction of PLC2-silencedplants with two different pathogens. First, we selectedPseudomonas syringae pv tomato (Pst) strain DC3000 as ahemibiotrophic pathogen that infects Arabidopsis (Xinand He, 2013). The virulence of Pst DC3000 on Arabi-dopsis depends on the type III secretion system (TTSS),which allows MTI suppression (Block and Alfano, 2011).Thus, proliferation of the PstDC3000mutant strain hrcC2

lacking a functional TTSS is restricted in this plant (Haucket al., 2003).WeusedPstDC3000 hrcC2 to evaluateMTI inPLC2-silenced plants. After spraying adult plants withthis bacterium, pathogen proliferationwas assessed 1 and3 d postinoculation. PLC2-silenced plants were moresusceptible to Pst DC3000 hrcC2 than wild-type plants(Fig. 2A). Under natural conditions, Pst enters host plants,

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through wounds or natural openings such as stomata,and then spreads and multiplies in intercellular spaces(Beattie and Lindow, 1995). The infiltration of bacteriawith a syringe bypasses the first steps of the natural in-fection process. When Pst DC3000 hrcC2 was infiltrated,no significant difference was detected between PLC2-silenced and nonsilenced plants (Fig. 2B). These ex-periments indicate that PLC2 is likely involved instomata-related MTI responses.

We further studied the growth of the virulent wild-type PstDC3000, whose TTSS effectors do interfere withMTI (Block and Alfano, 2011). No significant difference

in the proliferation of this adapted pathogenwas detectedbetween both plant genotypes (Fig. 2C), indicating thatPLC2 silencing does not have an effect when the virulentpathogen is infiltrated.

In order to study if PLC2 also played a role duringETI, we infiltrated Arabidopsis leaves with an avirulentstrain of Pst DC3000 expressing the type III secretedeffector AvrRpm1, which is recognized by the NLRRPM1 (Block and Alfano, 2011). PLC2-silenced plantsshowed the same ability as the wild type in constrain-ing the growth of this strain (Fig. 2D), indicating thatthe lack of PLC2 does not affect AvrRpm1 recognition-triggered growth restriction. Moreover, the hypersen-sitive response (HR) cell deathmeasured by ion leakageinduced by PstDC3000AvrRpm1was identical inwild-type and PLC2-silenced plants (Supplemental Fig. S2A).The effect of PLC2 silencing on HR also was tested byion leakage usingArabidopsis plants expressingAvrRpm1under the control of a dexamethasone-inducible promoter(DEX::AvrRpm1; Andersson et al., 2006). As a negativecontrol, AvrRpm1 was expressed in an RPM1 knockoutbackground (rpm1-3; DEX::AvrRpm1/rpm1-3; Mackeyet al., 2002, 2003).We stably silencedPLC2 by transformingboth backgrounds with ubi::amiR-PLC2 (SupplementalFig. S2C). Leaf discs from DEX::AvrRpm1/Col-0 orDEX::AvrRpm1/rpm1-3 silenced and nonsilenced plantswere induced with dexamethasone, and ion leakage wasmeasured at different time points. This experiment dem-onstrated no significant difference between the PLC2-silenced plants and wild-type plants (SupplementalFig. S2B), confirming that PLC2 is not required forAvrRpm1-induced HR.

Finally, we tested the ability of PLC2-silenced plantsto restrict the entry of the nonadapted pathogen E. pisi,the causal agent of pea (Pisum sativum) powdery mil-dew. Arabidopsis displays nonhost resistance towardE. pisi (Kuhn et al., 2016), whose spores are restrictedfrom penetrating the epidermal cell wall. This resis-tance relies on basal defenses and MAMP recognitionthat function also against powdery mildews adapted toArabidopsis (Kuhn et al., 2016). We assayed the epi-dermal penetration of the pathogen on wild-type andPLC2-silenced plants (Fig. 2E). We observed a signifi-cantly increased success in penetration of the epidermisby E. pisi spores on PLC2-silenced plants comparedwith wild-type plants, indicating that PLC2 is involvedin nonhost resistance. Altogether, the above-presentedresults suggest that PLC2 might play a role in MTIestablishment.

PLC2 Is Involved in ROS-Regulated Processes during MTI

In order to study the function of PLC2 in MTI, weused the MAMP flg22, a 22-amino acid sequence of theconserved N-terminal part of flagellin that is recog-nized by the FLS2 receptor (Gómez-Gómez and Boller,2000), and studied the two distinct MAPK- and ROS-dependent branches of MTI signaling (Bigeard et al.,2015).

Figure 1. PLC2 silencing by artificial microRNAs in Arabidopsis. A,Total RNA was isolated from leaves of 4- to 5-week-old Columbia-0(Col-0) or PLC2-silenced plants (T4 homozygous lines amiR-PLC2-4,amiR-PLC2-7, and amiR-PLC2-11). Relative transcript levels of PLC2were determined by RT-qPCR. Transcript levels were normalized toACT2. Error bars represent SD of three to nine individual plants. Differentletters indicate significant differences (ANOVA for unbalanced samples,posthoc Tukey-Kramer test at P , 0.001). B, PLC2 protein levels wereanalyzed by western blot using anti-AtPLC2 antibody in leaves of 4- to5-week-old Col-0, empty vector (EV), and amiR-PLC2-11 and amiR-PLC2-4 independent silenced lines. Ponceau S staining of Rubiscosubunit L was included as a loading control.

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Figure 2. Growth of P. syringae and E. pisi in Arabidopsis PLC2-silenced plants. Wild-type (Col-0), empty vector (EV), and PLC2-silenced lines (amiR-PLC2-11 and amiR-PLC2-4) were used. A, PLC2-silenced plants are more susceptible to the Pst DC3000

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Flg22-induced activation of a particular MAPK cas-cade is an early event that regulates transcriptionalreprogramming, which finally results in resistance (Bethkeet al., 2012). Western-blot analysis of Arabidopsis wild-type seedlings treated with flg22 using an antibodydirected against the conserved phosphorylated motifon the activation loop of MAPKs recognized threeimmunoreactive bands 15 min after treatment corre-sponding to at least MPK6,MPK3, andMPK4/11 (Bethkeet al., 2012; Supplemental Fig. S3). PLC2-silenced linesshowed a similar MAPK activation to wild-type plants(Supplemental Fig. S3). Similarly, flg22-induced expres-sion of FRK1, PHI1, andWRKY33, which are MAPK- andCDPK-dependent MAMP-activated immune markergenes (Boudsocq et al., 2010), showed no significantdifferences between wild-type and PLC2-silenced seed-lings (Supplemental Fig. S4). These results suggest thatPLC2 is not required for this particular branch of MTIsignaling.

Since oxidative burst is a MAPK-independent sig-naling event occurring after flg22 recognition in plantimmunity (Zhang et al., 2007; Segonzac and Zipfel,2011; Xu et al., 2014), we further studied the role ofPLC2 in ROS-dependent processes. First, we analyzedflg22-induced callose deposition (Luna et al., 2011). Tothis end, leaves were infiltrated with flg22 and, 18 hlater, stainedwith Aniline Blue for callose visualization.PLC2-silenced lines showed significantly less callosedeposition upon flg22 treatment compared with leavesof control plants, which were either transformed withthe empty vector or nontransformed wild-type plants(Fig. 3).

An earlier response of active immunity at the pre-invasive level is the closure of the stomata uponMAMPperception, which is also a ROS-dependent defenseresponse (Mersmann et al., 2010; Kadota et al., 2014; Liet al., 2014). In order to evaluate if stomatal closure wasaffected in PLC2-silenced plants, epidermal peels weretreated with flg22. As shown in Figure 4, flg22-mediatedinduction of stomatal closurewas impaired in epidermalpeels of PLC2-silenced plants, whereas ABA-induced

stomatal closure was unaffected. Together, these resultsimply that PLC2 is required for the full activation ofstomatal ROS-dependent immune responses.

PLC2 Is Involved in the flg22-Induced ROS Burst

Flg22 perception triggers a fast and transient increaseof apoplastic ROS (Felix et al., 1999). Using a luminol/peroxidase-based method, apoplastic ROS levels werequantified in flg22-treated leaf discs. A representativeexperiment is shown in Figure 5A, indicating that inPLC2-silenced line 11 (amiR-PLC2-11), ROS accumula-tion had similar kinetics but significantly lower levelsthan in control plants. To estimate such reduction, wequantified apoplastic ROS in additional independentexperiments including three different silenced linesas well as a control line carrying an empty vector. AllPLC2-silenced lines showed a reduction in ROS levels inresponse to flg22 (40%–75% compared with controlplants; Fig. 5B). Thus, our results demonstrated that PLC2is required for the full ROS accumulation followingflg22 recognition in Arabidopsis leaf discs.

PLC2 Associates with RBOHD

The flg22-induced ROS burst is generated via acti-vation of theplasmamembraneNADPHoxidaseRBOHD(Nühse et al., 2007; Zhang et al., 2007). Our results showthat PLC2 is required for the flg22-mediated ROS burstthat is generated via RBOHD activation (Fig. 5). Asmentioned earlier, PLC2 is localized at the plasma mem-brane, where RBOHD also exists in a complex with FLS2andBIK1 (Kadota et al., 2014; Li et al., 2014). To investigatewhether PLC2 associates with RBOHD, we immunopre-cipitated N-terminally FLAG-tagged RBOHD (stablyexpressed in Arabidopsis under the control of its ownpromoter) using anti-FLAG affinity beads. In three inde-pendent biological experiments, PLC2 coimmunoprecipi-tated with RBOHD in planta (Fig. 6; Supplemental Fig.S5). PLC2 could not be immunoprecipitated in wild-type

Figure 2. (Continued.)hrcC2 mutant. Bacteria were inoculated by spray at OD600 = 0.1, and the number of colony-forming units (CFU) per cm2 of leafextracts was determined. Data from three biological replicates each with three technical replicates were averaged (n = 9), andANOVA was performed considering each replicate as a factor. Error bars represents SE. Different letters indicate significant dif-ferences between genotypes (ANOVA, P , 0.001, posthoc Tukey’s test). dpi, Days postinoculation. B, PLC2-silenced plants donot show increased susceptibility to the PstDC3000 hrcC2mutant when the bacteria are syringe inoculated into the leaf apoplast.Bacterial suspension was inoculated at OD600 = 0.0001, and the number of CFU per cm2 of leaf extracts was determined. Datafrom three biological replicates each with three technical replicates were averaged (n = 9), and ANOVA was performed con-sidering each replicate as a factor. No significant differences were observed between genotypes. Error bars represent SE. C and D,PLC2-silenced lines showed no differences in susceptibility to virulent (C) and avirulent (D) Pst DC3000 infections. Pst DC3000(virulent) and Pst DC3000:AvrRpm1 (avirulent) were inoculated by infiltration at OD600 = 0.0002, and CFU per cm2 of leaf wascalculated. A representative experiment of four biological replicates is depicted. No significant differences were observed re-garding the EV control according to Student’s t test (P, 0.05). E, PLC2-silenced plants aremore susceptible to the nonadapted peapowderymildew E. pisi. The penetration rate at 3 d after inoculation was calculated as the percentage of successful penetration ofat least 50 germinated spores on three independent leaves. Error bars represent SE. Different letters indicate significant differences(multiple comparison using one-way ANOVA, posthoc Tukey’s test at P , 0.05). One representative experiment of four bio-logically independent replicates is depicted.

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plants that did not express FLAG-RBOHD. Notably, thebrassinosteroid receptor BRI1 (used here as an unrelatedplasma membrane-located protein control) was notdetected in anti-FLAG immunoprecipitates (Fig. 6). Inaddition, experiments in the presence of flg22 revealedthat the association was independent of the ligandbinding to FLS2, since the same amount of PLC2 wasimmunoprecipitated in treated as in nontreated plants(Fig. 6).

DISCUSSION

The participation of PI-PLC activity in signaling afterthe recognition of different MAMPs such as xylanase,flg22, and chitosan (van der Luit et al., 2000; Laxalt et al.,2007; Raho et al., 2011) or pathogen effector proteins (deJong et al., 2004; Andersson et al., 2006) has been de-scribed previously (Laxalt and Munnik, 2002; Munnik,2014). Here, we show genetic evidence that PLC2 is par-ticularly involved inMTI signaling. The molecular detailsof PI-PLC signaling in plants are still unclear, but there isevidence that (1) phosphatidylinositol 4-phosphate andPIP2 are most likely the substrates and (2) the phosphor-ylated products of IP2, IP3, and DAG, including variousinositol polyphosphates, PA, and DAG pyrophosphate,have roles as secondarymessengers (Munnik, 2014). PA isinvolved in the modulation of immune signaling com-ponents, such as MAPKs, PDK1, and RBOHD (Farmerand Choi, 1999; Lee et al., 2001; Szczegielniak et al., 2005;Anthony et al., 2006; Zhang et al., 2009). Unfortunately, in

Arabidopsis, we were not able to detect in vivo flg22-induced PA increase by radiolabeling with 32Pi on seed-lings, cotyledons, or leaf discs (Supplemental Fig. S6). Wecan envisage different plausible explanations: (1) flg22triggers a very local increase of PA in specialized cells ortissues, sowhenwemeasure overall in vivo production inorgans with different tissues, the signal gets diluted; (2)PA may be rapidly produced and metabolized and, thus,difficult to detect; or (3) 32Pi labeling conditions are notsensitive enough to distinguish small PA differences inArabidopsis. However, those undetectable changes in thelevels of signaling lipids can have strong effects on plantresponses.We cannot exclude that IP2 and IP3 can be veryrapidly phosphorylated to IP6 and, thus, probably in-crease Ca2+ in the cytosol or also participate in auxinsignaling via TIR1 and COI1-JA signaling, among others(Xue et al., 2009; Munnik, 2014; Williams et al., 2015).Indeed, mutants with altered inositol polyphosphatelevels showed altered defense responses (Murphyet al., 2008; Donahue et al., 2010; Mosblech et al., 2011;Hung et al., 2014; Laha et al., 2015). Whether thesecompounds are generated downstream of PLC2 remainsto be demonstrated.

PLC2 Is Required for Full Activation of Plant Immunity

In tomato (Solanum lycopersicum), using virus induced-gene silencing of different PLCs, SlPLC4 was found tobe involved specifically in the HR upon AVR4 per-ception, while SlPLC6 is required for multiple NLR-mediated responses (Vossen et al., 2010), suggestingthat, in tomato, both PLCs participate in ETI responses.Similarly, overexpression of SlPLC3 enhanced the Cf-4/Avr4-triggered HR (Abd-El-Haliem et al., 2016). Furtherstudies in tomato showed that SlPLC2 is required for

Figure 4. PLC2-silenced plants exhibit impaired flg22-induced sto-matal closure. Epidermal peels from Col-0 and PLC2-silenced plantswere incubated in opening buffer under light for 3 h. The peels weretreated with water, 1 mM flg22, 50 mM ABA, or 50 mM ABA + 1 mM flg22for 1 h. The results show means of 90 to 120 stomata measured fromthree independent experiments. Error bars represent SE. Different lettersdenote statistical differences (ANOVA for unbalanced samples, posthocTukey-Kramer test at P , 0.05). EV, Empty vector.

Figure 3. PLC2-silenced plants exhibit impaired flg22-induced callosedeposition. Leaves from 4- to 5-week-old Col-0 or amiR-PLC2 plantswere infiltrated with 1 mM flg22 or water as a control and incubated for18 h, and callose depositionwasmeasured as dots per area. Six differentmicroscopic areas (1 mm2) were taken per leaf. Two different leaves perindividual were analyzed. Three independent plants were analyzed perline per experiment. Three independent experiments were performed.Error bars represent SE. Different letters indicate significant differences(ANOVA for unbalanced samples, posthoc Tukey-Kramer test at P, 0.001).EV, Empty vector.

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xylanase-induced gene expression, ROS production, andplant susceptibility against Botrytis cinerea (Gonorazkyet al., 2014, 2016).

Here, we assayed three different strains of the hemi-biotrophic pathogen Pst DC3000: the virulent wild-typestrain to study the role of PLC2 in effector-triggeredsusceptibility, the avirulent strain expressing AvrRpm1 todetermine if PLC2 played a role during ETI, and the hrcC2

strain mutated in the type III secretion system to investi-gate if PLC2 was required for MTI. PLC2-silenced plants

showed increased susceptibility to Pst DC3000 hrcC2

but not to the virulent or avirulent strain, suggestingthat this protein is mostly involved in MTI. Whenthe hrcC2 strain was infiltrated, no differences in sus-ceptibility were found between PLC2-silenced andwild-type plants, indicating that the differences found

Figure 6. PLC2 associates with RBOHD. Coimmunoprecipitation ofPLC2 and ROBHD was performed in stable transgenic Arabidopsisseedlings (T3) expressing FLAG-RBOHD (pRBOHD:FLAG-RBOHD)treated (+) or not (2) with 1 mM flg22 for 15 min. Total protein extracts(Input) were subjected to immunoprecipitation (IP) with anti-FLAGbeads followed by immunoblot analysis with anti-PLC2 (a-PLC2) andanti-FLAG (a-FLAG) antibodies as indicated. Protein extracts of Col-0plants were used as negative controls. Anit-BRI1 (a-BRI1) antibodieswere used as plasma membrane protein not associated with RBOHD.CBB, Coomassie Brilliant Blue. These experiments were performedthree times with similar results.

Figure 5. PLC2-silenced plants exhibit impaired flg22-induced oxida-tive burst. The production of ROS was measured with a luminol-basedassay in Col-0 or amiR-PLC2 plants. A, Leaf discs from 4- to 5-week-oldplants were incubated with 100 nM flg22, and the luminescence wasmeasured every 1 min for 30 min and expressed as relative light units(RLU). A representative experiment is shown using wild-type (Col-0)and a PLC2-silenced line (amiR-PLC2-11) plants. B, Total ROS pro-duction was calculated integrating the areas under the curves andreferring to the Col-0 wild type treated with flg22 as 100%. Averagesof four independent experiments are shown. Error bars represent SE.Asterisks indicate statistically significant differences compared withthe flg22-treated Col-0 plant (ANOVA for unbalanced samples, mul-tiple comparisons versus control group posthoc Dunnett’s method atP , 0.05). EV, Empty vector.

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when the strain was sprayed could be explained by therole of stomata closure during infection. In order to furthercorroborate that PLC2 does not affect MTI final outputwhen the bacteria are syringe infiltrated into the apoplast,we studiedwhetherflg22 leads to inducedplant resistancein PLC2-silenced plants. Supplemental Figure S7 showsthat treatment of plants with flg22 triggers resistance tosyringe-infiltrated Pst DC3000 in wild-type as well asPLC2-silenced plants. These results suggest that PLC2controls stomatal preinvasive but not postinvasive im-munity. Accordingly, PLC2-silenced plants are impairedin stomatal closure upon flg22 treatment. These resultssuggest a role of PLC2 in stomatal immunity.Further studies also indicated that basal resistance

against the nonadapted pathogen pea powdery mildew,E. pisi, was impaired in PLC2-silenced plants. In thisnonhost interaction with Arabidopsis, the first line ofdefense is the recognition of MAMPs, such as chitin bythe LYK5-CERK1 receptor complex, triggering a series ofimmune responses includingMAPK activation and ROSburst mediated by NADPH oxidases (Kuhn et al., 2016).

PLC2 Participates in RBOHD-Dependent PlantDefense Responses

Callose accumulation is an MTI response that re-quires RBOHD (Luna et al., 2011), and flg22-inducedcallose deposition is reduced in PLC2-silenced plants.Another RBOHD-dependent response is flg22-inducedstomatal closure (Mersmann et al., 2010; Kadota et al.,2014; Li et al., 2014). The restriction of microbial entryby stomatal closure is one of the first MTI responses(Melotto et al., 2006). fls2mutant plants are impaired instomatal closure in response to flg22 and show in-creased susceptibility to Pst DC3000 when sprayedonto the leaf surface but not when infiltrated into leaves(Gómez-Gómez et al., 2001; Zipfel et al., 2004; Chinchillaet al., 2006; Zeng andHe, 2010). Importantly, the action ofABA on stomatal immunity seems to occur downstreamor independently of the pattern recognition receptorcomplex, because fls2, bik1, and rbohD mutants exhibitwild-type stomatal closure in response to exogenousABA (Macho et al., 2012; Kadota et al., 2014). Accordingly,we demonstrate that PLC2 is involved in flg22-inducedstomatal closure, whereas ABA-dependent stomatalclosure is unaffected. These results show that PLC2 isinvolved in callose deposition and stomatal closurefollowing flg22 perception in Arabidopsis plants.

PLC2 Acts Upstream of RBOHD Activation

We have demonstrated that PLC2 is required for thefull activation of flg22-induced ROS production. ROSproduction upon flg22 perception in Arabidopsis is de-pendent on the NADPH oxidase RBOHD (Kadota et al.,2014; Li et al., 2014). Posttranslational regulation ofRBOHDactivation involves Ca2+ via direct binding to EFhand motifs and phosphorylation by Ca2+-dependent(i.e. CPKs) and Ca2+-independent (i.e. BIK1) protein ki-nases (Logan et al., 1997; Boudsocq et al., 2010; Kadota

et al., 2014, 2015; Li et al., 2014). By using PLC inhibitors,PLC activation has been suggested to be required forROS production upon xylanase, chitosan, and Avr4 (deJong et al., 2004; Laxalt et al., 2007; Raho et al., 2011). PAalso has been shown to interact directly with RBOHDand enhance ROS production (Zhang et al., 2009). Uponcryptogein treatment of tobacco (Nicotiana tabacum) BY2cells, PLC and DGK inhibitors or silencing of the clusterIII of the tobacco DGK family resulted in reduced PAand ROS production (Cacas et al., 2017). Therefore, itcould be speculated that the second messengers derivedfrom PLC2 activation, PA and/or increased cytosolicCa2+ via IP6, for example, could positively regulate theNADPH oxidase activity, since PLC2-silenced plantsshowed reduced ROS production in response to flg22.

Flg22 activates MAPK signaling pathways leadingto the induction of immune gene expression. MPK3,MPK4/11, and MPK6 activation act independently ofthe RBOHD-mediated ROS burst (Zhang et al., 2012;Xu et al., 2014). Flg22-treated PLC2-silenced plantsshowed similar levels of MAPK activation and immunegene expression as the wild type, suggesting that MAPKsignaling is independent of PLC2.

RBOHD exists in a complex with the receptor kinaseFLS2, interacting directlywith BIK1 (Kadota et al., 2014; Liet al., 2014). Our results show that PLC2 is associatedwithRBOHD and that this association is ligand independent.In Arabidopsis, the receptor complex FLS2-BAK1 per-ceives flg22 and activates the downstream kinases BIK1and PBL1 by phosphorylation, which induce an influx ofextracellular Ca2+ in the cytosol (Li et al., 2014; Ranf et al.,2014). PLC2 contains a Ca2+-dependent phospholipid-binding domain (C2) and EF hand domains (Otterhaget al., 2001). In addition, PLC2 is localized to the plasmamembrane and is rapidly phosphorylated upon flg22treatment (Niittylä et al., 2007;Nühse et al., 2007). One canenvisage that PLC2 is part of the FLS2, BIK1, and RBOHDcomplex and that BIK1 or another component of thereceptor complex phosphorylates PLC2, leading to thegeneration of secondmessengers like PA or IP6, which,in turn, positively regulate or are required to sustain/reinforce the activity of RBOHD.

Other Roles of PLC2

Seeking knockout mutant plants for PLC2, we couldnot recover homozygous mutants and, therefore, de-cided to silence PLC2. Nevertheless, further character-ization showed that this gene is expressed during earlymegagametogenesis and in the embryo after fertiliza-tion, being required for both reproductive and embryodevelopment, presumably by controlling mitosis and/or the formation of cell-division planes (Li et al., 2015;Di Fino et al., 2017). The fact that we were able to obtainPLC2-silenced lines could be related to (1) low expres-sion levels of the 35S::amiR-PLC2 in the reproductiveorgans and embryos or (2) the silencing not being fullyeffective, with low levels of PLC2 in the gametophyteand/or embryos being sufficient for correct develop-ment. The requirement for PLC2 during development

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suggests that the mechanisms for PLC2 activation and/or its downstream targets, such as RBOHD, could besimilar in both the sporophyteduringflg22perception andthe gametophyte during development. Arabidopsis hasfive SERK proteins. SERK3/BAK1 and SERK4/BKK1 as-sociate with FLS2 and BIK1 (Chinchilla et al., 2007; Luet al., 2010; Zhang et al., 2010; Roux et al., 2011). SERK1and SERK2 are crucial in regulating male fertility and areexpressed in the ovule, female gametophyte, early em-bryos, and vascular cells (Hecht et al., 2001; Albrecht et al.,2005; Colcombet et al., 2005; Kwaaitaal et al., 2005). Wespeculate that PLC2 has a role in gametogenesis and em-bryo development, probably by signaling downstream ofLRR-RKs like SERKs. Nonetheless, whether PLC2 is spe-cific for the FLS2-BAK1-BIK1 receptor complex or partic-ipates in the signaling of other receptor complexes, likeLYK5-CERK1, as suggested by the results obtained withE. pisi, remains to be elucidated.

CONCLUSION

The activity of PI-PLC in signaling after the recognitionofdifferent MAMPs was described earlier. The Arabidopsisgenome contains nine PI-PLC genes; however, until thiswork, it was not known which one was specifically linkedto the plant defense response. We here present genetic ev-idence that PLC2 participates in MTI. PLC2 is required forthe full activation of ROS production and ROS-dependentresponses elicited by the MAMP flg22. PLC2 associateswith RBOHD, suggesting a positive regulation of the Ara-bidopsis NADPH oxidase activity by PLC2.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Seeds from Arabidopsis (Arabidopsis thaliana Col-0) transformed with an artifi-cial microRNA targeting specifically PLC2 (amiR-PLC2) under the control of thecauliflower mosaic virus 35S promoter or with the empty vector were germinatedin soil (soil:vermiculite:perlite, 3:1:1) and kept at 4°C for 2 d. Then, theywere grownat 25°C using a 16-h-light/8-h-dark photoperiod. In the case of infections (bacterialand fungal), plants were grown at 22°C in an 8-h-light/16-h-dark photoperiod.

For ion leakage experiments, Col-0 or rpm1.3 mutant plants transformed withthe coding sequence for Pseudomonas syringae pv tomato AvrRpm1 under the controlof a dexamethasone-inducible promoter (Aoyama and Chua, 1997) were grown asdescribed at 22°C in an 8-h-light/16-h-dark cycle. Both backgrounds were trans-formed with amiR-PLC2 under the control of the Ubiquitin10 promoter (pUBQ10).

amiR-PLC2-Silencing Constructs

AtPLC2 (At3g08510) silencing was performed using a specific artificialmicroRNA designed with WMD3 Web microRNA designer (http://wmd3.weigelworld.org). Arabidopsis miR319was used as a template, and the cloningstrategy was according to Ossowski et al. (2008).

Primers for Artificial MicroRNA Cloning

The following primers were used: PLC2 miR-s, 59-gaTTAAACACTCAG-TAATTGCGCtctctcttttgtattcc-39; PLC2 miR-a, 59-gaGCGCAATTACTGAGTGTTT-AAtcaaagagaatcaatga-39; PLC2 miR*s, 59-gaGCACAATTACTGACT-GTTTATtcacaggtcgtgatatg-39; and PLC2 miR*a, 59-gaATAAACAGTCAGTA-ATTGTGCtctacatatatattcct-39. Uppercase letters denote AtPLC2-targeted sites. TheamiR-PLC2 was cloned into pCHF3 vector (kanamycin resistance in plants)

driven by the cauliflower mosaic virus 35S promoter or into pUBQ10 desti-nation vector driven by the Ubiquitin10 promoter (Basta resistance in plants).

Arabidopsis Transformation

Arabidopsisplantswere transformedusing thefloraldipmethod (Zhangetal.,2006). T1 plants were sown on Murashige and Skoog (MS) agar (MS mediumwith Gamborg’s vitamins and 1% agar) plates with kanamycin (50 mg mL21 forpCHF3:amiR-PLC2) or BASTA (10 mg mL21 for pUBQ10:amiR-PLC2). After2 weeks, resistant plants were transferred to soil. T3 or T4 homozygous plants onwhich silencing levels were checked by qPCR were used for experiments.

Expression Analysis by RT-qPCR

TotalRNAwas extracted from10-d-old seedlings or leaves from4- to 5-week-old plants using the Trizol method according to the manufacturer’s instructions(Invitrogen). cDNA was synthesized on 1 mg of total RNA by MMLV reversetranscriptase from Promega using oligo(dT) primer in a final volume of 20 mL.The cDNA was diluted to a final volume of 100 mL, and 2.5 mL was used forqPCR. The Fast Universal SYBR Green Master Mix from Roche was employed,using a Step-One Real-Time PCR machine from Applied Biosystems. Thestandard amplification program was used. The expression levels of the gene ofinterest were normalized to those of the constitutive ACT2 (At3g18780) gene bysubtracting the cycle threshold value of ACT2 from the cycle threshold value ofthe gene (DCT). The nucleotide sequences of the specific primers for qPCRanalysis are listed in Supplemental Table S1. The annealing temperature foreach primer was 60°C. LinRegPCR was the program employed for the analysisof real-time qPCR data (Ruijter et al., 2009).

Western-Blot Analysis

Polyclonal antibodies were prepared as described (Otterhag et al., 2001). Apeptide, KDLGDEEVWGREVPSFIQR, corresponding to residues 266 to 284 ofAtPLC2 was synthesized. One rabbit was immunized at 2-week intervals, andserum was collected after the second boost. Protein extraction buffer (100 mM

NaPi, pH 7.5, 150 mM NaCl, 1 mM EDTA, and Sigma-Aldrich proteinase in-hibitor cocktail) was added to an equal volume of 4- to 5-week-old ground leaftissue, mixed, and centrifuged for 10 min at 10,000g. Protein concentrationin the supernatant was determined. Samples were loaded onto a 10% SDS-polyacrylamide gel, blotted onto nitrocellulose membranes, and stained withPonceau S for a loading control. Membranes were incubated overnight inphosphate-buffered saline plus Tween 20 containing polyclonal anti-PLC2antibody (1:2,000). The blot was washed three times with phosphate-bufferedsaline plus Tween 20 and revealed using a secondary anti-rabbit IgG antibodycoupled to alkaline phosphatase according to the manufacturer’s instructions(Sigma-Aldrich).

Bacterial Infection Assays

Six- to 8-week-old plants were used for bacterial inoculations. Strains PstDC3000 (virulent), Pst DC3000 AvrRpm1 (avirulent), and Pst DC3000 hrcC2

mutant were maintained on solid Pseudomonas agar F (King’s B medium;Biolife) supplemented with 50mg L21 rifampicin (for PstDC3000 hrcC2) or plus50 mg L21 kanamycin (for virulent and avirulent Pst strains). Bacterial sus-pensions of virulent and avirulent strains were inoculated into the abaxial sideof leaveswith a needleless syringe (10mMMgCl2, OD600 = 0.00002). The bacteriawere extracted at 1 or 3 d postinfiltration, and the number of colony-formingunits was determined after serial dilution and plating as described (Johanssonet al., 2014). The strain Pst DC3000 hrcC2 was inoculated either by spraying(10 mM MgCl2, OD600 = 0.1; 0.02% Silwet) or by infiltration with a needlesssyringe (10 mM MgCl2, OD600 = 0.0001). Following spray inoculations, plantswere kept covered with a transparent lid for 6 h. For spray- and syringe-inoculated plants, samples were taken at day 0, 1, or 3 postinoculation with anumber 1 cork borer. Bacterial growth was evaluated as described previously(Katagiri et al., 2002). Data shown in Figure 2, A, C, D, and E, correspond toone experiment representative of four independent biological assays per-formed. Again, in Figure 2B, one of three biological replicates that showedsimilar results is depicted. For all cases, each value in the graphs represents theaverage 6 SD of three technical replicates (three pools of four leaf discs col-lected from four independent plants at each time point) that were ground,diluted, and plated separately.

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Ion Leakage

Ion leakagewasmeasured in leaf discs after infiltration ofPstDC3000AvrRpm1(OD600 = 0.1), as well as in leaf discs of Col-0 plants expressing the coding sequenceof P. syringae AvrRpm1, under the control of a dexamethasone-inducible promoter(Andersson et al., 2006) as described (Johansson et al., 2014). Leaf discs from 4- to5-week-old empty vector or PLC2-silenced plants in the wild-type or rpm1-3background were placed in deionized water during 1 to 2 h and then washed andtransferred to six-well cultivation plates containing 10 mL of water (four discs perwell). For the dexamethasone-inducible AvrRpm1 plants, leaf discs were treatedwith 20 mM dexamethasone. The release of electrolytes from the leaf discs wasdetermined every 30 min for 5 h using a conductivity meter (Orion; Thermo Sci-entific) as described (Johansson et al., 2014). The experiment was repeated twice.

Fungal Inoculation and Scoring of Fungal Penetration

The nonhost powdery mildew fungus Erysiphe pisi (isolate CO-01) waspropagated on pea (Pisum sativum ‘Kelvedon Wonder’) plants. Inoculationswere carried out by powdering spores on leaves of 4-week-old Arabidopsiswild-type and PLC2-silenced plants. At 3 d postinoculation, leaves were stainedwith Trypan Blue as described (Koch and Slusarenko, 1990). The penetrationrate after inoculation was calculated as the percentage of successful penetrationattempts (penetration ending in plant cell death) as described (Pinosa et al.,2013) on at least 50 germinated spores on three independent leaves per geno-type. The experiment was repeated four times.

MAPK Activation

MAPK assays were performed on six 2-week-old seedlings grown in liquidMS medium (including vitamins; Duchefa) and 1% Suc. Seedlings were elicitedwith 1 mM flg22 for 5, 15, or 30 min and frozen in liquid nitrogen. MAPK ac-tivation was monitored by western blot with antibodies that recognize the dualphosphorylation of the activation loop of MAPK (pTEpY). Phospho-p44/42MAPK (Erk1/2; Thr-202/Tyr-204) rabbit monoclonal antibodies from CellSignaling were used according to the manufacturer’s protocol (1:5,000). Blotswere stained with Coomassie Brilliant Blue to verify equal loading.

Callose Deposition

Leaves from4- to5-week-oldplantswere fully infiltratedwith1mMflg22orwaterfor 18 h. Leaves were then incubated in 96% ethanol until all tissue was transparent,washed in 0.07 M phosphate buffer (pH 9), and incubated for 2 h in 0.07 M phosphatebuffer containing 0.01% Aniline Blue. Observations were performed with an epi-fluorescence microscope with UV filter (excitation, 365/10 nm; emission, 460/50 nm). The number of callose dotswas calculated using ImageJ software (Schneideret al., 2012). Six different microscopic areas (1 mm2) were taken per leaf. Two dif-ferent leavesper individualwere analyzed. Three independentplantswere analyzedper line per experiment. Three independent experiments were performed.

Epidermal Peel Preparation and StomatalAperture Measurement

Epidermal peelswere obtained from the abaxial surface of fully expanded leaves.The peelswere preincubated in opening buffer (10mMMES, pH 6.1 [MES titrated toits pKa with KOH], and 10 mM KCl) under white light at 25°C to promote stomatalopening.After 3 h of preincubation,flg22 (1mM) orABA (50mM; Sigma-Aldrich)wasadded to the opening buffer and incubated for 1 h. Stomatal apertures were mea-sured from digital images taken with a Nikon Coolpix 990 camera coupled to anopticalmicroscope (NikonEclipse 2000). Then, the stomatal porewidthwasdigitallydeterminedusing the image-analysis software ImageJ.Aperture values aremeans of90 to 120 stomata measured from at least three independent experiments.

ROS Detection

Leaf discs from 4- to 5-week-old plants were placed on 96-well black platesfloating in 200 mL of deionized water overnight. ROS production was triggeredwith 100 nM flg22 (N-QRLSTGSRINSAKDDAAGLQIA-C; Genbiotech) appliedtogether with 20 mM luminol (Sigma-Aldrich; catalog no. A8511) and 0.02 mgmL21 horseradish peroxidase (Sigma-Aldrich; catalog no. P6782). Luminescencewas measured with a luminometer (Thermo Scientific Luminoskan AscentMicroplate). Each plate contained 36 leaf discs for flg22 treatment and 12 leaf

discs formock treatment of the sameArabidopsis line. Every plate wasmeasuredover a period of 30 min with an interval of 1 min and repeated in four inde-pendent experiments.

Seedling Protein Extraction and Immunoprecipitation

For immunoprecipitation studies in seedlings, Arabidopsis rbohd/pRBOHD::FLAG-RBOHD (Kadota et al., 2014) seedswere surface sterilizedwith chlorine gasand germinated on plates containing MS medium (with Gamborg’s vitamins;Duchefa) and 1% Suc and 0.8% agar for the first 7 d at 22°C and with a 16-h lightperiod. Seedlings were transferred to liquid MS medium supplemented with 1%Suc and grown under the same conditions for an additional 7 d.

Two-week-old seedlingswere treatedwithflg22 (1mM)orwater andground toa fine powder in liquid nitrogen with sand (Sigma-Aldrich). Proteins were iso-lated in extraction buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10%glycerol, 5 mM DTT, 1 mM NaF, 1 mM Na2MoO4∙

2H2O, 1% phosphatase inhibitorcocktails 2 and 3 (Sigma-Aldrich), 1% (v/v) P9599 protease inhibitor cocktail(Sigma-Aldrich), 100 mM phenylmethylsulfonyl fluoride, and 1% (v/v) IGEPALCA-630 (Sigma-Aldrich). Extracts were incubated 30 min at 4°C and centrifugedfor 20min at 16,000g at 4°C. Supernatants were incubated for 1 to 2 h at 4°CwithANTI-FLAG M2 Affinity Gel (Sigma-Aldrich) and washed five times with ex-traction buffer. Beads were heated at 55°C in SDS loading buffer for 20 min torelease proteins. For immunoblotting, antibodies were used at the following di-lutions: a-PLC2 (1:5,000), a-FLAG-HRP (Sigma-Aldrich; 1:5,000), a-rabbit-HRP(Sigma-Aldrich; 1:10,000), and anti-BRI1 (1:5,000).

Accession Numbers

Accession numbers are as follows: AtPLC2 (At3g08510), AtRBOHD(At5G47910), and AtBRI1 (At4g39400).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. PLC2 silencing specificity and phenotypes ofsilenced plants.

Supplemental Figure S2. PLC2 is not involved in programmed cell deathduring ETI upon recognition of AvrRpm1 from P. syringae.

Supplemental Figure S3. PLC2 is not required for flg22-induced MAPKactivation.

Supplemental Figure S4. MAMP-activated gene expression is not deregu-lated in PLC2-silenced seedlings.

Supplemental Figure S5. PLC2 associates with RBOHD.

Supplemental Figure S6. Effect of flg22 on the formation of PA

Supplemental Figure S7. Flg22-induced resistance.

Supplemental Table S1. Primer sequences.

ACKNOWLEDGMENTS

We thank Alexandra Leschnin andDr. Oskar Johansson for helpingwith ionleakage measurements and bacterial and fungal infections, Dr. Jesús Nuñez forstatistical analysis, Sergio Batista for assistance in the greenhouse, and TeresaQuattrini for technical assistance.

Received February 7, 2017; accepted August 18, 2017; published August 21,2017.

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