modiﬁcations of sphingolipid content affect …...oxidative burst, is characteristic of successful...

Modifications of Sphingolipid Content AffectTolerance to Hemibiotrophic and NecrotrophicPathogens by Modulating Plant DefenseResponses in Arabidopsis1[OPEN]

Maryline Magnin-Robert, Doriane Le Bourse, Jonathan Markham, Stéphan Dorey, Christophe Clément,Fabienne Baillieul, and Sandrine Dhondt-Cordelier*

Unité de Recherche Vigne et Vin de Champagne Equipe d’Accueil 4707, Laboratoire Stress Défenses etReproduction des Plantes, Structure Fédérative de Recherche Condorcet Fédération de Recherche, CentreNational de la Recherche Scientifique 3417, Université de Reims Champagne-Ardenne, F–51687 Reims cedex 2,France (M.M.-R., S.D., C.C., F.B., S.D.-C.); and Center for Plant Science Innovation and Department ofBiochemistry, University of Nebraska, Lincoln, Nebraska 68588 (D.L.B., J.M.)

ORCID ID: 0000-0002-7142-1035 (F.B.).

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role inplant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defenseresponses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by thedihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibittolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pvtomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogensdiffering in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]),hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levelsare higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more stronglyin Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death andreactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. Inaddition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, andjasmonic acid-dependent gene expression in Atdpl1-1mutants. Our results demonstrate that sphingolipids play an important role inplant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signalingpathway, cell death, and sphingolipids.

Plants have evolved a complex array of defenses whenattacked by microbial pathogens. The success of plantresistance first relies on the capacity of the plant to rec-ognize its invader. Among early events, a transient pro-duction of reactive oxygen species (ROS), known as theoxidative burst, is characteristic of successful pathogenrecognition (Torres, 2010). Perception of pathogen attack

then initiates a large array of immune responses, in-cluding modification of cell walls, as well as the pro-duction of antimicrobial proteins and metabolites likepathogenesis-related (PR) proteins and phytoalexins,respectively (Schwessinger and Ronald, 2012). The planthormones salicylic acid (SA), jasmonic acid (JA), andethylene (ET) are key players in the signaling networksinvolved in plant resistance (Bari and Jones, 2009; Tsudaand Katagiri, 2010; Robert-Seilaniantz et al., 2011). In-teractions between these signal molecules allow theplant to activate and/or modulate an appropriate ar-ray of defense responses, depending on the pathogenlifestyle, necrotroph or biotroph (Glazebrook, 2005;Koornneef and Pieterse, 2008). Whereas SA is consid-ered essential for resistance to (hemi)biotrophic patho-gens, it is assumed that JA and ET signaling pathwaysare important for resistance to necrotrophic pathogens inArabidopsis (Arabidopsis thaliana; Thomma et al., 2001;Glazebrook, 2005). A successful innate immune responseoften includes the so-called hypersensitive response(HR), a form of rapid programmed cell death (PCD)

1 This work was supported by the Region Champagne-Ardenne(grant no. A2101–03).

* Address correspondence to [email protected] 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:Sandrine Dhondt-Cordelier ([email protected]).

S.D., C.C., F.B., and S.D.-C. designed the research; M.M.-R., D.L.B.,J.M., and S.D.-C. performed the experiments; M.M.-R., D.L.B., J.M.,and S.D.-C. analyzed the data; M.M.-R., D.L.B., J.M., S.D., C.C., F.B.,and S.D.-C. wrote the article.

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occurring in a limited area at the site of infection. Thissuicide of infected cells is thought to limit the spread ofbiotrophic pathogens, including viruses, bacteria, fungi,and oomycetes (Mur et al., 2008).

During the past decade, significant progress has beenmade in our understanding of the cellular function ofplant sphingolipids. Besides being structural compo-nents of cell membranes, sphingolipids are bioactivemetabolites that regulate important cellular processessuch as cell survival and PCD, occurring during eitherplant development or plant defense (Dunn et al., 2004;Berkey et al., 2012; Markham et al., 2013). The first evi-dence of the role of sphingolipids in these processescame from the use of the fungal toxins fumonisin B1(FB1) and AAL, produced by the necrotrophic agentAlternaria alternata f. sp. lycopersici. These toxins arestructural sphingosine (d18:1) analogs and function asceramide synthase inhibitors. They triggered PCDwhenexogenously applied to plants. Mutant strains in whichthe production of such toxins is abrogated failed to infectthe host plant, implying that toxin accumulation is

required for pathogenicity and that the induction ofplant PCD could be considered a virulence tool used bynecrotrophic pathogens (Berkey et al., 2012). Moreover,several studies revealed that ceramides (Cers) and long-chain bases (LCBs) are also potent inducers of PCD inplants. For example, exogenously applied Cers andLCBs (d18:0, d18:1, or t18:0) induced PCD either in cellsuspension cultures (Liang et al., 2003; Lachaud et al.,2010, 2011; Alden et al., 2011) or in whole seedlings(Shi et al., 2007; Takahashi et al., 2009; Saucedo-Garcíaet al., 2011). AAL- and FB1-induced PCD seemed to bedue to the accumulation of free sphingoid bases (dihy-drosphingosine [d18:0] and phytosphingosine [t18:0];Abbas et al., 1994; Brandwagt et al., 2000; Shi et al., 2007).Spontaneous cell death in lag one homolog1or L-myoinositol1-phosphate synthase mutant could be due to trihydroxy-LCB and/or Cer accumulation (Donahue et al., 2010;Ternes et al., 2011). Deciphering of Cer participation inthe induction of HR and associated PCD also came fromstudies on accelerated cell death5 (acd5) and enhancing re-sistance to powdery mildew8 (RPW8)-mediated hypersensitive

Figure 1. Atdpl1 mutants are moretolerant to B. cinerea but more suscep-tible to Pst than the wild type. B. cine-rea conidia suspension was depositedby using drop inoculation (A and B) orspray inoculation (E) on leaves of wild-type (WT) andAtdpl1mutant plants. Pstsolution was infiltrated into wild-typeand Atdpl1mutant leaves (A, C, andD).A, Photographs represent disease symp-toms observed 60 or 72 h after infectionby the fungus or Pst, respectively. B,Symptoms due to B. cinerea infectionwere scored by defining three lesiondiameter (d; in mm) classes. Statisticaldifferences of the mean lesion diame-ters between wild-type and Atdpl1plants were calculated with a Kruskal-Wallis test: **, P , 0.01; and ***, P ,0.005. C and D, Bacterial growth ofvirulent Pst strain DC3000 (C) andavirulent Pst strain AvrRPM1 (D) at 0, 6,24, 48, and 54 hpi. E, B. cinerea and Pstgrowth was quantified by qRT-PCR 3and 48 h after pathogen infection inleaves of wild-type and Atdpl1 mutantplants. Asterisks indicate significant dif-ferences between wild-type and Atdpl1samples according to Student’s t test:***, P , 0.005. Results are representa-tive of three independent experiments.

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response (erh1) mutants, which displayed overaccumula-tion of Cers. These mutants exhibited spontaneous celldeath and resistance to biotrophic pathogens, whichseemed to be linked with SA and PR protein accumula-tion (Liang et al., 2003; Wang et al., 2008).Altogether, these data provide evidence of a link be-

tween PCD, defense, and sphingolipid metabolism.However, the fatty acid hydroxylase1/2 (atfah1/atfah2) dou-ble mutant that accumulates SA and Cers was moretolerant to the obligate biotrophic fungus Golovinomycescichoracearum but did not display a PCD-like phenotype,suggesting that Cers alone are not involved in the in-duction of PCD (König et al., 2012). Moreover, Saucedo-García et al. (2011) postulated that dihydroxy-LCBs, butnot trihydroxy-LCBs, might be primary mediators forLCB-induced PCD. The sphingoid base hydroxylasesbh1/sbh2 double mutant completely lacking trihydroxy-LCBs showed enhanced expression of PCD markergenes (Chen et al., 2008). On the contrary, increase int18:0 was specifically sustained in plant interaction withthe avirulent Pseudomonas syringae pv tomato (Pst) strainand correlated with a strong PCD induction in leaves(Peer et al., 2010). Thus, the nature of sphingolipids ableto induce PCD is still under debate and may evolvedepending on plants and their environment. The phos-phorylated form of LCBs (LCB-Ps) could abrogatePCD induced by LCBs, Cers, or heat stress in a dose-dependent manner (Shi et al., 2007; Alden et al., 2011).

Furthermore, blocking the conversion of LCBs to LCB-Psby using specific inhibitors induced PCD in cell sus-pension culture (Alden et al., 2011). Recently, over-expression of rice (Oryza sativa) LCB kinase in transgenictobacco (Nicotiana tabacum) plants reduced PCD aftertreatment with FB1 (Zhang et al., 2013). Genetic muta-tion on LCB-P lyase encoded by the AtDPL1 gene,modifying the LCB-LCB-P ratio, could impact PCDlevels after treatment with FB1 (Tsegaye et al., 2007).Altogether, these data point to the existence of a rheostatbetween LCBs and their phosphorylated forms thatcontrols plant cell fate toward cell death or survival.

Data on plant sphingolipid functions are still frag-mentary. Only a few reports have described intercon-nections between sphingolipids, cell death, and plantdefense responses, almost exclusively in response to(hemi)biotrophic pathogens. Knowledge about such re-lations in response to necrotrophic pathogens is still in itsinfancy (Rivas-San Vicente et al., 2013; Bi et al., 2014). Inthis report, the link between sphingolipids, cell death,and plant defense has been explored in response to Bo-trytis cinerea infection and in comparison with Pst infec-tion. For this purpose,Atdpl1mutant plants, disturbed inLCB/LCB-P accumulation without displaying any phe-notype under standard growth conditions (Tsegaye et al.,2007), have been analyzed after pathogen infection. Ourresults revealed that modification of sphingolipid con-tents not only impacted plant tolerance to hemibiotrophs

Figure 2. Free LCB and LCB-P accumulation after challenge with pathogen. Leaves of wild-type (WT) or Atdpl1-1mutant plantswere sprayed with B. cinerea spore suspension (Bc) or potato dextrose broth (PDB; Control; A, B, E, and F) or infiltrated with PstDC3000, Pst AvrRPM1, or MgCl2 (Control; C, D, G, and H). Quantifications of LCBs (A–D) and LCB-Ps (E–H) were performed 48hpi. Asterisks on wild-type bars indicate significant differences between the pathogen-treated wild-type sample and the controlsample, and asterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated wild-type sample and thepathogen-treatedAtdpl1-1 sample according to Student’s t test: *, P, 0.05; **, P, 0.01; and ***, P, 0.005. Results aremeans offour to five independent biological experiments6 SD. Notice the different scale of LCB-P levels between wild-type and Atdpl1-1plants. DW, Dry weight.

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Sphingolipids in Plant Defense Responses



Figure 3. GIPC contents after B. cinerea or Pst infection. Leaves of wild-type (WT; left) or Atdpl1-1 mutant (right) plants weresprayed with PDB (Control; A and B) or B. cinerea spore suspension (Bc; C and D) or infiltrated with MgCl2 (Control; E and F), PstDC3000 (G and H), or Pst AvrRPM1 (I and J). Quantifications were performed 48 hpi. Asterisks on wild-type bars indicate sig-nificant differences between the pathogen-treated wild-type sample and the control sample, and asterisks on Atdpl1-1 bars in-dicate significant differences in total species between the pathogen-treated wild-type sample and the pathogen-treated Atdpl1-1sample according to Student’s t test: *, P, 0.05; **, P, 0.01; and ***, P, 0.005. Asterisks have only been considered for the totalspecies displaying the same fatty acid or hydroxylation/unsaturation degree. Results are means of four to five independent bio-logical experiments 6 SD. DW, Dry weight.





Figure 4. Cer species produced by wild-type andAtdpl1-1mutant plants upon pathogen infection. Leaves of wild-type (WT; left) orAtdpl1-1mutant (right) plants were sprayed with PDB (Control; A and B) or B. cinerea spore suspension (Bc; C and D) or infiltratedwith MgCl2 (Control; E and F), PstDC3000 (G andH), or Pst AvrRPM1 (I and J). Quantifications were performed 48 hpi. Asterisks onwild-type bars indicate significant differences between the pathogen-treated wild-type sample and the control sample, and asterisksonAtdpl1-1 bars indicate significant differences between the pathogen-treated wild-type sample and the pathogen-treated Atdpl1-1sample according to Student’s t test: *, P, 0.05; **, P, 0.01; and ***, P, 0.005. Asterisks have only been considered for the totalspecies displaying the same fatty acid or hydroxylation/unsaturation degree. Results are means of four to five independent biologicalexperiments 6 SD. DW, Dry weight.





but also greatly affected resistance to necrotrophs.Whereasthe SA signaling pathway is globally repressed in Atdpl1-1compared with wild-type plants, the JA signaling path-way is significantly enhanced. Cell death and ROS ac-cumulation are markedly modified in Atdpl1-1 mutantplants.We further demonstrated that phytosphingosine-1-phosphate (t18:0-P) and d18:0 are key players in pathogen-induced cell death and ROS generation. Here, we thusestablished a link between JA signaling, PCD, and sphin-golipid metabolism.

RESULTS

Necrotrophic and Hemibiotrophic Infection DifferentlyAffect the Atdpl1 Mutant Plant Response

In order to assess the role of sphingolipids in plantimmune responses to necrotrophic and hemibiotrophicpathogens, we used the Atdpl1mutant, which is affectedin the LCB/LCB-P rheostat by accumulating t18:1-P(Tsegaye et al., 2007). Whereas Atdpl1 shows no devel-opmental phenotype compared with wild-type plantsunder standard conditions, it exhibits a higher sensitivityto FB1 (Tsegaye et al., 2007). B. cinerea and Pst havebeen widely used to decipher defense mechanisms tonecrotrophic and hemibiotrophic pathogens in Arabi-dopsis (Glazebrook, 2005). To get some informationabout the susceptibility of the Atdpl1mutant to B. cinereaor Pst (either virulent [Pst DC3000] or avirulent [PstAvrRPM1] strain), three independentAtdpl1mutant lineswere challenged with these pathogens. The three Atdpl1mutant lines displayed similar responses upon pathogenchallenge (Fig. 1). In B. cinerea-infected wild-type plants,disease symptoms, showing chlorosis and necrosis, in-creased more rapidly than in B. cinerea-infected Atdpl1plants (Fig. 1A). On the contrary, symptomsdeveloped inresponse to Pst infection were more pronounced in mu-tant plants than in wild-type plants (Fig. 1A). The lesiondiameters were scored 48 and 60 h after drop inoculationwith B. cinerea and classified into size categories (Fig. 1B).Interestingly, Atdpl1 plants did not display necroticlesions of the largest size, whereas wild-type plantsshowed 10% of these lesions 48 h post inoculation (hpi).Only 2% of the largest lesions were observed in Atdpl1plants compared with 12% for wild-type plants 60 hpi.Furthermore, Atdpl1 mutants displayed a greater per-centage of small necrotic lesions than wild-type plants.Atdpl1 lines displayed approximately 45% and 65% ofsmall lesions,whereaswild-type plants showedonly 17%and 24% of small lesions 48 and 60 hpi, respectively.Consequently, fewer lesions of medium size were ob-served in Atdpl1 lines than in wild-type plants (Fig. 1B).

The average lesion diameter in the Atdpl1 mutantwas significantly lower than that in wild-type plants(**, P, 0.01 and ***, P, 0.005; Fig. 1B). Plants were alsoinfiltratedwithPstDC3000 orPstAvrRPM1at 107 colony-forming units (cfu) mL21, and bacterial populations wereevaluated 0, 6, 24, 30, 48, and 54 hpi. As alreadydescribed,avirulent strain growth was less important comparedwith the virulent strain in wild-type plants (Fig. 1, C and

D). Interestingly, infection with both bacterial strainsrevealed an increased susceptibility of Atdpl1 plants,allowing about 10-fold more bacterial growth as com-pared with wild-type plants (Fig. 1, C and D). Theseresults were also correlated by fungal and bacterialpopulation quantification in infected leaves by quanti-tative reverse transcription (qRT)-PCR (Fig. 1E). Inter-estingly, the AtDPL1 expression profile was similar aftereither B. cinerea or Pst infection (Supplemental Fig. S1).Until 12 hpi, no AtDPL1 transcript accumulation couldbe observed. AtDPL1 expression increased significantlybetween 12 and 24 hpi and rose continuously until thelater stages of infection. Symptoms due to either B. ci-nerea invasion or infection with the virulent or avirulentstrain of Pst visually appeared between 24 and 30 hpi(data not shown) and thus are delayed slightly com-pared with AtDPL1 expression. Deregulation of photo-synthesis is considered a tool for evaluating the first signof pathogen infection (Berger et al., 2007; Bolton, 2009).Repression of the RbcS gene (encoding the small subunitof ribulose-1,5-bisphosphate carboxylase) after pathogeninfection occurred at the same time (B. cinerea) or slightlyearlier (Pst) compared with AtDPL1 expression andsymptom appearance (Supplemental Fig. S1), suggestingthat an immediate consequence of pathogen perceptionincludes the induction of AtDPL1 gene expression. Col-lectively, these data indicate that lack of AtDPL1 activityin mutant plants significantly delays the development oflesions triggered by B. cinerea infection but renders plantsmore susceptible to Pst infection.

Sphingolipid Profiles in Wild-Type and Atdpl1-1 PlantsAre Affected Differently upon Pathogen Infection

To determine whether changes in the levels of certainsphingolipids are responsible for the delayed develop-ment of B. cinerea infection in the Atdpl1 mutant, sphin-golipid profiles were analyzed. The main sphingolipidspecies in Arabidopsis, LCBs and LCB-Ps (Fig. 2), gly-cosylinositol phosphoceramides (GIPCs; Fig. 3), Cers(Fig. 4), hydroxyceramides (hCers; Fig. 5), and gluco-sylceramides (GlcCers; Supplemental Fig. S2), were firstquantified in both the wild type and theAtdpl1-1mutantat 0 hpi (Supplemental Fig. S3). In wild-type andAtdpl1-1plants, LCB/LCB-P basal levels were almost in the samerange as those already described (Tsegaye et al., 2007;Supplemental Fig. S3). As described previously, the onlysignificant alteration in sphingolipid basal levels ob-served in the Atdpl1-1 mutant compared with the wildtype under typical growth conditions was an increase inone specific LCB-P (4-hydroxy-8-sphingenine-1-phosphate[t18:1-P]; Tsegaye et al., 2007; Supplemental Fig. S3).Next, we investigated the influence of B. cinerea infectionon the sphingolipid profile in wild-type plants. B. cinereainfection triggered LCB accumulation (from 63 for4,8-sphingadienine [d18:2] to 203 for d18:0; Fig. 2A) butalso a moderate increase in sphingosine-1-phosphate(d18:1-P) and t18:1-P amount (43 and 2.53, respec-tively) comparedwithmock-inoculatedwild-type plants




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Figure 5. hCer species produced bywild-type andAtdpl1-1mutant plants upon pathogen infection. Leaves of wild-type (WT; left)or Atdpl1-1 mutant (right) plants were sprayed with PDB (Control; A and B) or B. cinerea spore suspension (Bc; C and D) orinfiltrated with MgCl2 (Control; E and F), PstDC3000 (G and H), or Pst AvrRPM1 (I and J). Quantifications were performed 48 hpi.Asterisks on wild-type bars indicate significant differences between the pathogen-treated sample and the control sample, andasterisks on Atdpl1-1 bars indicate significant differences between the pathogen-treated wild-type sample and the pathogen-treated Atdpl1-1 sample according to Student’s t test: *, P , 0.05; **, P , 0.01; and ***, P , 0.005. Asterisks have only beenconsidered for the total species displaying the same fatty acid or hydroxylation/unsaturation degree. Results are means of four tofive independent biological experiments 6 SD. DW, Dry weight.





(Fig. 2E). The amount of total GIPCs and, more pre-cisely, saturated a-hydroxylated very-long-chain fatty acid(VLCFA)-containing GIPCs (C24 and C26; Fig. 3, A andC) was significantly lower after B. cinerea infection than inmock-treated plants (200 and 300 nmol g21 dry weight,respectively; Supplemental Fig. S4). Moreover, d18:0-,d18:1-, and t18:1-GIPC levels were also reduced after B.cinerea infection (Fig. 3,A andC). The amount of total Cersis 4 times higher in B. cinerea- than in mock-inoculatedwild-type plants (84 versus 21 nmol g21 dry weight;Supplemental Fig. S4). Most Cer molecules were affectedby the presence of B. cinerea (Fig. 4, A and C). Finally, thelevel of total hCers was not modified (SupplementalFig. S4); however, significant accumulation of saturateda-hydroxylated C16-, C18-, and C26-containing hCersand d18:0-hCer was observed after challenge with B. ci-nerea (Fig. 5, A and C). No change could be noticed inGlcCer levels (Supplemental Figs. S2 and S4).

To better understand the role of sphingolipids inplant resistance to the necrotrophic fungus, a compar-ison between sphingolipid profiles in B. cinerea-infectedAtdpl1-1 mutant and wild-type plants was then per-formed. With respect to the LCB/LCB-P pool, wild-type plants contained more LCBs (Supplemental Fig.S4), especially d18:0 and d18:2 (Fig. 2, A and B),whereas the Atdpl1-1 mutant accumulated more LCB-Ps(Supplemental Fig. S4), especially t18:0-P and t18:1-P(9- and 18-fold, respectively), when compared withwild-type plants (Fig. 2, E and F). The amount of totalGIPCs and, more precisely, saturated a-hydroxylatedVLCFA-containing GIPCs (C22, C24, and C26; Fig. 3, Cand D) was significantly higher inAtdpl1-1mutant thanin wild-type plants after B. cinerea infection (370 versus220 nmol g21 dry weight, respectively; SupplementalFig. S4). Total Cer amount was similar in both types ofplants (Fig. 4, C and D; Supplemental Fig. S4), but B.cinerea infection triggered an increase in hCer contents,especially saturated and monounsaturated VLCFA-containing hCers (Fig. 5, C and D), in Atdpl1-1 mutantcompared with wild-type plants (75 versus 27 nmol g21

dry weight, respectively; Supplemental Fig. S4). More-over, trihydroxy-hCers also accumulated three times inthemutant comparedwithwild-type plants in responseto the fungus (Fig. 5, C and D). No significant changewas observed in total GlcCer amount (SupplementalFigs. S2 and S4).

In order to compare sphingolipid profiles in responseto a hemibiotrophic pathogen, analyses were performed48 h after wild-type plant inoculation with avirulent orvirulent strains of Pst. Our data confirmed previous re-sults showing that sphingolipid increase was more sus-tained during the incompatible than the compatibleinteraction (Peer et al., 2010). Increase in t18:0 was ob-served in response to both types of bacteria, but infectionwith only Pst AvrRPM1 triggered a significant decreaseof d18:1 (Fig. 2C). After infection with Pst AvrRPM1, anincrease in d18:2-P, t18:0-P, and t18:1-P was observed,whereas only t18:0-P level was increased in response toPst DC3000 (Fig. 2G). GIPC levels were also not signifi-cantly modified in response to both types of bacteria

(Fig. 3, E, G, and I; Supplemental Fig. S4). Total contentsof d18:0-, d18:1-, t18:0-, and t18:1-Cers were increasedafter infection with Pst AvrRPM1 (Fig. 4, E and I). Onlyan increase in trihydroxy-Cers could be noticed in re-sponse to Pst DC3000 (Fig. 4, E and G). Moreover, thet18:0-Cer level was higher in the case of the incom-patible interaction than in the case of the compatibleone (40 versus 24 nmol g21 dry weight, respectively;Supplemental Fig. S4). C16-, C24-, and C26-Cersalso accumulated in response to both strains of Pst(Fig. 4, E, G, and I), and only C16-Cer accumulationwas more pronounced in the case of interaction withPst AvrRPM1 compared with Pst DC3000 (45 versus18 nmol g21 dry weight, respectively; Fig. 4, E, G, andI). Total contents of d18:0-hCers were increased inresponse to Pst (Fig. 5, E, G, and I). t18:0-hCers accu-mulated after challenge with the virulent strain andt18:1-hCers after challenge with the avirulent strain(Fig. 5, E, G, and I). Similar to B. cinerea infection,no regulation of GlcCer content could be noticed(Supplemental Figs. S2 and S4). Comparison of sphin-golipid profiles between Pst-infected wild-type andAtdpl1-1 mutant plants revealed an increase in d18:0(1.53) in Atdpl1-1 plants certainly due to infiltration,since it was also observed in control plants. An increasein t18:0-P level (53) was detected in Atdpl1-1 mutantplants compared with the wild type only in response tothe avirulent strain (Fig. 2H). No significant regulationof GIPC, Cer, hCer, or GlcCer pools was observed inresponse to either the virulent or avirulent strain (Figs.3–5; Supplemental Fig. S2).

Changes in Sphingolipid Profiles Affect Pathogen-InducedCell Death

Recently, several reports have revealed that somesphingolipids are important players in HR and associ-ated PCD (Berkey et al., 2012;Markham et al., 2013). HRis an effective strategy of plants to protect themselvesagainst (hemi)biotrophic microorganisms (Coll et al.,2011). In contrast, PCD processes promote the spread ofnecrotrophic pathogens such as B. cinerea (Govrin andLevine, 2000; Govrin et al., 2006). Thus, changes insphingolipid profiles and differences in tolerance uponB. cinerea or Pst infection prompted us to examine thecell death response upon pathogen attack. We thusmeasured electrolyte leakage to detect changes in lossof ions caused by plasma membrane damage charac-teristic of plant cell death (Dellagi et al., 1998; Kawasakiet al., 2005). Ion leakage measured after the inoculationof Atdpl1-1 plants with B. cinerea or Pst was reducedcompared with wild-type plants (Fig. 6). These resultssuggested that modification in sphingolipid contentcould play a role in modulating cell death processes inresponse to pathogen infection.

Expression levels of PCD marker genes, such asthe flavin-containing monooxygenase FMO and thesenescence-associatedgenesSAG12 andSAG13 (Brodersenet al., 2002), were also evaluated in order to verify if cell























death responses are modified in Atdpl1-1mutant plants(Fig. 7). FMO and SAG13were induced in both types ofplants with increasing infection spread of B. cinerea.Interestingly, these inductions occurred earlier andstronger in the wild type (between 12 and 24 hpi) thanin the Atdpl1-1mutant (between 24 and 30 hpi; Fig. 7, AandC). SAG12was only induced 48 hpi in both the wildtype and theAtdpl1-1mutant, and similar to SAG13 andFMO, its expression was stronger in the wild type(10,0003) than in the Atdpl1-1mutant (2,0003; Fig. 7E).As expected in the case of Pst infection, SAG13 and

FMO gene expression were induced earlier and strongerduring the incompatible interaction than during thecompatible interaction (Fig. 7, B and D). Wild-type andmutant plants displayed similar expression profileswith both types of bacteria; however, induction was lesspronounced in Atdpl1-1 mutant plants. Similar to B. ci-nerea infection, SAG12 transcript accumulation occurredonly at the later stages of infection (Fig. 7F). It is note-worthy that induction of these PCD marker genes fol-lowed a pattern similar to AtDPL1 gene expression inwild-type plants in response to either B. cinerea or Pstinfection (Supplemental Fig. S1, A and B).SAG12 is only expressed in senescent tissues. In

contrast, SAG13 and FMO are expressed in differentPCD processes (Lohman et al., 1994; Brodersen et al.,2002). Collectively, our data suggest that the induc-tion of SAG13 and FMO after either B. cinerea or Pstinfection could result from an HR-like PCD, whereasa senescence program is activated later. This couldalso explain the tolerance of Atdpl1 mutant plantstoward B. cinerea and their higher susceptibility to-ward Pst.

Modification of Sphingolipid Contents Affect ROSProduction in Response to Pathogen Infection

Transient production of ROS is a hallmark of suc-cessful pathogen recognition (Torres, 2010). To inves-tigate whether sphingolipid content perturbation inAtdpl1-1 plants affected pathogen recognition, we com-pared ROS production in the mutant versus wild-typeplants. Wild-type plants displayed a transient oxidativeburst, peaking around 300 min (B. cinerea) or 40 min(Pst) after inoculation (Fig. 8). This transient burst wassignificantly induced by 2.5 times in B. cinerea-infectedAtdpl1-1 plants compared with wild-type plants (Fig.8A). On the contrary, ROS levels were significantly re-duced in Pst-infected Atdpl1-1 plants compared withPst-infected wild-type plants (Fig. 8, B and C). Ourresults thus demonstrated that signaling events linkedto pathogen recognition are affected by sphingolipidperturbation in Atdpl1-1 plants.

Exogenous t18:0-P and d18:0 Differently ModifyPathogen-Induced Cell Death and ROS Production

Major changes in LCB-P contents in B. cinerea-inoculatedAtdpl1-1 mutant plants are an increase in t18:0-P levels

and a decrease in d18:0 amounts (Fig. 2).We thus tested theabilityof these sphingolipids tomodulatepathogen-inducedcell death (Fig. 9) and ROS production (Fig. 10). Ourdata showed that exogenous t18:0-P or d18:0 alone did notaffect cell death or ROS production, a finding consistentwith data obtained by Coursol et al. (2015). In t18:0-P-treated wild-type plants, symptoms and ion leakagetriggered by B. cinerea or Pst infection were reduced sig-nificantly (Fig. 9,A, C, and E). Exogenously applied d18:0did notmodify disease symptoms and electrolyte leakagein wild type-infected plants by B. cinerea and slightly re-duced electrolyte leakage triggered by the virulent Pststrain (Fig. 9, B, D, and F). Interestingly, disease symp-toms and electrolyte leakage were strongly reducedwhen wild-type plants were coinfiltrated with d18:0 andPst AvrRPM1 (Fig. 9, B and F).

Whereas the addition of t18:0-P increased anddelayed ROS production upon challenge with B. ci-nerea, it reduced the Pst-induced oxidative burst(Fig. 10, A–C). d18:0 had no significant effect on ROSaccumulation triggered by B. cinerea (Fig. 10D).However, it dramatically reduced the Pst-inducedoxidative burst (Fig. 10, E and F). These data indicatethat exogenously applied t18:0-P and d18:0 modify

Figure 6. Electrolyte leakage in Atdpl1-1 mutants after pathogen in-oculation. Conductivity (mS cm21) is shown for solution containing leafdiscs from either the wild type (WT) or the Atdpl1-1 mutant inoculatedwith B. cinerea (Bc) or PDB (Control) solution (A) or Pst DC3000, PstAvrRPM1, or 10 mM MgCl2 (B). Each value represents the mean6 SD ofthree replicates per experiment. The experiment was repeated threetimes with similar results.






signaling events and cell death triggered by infectionwith these two pathogens.

SA and ET/JA Signaling Pathways Are Modified inAtdpl1-1 Mutant Plants after Pathogen Challenge

Disruption of sphingolipid contents between wild-type and Atdpl1 plants could result in the differentialactivation of defense responses after pathogen infec-tion. PR1 and PR5 are well-known SA-dependent de-fense marker genes, NONEXPRESSED PATHOGENRELATED1 (NPR1) was shown to be a key regulator ofSA-mediated suppression of JA signaling (Spoel et al.,2003), and DEFENSIN (PDF1.2), CHITINASE (CHIT),and ETHYLENE RESPONSE FACTOR1 (ERF1) expres-sion is regulated by JA and ET whereas VEGETATIVESTORAGE PROTEIN1 (VSP1) and JASMONATE ZIM-DOMAIN8 (JAZ8) aremostly responsive to JA (Glazebrook,2005; Pieterse et al., 2009). First, the expression patternof these defense genes was monitored in wild-type andAtdpl1-1 mutant plants. No significant difference in the

expression of these defense genes was detected in wild-type andAtdpl1-1mutant plants grown under standardconditions (Figs. 11 and 12). These results indicated thatinactivation of the gene encoding LCB-P lyase itself didnot result in any defense response changes in plants. Theexpression levels of defense-related genes in Atdpl1-1mu-tant plants were then compared with wild-type plants inresponse toB. cinerea infection (Fig. 11).WhereasPR1,PR5,NPR1, and VSP1 expression showed similar inductionlevels in both genotypes, the expression of PDF1.2,CHIT, ERF1, and JAZ8 was markedly enhanced inAtdpl1-1mutant compared with wild-type plants. At 48hpi, there was a 12-fold increase for PDF1.2 and a 2-foldincrease for CHIT, ERF1, and JAZ8 compared withwild-type plants (Fig. 11). Since JA-responsive geneswere up-regulated in the Atdpl1-1 mutant, the expres-sion of three genes encoding key enzymes in JA bio-synthesis, LIPOXYGENASE2 (LOX2), ALLENE OXIDECYCLASE2 (AOC2), and 12-OXOPHYTODIENOICACID REDUCTASE3 (OPR3) as well as JAR1, encodingthe enzyme that converts JA to the jasmonoyl-isoleucine(JA-Ile) conjugate (Staswick and Tiryaki, 2004), was

Figure 7. Time course of PCDmarker geneexpression after B. cinerea or Pst infection.Leaves were sprayed with B. cinerea sporesuspension (Bc) or PDB (Control; A, C, andE) or infiltrated with a bacterial solution(Pst DC3000 or Pst AvrRPM1) or MgCl2(Control; B, D, and F). Themean values6 SD

from one representative experiment areshown. qRT-PCR of FMO (A and B), SAG13(C andD), and SAG12 (E and F) expressionwas performed in wild type (WT) andAtdpl1-1 mutant plants with five biologicalreplicates with comparable results.





also followed. The results showed that LOX2 andAOC2were significantly up-regulated up to 24 hpi in theAtdpl1-1 mutant but transcripts returned to a levelcomparable to the wild type thereafter (Fig. 11). Incontrast, the expression of OPR3 was similar in bothgenotypes. JAR1 expression was not affected by thefungus inoculation (Fig. 11). These results indicated

that both JA synthesis and signaling pathways wereenhanced in Atdpl1-1 mutant plants.

When infected with Pst, wild-type plants displayed astrong induction of PR1 expression, and, as expected,this induction was more pronounced (4-fold at 48 hpi)in the case of the incompatible interaction (Fig. 12).Surprisingly, a significant repression of this gene wasobserved 30 hpi in the Atdpl1-1 mutant compared withwild-type plants (63 for Pst DC3000 and 43 for PstAvrRPM1), but the level of PR1 expression was stillhigher in incompatible compared with compatible in-teractions. Accumulation of PR5 transcripts was alsoslightly more important in wild-type plants, but ex-pression levels were more important in the case of thecompatible interaction (Fig. 12). Under Pst attack,NPR1was slightly induced, but no difference between thewild type and the Atdpl1-1mutant was observed. CHITexpression was also more induced in response to PstAvrRPM1 (903) than Pst DC3000 (203) in wild-typeplants, and this induction profile was similar inAtdpl1-1 mutant plants (Fig. 12). As already described,inoculation with the bacterial pathogen (Pst DC3000 orPst AvrRPM1) led to a dramatic repression of PDF1.2expression, either in wild-type or in Atdpl1-1 mutantplants. In contrast, ERF1 and JAZ8were induced duringPst infection, andVSP1 expressionwas slightly inducedwhen challenged by Pst DC3000 but repressed after PstAvrRPM1 infection (Fig. 12). Expression of these threegenes was markedly enhanced in the Atdpl1-1 mutantcompared with wild-type plants. At the end of the timecourse, VSP1, ERF1, and JAZ8mRNA levels were 2-, 3-,and 6-fold higher in Atdpl1-1 than in wild-type plantsafter infection with either virulent or avirulent strains,respectively. Similar to B. cinerea infection, JAR1 ex-pression was not affected by inoculation with Pst. Re-garding genes involved in the JA biosynthetic pathway,LOX2was repressed,AOC2was not induced during Pstchallenge, and OPR3 was induced slightly, but no dif-ference between the two genotypes was observed (Fig.12). These data suggested that only the JA signalingpathway is positively affected in mutant plants uponchallenge with Pst.

To get further information on Atdpl1-1mutant defenseresponses, some defense-related phytohormones werealso quantified (Fig. 13). No change in phytohormonebasal levels was observed between wild-type andAtdpl1-1mutant plants (Fig. 13). This implied that Atdpl1-1 mu-tant plants, in contrast to other mutants with modifiedsphingolipid contents, do not display high constitutiveSA amounts (Greenberg et al., 2000; Wang et al., 2008;Ternes et al., 2011; König et al., 2012). Following patho-gen attack, all phytohormone levels were enhanced. SAaccumulation was essentially unchanged in the mutantcompared with wild-type plants, whatever the pathogenconsidered. Interestingly, levels of JA and its biologicallyactive conjugate, JA-Ile, were 2 to 3 times higher in theAtdpl1-1mutant compared with wild-type plants after B.cinerea or Pst infection, respectively. However, no differ-ence in JA levels between virulent and avirulent inter-action was noticed, but JA-Ile accumulation was slightly

Figure 8. Transient ROS production in response to pathogen infec-tion in wild-type and Atdpl1-1 mutant plants. The time course ofROS production in wild type (WT) and Atdpl1-1 mutant plants isshown in response to B. cinerea (Bc; A), Pst DC3000 (B), or PstAvrRPM1 (C) infection. Leaf discs were immersed in a solutioncontaining either 105 spores mL21 B. cinerea or 108 cfu mL21 Pst.Error bars represent SE from 12 biological repetitions. Three inde-pendent experiments were performed with similar results. RLUs,Relative light units.





higher in the case of the avirulent interaction in Atdpl1-1plants. Together, our data suggest that the JA-dependentsignaling pathway is preferentially activated in the Atdpl1-1mutant in response to pathogen infection.

DISCUSSION

Only a few articles have described a connection be-tween sphingolipid content, PCD, and defense reactionsduringbiotic stress (Berkey et al., 2012). Furthermore,mostof them focused on responses against (hemi)biotrophicpathogens, the role of sphingolipid in plant defenseagainst necrotrophs being largely unsolved (Rivas-SanVicente et al., 2013; Bi et al., 2014). Moreover, nearly allstudies revealed basal sphingolipid levels, and data of

sphingolipid contents during pathogen infection wereoften not available (Peer et al., 2010; Bi et al., 2014). Thiswork describes a comparison of sphingolipid contentduring hemibiotrophic and necrotrophic infection. Inthis study, we investigated the consequences of thedisruption of the sphingolipid profiles on plant im-munity responses such as cell death, ROS production,and signaling of plant defense responses during path-ogen infection.

Interplays between Sphingolipids and PCD

As in animal systems, new emerging evidence showedthat bioactive sphingolipids play a critical role as mod-ulators of plant PCD (Berkey et al., 2012; Saucedo-Garcíaet al., 2015). Here, sphingolipid content analyses showed

Figure 9. Exogenous effects of t18:0-P and d18:0 on electrolyte leakage in response to pathogen infection in wild-type plants. Aand B, B. cinerea conidia suspension was deposited on leaves of wild-type and Atdpl1-1mutant plants 15 min after infiltration ofeither t18-0-P or d18:0 solution. Pst and either t18-0-P or d18:0 solution were coinfiltrated into wild-type and Atdpl1-1 leaves.Photographs represent symptoms observed 60 or 72 h after infection by the fungus or Pst, respectively. C to F, Conductivity(mS cm21) of solution containing t18:0-P- or d18:0-infiltrated leaf discs from the wild type inoculated by spraying B. cinerea (Bc)or PDB (Control) solution (C and D) or by infiltration of Pst DC3000, Pst AvrRPM1, or 10 mM MgCl2 (E and F). Each value rep-resents the mean 6 SD of three replicates per experiment. The experiment was repeated three times with similar results.





that infection by B. cinerea or Pst triggered the accumu-lation of some species known to act in favor of cell sur-vival (LCB-Ps and hCers) or cell death (LCBs and Cers).Interestingly, the Atdpl1-1 mutant displayed higherlevels of d18:0 in response to infiltration (Fig. 2). More-over, this LCB reduced Pst-induced cell death andsymptoms, especially in the case of the incompatibleinteraction (Fig. 9). Since the HR often contributes toresistance to (hemi)biotrophic pathogens, our resultssuggested that a modification in d18:0 levels could im-pact plant cell death and, thus, resistance to such path-ogens. Recently, Coursol et al. (2015) showed that theaddition of d18:0 had no significant effect on the viabil-ity of cryptogein-treated cells, indicating that distinctmechanisms of regulation are involved in cell death ofcell cultures or plant tissue or after treatment by anelicitor or a pathogen. Necrotrophs are pathogens thatderive nutrients from dead or dying cells. PCD, includ-ing HR, can be beneficial to this kind of pathogen and,thus, could facilitate their infection and spread of disease(Govrin and Levine, 2000; Mayer et al., 2001; Govrinet al., 2006). Plants that are less potent to activate HR orwith reduced cell death present enhanced tolerance to B.cinerea infection and vice versa (Govrin and Levine,2000; van Baarlen et al., 2007). Similarly, antiapoptoticgenes conferred resistance to necrotrophic fungi intransgenic plants (Dickman et al., 2001; El Oirdi and

Bouarab, 2007). A general pattern established that in-fection of Arabidopsis by B. cinerea is promoted by andrequires an active cell death program in the host (vanKan, 2006), and resistance against this fungus dependson the balance between cell death and survival (vanBaarlen et al., 2007).

Interestingly, the Cer-accumulating acd5 mutant orCer-infiltrated plants were more susceptible to severalBotrytis spp. (van Baarlen et al., 2004, 2007). Moreover,myriocin, a potent inhibitor of SERINE PALMITOYL-TRANSFERASE (SPT), the first enzyme of sphingolipidbiosynthesis, had a death-antagonistic effect during theBotrytis elliptica-Lilium interaction (van Baarlen et al.,2004). This suggests that sphingolipid metabolism isinvolved in cell death triggered by Botrytis spp. Celldeath activation could thus be disturbed in Atdpl1plants, leading to a higher susceptibility toward (hemi)biotrophs and higher tolerance toward necrotrophs. Inthis work, B. cinerea infection triggered Cer and LCBaccumulation in wild-type plants. It is thus possiblethat the necrotrophic fungus promoted plant PCD-inducing factors (e.g. sphingolipids) in order to facilitateits penetration and spread inside plant cells. However,exogenous d18:0 did not modify ion leakage in thepresence of B. cinerea, suggesting that this LCB alone isnot involved in such a mechanism. Sphingolipid anal-ysis revealed that B. cinerea-infected Atdpl1-1 plants

Figure 10. Exogenous effects of t18:0-Pand d18:0 on ROS production in response to pathogen infection in wild-type plants. Thetime course of ROS production in t18:0-1-P- or d18:0-treatedwild-type plants is shown in response to B. cinerea (Bc; A andD), PstDC3000 (B and E), or Pst AvrRPM1 (C and F) infection. Leaf discs were immersed in a solution containing 100 mM t18:0-1-P ord18:0 and either 105 spores mL21 B. cinerea or 108 cfu mL21 Pst. Error bars represent SE from 12 biological repetitions. Threeindependent experiments were performed with similar results. RLUs, Relative light units.





accumulated more VLCFA-hCers and t18:0-P andt18:1-P but fewer Cers and LCBs compared with wild-type plants. Interestingly, our data showed that exog-enous t18:0-P reduced B. cinerea- and Pst-induced celldeath (Fig. 9). Thus, t18:0-P appears to be essential tomodulate plant cell death and, thus, plant resistancein response to pathogen infection. Moreover, it wasrecently demonstrated that AtFAH1 or 2-hydroxyVLCFAs, and thereby VLCFA-hCers, were key factorsin BAX INHIBITOR1-mediated cell death suppression(Nagano et al., 2012). These results confirmed thatsphingolipids play an important role in plant defenseresponses and that the plant is able to adjust its re-sponse by regulating a dynamic balance between celldeath (e.g. HR)- or cell survival-related sphingolipids.However, in contrast to infected Atdpl1 mutant, thefah1/fah2 double mutant presented a reduced amount ofhCers and elevated levels of Cers and LCBs but showedno lesion phenotype (König et al., 2012). Thus, it seemsthat the connection between sphingolipids and PCD isregulated by a fine-tuned process and, thus, could bemore complex than expected. Other parameters, such

as defense signaling pathways, could be involved insuch a mechanism.

Interconnections between Sphingolipids andDefense Mechanisms

Sphingolipids (e.g. LCBs and Cers) participate in theinduction and/or control of plant cell death. Moreover,plant cell death processes, such as HR, are also associ-atedwith plant defense or disease. It is thus conceivablethat some sphingolipids play key roles in plant innateimmunity. Recent studies brought to light intercon-nections between sphingolipids and defense mecha-nisms. Resistance to biotrophic pathogens often requiredROS production (Torres et al., 2002). Consistent withthis, Pst-infected Atdpl1-1 mutant displayed a reducedaccumulation of ROS and was more sensitive to thebacterial attack. In addition, Atdpl1-1 mutant accumu-lated more d18:0 in response to infiltration (Fig. 2), andd18:0 strongly reduced ROS production upon chal-lenge with this bacterium (Fig. 10). B. cinerea-infected

Figure 11. Expression levels of JA and SApathway-associated genes in wild-type (WT)and Atdpl1-1 mutant plants during B. cinerea(Bc) infection. Results are expressed as the foldincrease in transcript level compared with theuntreated control (0 h), referred to as the 13expression level. Values shown are means6 SD

of duplicate data from one representative ex-periment among five independent repetitions.





Atdpl1-1 plants displayed a higher production of ROS(Fig. 8). Several studies demonstrated that resistanceagainst B. cinerea (and other necrotrophs) is accompa-nied by the generation of ROS, and mutants impaired inROS production failed to resist the necrotrophic patho-gen (Contreras-Cornejo et al., 2011; Kraepiel et al., 2011;L’Haridon et al., 2011; Rasul et al., 2012; Savatin et al.,2014; Zhang et al., 2014). It has been shown that LCBs butnot LCB-Ps alone are able to induce ROS production(Peer et al., 2011). In this study, exogenously appliedt18:0-P increased B. cinerea-inducedROS generation (Fig.10). Accordingly, cryptogein-induced ROS accumula-tion is enhanced by a pretreatment with some LCB-Ps,especially t18:0-P (Coursol et al., 2015). This suggeststhat sphingolipids may interact differently with ROSproduction depending on the presence or not of anelicitor or pathogen. Interestingly, the similarity of ROSaccumulation upon infection between Atdpl1-1 plantsand t18:0-P- or d18:0-treated wild-type plants indicatedthat t18:0-P and d18:0 could have key roles in pathogenperception and, thus, in plant resistance toward hemi-biotrophic and necrotrophic pathogens.Several lines of evidence showed that plants dis-

rupted in sphingolipid metabolism often displayed a

spontaneous enhanced SA pathway (Greenberg et al.,2000; Brodersen et al., 2002; Wang et al., 2008; Terneset al., 2011; König et al., 2012; Mortimer et al., 2013;Rivas-San Vicente et al., 2013;Wu et al., 2015). Recently,it was shown that SA and its analog benzothiadiazoleaffect sphingolipid metabolism (Shi et al., 2015), in-cluding AtDPL1 gene expression (Wang et al., 2006).Since activation of the SA-dependent pathway is effec-tive against biotrophic and hemibiotrophic pathogens, ithas been postulated that sphingolipids played a key rolein defense against such pathogens in an SA-dependentpathway (Sánchez-Rangel et al., 2015). However,whereas acd5, erh1, and the double mutant fah1/fah2exhibited enhanced resistance to powdery mildew, theydisplayed a similar phenotype to wild-type plants uponinfection with P. syringae pv maculicola or Verticilliumlongisporum (Wang et al., 2008; König et al., 2012). Thissuggests that SA, sphingolipid-triggered cell death, andplant resistance could be independent regarding theplant/pathogen pair. Unfortunately, only basal levelsof sphingolipid were described, and no sphingolipidquantification during pathogen infection is available,making difficult a direct link between sphingolipidmetabolism and plant defense. In this work, infection

Figure 12. Expression levels of JA and SApathway-associated genes in wild-type (WT)and Atdpl1-1 mutant plants during Pst infec-tion. Results are expressed as the fold increasein transcript level comparedwith the untreatedcontrol (0 h), referred to as the 13 expressionlevel. Values shown are means 6 SD of dupli-cate data from one representative experimentamong five independent repetitions.





with either necrotrophic or hemibiotrophic pathogeninduced the production of all quantified phytohor-mones. It has been reported that several pathogens, in-cluding B. cinerea and Pst, activated both SA and JAaccumulation (Zimmerli et al., 2001; Govrin and Levine,2002; Schmelz et al., 2003; Spoel et al., 2003; Block et al.,2005; Glazebrook, 2005; Veronese et al., 2006), and crosstalk is thus used by the plant to adjust its response infavor of the most effective pathway. Interestingly, SPT-silenced tobacco plants displayed higher basal SA levelsand were more susceptible to A. alternata infection.However, no information concerning SA, JA, or sphin-golipid levels in response to infection is available, es-pecially as transgenic plants still displayed residualNbLCB2 gene expression (Rivas-SanVicente et al., 2013).In Arabidopsis, the acd5 mutant displayed constitutivehigh SA levels and expression of the PR1 gene. Thismutant was also more susceptible to B. cinerea andcontained higher Cer levels but reduced apoplastic ROSand PR1 and CHIT transcript accumulation upon in-fection (Greenberg et al., 2000; Bi et al., 2014). Consistentwith this, Atdpl1-1 mutant plants displayed similarCer levels and PR1 expression, higher apoplastic ROS

accumulation, and CHIT up-regulation in response toinfection but was more resistant to the necrotrophicfungus.

In Arabidopsis, it is now well admitted that SA has anantagonistic effect on JA signaling and reciprocally(Bostock, 2005; Glazebrook, 2005; Spoel et al., 2007; Thaleret al., 2012; Derksen et al., 2013). In tomato (Solanumlycopersicum), B. cinerea produces an exopolysaccharidethat activates the SA pathway, which, through NPR1,antagonizes the JA signaling pathway, thereby allowingthe fungus to enhance its disease (El Oirdi et al., 2011).Moreover, NPR1 needs to be activated by SA (Cao et al.,1998; Spoel et al., 2003). Here, SA accumulated in wild-type plants andNPR1was also stimulated upon infectionwith B. cinerea. However, the SA signaling pathway wassimilar in Atdpl1-1 plants. Moreover, JA biosynthetic andsignaling pathways were enhanced in the Atdpl1-1 mu-tant in response to B. cinerea inoculation. In the Atdpl1-1mutant, it thus seems that perturbation in sphingolipidmetabolism rendered either SA unable to activate NPR1orNPR1unable to antagonize JA accumulation. Thus, ourresults highlighted that disturbance of sphingolipid me-tabolism could impact not only the cell death programbutalso the JA signaling pathway, leading to plant tolerancetoward necrotrophic pathogens such as B. cinerea. In thatcase, the relationship between sphingolipids and JA could

Figure 13. Analysis of phytohormone accumulation in stressedwild-typeand Atdpl1-1mutant plants. JA, JA-Ile, and SA accumulation is shown inwild-type (WT) andAtdpl1-1mutant plants 0 or 30 h following B. cinerea(A) or Pst DC3000 or Pst AvrRPM1 (B) infection. Asterisks indicate sig-nificant differences between wild-type and Atdpl1-1 samples accordingto Student’s t test: *, P , 0.05; **, P , 0.01; and ***, P , 0.005. Valuesshown are means 6 SD from one representative experiment among fiveindependent repetitions. FW, Fresh weight.

Figure 14. Schematic overview of interconnections between sphingo-lipid metabolism, cell death, and defense signaling pathways in Atdpl1mutant plants upon pathogen attack. Upon disruption of the AtDPL1gene, infected plants accumulate some LCB-P, hCer, and GIPC species,thus reducing cell death. In theAtdpl1mutant, sphingolipidmetabolismmay also indirectly modulate cell death through its tight connection(double-headed dashed arrow) as a positive and/or negative regulator tojasmonate and/or SA signaling pathways, respectively. Reduced celldeath and high levels of jasmonates could thus explain that Atdpl1mutant plants are more tolerant to B. cinerea but more susceptible toPst. T-bars indicate inhibition; single-headed arrows indicate activation;double-headed arrows indicate unknown regulatory mechanisms. Ald,Aldehyde; Ethan-P, phosphoethanolamine; LCBK, LCB kinase; LCB-PPase, LCB-P phosphatase; LOH, LAG ONE HOMOLOG; SPHK1,SPHINGOSINE KINASE1.





be either indirect, implying that changes in sphingolipidsoperate in the cross talk between SA and JApathways butin an NPR1-independent manner, or direct, as some keygenes involved in JA biosynthesis are up-regulated inAtdpl1-1 plants. Similar to B. cinerea, a virulent strain ofPst, via its toxin coronatine, exerts its virulence by stim-ulating the JA signaling pathway in order to inhibit the SAsignaling pathway and, thus, facilitate its growth anddevelopment (Zhao et al., 2003; Brooks et al., 2005; Laurie-Berry et al., 2006; Uppalapati et al., 2007; Geng et al., 2012;Zheng et al., 2012; Xin and He, 2013). The dramatic re-duction in the expression of the SA-dependent markergene PR1 in Atdpl1-1 mutant plants could thus be ex-plained by the overaccumulation of jasmonates in theseplants. Whereas the VSP1 and JAZ8 expression profilecorrelated the JA and JA-Ile accumulation profile in re-sponse to infection with virulent or avirulent Pst, PDF1.2and CHIT expression did not. PDF1.2 and CHIT requireboth JA and ET signaling pathways but also the func-tion of MITOGEN-ACTIVATED PROTEIN KINASE4(MPK4), as JA-treated mpk4 mutants fail to expressPDF1.2 (Petersen et al., 2000). A discrepancy betweenPDF1.2 expression and JA accumulation has also beenobserved during the induced systemic resistance trig-gered by Pseudomonas fluorescens, which is regulatedthrough the JA signaling pathway (van Wees et al.,1999). This suggested that a component in the JA or ETsignaling pathway might be deficient/nonfunctional inAtdpl1-1 mutant plants in response to Pst infection orthat defense against Pst in theAtdpl1-1mutant might beregulated through a pathway that does not includePDF1.2 or CHIT. Collectively, our results suggestedthat AtDPL1 could be a negative and/or a positiveregulator of the JA- and SA-regulated defense pathway,respectively. Whereas a relationship between SA sig-naling and sphingolipids has often been described(Sánchez-Rangel et al., 2015), our results highlight, toour knowledge for the first time, that sphingolipidscould also play a key role in the JA signaling pathway.In conclusion, we propose a model in which plant

cells of the Atdpl1 mutant select the most appropriateresponse to defend themselves against pathogen attackby acting on sphingolipid metabolism in order tomodulate the cell death/survival balance, in close co-operation with the JA and/or SA signaling pathways(Fig. 14). Whereas SA involvement in PCD is wellknown, the relationship between JA and cell death isless understood. Plants treated with coronatine, whichshares structural similarities with JA-Ile and functionalsimilarities with JA, develop chlorosis (Bender et al.,1999; Overmyer et al., 2003). Coronatine-deficient mu-tants of Pst DC3000 are reduced in disease-associatednecrosis and chlorosis (Brooks et al., 2004, 2005). It hasbeen reported that JA is also essential in FB1- and AAL-induced cell death (Asai et al., 2000; Zhang et al., 2011).Interestingly, theAtdpl1mutant is more sensitive to FB1treatment (Tsegaye et al., 2007). Thus, sphingolipidmetabolism seemed to be intimately connected to de-fense processes to regulate plant responses to bioticstresses. In Arabidopsis, MPK6, which is involved in

the plant defense response (Ren et al., 2008; Beckerset al., 2009), has recently been described as an importantcontributor to the LCB-mediated PCD (Saucedo-Garcíaet al., 2011). However, the deciphering of the precisepathway leading to sphingolipid-induced cell death isfar from being totally elucidated. Further identificationof target genes and their functions will provide newinsights into how sphingolipids could be linked to celldeath and defense processes.

MATERIALS AND METHODS

Chemicals

Phytosphingosine-1-phosphate (t18:0-P) and dihydrosphingosine (d18:0) were pur-chased from Avanti Polar Lipids. Stock solutions were prepared in ethanol:dimethylsulfoxide (2:1, v/v; t18:0-P) or ethanol (d18:0) and dissolved to a final concentration of100 mM. Luminol and horseradish peroxidase were obtained from Sigma-Aldrich.

Plant Material and Growth Conditions

Seeds of the Arabidopsis (Arabidopsis thaliana) SALK lines 020151 (referred toas Atdpl1-1), 093662 (Atdpl1-2), and 078119 (Atdpl1-3) containing a transfer DNA(T-DNA) insertion in the At1g27980 locus were obtained from the NottinghamArabidopsis Stock Centre (http://arabidopsis.info). The SALK_020151 mutantwas chosen for the performed experiments because it exhibits the same phenotypeas the other Atdpl1 mutants but displays a complete lack of mRNA and a higherLCB/LCB-P accumulation in response to FB1 treatment (Tsegaye et al., 2007).Mutant and wild-type (Columbia-0) plants were grown and maintained under12-h-light/12-h-dark conditions (150mmolm22 s21, 20°C, and60%humidity) for 35d.

Isolation of the T-DNA Insertion Mutant andGenotype Characterization

The mutants SALK_020151, SALK_093662 (Tsegaye et al., 2007), andSALK_078119 were isolated according to the published procedure in SIGnAL(Alonso et al., 2003). The genotype of the knockout mutant line was analyzed byPCR using primers specific for the AtDPL1 gene (forward, 59-AGAAAGGCCT-CAAAGCTTGTC-39; and reverse, 59-TGCCAAATAGCATCATTCCTC-39) and aprimer specific for the T-DNA (LB1a, 59-TGGTTCACGTAGTGGGCCATCG-39).

Sphingolipidomic Analysis

Sphingolipid extraction from 10 to 20mg of lyophilized tissue and profiling byliquid chromatography-electrospray ionization-tandem mass spectrometry wereperformed as described (Markham and Jaworski, 2007) withmodifications, usingthe Shimadzu Prominence ultra-HPLC system and a 4000QTRAP mass spec-trometer (AB Sciex). Sphingolipids were separated on a 100-mmDionex AcclaimC18 column. Data analysis was performed using Analyst 1.6 and Multiquant 2.1software (AB Sciex). Four to five biologically independent repeats were per-formed, and a minimum of three technical replicates were run from each sample.

RNA Extraction and Real-Time qRT-PCR

Isolation of total RNA and real-time PCRwere performed as described by LeHénanff et al. (2013). Gene-specific primers are described in SupplementalTable S1. For each experiment, PCR was performed in duplicate, and at leastthree independent experiments were analyzed. Transcript levels were nor-malized against those of the ACTIN gene as an internal control. Fold inductioncompared with mock-treated sample was calculated using the DDCt method(Ct GI [unknown sample] – Ct GI [reference sample]) – (Ct actin [unknownsample] – Ct actin [reference sample]). GI is the gene of interest.

Pathogen Growth and Inoculation

Botrytis cinerea strain B05.10 was grown on solid tomato (Solanum lyco-persicum) medium (25% [v/v] tomato juice and 2.5% [w/v] agar) during 21 d at




http://arabidopsis.info




22°C. Collected conidia were resuspended in PDB supplemented by 0.02% (v/v)Silwet L-77 to a final density of 105 conidia mL21. After incubation for 3 h at22°C and 150 rpm, germinated spores were used for plant inoculation byspraying the upper face of the leaves. Control inoculations were performedwith 0.02% PDB Silwet L-77.

The bacterial leaf pathogen Pseudomonas syringae pv tomato strain DC3000 orPst AvrRPM1 was cultured overnight at 28°C in liquid King’s B medium, sup-plemented with rifampicin (50 mg mL21) and kanamycin (50 mg mL21). Subse-quently, bacterial cells were collected by centrifugation and resuspended in10 mM MgCl2 to a final density of 107 cfu mL21 (optical density = 0.01). The bacte-rial solutions were thus infiltrated from the abaxial side into leaf using a 1-mL sy-ringe without a needle. Control inoculations were performed with 10 mM MgCl2.

Leaves were collected from 0 to 48 hpi, frozen in liquid nitrogen, and storedat 280°C until use.

Pathogen Assay in Planta

B. cinerea infections were performed as described previously (Le Hénanffet al., 2013). Plants were placed in translucent boxes under high humidity at150 mE m22 s21. Five or six leaves per plant were drop inoculated with 5 mL ofthe conidia suspension adjusted at 105 conidia mL21 in PDB. Lesion diameterswere measured 48 and 60 hpi. Forty to 60 leaves were inoculated per treatmentand per genotype, and experiments were independently repeated four times.

Bacterial infections were performed as described previously (Sanchez et al.,2012). Briefly, eight foliar discs from four leaves were excised using a cork borerand ground in 1mL of 10mMMgCl2 with a plastic pestle. Appropriate dilutionswere plated on King’s B medium with appropriate antibiotics, and bacterialcolonies were counted. Data are reported as means and SD of the log (cfu cm22)of three replicates. Growth assays were performed four times with similarresults.

Electrolyte Leakage

Tenminutes after bacteria injection (Torres et al., 2002) or 20 h after B. cinereainfection (Govrin and Levine, 2002), 9-mm-diameter leaf discs were collectedfrom the infected area and washed extensively with water for 50 min, and theneight discs were placed in a tube with 15 mL of fresh water. To test thesphingolipid effect on ion leakage, pathogen inoculum was supplemented ornot with 100 mM t18:0-P or d18:0. Conductivity measurements (three to fourreplicates for each treatment) were then conducted over time using a B-771LaquaTwin (Horiba) conductivity meter.

ROS Production

Measurements of ROS production were performed as described previously(Smith and Heese, 2014). Briefly, single leaf disc halves were placed in wells of a96-well plate containing 150 mL of distilled water and then incubated overnight atroom temperature. Just before ROS quantification, distilled water was replaced by150mLof an elicitation solution containing 20mgmL21 horseradish peroxidase and0.2 mM luminol. For tests involving bacteria, Pst was added to the elicitation so-lution to a final bacterial concentration of 108 cfu mL21. For tests involving B. ci-nerea, germinated spores were added to the elicitation solution to a final density of105 conidia mL21. For tests involving sphingolipids, 100 mM t18:0-P or d18:0 wasadded concomitantly with bacterium or fungus to the elicitation solution.

Phytohormone Analysis

Phytohormones were quantified using ultra-HPLC-tandem mass spec-trometry according to Glauser et al. (2014).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Time course of AtDPL1 and RbcS expression afterB. cinerea or Pst infection.

Supplemental Figure S2. Glucosylceramide contents after B. cinerea or Pstinfection.

Supplemental Figure S3. Total content in major sphingolipid classes in WTand Atdpl1-1 mutant plants before infection with B. cinerea or Pst.

Supplemental Figure S4. Total content in major sphingolipid classes inwild-type and Atdpl1-1 mutant plants after infection with B. cinerea or Pst.

Supplemental Table S1. Gene-specific primers used in real-time reverse-transcription PCR.

ACKNOWLEDGMENTS

We thank Gaetan Glauser and Neil Villard (Neuchâtel Platform of Analyt-ical Chemistry, University of Neuchâtel) for excellent technical assistance inphytohormone quantification.

Received July 21, 2015; accepted September 11, 2015; published September 16,2015.

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