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Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis

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

Published November 2015. DOI: https://doi.org/10.1104/pp.15.01126

Abstract

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing 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]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in 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 and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids.

Plants have evolved a complex array of defenses when attacked by microbial pathogens. The success of plant resistance first relies on the capacity of the plant to recognize its invader. Among early events, a transient production of reactive oxygen species (ROS), known as the oxidative burst, is characteristic of successful pathogen recognition (Torres, 2010). Perception of pathogen attack then initiates a large array of immune responses, including modification of cell walls, as well as the production of antimicrobial proteins and metabolites like pathogenesis-related (PR) proteins and phytoalexins, respectively (Schwessinger and Ronald, 2012). The plant hormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are key players in the signaling networks involved in plant resistance (Bari and Jones, 2009; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate array of defense responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). Whereas SA is considered essential for resistance to (hemi)biotrophic pathogens, it is assumed that JA and ET signaling pathways are important for resistance to necrotrophic pathogens in Arabidopsis (Arabidopsis thaliana; Thomma et al., 2001; Glazebrook, 2005). A successful innate immune response often includes the so-called hypersensitive response (HR), a form of rapid programmed cell death (PCD) occurring in a limited area at the site of infection. This suicide of infected cells is thought to limit the spread of biotrophic pathogens, including viruses, bacteria, fungi, and oomycetes (Mur et al., 2008).

During the past decade, significant progress has been made in our understanding of the cellular function of plant sphingolipids. Besides being structural components of cell membranes, sphingolipids are bioactive metabolites that regulate important cellular processes such as cell survival and PCD, occurring during either plant development or plant defense (Dunn et al., 2004; Berkey et al., 2012; Markham et al., 2013). The first evidence of the role of sphingolipids in these processes came from the use of the fungal toxins fumonisin B1 (FB1) and AAL, produced by the necrotrophic agent Alternaria alternata f. sp. lycopersici. These toxins are structural sphingosine (d18:1) analogs and function as ceramide synthase inhibitors. They triggered PCD when exogenously applied to plants. Mutant strains in which the production of such toxins is abrogated failed to infect the host plant, implying that toxin accumulation is required for pathogenicity and that the induction of plant PCD could be considered a virulence tool used by necrotrophic pathogens (Berkey et al., 2012). Moreover, several studies revealed that ceramides (Cers) and long-chain bases (LCBs) are also potent inducers of PCD in plants. For example, exogenously applied Cers and LCBs (d18:0, d18:1, or t18:0) induced PCD either in cell suspension 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-García et al., 2011). AAL- and FB1-induced PCD seemed to be due to the accumulation of free sphingoid bases (dihydrosphingosine [d18:0] and phytosphingosine [t18:0]; Abbas et al., 1994; Brandwagt et al., 2000; Shi et al., 2007). Spontaneous cell death in lag one homolog1 or 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 in the induction of HR and associated PCD also came from studies on accelerated cell death5 (acd5) and enhancing resistance to powdery mildew8 (RPW8)-mediated hypersensitive response (erh1) mutants, which displayed overaccumulation of Cers. These mutants exhibited spontaneous cell death and resistance to biotrophic pathogens, which seemed to be linked with SA and PR protein accumulation (Liang et al., 2003; Wang et al., 2008).

Altogether, these data provide evidence of a link between PCD, defense, and sphingolipid metabolism. However, the fatty acid hydroxylase1/2 (atfah1/atfah2) double mutant that accumulates SA and Cers was more tolerant to the obligate biotrophic fungus Golovinomyces cichoracearum but did not display a PCD-like phenotype, suggesting that Cers alone are not involved in the induction of PCD (König et al., 2012). Moreover, Saucedo-García et al. (2011) postulated that dihydroxy-LCBs, but not trihydroxy-LCBs, might be primary mediators for LCB-induced PCD. The sphingoid base hydroxylase sbh1/sbh2 double mutant completely lacking trihydroxy-LCBs showed enhanced expression of PCD marker genes (Chen et al., 2008). On the contrary, increase in t18:0 was specifically sustained in plant interaction with the avirulent Pseudomonas syringae pv tomato (Pst) strain and correlated with a strong PCD induction in leaves (Peer et al., 2010). Thus, the nature of sphingolipids able to induce PCD is still under debate and may evolve depending on plants and their environment. The phosphorylated form of LCBs (LCB-Ps) could abrogate PCD 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-Ps by using specific inhibitors induced PCD in cell suspension culture (Alden et al., 2011). Recently, overexpression of rice (Oryza sativa) LCB kinase in transgenic tobacco (Nicotiana tabacum) plants reduced PCD after treatment with FB1 (Zhang et al., 2013). Genetic mutation on LCB-P lyase encoded by the AtDPL1 gene, modifying the LCB-LCB-P ratio, could impact PCD levels after treatment with FB1 (Tsegaye et al., 2007). Altogether, these data point to the existence of a rheostat between LCBs and their phosphorylated forms that controls plant cell fate toward cell death or survival.

Data on plant sphingolipid functions are still fragmentary. Only a few reports have described interconnections between sphingolipids, cell death, and plant defense responses, almost exclusively in response to (hemi)biotrophic pathogens. Knowledge about such relations in response to necrotrophic pathogens is still in its infancy (Rivas-San Vicente et al., 2013; Bi et al., 2014). In this report, the link between sphingolipids, cell death, and plant defense has been explored in response to Botrytis cinerea infection and in comparison with Pst infection. For this purpose, Atdpl1 mutant plants, disturbed in LCB/LCB-P accumulation without displaying any phenotype under standard growth conditions (Tsegaye et al., 2007), have been analyzed after pathogen infection. Our results revealed that modification of sphingolipid contents not only impacted plant tolerance to hemibiotrophs but also greatly affected resistance to necrotrophs. Whereas the SA signaling pathway is globally repressed in Atdpl1-1 compared with wild-type plants, the JA signaling pathway is significantly enhanced. Cell death and ROS accumulation are markedly modified in Atdpl1-1 mutant plants. 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 thus established a link between JA signaling, PCD, and sphingolipid metabolism.

RESULTS

Necrotrophic and Hemibiotrophic Infection Differently Affect the Atdpl1 Mutant Plant Response

In order to assess the role of sphingolipids in plant immune responses to necrotrophic and hemibiotrophic pathogens, we used the Atdpl1 mutant, which is affected in the LCB/LCB-P rheostat by accumulating t18:1-P (Tsegaye et al., 2007). Whereas Atdpl1 shows no developmental phenotype compared with wild-type plants under standard conditions, it exhibits a higher sensitivity to FB1 (Tsegaye et al., 2007). B. cinerea and Pst have been widely used to decipher defense mechanisms to necrotrophic and hemibiotrophic pathogens in Arabidopsis (Glazebrook, 2005). To get some information about the susceptibility of the Atdpl1 mutant to B. cinerea or Pst (either virulent [Pst DC3000] or avirulent [Pst AvrRPM1] strain), three independent Atdpl1 mutant lines were challenged with these pathogens. The three Atdpl1 mutant lines displayed similar responses upon pathogen challenge (Fig. 1). In B. cinerea-infected wild-type plants, disease symptoms, showing chlorosis and necrosis, increased more rapidly than in B. cinerea-infected Atdpl1 plants (Fig. 1A). On the contrary, symptoms developed in response to Pst infection were more pronounced in mutant plants than in wild-type plants (Fig. 1A). The lesion diameters were scored 48 and 60 h after drop inoculation with B. cinerea and classified into size categories (Fig. 1B). Interestingly, Atdpl1 plants did not display necrotic lesions of the largest size, whereas wild-type plants showed 10% of these lesions 48 h post inoculation (hpi). Only 2% of the largest lesions were observed in Atdpl1 plants compared with 12% for wild-type plants 60 hpi. Furthermore, Atdpl1 mutants displayed a greater percentage of small necrotic lesions than wild-type plants. Atdpl1 lines displayed approximately 45% and 65% of small lesions, whereas wild-type plants showed only 17% and 24% of small lesions 48 and 60 hpi, respectively. Consequently, fewer lesions of medium size were observed in Atdpl1 lines than in wild-type plants (Fig. 1B).

Figure 1.
Figure 1.

Atdpl1 mutants are more tolerant to B. cinerea but more susceptible to Pst than the wild type. B. cinerea conidia suspension was deposited by using drop inoculation (A and B) or spray inoculation (E) on leaves of wild-type (WT) and Atdpl1 mutant plants. Pst solution was infiltrated into wild-type and Atdpl1 mutant leaves (A, C, and D). A, Photographs represent disease symptoms observed 60 or 72 h after infection by the fungus or Pst, respectively. B, Symptoms due to B. cinerea infection were scored by defining three lesion diameter (d; in mm) classes. Statistical differences of the mean lesion diameters between wild-type and Atdpl1 plants were calculated with a Kruskal-Wallis test: **, P < 0.01; and ***, P < 0.005. C and D, Bacterial growth of virulent Pst strain DC3000 (C) and avirulent Pst strain AvrRPM1 (D) at 0, 6, 24, 48, and 54 hpi. E, B. cinerea and Pst growth was quantified by qRT-PCR 3 and 48 h after pathogen infection in leaves of wild-type and Atdpl1 mutant plants. Asterisks indicate significant differences between wild-type and Atdpl1 samples according to Student’s t test: ***, P < 0.005. Results are representative of three independent experiments.

The average lesion diameter in the Atdpl1 mutant was significantly lower than that in wild-type plants (**, P < 0.01 and ***, P < 0.005; Fig. 1B). Plants were also infiltrated with Pst DC3000 or Pst AvrRPM1 at 107 colony-forming units (cfu) mL−1, and bacterial populations were evaluated 0, 6, 24, 30, 48, and 54 hpi. As already described, avirulent strain growth was less important compared with the virulent strain in wild-type plants (Fig. 1, C and D). Interestingly, infection with both bacterial strains revealed an increased susceptibility of Atdpl1 plants, allowing about 10-fold more bacterial growth as compared with wild-type plants (Fig. 1, C and D). These results were also correlated by fungal and bacterial population quantification in infected leaves by quantitative reverse transcription (qRT)-PCR (Fig. 1E). Interestingly, the AtDPL1 expression profile was similar after either B. cinerea or Pst infection (Supplemental Fig. S1). Until 12 hpi, no AtDPL1 transcript accumulation could be observed. AtDPL1 expression increased significantly between 12 and 24 hpi and rose continuously until the later stages of infection. Symptoms due to either B. cinerea invasion or infection with the virulent or avirulent strain of Pst visually appeared between 24 and 30 hpi (data not shown) and thus are delayed slightly compared with AtDPL1 expression. Deregulation of photosynthesis is considered a tool for evaluating the first sign of pathogen infection (Berger et al., 2007; Bolton, 2009). Repression of the RbcS gene (encoding the small subunit of ribulose-1,5-bisphosphate carboxylase) after pathogen infection occurred at the same time (B. cinerea) or slightly earlier (Pst) compared with AtDPL1 expression and symptom appearance (Supplemental Fig. S1), suggesting that an immediate consequence of pathogen perception includes the induction of AtDPL1 gene expression. Collectively, these data indicate that lack of AtDPL1 activity in mutant plants significantly delays the development of lesions triggered by B. cinerea infection but renders plants more susceptible to Pst infection.

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

To determine whether changes in the levels of certain sphingolipids are responsible for the delayed development of B. cinerea infection in the Atdpl1 mutant, sphingolipid profiles were analyzed. The main sphingolipid species in Arabidopsis, LCBs and LCB-Ps (Fig. 2), glycosylinositol phosphoceramides (GIPCs; Fig. 3), Cers (Fig. 4), hydroxyceramides (hCers; Fig. 5), and glucosylceramides (GlcCers; Supplemental Fig. S2), were first quantified in both the wild type and the Atdpl1-1 mutant at 0 hpi (Supplemental Fig. S3). In wild-type and Atdpl1-1 plants, LCB/LCB-P basal levels were almost in the same range as those already described (Tsegaye et al., 2007; Supplemental Fig. S3). As described previously, the only significant alteration in sphingolipid basal levels observed in the Atdpl1-1 mutant compared with the wild type under typical growth conditions was an increase in one 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 infection on the sphingolipid profile in wild-type plants. B. cinerea infection triggered LCB accumulation (from 6× for 4,8-sphingadienine [d18:2] to 20× for d18:0; Fig. 2A) but also a moderate increase in sphingosine-1-phosphate (d18:1-P) and t18:1-P amount (4× and 2.5×, respectively) compared with mock-inoculated wild-type plants (Fig. 2E). The amount of total GIPCs and, more precisely, saturated α-hydroxylated very-long-chain fatty acid (VLCFA)-containing GIPCs (C24 and C26; Fig. 3, A and C) was significantly lower after B. cinerea infection than in mock-treated plants (200 and 300 nmol g−1 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 and C). The amount of total Cers is 4 times higher in B. cinerea- than in mock-inoculated wild-type plants (84 versus 21 nmol g−1 dry weight; Supplemental Fig. S4). Most Cer molecules were affected by the presence of B. cinerea (Fig. 4, A and C). Finally, the level of total hCers was not modified (Supplemental Fig. S4); however, significant accumulation of saturated α-hydroxylated C16-, C18-, and C26-containing hCers and d18:0-hCer was observed after challenge with B. cinerea (Fig. 5, A and C). No change could be noticed in GlcCer levels (Supplemental Figs. S2 and S4).

Figure 2.
Figure 2.

Free LCB and LCB-P accumulation after challenge with pathogen. Leaves of wild-type (WT) or Atdpl1-1 mutant plants were sprayed with B. cinerea spore suspension (Bc) or potato dextrose broth (PDB; Control; A, B, E, and F) or infiltrated with Pst DC3000, Pst AvrRPM1, or MgCl2 (Control; C, D, G, and H). Quantifications of LCBs (A–D) and LCB-Ps (E–H) were performed 48 hpi. Asterisks on wild-type bars indicate significant differences between the pathogen-treated wild-type sample and the control sample, and asterisks 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. Results are means of four to five independent biological experiments ± sd. Notice the different scale of LCB-P levels between wild-type and Atdpl1-1 plants. DW, Dry weight.

Figure 3.
Figure 3.

GIPC contents after B. cinerea or Pst 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) or infiltrated with MgCl2 (Control; E and F), Pst DC3000 (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 wild-type sample and the control sample, and asterisks on Atdpl1-1 bars indicate significant differences in total species 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 been considered for the total species displaying the same fatty acid or hydroxylation/unsaturation degree. Results are means of four to five independent biological experiments ± sd. DW, Dry weight.

Figure 4.
Figure 4.

Cer species produced by wild-type and Atdpl1-1 mutant 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) or infiltrated with MgCl2 (Control; E and F), Pst DC3000 (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 wild-type sample and the control sample, and asterisks 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 been considered for the total species displaying the same fatty acid or hydroxylation/unsaturation degree. Results are means of four to five independent biological experiments ± sd. DW, Dry weight.

Figure 5.
Figure 5.

hCer species produced by wild-type and Atdpl1-1 mutant 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) or infiltrated with MgCl2 (Control; E and F), Pst DC3000 (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, and asterisks 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 been considered for the total species displaying the same fatty acid or hydroxylation/unsaturation degree. Results are means of four to five independent biological experiments ± sd. DW, Dry weight.

To better understand the role of sphingolipids in plant resistance to the necrotrophic fungus, a comparison between sphingolipid profiles in B. cinerea-infected Atdpl1-1 mutant and wild-type plants was then performed. 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 with wild-type plants (Fig. 2, E and F). The amount of total GIPCs and, more precisely, saturated α-hydroxylated VLCFA-containing GIPCs (C22, C24, and C26; Fig. 3, C and D) was significantly higher in Atdpl1-1 mutant than in wild-type plants after B. cinerea infection (370 versus 220 nmol g−1 dry weight, respectively; Supplemental Fig. S4). Total Cer amount was similar in both types of plants (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 mutant compared with wild-type plants (75 versus 27 nmol g−1 dry weight, respectively; Supplemental Fig. S4). Moreover, trihydroxy-hCers also accumulated three times in the mutant compared with wild-type plants in response to the fungus (Fig. 5, C and D). No significant change was observed in total GlcCer amount (Supplemental Figs. S2 and S4).

In order to compare sphingolipid profiles in response to a hemibiotrophic pathogen, analyses were performed 48 h after wild-type plant inoculation with avirulent or virulent strains of Pst. Our data confirmed previous results showing that sphingolipid increase was more sustained during the incompatible than the compatible interaction (Peer et al., 2010). Increase in t18:0 was observed in response to both types of bacteria, but infection with only Pst AvrRPM1 triggered a significant decrease of d18:1 (Fig. 2C). After infection with Pst AvrRPM1, an increase in d18:2-P, t18:0-P, and t18:1-P was observed, whereas only t18:0-P level was increased in response to Pst DC3000 (Fig. 2G). GIPC levels were also not significantly modified in response to both types of bacteria (Fig. 3, E, G, and I; Supplemental Fig. S4). Total contents of d18:0-, d18:1-, t18:0-, and t18:1-Cers were increased after infection with Pst AvrRPM1 (Fig. 4, E and I). Only an increase in trihydroxy-Cers could be noticed in response to Pst DC3000 (Fig. 4, E and G). Moreover, the t18:0-Cer level was higher in the case of the incompatible interaction than in the case of the compatible one (40 versus 24 nmol g−1 dry weight, respectively; Supplemental Fig. S4). C16-, C24-, and C26-Cers also accumulated in response to both strains of Pst (Fig. 4, E, G, and I), and only C16-Cer accumulation was more pronounced in the case of interaction with Pst AvrRPM1 compared with Pst DC3000 (45 versus 18 nmol g−1 dry weight, respectively; Fig. 4, E, G, and I). Total contents of d18:0-hCers were increased in response to Pst (Fig. 5, E, G, and I). t18:0-hCers accumulated after challenge with the virulent strain and t18:1-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 sphingolipid profiles between Pst-infected wild-type and Atdpl1-1 mutant plants revealed an increase in d18:0 (1.5×) in Atdpl1-1 plants certainly due to infiltration, since it was also observed in control plants. An increase in t18:0-P level (5×) was detected in Atdpl1-1 mutant plants compared with the wild type only in response to the avirulent strain (Fig. 2H). No significant regulation of GIPC, Cer, hCer, or GlcCer pools was observed in response to either the virulent or avirulent strain (Figs. 3–5; Supplemental Fig. S2).

Changes in Sphingolipid Profiles Affect Pathogen-Induced Cell Death

Recently, several reports have revealed that some sphingolipids are important players in HR and associated PCD (Berkey et al., 2012; Markham et al., 2013). HR is an effective strategy of plants to protect themselves against (hemi)biotrophic microorganisms (Coll et al., 2011). In contrast, PCD processes promote the spread of necrotrophic pathogens such as B. cinerea (Govrin and Levine, 2000; Govrin et al., 2006). Thus, changes in sphingolipid profiles and differences in tolerance upon B. cinerea or Pst infection prompted us to examine the cell death response upon pathogen attack. We thus measured electrolyte leakage to detect changes in loss of ions caused by plasma membrane damage characteristic of plant cell death (Dellagi et al., 1998; Kawasaki et al., 2005). Ion leakage measured after the inoculation of Atdpl1-1 plants with B. cinerea or Pst was reduced compared with wild-type plants (Fig. 6). These results suggested that modification in sphingolipid content could play a role in modulating cell death processes in response to pathogen infection.

Figure 6.
Figure 6.

Electrolyte leakage in Atdpl1-1 mutants after pathogen inoculation. Conductivity (μS cm−1) is shown for solution containing leaf discs from either the wild type (WT) or the Atdpl1-1 mutant inoculated with B. cinerea (Bc) or PDB (Control) solution (A) or Pst DC3000, Pst AvrRPM1, or 10 mm MgCl2 (B). Each value represents the mean ± sd of three replicates per experiment. The experiment was repeated three times with similar results.

Expression levels of PCD marker genes, such as the flavin-containing monooxygenase FMO and the senescence-associated genes SAG12 and SAG13 (Brodersen et al., 2002), were also evaluated in order to verify if cell death responses are modified in Atdpl1-1 mutant plants (Fig. 7). FMO and SAG13 were induced in both types of plants with increasing infection spread of B. cinerea. Interestingly, these inductions occurred earlier and stronger in the wild type (between 12 and 24 hpi) than in the Atdpl1-1 mutant (between 24 and 30 hpi; Fig. 7, A and C). SAG12 was only induced 48 hpi in both the wild type and the Atdpl1-1 mutant, and similar to SAG13 and FMO, its expression was stronger in the wild type (10,000×) than in the Atdpl1-1 mutant (2,000×; Fig. 7E).

Figure 7.
Figure 7.

Time course of PCD marker gene expression after B. cinerea or Pst infection. Leaves were sprayed with B. cinerea spore suspension (Bc) or PDB (Control; A, C, and E) or infiltrated with a bacterial solution (Pst DC3000 or Pst AvrRPM1) or MgCl2 (Control; B, D, and F). The mean values ± sd from one representative experiment are shown. qRT-PCR of FMO (A and B), SAG13 (C and D), and SAG12 (E and F) expression was performed in wild type (WT) and Atdpl1-1 mutant plants with five biological replicates with comparable results.

As expected in the case of Pst infection, SAG13 and FMO gene expression were induced earlier and stronger during the incompatible interaction than during the compatible interaction (Fig. 7, B and D). Wild-type and mutant plants displayed similar expression profiles with both types of bacteria; however, induction was less pronounced in Atdpl1-1 mutant plants. Similar to B. cinerea infection, SAG12 transcript accumulation occurred only at the later stages of infection (Fig. 7F). It is noteworthy that induction of these PCD marker genes followed a pattern similar to AtDPL1 gene expression in wild-type plants in response to either B. cinerea or Pst infection (Supplemental Fig. S1, A and B).

SAG12 is only expressed in senescent tissues. In contrast, SAG13 and FMO are expressed in different PCD processes (Lohman et al., 1994; Brodersen et al., 2002). Collectively, our data suggest that the induction of SAG13 and FMO after either B. cinerea or Pst infection could result from an HR-like PCD, whereas a senescence program is activated later. This could also explain the tolerance of Atdpl1 mutant plants toward B. cinerea and their higher susceptibility toward Pst.

Modification of Sphingolipid Contents Affect ROS Production in Response to Pathogen Infection

Transient production of ROS is a hallmark of successful pathogen recognition (Torres, 2010). To investigate whether sphingolipid content perturbation in Atdpl1-1 plants affected pathogen recognition, we compared ROS production in the mutant versus wild-type plants. Wild-type plants displayed a transient oxidative burst, peaking around 300 min (B. cinerea) or 40 min (Pst) after inoculation (Fig. 8). This transient burst was significantly induced by 2.5 times in B. cinerea-infected Atdpl1-1 plants compared with wild-type plants (Fig. 8A). On the contrary, ROS levels were significantly reduced in Pst-infected Atdpl1-1 plants compared with Pst-infected wild-type plants (Fig. 8, B and C). Our results thus demonstrated that signaling events linked to pathogen recognition are affected by sphingolipid perturbation in Atdpl1-1 plants.

Figure 8.
Figure 8.

Transient ROS production in response to pathogen infection in wild-type and Atdpl1-1 mutant plants. The time course of ROS production in wild type (WT) and Atdpl1-1 mutant plants is shown in response to B. cinerea (Bc; A), Pst DC3000 (B), or Pst AvrRPM1 (C) infection. Leaf discs were immersed in a solution containing either 105 spores mL−1 B. cinerea or 108 cfu mL−1 Pst. Error bars represent se from 12 biological repetitions. Three independent experiments were performed with similar results. RLUs, Relative light units.

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

Major changes in LCB-P contents in B. cinerea-inoculated Atdpl1-1 mutant plants are an increase in t18:0-P levels and a decrease in d18:0 amounts (Fig. 2). We thus tested the ability of these sphingolipids to modulate pathogen-induced cell death (Fig. 9) and ROS production (Fig. 10). Our data showed that exogenous t18:0-P or d18:0 alone did not affect cell death or ROS production, a finding consistent with data obtained by Coursol et al. (2015). In t18:0-P-treated wild-type plants, symptoms and ion leakage triggered by B. cinerea or Pst infection were reduced significantly (Fig. 9, A, C, and E). Exogenously applied d18:0 did not modify disease symptoms and electrolyte leakage in wild type-infected plants by B. cinerea and slightly reduced electrolyte leakage triggered by the virulent Pst strain (Fig. 9, B, D, and F). Interestingly, disease symptoms and electrolyte leakage were strongly reduced when wild-type plants were coinfiltrated with d18:0 and Pst AvrRPM1 (Fig. 9, B and F).

Figure 9.
Figure 9.

Exogenous effects of t18:0-P and d18:0 on electrolyte leakage in response to pathogen infection in wild-type plants. A and B, B. cinerea conidia suspension was deposited on leaves of wild-type and Atdpl1-1 mutant plants 15 min after infiltration of either 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 (μS cm−1) 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 represents the mean ± sd of three replicates per experiment. The experiment was repeated three times with similar results.

Figure 10.
Figure 10.

Exogenous effects of t18:0-P and d18:0 on ROS production in response to pathogen infection in wild-type plants. The time course of ROS production in t18:0-1-P- or d18:0-treated wild-type plants is shown in response to B. cinerea (Bc; A and D), Pst DC3000 (B and E), or Pst AvrRPM1 (C and F) infection. Leaf discs were immersed in a solution containing 100 µm t18:0-1-P or d18:0 and either 105 spores mL−1 B. cinerea or 108 cfu mL−1 Pst. Error bars represent se from 12 biological repetitions. Three independent experiments were performed with similar results. RLUs, Relative light units.

Whereas the addition of t18:0-P increased and delayed ROS production upon challenge with B. cinerea, it reduced the Pst-induced oxidative burst (Fig. 10, A–C). d18:0 had no significant effect on ROS accumulation triggered by B. cinerea (Fig. 10D). However, it dramatically reduced the Pst-induced oxidative burst (Fig. 10, E and F). These data indicate that exogenously applied t18:0-P and d18:0 modify signaling events and cell death triggered by infection with these two pathogens.

SA and ET/JA Signaling Pathways Are Modified in Atdpl1-1 Mutant Plants after Pathogen Challenge

Disruption of sphingolipid contents between wild-type and Atdpl1 plants could result in the differential activation of defense responses after pathogen infection. PR1 and PR5 are well-known SA-dependent defense marker genes, NONEXPRESSED PATHOGEN RELATED1 (NPR1) was shown to be a key regulator of SA-mediated suppression of JA signaling (Spoel et al., 2003), and DEFENSIN (PDF1.2), CHITINASE (CHIT), and ETHYLENE RESPONSE FACTOR1 (ERF1) expression is regulated by JA and ET whereas VEGETATIVE STORAGE PROTEIN1 (VSP1) and JASMONATE ZIM-DOMAIN8 (JAZ8) are mostly responsive to JA (Glazebrook, 2005; Pieterse et al., 2009). First, the expression pattern of these defense genes was monitored in wild-type and Atdpl1-1 mutant plants. No significant difference in the expression of these defense genes was detected in wild-type and Atdpl1-1 mutant plants grown under standard conditions (Figs. 11 and 12). These results indicated that inactivation of the gene encoding LCB-P lyase itself did not result in any defense response changes in plants. The expression levels of defense-related genes in Atdpl1-1 mutant plants were then compared with wild-type plants in response to B. cinerea infection (Fig. 11). Whereas PR1, PR5, NPR1, and VSP1 expression showed similar induction levels in both genotypes, the expression of PDF1.2, CHIT, ERF1, and JAZ8 was markedly enhanced in Atdpl1-1 mutant compared with wild-type plants. At 48 hpi, there was a 12-fold increase for PDF1.2 and a 2-fold increase for CHIT, ERF1, and JAZ8 compared with wild-type plants (Fig. 11). Since JA-responsive genes were up-regulated in the Atdpl1-1 mutant, the expression of three genes encoding key enzymes in JA biosynthesis, LIPOXYGENASE2 (LOX2), ALLENE OXIDE CYCLASE2 (AOC2), and 12-OXOPHYTODIENOIC ACID REDUCTASE3 (OPR3) as well as JAR1, encoding the enzyme that converts JA to the jasmonoyl-isoleucine (JA-Ile) conjugate (Staswick and Tiryaki, 2004), was also followed. The results showed that LOX2 and AOC2 were significantly up-regulated up to 24 hpi in the Atdpl1-1 mutant but transcripts returned to a level comparable to the wild type thereafter (Fig. 11). In contrast, the expression of OPR3 was similar in both genotypes. JAR1 expression was not affected by the fungus inoculation (Fig. 11). These results indicated that both JA synthesis and signaling pathways were enhanced in Atdpl1-1 mutant plants.

Figure 11.
Figure 11.

Expression levels of JA and SA pathway-associated genes in wild-type (WT) and Atdpl1-1 mutant plants during B. cinerea (Bc) infection. Results are expressed as the fold increase in transcript level compared with the untreated control (0 h), referred to as the 1× expression level. Values shown are means ± sd of duplicate data from one representative experiment among five independent repetitions.

Figure 12.
Figure 12.

Expression levels of JA and SA pathway-associated genes in wild-type (WT) and Atdpl1-1 mutant plants during Pst infection. Results are expressed as the fold increase in transcript level compared with the untreated control (0 h), referred to as the 1× expression level. Values shown are means ± sd of duplicate data from one representative experiment among five independent repetitions.

When infected with Pst, wild-type plants displayed a strong 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 was observed 30 hpi in the Atdpl1-1 mutant compared with wild-type plants (6× for Pst DC3000 and 4× for Pst AvrRPM1), but the level of PR1 expression was still higher in incompatible compared with compatible interactions. Accumulation of PR5 transcripts was also slightly more important in wild-type plants, but expression levels were more important in the case of the compatible interaction (Fig. 12). Under Pst attack, NPR1 was slightly induced, but no difference between the wild type and the Atdpl1-1 mutant was observed. CHIT expression was also more induced in response to Pst AvrRPM1 (90×) than Pst DC3000 (20×) in wild-type plants, and this induction profile was similar in Atdpl1-1 mutant plants (Fig. 12). As already described, inoculation with the bacterial pathogen (Pst DC3000 or Pst AvrRPM1) led to a dramatic repression of PDF1.2 expression, either in wild-type or in Atdpl1-1 mutant plants. In contrast, ERF1 and JAZ8 were induced during Pst infection, and VSP1 expression was slightly induced when challenged by Pst DC3000 but repressed after Pst AvrRPM1 infection (Fig. 12). Expression of these three genes was markedly enhanced in the Atdpl1-1 mutant compared with wild-type plants. At the end of the time course, VSP1, ERF1, and JAZ8 mRNA levels were 2-, 3-, and 6-fold higher in Atdpl1-1 than in wild-type plants after infection with either virulent or avirulent strains, respectively. Similar to B. cinerea infection, JAR1 expression was not affected by inoculation with Pst. Regarding genes involved in the JA biosynthetic pathway, LOX2 was repressed, AOC2 was not induced during Pst challenge, and OPR3 was induced slightly, but no difference between the two genotypes was observed (Fig. 12). These data suggested that only the JA signaling pathway is positively affected in mutant plants upon challenge with Pst.

To get further information on Atdpl1-1 mutant defense responses, some defense-related phytohormones were also quantified (Fig. 13). No change in phytohormone basal levels was observed between wild-type and Atdpl1-1 mutant plants (Fig. 13). This implied that Atdpl1-1 mutant plants, in contrast to other mutants with modified sphingolipid contents, do not display high constitutive SA amounts (Greenberg et al., 2000; Wang et al., 2008; Ternes et al., 2011; König et al., 2012). Following pathogen attack, all phytohormone levels were enhanced. SA accumulation was essentially unchanged in the mutant compared with wild-type plants, whatever the pathogen considered. Interestingly, levels of JA and its biologically active conjugate, JA-Ile, were 2 to 3 times higher in the Atdpl1-1 mutant compared with wild-type plants after B. cinerea or Pst infection, respectively. However, no difference in JA levels between virulent and avirulent interaction was noticed, but JA-Ile accumulation was slightly higher in the case of the avirulent interaction in Atdpl1-1 plants. Together, our data suggest that the JA-dependent signaling pathway is preferentially activated in the Atdpl1-1 mutant in response to pathogen infection.

Figure 13.
Figure 13.

Analysis of phytohormone accumulation in stressed wild-type and Atdpl1-1 mutant plants. JA, JA-Ile, and SA accumulation is shown in wild-type (WT) and Atdpl1-1 mutant plants 0 or 30 h following B. cinerea (A) or Pst DC3000 or Pst AvrRPM1 (B) infection. Asterisks indicate significant differences between wild-type and Atdpl1-1 samples according to Student’s t test: *, P < 0.05; **, P < 0.01; and ***, P < 0.005. Values shown are means ± sd from one representative experiment among five independent repetitions. FW, Fresh weight.

DISCUSSION

Only a few articles have described a connection between sphingolipid content, PCD, and defense reactions during biotic stress (Berkey et al., 2012). Furthermore, most of them focused on responses against (hemi)biotrophic pathogens, the role of sphingolipid in plant defense against necrotrophs being largely unsolved (Rivas-San Vicente et al., 2013; Bi et al., 2014). Moreover, nearly all studies revealed basal sphingolipid levels, and data of sphingolipid contents during pathogen infection were often not available (Peer et al., 2010; Bi et al., 2014). This work describes a comparison of sphingolipid content during hemibiotrophic and necrotrophic infection. In this study, we investigated the consequences of the disruption of the sphingolipid profiles on plant immunity responses such as cell death, ROS production, and signaling of plant defense responses during pathogen infection.

Interplays between Sphingolipids and PCD

As in animal systems, new emerging evidence showed that bioactive sphingolipids play a critical role as modulators of plant PCD (Berkey et al., 2012; Saucedo-García et al., 2015). Here, sphingolipid content analyses showed that infection by B. cinerea or Pst triggered the accumulation of some species known to act in favor of cell survival (LCB-Ps and hCers) or cell death (LCBs and Cers). Interestingly, the Atdpl1-1 mutant displayed higher levels of d18:0 in response to infiltration (Fig. 2). Moreover, this LCB reduced Pst-induced cell death and symptoms, especially in the case of the incompatible interaction (Fig. 9). Since the HR often contributes to resistance to (hemi)biotrophic pathogens, our results suggested that a modification in d18:0 levels could impact plant cell death and, thus, resistance to such pathogens. Recently, Coursol et al. (2015) showed that the addition of d18:0 had no significant effect on the viability of cryptogein-treated cells, indicating that distinct mechanisms of regulation are involved in cell death of cell cultures or plant tissue or after treatment by an elicitor or a pathogen. Necrotrophs are pathogens that derive nutrients from dead or dying cells. PCD, including 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; Govrin et al., 2006). Plants that are less potent to activate HR or with reduced cell death present enhanced tolerance to B. cinerea infection and vice versa (Govrin and Levine, 2000; van Baarlen et al., 2007). Similarly, antiapoptotic genes conferred resistance to necrotrophic fungi in transgenic plants (Dickman et al., 2001; El Oirdi and Bouarab, 2007). A general pattern established that infection of Arabidopsis by B. cinerea is promoted by and requires an active cell death program in the host (van Kan, 2006), and resistance against this fungus depends on the balance between cell death and survival (van Baarlen et al., 2007).

Interestingly, the Cer-accumulating acd5 mutant or Cer-infiltrated plants were more susceptible to several Botrytis spp. (van Baarlen et al., 2004, 2007). Moreover, myriocin, a potent inhibitor of SERINE PALMITOYLTRANSFERASE (SPT), the first enzyme of sphingolipid biosynthesis, had a death-antagonistic effect during the Botrytis elliptica-Lilium interaction (van Baarlen et al., 2004). This suggests that sphingolipid metabolism is involved in cell death triggered by Botrytis spp. Cell death activation could thus be disturbed in Atdpl1 plants, leading to a higher susceptibility toward (hemi)biotrophs and higher tolerance toward necrotrophs. In this work, B. cinerea infection triggered Cer and LCB accumulation in wild-type plants. It is thus possible that the necrotrophic fungus promoted plant PCD-inducing factors (e.g. sphingolipids) in order to facilitate its penetration and spread inside plant cells. However, exogenous d18:0 did not modify ion leakage in the presence of B. cinerea, suggesting that this LCB alone is not involved in such a mechanism. Sphingolipid analysis revealed that B. cinerea-infected Atdpl1-1 plants accumulated more VLCFA-hCers and t18:0-P and t18:1-P but fewer Cers and LCBs compared with wild-type plants. Interestingly, our data showed that exogenous t18:0-P reduced B. cinerea- and Pst-induced cell death (Fig. 9). Thus, t18:0-P appears to be essential to modulate plant cell death and, thus, plant resistance in response to pathogen infection. Moreover, it was recently demonstrated that AtFAH1 or 2-hydroxy VLCFAs, and thereby VLCFA-hCers, were key factors in BAX INHIBITOR1-mediated cell death suppression (Nagano et al., 2012). These results confirmed that sphingolipids play an important role in plant defense responses and that the plant is able to adjust its response by regulating a dynamic balance between cell death (e.g. HR)- or cell survival-related sphingolipids. However, in contrast to infected Atdpl1 mutant, the fah1/fah2 double mutant presented a reduced amount of hCers and elevated levels of Cers and LCBs but showed no lesion phenotype (König et al., 2012). Thus, it seems that the connection between sphingolipids and PCD is regulated by a fine-tuned process and, thus, could be more complex than expected. Other parameters, such as defense signaling pathways, could be involved in such a mechanism.

Interconnections between Sphingolipids and Defense Mechanisms

Sphingolipids (e.g. LCBs and Cers) participate in the induction and/or control of plant cell death. Moreover, plant cell death processes, such as HR, are also associated with plant defense or disease. It is thus conceivable that some sphingolipids play key roles in plant innate immunity. Recent studies brought to light interconnections between sphingolipids and defense mechanisms. Resistance to biotrophic pathogens often required ROS production (Torres et al., 2002). Consistent with this, Pst-infected Atdpl1-1 mutant displayed a reduced accumulation of ROS and was more sensitive to the bacterial attack. In addition, Atdpl1-1 mutant accumulated more d18:0 in response to infiltration (Fig. 2), and d18:0 strongly reduced ROS production upon challenge with this bacterium (Fig. 10). B. cinerea-infected Atdpl1-1 plants displayed a higher production of ROS (Fig. 8). Several studies demonstrated that resistance against B. cinerea (and other necrotrophs) is accompanied by the generation of ROS, and mutants impaired in ROS production failed to resist the necrotrophic pathogen (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 but not LCB-Ps alone are able to induce ROS production (Peer et al., 2011). In this study, exogenously applied t18:0-P increased B. cinerea-induced ROS generation (Fig. 10). Accordingly, cryptogein-induced ROS accumulation is enhanced by a pretreatment with some LCB-Ps, especially t18:0-P (Coursol et al., 2015). This suggests that sphingolipids may interact differently with ROS production depending on the presence or not of an elicitor or pathogen. Interestingly, the similarity of ROS accumulation upon infection between Atdpl1-1 plants and t18:0-P- or d18:0-treated wild-type plants indicated that t18:0-P and d18:0 could have key roles in pathogen perception and, thus, in plant resistance toward hemibiotrophic and necrotrophic pathogens.

Several lines of evidence showed that plants disrupted in sphingolipid metabolism often displayed a spontaneous enhanced SA pathway (Greenberg et al., 2000; Brodersen et al., 2002; Wang et al., 2008; Ternes et 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 benzothiadiazole affect sphingolipid metabolism (Shi et al., 2015), including AtDPL1 gene expression (Wang et al., 2006). Since activation of the SA-dependent pathway is effective against biotrophic and hemibiotrophic pathogens, it has been postulated that sphingolipids played a key role in defense against such pathogens in an SA-dependent pathway (Sánchez-Rangel et al., 2015). However, whereas acd5, erh1, and the double mutant fah1/fah2 exhibited enhanced resistance to powdery mildew, they displayed a similar phenotype to wild-type plants upon infection with P. syringae pv maculicola or Verticillium longisporum (Wang et al., 2008; König et al., 2012). This suggests that SA, sphingolipid-triggered cell death, and plant resistance could be independent regarding the plant/pathogen pair. Unfortunately, only basal levels of sphingolipid were described, and no sphingolipid quantification during pathogen infection is available, making difficult a direct link between sphingolipid metabolism and plant defense. In this work, infection with either necrotrophic or hemibiotrophic pathogen induced the production of all quantified phytohormones. It has been reported that several pathogens, including B. cinerea and Pst, activated both SA and JA accumulation (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 cross talk is thus used by the plant to adjust its response in favor of the most effective pathway. Interestingly, SPT-silenced tobacco plants displayed higher basal SA levels and were more susceptible to A. alternata infection. However, no information concerning SA, JA, or sphingolipid levels in response to infection is available, especially as transgenic plants still displayed residual NbLCB2 gene expression (Rivas-San Vicente et al., 2013). In Arabidopsis, the acd5 mutant displayed constitutive high SA levels and expression of the PR1 gene. This mutant was also more susceptible to B. cinerea and contained higher Cer levels but reduced apoplastic ROS and PR1 and CHIT transcript accumulation upon infection (Greenberg et al., 2000; Bi et al., 2014). Consistent with this, Atdpl1-1 mutant plants displayed similar Cer levels and PR1 expression, higher apoplastic ROS accumulation, and CHIT up-regulation in response to infection but was more resistant to the necrotrophic fungus.

In Arabidopsis, it is now well admitted that SA has an antagonistic effect on JA signaling and reciprocally (Bostock, 2005; Glazebrook, 2005; Spoel et al., 2007; Thaler et al., 2012; Derksen et al., 2013). In tomato (Solanum lycopersicum), B. cinerea produces an exopolysaccharide that activates the SA pathway, which, through NPR1, antagonizes the JA signaling pathway, thereby allowing the 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 and NPR1 was also stimulated upon infection with B. cinerea. However, the SA signaling pathway was similar in Atdpl1-1 plants. Moreover, JA biosynthetic and signaling pathways were enhanced in the Atdpl1-1 mutant in response to B. cinerea inoculation. In the Atdpl1-1 mutant, it thus seems that perturbation in sphingolipid metabolism rendered either SA unable to activate NPR1 or NPR1 unable to antagonize JA accumulation. Thus, our results highlighted that disturbance of sphingolipid metabolism could impact not only the cell death program but also the JA signaling pathway, leading to plant tolerance toward necrotrophic pathogens such as B. cinerea. In that case, the relationship between sphingolipids and JA could be either indirect, implying that changes in sphingolipids operate in the cross talk between SA and JA pathways but in an NPR1-independent manner, or direct, as some key genes involved in JA biosynthesis are up-regulated in Atdpl1-1 plants. Similar to B. cinerea, a virulent strain of Pst, via its toxin coronatine, exerts its virulence by stimulating the JA signaling pathway in order to inhibit the SA signaling pathway and, thus, facilitate its growth and development (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 reduction in the expression of the SA-dependent marker gene PR1 in Atdpl1-1 mutant plants could thus be explained by the overaccumulation of jasmonates in these plants. Whereas the VSP1 and JAZ8 expression profile correlated the JA and JA-Ile accumulation profile in response to infection with virulent or avirulent Pst, PDF1.2 and CHIT expression did not. PDF1.2 and CHIT require both JA and ET signaling pathways but also the function of MITOGEN-ACTIVATED PROTEIN KINASE4 (MPK4), as JA-treated mpk4 mutants fail to express PDF1.2 (Petersen et al., 2000). A discrepancy between PDF1.2 expression and JA accumulation has also been observed during the induced systemic resistance triggered by Pseudomonas fluorescens, which is regulated through the JA signaling pathway (van Wees et al., 1999). This suggested that a component in the JA or ET signaling pathway might be deficient/nonfunctional in Atdpl1-1 mutant plants in response to Pst infection or that defense against Pst in the Atdpl1-1 mutant might be regulated through a pathway that does not include PDF1.2 or CHIT. Collectively, our results suggested that AtDPL1 could be a negative and/or a positive regulator of the JA- and SA-regulated defense pathway, respectively. Whereas a relationship between SA signaling and sphingolipids has often been described (Sánchez-Rangel et al., 2015), our results highlight, to our knowledge for the first time, that sphingolipids could 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 appropriate response to defend themselves against pathogen attack by acting on sphingolipid metabolism in order to modulate the cell death/survival balance, in close cooperation with the JA and/or SA signaling pathways (Fig. 14). Whereas SA involvement in PCD is well known, the relationship between JA and cell death is less understood. Plants treated with coronatine, which shares structural similarities with JA-Ile and functional similarities with JA, develop chlorosis (Bender et al., 1999; Overmyer et al., 2003). Coronatine-deficient mutants of Pst DC3000 are reduced in disease-associated necrosis and chlorosis (Brooks et al., 2004, 2005). It has been reported that JA is also essential in FB1- and AAL-induced cell death (Asai et al., 2000; Zhang et al., 2011). Interestingly, the Atdpl1 mutant is more sensitive to FB1 treatment (Tsegaye et al., 2007). Thus, sphingolipid metabolism seemed to be intimately connected to defense processes to regulate plant responses to biotic stresses. In Arabidopsis, MPK6, which is involved in the plant defense response (Ren et al., 2008; Beckers et al., 2009), has recently been described as an important contributor to the LCB-mediated PCD (Saucedo-García et al., 2011). However, the deciphering of the precise pathway leading to sphingolipid-induced cell death is far from being totally elucidated. Further identification of target genes and their functions will provide new insights into how sphingolipids could be linked to cell death and defense processes.

Figure 14.
Figure 14.

Schematic overview of interconnections between sphingolipid metabolism, cell death, and defense signaling pathways in Atdpl1 mutant plants upon pathogen attack. Upon disruption of the AtDPL1 gene, infected plants accumulate some LCB-P, hCer, and GIPC species, thus reducing cell death. In the Atdpl1 mutant, sphingolipid metabolism may also indirectly modulate cell death through its tight connection (double-headed dashed arrow) as a positive and/or negative regulator to jasmonate and/or SA signaling pathways, respectively. Reduced cell death and high levels of jasmonates could thus explain that Atdpl1 mutant plants are more tolerant to B. cinerea but more susceptible to Pst. 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-P Pase, LCB-P phosphatase; LOH, LAG ONE HOMOLOG; SPHK1, SPHINGOSINE KINASE1.

MATERIALS AND METHODS

Chemicals

Phytosphingosine-1-phosphate (t18:0-P) and dihydrosphingosine (d18:0) were purchased from Avanti Polar Lipids. Stock solutions were prepared in ethanol:dimethyl sulfoxide (2:1, v/v; t18:0-P) or ethanol (d18:0) and dissolved to a final concentration of 100 µm. Luminol and horseradish peroxidase were obtained from Sigma-Aldrich.

Plant Material and Growth Conditions

Seeds of the Arabidopsis (Arabidopsis thaliana) SALK lines 020151 (referred to as Atdpl1-1), 093662 (Atdpl1-2), and 078119 (Atdpl1-3) containing a transfer DNA (T-DNA) insertion in the At1g27980 locus were obtained from the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info). The SALK_020151 mutant was chosen for the performed experiments because it exhibits the same phenotype as the other Atdpl1 mutants but displays a complete lack of mRNA and a higher LCB/LCB-P accumulation in response to FB1 treatment (Tsegaye et al., 2007). Mutant and wild-type (Columbia-0) plants were grown and maintained under 12-h-light/12-h-dark conditions (150 μmol m−2 s−1, 20°C, and 60% humidity) for 35 d.

Isolation of the T-DNA Insertion Mutant and Genotype Characterization

The mutants SALK_020151, SALK_093662 (Tsegaye et al., 2007), and SALK_078119 were isolated according to the published procedure in SIGnAL (Alonso et al., 2003). The genotype of the knockout mutant line was analyzed by PCR using primers specific for the AtDPL1 gene (forward, 5′-AGAAAGGCCTCAAAGCTTGTC-3′; and reverse, 5′-TGCCAAATAGCATCATTCCTC-3′) and a primer specific for the T-DNA (LB1a, 5′-TGGTTCACGTAGTGGGCCATCG-3′).

Sphingolipidomic Analysis

Sphingolipid extraction from 10 to 20 mg of lyophilized tissue and profiling by liquid chromatography-electrospray ionization-tandem mass spectrometry were performed as described (Markham and Jaworski, 2007) with modifications, using the Shimadzu Prominence ultra-HPLC system and a 4000QTRAP mass spectrometer (AB Sciex). Sphingolipids were separated on a 100-mm Dionex Acclaim C18 column. Data analysis was performed using Analyst 1.6 and Multiquant 2.1 software (AB Sciex). Four to five biologically independent repeats were performed, 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 PCR were performed as described by Le Hénanff et al. (2013). Gene-specific primers are described in Supplemental Table S1. For each experiment, PCR was performed in duplicate, and at least three independent experiments were analyzed. Transcript levels were normalized against those of the ACTIN gene as an internal control. Fold induction compared with mock-treated sample was calculated using the ΔΔCt method (Ct GI [unknown sample] – Ct GI [reference sample]) – (Ct actin [unknown sample] – 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 lycopersicum) medium (25% [v/v] tomato juice and 2.5% [w/v] agar) during 21 d at 22°C. Collected conidia were resuspended in PDB supplemented by 0.02% (v/v) Silwet L-77 to a final density of 105 conidia mL−1. After incubation for 3 h at 22°C and 150 rpm, germinated spores were used for plant inoculation by spraying the upper face of the leaves. Control inoculations were performed with 0.02% PDB Silwet L-77.

The bacterial leaf pathogen Pseudomonas syringae pv tomato strain DC3000 or Pst AvrRPM1 was cultured overnight at 28°C in liquid King’s B medium, supplemented with rifampicin (50 µg mL−1) and kanamycin (50 µg mL−1). Subsequently, bacterial cells were collected by centrifugation and resuspended in 10 mm MgCl2 to a final density of 107 cfu mL−1 (optical density = 0.01). The bacterial solutions were thus infiltrated from the abaxial side into leaf using a 1-mL syringe without a needle. Control inoculations were performed with 10 mm MgCl2.

Leaves were collected from 0 to 48 hpi, frozen in liquid nitrogen, and stored at −80°C until use.

Pathogen Assay in Planta

B. cinerea infections were performed as described previously (Le Hénanff et al., 2013). Plants were placed in translucent boxes under high humidity at 150 µE m−2 s−1. Five or six leaves per plant were drop inoculated with 5 µL of the conidia suspension adjusted at 105 conidia mL−1 in PDB. Lesion diameters were measured 48 and 60 hpi. Forty to 60 leaves were inoculated per treatment and 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 borer and ground in 1 mL of 10 mm MgCl2 with a plastic pestle. Appropriate dilutions were plated on King’s B medium with appropriate antibiotics, and bacterial colonies were counted. Data are reported as means and sd of the log (cfu cm−2) of three replicates. Growth assays were performed four times with similar results.

Electrolyte Leakage

Ten minutes after bacteria injection (Torres et al., 2002) or 20 h after B. cinerea infection (Govrin and Levine, 2002), 9-mm-diameter leaf discs were collected from the infected area and washed extensively with water for 50 min, and then eight discs were placed in a tube with 15 mL of fresh water. To test the sphingolipid effect on ion leakage, pathogen inoculum was supplemented or not with 100 µm t18:0-P or d18:0. Conductivity measurements (three to four replicates for each treatment) were then conducted over time using a B-771 LaquaTwin (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 a 96-well plate containing 150 μL of distilled water and then incubated overnight at room temperature. Just before ROS quantification, distilled water was replaced by 150 μL of an elicitation solution containing 20 μg mL−1 horseradish peroxidase and 0.2 μm luminol. For tests involving bacteria, Pst was added to the elicitation solution to a final bacterial concentration of 108 cfu mL−1. For tests involving B. cinerea, germinated spores were added to the elicitation solution to a final density of 105 conidia mL−1. For tests involving sphingolipids, 100 µm t18:0-P or d18:0 was added concomitantly with bacterium or fungus to the elicitation solution.

Phytohormone Analysis

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

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Gaetan Glauser and Neil Villard (Neuchâtel Platform of Analytical Chemistry, University of Neuchâtel) for excellent technical assistance in phytohormone quantification.

Footnotes

  • www.plantphysiol.org/cgi/doi/10.1104/pp.15.01126

  • The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Sandrine Dhondt-Cordelier (sandrine.cordelier{at}univ-reims.fr).

  • 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.

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

  • ↵[OPEN] Articles can be viewed without a subscription.

Glossary

ROS
reactive oxygen species
SA
salicylic acid
JA
jasmonic acid
ET
ethylene
HR
hypersensitive response
PCD
programmed cell death
FB1
fumonisin B1
AAL
toxin produced by Alternaria alternata f. sp. lycopersici
Cer
ceramide
LCB
long-chain base
LCB-P
long-chain base phosphate
hpi
hours post inoculation
cfu
colony-forming units
qRT
quantitative reverse transcription
GIPC
glycosylinositol phosphoceramide
hCer
hydroxyceramide
GlcCer
glucosylceramide
VLCFA
very-long-chain fatty acid
JA-Ile
jasmonoyl-isoleucine
T-DNA
transfer DNA
PDB
potato dextrose broth
  • Received July 21, 2015.
  • Accepted September 11, 2015.
  • Published September 16, 2015.

REFERENCES

  1. ↵
    1. Abbas HK,
    2. Tanaka T,
    3. Duke SO,
    4. Porter JK,
    5. Wray EM,
    6. Hodges L,
    7. Sessions AE,
    8. Wang E,
    9. Merrill AH Jr.,
    10. Riley RT
    (1994) Fumonisin- and AAL-toxin-induced disruption of sphingolipid metabolism with accumulation of free sphingoid bases. Plant Physiol 106: 1085–1093
    OpenUrlAbstract
  2. ↵
    1. Alden KP,
    2. Dhondt-Cordelier S,
    3. McDonald KL,
    4. Reape TJ,
    5. Ng CK,
    6. McCabe PF,
    7. Leaver CJ
    (2011) Sphingolipid long chain base phosphates can regulate apoptotic-like programmed cell death in plants. Biochem Biophys Res Commun 410: 574–580
    OpenUrlCrossRefPubMed
  3. ↵
    1. Alonso JM,
    2. Stepanova AN,
    3. Leisse TJ,
    4. Kim CJ,
    5. Chen H,
    6. Shinn P,
    7. Stevenson DK,
    8. Zimmerman J,
    9. Barajas P,
    10. Cheuk R, et al.
    (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Asai T,
    2. Stone JM,
    3. Heard JE,
    4. Kovtun Y,
    5. Yorgey P,
    6. Sheen J,
    7. Ausubel FM
    (2000) Fumonisin B1-induced cell death in Arabidopsis protoplasts requires jasmonate-, ethylene-, and salicylate-dependent signaling pathways. Plant Cell 12: 1823–1836
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Bari R,
    2. Jones JD
    (2009) Role of plant hormones in plant defence responses. Plant Mol Biol 69: 473–488
    OpenUrlCrossRefPubMed
  6. ↵
    1. Beckers GJ,
    2. Jaskiewicz M,
    3. Liu Y,
    4. Underwood WR,
    5. He SY,
    6. Zhang S,
    7. Conrath U
    (2009) Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana. Plant Cell 21: 944–953
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Bender CL,
    2. Alarcón-Chaidez F,
    3. Gross DC
    (1999) Pseudomonas syringae phytotoxins: mode of action, regulation, and biosynthesis by peptide and polyketide synthetases. Microbiol Mol Biol Rev 63: 266–292
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Berger S,
    2. Sinha AK,
    3. Roitsch T
    (2007) Plant physiology meets phytopathology: plant primary metabolism and plant-pathogen interactions. J Exp Bot 58: 4019–4026
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Berkey R,
    2. Bendigeri D,
    3. Xiao S
    (2012) Sphingolipids and plant defense/disease: the “death” connection and beyond. Front Plant Sci 3: 68
    OpenUrlPubMed
  10. ↵
    1. Bi FC,
    2. Liu Z,
    3. Wu JX,
    4. Liang H,
    5. Xi XL,
    6. Fang C,
    7. Sun TJ,
    8. Yin J,
    9. Dai GY,
    10. Rong C, et al.
    (2014) Loss of ceramide kinase in Arabidopsis impairs defenses and promotes ceramide accumulation and mitochondrial H2O2 bursts. Plant Cell 26: 3449–3467
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Block A,
    2. Schmelz E,
    3. O’Donnell PJ,
    4. Jones JB,
    5. Klee HJ
    (2005) Systemic acquired tolerance to virulent bacterial pathogens in tomato. Plant Physiol 138: 1481–1490
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Bolton MD
    (2009) Primary metabolism and plant defense: fuel for the fire. Mol Plant Microbe Interact 22: 487–497
    OpenUrlCrossRefPubMed
  13. ↵
    1. Bostock RM
    (2005) Signal crosstalk and induced resistance: straddling the line between cost and benefit. Annu Rev Phytopathol 43: 545–580
    OpenUrlCrossRefPubMed
  14. ↵
    1. Brandwagt BF,
    2. Mesbah LA,
    3. Takken FL,
    4. Laurent PL,
    5. Kneppers TJ,
    6. Hille J,
    7. Nijkamp HJ
    (2000) A longevity assurance gene homolog of tomato mediates resistance to Alternaria alternata f. sp. lycopersici toxins and fumonisin B1. Proc Natl Acad Sci USA 97: 4961–4966
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Brodersen P,
    2. Petersen M,
    3. Pike HM,
    4. Olszak B,
    5. Skov S,
    6. Odum N,
    7. Jørgensen LB,
    8. Brown RE,
    9. Mundy J
    (2002) Knockout of Arabidopsis accelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defense. Genes Dev 16: 490–502
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Brooks DM,
    2. Bender CL,
    3. Kunkel BN
    (2005) The Pseudomonas syringae phytotoxin coronatine promotes virulence by overcoming salicylic acid-dependent defences in Arabidopsis thaliana. Mol Plant Pathol 6: 629–639
    OpenUrlCrossRefPubMed
  17. ↵
    1. Brooks DM,
    2. Hernández-Guzmán G,
    3. Kloek AP,
    4. Alarcón-Chaidez F,
    5. Sreedharan A,
    6. Rangaswamy V,
    7. Peñaloza-Vázquez A,
    8. Bender CL,
    9. Kunkel BN
    (2004) Identification and characterization of a well-defined series of coronatine biosynthetic mutants of Pseudomonas syringae pv. tomato DC3000. Mol Plant Microbe Interact 17: 162–174
    OpenUrlCrossRefPubMed
  18. ↵
    1. Cao H,
    2. Li X,
    3. Dong X
    (1998) Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc Natl Acad Sci USA 95: 6531–6536
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Chen M,
    2. Markham JE,
    3. Dietrich CR,
    4. Jaworski JG,
    5. Cahoon EB
    (2008) Sphingolipid long-chain base hydroxylation is important for growth and regulation of sphingolipid content and composition in Arabidopsis. Plant Cell 20: 1862–1878
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Coll NS,
    2. Epple P,
    3. Dangl JL
    (2011) Programmed cell death in the plant immune system. Cell Death Differ 18: 1247–1256
    OpenUrlCrossRefPubMed
  21. ↵
    1. Contreras-Cornejo HA,
    2. Macías-Rodríguez L,
    3. Beltrán-Peña E,
    4. Herrera-Estrella A,
    5. López-Bucio J
    (2011) Trichoderma-induced plant immunity likely involves both hormonal- and camalexin-dependent mechanisms in Arabidopsis thaliana and confers resistance against necrotrophic fungi Botrytis cinerea. Plant Signal Behav 6: 1554–1563
    OpenUrlCrossRefPubMed
  22. ↵
    1. Coursol S,
    2. Fromentin J,
    3. Noirot E,
    4. Brière C,
    5. Robert F,
    6. Morel J,
    7. Liang YK,
    8. Lherminier J,
    9. Simon-Plas F
    (2015) Long-chain bases and their phosphorylated derivatives differentially regulate cryptogein-induced production of reactive oxygen species in tobacco (Nicotiana tabacum) BY-2 cells. New Phytol 205: 1239–1249
    OpenUrlCrossRefPubMed
  23. ↵
    1. Dellagi A,
    2. Brisset MN,
    3. Paulin JP,
    4. Expert D
    (1998) Dual role of desferrioxamine in Erwinia amylovora pathogenicity. Mol Plant Microbe Interact 11: 734–742
    OpenUrlCrossRefPubMed
  24. ↵
    1. Derksen H,
    2. Rampitsch C,
    3. Daayf F
    (2013) Signaling cross-talk in plant disease resistance. Plant Sci 207: 79–87
    OpenUrlCrossRefPubMed
  25. ↵
    1. Dickman MB,
    2. Park YK,
    3. Oltersdorf T,
    4. Li W,
    5. Clemente T,
    6. French R
    (2001) Abrogation of disease development in plants expressing animal antiapoptotic genes. Proc Natl Acad Sci USA 98: 6957–6962
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Donahue JL,
    2. Alford SR,
    3. Torabinejad J,
    4. Kerwin RE,
    5. Nourbakhsh A,
    6. Ray WK,
    7. Hernick M,
    8. Huang X,
    9. Lyons BM,
    10. Hein PP, et al.
    (2010) The Arabidopsis thaliana Myo-inositol 1-phosphate synthase1 gene is required for Myo-inositol synthesis and suppression of cell death. Plant Cell 22: 888–903
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Dunn TM,
    2. Lynch DV,
    3. Michaelson LV,
    4. Napier JA
    (2004) A post-genomic approach to understanding sphingolipid metabolism in Arabidopsis thaliana. Ann Bot (Lond) 93: 483–497
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. El Oirdi M,
    2. Bouarab K
    (2007) Plant signalling components EDS1 and SGT1 enhance disease caused by the necrotrophic pathogen Botrytis cinerea. New Phytol 175: 131–139
    OpenUrlCrossRefPubMed
  29. ↵
    1. El Oirdi M,
    2. El Rahman TA,
    3. Rigano L,
    4. El Hadrami A,
    5. Rodriguez MC,
    6. Daayf F,
    7. Vojnov A,
    8. Bouarab K
    (2011) Botrytis cinerea manipulates the antagonistic effects between immune pathways to promote disease development in tomato. Plant Cell 23: 2405–2421
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Geng X,
    2. Cheng J,
    3. Gangadharan A,
    4. Mackey D
    (2012) The coronatine toxin of Pseudomonas syringae is a multifunctional suppressor of Arabidopsis defense. Plant Cell 24: 4763–4774
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. JJ Sanchez-Serrano,
    2. J Salinas
    1. Glauser G,
    2. Vallat A,
    3. Balmer D
    (2014) Hormone profiling. In JJ Sanchez-Serrano, J Salinas, eds, Arabidopsis Protocols: Methods in Molecular Biology, Vol 1062. Springer Science + Business Media, New York, pp 597–608
  32. ↵
    1. Glazebrook J
    (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43: 205–227
    OpenUrlCrossRefPubMed
  33. ↵
    1. Govrin EM,
    2. Levine A
    (2000) The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr Biol 10: 751–757
    OpenUrlCrossRefPubMed
  34. ↵
    1. Govrin EM,
    2. Levine A
    (2002) Infection of Arabidopsis with a necrotrophic pathogen, Botrytis cinerea, elicits various defense responses but does not induce systemic acquired resistance (SAR). Plant Mol Biol 48: 267–276
    OpenUrlCrossRefPubMed
  35. ↵
    1. Govrin EM,
    2. Rachmilevitch S,
    3. Tiwari BS,
    4. Solomon M,
    5. Levine A
    (2006) An elicitor from Botrytis cinerea induces the hypersensitive response in Arabidopsis thaliana and other plants and promotes the gray mold disease. Phytopathology 96: 299–307
    OpenUrlCrossRefPubMed
  36. ↵
    1. Greenberg JT,
    2. Silverman FP,
    3. Liang H
    (2000) Uncoupling salicylic acid-dependent cell death and defense-related responses from disease resistance in the Arabidopsis mutant acd5. Genetics 156: 341–350
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Kawasaki T,
    2. Nam J,
    3. Boyes DC,
    4. Holt BF III.,
    5. Hubert DA,
    6. Wiig A,
    7. Dangl JL
    (2005) A duplicated pair of Arabidopsis RING-finger E3 ligases contribute to the RPM1- and RPS2-mediated hypersensitive response. Plant J 44: 258–270
    OpenUrlCrossRefPubMed
  38. ↵
    1. König S,
    2. Feussner K,
    3. Schwarz M,
    4. Kaever A,
    5. Iven T,
    6. Landesfeind M,
    7. Ternes P,
    8. Karlovsky P,
    9. Lipka V,
    10. Feussner I
    (2012) Arabidopsis mutants of sphingolipid fatty acid α-hydroxylases accumulate ceramides and salicylates. New Phytol 196: 1086–1097
    OpenUrlCrossRefPubMed
  39. ↵
    1. Koornneef A,
    2. Pieterse CM
    (2008) Cross talk in defense signaling. Plant Physiol 146: 839–844
    OpenUrlFREE Full Text
  40. ↵
    1. Kraepiel Y,
    2. Pédron J,
    3. Patrit O,
    4. Simond-Côte E,
    5. Hermand V,
    6. Van Gijsegem F
    (2011) Analysis of the plant bos1 mutant highlights necrosis as an efficient defence mechanism during D. dadantii/Arabidopsis thaliana interaction. PLoS One 6: e18991
    OpenUrlCrossRefPubMed
  41. ↵
    1. Lachaud C,
    2. Da Silva D,
    3. Amelot N,
    4. Béziat C,
    5. Brière C,
    6. Cotelle V,
    7. Graziana A,
    8. Grat S,
    9. Mazars C,
    10. Thuleau P
    (2011) Dihydrosphingosine-induced programmed cell death in tobacco BY-2 cells is independent of H2O2 production. Mol Plant 4: 310–318
    OpenUrlCrossRefPubMed
  42. ↵
    1. Lachaud C,
    2. Da Silva D,
    3. Cotelle V,
    4. Thuleau P,
    5. Xiong TC,
    6. Jauneau A,
    7. Brière C,
    8. Graziana A,
    9. Bellec Y,
    10. Faure JD, et al.
    (2010) Nuclear calcium controls the apoptotic-like cell death induced by d-erythro-sphinganine in tobacco cells. Cell Calcium 47: 92–100
    OpenUrlCrossRefPubMed
  43. ↵
    1. Laurie-Berry N,
    2. Joardar V,
    3. Street IH,
    4. Kunkel BN
    (2006) The Arabidopsis thaliana JASMONATE INSENSITIVE 1 gene is required for suppression of salicylic acid-dependent defenses during infection by Pseudomonas syringae. Mol Plant Microbe Interact 19: 789–800
    OpenUrlCrossRefPubMed
  44. ↵
    1. Le Hénanff G,
    2. Profizi C,
    3. Courteaux B,
    4. Rabenoelina F,
    5. Gérard C,
    6. Clément C,
    7. Baillieul F,
    8. Cordelier S,
    9. Dhondt-Cordelier S
    (2013) Grapevine NAC1 transcription factor as a convergent node in developmental processes, abiotic stresses, and necrotrophic/biotrophic pathogen tolerance. J Exp Bot 64: 4877–4893
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. L’Haridon F,
    2. Besson-Bard A,
    3. Binda M,
    4. Serrano M,
    5. Abou-Mansour E,
    6. Balet F,
    7. Schoonbeek HJ,
    8. Hess S,
    9. Mir R,
    10. Léon J, et al.
    (2011) A permeable cuticle is associated with the release of reactive oxygen species and induction of innate immunity. PLoS Pathog 7: e1002148
    OpenUrlCrossRefPubMed
  46. ↵
    1. Liang H,
    2. Yao N,
    3. Song JT,
    4. Luo S,
    5. Lu H,
    6. Greenberg JT
    (2003) Ceramides modulate programmed cell death in plants. Genes Dev 17: 2636–2641
    OpenUrlAbstract/FREE Full Text
  47. ↵
    1. Lohman KN,
    2. Gan S,
    3. John MC,
    4. Amasino RM
    (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiol Plant 92: 322–328
    OpenUrlCrossRef
  48. ↵
    1. Markham JE,
    2. Jaworski JG
    (2007) Rapid measurement of sphingolipids from Arabidopsis thaliana by reversed-phase high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 21: 1304–1314
    OpenUrlCrossRefPubMed
  49. ↵
    1. Markham JE,
    2. Lynch DV,
    3. Napier JA,
    4. Dunn TM,
    5. Cahoon EB
    (2013) Plant sphingolipids: function follows form. Curr Opin Plant Biol 16: 350–357
    OpenUrlCrossRefPubMed
  50. ↵
    1. Mayer AM,
    2. Staples RC,
    3. Gil-ad NL
    (2001) Mechanisms of survival of necrotrophic fungal plant pathogens in hosts expressing the hypersensitive response. Phytochemistry 58: 33–41
    OpenUrlCrossRefPubMed
  51. ↵
    1. Mortimer JC,
    2. Yu X,
    3. Albrecht S,
    4. Sicilia F,
    5. Huichalaf M,
    6. Ampuero D,
    7. Michaelson LV,
    8. Murphy AM,
    9. Matsunaga T,
    10. Kurz S, et al.
    (2013) Abnormal glycosphingolipid mannosylation triggers salicylic acid-mediated responses in Arabidopsis. Plant Cell 25: 1881–1894
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Mur LA,
    2. Kenton P,
    3. Lloyd AJ,
    4. Ougham H,
    5. Prats E
    (2008) The hypersensitive response: the centenary is upon us but how much do we know? J Exp Bot 59: 501–520
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Nagano M,
    2. Takahara K,
    3. Fujimoto M,
    4. Tsutsumi N,
    5. Uchimiya H,
    6. Kawai-Yamada M
    (2012) Arabidopsis sphingolipid fatty acid 2-hydroxylases (AtFAH1 and AtFAH2) are functionally differentiated in fatty acid 2-hydroxylation and stress responses. Plant Physiol 159: 1138–1148
    OpenUrlAbstract/FREE Full Text
  54. ↵
    1. Overmyer K,
    2. Brosché M,
    3. Kangasjärvi J
    (2003) Reactive oxygen species and hormonal control of cell death. Trends Plant Sci 8: 335–342
    OpenUrlCrossRefPubMed
  55. ↵
    1. Peer M,
    2. Bach M,
    3. Mueller MJ,
    4. Waller F
    (2011) Free sphingobases induce RBOHD-dependent reactive oxygen species production in Arabidopsis leaves. FEBS Lett 585: 3006–3010
    OpenUrlCrossRefPubMed
  56. ↵
    1. Peer M,
    2. Stegmann M,
    3. Mueller MJ,
    4. Waller F
    (2010) Pseudomonas syringae infection triggers de novo synthesis of phytosphingosine from sphinganine in Arabidopsis thaliana. FEBS Lett 584: 4053–4056
    OpenUrlCrossRefPubMed
  57. ↵
    1. Petersen M,
    2. Brodersen P,
    3. Naested H,
    4. Andreasson E,
    5. Lindhart U,
    6. Johansen B,
    7. Nielsen HB,
    8. Lacy M,
    9. Austin MJ,
    10. Parker JE, et al.
    (2000) Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell 103: 1111–1120
    OpenUrlCrossRefPubMed
  58. ↵
    1. Pieterse CM,
    2. Leon-Reyes A,
    3. Van der Ent S,
    4. Van Wees SC
    (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5: 308–316
    OpenUrlCrossRefPubMed
  59. ↵
    1. Rasul S,
    2. Dubreuil-Maurizi C,
    3. Lamotte O,
    4. Koen E,
    5. Poinssot B,
    6. Alcaraz G,
    7. Wendehenne D,
    8. Jeandroz S
    (2012) Nitric oxide production mediates oligogalacturonide-triggered immunity and resistance to Botrytis cinerea in Arabidopsis thaliana. Plant Cell Environ 35: 1483–1499
    OpenUrlCrossRefPubMed
  60. ↵
    1. Ren D,
    2. Liu Y,
    3. Yang KY,
    4. Han L,
    5. Mao G,
    6. Glazebrook J,
    7. Zhang S
    (2008) A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis. Proc Natl Acad Sci USA 105: 5638–5643
    OpenUrlAbstract/FREE Full Text
  61. ↵
    1. Rivas-San Vicente M,
    2. Larios-Zarate G,
    3. Plasencia J
    (2013) Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici. Planta 237: 121–136
    OpenUrlCrossRefPubMed
  62. ↵
    1. Robert-Seilaniantz A,
    2. Grant M,
    3. Jones JD
    (2011) Hormone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism. Annu Rev Phytopathol 49: 317–343
    OpenUrlCrossRefPubMed
  63. ↵
    1. Sanchez L,
    2. Courteaux B,
    3. Hubert J,
    4. Kauffmann S,
    5. Renault JH,
    6. Clément C,
    7. Baillieul F,
    8. Dorey S
    (2012) Rhamnolipids elicit defense responses and induce disease resistance against biotrophic, hemibiotrophic, and necrotrophic pathogens that require different signaling pathways in Arabidopsis and highlight a central role for salicylic acid. Plant Physiol 160: 1630–1641
    OpenUrlAbstract/FREE Full Text
  64. ↵
    1. Sánchez-Rangel D,
    2. Rivas-San Vicente M,
    3. de la Torre-Hernández ME,
    4. Nájera-Martínez M,
    5. Plasencia J
    (2015) Deciphering the link between salicylic acid signaling and sphingolipid metabolism. Front Plant Sci 6: 125
    OpenUrlPubMed
  65. ↵
    1. Saucedo-García M,
    2. Gavilanes-Ruíz M,
    3. Arce-Cervantes O
    (2015) Long-chain bases, phosphatidic acid, MAPKs, and reactive oxygen species as nodal signal transducers in stress responses in Arabidopsis. Front Plant Sci 6: 55
    OpenUrlPubMed
  66. ↵
    1. Saucedo-García M,
    2. Guevara-García A,
    3. González-Solís A,
    4. Cruz-García F,
    5. Vázquez-Santana S,
    6. Markham JE,
    7. Lozano-Rosas MG,
    8. Dietrich CR,
    9. Ramos-Vega M,
    10. Cahoon EB, et al.
    (2011) MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis. New Phytol 191: 943–957
    OpenUrlCrossRefPubMed
  67. ↵
    1. Savatin DV,
    2. Bisceglia NG,
    3. Marti L,
    4. Fabbri C,
    5. Cervone F,
    6. De Lorenzo G
    (2014) The Arabidopsis NUCLEUS- AND PHRAGMOPLAST-LOCALIZED KINASE1-related protein kinases are required for elicitor-induced oxidative burst and immunity. Plant Physiol 165: 1188–1202
    OpenUrlAbstract/FREE Full Text
  68. ↵
    1. Schmelz EA,
    2. Engelberth J,
    3. Alborn HT,
    4. O’Donnell P,
    5. Sammons M,
    6. Toshima H,
    7. Tumlinson JH III.
    (2003) Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants. Proc Natl Acad Sci USA 100: 10552–10557
    OpenUrlAbstract/FREE Full Text
  69. ↵
    1. Schwessinger B,
    2. Ronald PC
    (2012) Plant innate immunity: perception of conserved microbial signatures. Annu Rev Plant Biol 63: 451–482
    OpenUrlCrossRefPubMed
  70. ↵
    1. Shi C,
    2. Yin J,
    3. Liu Z,
    4. Wu JX,
    5. Zhao Q,
    6. Ren J,
    7. Yao N
    (2015) A systematic simulation of the effect of salicylic acid on sphingolipid metabolism. Front Plant Sci 6: 186
    OpenUrlPubMed
  71. ↵
    1. Shi L,
    2. Bielawski J,
    3. Mu J,
    4. Dong H,
    5. Teng C,
    6. Zhang J,
    7. Yang X,
    8. Tomishige N,
    9. Hanada K,
    10. Hannun YA, et al.
    (2007) Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis. Cell Res 17: 1030–1040
    OpenUrlCrossRefPubMed
  72. ↵
    1. Smith JM,
    2. Heese A
    (2014) Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae. Plant Methods 10: 6
    OpenUrlCrossRefPubMed
  73. ↵
    1. Spoel SH,
    2. Johnson JS,
    3. Dong X
    (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestyles. Proc Natl Acad Sci USA 104: 18842–18847
    OpenUrlAbstract/FREE Full Text
  74. ↵
    1. Spoel SH,
    2. Koornneef A,
    3. Claessens SM,
    4. Korzelius JP,
    5. Van Pelt JA,
    6. Mueller MJ,
    7. Buchala AJ,
    8. Métraux JP,
    9. Brown R,
    10. Kazan K, et al.
    (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 15: 760–770
    OpenUrlAbstract/FREE Full Text
  75. ↵
    1. Staswick PE,
    2. Tiryaki I
    (2004) The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis. Plant Cell 16: 2117–2127
    OpenUrlAbstract/FREE Full Text
  76. ↵
    1. Takahashi Y,
    2. Berberich T,
    3. Kanzaki H,
    4. Matsumura H,
    5. Saitoh H,
    6. Kusano T,
    7. Terauchi R
    (2009) Unraveling the roles of sphingolipids in plant innate immunity. Plant Signal Behav 4: 536–538
    OpenUrlCrossRefPubMed
  77. ↵
    1. Ternes P,
    2. Feussner K,
    3. Werner S,
    4. Lerche J,
    5. Iven T,
    6. Heilmann I,
    7. Riezman H,
    8. Feussner I
    (2011) Disruption of the ceramide synthase LOH1 causes spontaneous cell death in Arabidopsis thaliana. New Phytol 192: 841–854
    OpenUrlCrossRefPubMed
  78. ↵
    1. Thaler JS,
    2. Humphrey PT,
    3. Whiteman NK
    (2012) Evolution of jasmonate and salicylate signal crosstalk. Trends Plant Sci 17: 260–270
    OpenUrlCrossRefPubMed
  79. ↵
    1. Thomma BP,
    2. Penninckx IA,
    3. Broekaert WF,
    4. Cammue BP
    (2001) The complexity of disease signaling in Arabidopsis. Curr Opin Immunol 13: 63–68
    OpenUrlCrossRefPubMed
  80. ↵
    1. Torres MA
    (2010) ROS in biotic interactions. Physiol Plant 138: 414–429
    OpenUrlCrossRefPubMed
  81. ↵
    1. Torres MA,
    2. Dangl JL,
    3. Jones JD
    (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 99: 517–522
    OpenUrlAbstract/FREE Full Text
  82. ↵
    1. Tsegaye Y,
    2. Richardson CG,
    3. Bravo JE,
    4. Mulcahy BJ,
    5. Lynch DV,
    6. Markham JE,
    7. Jaworski JG,
    8. Chen M,
    9. Cahoon EB,
    10. Dunn TM
    (2007) Arabidopsis mutants lacking long chain base phosphate lyase are fumonisin-sensitive and accumulate trihydroxy-18:1 long chain base phosphate. J Biol Chem 282: 28195–28206
    OpenUrlAbstract/FREE Full Text
  83. ↵
    1. Tsuda K,
    2. Katagiri F
    (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity. Curr Opin Plant Biol 13: 459–465
    OpenUrlCrossRefPubMed
  84. ↵
    1. Uppalapati SR,
    2. Ishiga Y,
    3. Wangdi T,
    4. Kunkel BN,
    5. Anand A,
    6. Mysore KS,
    7. Bender CL
    (2007) The phytotoxin coronatine contributes to pathogen fitness and is required for suppression of salicylic acid accumulation in tomato inoculated with Pseudomonas syringae pv. tomato DC3000. Mol Plant Microbe Interact 20: 955–965
    OpenUrlCrossRefPubMed
  85. ↵
    1. van Baarlen P,
    2. Staats M,
    3. van Kan JA
    (2004) Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica. Mol Plant Pathol 5: 559–574
    OpenUrlCrossRefPubMed
  86. ↵
    1. van Baarlen P,
    2. Woltering EJ,
    3. Staats M,
    4. van Kan JA
    (2007) Histochemical and genetic analysis of host and non-host interactions of Arabidopsis with three Botrytis species: an important role for cell death control. Mol Plant Pathol 8: 41–54
    OpenUrlCrossRefPubMed
  87. ↵
    1. van Kan JA
    (2006) Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trends Plant Sci 11: 247–253
    OpenUrlCrossRefPubMed
  88. ↵
    1. van Wees SC,
    2. Luijendijk M,
    3. Smoorenburg I,
    4. van Loon LC,
    5. Pieterse CM
    (1999) Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge. Plant Mol Biol 41: 537–549
    OpenUrlCrossRefPubMed
  89. ↵
    1. Veronese P,
    2. Nakagami H,
    3. Bluhm B,
    4. Abuqamar S,
    5. Chen X,
    6. Salmeron J,
    7. Dietrich RA,
    8. Hirt H,
    9. Mengiste T
    (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens. Plant Cell 18: 257–273
    OpenUrlAbstract/FREE Full Text
  90. ↵
    1. Wang D,
    2. Amornsiripanitch N,
    3. Dong X
    (2006) A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants. PLoS Pathog 2: e123
    OpenUrlCrossRefPubMed
  91. ↵
    1. Wang W,
    2. Yang X,
    3. Tangchaiburana S,
    4. Ndeh R,
    5. Markham JE,
    6. Tsegaye Y,
    7. Dunn TM,
    8. Wang GL,
    9. Bellizzi M,
    10. Parsons JF, et al.
    (2008) An inositolphosphorylceramide synthase is involved in regulation of plant programmed cell death associated with defense in Arabidopsis. Plant Cell 20: 3163–3179
    OpenUrlAbstract/FREE Full Text
  92. ↵
    1. Wu JX,
    2. Li J,
    3. Liu Z,
    4. Yin J,
    5. Chang ZY,
    6. Rong C,
    7. Wu JL,
    8. Bi FC,
    9. Yao N
    (2015) The Arabidopsis ceramidase AtACER functions in disease resistance and salt tolerance. Plant J 81: 767–780
    OpenUrlCrossRefPubMed
  93. ↵
    1. Xin XF,
    2. He SY
    (2013) Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants. Annu Rev Phytopathol 51: 473–498
    OpenUrlCrossRefPubMed
  94. ↵
    1. Zhang H,
    2. Li L,
    3. Yu Y,
    4. Mo J,
    5. Sun L,
    6. Liu B,
    7. Li D,
    8. Song F
    (2013) Cloning and characterization of two rice long-chain base kinase genes and their function in disease resistance and cell death. Mol Biol Rep 40: 117–127
    OpenUrlCrossRefPubMed
  95. ↵
    1. Zhang L,
    2. Jia C,
    3. Liu L,
    4. Zhang Z,
    5. Li C,
    6. Wang Q
    (2011) The involvement of jasmonates and ethylene in Alternaria alternata f. sp. lycopersici toxin-induced tomato cell death. J Exp Bot 62: 5405–5418
    OpenUrlAbstract/FREE Full Text
  96. ↵
    1. Zhang Y,
    2. Yang X,
    3. Zeng H,
    4. Guo L,
    5. Yuan J,
    6. Qiu D
    (2014) Fungal elicitor protein PebC1 from Botrytis cinerea improves disease resistance in Arabidopsis thaliana. Biotechnol Lett 36: 1069–1078
    OpenUrlCrossRefPubMed
  97. ↵
    1. Zhao Y,
    2. Thilmony R,
    3. Bender CL,
    4. Schaller A,
    5. He SY,
    6. Howe GA
    (2003) Virulence systems of Pseudomonas syringae pv. tomato promote bacterial speck disease in tomato by targeting the jasmonate signaling pathway. Plant J 36: 485–499
    OpenUrlCrossRefPubMed
  98. ↵
    1. Zheng XY,
    2. Spivey NW,
    3. Zeng W,
    4. Liu PP,
    5. Fu ZQ,
    6. Klessig DF,
    7. He SY,
    8. Dong X
    (2012) Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation. Cell Host Microbe 11: 587–596
    OpenUrlCrossRefPubMed
  99. ↵
    1. Zimmerli L,
    2. Métraux JP,
    3. Mauch-Mani B
    (2001) β-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophic fungus Botrytis cinerea. Plant Physiol 126: 517–523
    OpenUrlAbstract/FREE Full Text
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Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis
Maryline Magnin-Robert, Doriane Le Bourse, Jonathan Markham, Stéphan Dorey, Christophe Clément, Fabienne Baillieul, Sandrine Dhondt-Cordelier
Plant Physiology Nov 2015, 169 (3) 2255-2274; DOI: 10.1104/pp.15.01126
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