SOBER1 activity suppresses phosphatidic acid accumulation and plant immunity in response to bacterial effector AvrBsT

Angela Kirik and Mary Beth Mudgett1

Department of Biology, Stanford University, 228A Gilbert Bioscience, Stanford, CA 94305-5020

Edited by Brian J. Staskawicz, University of California, Berkeley, CA, and approved October 2, 2009 (received for review April 7, 2009) Arabidopsis thaliana ecotype Pi-0 is resistant to Pseudomonas syrin- Biochemical studies, however, show that YopJ-like effectors gae pathovar tomato (Pst) strain DC3000 expressing the T3S effector have acetylation, deubiquitination, and/or desumolyation activ- protein AvrBsT. Resistance is due to a loss of function mutation ities (9–11). Thus, these proteins have likely evolved distinct (sober1–1) in a conserved ␣/␤ , SOBER1 (Suppressor of activities to modulate signaling in different hosts. How eu- AvrBsT Elicited Resistance1). Members of this superfamily possess karyotes recognize these effectors and mount defense is an phospholipase and activity with diverse substrate outstanding question. specificity. The nature of SOBER1 enzymatic activity and substrate We used Arabidopsis thaliana as a model genetic system to specificity was not known. SOBER1-dependent suppression of the study resistance to AvrBsT, a YopJ-like effector from Xan- hypersensitive response (HR) in Pi-0 suggested that it might hydrolyze thomonas campestris pathovar vesicatoria (Xcv) (12). AvrBsT’s a plant or precursor required for HR induction. Here, we show virulence function remains elusive; however, its putative cata- that Pi-0 leaves infected with Pst DC3000 expressing AvrBsT accumu- lytic residues are required to induce the HR inside resistant lated higher levels of phosphatidic acid (PA) compared to leaves plants (8, 13). This suggests that AvrBsT-dependent proteolysis infected with Pst DC3000. (PLD) activity was required or modification of plant proteins triggers immune signaling. for high PA levels and AvrBsT-dependent HR in Pi-0. Overexpression Because Xcv is not a pathogen of Arabidopsis, Pseudomonas of SOBER1 in Pi-0 reduced PA levels and inhibited HR. These data syringae pathovar tomato (Pst) strain DC3000 was engineered to implicated PA, phosphatidylcholine (PC) and lysophosphatidylcholine deliver AvrBsT into Arabidopsis cells by its T3S system (13). Of (LysoPC) as potential SOBER1 substrates. Recombinant His6-SOBER1 71 ecotypes examined, only one ecotype (Pi-0) was resistant to hydrolyzed PC but not PA or LysoPC in vitro indicating that the Pst DC3000 expressing AvrBsT (i.e., Pst DC3000 AvrBsT). Pi-0 has (PLA2) activity. Chemical inhibition of resistance is due to a recessive allele (sober1–1) that inactivates PLA2 activity in leaves expressing SOBER1 resulted in HR in response a conserved ␣/␤ hydrolase, designated as SOBER1 (Suppressor to Pst DC3000 AvrBsT. These data are consistent with the model that of AvrBsT Elicited Resistance1) (13). Members of this super- SOBER1 PLA2 activity suppresses PLD-dependent production of PA in family possess phospholipase and carboxylesterase activity with response to AvrBsT elicitation. This work highlights an important role diverse substrate specificity. SOBER1 orthologs from yeast for SOBER1 in the regulation of PA levels generated in plants in and humans exhibit (LysoPLA) and acyl response to biotic stress. protein thioesterase (APT) activity (14, 15). SOBER1 sub- strate specificity has remained elusive; however, in vitro assays disease resistance ͉ hypersensitive response ͉ lipid profiling ͉ pathogen showed that it has carboxylesterase activity characteristic of its superfamily (13). acterial pathogens of plants and animals use the type III The goal of this study was to identify SOBER1 substrates and Bsecretion (T3S) system to introduce protein substrates (i.e., determine how this enzyme negatively regulates AvrBsT- effectors) into their host cells during infection (1, 2). T3S triggered immunity in Arabidopsis. SOBER1 enzyme activity is effectors are important virulence factors that alter eukaryotic required to suppress Pst DC3000 AvrBsT growth and HR in signaling pathways to promote bacterial growth and coloniza- planta (13). Therefore, we hypothesized that AvrBsT action tion. In plant cells, T3S effectors inhibit basal and resistance (R) within sober1–1 leaves might lead to the accumulation of a protein-mediated immunity, although the mechanisms by which phospholipid species that triggers immunity during infection. they do so are mostly unknown (3, 4). Such a lipid species could be a SOBER1 substrate. Alternatively, Plant basal immune responses are initiated by host pattern- the lipid species could be the hydrolyzed product of a precursor recognition receptors following recognition of pathogen-associated lipid that is a SOBER1 substrate. Resistant Pi-0 (sober1–1) leaves molecular patterns (PAMPs) at the cell surface (5). PAMP- lacking SOBER1 activity would then be expected to accumulate triggered immunity (PTI) results from the production of reactive the phospholipid species whereas susceptible Col-0 (SOBER1) oxygen species, activation of MAP kinase signaling, modulation of leaves would not. To test this hypothesis, we determined the host transcription, and deposition of callose at the plant wall, events phospholipid composition in Arabidopsis leaves infected with Pst that lead to inhibition of pathogen growth (3). R proteins specifi- DC3000 AvrBsT versus Pst DC3000. cally recognize T3S effector perturbation within plant cells and We report that phosphatidic acid (PA) is the major phospho- robustly activate similar defense cascades, often referred to as lipid that accumulates in Pi-0 leaves in a sober1–1/AvrBsT- effector-triggered immunity (ETI) (6). To suppress plant immunity, dependent manner. Inhibition of phospholipase D (PLD) activ- bacterial pathogens use multiple T3S effectors to inactivate key signaling components of PTI and ETI (3). T3S effectors found in animal pathogens are usually absent in Author contributions: A.K. and M.B.M. designed research; A.K. performed research; A.K. plant pathogens. One exception is the Yersinia YopJ effector and M.B.M. analyzed data; and A.K. and M.B.M. wrote the paper. family (7). Several YopJ-like homologs are found in Xanthomo- The authors declare no conflict of interest. nas, Pseudomonas, Ralstonia, Erwinia, and a plant symbiont This article is a PNAS Direct Submission. Rhizobium (7). Each protein has a putative catalytic core re- 1To whom correspondence should be addressed. E-mail: [email protected]. sembling C55 cysteine proteases (8), suggesting that YopJ-like This article contains supporting information online at www.pnas.org/cgi/content/full/ effectors might use similar chemistry to alter eukaryotic targets. 0903859106/DCSupplemental.

20532–20537 ͉ PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903859106 Downloaded by guest on September 26, 2021 ity or overexpression of SOBER1 was sufficient to reduce PA levels and inhibit HR in Pi-0 leaves inoculated with Pst DC3000 AvrBsT. In vitro enzyme assays revealed that SOBER1 has phospholipase A2 (PLA2) activity and hydrolyzes the major structural lipid PC, but not PA or LysoPC. These data are consistent with the model that SOBER1 PLA2 activity sup- presses PLD-dependent production of PA in response to AvrBsT elicitation. This work highlights an important role for SOBER1 in the regulation of PA levels generated in plants in response to biotic stress. Results PA Accumulates in Pi-0 Leaves in Response to AvrBsT. The goal of this work was 2-fold. First, we wanted to determine if AvrBsT alters the phospholipid composition in leaves infected with Pst DC3000 AvrBsT, because SOBER1 activity is required to suppress AvrBsT-induced immunity in Arabidopsis (13). Second, we wanted to determine if SOBER1 has phospholipase activity and identify potential substrates in planta. Our hypothesis was that SOBER1 might hydrolyze a phospholipid species, or its precur- sor, that accumulates in response to AvrBsT perturbation. To test this hypothesis, we determined the phospholipid composition in two well characterized Arabidopsis genetic back- grounds: Pi-0 (sober1–1) and Col-0 (SOBER1) (13). Pi-0 plants lacking SOBER1 activity are resistant to Pst DC3000 AvrBsT infection, whereas Col-0 plants containing wild type SOBER1 activity are susceptible (13). Pi-0 and Col-0 leaves were inocu- Fig. 1. Total phospholipid composition in Pi-0 and Col-0 leaves inoculated ϫ 7 lated with a 5 10 cells/mL suspension of Pst DC3000 AvrBsT with Pst DC3000 vector or Pst DC3000 AvrBsT. Leaves were inoculated with 5 ϫ or Pst DC3000 vector. were extracted at 16 h postinocu- 107 cells/mL of bacteria. Lipids were extracted 16 HPI and analyzed by MS. lation (HPI). This time point was selected because Pi-0 leaves Molecular species analyzed: PS, phosphatidylserine; PA, phosphatidic acid; PC, infected with Pst DC3000 AvrBsT are still turgid and contain the phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglyc- highest mRNA levels of the PATHOGENESIS-RELATED1 erol; and PI, phosphatidylinositol. Values are means Ϯ SD (n ϭ 5). * ϭ (PR1) gene (Fig. S1). High PR1 expression correlates with Significant difference (t test, P Ͻ 0.05) in lipid composition when comparing reduced Pst DC3000 AvrBsT growth and symptom production in leaves of the same ecotype. Pi-0 leaves (13). We suspected that the concentration of the putative lipid species would be most abundant when defense AvrBsT. The total PA pool in Pi-0 Pro ::FLAG-SOBER1 leaves gene expression was highest under these conditions. Electros- 35S inoculated with Pst DC3000 AvrBsT was similar to that found in pray ionization triple quadrupole mass spectrometry (MS) was then performed to quantify individual phospholipid and galac- leaves inoculated with Pst DC3000 vector (Fig. 2). These data tolipid species. The level of each lipid species detected in the suggest that SOBER1 negatively regulates PA levels in plant cells infected leaves is shown in Figs. S2 and S3. during Pst DC3000 AvrBsT infection and further supports our Lipid profiling revealed that only one phospholipid accumu- finding that elevated PA levels in sober1–1 leaves correlate with lated significantly in leaves in a sober1–1/AvrBsT-dependent AvrBsT-triggered immunity (Fig. 1). manner. Total PA levels were higher in Pi-0 (sober1–1) leaves inoculated with Pst DC3000 AvrBsT compared to Pi-0 leaves PLD Activity Is Required for AvrBsT-Dependent HR and PA Accumula- inoculated with Pst DC3000 vector (Fig. 1). By contrast, PA tion in Pi-0. We next determined the major enzymatic source of levels were similar in Col-0 leaves inoculated with Pst DC3000 PA in Pi-0 leaves. PA pools are generated directly and indirectly vector or Pst DC3000 AvrBsT (Fig. 1). Notably, total LysoPC by the hydrolytic activity of PLD and (PLC), levels were significantly lower in leaves in a sober1–1/AvrBsT- respectively (Fig. 3A) (16). For example, PLD cleaves phospho- dependent manner (Fig. S4A). The relative change of the other phospholipids (Fig. 1), lysophospholipids (Fig. S4A), and galac- tolipids (Fig. S4B) in Pi-0 compared to Col-0 in response to Pst DC3000 AvrBsT versus Pst DC3000 vector were remarkably similar, although the total concentration of lipids varied between ecotypes. MS analysis was repeated with an independent set of biological samples and similar results were obtained. In general, total PA levels were approximately 1.6- to 2.1-fold higher in Pi-0 leaves at 16 HPI in response to AvrBsT perturbation under these inoculation conditions. These data indicate that the elevated PA levels in Pi-0 leaves infected with Pst DC3000 AvrBsT correlate with HR induction and disease resistance (13). Moreover, they suggest that PA or a PA precursor is a SOBER1 substrate.

SOBER1 Overexpression Suppresses PA Accumulation. Transgenic Fig. 2. Total PA levels in Pi-0 and Pi-0 Pro35S::FLAG-SOBER1 (Pi-0 compl) Pi-0 lines (i.e., Pi-0 Pro35S::FLAG-SOBER1) overexpressing SO- leaves inoculated with Pst DC3000 vector or Pst DC3000 AvrBsT. Lipids were BER1 are susceptible to Pst DC3000 AvrBsT and do not produce extracted 16 HPI from inoculated leaves (5 ϫ 107 cells/mL) and analyzed by MS

HR (13). These lines were used to determine if SOBER1 to quantify PA. Values are means Ϯ SD (n ϭ 5). * ϭ Significant difference (t test, PLANT BIOLOGY overexpression abrogates PA accumulation in response to P Ͻ 0.05) in lipid composition when comparing leaves of the same ecotype.

Kirik and Mudgett PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 ͉ 20533 Downloaded by guest on September 26, 2021 (Fig. 1 and Fig. S2), we suspected that PA production in Pi-0 leaves was primarily due to a PLC-independent pathway. To verify this, we used a chemical approach to determine which enzyme class is required for AvrBsT-dependent PA accumula- tion and HR in Pi-0. Pi-0 leaves (n ϭ 100) were inoculated with a high concentration (3 ϫ 108 cells/mL) of each bacterial strain containing 1-butanol and/or neomycin. Typically, AvrBsT- elicited HR occurs between 8–12 HPI. HR symptoms were recorded at 8 HPI and are summarized in Table S1. Represen- tative HR phenotypes observed are shown in Fig. 3B. We also quantified PA levels in lipid extracts isolated from similarly infected leaves at 6 HPI, just before HR. Pi-0 leaves inoculated with Pst DC3000 AvrBsT produced HR (98%: Fig. 3B and Table S1) and contained high levels of PA (Fig. 3C). PA accumulated to much higher levels under these conditions (i.e., high titer and short time point) and was approx- imately 10-fold higher in Pi-0 leaves inoculated with Pst DC3000 AvrBsT compared to those inoculated with Pst DC3000 vector (Fig. 3C). By contrast, 68% of Pi-0 leaves inoculated with Pst DC3000 AvrBsT and 1-butanol did not produce HR (Fig. 3B and Table S1) or accumulate PA (Fig. 3C), indicating that PLD activity is required for both responses. Primary alcohols like 1-butanol antagonize PLD activity by stimulating PLD transphosphatidylation activity resulting in an increase in phos- phatidylalcohol products and a decrease in PA (16). Tert- butanol, an inactive analog of 1-butanol, did not affect AvrBsT- dependent HR production (Fig. 3B and Table S1)orPA accumulation (Fig. 3C). We also tested N-acylethanolamine (NAE), a PLD␣-specific inhibitor (17). NAE did not alter the HR response, suggesting that the activity of other PLD isoforms likely contribute to PA production and HR activation. Neomy- cin, which binds phosphatidylinositol bisphosphate (PIP2) and inhibits PIP2-activated PLC and PLD activity (16, 18), did not alter AvrBsT-dependent HR or PA levels (Fig. 3B and Table S1). A mixture of neomycin and 1-butanol was required to inhibit HR induced by Pst DC3000 AvrRpt2 (Fig. 3 B and C and Table S1), consistent with the finding that both PLD and PLC activity contribute to RPS2-mediated disease resistance (19). These data suggest that distinct PLD isoform activity is required for AvrBsT-dependent PA accumulation and HR in Pi-0. Moreover, these data suggest that SOBER1 suppresses PA levels in AvrBsT-infected leaves by hydrolyzing PA or PC, a precursor lipid that is hydrolyzed by PLD to produce PA.

SOBER1 Has PLA2 Activity and Hydrolyzes PC. We next tested if Fig. 3. PLD activity is required for AvrBsT-dependent HR in Pi-0. (A) Sche- SOBER1 possessed phospholipase activity in vitro using three matic of phospholipase-dependent reactions that generate PA in plant cells. candidate substrates: PA, PC, and LysoPC. Recombinant His6- Lipid : PLD ϭ phospholipase D; PLC ϭ phospholipase C; LysoPLA ϭ SOBER1, mutant His6-SOBER1(H192A), and yeast His6- Lysophospholipase A; PLA ϭ phospholipase A; and DGK ϭ diacylglycerol APT1/LysoPLA were purified and incubated with vesicles con- kinase. Lipid molecules: G3P ϭ glycerol 3-phosphate; LysoPC ϭ lysophospho- taining fluorescent NBD-labeled analogues of PA, PC, and choline; PC ϭ phosphatidylcholine; PA ϭ phosphatidic acid; FA ϭ fatty acids; LysoPC. A plant enzyme extract capable of hydrolyzing all of the ϭ ϭ ϭ PIP2 phosphatidylinositol bisphosphate; DAG diacylglycerol; IP3 inositol substrates was used as a positive control (Fig. 4). None of the triphosphate. 1-butanol and neomycin modulate PLD and PLC activity, respec- purified enzymes hydrolyzed NBD-PA labeled at the sn-2 posi- tively. (B) HR symptoms in inoculated Pi-0 leaves after chemical treatment. tion above the level of spontaneous substrate hydrolysis detected Leaves were infiltrated with a high titer of bacteria (3 ϫ 108 cells/mL) Ϯ chemical(s). Bacteria ϭ Pst DC3000 vector, Pst DC3000 AvrBsT or Pst DC3000 in the buffer control (Fig. 4) or NBD-PA labeled at the sn-1 AvrRpt2. Chemicals ϭ 0.4% 1-butanol, 0.4% tert-butanol, 0.01% neomycin, or position. However, both His6-SOBER1 and yeast His6-APT1/ 0.4% 1-butanol ϩ 0.01% neomycin. HR symptoms were photographed 8 HPI. LysoPLA hydrolyzed the sn-2 acylester bond of NBD-PC result- Experiment was repeated three times. (C) Total PA levels in Pi-0 leaves inoc- ing in a 7-fold increase in fluorescence relative to the buffer ulated with bacteria and test chemicals. Leaves were inoculated as described control (Fig. 4). Incubation of mutant His6-SOBER1(H192A) in (B). At 6 HPI, lipids were extracted and analyzed by MS to quantify PA. with NBD-PC generated 3- to 4-fold less fluorescence (Fig. 4), Values are means Ϯ SD (n ϭ 3). * ϭ Significant difference (t test, P Ͻ 0.05). demonstrating that active site His residue is required for catal- ysis. Furthermore, hydrolysis of PC by His6-SOBER1 was opti- mal in buffer lacking Ca2ϩ indicating that SOBER1 lipids like PC to produce PA and choline. PLC cleaves phos- activity is Ca2ϩ-independent. By contrast, 1-acyl-NBD-LysoPC phatidylinositol bisphosphate (PIP2) to produce diacylglycerol was a poor substrate for both His6-SOBER1 and yeast His6- (DAG). Diacylglycerol kinase (DGK) then cleaves DAG to APT1/LysoPLA under the conditions tested (Fig. 4). These data produce PA. Because phosphatidylinositol (PI) levels did not show that SOBER1 has PLA2 activity (20) and hydrolyzes PC but significantly change in a sober1–1/AvrBsT-dependent manner not PA in vitro.

20534 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903859106 Kirik and Mudgett Downloaded by guest on September 26, 2021 signaling is poorly understood, in part because multiple PLD and PLC isoforms influence the local and transient accumulation of PA (24). Using Arabidopsis as a model genetic system to understand the molecular basis of AvrBsT-specific immunity, we discovered that SOBER1 plays a role in the regulation of PA in planta in response to AvrBsT perturbation. In plants naturally lacking SOBER1 activity (i.e., the Pi-0 ecotype), AvrBsT action within the plant cell resulted in elevated PA levels (Fig. 1). High PA levels correlated with HR (Fig. 3) and bacterial disease resis- tance (13) in a sober1–1/AvrBsT dependent manner. Pi-0 lines expressing SOBER1 by genetic complementation did not accu- mulate PA (Fig. 2) or produce HR (13). By contrast, SOBER1 did not affect AvrRpt2-elicited HR (Table S1), a response Fig. 4. SOBER1 hydrolyzes PC exhibiting PLA2 activity. Purified His6-SOBER1, mediated by PA produced by both the PLD- and PLC-dependent mutant His6-SOBER1(H192A), yeast His6-APT1/LysoPLA protein, or plant ex- ␮ pathways (19). This suggests that SOBER1 is not a general tract was incubated with 10 M of each phospholipid substrate (NBD-PA, suppressor of PA-mediated immune responses during infection. NBD-PC, NBD-LysoPC) for 20 min. Relative fluorescence Ϯ SD (n ϭ 3) is shown. Experiment was repeated four times. Rather SOBER1 appears to suppress PLD-dependent PA pro- duction in response to AvrBsT perturbation (see working model, Fig. S5). Whether or not SOBER1 functions as a global regulator

Inhibition of PLA2 Activity Activates AvrBsT-Dependent HR in SOBER1 of PA-mediated immunity triggered by other microbial elicitors Plants. We next determined if PLA2 activity was required to remains to be determined. suppress HR in Col-0 (SOBER1) and transgenic Pi-0 At this point, we cannot rule out that other lipid mediators might Pro35S::FLAG-SOBER1 leaves in response to Pst DC3000 also be involved in HR induction in the Pi-0 background. It is AvrBsT. For each line, leaves (n ϭ 50) were inoculated with a 3 ϫ possible that an unstable or short-lived PA-derived lipid mediates 108 cells/mL suspension of bacteria containing bromoenol lac- AvrBsT-elicited HR. Very-long-chain fatty acids (VLCFAs) and 2ϩ sphingolipids are known to function in lipid signaling associated tone (BEL), a Ca -independent, irreversible inhibitor of PLA2 activity (21). HR symptoms determined at 8 HPI are shown in with HR and disease resistance in plants (25, 26). Significant Table S1. Fifty-six percent of Col-0 leaves inoculated with Pst changes in the distinct lengths, however, were not detected DC3000 AvrBsT ϩ BEL produced HR (Fig. 5A and Table S1), in our MS data. whereas very few leaves (6%) inoculated with Pst DC3000 The biochemical mechanism by which AvrBsT increases PA AvrBsT ϩ DMSO (the BEL solvent) produced HR. BEL or levels in sober1–1 plants is not clear. Activation of PLD isoforms DMSO treatment; however, did not affect the timing or intensity is likely involved considering that PLD activity was required for of Pst DC3000 AvrRpt2-dependent HR (Fig. 5 and Table S1). elevated PA levels and HR induction in sober1–1 leaves in response to AvrBsT (Fig. 3). Arabidopsis contains 12 isoforms The majority (72%) of Pi-0 Pro ::FLAG-SOBER1 leaves inoc- 35S partitioned into six classes depending on sequence and biochem- ulated with Pst DC3000 AvrBsT ϩ BEL produced HR (Fig. 5B ical properties (27). Different PLD isoforms are activated by and Table S1). These data suggest that SOBER1’s PLA activity 2 pathogens (28), participate in salicylic acid-dependent signaling is required to suppress AvrBsT-dependent HR in Arabidopsis. (29) and mediate PA-dependent oxidative stress responses (30). Discussion Determining which PLD enzymes are involved in AvrBsT trig- gered immunity should reveal where PA is produced within the PA is an important lipid second messenger associated with stress plant cell and help to identify which host target(s) and pathways signaling in both plants and animals (22). In plants, PA accu- are activated during this interaction. The recent link established mulates in response to wounding, pathogen attack, osmotic between PA-binding proteins and MAPK cascades that control stress, and thermal stress (22, 23). The activation of two phos- oxidative stress (31) and plant senescence (32) suggest potential pholipases, PLD and PLC, is the initial step that leads to the host targets. formation of PA. How plant cells regulate PA levels and A major PA pool in cells comes from PLD-dependent hydro- lysis of the structural phospholipid PC (27). Thus, it was con- ceivable that SOBER1 might compete with PLD for PC avail- ability and directly affect PA accumulation during Pst DC3000 AvrBsT infection. Consistent with this hypothesis, SOBER1 can hydrolyze PC in vitro at the sn-2 position indicating that it has PLA2 activity (Fig. 4). Furthermore, chemical inhibition of enzymes with PLA2 activity in Col-0 (SOBER1) and transgenic Pi-0 Pro35S::FLAG-SOBER1 leaves inoculated with Pst DC3000 AvrBsT resulted in HR (Fig. 5), phenocopying the sober1–1 mutation. These studies suggest that SOBER1 PLA2 activity is required to suppress AvrBsT-specific immune responses. A number of PLA2 enzymes have been linked to plant defense signaling (33). The majority of theses proteins are patatin- related plant PLAs (34–37). Interestingly, AtPLP2 is a plant susceptibility factor during fungal and bacterial infection (37). By contrast, other PLA s promote cell death associated with de- Fig. 5. PLA activity is required to suppress AvrBsT-induced HR in Arabidop- 2 2 fense and development (34–36, 38). SOBER1, however, is not sis. Col-0 (SOBER1) (A) and Pi-0 Pro35S::FLAG-SOBER1 complemented line (Pi-0 sober1–1 compl) (B) were inoculated with a 3 ϫ 108 cells/mL suspension of Pst homologous to any members in the Arabidopsis PLA family (33). DC3000 vector, Pst DC3000 AvrBsT, or Pst DC3000 AvrRpt2 containing DMSO This implies that enzymes with PLA2-like activity from different

or 80 ␮M BEL in DMSO. HR symptoms were recorded at 8 HPI. Experiment was lipase families may play distinct roles as positive and negative PLANT BIOLOGY repeated three times. regulators of phospholipid-mediated cell death in plants.

Kirik and Mudgett PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 ͉ 20535 Downloaded by guest on September 26, 2021 ␮ How SOBER1 PLA2 activity negatively regulates PA accu- icals (0.4% 1-butanol, 0.01% neomycin, or 80 M BEL in DMSO) were added mulation and defense signaling remains to be determined. to the suspensions to modulate PLD, PLC, and PLA2 activity, respectively. SOBER1 could compete with distinct PLD isoforms for sub- Symptoms were recorded 8 HPI. strate availability (e.g., PC) at or near the plasma membrane, controlling the flux of phospholipid metabolism during infec- Plant Growth Conditions. Arabidopsis thaliana was grown in Metro-Mix 200 ␮ ⅐ Ϫ2⅐ Ϫ1 tion. Alternatively, SOBER1 could regulate PLD activity by soil (Premier Horticulture) in growth chambers (22 °C, 60% RH, 125 E m s fluorescent illumination) on an 8-h light/16-h dark cycle. product feedback inhibition, as observed in animal cells (39). The fact that SOBER1 orthologs possess acyl protein thioester- Lipid Profiling. Lipid extraction, analysis and quantification of phospholipid ase activity (14, 15) also suggests that SOBER1 could impact the and galactolipid species were performed as described (34, 42). Col-0 and Pi-0 assembly and/or function of PLD complexes at the plasma leaves were infiltrated with a 5 ϫ 107 cells/mL suspension of Pst DC3000 vector membrane. or Pst DC3000 AvrBsT in 1 mM MgCl2. Lipids were extracted from leaves at 16 In addition to PA, AvrBsT-induced disease resistance in HPI and analyzed on an electrospray ionization triple quadrupole mass spec- Arabidopsis requires NDR1, SID2, and NPR1 (13). Involvement trometer (API 4000, Applied Biosystems). Five replicates for each treatment of a specific R-gene is likely, although no candidates have were performed. Paired values were subjected to a Student’s t test to deter- emerged yet from our genetic analyses. Alternatively, an R-gene mine statistical significance. To specifically quantify PA levels, Pi-0 leaves were independent pathway(s) could result in immunity considering infiltrated with a high titer (3 ϫ 108 cells/mL) of Pst DC3000 vector or Pst Ϯ that PA is a multifunctional stress signal (22). AvrBsT-specific DC3000 AvrBsT in 1 mM MgCl2 0.4% 1-butanol and/or 0.01% neomycin. immune responses resembling R-gene mediated disease resis- Lipids were extracted at 6 HPI before the onset of HR and processed, as tance have been reported for the Solanaceae, the natural host for described above. Xanthomonas strains that carry AvrBsT (40). All pepper culti- Enzyme Assays. His6-SOBER1, His6-SOBER1(H192A), and yeast His6-APT1/ vars tested to date are resistant to Xcv expressing AvrBsT, LysoPLA were purified as described in ref. 13. Protein (0.1 ␮g) was incubated whereas tomato plants are generally susceptible (40). The R- for 20 min with vesicles containing 10 ␮M of NBD-labeled phospholipids gene in pepper (designated as BST) has not been cloned. It is (Avanti) as described (34, 43). NBD-PA ϭ 1-acyl-2-[12-[(7-nitro-2–1,3- unlikely that resistance in pepper is due to a mutation in a benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphate (monoam- SOBER1 ortholog because reported SOBER1-like ESTs are monium salt). NBD-PC ϭ 1-acyl-2-[12-[(7-nitro-2–1,3-benzoxadiazol-4- predicted to encode wild type proteins. Progress in dissecting yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine. NBD-LysoPC ϭ 1-[12-[(7- AvrBsT-triggered immune signaling in Arabidopsis provides a nitro-2–1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-2-hydroxy-sn-glycero-3- foundation to elucidate the molecular basis of disease resistance phosphocholine. Fluorescence was measured with a fluorometer (excitation ϭ in pepper. 485 nm; emission ϭ 535 nm).

Materials and Methods ACKNOWLEDGMENTS. Thanks to Ruth Welti and Mary Roth at the Kansas Lipidomics Research Center Analytical Laboratory (supported by National Pathogen Strains and HR Assays. Strains used: Pseudomonas syringae pv. Science Foundation Experimental Program to Stimulate Competitive Research tomato (Pst) DC3000; Pst DC3000 pDD62 (empty vector); Pst DC3000 program EPS-0236913 and the State of Kansas) for lipid analysis, Kent Chap- pVSP61(avrRpt2); and Pst DC3000 pVSP61(avrRpt21–100::avrBsT11–350-HA) (13). man and Aruna Kilaru (University of North Texas, Denton, TX) for NAE Pst DC3000 was grown on nutrient yeast glycerol agar (NYGA) medium (41) at inhibitor, and colleagues (S. Long, D. Bergmann, C. Yanofsky and D. Ehrhardt) 8 28 °C. For HR assays, a bacterial suspension (3 ϫ 10 cells/mL) in 1 mM MgCl2 for equipment use. This work was funded by the National Institutes of Health was hand-infiltrated into 4- to 5-week-old leaves using a 1-cc syringe. Chem- grant 1RO1 GM-068886 awarded to M.B.M.

1. Alfano JR, Collmer A (2004) Type III secretion system effector proteins: Double agents 18. Qin W, Pappan K, Wang X (1997) Molecular heterogeneity of phospholipase D (PLD). in bacterial disease and plant defense. Annu Rev Phytopathol 42:385–414. Cloning of PLDgamma and regulation of plant PLDgamma, -beta, and -alpha by 2. Coburn B, Sekirov I, Finlay BB (2007) Type III secretion systems and disease. Clin polyphosphoinositides and calcium. J Biol Chem 272:28267–28273. Microbiol Rev 20:535–549. 19. Andersson MX, Kourtchenko O, Dangl JL, Mackey D, Ellerstrom M (2006) Phospho- 3. Gohre V, Robatzek S (2008) Breaking the barriers: Microbial effector molecules subvert lipase-dependent signalling during the AvrRpm1- and AvrRpt2-induced disease resis- plant immunity. Annu Rev Phytopathol 46:189–215. tance responses in Arabidopsis thaliana. Plant J 47:947–959. 4. Abramovitch RB, Anderson JC, Martin GB (2006) Bacterial elicitation and evasion of 20. Wang X (2001) Plant . Annu Rev Plant Physiol Plant Mol Biol 52:211– plant innate immunity. Nat Rev Mol Cell Biol 7:601–611. 231. 5. Zipfel C (2008) Pattern-recognition receptors in plant innate immunity. Curr Opin 21. Hazen SL, Zupan LA, Weiss RH, Getman DP, Gross RW (1991) Suicide inhibition of canine Immunol 20:10–16. myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based dis- 6. Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323–329. crimination between calcium-dependent and -independent phospholipases A2. J Biol 7. Staskawicz BJ, Mudgett MB, Dangl JL, Galan JE (2001) Common and contrasting themes Chem 266:7227–7232. of plant and animal diseases. Science 292:2285–2289. 22. Testerink C, Munnik T (2005) Phosphatidic acid: A multifunctional stress signaling lipid 8. Orth K, et al. (2000) Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like in plants. Trends Plants Sci 10:368–375. protein protease. Science 290:1594–1597. 23. Mishkind M, Vermeer JE, Darwish E, Munnik T (2009) Heat stress activates phospho- 9. Mukherjee S, et al. (2006) Yersinia YopJ acetylates and inhibits kinase activation by lipase D and triggers PIP accumulation at the plasma membrane and nucleus. Plant J blocking phosphorylation. Science 312:1211–1214. (in press). 10. Rytkonen A, et al. (2007) SseL, a Salmonella deubiquitinase required for macrophage 24. Munnik T, Testerink C (2009) Plant phospholipid signaling: ‘‘In a nutshell.’’ J Lipid Res killing and virulence. Proc Natl Acad Sci USA 104:3502–3507. 50 Suppl:S260–265. 11. Roden JA, et al. (2004) A genetic screen to isolate type III effectors translocated into 25. Raffaele S, et al. (2008) A MYB transcription factor regulates very-long-chain fatty acid pepper cells during Xanthomonas infection. Proc Natl Acad Sci USA 101:16624– for activation of the hypersensitive cell death response in Arabidopsis. 16629. Plant Cell 20:752–767. 12. Ciesiolka LD, et al. (1999) Regulation of expression of avirulence gene avrRxv and 26. Shi L, et al. (2007) Involvement of sphingoid bases in mediating reactive oxygen interme- identification of a family of host interaction factors by sequence analysis of avrBsT. Mol diate production and programmed cell death in Arabidopsis. Cell Res 17:1030–1040. Plant Microbe Interact 12:35–44. 27. Li M, Hong Y, Wang X (2009) Phospholipase D- and phosphatidic acid-mediated 13. Cunnac S, et al. (2007) A conserved carboxylesterase is a suppressor of avrbst-elicited signaling in plants. Biochim Biophys Acta (in press). resistance in Arabidopsis. Plant Cell 19:688–705. 28. de Torres Zabela M, Fernandez-Delmond I, Niittyla T, Sanchez P, Grant M (2002) Differ- 14. Duncan JA, Gilman AG (2002) Characterization of Saccharomyces cerevisiae acyl- ential expression of genes encoding Arabidopsis phospholipases after challenge with protein thioesterase 1, the enzyme responsible for G protein alpha subunit deacylation virulent or avirulent Pseudomonas isolates. Mol Plant Microbe Interact 15:808–816. in vivo. J Biol Chem 277:31740–31752. 29. Krinke O, et al. (2009) Phospholipase D activation is an early component of the salicylic 15. Zhang YY, Dennis EA (1988) Purification and characterization of a lysophospholipase acid signaling pathway in Arabidopsis cell suspensions. Plant Physiol 150:424–436. from a macrophage-like cell line P388D1. J Biol Chem 263:9965–9972. 30. Sang Y, Cui D, Wang X (2001) Phospholipase D and phosphatidic acid-mediated 16. Meijer HJ, Munnik T (2003) Phospholipid-based signaling in plants. Annu Rev Plant Biol generation of superoxide in Arabidopsis. Plant Physiol 126:1449–1458. 54:265–306. 31. Anthony RG, Khan S, Costa J, Pais MS, Bogre L (2006) The Arabidopsis protein kinase 17. Austin-Brown SL, Chapman KD (2002) Inhibition of phospholipase D alpha by N- PTI1–2 is activated by convergent phosphatidic acid and oxidative stress signaling acylethanolamines. Plant Physiol 129:1892–1898. pathways downstream of PDK1 and OXI1. J Biol Chem 281:37536–37546.

20536 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903859106 Kirik and Mudgett Downloaded by guest on September 26, 2021 32. Testerink C, Larsen PB, van der Does D, van Himbergen JA, Munnik T (2007) Phospha- 38. Reina-Pinto JJ, et al. (2009) Misexpression of fatty acid elongation1 in the Arabidopsis tidic acid binds to and inhibits the activity of Arabidopsis CTR1. J Exp Bot 58:3905–3914. epidermis induces cell death and suggests a critical role for phospholipase A2 in this 33. Ryu SB (2004) Phospholipid-derived signaling mediated by phospholipase A in plants. process. Plant Cell 21:1252–1272. Trends Plants Sci 9:229–235. 39. Ryu SB, Palta JP (2000) Specific inhibition of rat brain phospholipase D by lysophos- 34. Yang W, et al. (2007) AtPLAI is an acyl hydrolase involved in basal jasmonic acid production pholipids. J Lipid Res 41:940–944. and Arabidopsis resistance to Botrytis cinerea. J Biol Chem 282:18116–18128. 40. Stall RE, Jones JB, Minsavage GV (2009) Durability of resistance in tomato and pepper 35. Holk A, Rietz S, Zahn M, Quader H, Scherer GF (2002) Molecular identification of to xanthomonads causing bacterial spot. Annu Rev Phytopathol 47:265–284. cytosolic, patatin-related phospholipases A from Arabidopsis with potential functions 41. Turner P, Barber C, Daniels M (1984) Behaviour of the transposons Tn5 and Tn7 in in plant signal transduction. Plant Physiol 130:90–101. Xanthomonas campestris pv. campestris. Mol Gen Genet 195:101–107. 36. Dhondt S, Geoffroy P, Stelmach BA, Legrand M, Heitz T (2000) Soluble phospholipase 42. Welti R, et al. (2002) Profiling membrane lipids in plant stress responses. Role of A2 activity is induced before oxylipin accumulation in tobacco mosaic virus-infected phospholipase D alpha in freezing-induced lipid changes in Arabidopsis. J Biol Chem tobacco leaves and is contributed by patatin-like enzymes. Plant J 23:431–440. 277:31994–32002. 37. La Camera S, et al. (2005) A pathogen-inducible patatin-like lipid acyl hydrolase 43. Moreau RA (1989) An evaluation o NBD-phospholipids as substrates for the measure- facilitates fungal and bacterial host colonization in Arabidopsis. Plant J 44:810–825. ment of phospholipase and lipase activities. Lipids 24:691–699. PLANT BIOLOGY

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