SOBER1 phospholipase activity suppresses phosphatidic acid accumulation and plant immunity in response to bacterial effector AvrBsT.
ABSTRACT: Arabidopsis thaliana ecotype Pi-0 is resistant to Pseudomonas syringae pathovar tomato (Pst) strain DC3000 expressing the T3S effector protein AvrBsT. Resistance is due to a loss of function mutation (sober1-1) in a conserved alpha/beta hydrolase, SOBER1 (Suppressor of AvrBsT Elicited Resistance1). Members of this superfamily possess phospholipase and carboxylesterase activity with diverse substrate specificity. The nature of SOBER1 enzymatic activity and substrate specificity was not known. SOBER1-dependent suppression of the hypersensitive response (HR) in Pi-0 suggested that it might hydrolyze a plant lipid or precursor required for HR induction. Here, we show that Pi-0 leaves infected with Pst DC3000 expressing AvrBsT accumulated higher levels of phosphatidic acid (PA) compared to leaves infected with Pst DC3000. Phospholipase D (PLD) activity was required for high PA levels and AvrBsT-dependent HR in Pi-0. Overexpression of SOBER1 in Pi-0 reduced PA levels and inhibited HR. These data implicated PA, phosphatidylcholine (PC) and lysophosphatidylcholine (LysoPC) as potential SOBER1 substrates. Recombinant His(6)-SOBER1 hydrolyzed PC but not PA or LysoPC in vitro indicating that the enzyme has phospholipase A(2) (PLA(2)) activity. Chemical inhibition of PLA(2) activity in leaves expressing SOBER1 resulted in HR in response to Pst DC3000 AvrBsT. These data are consistent with the model that SOBER1 PLA(2) activity suppresses 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.
Project description:Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1's defense function, which is linked to the activation of ETI.
Project description:Pathogen infection of higher plants often induces rapid production of phosphatidic acid (PA) and changes in lipid profiles, but the enzymatic basis and the function of the lipid change in pathogen-plant interactions are not well understood. Infection of phospholipase D ?1 (PLD?1)-deficient plants by Pseudomonas syringae tomato pv DC3000 (Pst DC30000) resulted in less bacterial growth than in wild-type plants, and the effect was more profound in virulent Pst DC3000 than avirulent Pst DC3000 (carrying the avirulence gene avrRpt2) infection. The expression levels of salicylic acid (SA)-inducible genes were higher, but those inducible by jasmonic acid (JA) showed lower expression in PLD?1 mutants than in wild-type plants. However, PLD?1-deficient plants were more susceptible than wild-type plants to the fungus Botrytis cinerea. The PLD?1-deficient plants had lower levels of PA, JA and JA-related defense gene expression after B. cinerea inoculation. PLD?1 plays a positive role in pathogen-induced JA production and plant resistance to the necrotrophic fungal pathogen B. cinerea, but a negative role in the SA-dependent signaling pathway and plant tolerance to infection with biotrophic Pst DC3000. PLD?1 is responsible for most of the increase in PA production in response to necrotrophic B. cinerea and virulent Pst DC3000 infection, but contributes less to avirulent Pst DC3000 (avrRpt2)-induced PA production.
Project description:<h4>Background</h4>Plants have evolved an array of constitutive and inducible defense strategies to restrict pathogen ingress. However, some pathogens still manage to invade plants and impair growth and productivity. Previous studies have revealed several key regulators of defense responses, and efforts have been made to use this information to develop disease resistant crop plants. These efforts are often hampered by the complexity of defense signaling pathways. To further elucidate the complexity of defense responses, we screened a population of T-DNA mutants in Colombia-0 background that displayed altered defense responses to virulent Pseudomonas syringae pv. tomato DC3000 (Pst DC3000).<h4>Results</h4>In this study, we demonstrated that the Arabidopsis Purple Acid Phosphatse5 (PAP5) gene, induced under prolonged phosphate (Pi) starvation, is required for maintaining basal resistance to certain pathogens. The expression of PAP5 was distinctly induced only under prolonged Pi starvation and during the early stage of Pst DC3000 infection (6 h.p.i). T-DNA tagged mutant pap5 displayed enhanced susceptibility to the virulent bacterial pathogen Pst DC3000. The pap5 mutation greatly reduced the expression of pathogen inducible gene PR1 compared to wild-type plants. Similarly, other defense related genes including ICS1 and PDF1.2 were impaired in pap5 plants. Moreover, application of BTH (an analog of SA) restored PR1 expression in pap5 plants.<h4>Conclusion</h4>Taken together, our results demonstrate the requirement of PAP5 for maintaining basal resistance against Pst DC3000. Furthermore, our results provide evidence that PAP5 acts upstream of SA accumulation to regulate the expression of other defense responsive genes. We also provide the first experimental evidence indicating the role PAP5 in plant defense responses.
Project description:To reveal the underlying molecular mechanism of GH3.5 action in modulating the SA and auxin pathways, we performed transcriptional profiling of gh3.5-1D plants after infection with or without Pst DC3000(avrRpt2) on a global scale using the Affymetrix Arabidopsis ATH1 GeneChip Experiment Overall Design: Each 3 leaves of plants of 5-week-old wild type Columbia-0 and mutant gh3.5-1D (heterozygous) were inoculated with Pst DC3000(avrRpt2)at 105 cfu mL-1. Leaves were harvested at 0 h (uninoculated) and 48 hours after inoculation. Three biological repeats were performed on Columbia-0 (wild-type, Col-0) and gh3.5-1D (mutant) after infection with or without Pst DC3000(avrRpt2), respectively.
Project description:During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A<sub>2</sub> (PLA<sub>2</sub>), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA<sub>2</sub> in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA<sub>2</sub> enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA<sub>2</sub> involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA<sub>2</sub>α, cPLA<sub>2</sub>γ, iPLA<sub>2</sub>β, or iPLA<sub>2</sub>γ. Downregulation of the β isoform of iPLA<sub>2</sub> blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.
Project description:Lysophosphatidic acids (LysoPAs) and lysophosphatidylserine (LysoPS) are emerging lipid mediators proposed to be involved in the pathogenesis of acute coronary syndrome (ACS). In this study, we attempted to elucidate how LysoPA and LysoPS become elevated in ACS using human blood samples collected simultaneously from culprit coronary arteries and peripheral arteries in ACS subjects. We found that: 1) the plasma LysoPA, LysoPS, and lysophosphatidylglycerol levels were not different, while the lysophosphatidylcholine (LysoPC), lysophosphatidylinositol, and lysophosphatidylethanolamine (LysoPE) levels were significantly lower in the culprit coronary arteries; 2) the serum autotaxin (ATX) level was lower and the serum phosphatidylserine-specific phospholipase A<sub>1</sub> (PS-PLA<sub>1</sub>) level was higher in the culprit coronary arteries; 3) the LysoPE and ATX levels were significant explanatory factors for the mainly elevated species of LysoPA, except for 22:6 LysoPA, in the peripheral arteries, while the LysoPC and LysoPE levels, but not the ATX level, were explanatory factors in the culprit coronary arteries; and 4) 18:0 and 18:1 LysoPS were significantly correlated with PS-PLA<sub>1</sub> only in the culprit coronary arteries. In conclusion, the origins of LysoPA and LysoPS might differ between culprit coronary arteries and peripheral arteries, and substrates for ATX, such as LysoPC and LysoPE, might be important for the generation of LysoPA in ACS.
Project description:Plant 9-lipoxygenases (9-LOX) and α-dioxygenases (α-DOX) initiate the synthesis of oxylipins after bacterial infection. Here, the role of these enzymes in plant’s defense was investigated using individual Arabidopsis thaliana lox1 and dox1 mutants and a double lox1 dox1 mutant. Studies with Pseudomonas syringae pv tomato (Pst) revealed the enhanced susceptibility of lox1 to the virulent strain Pst DC3000 and the partial impairment of lox1 and dox1 mutants to activate systemic acquired resistance. Notably, both defects were enhanced in the lox1 dox1 plants as compared with individual mutants. We found that pre-treatment with 9-LOX- and -DOX-generated oxylipins protected plant tissues against bacterial infection. The strongest effect in this respect was exerted by 9-ketooctadecatrienoic acid (9-KOT), which is produced from linolenic acid by 9-LOX. Quantification of 9-KOT revealed its accumulation after bacterial infection. The levels were reduced in lox1 and lox1 dox1 plants but strongly increased in the dox1 mutant due to metabolic interaction of the two pathways. Transcriptional analyses indicated that 9-KOT pre-treatment modifies hormone homeostasis during bacterial infection. The nature of the changes detected suggested that 9-KOT interfers the hormonal changes caused by bacterial effectors. This notion was substantiated by the finding that 9-KOT failed to reduce the growth of PstDC3000hrpA, a mutant compromised in effector secretion, and of the avirulent strain Pst DC3000 avrRpm1. Further support to the action of the 9-LOX- and -DOX-oxylipin pathways as modulators of hormone homeostasis was the observation that lox1 dox1 seedlings are hypersensitive to the growth-inhibitory effect of ABA and showed enhanced activation of ABA-inducible marker genes. Two experiments, Col-0 treated with 9-KOT vs. Col-0 treated with water and Col-0 treated with 9-KOT vs. Col-0 treated with water after treatment with Pst DC3000. Biological replicates: 4 control replicates treated , 4 9-KOT treated replicates; 4 control treated and Pst DC3000 replicates and 4 9-KOT treated and Pst DC3000 replicates
Project description:The intrinsic defense mechanisms of plants toward pathogenic bacteria have been widely investigated for years and are still at the center of interest in plant biosciences research. This study investigated the role of the <i>AtbZIP62</i> gene encoding a transcription factor (TF) in the basal defense and systemic acquired resistance in Arabidopsis using the reverse genetics approach. To achieve that, the <i>atbzip62</i> mutant line (lacking the <i>AtbZIP62</i> gene) was challenged with <i>Pseudomonas syringae</i> pv. <i>tomato</i> (<i>Pst DC3000</i>) inoculated by infiltration into Arabidopsis leaves at the rosette stage. The results indicated that <i>atbzip62</i> plants showed an enhanced resistance phenotype toward <i>Pst DC3000 vir</i> over time compared to Col-0 and the susceptible disease controls, <i>atgsnor1-3</i> and <i>atsid2.</i> In addition, the transcript accumulation of pathogenesis-related genes, <i>AtPR1</i> and <i>AtPR2</i>, increased significantly in <i>atbzip62</i> over time (0-72 h post-inoculation, hpi) compared to that of <i>atgsnor1-3</i> and <i>atsid2</i> (susceptible lines), with <i>AtPR1</i> prevailing over <i>AtPR2</i>. When coupled with the recorded pathogen growth (expressed as a colony-forming unit, CFU mL<sup>-1</sup>), the induction of <i>PR</i> genes, associated with the salicylic acid (SA) defense signaling, in part explained the observed enhanced resistance of <i>atbzip62</i> mutant plants in response to <i>Pst DC3000 vir</i>. Furthermore, when <i>Pst DC3000 avrB</i> was inoculated, the expression of <i>AtPR1</i> was upregulated in the systemic leaves of Col-0, while that of <i>AtPR2</i> remained at a basal level in Col-0. Moreover, the expression of <i>AtAZI</i> (a systemic acquired resistance -related) gene was significantly upregulated at all time points (0-24 h post-inoculation, hpi) in <i>atbzip62</i> compared to Col-0 and <i>atgsnor1-3</i> and <i>atsid2</i>. Under the same conditions, <i>AtG3DPH</i> exhibited a high transcript accumulation level 48 hpi in the <i>atbzip62</i> background. Therefore, all data put together suggest that <i>AtPR1</i> and <i>AtPR2</i> coupled with <i>AtAZI</i> and <i>AtG3DPH</i>, with <i>AtAZI</i> prevailing over <i>AtG3DPH</i>, would contribute to the recorded enhanced resistance phenotype of the <i>atbzip62</i> mutant line against <i>Pst DC3000</i>. Thus, the <i>AtbZIP62</i> TF is proposed as a negative regulator of basal defense and systemic acquired resistance in plants under <i>Pst DC3000</i> infection.
Project description:The nonhost-specific phytotoxin coronatine (COR) produced by several pathovars of Pseudomonas syringae functions as a jasmonic acid-isoleucine (JA-Ile) mimic and contributes to disease development by suppressing plant defense responses and inducing reactive oxygen species in chloroplast. It has been shown that the F-box protein CORONATINE INSENSITIVE 1 (COI1) is the receptor for COR and JA-Ile. JASMONATE ZIM DOMAIN (JAZ) proteins act as negative regulators for JA signaling in Arabidopsis. However, the physiological significance of JAZ proteins in P. syringae disease development and nonhost pathogen-induced hypersensitive response (HR) cell death is not completely understood. In this study, we identified JAZ genes from tomato, a host plant for P. syringae pv. tomato DC3000 (Pst DC3000), and examined their expression profiles in response to COR and pathogens. Most JAZ genes were induced by COR treatment or inoculation with COR-producing Pst DC3000, but not by the COR-defective mutant DB29. Tomato SlJAZ2, SlJAZ6 and SlJAZ7 interacted with SlCOI1 in a COR-dependent manner. Using virus-induced gene silencing (VIGS), we demonstrated that SlJAZ2, SlJAZ6 and SlJAZ7 have no effect on COR-induced chlorosis in tomato and Nicotiana benthamiana. However, SlJAZ2-, SlJAZ6- and SlJAZ7-silenced tomato plants showed enhanced disease-associated cell death to Pst DC3000. Furthermore, we found delayed HR cell death in response to the nonhost pathogen Pst T1 or a pathogen-associated molecular pattern (PAMP), INF1, in SlJAZ2- and SlJAZ6-silenced N. benthamiana. These results suggest that tomato JAZ proteins regulate the progression of cell death during host and nonhost interactions.
Project description:The goal of the microarray experiment was to determine the induction kinetics of transcriptome changes in the Arabidopsis mutant Atelp2, npr1 and wild type in response to infection of the avirulent bacterial pathogen Pst DC3000/avrRpt2. Results indicated that Atelp2 exhibited slower kinetcis of transcriptional changes than the wild type after Pst DC3000/avrRpt2 infection, whereas npr1 did not show significant alteration in the induction kinetics. Three biological replicates with leaves from 8 plants per sample were collected at 0, 4, 8, and 12 hours after inoculation with the avirulent bacterial pathogen Pst DC3000/avrRpt2. After extraction, RNA concentration was determined on a NanoDrop Spectrophotometer (Thermofisher Scientific, Waltham, MA) and sample quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Each replicate was used as one sample for microarray analysis.