Host-specific plant signal and G-protein activator, mastoparan, trigger differentiation of zoospores of the phytopathogenic oomycete Aphanomyces cochlioides

We found that the gradient of a host-specific attractant, cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone) isolated from the roots of spinach triggered encystment followed by germination of zoospores of Aphanomyces cochlioidesat a concentration less than micromolar order. This compound did not affect the growth and reproduction of this phytopathogen up to 10^–6 M concentration in the culture medium. We also observed that mastoparan, an activator of heterotrimeric G-protein could inhibit the motility of zoospores and then strikingly effect encystment followed by 60–80% germination of cysts. Concomitant application of cochliophilin A and mastoparan showed stronger encystment followed by 100% germination of cysts. In addition, we have observed that chemicals interfering with phospholipase C activity (neomycin) and Ca²⁺ influx/release (EGTA and loperamide) suppress cochliophilin A or mastoparan induced encystment and germination. These results suggest that G-protein mediated signal transduction mechanism may be involved in the differentiation of the A. cochlioides zoospores. This is the first report on the differentiation of oomycete zoospores initiated by a host-specific plant signal or a G-protein activator.     

Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation

Rhizobium-secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [(32)P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.     

几种真菌发酵液对致病疫霉的抑制作用

测定了８种真菌发酵液在５种不同浓度下对致病疫霉菌丝生长、游动孢子静止、静止胞萌发、附着胞形成和侵入丝形成等不同阶段的影响。结果表明,供试真菌不同浓度的发酵液,对致病疫霉上述各个阶段均有一定程度的抑制作用,并均随发酵液浓度增加,抑制作用逐渐增强,浓度为１００％时,抑制作用均达到最高。其中,立枯丝核菌发酵液的抑制作用最强,浓度为１００％时,对致病疫霉菌丝生长的抑制率达到９０．４％,而静止胞萌发率仅为２．４％,附着胞及侵入丝均未见形成。     

The Ang II-induced growth of vascular smooth muscle cells involves a phospholipase D-mediated signaling mechanism

Angiotensin (Ang) II acts as a mitogen in vascular smooth muscle cells (VSMC) via the activation of multiple signaling cascades, including phospholipase C, tyrosine kinase, and mitogen-activated protein kinase pathways. However, increasing evidence supports signal-activated phospholipases A(2) and D (PLD) as additional mechanisms. Stimulation of PLD results in phosphatidic acid (PA) formation, and PA has been linked to cell growth. However, the direct involvement of PA or its metabolite diacylglycerol (DAG) in Ang II-induced growth is unclear. PLD activity was measured in cultured rat VSMC prelabeled with [(3)H]oleic acid, while the incorporation of [(3)H]thymidine was used to monitor growth. We have previously reported the Ang II-dependent, AT(1)-coupled stimulation of PLD and growth in VSMC. Here, we show that Ang II (100 nM) and exogenous PLD (0.1-100 units/mL; Streptomyces chromofuscus) stimulated thymidine incorporation (43-208% above control). PA (100 nM-1 microM) also increased thymidine incorporation to 135% of control. Propranolol (100 nM-10 microM), which inhibits PA phosphohydrolase, blocked the growth stimulated by Ang II, PLD, or PA by as much as 95%, an effect not shared by other beta-adrenergic antagonists. Propranolol also increased the production of PA in the presence of Ang II by 320% and reduced DAG and arachidonic acid (AA) accumulation. The DAG lipase inhibitor RHC-80267 (1-10 microM) increased Ang II-induced DAG production, while attenuating thymidine incorporation and release of AA. Thus, it appears that activation of PLD, formation of PA, conversion of PA to DAG, and metabolism of DAG comprise an important signaling cascade in Ang II-induced growth of VSMC.     

Secretions of MMP-9 by soluble glucocorticoid-induced tumor necrosis factor receptor (sGITR) mediated by protein kinase C (PKC)delta and phospholipase D (PLD) in murine macrophage

The secretion of matrix metalloproteinase (MMP-9) is stimulated by the glucocorticoid-induced tumor necrosis factor receptor (GITR), a new tumor necrosis factor receptor (TNFR) family, in murine macrophages via an activation of protein kinase C (PKC)delta and phospholipase D (PLD). Secretions of MMP-9 are stimulated by the phosphatidic acid (PA), a product of PLD activity and an inhibition of PA production by a 1-propanol inhibited secretion of MMP-9 by soluble GITR (sGITR). MMP-9 is not secreted by diacylglycerol (DAG) and an inhibitor of PA phosphatase has no effect on the secretion induced by sGITR, indicating that PA is responsible for MMP-9 secretion in murine macrophages. Our data indicates that sGITR-induced activation of PKCdelta and PLD increases MMP-9 secretions in macrophages.     

Nod factor and elicitors activate different phospholipid signaling pathways in suspension-cultured alfalfa cells

Lipo-chitooligosaccharides (Nod factors) are produced by symbiotic Rhizobium sp. bacteria to elicit Nod responses on their legume hosts. One of the earliest responses is the formation of phosphatidic acid (PA), a novel second messenger in plant cells. Remarkably, pathogens have also been reported to trigger the formation of PA in nonlegume plants. To investigate how host plants can distinguish between symbionts and pathogens, the effects of Nod factor and elicitors (chitotetraose and xylanase) on the formation of PA were investigated in suspension-cultured alfalfa (Medicago sativa) cells. Theoretically, PA can be synthesized via two signaling pathways, i.e. via phospholipase D (PLD) and via phospholipase C in combination with diacylglycerol (DAG) kinase. Therefore, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used to determine the contribution of each pathway to PA formation. In support, PLD activity was specifically measured by using the ability of the enzyme to transfer the phosphatidyl group of its substrate to a primary alcohol. In practice, Nod factor, chitotetraose, and xylanase induced the formation of PA and its phosphorylated product DAG pyrophosphate within 2 min of treatment. However, whereas phospholipase C and DAG kinase were activated during treatment with all three different compounds, PLD was only activated by Nod factor. No evidence was obtained for the activation of phospholipase A(2).     

Role of phospholipase C and D signalling pathways in vasopressin-dependent myogenic differentiation

Arg⁸-vasopressin (AVP) is a potent inducer of myogenic differentiation stimulating the expression of myogenic regulatory factors. To understand the mechanism of its effect on myogenesis, we investigated the early signals induced by AVP in myogenic target cells. In the rat skeletal muscle cell line L6, AVP selectively stimulates phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) breakdown, through the activation of phospholipases C and D (PLC, PLD), as shown by the generation of Ins(1,4,5)P₃ and phosphatidylethanol (PtdEtOH), respectively. AVP induces the biphasic increase of sn-1,2-diacylglycerol (DAG) consisting in a rapid peak followed by a sustained phase, and the monophasic generation of phosphatidic acid (PA). Propranolol (a PA phosphatase inhibitor) and Zn²⁺ (a PLD inhibitor), abolish the sustained phase of DAG generation. Our data indicate that PtdIns-PLC activity is mainly responsible for the rapid phase of AVP-dependent DAG generation, whereas the sustained phase is dependent upon PtdCho-PLD activity and PA dephosphorylation, ruling out any significant role of DAG kinase. Modifications of PA level correlate with parallel changes of PLC activity, indicating a possible cross-talk between the two signal transduction pathways in the intact cell. PLD activation is elicited at AVP concentrations two orders of magnitude lower than those required for PLC activation. The differentiation of L6 myoblasts into multinucleated fibers is stimulated significantly by AVP at concentrations at which PLD, but not PLC, is activated. These data provide the first evidence for an important role of PLD in the mechanism of AVP-induced muscle differentiation. J. Cell. Physiol. 171:34–42, 1997. © 1997 Wiley-Liss, Inc.     

Ceramide does not inhibit protein kinase C beta-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) betaI and betaII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC beta-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.     

Activation of actin polymerization by phosphatidic acid derived from phosphatidylcholine in IIC9 fibroblasts

alpha-Thrombin induced a change in the cell morphology of IIC9 fibroblasts from a semiround to an elongated form, accompanied by an increase in stress fibers. Incubation of the cells with phospholipase D (PLD) from Streptomyces chromofuscus and exogenous phosphatidic acid (PA) caused similar morphological changes, whereas platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) induced different changes, e.g., disruption of stress fibers and cell rounding. alpha-Thrombin, PDGF, and exogenous PLD increased PA by 20-40%, and PMA produced a smaller increase. alpha-Thrombin and exogenous PLD produced rapid increases in the amount of filamentous actin (F-actin) that were sustained for at least 60 min. However, PDGF produced a transient increase of F-actin at 1 min and PMA caused no significant change. Dioctanoylglycerol was ineffective except at 50 micrograms/ml. Phospholipase C from Bacillus cereus, which increased diacylglycerol (DAG) but not PA, did not change F-actin content. Down-regulation of protein kinase C (PKC) did not block actin polymerization induced by alpha-thrombin. H-7 was also ineffective. Exogenous PA activated actin polymerization with a significant effect at 0.01 microgram/ml and a maximal increase at 1 microgram/ml. No other phospholipids tested, including polyphosphoinositides, significantly activated actin polymerization. PDGF partially inhibited PA-induced actin polymerization after an initial increase at 1 min. PMA completely or largely blocked actin polymerization induced by PA or PLD. These results show that PC-derived PA, but not DAG or PKC, activates actin polymerization in IIC9 fibroblasts, and indicate that PDGF and PMA have inhibitory effects on PA-induced actin polymerization.     

Lack of Phospholipase D Activity in Chromaffin Cells: Bradykinin-Stimulated Phosphatidic Acid Formation Involves Phospholipase C in Chromaffin Cells but Phospholipase D in PC12 Cells

The role of lipid-bound second messengers in the regulation of neurotransmitter secretion is an important but poorly understood subject. Both bovine adrenal chromaffin cells and rat phoeochromocytoma (PC12) cells, two widely studied models of neuronal function, respond to bradykinin by generating phosphatidic acid (PA). This putative second messenger may be produced by two receptor-linked pathways: sequential action of phospholipase C (PLC) and diacylglycerol kinase (DAG kinase), or directly by phospholipase D (PLD). Here we show that bradykinin stimulation of chromaffin cells prelabelled (24 h) with 32Pi leads to production of [32P]PA which is not affected by 50 mM butanol. However, bradykinin stimulation of PC12 cells leads to [32P]PA formation, all of which is converted to phosphatidylbutanol in the presence of butanol. When chromaffin cells prelabelled with [3H]choline were stimulated with bradykinin there was no enhancement of formation of water soluble products of phosphatidylcholine hydrolysis. When chromaffin cells were permeabilised with pneumolysin and incubated in the presence of [gamma-32P]ATP, the formation of [32P]PA was still stimulated by bradykinin. These results show that, although both neuronal models synthesize PA in response to bradykinin, they do so by quite different routes: PLC/DAG kinase for chromaffin cells and PLD for PC12 cells. The observation that neither bradykinin nor tetradecanoyl phorbol acetate stimulate PLD in chromaffin cells suggests that these cells lack PLD activity. The conservation of PA formation, albeit by different routes, may indicate an essential role of PA in the regulation of cellular events by bradykinin.     

A Phytophthora sojae G-protein alpha subunit is involved in chemotaxis to soybean isoflavones

For the soybean pathogen Phytophthora sojae, chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named PsGPA1, is a single-copy gene in P. sojae and encodes a G-protein alpha subunit that shares 96% identity in amino acid sequence with that of Phytophthora infestans. To elucidate the function, expression of PsGPA1 was silenced by introducing antisense constructs into P. sojae. PsGPA1 silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the PsGPA1-silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene, PsCAM1, and two calcium- and calmodulin-dependent protein kinase genes, PsCMK3 and PsCMK4, were increased in the mutant zoospores, suggesting that PsGPA1 negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis.     

Differential changes in phospholipase D and phosphatidate phosphohydrolase activities in ischemia-reperfusion of rat heart

Phospholipase D (PLD2) produces phosphatidic acid (PA), which is converted to 1,2 diacylglycerol (DAG) by phosphatidate phosphohydrolase (PAP2). Since PA and DAG regulate Ca(2+) movements, we examined PLD2 and PAP2 in the sarcolemma (SL) and sarcoplasmic reticular (SR) membranes from hearts subjected to ischemia and reperfusion (I-R). Although SL and SR PLD2 activities were unaltered after 30 min ischemia, 5 min reperfusion resulted in a 36% increase in SL PLD2 activity, whereas 30 min reperfusion resulted in a 30% decrease in SL PLD2 activity, as compared to the control value. SR PLD2 activity was decreased (39%) after 5 min reperfusion, but returned to control levels after 30 min reperfusion. Ischemia for 60 min resulted in depressed SL and SR PLD2 activities, characterized with reduced V(max) and increased K(m) values, which were not reversed during reperfusion. Although the SL PAP2 activity was decreased (31%) during ischemia and at 30 min reperfusion (28%), the SR PAP2 activity was unchanged after 30 min ischemia, but was decreased after 5 min reperfusion (25%) and almost completely recovered after 30 min reperfusion. A 60 min period of ischemia followed by reperfusion caused an irreversible depression of SL and SR PAP2 activities. Our results indicate that I-R induced cardiac dysfunction is associated with subcellular changes in PLD2 and PAP2 activities.     

Mastoparan selectively activates phospholipase D2 in cell membranes

Both known isoforms of phospholipase (PL) D, PLD1 and PLD2, require phosphatidylinositol 4,5-bisphosphate for activity. However, PLD2 is fully active in the presence of this phospholipid, whereas PLD1 activation is dependent on additional factors such as ADP-ribosylation factor-1 (ARF-1) and protein kinase Calpha. We find that mastoparan, an activator of G(i) and mast cells, stimulates an intrinsic PLD activity, most likely PLD2, in fractions enriched in plasma membranes from rat basophilic leukemia 2H3 mast cells. Overexpression of PLD2, but not of PLD1, results in a large increase in the mastoparan-inducible PLD activity in membrane fractions, particularly those enriched in plasma membranes. As in previous studies, expressed PLD2 is localized primarily in the plasma membrane and PLD1 in granule membranes. Studies with pertussis toxin and other agents indicate that mastoparan stimulates PLD2 independently of G(i), ARF-1, protein kinase C, and calcium. Kinetic studies indicate that mastoparan interacts synergistically with phosphatidylinositol 4,5-bisphosphate and that oleate, itself a weak stimulant of PLD2 at low concentrations, is a competitive inhibitor of mastoparan stimulation of PLD2. Therefore, mastoparan may be useful for investigating the regulation of PLD2, particularly in view of the well studied molecular interactions of mastoparan with certain other strategic signaling proteins.     

Ceramide does not inhibit protein kinase C β-dependent phospholipase D activity stimulated by anti-Fas monoclonal antibody in A20 cells

We have investigated the roles of ceramide in Fas signalling leading to phospholipase D (PLD) activation in A20 cells. Upon stimulation of Fas signalling by anti-Fas monoclonal antibody, sphingomyelin hydrolysis and activation of PLD were induced. Also, the translocation of protein kinase C (PKC) βI and βII and the elevation of diacylglycerol (DAG) content were induced by Fas cross-linking. When phosphatidylcholine-specific phospholipase C (PC-PLC) was inhibited by D609, the Fas-induced changes in PLD activity, DAG content, and PKC translocation were inhibited. In contrast, D609 had no effect on Fas-induced alterations in sphingolipid metabolism, suggesting that changes in ceramide content do not account for Fas-induced PLD activation. Furthermore, C6-ceramide had no effect on Fas-induced PLD activation and PKC translocation. Taken together, these data might suggest that ceramide generated by Fas cross-linking does not affect PKC β-dependent PLD activity stimulated by anti-Fas monoclonal antibody in A20 cells.     

Monoclonal antibodies to cell surface components of zoospores and cysts of the fungusPythium aphanidermatum reveal species-specific antigens

A panel of monoclonal antibodies (MAbs) designated PA1 to PA8 has been raised against cell surface components of zoospores and cysts of the pathogenic fungusPythium aphanidermatum. The antibodies were selected on the basis of binding assays using indirect immunofluorescence. Four binding patterns were observed: PA1 labeled the entire zoospore surface including both flagella, PA2 binding was restricted to the anterior flagellum, PA3–PA6 bound to the adhesive cell coat secreted by zoospores during encystment, and PA7 and PA8 labeled zoospores and the cyst cell wall. Electron microscopic immunogold labeling of zoospores showed that PA2 bound to the mastigonemes on the anterior flagellum. The MAbs were tested for binding to zoospores and cysts of several isolates ofP. aphanidermatum, and to zoospores and cysts of several species ofPythium, Phystophthora, Aphanomyces, andSaprolegnia. The results showed that the antigens recognized by MAbs PA1–PA6 were restricted toP. aphanidermatum, whereas those recognized by PA7 and PA8 occurred on all species tested.     

Phospholipase D/phosphatidic acid signal transduction: role and physiological significance in lung

Phospholipase D (PLD), a phospholipid phosphohydrolase, catalyzes the hydrolysis of phosphatidylcholine and other membrane phospholipids to phosphatidic acid (PA) and choline. PLD, ubiquitous in mammals, is a critical enzyme in intracellular signal transduction. PA generated by agonist- or reactive oxygen species (ROS)-mediated activation of the PLDI and PLD2 isoforms can be subsequently converted to lysoPA (LPA) or diacylglycerol (DAG) by phospholipase A1/A2 or lipid phosphate phosphatases. In pulmonary epithelial and vascular endothelial cells, a wide variety of agonists stimulate PLD and involve Src kinases, p-38 mitogen activated protein kinase, calcium and small G proteins. PA derived from the PLD pathway has second-messenger functions. In endothelial cells, PA regulates NAD[P]H oxidase activity and barrier function. In airway epithelial cells, sphingosine-1-phosphate and PA-induced IL-8 secretion and ERKI/2 phosphorylation is regulated by PA. PA can be metabolized to LPA and DAG, which function as first- and second-messengers, respectively. Signaling enzymes such as Raf 1, protein kinase Czeta and type I phosphatidylinositol-4-phosphate 5-kinase are also regulated by PA in mammalian cells. Thus, PA and its metabolic products play a central role in modulating endothelial and epithelial cell functions.     

The Role of Diacylglycerol Kinase ζ and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy

The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.     

A HPLC-fluorescence detection method for determination of cardiac phospholipase D activity in vitro

A nonradioactive assay for the investigation of phospholipase D (PLD) activity in cardiac membranes has been developed. A fluorescent derivative of phosphatidylcholine [2-decanoyl-1-(O-(11-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3proprionyl)amino) undecyl) sn-glycero-3-phosphocholine] was utilized as substrate in an in vitro PLD-catalyzed transphosphatidylation reaction utilizing ethanol as second substrate. Unreacted phosphatidylcholine and the products of phospholipase activity (PEtOH, phosphatidylethanol; PA, phosphatidic acid; DAG, diacylglycerol) were separated by a binary gradient HPLC system and detected by fluorometry. The detection limit of this assay is approximately 0.6 pmol PEtOH. The reaction proceeded at a linear rate for up to 45 min and increased linearly with increasing amounts of rat cardiac membrane protein in a range of 0.625 microg up to at least 25 microg. In the presence of potassium fluoride, formation of fluorescent PA increased at the expense of DAG generation, demonstrating the presence of PA phosphohydrolase activity in rat cardiac membranes. PEtOH formation was unchanged in the presence of the PA phosphohydrolase inhibitor, indicating that the phosphatidylalcohol is not subject to further metabolism by this enzyme. Activation of protein kinase C by phorbol ester significantly increased PLD activity in cardiac membranes. This assay proved to be sensitive for accurate and rapid assessment of PLD activity in cardiac membranes permitting further characterization of the regulation of PLD signal transduction in the heart.