Arabidopsis phospholipase Dα1 and Dδ oppositely modulate EDS1- and SA-independent basal resistance against adapted powdery mildew.

Plants use a tightly regulated immune system to fight off various pathogens. Phospholipase D (PLD) and its product, phosphatidic acid, have been shown to influence plant immunity; however, the underlying mechanisms remain unclear. Here, we show that the Arabidopsis mutants pldα1 and pldδ, respectively, exhibited enhanced resistance and enhanced susceptibility to both well-adapted and poorly adapted powdery mildew pathogens, and a virulent oomycete pathogen, indicating that PLDα1 negatively while PLDδ positively modulates post-penetration resistance. The pldα1δ double mutant showed a similar infection phenotype to pldα1, genetically placing PLDα1 downstream of PLDδ. Detailed genetic analyses of pldδ with mutations in genes for salicylic acid (SA) synthesis (SID2) and/or signaling (EDS1 and PAD4), measurement of SA and jasmonic acid (JA) levels, and expression of their respective reporter genes indicate that PLDδ contributes to basal resistance independent of EDS1/PAD4, SA, and JAsignaling. Interestingly, while PLDα1-enhanced green fluorescent protein (eGFP) was mainly found in the tonoplast before and after haustorium invasion, PLDδ-eGFP's focal accumulation to the plasma membrane around the fungal penetration site appeared to be suppressed by adapted powdery mildew. Together, our results demonstrate that PLDα1 and PLDδ oppositely modulate basal, post-penetration resistance against powdery mildew through a non-canonical mechanism that is independent of EDS1/PAD4, SA, and JA.

Low molecular weight GTP-binding proteins in HL-60 granulocytes. Assessment of the role of ARF and of a 50-kDa cytosolic protein in phospholipase D activation.

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol. HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromatography. Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution assay consisting of 3H-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in the region of 50 kDa, and a second discrete peak of PLD reconstitution activity was observed in the region of 18 kDa. Rho GDP/GTP exchange inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rac1. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography. The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of reconstituting activity was not consistent with the participation of cytosolic Rho-related GTPases in the activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP-binding proteins, reduced GTP gamma S-stimulated PLD activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac, could be partially separated from the PLD-stimulating factor(s) by gel-permeation chromatography. Moreover, recombinant Smg GDS failed to stimulate GTP-dependent PLD activity. Cytosolic ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa peak of reconstitution activity. Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1D9. The effects of the 50-kDa and 18-kDa PLD-inducing factors on the salt-extracted PLD activity were synergistic. The weak stimulatory effect of ARF alone suggested that the GTP gamma S-stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-regulatory proteins. We propose that a membrane-bound GTP-binding protein, possibly ARF, may be involved in the activation of PLD when combined with the component(s) of the 50-kDa fraction.

Low molecular weight GTP-binding proteins in hepatocytes and an assessment of the role of p21ras proteins in the activation of phospholipase D.

A GTP-binding protein with an apparent molecular weight of 25 kDa was detected in hepatocyte extracts using SDS-PAGE and [alpha-32P]GTP. p21ras proteins could only be detected by immunological analysis. The amounts of p21ras proteins present in isolated hepatocytes and in a highly purified preparation of liver plasma membrane vesicles were 0.3 and 4 ng p21ras protein/micrograms membrane protein, respectively. In comparison with the total cell extract, the degree of enrichment of plasma membrane vesicles with p21ras was similar to that of 5'-nucleotidase. The p21ras proteins were tightly associated with the membrane. Treatment of [3H]choline-labelled plasma membranes with an excess concentration of the anti-p21ras antibody Y13-259 failed to inhibit either basal or guanosine 5'-[gamma-thio]triphosphate (GTP[S])-stimulated [3H]choline release. It is concluded that in hepatocytes (a) the majority of p21ras is bound to the plasma membrane and (b) p21ras is not directly involved in the activation by GTP[S] of phospholipase D.

Assessment of receptor-dependent activation of phosphatidylcholine hydrolysis by both phospholipase D and phospholipase C.

Enhancement of cellular phospholipase D (PLD)-1 and phospholipase C (PLC)-mediated hydrolysis of endogenous phosphatidylcholine (PC) during receptor-mediated cell activation has received increasing attention inasmuch as both enzymes can result in the formation of 1,2-diacylglycerol (DAG). The activities of PLD and PLC were examined in purified mast cells by quantitating the mass of the water-soluble hydrolysis products choline and phosphorylcholine, respectively. Using an assay based on choline kinase-mediated phosphorylation of choline that is capable of measuring choline and phosphorylcholine in the low picomole range, we quantitated the masses of both cell-associated and extracellular choline and phosphorylcholine. Activating mast cells by crosslinking its immunoglobulin E receptor (Fc epsilon-RI) resulted in an increase in cellular choline from 13.1 +/- 1.2 pmol/10(6) mast cells (mean +/- SE in unstimulated cells) to levels 5- to 10-fold higher, peaking 20 s after stimulation and rapidly returning toward baseline. The increase in cellular choline mass paralleled the increase in labeled phosphatidic acid accumulation detected in stimulated cells prelabeled with [3H]palmitic acid and preceded the increase in labeled DAG. Although intracellular phosphorylcholine levels were approximately 15-fold greater than choline in unstimulated cells (182 +/- 19 pmol/10(6) mast cells), stimulation resulted in a significant fall in phosphorylcholine levels shortly after stimulation. Pulse chase experiments demonstrated that the receptor-dependent increase in intracellular choline and the fall in phosphorylcholine were not due to hydrolysis of intracellular phosphorylcholine and suggested a receptor-dependent increase in PC resynthesis. When the extracellular medium was examined for the presence of water-soluble products of PC hydrolysis, receptor-dependent increases in the mass of both choline and phosphorylcholine were observed. Labeling studies demonstrated that these extracellular increases were not the result of leakage of these compounds from the cytosol. Taken together, these data lend support for a quantitatively greater role for receptor-mediated PC-PLD compared with PC-PLC during activation of mast cells.