The role of reversible phosphorylation of the host plasma membrane H+-ATPase i n signal transduction during the incompatible interaction between tomato cells and the fungal pathogen Cladosporium fulvum was investigated. Tomato cells (with the Cf-5 resistance gene) or isolated plasma membranes from Cf-5 cells treated with elicitor preparations from race 2.3 or 4 of C. fulvum (containing the avr5 gene product) showed a marked dephosphorylation of plasma membrane H+-ATPase. Similar treatment with elicitor preparations from races 5 and 2.4.5.9.11 (lacking the avr5 gene product) showed no change in dephosphorylation. Elicitor (race 4) treatment of cells, but not of isolated plasma membranes, for 2 hr resulted i n rephosphorylation of the ATPase via Ca2+-dependent protein kinases. The initial (first hour) rephosphorylation was enhanced by protein kinase C (PKC) activators and was prevented by PKC inhibitors. Activity of a second kinase appeared after 1 hr and was responsible for the continuing phosphorylation of the H+-ATPase. This latter Ca2+-dependent kinase was inhibited by a calmodulin (CaM) antagonist and by an inhibitor of Ca2+/CaM-dependent protein kinase II. The activation of the Ca2+/CaM-dependent protein kinase depended on the prior activation of the PKC-like kinase.

The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase t o the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components t o the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca 2+-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation t o the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants.

The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components to the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca(2+)-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation to the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants.

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Calcium-transporting ATPases were compared in endoplasmic reticulum (ER)-and plasma membrane-enriched fractions of red beet (Beta vulgaris 1.) storage tissue by measuring "Ca uptake and calcium-dependent phosphoenzyme formation. l h e plasma membrane fraction was prepared by aqueous two-phase partitioning of a microsomal fraction and collecting the upper phase. l h e ERenriched fraction was obtained by submitting a sucrose-gradient ER-enriched fradion to aqueous two-phase partitioning and collecting the lower phase; this reduced contaminating plasma membrane, which partitioned into the upper phase. l h e ATP-dependent calcium uptake observed in both fractions was released by the calcium ionophore A23187. Calcium uptake showed saturation kinetics for calcium with K, values of 0.92 mmol m-3 for the ER fraction and 1.24 mmol m-3 for the plasma membrane fraction. Uptake into both fractions was inhibited by vanadate and erythrosin 6, although the plasma membrane system was slightly more sensitive to both inhibitors. Cyclopiazonic acid and thapsigargin, at low concentrations, had no marked effect on uptake. The plasma membrane system was less substrate-specific for ATP than the ER system, since it was able to use CTP and ITP to drive calcium transport at up to 50% of the leve1 obtained with ATP. Following phosphorylation with [-p3'P]ATP, two high molecular mass, calcium-dependent phosphoproteins (119 and 124 kD) and a low molecular mass, calcium-independent phosphoprotein (1 7 kD) were observed in the plasma membrane fraction. The ER fraction showed one high molecular mass phosphoprotein (119 kD) in the presence of calcium and two low molecular mass phosphoproteins (17 and 20 kD) that showed no calcium dependence. l h e low molecular mass phosphoproteins were insensitive to hydroxylamine, but they did show turnover. l h e identity of these proteins is unknown, but they do not have the properties of phosphorylated intermediates of calcium-ATPases. In contrast, the high molecular mass phosphoproteins displayed properties consistent with their representing phosphorylated intermediates of E,Ez-type ATPases; they were hydroxylamine-sensitive, showed rapid turnover, and were inhibited by vanadate. Because they showed calcium-dependent phosphorylation and were sensitive to erythrosin B, the 119-and 124-kD phosphoproteins may be phosphorylated intermediates of the ER and plasma membrane calcium ATPases. These phosphoproteins were characterized further with respect to inhibitor sensitivity, responses to ions, and substrate specificity.