Induction of plant gp91 phox homolog by fungal cell wall, arachidonic acid, and salicylic acid in potato.

The oxidative burst has been suggested to be a primary event responsible for triggering the cascade of defense responses in various plant species against infection with avirulent pathogens or pathogen-derived elicitors. The molecular mechanisms of rapid production of active oxygen species (AOS), however, are not well known. We isolated homologs of gp91 phox, a plasma membrane protein of the neutrophil NADPH oxidase, from a potato cDNA library. Molecular cloning of the cDNA showed that there are two isogenes, designated StrbohA and StrbohB, respectively. The RNA gel blot analyses showed that StrbohA was constitutively expressed at a low level, whereas StrbohB was induced by hyphal wall components (HWC elicitor) from Phytophthora infestans in potato tubers. Treatment of potato tubers with HWC elicitor caused a rapid but weak transient accumulation of H2O2 (phase I), followed by a massive oxidative burst 6 to 9 h after treatment (phase II). Diphenylene iodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, blocked both bursts, whereas pretreatment of the protein synthesis inhibitor cycloheximide with the tuber abolished only the second burst. These results suggest that the expression of StrbohA and StrbohB contributes to phase I and II bursts, respectively. The same is true for arachidonic acid, a lipid component of P. infestans-stimulated biphasic oxidative burst, whereas an endogenous signaling molecule, salicylic acid, only induced a weak phase II burst. Both molecules induced the StrbohB expression, which is in agreement with the second burst. To characterize the signal transduction pathway leading to the oxidative burst, we examined the role of protein phosphorylation in HWC-stimulated StrbohB gene expression. K252a and staurosporine, two protein kinase inhibitors, blocked the transcript accumulation. Two inhibitors of extracellular Ca2+ movement, however, did not abolish the transcript accumulation of StrbohB, suggesting that certain calcium-independent protein kinases are involved in the process of StrbohB gene expression. Additionally, we examined a causal relationship between the oxidative burst and expression of defense genes induced by the HWC elicitor. The transcript accumulation of genes related to sesquiterpenoid phytoalexin synthesis (lubimin and rishitin) and phenylpropanoid pathway was inhibited slightly by the DPI treatment, suggesting that the oxidative burst is not essential to activate these genes. Interestingly, the concomitant presence of DPI with the elicitor resulted in an increase in lubimin accumulation and a decrease in rishitin accumulation. Because it is known that lubimin is metabolized into rishitin via oxylubimin, we propose that AOS mediates the synthesis of rishitin from lubimin.

cDNA cloning of sesquiterpene cyclase and squalene synthase, and expression of the genes in potato tuber infected with Phytophthora infestans.

Sesquiterpene cyclase and squalene synthase are key branch point enzymes in isoprenoid pathway for the synthesis of sesquiterpenoid phytoalexins and sterols/steroid glycoalkaloids, respectively. cDNA clones encoding these enzymes were isolated from potato. A phylogenetic tree showed that the sesquiterpene cyclase is vetispiradiene synthase. Infection of Phytophthora infestans with potato tubers caused transient increases in the transcript level of vetispiradiene synthase in a compatible and an incompatible interactions. On the other hand, wound-induced expression of the squalene synthase was suppressed in favor of the expression of vetispiradiene synthase regardless of inoculated races.

Identification of chitinase and osmotin-like protein as actin-binding proteins in suspension-cultured potato cells.

Cytoplasmic aggregation is an early resistance-associated event that is observed in potato tissues either after penetration of an incompatible race of Phytophthora infestans, the potato late blight fungus, or after treatment with hyphal wall components (HWC) prepared from P. infestans. In potato cells in suspension culture, the number of cells with cytoplasmic aggregation increased upon treatment with HWC, but such an increase was suppressed by treatment with cytochalasin D prior to treatment with HWC. This result suggested that cytoplasmic aggregation in cultured potato cells might be connected with the association of actin filaments. To identify the molecular basis of cytoplasmic aggregation, we purified actin and actin-related proteins by affinity chromatography on a column of immobilized DNase I from cultured potato cells and isolated proteins of 43 kDa, 32 kDa and 22 kDa. Analysis of the amino-terminal amino acid sequences indicated that the 43 kDa, 32 kDa and 22 kDa proteins were potato actin, basic chitinase and osmotin-like protein, respectively. This conclusion was supported by the results of Western blotting analysis of the 43 kDa and 32 kDa proteins with antibodies against actin and basic chitinase. Binding analysis with actin coupled to actin-specific antibodies and biotinylated actin suggested that the 32 kDa and 22 kDa proteins had actin-binding activity. In addition, examination of biomolecular interactions using an optical biosensor confirmed the binding of chitinase to actin. These results imply the possibility that basic chitinase and osmotin-like protein might be involved in cytoplasmic aggregation, hereby participating. In the potato cell's defense against attack by pathogen.

The oxidative burst protects plants against pathogen attack: mechanism and role as an emergency signal for plant bio-defence--a review.

Various aspects, mechanisms and functions of the oxidative burst with generation of O2- superoxide anions in plant cells, which is stimulated by active defence-inducing agents such as fungal infection or elicitor treatment, were reviewed mainly on the basis of experimental evidence obtained in a system of Solanaceae plants and Phytophthora spp. The oxidative burst may be due to an O(2-)generating NADPH oxidase in the plasma membrane, which is activated with combinations of cytosolic proteins, Ca2+, calmodulin and protein kinase, following stimulation by elicitor molecules. The oxidative burst may play the role of an internal emergency signal for induction of the metabolic cascade for active defence.

A cytosolic phospholipase A2 from potato tissues appears to be patatin.

Phospholipase (PL) A2 is involved in signal transduction in the resistance reaction that is induced in potato by inoculation of an incompatible race of Phytophthora infestans, the late blight fungus, or by treatment with fungal elicitor hyphal wall components (Kawakita et al. 1993). In this study, PLA2 in the soluble fraction from potato tuber was purified. The following results suggested that the enzyme was, in fact, patatin: (1) the molecular mass of the purified enzyme was 40 kDa, the same as that of patatin; (2) the pI of the purified enzyme was approximately 4.75, which corresponds to that of patatin; and (3) the amino-terminal amino acid sequence of the purified enzyme showed a high degree of homology to that of patatin. Patatin is known as a storage protein of the potato tuber and it has been shown to have esterase activity. However, other enzymatic activities and the function(s) of patatin are unknown. We investigated the PLA activities of the purified patatin. The PLA2 activity of the patatin was much higher than the PLA1 activity, even though the protein exhibited both activities. The PLA2 activity of the enzyme was particularly apparent when phosphatidylcholine with linoleic acid at the sn-2 position was used as substrate. Lower activity was observed with phosphatidylcholine with palmitic acid, oleic acid and arachidonic acid at the sn-2 position.