Date of Award:

5-1-1987

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Biology

Committee Chair(s)

Jon Y. Takemoto

Committee

Jon Y. Takemoto

Abstract

Syringomycin is a peptide toxin produced by the phytopathogen Pseudomonas syringae pv syringae. It is a major virulence factor produced by this organism and is involved in the development of a number of destructive plant diseases. Syringomycin preferentially stimulated the vanadate sensitive ATPase associated with the plasma membrane of red beet. The stimulation of ATPase activity was not due to a collapse of the ion gradients or to a detergent like breakdown of the membranes. Ionophores did not affect the stimulation of ATPase activity. Deoxycholate eliminated the ability of the toxin to stimulate ATPase activity, and the Zwittergent 3-14 solubilized ATPase was insensitive to the toxin. Steady-state, proton pumping by membrane vesicles was not affected by syringomycin. Phosphorylation of the membranes revealed that the enzyme displayed an altered Vmax but the Km was unaffected. Syringomycin, therefore, affected the ATPase through covalent modification of the enzyme. Syringomycin stimulated the phosphorylation of several plasma membrane polypeptides of red beet storage tissue. Among the many proteins phosphorylated was a protein with a Mr 100K which corresponded in size to the catalytic subunit of the plasma membrane H+-ATPase. The phosphorylations were insensitive to hydroxylamine Addition of the calcium chelator EGTA eliminated the ability of the toxin to stimulate phosphorylation, but adding excess calcium restored the phosphorylation. Increased phosphorylation of the proteins was not due to an inhibition of phosphoprotein phosphatases. Phosphorylation of the polypeptides was reduced upon extraction of membranes with deoxycholate, a treatment which also eliminated the ability of the toxin to stimulate ATPase activity. The phorbol ester, 12-otetradecanoyl phorbol 13-acetate, and kinase inhibitors 1-(5-Isoquinolinesulfonyl)-2-2-methylpiperazinedihydrochloride (H-7) and N-(2-aminoethyl)-5- isoquinolinesulfonamidedihydrochloride (H-9) did not affect toxin induced kinase activity. Immunodetection using anti-ATPase antibodies demonstrated that the H+-ATPase was one of the proteins phosphorylated in the presence of syringomyc in. It is conceivable that phosphorylation of other plasma membrane proteins, such as ion pumps and channels are involved in the toxin's mode of action. These observations open new avenues to explore the regulation of plasma membrane transport processes, and their alteration during plant diseases.

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