Activity of Ptr ToxA fromPyrenophora tritici-repentisrequires host metabolism

Abstract

Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces a cultivar-specific toxin (Ptr ToxA) required for high virulence. Sensitive wheat lines develop necrosis within 48 h of infiltration with nM levels of purified toxin, but the mechanism by which this occurs is not understood. The involvement of wheat metabolism in the action of Ptr ToxA was investigated here with a qualitative assay based on necrosis development and a more rapid and quantitative assay based on electrolyte leakage. Necrosis and electrolyte leakage failed to develop when toxin-treated wheat was incubated at 4 °C, but the inhibition of toxin action by cold was reversible. Necrosis developed when wheat was exposed to toxin for 8 h at 4 °C, then shifted to 20 °C for an additional 40 h. Electrolyte leakage was delayed by only 2 h in toxin-treated leaves held at 4 °C for 6 h then shifted to 20 °C, relative to leaves held at 20 °C throughout. Toxin-induced electrolyte leakage was reduced by the co-application of sodium vanadate, an inhibitor of H+-ATPases. However, the level of protection from electrolyte leakage by a single application of vanadate decreased as time increased, and the inhibitor failed to prevent necrosis development. Inhibitors of transcription (α-amanitin and cordycepin) and of translation (cycloheximide) each reduced the level of toxin-induced electrolyte leakage. Cycloheximide was the only inhibitor that prevented necrosis development when co-infiltrated with toxin. The data indicate that the action of Ptr ToxA requires active host processes including transcription, translation, and functional H+-ATPases.