Purification and mode of action of PTR, Pyrenophora tritici-repentis, chlorosis toxin

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Strelkov, Stephen E.
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Pyrenophora tritici-repentis differentially induces tan necrosis and extensive chlorosis in its hexaploid wheat host. A chlorosis inducing host-specific toxin, termed the Ptr chlorosis toxin, has been identified from race 5 of P. tritici-repentis. Ptr chlorosis toxin was purified from the culture filtrates of race 5 isolates, and the physiological development of chlorosis was investigated. Partial purification was performed by 25-80% ammonium sulfate precipitation and passage through a CM-S C25 cation exchange column. Final purification was performed on fast performance liquid chromatogra hy (FPLC), using a MonoS 5/5 cation exchanger, followed by size fractionation on a Superose 12 HR 10/30 column. Purity was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular weight of the toxin determined to be 6.61 kDa by mass spectrometry. The physiological development of chlorosis in sensitive wheat was investigated using partially purified toxin. Treatment with the toxin had no effect on the greening of etiolated tissue, suggesting that chlorosis results from chlorophyll degradation, rather than inhibition of chlorophyll synthesis. Development of chlorosis was light-dependent, suggesting that it may be a consequence of photochemical bleaching. To test for the involvement of active oxygen (AO) species in photobleaching, toxin-treated tissue was floated in solutions of various AO scavengers. The compound p-benzoquinone, which quenches singlet oxygen and triplet chlorophyll, prevented the development of chlorosis, suggesting that AO species are involved in chlorophyll degradation. High performance liquid chromatography (HPLC) chlorophyll degradation profiles were also consistent with photooxidation. Decreases in carotenoid levels were smaller than and concurrent with the declines in chlorophyll, indicating that toxin-induced chlorosis was not the result of a carotenoid deficiency. It appears that Ptr chlorosis toxin, directly or indirectly, inhibits photosynthesis, leading to chlorophyll photodestruction as illuminated thylakoid membranes become unable to dissipate excitation energy normally used in photosynthesis.