Purification and partial characterization of the major "pathogenesis-related" tomato leaf protein P14 from potato spindle tuber viroid (PSTV)-infected tomato leaves

Abstract

The acid-extractable leaf proteins of potato spindle tuber viroid (PSTV) infected tomato plants were analysed electrophoretically on polyacrylamide gels. The most prominent alteration found during disease development was the appearance of a "pathogenesis-related" protein with an apparent molecular weight of 14,000 (called P14) which is drastically increased in concentration. Its induction, however, is not viroid-specific because it is also accumulating after viral and fungal infections. The degree of P14 accumulation could be directly correlated with the severity of the disease symptoms and its concentration was found to be highest in leaves of the tomato cultivar "Rutgers" four weeks after infection. P14 was isolated from such leaf material by acid-extraction of the leaf proteins, which were concentrated from the clarified homogenates by ultrafiltration through hollow fiber systems or by precipitation at 60 per cent ammonium sulphate saturation. P14 was finally purified by ion exchange chromatography on sulfopropyl (SP-C25) Sephadex and on DEAE cellulose. A protein with properties similar to those of P14 could also be isolated from healthy tomato leaves, where its concentration is about forty to fifty times lower than PSTV-infected tissue. P14 can be stained with Coomassie Brilliant Blue, silver and ethidium bromide, it is sensitive to digestion with pronase and not altered when treated with RNase and DNase. P14 is a basic protein with an estimated isoelectric point of 10.7 and its unusual behaviour during ultrafiltration indicates that it represents an elongated rather than a globular molecule in solution. P14 seems to be different from any of the so-called "pathogenesis-related" proteins described so far in Gynura aurantiaca, "Etrog" citron, potato and tomato after viroid-infection and in tobacco, cucumber and bean leaves after virus- or fungus-induced hypersensitive reactions.