Pathogenesis-related proteins of tomato: II. Biochemical and immunological characterization

Abstract

Acid soluble apoplastic proteins were isolated from leaves of Lycopersicon esculentum (Mill) plants infected by Phytophthora infestans and separated by two-dimensional gel electrophoresis. Two groups of proteins with either basic (pI ⩾ 8) or acidic (pI ⩽ 6) pls were distinguished. Some of the proteins, including P45 and P70, were identified as glycoproteins by Con A affinity chromatography. P14 and P70 were purified and a partial amino acid sequence obtained. The purified proteins were also used to raise antibodies in rabbits. The partial sequence obtained for P14 was fully homologous between positions 24 and 43 with a sequence published for a P14 protein isolated from potato spindle tuber virus infected tomato plants. No homologies with this sequence or the N-terminal sequence of P70 could be found in the National Biomedicai Research Foundation data bank, except for the published homology of P14 with PR-1a from tobacco. The antisera raised against P14 and P70 showed that the proteins shared no common antigenic determinants. The anti-P70 antibodies recognized many other proteins besides P70—most probably glycoproteins. Antibodies directed against tobacco chitinase showed cross reactions with the acid soluble proteins P26, 30 and 31' and antibodies directed against tobacco β-1,3-glucanase showed cross reactions with P31 and P36. These acid soluble proteins, as well as the respective enzyme activities, were strongly enhanced in infected tomato plants. By means of chromatofocusing, P36 was strongly enriched and P30 was purified to apparent purity. P45 proved to have peroxidase activity. No phenoloxidase activity was found associated with the acid soluble protein fraction.