Detection of 10 additional pathogenesis-related (b) proteins in intercellular fluid extracts from stressed 'Xanthi-nc' tobacco leaf tissue

Abstract

By using two-dimensional polyacrylamide gel electrophoresis, 10 additional pathogenesis-related proteins (b6c, b9c, b10a, b10b, b11a, b11b, b12, b13, b14, b15) were found in intercellular fluid extracts of stressed 'Xanthi-nc' tobacco leaf tissue. Proteins were identified as extracellular pathogenesis-related stress proteins by polyacrylamide gel electrophoresis analysis of three successive intercellular fluid extracts compared with homogenates before and after making intercellular fluid extracts. Four proteins (b12, b13, b14, b15) were only resolved by using native polyacrylamide gel electrophoresis for basic proteins in the first dimension gel and they were best extracted in 0.05 M Tris–HCl (pH 7.5) and 0.05 M CaCl2 as the infiltration buffer. The four basic proteins were found in intercellular fluid extracts from leaf tissue subjected to the same types of chemical inducers as previously described for tobacco pathogenesis-related proteins. Their accumulation was inhibited by basic amino acids or spermidine (1 mM) and they were resistant to endogenous and exogenous (trypsin, subtilisin) proteolysis. They did not bind to concanavalin A – Sepharose. These findings indicate that at least 23 proteins accumulate extracellularly after various types of stress in 'Xanthi-nc' tobacco green tissue. These proteins probably represent several groups or families of plant stress proteins.