The pathogenesis-related proteins of tobacco: their labelling from [14C] amino acids in leaves reacting hypersensitively to infection by tobacco mosaic virus

Abstract

Extracts from leaves of Nicotiana tabacum cv. Samsun NN which have developed a hypersensitive response to infection by tobacco mosaic virus (TMV), contain at least 10 pathogenesis-related (PR) proteins which are absent from or present in very small amounts in uninfected leaves. When [¹⁴C] amino acids were injected into leaves which were still attached to the plants and which had been inoculated with TMV 3 days earlier, a significant radioactivity became associated with all PR-proteins that were resolvable from other host proteins on non-denaturing gels. The incorporation of labelled amino acids into the individual polypeptides was investigated by a procedure involving two successive electrophoretic migrations, first under non-denaturing and then under denaturing conditions. This procedure, when applied to those PR-proteins whose composition is known, PR-1a, PR-1b, PR-1c, PR-2 and PR-N showed that they all accumulated significant radiolabel within 3 h of feeding the leaves with the [¹⁴C] amino acids. Significant radioactivity was also associated with PR-proteins in inoculated leaves within a few hours of feeding the [¹⁴C] amino acids to detached leaves through the petiole, but this method was much less efficient than the injection procedure. Specific radioactivities of the PR-proteins were compared with those of other host proteins and changes were followed during further incubation with unlabelled amino acids in order to investigate the possibility that the PR-proteins are stable end-products from proteolytic cleavage of constitutive proteins. The results indicate that de novo synthesis rather than proteolytic cleavage is responsible for the production and accumulation of PR-proteins in hypersensitively reacting leaves of tobacco.