Purification and characterization of 8 of the pathogenesis-related proteins in tobacco leaves reacting hypersensitively to tobacco mosaic virus

Abstract

Leaves of tobacco plants (Nicotiana tabacum cv. Samsun NN) which are reacting hypersensitively to infection with tobacco mosaic virus contain 10 major pathogenesis-related (PR) proteins which are absent, or present in small amounts in uninfected leaves. We describe here a preparative procedure of purification of the tobacco PR-proteins which involves a combination of conventional and high-performance liquid chromatography. The separation and isolation of the proteins were based on differences in net charge at different pH values, in isoelectric point and in apparent molecular weight. This procedure led to the purification to homogeneity of 8 PR-proteins, as shown by polyacrylamide slab gel electrophoresis (PAGE) of the purified proteins under denaturing and non-denaturing conditions. These were the 3 well-known proteins PR-1a,-1b and-1c, and 5 other major PR-proteins, called PR-2,-N,-O,-P and-Q, according to the nomenclature of Van Loon (39). None of the purified PR-proteins gave a positive Schiff reaction for carbohydrate content. Molecular weight determinations from gel permeation chromatography and from sodium dodecyl sulphate (SDS)-PAGE indicated that all 8 PR-proteins were monomers and that three groups could be distinguished among them. The first group is the PR-1 group containing PR-1a,-1b and-1c (12000 MW), the second consists of PR-P and PR-Q (14000 MW) and the third of PR-2, PR-N and PR-O (25000 MW). In the PR-1 group, PR-1a can be distinguished clearly from the two other members on denaturing slab gels containing both SDS and urea.

Pathogenesis-Related PR-1 Proteins Are Antifungal (Isolation and Characterization of Three 14-Kilodalton Proteins of Tomato and of a Basic PR-1 of Tobacco with Inhibitory Activity against Phytophthora infestans)

“…Electrophoresis was performed on slab gels according to Laemmli (1970) for SDS gels and according to Jamet and Fritig (1986) for native basic gels. Gel fixation and staining were carried out as previously described Geoffroy et al, 1990).…”

“…The three acidic PR-1 proteins were purified from virusinfected Samsun-NN leaves by a procedure already described (Jamet and Fritig, 1986) and appeared homogeneous upon SDS-PAGE (Fig. 6B).…”

Biological function of pathogenesis-related proteins: Four tobacco pathogenesis-related proteins are chitinases

“…Proteins PR-P and PR-Q extracted and purified to homogeneity as described (24) were administered (1.75 mg each) to rabbits in six intramuscular injections. Ten days after each boost, immunization serum was collected, clarified by centrifugation, and stored at -20°C.…”

Several “pathogenesis-related” proteins in potato are 1,3-β-glucanases and chitinases

“…The most widely used operational definition of PR proteins is that of polypeptides with relatively low molecular weights (Mr, 10,000- 40,000) that accumulate extracellularly in infected plant tissue, exhibit high resistance to proteolytic degradation, and often, but not always, possess extreme isoelectric points (1). PR proteins have been studied in several systems with respect to physical properties, relationship to the corresponding mRNAs and cDNAs, and gene activation following pathogen infection or elicitor treatment (1)(2)(3)(4)(5)(6)(7)(8)(9). However, the biochemical functions of PR proteins have not been reported.…”