A protease was isolated from potato (Solanum tuberosum L. cv. Huinkul) tuber disks after 24 h of aeration when proteolysis is markedly increased. Purification was performed by ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography. A size of 40 kDa was estimated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and gel filtration, it is monomeric and its properties are consistent with those of aspartic proteinases (EC 3.4.23): it had a pH optimum between 4 and 5 and it was inhibited by pepstatin. Partial homology with other plant aspartic proteinases was observed in two sequenced tryptic fragments. It binds to Sepharose‐concanavalin A and can be eluted with α‐methyl mannoside, indicating that it is possibly glycosylated. Unlike other aspartic proteinases from Solanaceae that degrade pathogenesis‐related proteins, it is unable to cleave a basic chitinase from potato. Moreover, this aspartic protease is strongly inhibited by the basic chitinase; the 50% inhibition is obtained when the molar ratio approaches 1, the same as with pepstatin. The interaction between this aspartic protease and a new type of endogenous inhibitor may be an interesting starting point to study the regulation of these aspartic proteases during stress.

No abstract

The mechanism involved in systemic acquired resistance (SAR) can be non-specifically induced in susceptible plants. In response to pathogens, plants' natural defence mechanisms include the production of lignin and phytoalexins and the induction of plant enzymes. The aim of this research was to study the induction of SAR mediated by the chemical activator DL-3-aminobutyric acid (BABA) and the fungicide fosetyl-aluminium in potato cultivars with different levels of resistance against Phytophthora infestans (Mont) de Bary. To study the chemical induction of the resistance, the foliage of several potato cultivars was sprayed with BABA, fosetyl-aluminium or water (as a control treatment). After 3 days the foliage was inoculated with P. infestans. Seven days after inoculation, development of disease symptoms in the foliage was assessed. In postharvest tuber samples, evidence for enhancement of the defence response was evaluated by measuring the protein content of several hydrolytic enzymes as well as the phenol and phytoalexin content. The highest level of protection against late blight was observed when the chemicals were applied at early stages of crop development. An increase in resistance to late blight was also detected in tubers after harvest. There was also an increase in the protein level of beta-1,3-glucanase and aspartic protease as well as in the phenol and phytoalexin content of potato tuber discs obtained from postharvest tubers of treated plants. Thus the protective effect seemed to persist throughout the whole crop cycle. This treatment may offer the possibility of controlling both foliage and tuber blight and could have a major impact in reducing over-winter survival of P. infestans in tubers.

Solanum tuberosum aspartic proteases (StAPs) with antimicrobial activity are induced after abiotic and biotic stress. In this study the ability of StAPs to produce a direct antimicrobial effect was investigated. Viability assays demonstrated that StAPs are able to kill spores of Fusarium solani and Phytophthora infestans in a dose-dependent manner. Localization experiments with FITC-labelled StAPs proved that the proteins interact directly with the surface of spores and hyphae of F. solani and P. infestans. Moreover, incubation of spores and hyphae with StAPs resulted in membrane permeabilization, as shown by the uptake of the fluorescent dye SYTOX Green. It is concluded that the antimicrobial effect of StAPs against F. solani and P. infestans is caused by a direct interaction with the microbial surfaces followed by membrane permeabilization.