Claudia R. Oliva

Characterization of an extracellular serine protease of Fusarium eumartii and its action on pathogenesis related proteins

Proteases have been proposed as part of the invasion strategies of some pathogenic fungi. In this work, a serine protease produced by the phytopathogenic fungus Fusarium solani f.sp. eumartii was purified and characterized. Purification of the enzyme was accomplished by gel filtration through a Superose 12 column, followed by hydrophobic interaction chromatography in Phenyl Superose and gel filtration chromatography through Superdex 75. Analysis of the purified enzyme by SDS/PAGE without heat treatment, revealed a single band, which corresponded to the proteolytic activity detected by zymogram. When this protein was subjected to denaturing conditions, two major polypeptides of approximately 30 and 33kDa were revealed. The N-terminal amino acid sequence of one of these polypeptides showed a high similarity with fungal mature serine proteases of the subtilisin family. This protease hydrolysed in vitro, specific polypeptides of potato intercellular washing fluids and cell walls. The protease was also able to degrade pathogenesis-related proteins from the intercellular washing fluids. The role of this serine protease as part of the fungal strategy to colonize potato tuber tissues is discussed.

An aspartic protease with antimicrobial activity is induced after infection and wounding in intercellular fluids of potato tubers

Aspartic proteases (APs) one of the main proteinase classes, have different physiological functions in animals, fungi and viruses. In plants, knowledge of the biological roles of APs is less well developed. An AP has been purified from potato tuber and leaves (Guevara et al., 1999, 2001). In this paper, the changes in the level of AP in response to infection by Phytophthora infestans (P. infestans) and wounding were studied in intercellular washing fluids (IWFs) from tuber disks of two potato cultivars differing in their susceptibility to P. infestans. A differential induction was observed between both cultivars: in the resistant cultivar, induction was higher and faster in infected tissues than in wounded ones. In the susceptible cultivar, a lower and later accumulation was observed than in the resistant cultivar. In addition, AP had a direct inhibitory effect on the germination of cysts of P. infestans and conidia of Fusarium solani. The pattern of accumulation and in vitro activity of AP suggest that this enzyme may have a role in the defense response of potato.

Production of phytoalexins, glycoalkaloids and phenolics in leaves and tubers of potato cultivars with different degrees of field resistance after infection withPhytophthora infestans

SummaryThe kinetics of accumulation of phytoalexins, glycoalkaloids and phenolics was studied in two potato cultivars differing in their degrees of field resistance when infected withPhytophthora infestans. Tuber slices and leaves of cvs Pampeana INTA (high degree of field resistance, free of R genes) and Bintje (susceptible) were infected with race C (complex race 1, 3, 5, 7, 11) ofPhytophthora infestans. Phytoalexins and phenolics accumulated in tuber and leaf tissues which had been inoculated. The levels of these compounds in the susceptible cv. Bintje were relatively low and similar to those found before inoculation. Leaves of cv. Pampeana INTA had a very high glycoalkaloid content, suggesting that glycoalkaloids may play a role in protection of leaves against the fungus. However, we could find no correlation between resistance and glycoalkaloid content of tubers. Our results suggest a major role of phytoalexins, phenolics and glycoalkaloids in the complex mechanisms of field resistance.

Purification and Characterization of an Endopolygalacturonase Produced by Sclerotinia Sclerotiorum

An endopolygalacturonase (endo-PG), was purified from the culture medium of a local isolate of Sclerotinia sclerotiorum with ammonium sulphate precipitation, cation exchange chromatography and gel filtration. The purified endo-PG had a molecular mass of approximately 18 kDa estimated by gel filtration. The isoelectric point was determined by isoelectric focusing to be approximately 8, suggesting that PG II possesses a net positive charge at physiological pHs. The pH optimum for the enzyme was at pH 4.5. The endo-PG showed essentially the same affinity for pectin and polygalacturonic acid as substrates.

Protease inhibitor activity is associated to a basic chitinase from potato but not to an acidic one

SummarySeveral pathogenesis-related proteins, which are produced in plants submitted to stress, have been identified as chitinases. We have previously described that a potato basic chitinase strongly inhibited the activity of an aspartic protease isolated from the same source. In this work, we have tested the activity of two potato chitinases as protease inhibitors. A basic chitinase (ChiB) inhibited trypsin, chymotrypsin, subtilisin, proteinase K and a serine protease ofFusarium sp. An acidic one (ChiA) did not show inhibitory activity. The kinetics of trypsin inhibition by ChiB revealed different patterns of inhibition with azocasein and BAPNA. Metal ions affected differentially both activities, suggesting that ChiB is a bifunctional protein. These results and those reported by other authors suggest a new physiological role for pathogenesis-related proteins. However, results presented in this paper suggest that the protease inhibitor activity is not a general characteristic of potato chitinases.