1.2.27
MECHANISM OF POTATO TUBER DEFENCE INDUCED BY FUSARIUM OXYSPORUM LIPIDS

O GOZIA, J CIOPRAGA, A HILLEBRAND, R TUDOR, F NITU, M LUNGU and VL BREZUICA

Institute of Biochemistry, Spi. lndependentei 296, 17-37, Bucharest 77700, Romania

Background and objectives
The role of the chitin-binding domains in the expression of Solanum tuberosum agglutinin (STA) antifungal properties was demonstrated by testing the purified STA preparation, devoid of contaminating chitinase, on early developmental stages of the fungus Fusarium oxysporum [1]. We found that the inhibitory effect of STA on fungus development was prevented by certain fungal compounds (identified as lipids), shown to have an elicitation function. In the present work we aimed to study the interaction between potato tuber and a pathogenic fungus, Fusarium oxysporum, by investigation of the signals involved in the induction of a defence response and the potential role of the chitin-binding protein in recognition and defence.

Materials and methods
The isolation of fungal compounds was performed in organic solvents by column chromatography, using different stationary phases. The various steps in the preparative procedure and the purity of the isolated compounds were checked by thin-layer chromatography (TLC). The interaction between glycolipids and STA was studied by overlay assay on TLC with radiolabelled lectin. The elicitation function of all the fractions obtained during purification was investigated by chitinase, peroxidase and free proline assays, using as a biological model a study of freshly prepared potato tuber disks. Also, STA was examined in vitro in respect of its participation in cell wall reinforcement, following its treatment with peroxidase. The signal transduction pathways involved in the potato tuber defence response were investigated using, as elicitors, phosphatidyl glycerol (PG) and phosphatidyl choline (PC), detected in the pool of phospholipids, extracted from the fungal mycelium. The potential involvement of lipoxygenase (LOX), Ca2+ and phosphorylation processes in the signal transduction pathway were tested by administration of salicylhydroxamic acid (SHAM), EGTA, staurosporine and ophiobolin (a calmodulin inhibitor).

Results and conclusions
The following compounds have been identified as elicitors in the processed Fusarium oxysporum lipid extract: arachidonic acid, phospholipids, particularly PG and PC, and non-acid glycolipids. Following elicitation, peroxidase activity increased 60-fold (PC and non-acid glycolipids) and 80-fold (PG) over the constitutive level. Maximum of activity was detected at 48 h after elicitor treatment, as a result of gene expression activation, as demonstrated by inhibition with cycloheximide. The increased levels of peroxidase were accompanied by a decrease in the free proline concentration after elicitation. In vitro studies also revealed that the STA, treated with peroxidase and H2O2, became more resistant to trypsin attack. Elicitation and H2O2 influenced lectin activity by diminishing the value of the haemagglutination titre. The experiments revealed that the non-acid glycolipid fraction bound the potato lectin, which exibited binding specificities not only for glycosphingolipids with terminal N-acetyl-galactosamine sequences, but also for lactosylceramide. This represents a new carbohydrate specificity for potato lectin reported so far. Nevertheless, during the separation procedure of glycolipids and phospholipids, some fractions acted as suppressors of the potato defence response, as revealed by a decrease in peroxidase accumulation. Regarding the elicitation with PG: PC, SHAM and staurosporine appeared to be very effective, demonstrating that jasmonic acid production and phosphorylation are two important processes in the tuber defence responses. The key enzyme seemed to be a protein kinase which was calmodulin-dependent because the ophiobolin was the most effective inhibitor of elicitation. These results provide evidence of the concerted participation of glycoconjugates in the defence mechanism of both partners: the host and the pathogen.

References
1. Gozia OO, Ciopraga J, Bentia T et al., 1993. CR Academy of Sciences, Paris, Sciences de la vie/Life sciences 316, 788-792.