Food Hydrocolloids vol.I no.5/6 pp.499-500, 1987

Apoplastic glycosylhydrolases as markers of earlypathogenesis

S.Green and S.C.Fry

Department of Botany, University of Edinburgh, King 's Buildings , MayfieldRoad, Edinburgh EH9 3JH, UK

Plant cell wall components have been shown to have regulatory effects on various planttissues (1) . Effects include elicitation of phytoalexins (2) and induction of systemic syn­thesis of proteinase inhibitors (3).

A further effect has been identified-certain cell wall fragments have been foundto be toxic to plant cells (4). It was speculated that hydrolytic enzymes produced byinvading micro-organisms or by the plant hydrolyse cell wall polymers and release oligo­saccharides. The latter may then have a toxic effect on resistant plants manifested ashypersensitive death. The aim of this work is to demonstrate the existence of hydrolyticenzymes and their products.

The model system used to test this hyothesis was the tomato Lycopersicon esculen­tum and its economically damaging fungal pathogen Cladosporiumfulvum. In collabora­tion with Dr PJ .G.M. de Wit at Wageningen, genetically defined infections have beenmade. Infected leaves, and uninfected controls , were used as a source of apoplasticfluid (from the intercellular spaces) which was extracted by an infiltration/centrifuga­tion method (5) . The fluid has already been shown to contain substances capable ofevoking the hypersensitive response (6). The apoplastic fluid was used in very sen­sitive radiochemical assays with uniformly 14C-labelled cell wall material; radioactivematerial solubilized is analysed by chromatographic methods.

Apoplastic fluid was extracted from the healthy cultivar Cf5 , from Cf5 infected withthe race 4 pathogen (incompatible interaction) and from Cf5 infected with the race 5pathogen (compatible interaction ) . After incubation of each fluid sample with 14C_labelled cell walls, the supernatant was assayed for radioactive material solubilized fromthis substrate. A considerable amount of radioactivity was solubilzed by apoplastic fluidfrom uninfected Cf5 showing that apoplastic hydrolases are naturally present inuninfected plants. Apoplastic fluid from the Cf5/race 4 interaction solubilized moreradioactivity from the cell wall substrate than the apoplastic fluid from uninfected Cf5and the Cf5/race 5 interaction. This suggests that enzyme activity is increased in thecourse of active defence.

Radioactive material solubilized by apoplastic fluid after 7 h incubation was analys­ed on Bio-Gel P2. In all cases the material contained a range of poly -, oligo- and mono­saccharides. Apoplastic fluid from the susceptible interaction hydrolysed moremonosaccharides than that from the healthy plant , otherwise the solubilized materialwas very similar. The major difference was with apoplastic fluid from the resistantinteraction , which hydrolysed fewer polysaccharides and significantly more monosac­charides.

Results so far suggest that enzyme activity is present in the apoplastic fluid and that

© IRL Press Limited, Oxford, England 499

S.Green and S.C.Fry

it is enhanced upon infection. The identify of the enzyme(s) is under investigation. In­itial results suggest there are differences between material solubilized from the cell wallby apoplastic fluid from healthy and infected plants. Further analysis is currently underway to try to identify any oligosaccharides characteristic of resistance and/or suscep­tibility.

Acknowledgements

This study is being supported by the Commission of European CommunitiesBiotechnology Action Programme.

References

1. AJbersheim,P., DarviJI,A.G., McNeil,M., Valent,B. and Sharp,J. (1983)In Ciferri,O. and Dure,L. (eds),Structure and Function of Plant Genomes. Plenum Press, New York, pp. 293-312.

2. Darvill,A.G. and AJbersheim,P. (1983) Annu. Rev. Plant Physiol., 35, 243 -275.3. Ryan,C.A., Bishop,P., Pearce,G., Darvill,A.G., McNeil,M. and Albersheim,P. (1981) Plant Physiol.,

68, 616-618.4. Yamazaki,N., Fry,S.C., Darvill,A.G. and AJbersheim,P. (1983) Plant Physiol., 72, 864-869.5.De Wit,P.J.G.M. and Spikman,G. (1982) Physiol. Plant Pathol., 21,1-11.6. De Wit,PJ.G.M., Hofman,A.E., Velthuis,G.C.M. and Kuc,J.A. (1985) Plant Physiol., 77, 642-647.

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