1.3.13
HOST-SPECIFIC EFFECT OF AM-TOXIN PRODUCED BY ALTERNARIA ALTERNATA APPLE PATHOTYPE ON CHLOROPLASTS

H OTANI1, Y OKUTANI2, A SHIBATA2, M KODAMA2 and K KOHMOTO2

1United Graduate School of Agricultural Sciences, and 2Faculty of Agriculture, Tottori University, Tottori 680-0945, Japan

Background and objectives
Alternaria alternata (apple pathotype), the causal fungus of Alternaria leaf blotch of apple, produces a host-specific toxin named AM-toxin [1]. AM-toxin has primary effects on chloroplasts and plasma membranes of susceptible apple cultivars [1]. Susceptible apple cultivars show a marked tissue specificity in relation to AM-toxin action [1]. Non-green tissues such as petals are completely insensitive to the toxin, suggesting that chloroplasts are necessary for toxin sensitivity. To elucidate the action of AM-toxin on chloroplasts, we isolated physiologically active chloroplasts from apple leaves and investigated the effect of AM-toxin on the isolated chloroplasts.

Materials and methods
Chloroplasts isolated from susceptible and resistant apple leaves cultured in vitro, and purified AM-toxin, were used in this experiment. Photosynthetic activity of the isolated chloroplasts was measured by oxygen evolution using a Gilson Oxygraph equipped with a Clark-type oxygen electrode. Proteins in the isolated chloroplasts were solubilized by detergent treatment, and interaction of the protein fractions and AM-toxin was analysed using a resonant mirror biosensor (BIAcore or IAsys).

Results and conclusions
The chloroplasts isolated from both types of apple leaves were photosynthetically active. When the chloroplasts were treated with AM-toxin, the toxin inhibited photosynthetic activity in susceptible chloroplasts, but not in resistant chloroplasts. Broken chloroplasts without envelopes were obtained by suspending intact chloroplasts in hypotonic solution, then treated with AM-toxin. AM-toxin did not then affect the photosynthetic activity in susceptible chloroplasts. Therefore the effect of AM-toxin on the activity of the magnesium ion-dependent ATPase located in chloroplast envelopes was investigated. The activity was inhibited in susceptible chloroplasts treated with AM-toxin. These results suggest that chloroplast envelopes may be involved in the host-specific action of AM-toxin. A carboxyl group was introduced in the structure of AM-toxin by the Friedel-Crafts reaction, and the toxin derivative was immobilized as a ligand on the tip of a biosensor. When protein fractions solubilized from isolated chloroplasts were added on the tip, a binding signal was detected in susceptible chloroplast protein fractions, but not in resistant chloroplast fractions. On the other hand, when the susceptible protein fractions were pre-treated with the toxin derivative, the binding signal disappeared. These results suggest that AM-toxin-binding proteins may exist in susceptible chloroplasts. Isolation of the proteins is presently being undertaken.

Reference
1. Otani H, Kohmoto K, Kodama M, 1995. Canadian Journal of Botany 73, S453-S458.