Evaluation of peracid formation as the basis for resistance to infection in plants transformed with haloperoxidase

Nonheme haloperoxidase (HPO-P) isolated from Pseudomonas pyrrocinia catalyzed the peroxiclation of alkyl acids to peracids. Among acids tested as substrates, acetic acid was most readily peroxidized, The reaction product peracetate possessed potent antifungal activity: 50% death (LD50) of Aspergillus flavus occurred at 25 muM peracetate. Viability of A. flavus was inhibited by up to 80% by leaf extracts of tobacco plants transformed with the HPO-P gene from P. pyrrocinia compared to viability of fungi exposed to extracts from controls. To elucidate if peracid formation by HPO-P was the basis for antifungal activity in transgenic leaf tissues, lethalities of hydrogen peroxide-acetate-H PO-P combinations against A. flavus were examined in vitro. LD50 of A. flavus exposed to the combinations occurred at 30 mM acetate when concentrations of hydrogen peroxide and HPO-P were held constant. This value was identical to the LD50 produced by 30 mM acetate in the absence of hydrogen peroxideHPO-P and therefore did not account for enhanced antifungal activity in transgenic plants. For clarification, kinetics of the enzymic reaction were examined. According to the concentration of acetate needed for enzyme saturation (K-m = 250 mM), acetate was lethal prior to its oxidation to peracetate. Results indicate that peracid generation by HPO-P was not the basis for enhanced antifungal activity in transgenic plants expressing the HPO-P gene.