Evaluation of Peracid Formation as the Basis for Resistance to Infection in Plants Transformed with Haloperoxidase T. J. JACKS,* ,² K. RAJASEKARAN, ² K. D. STROMBERG, ² A. J. DE LUCCA, ² AND K.-H. VAN PE Ä E ‡ Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Boulevard, New Orleans, Louisiana 70124, and Institut fu ¨r Biochemie, Technische Universita ¨t Dresden, D-01062 Dresden, Germany Nonheme haloperoxidase (HPO-P) isolated from Pseudomonas pyrrocinia catalyzed the peroxidation 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 (LD 50 ) of Aspergillus flavus occurred at 25 μM 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-HPO-P combinations against A. flavus were examined in vitro. LD 50 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 LD 50 produced by 30 mM acetate in the absence of hydrogen peroxide- HPO-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. KEYWORDS: Aspergillus flavus; chloroperoxidase; haloperoxidase; peracetate; peracid; plant disease; plant transformation; Pseudomonas pyrrocinia; tobacco INTRODUCTION Hydrogen peroxide is a microbicide generated by plants and animals in response to microbial invasion (1-3). It is also a reactant for the biosynthesis of deadlier microbicides in organ- isms that contain haloperoxidase (HPO) (EC 1.11.1.10) (3). For instance, animal and bacterial HPO catalyzes the peroxidation of chloride by hydrogen peroxide to produce hypochlorite that is 90-fold more lethal than is hydrogen peroxide to Aspergillus flaVus (4, 5). Vascular plants generate hydrogen peroxide but lack HPO and consequently lack the corresponding system of antimicrobial protection. To overcome this lack, we transformed plants with a gene for nonheme HPO from Pseudomonas pyrrocinia (HPO-P) (6, 7). The resultant transgenic plants exhibited greater fungal resistance in planta and greater anti- fungal activity in vitro than the corresponding amounts in control plants. HPO-P catalyzes the generation of peracetic acid, a potent antifungal agent (6), from acetic acid and hydrogen peroxide (8). Whether formation of peracids such as peracetic acid by HPO-P is the basis for enhanced antifungal activity in transgenic plants was examined in this study. MATERIALS AND METHODS Culture media were from Difco Laboratories (Detroit, MI). Hydrogen peroxide, obtained from Matheson Coleman & Bell (Norwood, OH), was quantified from its molar absorptivity of 67 M -1 cm -1 at 230 nm (9). Peracetic acid and monochlorodimedon were obtained from Sigma Chemical Co. (St. Louis, MO). Other chemicals were obtained from commercial sources supplying the highest grades available. HPO-P was purified from P. pyrrocinia as described previously (10) and was the enzyme source used in all experiments. HPO-P was not purified from leaves. The corresponding HPO-P gene was isolated from P. pyrrocinia as described earlier (11). Transformation of tobacco (Nicotiana tabacum cvs. Xanthi and SR-1) was accomplished using the Agrobacterium tumefaciens-mediated leaf disk transformation system (12) as described previously (7). Plants regenerated from parallel transformation experi- ments with pBI121 but lacking the HPO-P gene served as controls. Results from PCR, Southern, northern, and western blot analyses and from enzyme assays showed that transformations were successful. Antifungal activities of leaf extracts of transformed and control tobacco plants were assessed in vitro as described earlier (13). Briefly, conidial suspensions of A. flaVus were prepared from cultures grown on potato dextrose-agar slants for 7 days at 30 °C. Conidial suspensions * Author to whom correspondence should be addressed [telephone (504) 286-4380; fax (504) 286-4419; e-mail tjacks@srrc.ars.usda.gov]. ² Southern Regional Research Center. ‡ Technische Universita ¨t Dresden. 706 J. Agric. Food Chem. 2002, 50, 706-709 10.1021/jf011006q CCC: $22.00 © 2002 American Chemical Society Published on Web 01/18/2002