FIG 8.

Enhanced LOX activity in N. benthamiana agroinfiltrated with PVX P25/VSR. (A) Leaves were infiltrated with combinations of Agrobacterium cultures containing binary constructs expressing PVX P25 plus either GUS, PPV HCwt, PPV HCLH, or TBSV P19. LOX activity was assayed in leaf extracts at 7 days postagroinoculation (dpa) using linoleic acid (0.1 mM) as a substrate. Data represent the means ± standard errors for 6 replicates, each consisting of four plants that received the same treatment. Statistically significant differences between means were determined by employing Scheffé's multiple-range test. Different letters (a, b, c) indicate significant differences at P values of <0.05. (B) Salicylic acid (SA) or water solution as a control was applied to leaves that were agroinfiltrated with GUS alone or combinations of cultures containing P25wt-T7 plus either GUS, PPV HCwt, or TBSV P19. Leaf disks were excised and assayed for electrolyte leakage at 8 dpa (upper panel). Western blot analysis of extracts derived from leaf patches at 6 dpa, using antibodies against the T7 epitope (middle panel). The lower panel below the Western blot shows the Ponceau S-stained membrane after blotting, as loading control. (C) RT-PCR analysis of expression of the threonine deaminase (TD) and proteinase inhibitor I (PI-I) genes in SA-treated and control-treated plants. The same RT reactions were used to amplify Actin gene transcripts as a control. The numbers of PCR cycles are indicated below the treatments. −RT, control without RT. (D) SA or mock solution as a control was applied to wild-type (Wt) plants and plants expressing the salicylate hydroxylase gene (NahG). Leaf disks were excised and assayed for electrolyte leakage at 8 dpa. Data represent the means ± standard errors for 12 replicates, each consisting of four plants that received the same treatment in two independent experiments. Asterisks indicate significant differences in SA-treated plants compared with control-treated plants (Student's t test, P < 0.05).