Figure 4. Transformation with RPS4/RRS1 breaks restricted taxonomic functionality.

(A,B) R. solanacearum resistance analysis in RPS4/RRS1 dual R gene-transformed tomato (RR) and control plants. Two independent T2 transgenic lines carrying both RPS4 and RRS1 have been tested. Six-week-old tomato plants were inoculated with R. solanacearum expressing popP2. (A) Disease symptoms on tomato plants inoculated with R. solanacearum expressing popP2 at 15 dpi. (B) Plants were rated every other day on a 0 to 5 disease index scale from 0 (no visible wilt) to 5 (the whole plant is dead). Each point represents the mean disease index (± SE) for three independent experiments, each containing 5 to 10 plants per treatment. The asterisks indicate statistical significance from the controls (Dunnett’s method, P<0.05). The control plants (vector control) wilted after inoculation with R. solanacearum expressing popP2, while RPS4/RRS1 transformed plants (RR) were resistant. (C,D) Infection assays with Pseudomonas syringae pv. tomato DC3000 carrying the effector AvrRps4 (Pst-avrRps4) in RPS4/RRS1 dual R gene-transformed tomato (RR) and control plants. Two independent T2 transgenic lines carrying both RPS4 and RRS1 were tested. The right sides of leaves of six-week-old tomato plants were infiltrated with bacterial suspensions (5×10⁴ cfu ml⁻¹). There were 10–12 inoculation sites per leaf. Inoculation sites indicated by arrowheads. (C) Disease symptoms on tomato leaves inoculated with Pst-avrRps4 at 7 dpi. (D) Leaves were harvested at 0 and 3 dpi. Leaves infected with Pst-avrRps4 developed chlorotic lesions at 3 dpi. Bars indicate SE (n = 6). The asterisk indicates statistical significance from the control (3d) (Dunnett’s method, P<0.05). The experiment was repeated at least two times with similar results. (E,F) Colletotrichum orbiculare resistance in RPS4/RRS1 dual R gene-transgenic cucumber (RR) and control plants. Four independent T2 transgenic lines carrying both RPS4 and RRS1 were tested. (E) Mature leaves of 2.5 true leaf stage seedlings were inoculated with spotting 5 µl of a conidial suspension of C. orbiculare (5×10⁵ spores ml⁻¹) on the leaf. Photographs were taken at 6 dpi. (F) Mature leaves of 2.5 true leaf stage seedlings were spray-inoculated with a conidial suspension of C. orbiculare (5×10⁵ spores ml⁻¹). Pathogen growth was determined by measuring C. orbiculare-actin mRNA by qRT-PCR. Bars indicate SE. The asterisk indicates statistical significance from the control (Dunnett’s method, P<0.05). The experiment was repeated at least two times with similar results.