PMC:3577827 / 11742-16048 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3577827","sourcedb":"PMC","sourceid":"3577827","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3577827","text":"Transgenic Tomato Plants Expressing RPS4 and RRS1 Confer Resistance to Two Taxonomically Distinct Bacteria\nTo test whether the RPS4/RRS1 pair confer immunity in another solanaceous plant, we generated transgenic tomato plants. Seven transgenic tomato T1 plants carrying a 10.9 kbp genomic fragment containing RPS4 and RRS1 were obtained and shown to contain these transgenes by PCR. T2 progenies derived from selfing T1 plants were assessed for the presence of the transgenes by PCR (Figure S1). Expression of the transgenes in T2 plants was confirmed by qRT-PCR (Figure S2). The bacterial wilt phytopathogen R. solanacearum is a serious soilborne disease that attacks almost 200 plant species in 33 plant families, including Solanaceae. Tomato plants (Solanum lycopersicum) transformed with RPS4 and RRS1 were resistant to R. solanacearum expressing popP2 (Figure 4A, B), but susceptible to R. solanacearum without popP2, indicating that the conferred resistance is specific for the PopP2 effector (Figure S6A). Pseudomonas syringae pv. tomato DC3000 (Pst) causes bacterial speck on tomato, a disease characterized by defoliation, blossom blight, and lesions on developing fruit. Transgenic tomato plants were resistant to Pst-avrRps4 (Figure 4C, D) but not to Pst containing a vector control (Figure S6B), indicating that conferred resistance is specific for the AvrRps4 effector. In addition, two independent T2 progenies segregated 3∶1 for resistance versus susceptibility to R. solanacearum expressing popP2 and Pst-avrRps4. These transgenic plants grew normally and showed no significant constitutive expression of inducible defense-related gene PR1 (Figure S3D \u0026 S4).\n10.1371/journal.pone.0055954.g004 Figure 4 Transformation with RPS4/RRS1 breaks restricted taxonomic functionality.\n(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\u003c0.05). 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\u003c0.05). 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\u003c0.05). 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