PMC:3577827 / 5567-17345 JSONTXT

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and RRS1 Confer Resistance to C. higginsianum in Transgenic Brassica Rapa and Brassica Napus\nColletotrichum sp. are ascomycete fungi causing anthracnose diseases on a large number of agronomically important crops and vegetables. C. higginsianum causes anthracnose disease in Brassicaceae plants. To investigate whether the RPS4 and RRS1 pair functions in other Brassicaceae plants, we generated transgenic Brassica rapa (B. rapa) L. Perviridis Group (Japanese Mustard Spinach, Komatsuna) and Brassica napus (B. napus) L. plants expressing either RPS4 or RRS1, and RPS4 and RRS1 together under the control of the native promoter (Figure 1). Agrobacterium-mediated transformation produced four B. rapa primary transformants (T1) carrying both RPS4 (6.3 kbp) and RRS1 (8.2 kbp) genomic fragments under control of the native Arabidopsis promoters. Seventeen B. rapa T1 plants carrying only RRS1 genomic fragment, but only one for RPS4 were obtained. We also obtained four B. napus T1 plants carrying a 10.9 kbp genomic fragment containing RPS4 and RRS1. Secondary transformants (T2) derived from bud pollination of B. rapa and selfing B. napus T1 plants were assessed for the presence of the transgenes by PCR (Figure S1) (data not shown). Expression of the transgenes in T2 plants was confirmed by qRT-PCR (Figure S2). When inoculated with C. higginsianum, wild-type (WT) B. rapa and B. napus plants developed the brown necrotic lesions typical of anthracnose. B. rapa and B. napus plants transformed with the RPS4 and RRS1 pair were highly resistant to the pathogen, developing only small necrotic flecks at the inoculated sites (Figure 2A–D). However, transgenic B. rapa plants expressing either RPS4 or RRS1 alone were as susceptible to C. higginsianum as WT (Figure 2A, B). These results indicate that RPS4 and RRS1 are both needed to confer resistance to the pathogen in Brassicaceae. Transgenic plants expressing RPS4 and RRS1 grew normally and did not constitutively express inducible defense gene PR1, suggesting that no autoimmunity response was induced by introducing the R genes (Figure S3A, B \u0026 S4).\n10.1371/journal.pone.0055954.g002 Figure 2 Colletotrichum higginsianum resistance analysis in transgenic Brassica plants expressing RPS4 and RRS1.\n(A) Infection phenotypes of B. rapa plants leaves inoculated with C. higginsianum. Four, three, and one independent T2 transgenic B. rapa lines carrying both RPS4 and RRS1 (RR), either RRS1 or RPS4 alone, respectively, were tested. Mature leaves of 2.5 true leaf stage seedlings were inoculated with spotting 5 µl of a conidial suspension of C. higginsianum (5×105 spores ml−1) on the leaf. Photographs were taken at 6 dpi. Each picture shows a representative of three independent experiments. (B) Quantification of C. higginsianum in planta by qRT-PCR. Mature leaves of 2.5 true leaf stage seedlings were spray-inoculated with a conidial suspension of C. higginsianum (5×105 spores ml−1). Inoculated B. rapa leaves were harvested at 4 dpi, and total RNA was isolated. QRT-PCR was performed with C. higginsianum actin (Chin-ACT) primers for each sample. (C) Infection phenotypes of B. napus plants leaves inoculated with C. higginsianum. Four independent T2 transgenic lines carrying both RPS4 and RRS1 (RR) were tested. Mature leaves of 2.5 true leaf stage seedlings were spray-inoculated with a conidial suspension of C. higginsianum (5×105 spores ml−1). Photographs were taken at 6 dpi. Each picture shows a representative of three independent experiments. (D) Quantification of C. higginsianum in planta by qRT-PCR. Inoculated B. napus leaves were harvested at 4 dpi, and total RNA was isolated. QRT-PCR was performed with Chin-ACT primers for each sample. Bars indicate SE. The asterisks indicate statistical significance from the WT controls (Dunnett’s method, P\u003c0.05). This experiment was repeated three times with similar results.\n\nNicotiana benthamiana Plants Expressing RPS4 and RRS1 Recognize Bacterial Effectors AvrRps4 and PopP2\nBecause RPS4 and RRS1 function properly together in three different Brassicaceae plant species, we thought it is possible that this R gene pair would be able to break the restricted taxonomic functionality boundary when expressed in non-Brassicaceae plants. We generated transgenic Nicotiana benthamiana (N. benthamiana) (Solanaceae) plants expressing RPS4 and RRS1 under the control of their cognate promoters. We obtained thirteen N. benthamiana T1 plants carrying a 10.9 kbp genomic fragment containing RPS4 and RRS1 and two with only RPS4 and one with only RRS1. T2 and T3 progenies derived from selfing T1 and T2 plants, respectively, were assessed for the presence of the transgenes by PCR (Figure S1) and T3 homozygous lines were used in all assays described here. Expression of the transgenes in T3 plants was confirmed by qRT-PCR (Figure S2). To test functionality of the R genes, we used AvrRps4 or PopP2 effectors which are specifically recognized by the RPS4 and RRS1 pair in Arabidopsis. We found that AvrRps4 or PopP2 produced by Agrobacterium-mediated transient expression under control of a cauliflower mosaic virus (CaMV) 35S constitutive promoter induced cell death in N. benthamiana transformed with RPS4 and RRS1, but not in plants expressing only RPS4 or RRS1, indicating that the R gene pair is able to recognizes the AvrRps4 or PopP2 effectors in a non-Brassicaceae plant (Figure 3). We verified by qRT-PCR quantification that mRNAs for avrRps4 and popP2 accumulated to similar levels in transgenic plants. (Figure S5). No inappropriate autoimmune responses were induced by the introduced R gene pair in transgenic N. benthamiana plants (Figure S3C \u0026 S4).\n10.1371/journal.pone.0055954.g003 Figure 3 Transient expression assay in Nicotiana benthamiana transformed with RPS4 and/or RRS1.\nTransient expression assay of avrRps4 or popP2 was performed by Agrobacterium infiltration in four-week-old T3 homozygous transgenic N. benthamiana leaves expressing RPS4 and/or RRS1. Two, one, and two independent T2 transgenic lines carrying both RPS4 and RRS1, either RRS1 or RPS4 alone, respectively, were tested. Photographs were taken at 10 dpi.\n\nTransgenic 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). The experiment was repeated at least two times with similar results.\n\nTransgenic Cucumber Plants Expressing RPS4 and RRS1 are Resistant to Colletotrichum orbiculare \nTo investigate if the RPS4/RRS1 pair confers broad-range resistance, we tested Colletotrichum orbiculare (C. orbiculare) on cucumber (Cucumis sativus; Cucurbitaceae) (C. sativus). Six transgenic cucumber T1 plants carrying a 10.9 kbp genomic fragment containing RPS4 and RRS1 were obtained. 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). Wild type cucumber plants developed brown necrotic lesions surrounded by a yellow halo, a typical symptom of anthracnose disease (Figure 4E). In contrast, RPS4/RRS1 cucumber plants were highly resistant, developing only small necrotic flecks at the inoculated sites, indicative of an active defense reaction (Figure 4E, F). In addition, four independent T2 cucumber progenies segregated 3∶1 for resistance versus susceptibility to C. orbiculare. As in N. benthamiana and tomato, introduction of the RPS4/RRS1 pair did not induce autoimmunity, indicating that RPS4 and RRS1 are tightly regulated in cucumber (Figure S3E \u0026 S4). RPS4/RRS1 thus confers resistance to Colletotrichum sp. in taxonomically distinct families.\n"}