PMC:3577827 / 5576-9479
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3577827","sourcedb":"PMC","sourceid":"3577827","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3577827","text":"RPS4 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\nN","divisions":[{"label":"Title","span":{"begin":0,"end":97}},{"label":"Figure 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