15 research outputs found

    Reactions of soybean differentials carrying <i>Rps1a</i>, <i>1b</i>, <i>1c</i>, <i>1d</i>, <i>1k</i>, <i>2</i>, <i>3a</i>, <i>3b</i>, <i>3c</i>, <i>4</i>, <i>5</i>, <i>6</i>, <i>7</i>, and <i>8</i> genes to <i>Phytophthora sojae</i> isolates.

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    <p>Reactions of soybean differentials carrying <i>Rps1a</i>, <i>1b</i>, <i>1c</i>, <i>1d</i>, <i>1k</i>, <i>2</i>, <i>3a</i>, <i>3b</i>, <i>3c</i>, <i>4</i>, <i>5</i>, <i>6</i>, <i>7</i>, and <i>8</i> genes to <i>Phytophthora sojae</i> isolates.</p

    Physical map of NBS-LRR like genes present on the <i>Rps12</i> region.

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    <p>(A) Genetic map of the <i>Rps12</i> region (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169950#pone.0169950.g004" target="_blank">Fig 4B</a>). (B) Physical map of eight NBS-LRR-like genes identified from the <i>Rps12</i> region of the Williams 82 genome.</p

    Reactions of soybean differentials carrying <i>Rps1a</i>, <i>1b</i>, <i>1c</i>, <i>1d</i>, <i>1k</i>, <i>2</i>, <i>3a</i>, <i>3b</i>, <i>3c</i>, <i>4</i>, <i>5</i>, <i>6</i>, <i>7</i>, and <i>8</i> genes to <i>Phytophthora sojae</i> isolates.

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    <p>Reactions of soybean differentials carrying <i>Rps1a</i>, <i>1b</i>, <i>1c</i>, <i>1d</i>, <i>1k</i>, <i>2</i>, <i>3a</i>, <i>3b</i>, <i>3c</i>, <i>4</i>, <i>5</i>, <i>6</i>, <i>7</i>, and <i>8</i> genes to <i>Phytophthora sojae</i> isolates.</p

    Analysis of <i>NBSRps4/6</i>-specific molecular markers linked to a novel <i>Phytophthora</i> resistance gene.

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    <p>(A) The <i>NBSRps4/6</i> specific sequence (GenBank accession no. AY258630 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169950#pone.0169950.ref050" target="_blank">50</a>]) used for developing molecular markers. Primer sequences used for PCR are underlined and marked with half arrows. The PCR primers for amplified targets, NBSRps4/6-1272, NBSRps4/6-869, NBSRps4/6-533 and NBSRps4/6-130, are shown along the primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169950#pone.0169950.s003" target="_blank">S2 Table</a>). (B) The <i>NBSRps4/6</i> specific molecular markers linked to the novel <i>Rps</i> gene. L, 100 bp DNA Ladder (New England Biolabs, USA); SP, susceptible parent AR2; RP, resistant parent PI399036; RB, bulk of 10 resistant homozygous RILs; SB, bulk of 10 susceptible RILs. NBSRps4/6-533, <i>NBSRps4/6</i> specific NBSRps4/6-533 marker; NBSRps4/6-130, <i>NBSRps4/6</i> specific NBSRps4/6-130 marker.</p

    Expression of CaMV35S promoter and MSgt-FSgt promoter in different cell/tissue type of transgenic tobacco (<i>N. tabaccum</i> cv Samsun NN) lines assayed in confocal laser scanning microscope (CLSM).

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    <p>(a) The bar diagram denotes GUS activities (4- MU Fluorescence) of the different cell types of the leaf blade from wild type plant (<i>N. tabaccum</i> cv Samsun NN, as untransformed control), and transgenic plants generated for the GUS construct: pKYLXGUS (with CaMV35S promoter) and pKMSgt-FSgtGUS (with hybrid promoter). Different cell/tissue type of leaf blade is presented. T: Trichomes; UE: Upper epidermis; LE: Lower Epidermis; SC: Subsidiary cells; GC: Guard cells; SM: Spongy mesophyll; PM; Palisade mesophyll. (b) The bar diagram denotes GUS activities (4- MU Fluorescence) of the different cell types of the leaf midrib from wild type plant (<i>N. tabaccum</i> cv Samsun NN, as untransformed control), and transgenic plants generated for the GUS construct: pKYLXGUS (with CaMV35S promoter) and pKMSgt-FSgtGUS (with hybrid promoter). Different cell/tissue type of the leaf midrib is presented. T: Trichomes; UE: Upper epidermis; ChC: Collenchymatous cells; PC: Parenchymatous cells; IP: Internal Phloem; X: Xylem; EP: External Phloem; LE: Lower Epidermis. (c) The bar diagram denotes GUS activities (4- MU Fluorescence) of the different cell types of the stem from wild type plant (<i>N. tabaccum</i> cv Samsun NN, as untransformed control), and transgenic plants generated for the GUS construct: pKYLXGUS (with CaMV35S promoter) and pKMSgt-FSgtGUS (with hybrid promoter). Different cell/tissue type of stem is presented. T: Trichomes; E: Epidermal cells; CCl: Cortical collenchymas; CP: Cortical parenchyma; EP: External Phloem; X: Xylem; IP: Internal Phloem; PP: Pith parenchyma. (d) The bar diagram denotes GUS activities (4- MU Fluorescence) of the different cell types of the root from wild type plant (<i>N. tabaccum</i> cv Samsun NN, as untransformed control), and transgenic plants generated for the GUS construct: pKYLXGUS (with CaMV35S promoter) and pKMSgt-FSgtGUS (with hybrid promoter). Different cell/tissue type of root is presented. E: Epidermal cells; CC: Cortical cell; X: Xylem; P: Phloem. (e) The bar diagram denotes normalized GUS activities (4-MU Fluorescence) of the MSgt-FSgt promoter in different cell types of stem, leaf midrib, leaf blade and the root of transgenic plants expressing the MSgt-FSgt promoter. T: Trichomes; EP: External Phloem; X: Xylem; IP: Internal Phloem: ChC, Collenchymatous cells; PC: Parenchymatous cells; UE: Upper epidermis; LE: Lower Epidermis; SM: Spongy mesophyll; PM: Palisade mesophyll; SC: Subsidiary cells; GC: Guard cells; CCl: Cortical collenchymas.</p

    Fluorescence detection and transient assay of GUS (as 4-MU fluorescence) and GFP using a confocal laser scanning microscope (CLSM) for parental and hybrid promoters in tobacco protoplasts.

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    <p>(a) GUS activity as 4-MU fluorescence recorded in confocal microscope as described in methods for the promoter-GUS constructs: 1, pUCPMA (empty vector control, with no GUS); 2, pUCPMAGUS (with CaMV35S promoter) 3, pMS8GUS (with MS8 promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024627#pone.0024627-Dey1" target="_blank">[22]</a>); 4, pFS3GUS (with FS3 promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024627#pone.0024627-Bhattacharyya1" target="_blank">[23]</a>) and 5, pUPMSgt-FSgtGUS (recombinant promoter). (b) GFP fluorescence recorded in confocal microscope as described in methods for the promoter-GFP constructs: 1, pUCPMA (empty vector control, with no GFP); 2, pUCPMAGFP (with CaMV35S promoter); 3, pMS8GFP (with MS8 promoter); 4, pFS3GFP (with FS3 promoter); and 5, pUPMSgt-FSgtGFP (hybrid promoter). (c) Transient assay of 4-MU fluorescence (for GUS) and GFP using CLSM for parental and hybrid promoters in tobacco protoplasts, fluorescence intensities for GUS and GFP were measured as described in methods and presented in a bar diagram as average value ± SD of two independent experiments, each performed in triplicate, for the GUS and GFP constructs as described in panel a and b: empty vector with no GUS or GFP (Control), CaMV35S, MS8, FS3 and MSgt-FSgt promoter constructs. (CaMV35S: <i>Cauliflower mosaic virus</i>; MS8: <i>Mirabilis mosaic virus</i> sub-genomic transcript promoter; FS3: <i>Figwort mosaic virus</i> sub-genomic transcript promoter; MSgt-FSgt: the hybrid promoter).</p

    Expression analysis of CaMV35S and MSgt-FSgt promoters in transgenic <i>Arabidopsis</i> plants.

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    <p>Seventy five independent transgenic <i>Arabidopsis</i> lines were generated with the constructs pKYLXGUS and pKMSgt-FSgtGUS as described in methods. The average GUS activity ± SD from two independent experiments of 75 independent lines (21 days old) developed for each construct pKYLXGUS (35S-GUS), pKMSgt-FSgtGUS (MSgt-FSgtGUS) and empty vector pKYLX (control) are presented in the panel (a) line number 1 to 25, panel (b) line number 26 to 50 and panel (c) line number 51 to 75. Silenced (very low/basal level expression) lines were shown as different symbols for the CaMV35S and MSgt-FSgt promoters.</p

    Transient expression analysis of <i>in vitro</i> methylated CaMV35S and MSgt FSgt promoter linked with the GUS reporter gene.

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    <p>(a) At the top, the size in bp of the promoter sequence, relative position of TATA box, transcription start site (TSS) in the CaMV35S and MSgt-FSgt promoters are shown. Based on analysis using SVM tool (<a href="http://bio.dfci.harvard.edu/Methylator" target="_blank">http://bio.dfci.harvard.edu/Methylator</a>) the putative CG methylation sites were identified and their respective locations were shown. (b) Transient GUS-expression activities of demethylated (dam -ve) and <i>in vitro</i> methylated (<i>M.SssI</i>) CaMV35S and MSgt-FSgt promoter GUS constructs. The plasmids (pUCPMA-GUS and pUPMSgt-FSgtGUS) were grown in dam<sup>−</sup> dcm<sup>−</sup> bacteria and methylated in vitro with <i>M.SssI</i> enzyme as described in materials and methods. The data presented is an average ±SD of two experiments, each performed in duplicate, extracts from empty vector (pUCPMA) transformed protoplasts taken as control. The statistical unpaired student t test using Graph Pad Prism (version 5.01) showed significance at <i>P</i> value  = 0.0073.</p

    Tissue specific expression of the GUS reporter gene directed by either CaMV35S or MSgt-FSgt promoter in transgenic tobacco.

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    <p>(a) The superimposed images of transmitted and blue fluorescence of transverse sections of leaf blade from wild plants (<i>N. tabacum</i> cv. Samsun NN, as control) and transgenic plants for CaMV35S and MSgt-FSgt promoters are presented. SM: Spongy mesophyll; UE: Upper epidermis; PM: Palisade mesophyll; T: Trichomes; and LE: Lower Epidermis. (b) The superimposed image of transmitted and blue fluorescence of few representative S: stomata from the leaf blade of wild plants (as control) and transgenic plants raised for CaMV35S and MSgt-FSgt promoter are presented. SC: Subsidiary cells; GC: Guard cells. (c) The superimposed image of transmitted and blue fluorescence of transverse sections of the leaf midrib from wild type plants (as control) and transgenic plants raised for CaMV35S and MSgt-FSgt promoters are presented. UE: Upper epidermis; T: Trichomes; LE: Lower Epidermis; ChC: Collenchymatous cells; PC: Parenchymatous cells; IP: Internal Phloem; X: Xylem; EP: External Phloem. (d) The superimposed image of transmitted and blue fluorescence of transverse sections of the stem from wild type plants (as control) and transgenic plants raised by CaMV35S and MSgt-FSgt promoters are presented. T: Trichomes; CCl: Cortical collenchyma; PP: Pith parenchyma; CP: Cortical parenchyma; E: Epidermal cells; IP: Internal Phloem; X: Xylem; EP: External Phloem. (E) The superimposed image of transmitted and blue fluorescence of transverse sections of root from wild type plants (as control) and transgenic plants developed for CaMV35S and MSgt-FSgt promoter is presented. E: Epidermal cells; CC: Cortical cell; X: Xylem; P: Phloem.</p

    Comparative expression analysis of CaMV35S promoter and recombinant hybrid promoter in transgenic <i>Arabidopsis</i> plants.

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    <p>(a) Histochemical localization of GUS activity (blue coloration) in 21 days old transgenic <i>Arabidopsis</i> seedlings (magnification x 10.0) developed for the constructs: pKYLX (empty vector, Control), pKYLXGUS (with the CaMV35S promoter, 35S), and pKMSgt-FSgtGUS (hybrid promoter, MSgt-FSgt). (b) Promoter activity was assayed in 21 days old Arabidopsis seedlings (R1 progeny, 2nd generation, Kan<sup>R</sup>) grown aseptically on an MS-agar medium in presence of kanamycin (100 µg/ml) and 1% sucrose. Soluble protein extracts (5 µg) from whole seedlings were used for the GUS assay. The data present average ± SD of four independent experiments for each construct: pKYLX (empty vector control), pKYLXGUS (with the CaMV35S promoter), and pKMSgt-FSgtGUS (hybrid promoter); plasmids construction strategy described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024627#s2" target="_blank">methods</a> section. (c) Electrophoresis of RT-dependent PCR amplifications of GUS transcripts from total RNA isolated from transgenic <i>Arabidopsis</i> plant developed for pKYLX (empty vector control), pKYLXGUS (with CaMV35S promoter), and pKMSgt-FSgtGUS (hybrid promoter); plasmids construction strategy described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024627#s2" target="_blank">methods</a> section, the arrow indicating expected band. (d) Electrophoresis of RT-dependent PCR amplifications of GAPDH transcripts from total RNA obtained from transgenic plant developed for empty vector (1), CaMV35S (2), and MSgt-FSgt (3) promoter constructs, arrow indicating expected band. (e) Electrophoresis of RT-independent (-RT) PCR amplifications of GUS transcripts from total RNA obtained from transgenic plant developed for empty vector (1), CaMV35S (2), and MSgt-FSgt (3) promoter constructs showing no amplification (to check the presence of genomic DNA in the RNA preparation).</p
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