14 research outputs found

    Detection of siRNA in <i>Phytophthora parasitica</i> re-isolated from transgenic plants producing <i>GFP</i> siRNAs or <i>PnPMA1</i> siRNAs.

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    <p>dsPMA1 and dsGFP <i>Arabidopsis</i>, <i>P. parasitica</i> re-isolated from transgenic plants expressing <i>PnPMA1</i> dsRNA and <i>GFP</i> dsRNA, respectively; WT, <i>P. parasitica</i> isolated from diseased wild-type <i>A. thaliana</i>; lanes 1–3, <i>P. parasitica</i> cultures five days after being isolated from three independent transgenic <i>A. thaliana</i> lines producing <i>GFP</i> siRNAs or <i>PnPMA1</i> siRNAs; P1, A <i>P. parasitica</i> transformant accumulated <i>PnPMA1</i> siRNAs as a positive control for <i>PnPMA1</i> siRNA detection; P2, A transgenic <i>A. thaliana</i> line accumulated <i>GFP</i> siRNAs as a positive control for <i>GFP</i> siRNA detection; M, RNA size markers, from bottom up are 21 and 24 nt RNAs, respectively.</p

    Cytological characterization of infection of transgenic <i>Arabidopsis thaliana</i> producing <i>GFP</i> dsRNA with GFP-expressing <i>Phytophthora parasitica</i>.

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    <p>(<b>A</b>) Successful colonization of both transgenic (a) and wild-type (b) <i>A. thaliana</i> plants by <i>P. parasitica</i> as shown with the development of haustoria in the mesophyll cells (scale bar: 20 µm). The diseased leaves of 3 d post inoculation were stained with trypan blue and viewed under Olympus BX-51 microscope equipped with differential interference contrast (DIC) optics. (B) Colonization of root tissues of transgenic <i>A. thaliana</i> lines expressing <i>GFP</i> dsRNA by <i>P. parasitica</i> stably expressing GFP (strain 1-1–2-1). The infected roots of both wild type (a) and three independent transgenic lines (b–d) that accumulated GFP siRNAs were collected 12 h, 24 h and 48 h post inoculation with zoospores prepared from <i>P. parasitica</i> strain 1-1–2-1, respectively, and were viewed with an Olympus BX-51 fluorescent microscope with a GFP filter (images taken 12 h and 24 h post inoculation, scale bar: 20 µm; images taken 48 h post inoculation, scale bar: 50 µm) (C) Development of <i>P. parasitica</i> haustoria in transgenic <i>A. thaliana</i> roots expressing GFP dsRNA. The infected roots of both wild-type (a) and three independent transgenic lines (b–d) that accumulated GFP siRNAs were collected 12 h post inoculation of zoospores prepared from <i>P. parasitica</i> strain 1-1–2-1 and were viewed with an Olympus BX-51 fluorescent microscope with a GFP filter (image of wild-type, scale bar: 20 µm; images of transgenic lines, scale bar: 50 µm).</p

    Generation of transgenic <i>Arabidopsis thaliana</i> lines with accumulation of siRNAs derived from transgene constructs.

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    <p>(<b>A</b>) Schematic representation of the silencing constructs in the pART27 binary vector. The selected regions of <i>GFP</i> (612 bp) and <i>PnPMA1</i> (262 bp) were cloned in an inverted-repeat configuration using the appropriate restriction enzymes into the sites spanned by 35 S promoter and OCS terminator regions in pKANNIBAL, respectively. The expression cassettes were subsequently transferred into the binary vector pART27, respectively. Accumulation of siRNAs in independent T2 <i>A. thaliana</i> lines transformed with (<b>B</b>) <i>GFP</i> dsRNA construct (lanes 1–8, T2 individuals from each of 8 independent T1 transgenic plants) (b1) or (<b>C</b>) <i>PnPMA1</i> dsRNA (lanes 1–4, T2 individuals from each of 4 independent T1 transgenic plants) construct; WT, wild-type <i>A. thaliana</i>; M, RNA size marker, 21, 24 and 30 nt from bottom up, respectively. Lower panel indicates ethidium bromide staining of total RNAs to show equal loading of RNA samples.</p

    Analysis of <i>Phytophthora parasitica</i> transformants.

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    <p>(<b>A</b>) Detection of siRNA accumulation in transgenic <i>P. parasitica</i> lines transformed with a <i>PnPMA1</i> dsRNA construct. Northern blot of total RNA probed with <i>PnPMA1</i> specific probe. (<b>B</b>) Quantitative RT-PCR analyses of <i>PnPMA1</i> expression in <i>P. parasitica</i>. <i>P. parasitica WS041</i> was used as a reference gene for quantification and normalization of <i>P. parasitica</i> gene expression. The data presented are relative gene expression calculated after being normalized to <i>WS041</i> with the 2<sup>−ΔΔct</sup> method. The data represent the means of three replicates. Bars represent the standard errors of three independent measurements. WT, wild-type <i>P. parasitica</i>; CK, <i>P. parasitica</i> transformed with hygromycin resistance gene; lanes 1 and 2, two independent <i>P. parasitica</i> silenced transformants accumulated <i>PnPMA1</i> siRNAs; M, RNA size marker, from bottom up are 21 and 24 nt RNAs, respectively.</p

    Quantitative RT-PCR analyses of <i>GFP</i> and <i>PnPMA1</i> expression in <i>Phytophthora parasitica</i>.

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    <p>(<b>A</b>) <i>GFP</i> (a) and <i>PnPMA1</i> (b) abundance in <i>P. parasitica</i> strain 1-2–1-1 stably expressing ER-rendered GFP in infected detached leaves. (<b>B</b>) Quantification of <i>GFP</i> (a-c) and <i>PnPMA1</i> (d–f) in 1-1–2-1 in infected roots 12 h, 24 h and 48 h post inoculation, respectively. WT, wild-type <i>A. thaliana</i>; CK, <i>A. thaliana</i> transformed with pART27; 1, 2 and 3 are three independent transgenic <i>A. thaliana</i> lines that accumulated <i>GFP</i> or <i>PnPMA1</i> siRNAs (the same set of lines as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028114#pone-0028114-g002" target="_blank">Figure 2</a>). <i>P. parasitica WS041</i> was used as a reference gene for quantification and normalization of <i>P. parasitica</i> gene expression. The data presented are relative gene expression calculated after being normalized to <i>WS041</i> with the 2<sup>−ΔΔct</sup> method. The data represent the means of three replicates. Bars represent the standard errors of three independent measurements. Statistical significance of the target genes silencing effect was performed by Student's <i>t</i> test using <i>R</i> (one-tailed, transgenic lines versus wild-type).</p

    Table_1.xlsx

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    <p>Trichothecene mycotoxins, such as deoxynivalenol (DON) produced by the fungal pathogen, Fusarium graminearum, are not only important for plant infection but are also harmful to human and animal health. Trichothecene targets the ribosomal protein Rpl3 that is conserved in eukaryotes. Hence, a self-defense mechanism must exist in DON-producing fungi. It is reported that TRI (trichothecene biosynthesis) 101 and TRI12 are two genes responsible for self-defense against trichothecene toxins in Fusarium. In this study, however, we found that simultaneous disruption of TRI101 and TRI12 has no obvious influence on DON resistance upon exogenous DON treatment in F. graminearum, suggesting that other mechanisms may be involved in self-defense. By using RNA-seq, we identified 253 genes specifically induced in DON-treated cultures compared with samples from cultures treated or untreated with cycloheximide, a commonly used inhibitor of eukaryotic protein synthesis. We found that transporter genes are significantly enriched in this group of DON-induced genes. Of those genes, 15 encode major facilitator superfamily transporters likely involved in mycotoxin efflux. Significantly, we found that genes involved in the metabolism of gamma-aminobutyric acid (GABA), a known inducer of DON production in F. graminearum, are significantly enriched among the DON-induced genes. The GABA biosynthesis gene PROLINE UTILIZATION 2-2 (PUT2-2) is downregulated, while GABA degradation genes are upregulated at least twofold upon treatment with DON, resulting in decreased levels of GABA. Taken together, our results suggest that transporters influencing DON efflux are important for self-defense and that GABA mediates the balance of DON production and self-defense in F. graminearum.</p

    Protein architecture of subfamily A AtbZIP and BrbZIP proteins in Arabidopsis and Chinese cabbage (<i>Brassica rapa</i>).

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    <p>The distribution of conserved motifs identified from 23 BrbZIP and 13 AtbZIP proteins of the subfamily A are predicted by the MEME (Multiple Em for Motif Elicitation) tool. Each motif is represented by a number in colored box. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158966#pone.0158966.s008" target="_blank">S4 Table</a> for detailed motif information.</p

    Genome-Wide Analysis of the bZIP Gene Family Identifies Two ABI5-Like bZIP Transcription Factors, BrABI5a and BrABI5b, as Positive Modulators of ABA Signalling in Chinese Cabbage

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    <div><p>bZIP (basic leucine zipper) transcription factors coordinate plant growth and development and control responses to environmental stimuli. The genome of Chinese cabbage (<i>Brassica rapa</i>) encodes 136 putative bZIP transcription factors. The bZIP transcription factors in <i>Brassica rapa</i> (BrbZIP) are classified into 10 subfamilies. Phylogenetic relationship analysis reveals that subfamily A consists of 23 BrbZIPs. Two BrbZIPs within subfamily A, Bra005287 and Bra017251, display high similarity to ABI5 (ABA Insensitive 5). Expression of subfamily A BrbZIPs, like <i>BrABI5a</i> (Bra005287/BrbZIP14) and <i>BrABI5b</i> (Bra017251/BrbZIP13), are significantly induced by the plant hormone ABA. Subcellular localization assay reveal that both BrABI5a and BrABI5b have a nuclear localization. BrABI5a and BrABI5b could directly stimulate ABA Responsive Element-driven <i>HIS</i> (a <i>HIS3</i> reporter gene, which confers His prototrophy) or LUC (<i>LUCIFERASE</i>) expression in yeast and Arabidopsis protoplast. Deletion of the bZIP motif abolished BrABI5a and BrABI5b transcriptional activity. The ABA insensitive phenotype of Arabidopsis <i>abi5-1</i> is completely suppressed in transgenic lines expressing BrABI5a or BrABI5b. Overall, these results suggest that ABI5 orthologs, BrABI5a and BrABI5b, have key roles in ABA signalling in Chinese cabbage.</p></div

    Gene structure of the subfamily A <i>AtbZIP</i> and <i>BrbZIP</i> genes in Arabidopsis and Chinese cabbage (<i>Brassica rapa</i>).

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    <p>Exon/intron organization of subfamily A <i>AtbZIP</i> and <i>BrbZIP</i> genes was depicted with the online Gene Structure Display Server. The exons and introns are represented by green boxes and blue lines respectively. The purple box denotes the bZIP domain region.</p

    Domain structure and expression pattern of BrABI5a and BrABI5b.

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    <p>(A) Schematic diagram of domains in the BrABI5a and BrABI5b protein. Three N-terminal and one C-terminal conserved sequences (C1, C2, C3 and C4) are shown in the green box, the basic domain is shown in the blue box, the bipartite nuclear localization signal is shown in the black brown rectangle and the Leu residues defining the Leu zipper are shown in the red rectangle, #, the conserved sumoylation site is shown in brown. (B) Exon/intron organization of <i>BrABI5a</i> and <i>BrABI5b</i> genes. The exons and introns are represented by boxes and lines respectively. (C-D) qRT-PCR analysis of the expression patterns of <i>BrABI5a</i> and <i>BrABI5b</i> under various environmental stress conditions. The relative expression of <i>BrABI5a</i> or <i>BrABI5b</i> was normalized to the expression of cabbage <i>ACTIN2</i> (<i>BrACTIN2</i>) and expressed relative to the level in mock-treated seedlings.</p
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