Growth Promotion-Related miRNAs in <i>Oncidium</i> Orchid Roots Colonized by the Endophytic Fungus <i>Piriformospora indica</i>

<div><p><i>Piriformospora indica</i>, an endophytic fungus of Sebacinales, colonizes the roots of a wide range of host plants and establishes various benefits for the plants. In this work, we describe miRNAs which are upregulated in <i>Oncidium</i> orchid roots after colonization by the fungus. Growth promotion and vigorous root development were observed in <i>Oncidium</i> hybrid orchid, while seedlings were colonized by <i>P. indica</i>. We performed a genome-wide expression profiling of small RNAs in <i>Oncidium</i> orchid roots either colonized or not-colonized by <i>P. indica</i>. After sequencing, 24,570,250 and 24744,141 clean reads were obtained from two libraries. 13,736 from 17,036,953 unique sequences showed homology to either 86 miRNA families described in 41 plant species, or to 46 potential novel miRNAs, or to 51 corresponding miRNA precursors. The predicted target genes of these miRNAs are mainly involved in auxin signal perception and transduction, transcription, development and plant defense. The expression analysis of miRNAs and target genes demonstrated the regulatory functions they may participate in. This study revealed that growth stimulation of the <i>Oncidium</i> orchid after colonization by <i>P. indica</i> includes an intricate network of miRNAs and their targets. The symbiotic function of <i>P. indica</i> on <i>Oncidium</i> orchid resembles previous findings on Chinese cabbage. This is the first study on growth regulation and development of <i>Oncidium</i> orchid by miRNAs induced by the symbiotic fungus <i>P. indica</i>.</p></div

Target genes prediction for miRNAs in <i>P. indica</i> colonized- <i>Oncidium</i> roots.

<p>P/CK: ratio of miRNA TPM between roots with/without colonization by <i>P.indica.</i></p

Expression analyses of miRNAs in <i>Oncidium</i> orchid ± <i>P. indica</i> by qPCR.

<p>Roots colonized ± <i>P. indica</i> for 8 weeks were sampled. Data represent the mean ± SD of 3 replicates and normalization by 5.8S rRNA. <b>e</b> represents the decimal point. * indicates antisense strand; no <i>P. ind</i>i<i>ca</i> indicates that the novel miRNAs were specifically found in the<i>–P. indica</i> orchid library. <i>P. indica</i> indicates that the novel miRNAs were specifically found in the +<i>P. indica</i> orchid library.</p

Stage and Gene Specific Signatures Defined by Histones H3K4me2 and H3K27me3 Accompany Mammalian Retina Maturation In Vivo

<div><p>The epigenetic contribution to neurogenesis is largely unknown. There is, however, growing evidence that posttranslational modification of histones is a dynamic process that shows many correlations with gene expression. Here we have followed the genome-wide distribution of two important histone H3 modifications, H3K4me2 and H3K27me3 during late mouse retina development. The retina provides an ideal model for these studies because of its well-characterized structure and development and also the extensive studies of the retinal transcriptome and its development. We found that a group of genes expressed only in mature rod photoreceptors have a unique signature consisting of de-novo accumulation of H3K4me2, both at the transcription start site (TSS) and over the whole gene, that correlates with the increase in transcription, but no accumulation of H3K27me3 at any stage. By <em>in silico</em> analysis of this unique signature we have identified a larger group of genes that may be selectively expressed in mature rod photoreceptors. We also found that the distribution of H3K4me2 and H3K27me3 on the genes widely expressed is not always associated with their transcriptional levels. Different histone signatures for retinal genes with the same gene expression pattern suggest the diversities of epigenetic regulation. Genes without H3K4me2 and H3K27me3 accumulation at any stage represent a large group of transcripts never expressed in retina. The epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. In addition to the developmental patterns seen in wild type retina, the dramatic changes of histone modification in the retinas of mutant animals lacking rod photoreceptors provide a tool to study the epigenetic changes in other cell types and thus describe a broad range of epigenetic events in a solid tissue <em>in vivo</em>.</p> </div

Category distribution of small RNAs in root tissues of <i>Oncidium</i> orchid ± <i>P. indica</i> colonization.

<p>−<i>P. indica</i>: roots without colonization by <i>P. indica</i>;</p><p>+<i>P. indica</i>: roots colonized by <i>P. indica.</i></p

Expression analyses of the target genes of miRNAs by qPCR.

<p>Roots colonized with <i>P. indica</i> for 0, 1, 3 and 8 weeks were sampled and mRNA expression level was analyzed by qPCR. Data represents the mean ± SD of 3 replicates, and were normalized to the <i>Actin</i> mRNA level.</p

Changes in histone modifications during retina maturation.

<p>(<b>A</b>) Immunofluorescence microscopic images of sagittal sections of developmental mouse retina tissue array stained with anti-H3K4me2 (<b>A</b>, green, upper panels) and anti-Rhodopsin (<b>A</b>, red, upper panels), anti-H3K27me3 (<b>A</b>, green, lower panels) and anti-SVP38 (<b>A</b>, red, lower panels), and counterstained with Hoechst in cell nuclei (<b>A,</b> blue). ONBL, outer neuroblast layer; INBL, inner neuroblast layer; GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar represents 25 µm. (<b>B</b>) H3K4me2 labeling with high magnification for ONL from adult retina (Scale bar = 6 µm). (<b>C</b>) H3K27me3 labeling with high magnification for ONL from adult retina (Scale bar = 6 µm). (<b>D</b>) Averaged and normalized intensity profiles for fluorescence of each specific antibody or Hoechst staining across the nuclear centers (e.g. white bars in <b>B</b> and <b>C</b>) of rod photoreceptor nuclei (n = 5). (<b>E</b>) Adult retina outer nuclear layer labeled with an antibody recognizing Crx. (<b>F</b>) Control labeling of adult ONL showing lack of staining with secondary antibody alone. (<b>G</b>) Averaged and normalized intensity profile for Crx labeling (green) and Hoechst (blue) across rod nuclear centers (bar in <b>E</b>). (<b>H, I</b>) Confocal images with high magnification for RGCL from adult retina (Scale bar = 15 µm). Cells marked * or # show distinct cellular distribution with H3K4me2 antibody (<b>H</b>, green) and a′ or b′ show distinct cellular distribution with H3K27me3 antibody (<b>I</b>, green).</p

Growth effects of <i>P. indica</i> on <i>Oncidium</i> orchid.

<p>(A) Seedlings inoculated with <i>P. indica</i> for 8 weeks showed significantly enhanced growth and root development. (B–D) Anatomic structures of roots colonized by <i>P. indica</i> for 24 hours. Hyphae (green spots in B, arrow head) and penetration site (red spots in C, arrow head) were overlayed in bright field (D, arrow head). Bar = 50 µm. (E) Microscopic structure of roots colonized by <i>P. indica</i> for 5 days. A large number of hyphae were widespread over the root surface and tip, elongation zone and mature zone. Bar = 200 µm. (F, G) Microscopic structures of transverse sections and longitudinal sections of roots colonized by <i>P.indica</i> for 5 days. Hyphae fully colonized the velamen. Chloroplast autofluorescence (red) was also detected in cortex. Bar = 200 µm. (H) Microscopic structures of transverse section of roots colonized by <i>P. indica</i> for 5 days. <i>P. indica</i> was restricted in the velamen and not detectable in the exodermis of <i>Oncidium</i> roots. Chloroplast autofluorescence (red) was detected in cortex. Bar = 50 µm. (I) Micrograph of root cross sections from seedlings colonized with <i>P. indica</i> for 5 days. Without chlorophyll fluorescence in velamen, the penetration sites (red spot) were clearly detected. Bar = 20 µm. (J) Growth quantification of seedling colonized with <i>P. indica</i> for 8 weeks. Fresh weight, plant height, leaf number, leaf wide, stem diameter, root number and diameter were analyzed. Error bars represent SD for three independent experiments. *, <i>P</i> value<0.05; **, <i>P</i><0.001. Hyphae were stained with chitin-specific WGA-AF488 (green). Penetrated sites (Ps) were stained with lectin-specific conA-AF633 (red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084920#pone.0084920-Zuccaro1" target="_blank">[88]</a>. Samples were analyzed and photographed with an Olympus IX71 inverted microscope system (Japan). Ve, velamen; EX, exodermis; Cort: cortex; Ch: Chloroplast. Hy, hyphae; Ps, penetration site.</p

Genes with the same expression patterns show different histone signatures in retina.

<p>(<b>A</b>) H3K4me2 and H3K27 accumulation in examples of genes up-regulated during retina maturation. (<b>B</b>) H3K4me2 and H3K27 accumulation in examples of genes down-regulated during retina maturation. Closed arrowheads show TSS of each gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s006" target="_blank">Tables S4</a> &S5). (<b>C</b>) Cluster analysis of H3K4me2 and H3K27me3 occupancy around TSS (+/−2.5 Kb) at all developmental stages for the genes upregulated in mature retina. Tree-view shows 4 clusters (C1–C4) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). (<b>D</b>) Same analysis as in (<b>C</b>) for the genes downregulated in mature retina. Cluster analysis of H3K4me2 and Tree-view shows 3 clusters (C1′–C3′) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). For <b>A</b> and <b>B</b> y-axis is the number of reads in a 100 bp interval. For <b>C</b> and <b>D</b>, (upper panels) y-axis is the normalized occupancy, or the number of reads for a given histone modification in an interval +/−2.5 kb around the TSS of given gene, normalized for the total number of mapped reads in given experiment.</p

Genes with the same expression patterns show different histone signatures in retina.

<p>(<b>A</b>) H3K4me2 and H3K27 accumulation in examples of genes up-regulated during retina maturation. (<b>B</b>) H3K4me2 and H3K27 accumulation in examples of genes down-regulated during retina maturation. Closed arrowheads show TSS of each gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s006" target="_blank">Tables S4</a> &S5). (<b>C</b>) Cluster analysis of H3K4me2 and H3K27me3 occupancy around TSS (+/−2.5 Kb) at all developmental stages for the genes upregulated in mature retina. Tree-view shows 4 clusters (C1–C4) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). (<b>D</b>) Same analysis as in (<b>C</b>) for the genes downregulated in mature retina. Cluster analysis of H3K4me2 and Tree-view shows 3 clusters (C1′–C3′) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). For <b>A</b> and <b>B</b> y-axis is the number of reads in a 100 bp interval. For <b>C</b> and <b>D</b>, (upper panels) y-axis is the normalized occupancy, or the number of reads for a given histone modification in an interval +/−2.5 kb around the TSS of given gene, normalized for the total number of mapped reads in given experiment.</p