8 research outputs found
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
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
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
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
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
Additional file 2: Table S2. of WRKY6 restricts Piriformospora indica-stimulated and phosphate-induced root development in Arabidopsis
Genes which were regulated more than 2-fold (log2 value ≥ 1) in response to Pi limitation, P. indica and mutation of WRKY6. Table S3. Primers used in this study. Table S4. Microarray validation with Real-time PCR. (PDF 563 kb
Additional file 1: Table S1. of WRKY6 restricts Piriformospora indica-stimulated and phosphate-induced root development in Arabidopsis
Genes which are up-regulated more than 2-fold in wrky6 + P. indica in LP (log2 value ≥ 1 or ≤ −1). (XLS 200 kb
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