The RNA polymerase II subunit RPB-9 recruits the integrator complex to terminate Caenorhabditis elegans piRNA transcription.

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription

Sequencing and Analysis of Plastid Genome in Mycoheterotrophic Orchid Neottia nidus-avis

Plastids are the semiautonomous organelles that possess their own genome inherited from the cyanobacterial ancestor. The primary function of plastids is photosynthesis so the structure and evolution of plastid genomes are extensively studied in photosynthetic plants. In contrast, little is known about the plastomes of nonphotosynthetic species. In higher plants, plastid genome sequences are available for only three strictly nonphotosynthetic species, the liverwort Aneura mirabilis and two flowering plants, Epifagus virginiana and Rhizanthella gardneri. We report here the complete sequence of a plastid genome of nonphotosynthetic mycoheterotrophic orchid Neottia nidus-avis, determined using 454 pyrosequencing technology. It was found to be reduced in both genome size and gene content; this reduction is however not as drastic as in the other nonphotosynthetic orchid, R. gardneri. Neottia plastome lacks all genes encoding photosynthetic proteins, RNA polymerase subunits but retains most genes of translational apparatus. Those genes that are retained have an increased rate of both synonymous and nonsynonymous substitutions but do not exhibit relaxation of purifying selection either in Neottia or in Rhizanthella

Immune reaction and survivability of salmonella typhimurium and salmonella infantis after infection of primary avian macrophages.

Salmonella serovars are differentially able to infect chickens. The underlying causes are not yet fully understood. Aim of the present study was to elucidate the importance of Salmonella Pathogenicity Island 1 and 2 (SPI-1 and -2) for the virulence of two non-host-specific, but in-vivo differently invasive, Salmonella serovars in conjunction with the immune reaction of the host. Primary avian splenic macrophages were inoculated with Salmonella enterica sub-species enterica serovar (S.) Typhimurium and S. Infantis. The number and viability of intracellular bacteria and transcription of SPI-1 and -2 genes by the pathogens, as well as transcription of immune-related proteins, surface antigen expression and nitric oxide production by the macrophages, were compared at different times post inoculation. After infection, both of the Salmonella serovars were found inside the primary macrophages. Invasion-associated SPI-1 genes were significantly higher transcribed in S. Infantis- than S. Typhimurium-infected macrophages. The macrophages counteracted the S. Infantis and S. Typhimurium infection with elevated mRNA expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-12, IL-18 and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) as well as with an increased synthesis of nitric oxide. Despite these host cell attacks, S. Typhimurium was better able than S. Infantis to survive within the macrophages and transcribed higher rates of the SPI-2 genes spiC, ssaV, sifA, and sseA. The results showed similar immune reactions of primary macrophages after infection with both of the Salmonella strains. The more rapid and stronger transcription of SPI-2-related genes by intracellular S. Typhimurium compared to S. Infantis might be responsible for its better survival in avian primary macrophages

Quantification of <i>Salmonella</i>-containing macrophages (flow cytometry) and numbers of intracellular bacteria (microbiology).

<p>The percentage of avian macrophages containing intracellular <i>Salmonella</i> was analyzed by flow cytometry after staining with an anti-<i>Salmonella</i> LPS antibody. Additionally, the SSC fluorescence intensity, which correlates with the <i>Salmonella</i> invasion/uptake, was determined after <i>Salmonella</i> infection of the primary macrophages. Numbers of viable intracellular <i>Salmonella</i> per avian macrophages were determined using bacteriology. White columns—macrophages infected with <i>S</i>. Typhimurium (n = 8), Grey columns—macrophages infected with <i>S</i>. Infantis (n = 6) ** <i>P</i> ≤ 0.05 or * <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. <i>S</i>. Infantis; <b>a</b>) <i>P</i> ≤ 0.05 or a) <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. control; <b>b</b>) <i>P</i> ≤ 0.05 or b) <i>P</i> ≤ 0.1 <i>S</i>. Infantis vs. control.</p

Detection of nitric oxide concentration in supernatants from avian macrophages after <i>Salmonella</i> infection.

<p>White columns—macrophages infected with <i>S</i>. Typhimurium (n = 4), Grey columns—macrophages infected with <i>S</i>. Infantis (n = 6) <b>a</b>) <i>P</i> ≤ 0.05 or a) <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. control; <b>b</b>) <i>P</i> ≤ 0.05 or b) <i>P</i> ≤ 0.1 <i>S</i>. Infantis vs. control.</p

Flow cytometric analysis of primary avian macrophages after infection.

<p>The vitality of avian macrophages was defined by propidium iodide. Apoptotic cells were stained using Annexin V Fluos. Diagrams additionally show fold changes of fluorescence intensity of <i>S</i>. Typhimurium- and <i>S</i>. Infantis-infected avian macrophages after staining with different monoclonal antibodies. White columns—macrophages infected with <i>S</i>. Typhimurium (n = 4), Grey columns—macrophages infected with <i>S</i>. Infantis (n = 4) ** <i>P</i> ≤ 0.05 or * <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. <i>S</i>. Infantis; <b>a</b>) <i>P</i> ≤ 0.05 or a) <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. control; <b>b</b>) <i>P</i> ≤ 0.05 or b) <i>P</i> ≤ 0.1 <i>S</i>. Infantis vs. control.</p

Flow cytometric characterisation of primary avian macrophages.

<p>FSC-SSC dot plot diagram shows primary avian macrophages as large and granular cells at day 7 of culture. Dead cells were stained using propidium iodide and represented approximately 2.7% of avian macrophages. Representative histograms indicate expression of cell-surface molecules on primary avian macrophages (gate 1) cultured for 7 days. Multigraph overlay from flow cytometry analysis are shown. The black line represents the isotype control, the gray peak shows cells labeled with the appropriate monoclonal antibody. Cultured macrophages were positively stained with the antibodies against chicken monocytes/macrophages—KUL01, CD44, MHC class I and II. Cultured macrophages were negatively stained for B cells (BU1), γδ T cells (TCR1) and αβ T cells (TCR2). n = 8</p

Relative quantification of mRNA expression of immune-related proteins in primary avian macrophages infected with <i>S</i>. Typhimurium or <i>S</i>. Infantis.

<p>Data were presented as fold change compared to non-infected macrophages. White columns—macrophages infected with <i>S</i>. Typhimurium (n = 6), Grey columns—macrophages infected with <i>S</i>. Infantis (n = 6) ** <i>P</i> ≤ 0.05 or * <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. <i>S</i>. Infantis; <b>a</b>) <i>P</i> ≤ 0.05 or a) <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. control; <b>b</b>) <i>P</i> ≤ 0.05 or b) <i>P</i> ≤ 0.1 <i>S</i>. Infantis vs. control.</p

Phase contrast micrographs of primary avian macrophages.

<p><b>A)</b> Heterogeneous cell population at day 1 after isolation. <b>B)</b> Day 7 of primary cell culture, monocytes differentiated into flattened circular macrophages with extensive cytoplasmic veil. arrowhead: lymphocyte, arrow: primary macrophage, bar = 100 μm.</p