Additional file 1: of A Lotus japonicus E3 ligase interacts with the Nod Factor Receptor 5 and positively regulates nodulation

Table S1. Primers used for cloning into expression vectors. Table S2. Primers used in qRT-PCR. Table S3. Primers used for cloning into GoldenGate and Gateway vectors. Figure S1. Phylogenetic tree of amino acid sequences of LjPUB13 with previously characterized E3s of other species and L. japonicus uncharacterized PUBs. Figure S2. Amino acid sequence alignment of LjPUB13 with AtPUB13. Figure S3. Expression levels of LjPUB13 in pub13 LORE1 mutants. Figure S4. Formation of nodules in pub13.1 vs. har1 mutants. Figure S5. Inoculated 28-day-old wild type and homozygous pub13.3 mutants. Figure S6. Nodule sections of wild type, pub13.1 and pub13.2 plants. Figure S7. ROS accumulation in roots of pub13 mutants vs wt plants. Figure S8. Expression of defence genes in L. japonicus wild type and pub13 mutants. Figure S9. LjFLS2 ubiquitination tests. (DOCX 39900 kb

The “topology screen” assay.

<p>(<b>A</b>) Expression constructs. <i>hr3</i> enhancer, baculoviral (BmNPV) homologous region 3 enhancer sequence; pActin, <i>Bombyx mori</i> A3 cytoplasmic actin promoter; MCS, multiple cloning site; actin pA, 3′untranslated region of <i>B. mori</i> actin gene containing polyadenylation signals; Flag, epitope tag; OR; Odorant or opioid receptor ORF; THE, Tobacco etch virus protease recognition site; HR3, <i>Bombyx mori</i> hormone receptor 3. (<b>B</b>) Hypothetical model illustrating possible location of HR3 in both fusion constructs, with respect to the OR orientation (GPCR or not) in the membrane. RORE-bA, response element for retinoic acid receptor-related orphan receptor/basal actin promoter.</p

Co-localization of odorant receptors expressed in lepidopteran insect cells.

<p>(<b>A</b>) Expression constructs for N-terminally (mycOR1, mycOR2) or C-terminally tagged receptors (OR1myc) were transfected in Bm5 cells (in the absence/presence of OR7) and the localization of the expressed ORs was detected using anti-Myc antibody. Control indicates transfection with empty expression vector. (<b>B</b>) Co-localization of OR2 with the plasma membrane marker wheat germ agglutinin. Cells expressing OR2myc were double stained with WGA-Texas Red-X conjugate (b, e, f) and with anti-myc antibody (a, d, g) in the presence or absence of saponin (a-f and g-i, respectively). (<b>C</b>) Detection of ORs in the membrane fraction of stable cell lines coexpressing mycOR1 (lane 2) or mycOR2 (lane 3) along with flagOR7. Immunoblotting was performed with anti-Myc and anti-Flag antibodies (upper and lower panels, respectively). Membranes from Bm5 untransfected cells were used as a negative control (lane 1). (<b>D</b>) Co-localization of OR1 or OR2 with OR7. Bm5 cells were co-transfected with expression plasmids for N-terminally tagged mycOR1 or mycOR2 together with N-terminally tagged flagOR7 expression vector. Tagged ORs were detected with anti-Myc/anti-mouse fluorescein-labelled IgG and anti-Flag/anti-rabbit Alexa fluor-labelled IgG as indicated and counter-stained with DAPI. (<b>E</b>) Pull-down assays showing heteromerization between OR1 and OR7 or OR2 and OR7. Extracts containing C-terminally Myc-His-tagged OR7 were incubated with Ni²⁺-NTA beads and bound protein complexes were analyzed by Western blot by anti-Flag antibody (upper panel) for the presence of N-terminally Flag-tagged OR1 and OR2 or by anti-Myc antibody (lower panel) to detect OR7mychis.</p

Flow cytometric analysis of expression of Myc-tagged OR2 on the surface of Bm5 cells.

<p>(<b>A</b>) N- or (<b>B</b>) C-terminally Myc-tagged OR2 were stably expressed in Bm5 cells in the presence of flagOR7 and analyzed by FACS for the extracellular localization of the Myc tag. For each panel, the green tracing and number represent fluorescence values obtained from the staining of the cells with only the FITC labelled secondary antibodies, while the red tracing and number represent values obtained from cells incubated with both the primary anti-Myc and the FITC labelled secondary antibodies. Increased fluorescence intensity (2.59-fold over the background value) was observed for the C-terminally Myc-tagged receptor in comparison to the receptor that was Myc-tagged at the N-terminal end (1.43-fold over the background value). (<b>C</b>) Values (increases over background) indicated in this panel represent the mean ± S.E.M. of three independent experiments. (<b>D</b>) Western blot analyses of whole cell lysates from the stable cell lines used for FACS analysis and control, mock-transformed cells, probed with anti-Myc (upper) and anti- tubulin (lower) antibodies. (<b>E</b>) FACS analysis of cells expressing the N-terminally Myc-tagged µ-opioid receptor used as a positive control for the extracellular localization of the Myc tag. Green and red tracing/numbers are as in panels A and B. Inset shows the detection of µOR in these cells by western blotting with the anti-myc antibody. The arrowhead and the arrow point to major bands detected (putative monomer and dimer, respectively), while the positions of 50, 90 and 118-kDa molecular mass markers are indicated at left.</p

List of oligonucleotides used in PCR. Restriction sites are underlined; initiation and termination codons are in bold and italics, respectively.

<p>List of oligonucleotides used in PCR. Restriction sites are underlined; initiation and termination codons are in bold and italics, respectively.</p

Expression of <i>A. gambiae</i> OR1, OR2 and OR7 in insect cells.

<p>(<b>A</b>) Schematic representation of the basic backbone vector (pEIA) used for the heterologous expression of various forms (tagged and untagged) of ORs in lepidopteran cells. <i>hr3</i> enhancer, baculoviral (BmNPV) homologous region 3 enhancer sequence; pActin, <i>Bombyx mori</i> A3 cytoplasmic actin promoter; MCS, multiple cloning site; actin pA, 3′untranslated region of <i>B. mori</i> actin gene containing polyadenylation signals; IE1 cassette, baculoviral (BmNPV) DNA fragment containing the <i>ie-1</i> transactivator gene under the control of its native viral promoter; OR; <i>A. gambiae</i> odorant receptor ORF; Myc, Flag and MycHis, epitope tags. (<b>B</b>) Detection of heterologous expression of C-terminally MycHis-tagged ORs in transfected Hi5 cells using Myc monoclonal antibody. (<b>C</b>) Detailed western blot analysis of OR2. Hi5 cells were transfected with plasmids expressing different versions of OR2, and lysates were analyzed using a specific polyclonal antibody against OR2 (left panel, lanes 1–5) or monoclonal antibodies against the Myc (middle panel, lanes 6–9) or the Flag epitope (right panel, lanes 10–11). Arrowheads and arrows indicate major bands corresponding to monomers and putative dimers, respectively. (<b>D</b>) Detailed western blot analysis of OR1. Hi5 cells were transfected with plasmids expressing different versions of OR1, and lysates were analyzed using monoclonal antibodies against the Myc (middle panel, lanes 1–4) or the Flag epitope (right panel, lanes 5–6). In the left panel immunoreactivity of the specific polyclonal antibody against OR1 is shown, with lysates from cells expressing mycOR1 after treatment with the proteasome inhibitor MG132. Molecular weight markers are shown on the left. (<b>E</b>) and (<b>F</b>) Effect of coexpression of OR7 on the expression levels of OR1 and OR2. Hi5 cells were transfected with constant amounts (45% of total DNA) of Myc-tagged OR1 or OR2, along with equal amounts of Flag-tagged OR7 or empty vector (pEIA), and pEIA-GFP (10% of total DNA, for evaluation of the efficiency of transfection). Whole cell lysates (<b>E</b>) and membrane fractions (<b>F</b>) were analysed by SDS-PAGE and western blot. Detection of OR1, OR2 and OR7 was done using the anti-Myc and anti-Flag antibodies either consecutively (in <b>E</b>) or simultaneously (in <b>F</b>). To control for loading, the whole lysate fractions were also probed with an anti-tubulin antibody.</p

Topology assays for the mosquito OR1 and OR2.

<p>Chimeric receptor proteins fused at either their N- or the C- terminus to the TEV cleavage sequence (THE) and the HR3 transcription factor are co-expressed in Hi5 cells with a GFP reporter construct together with or without TEV protease. For both OR1 (<b>A</b>) and OR2 (<b>B</b>) N-terminal fusions (HR3-THE-OR1 and HR3-THE-OR2), expression of the TEV protease resulted in increase of fluorescence of the cells. No significant increase of fluorescence was detected when the C-terminal fusions of ORs were used (OR1-THE-HR3 and OR2-THE-HR3). Similar constructs of the human opioid receptor δ (δOR) that was used as control (<b>C</b>) give opposite results with an increased fluorescence for the C-terminal fusion (δOR-THE-HR3). For each chimeric receptor protein, both representative images (left) and quantitative results (right, with values representing the mean ± S.E.M. of four experiments) from the fluorometric analysis are shown.</p

AtHESPERIN: a novel regulator of circadian rhythms with poly(A)-degrading activity in plants

<p>We report the identification and characterization of a novel gene, <i>AtHesperin</i> (<i>AtHESP</i>) that codes for a deadenylase in <i>Arabidopsis thaliana</i>. The gene is under circadian clock-gene regulation and has similarity to the mammalian <i>Nocturnin</i>. AtHESP can efficiently degrade poly(A) substrates exhibiting allosteric kinetics. Size exclusion chromatography and native electrophoresis coupled with kinetic analysis support that the native enzyme is oligomeric with at least 3 binding sites. Knockdown and overexpression of <i>AtHESP</i> in plant lines affects the expression and rhythmicity of the clock core oscillator genes <i>TOC1</i> and <i>CCA1</i>. This study demonstrates an evolutionary conserved poly(A)-degrading activity in plants and suggests deadenylation as a mechanism involved in the regulation of the circadian clock. A role of <i>AtHESP</i> in stress response in plants is also depicted.</p