学会抄録

Genome annotation. (XLSX 9 kb

A stable monomeric form of human IL-10 has impaired activity.

<p>Bone marrow-derived macrophages, dendritic cells and mast cells from wild-type mice were tested for their response to human IL-10 and a stable monomeric form of IL-10 (IL-10m). Cells pre-treated with IL-10 were stimulated with 100 ng/ml LPS and TNF-α expression was determined to asses anti-inflammatory properties of IL-10 in macrophages (A) and dendritic cells (B) (<i>n</i> = 3, error bars indicate standard error). Mast cells were cultured for 48 hours in the presence of IL-10, where after cell viability was determined (C) (<i>n</i> = 3, error bars indicate standard error). <i>P</i><0.01 at all concentrations in all three assays.</p

The extracellular domain of IL-10R2 is not sufficient to maintain IL-10 activity.

<p>CHO-K1 cells were co-transfected with combinations of the expression vectors for IL-10R1, IL-10R2, IL-10R2^Δ230–330 (extracellular domain), IL-10R2^Δ1–190 (intracellular domain) or an empty vector (mcs) and cultured for 24 hours. Surface expression of IL-10R1 or IL-10R2 was analysed by flow cytometry and mean fluorescent intensity is plotted (<i>n</i> = 6, error bars represent standard error) (A). Phosphorylation of tyrosine 705 (Y705) of STAT3 by IL-10 (100 ng/ml) in CHO-K1 cells upon transient transfection with IL-10 receptors was analysed by western blot (B).</p

The intracellular domain of IL-10R2 mediates conformational changes in IL-10R1.

<p>CHO-K1 cells were co-transfected with combinations of the expression vectors for IL-10R1, IL-10R2, IL-10R2^Δ230–330, IL-10R2^Δ1–190 or an empty vector (mcs) and cultured for 24 hours. Cells were surface stained with the 1B1.3a anti-mouse IL-10R1 monoclonal antibody and analysed by flow cytometry (A). Mean fluorescent intensity for IL-10R1 binding is plotted in a dose-dependent manner (<i>n</i> = 6, error bars represent standard error). IL-10 was cross-linked to the surface of transfected cells and surface-bound IL-10 was detected by flow cytometry (B). Mean fluorescent intensity for IL-10 binding is plotted (<i>n</i> = 3, error bars represent standard error). Asterisk(s) indicate significant differences as determined by a Welch’s <i>t</i>-test (*<i>P</i><0.05; **<i>P</i><0.01).</p

Tyk2 affects early responses to IL-10.

<p>Bone marrow-derived macrophages and dendritic cells from Tyk2^-/- mice were investigated in more detail for their signaling. Phosphorylation of tyrosine 705 (Y705) of STAT3 by IL-10 (0, 1, 10 and 100 ng/ml) was analysed in wild-type and Tyk2^-/- macrophages and dendritic cells by western blot (A and B respectively). Relative up-regulation of SOCS3 mRNA expression by IL-10 was analysed by quantitative PCR in both macrophages and dendritic cells (C). Fold induction of SOCS3 expression was calculated using the 2^ΔCt method using HPRT as a reference gene. Macrophages and dendritic cells from wild-type and Tyk2^-/- transgenic mice were pre-treated with IL-10 and were stimulated with 100 ng/ml LPS and the inhibition of TNF-α expression was determined at 2 and 24 hours (D). Asterisk(s) indicate significant differences as determined by a Welch’s <i>t</i>-test (*<i>P</i><0.05; **<i>P</i><0.01).</p

The differential response of bone marrow-derived cells to IL-10.

<p>Bone marrow-derived macrophages, dendritic cells and mast cells were analysed by flow cytometry for the expression of cellular markers CD11b & F4/80 (A), CD11c & MHC-II (D) and FcεRI & c-kit (G), respectively. Bone marrow-derived cells were tested for their response to IL-10 by measuring suppression of TNF-α expression by macrophages (B) and dendritic cells (E) or proliferative/anti-apoptotic ability in mast cells (H) (<i>n</i> = 4, error bars indicate standard error). Dose dependent tyrosine phosphorylation of STAT transcription factors by IL-10 (0, 1, 10 or 100 ng/ml) was analysed by western blot in macrophages (C), dendritic cells (F) and mast cells (I).</p

IL-10R2 mediated signaling via Tyk2 plays a limited role in IL-10 activity.

<p>Bone marrow-derived macrophages, dendritic cells and mast cells from wild-type, IL-10R1^-/-, IL-10R2^-/- and Tyk2^-/- mice were tested for their response to IL-10. Macrophages and dendritic cells from wild-type and IL-10R^-/- mice were pre-treated with IL-10 and subsequently stimulated with 100 ng/ml LPS. The percentage of inhibition of TNF-α expression of macrophages and dendritic cells was determined after overnight incubation (A and B, respectively) (<i>n</i> = 3, error bars indicate standard error). Similarly, macrophages and dendritic cells from Tyk2^-/- mice were tested for their response to IL-10 (D and E, respectively) (<i>n</i> = 4, error bars indicate standard error). Mast cells from wild-type and transgenic mice were cultured for 48 hours in the presence of IL-10 and cell viability was determined (C and F) (<i>n</i> = 3 for IL-10R^-/- mice and <i>n</i> = 4 for Tyk2^-/- mice, error bars indicate standard error). Asterisk(s) indicate significant differences as determined by a Welch’s <i>t</i>-test (*<i>P</i><0.05; **<i>P</i><0.01).</p

Apoplastic Gr-VAP1 suppresses immunity of potato plants to <i>G. rostochiensis</i>.

<p>(A) Transgenic potato plants stably overexpressing Gr-VAP1 in the apoplast show enhanced susceptibility to <i>G. rostochiensis</i>. The number of nematodes per plant was compared at 6 weeks post inoculation for two independent transgenic potato lines harboring either Gr-VAP1 (<i>Gr-VAP1-A</i> and <i>Gr-VAP1-B</i>) or the corresponding T-DNA insert of the empty binary expression vector (<i>EV</i>). The expression constructs included native signal peptide for secretion of Gr-VAP1. Bars represent standard errors of the means. Different letters indicate statistically significant differences between plant genotypes as determined with ANOVA (with <i>P</i>-values <0.05). (B) Apoplastic Gr-VAP1 perturbs the active site of the extracellular defense-related papain-like cysteine protease C14^tub of potato (<i>S. tuberosum</i>). Image shows binding of the fluorescent activity-based probe DCG-04 to the active site of C14^tub and C14^lyc of tomato (<i>S. lycopersicum</i>) following treatment with Gr-VAP1 isolated from apoplastic fluids of agroinfiltrated leaves. Treatments with the Avr2, egg white cystatin, and apoplastic fluids from agroinfiltrations with the empty binary expression vector (Empty vector), and with buffer alone (Buffer) were included as controls.</p

Ectopic venom allergen-like proteins suppress basal immunity in <i>Arabidopsis thaliana</i>.

<p>(A) Heterologous expression of the venom allergen-like protein Gr-VAP1 from <i>G. rostochiensis</i>, and Hs-VAP1 and Hs-VAP2 from <i>Heterodera schachtii</i> in the apoplast of transgenic Arabidopsis lines enhances their susceptibility to <i>H. schachtii</i>. Two independent transgenic lines per construct (-A and -B) were compared with corresponding transgenic line harboring the T-DNA of the empty vector (EV) and wild type <i>A. thaliana</i> (Col-0). Bars represent mean number of nematodes per plants with standard errors of the means. Letters indicate statistical significance when using <i>P</i>-value <0.05 as threshold. (B) Ectopic Hs-VAP1 and Hs-VAP2 enhance development of disease symptoms of fungal and oomycete pathogens in leaves of transgenic Arabidopsis lines. Pictures show symptoms on leaves inoculated with <i>Botrytis cinerea</i>, <i>Plectosphaerella cucumeria</i>, and two isolates of <i>Phytophthora brassicae</i>, or mock inoculated. (C) Ectopic Hs-VAP1 and Hs-VAP2 suppress seedling growth response of Arabidopsis to the immunogenic peptide flg22. Bars represent mean root length of transgenic lines with standard error of mean after 10 days in the presence or absence of 10 µM flg22.</p

Ten most down-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis.

<p>Differentially expressed genes in transgenic <i>Arabidopsis thaliana</i> overexpressing <i>Hs-VAP1</i> and <i>Hs-VAP2</i> relative to the corresponding transgenic empty vector control plants (for full lists see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004569#ppat.1004569.s008" target="_blank">S1 Table</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004569#ppat.1004569.s009" target="_blank">S2 Table</a>).</p><p>* The fold change (FC) is calculated as standardized log2-transformed counts per million relative to transgenic plants harboring the corresponding empty vector.</p><p>Ten most down-regulated transcripts by ectopic venom allergen-like proteins in Arabidopsis.</p