The increased cytotoxicity of uNK cells by mifepristone was inhibited by PD98059 or U0126.

<p>Before the treatment with cortisol ± mifepristone, uNK cells were pretreated with PD98059 (A) or U0126 (B) for 30 min. They were then treated with mifepristone ± cortisol for 24 h. Cells stimulated with IL-15 were used as positive control. The cytotoxicity of uNK cells was detected by the MTS assay. Data were analyzed using ANOVA and expressed as means ± SEM. *, <i>P</i><0.05.</p

Upregulation of perforin expression by mifepristone was inhibited by PD98059 or U0126.

<p>Before the treatment with cortisol ± mifepristone, uNK cells were pretreated with PD98059 (A) or U0126 (B) for 30 min. They were then treated with mifepristone ± cortisol for 24 h. Cells stimulated with IL-15 were used as positive control. Perforin expression in uNK cells was examined by flow cytometry. The value is the percent of perforin-positive cells in the total number of uNK cells. Data were analyzed using ANOVA and expressed as means ± SEM. *, <i>P</i><0.05.</p

Effects of cortisol on mifepristone-induced uNK-cell cytotoxicity and perforin expression.

<p>Isolated uNK cells were treated with cortisol (1.0 µM) ± and mifepristone (1.0 µM) for 24 h. A, a representative flow cytometry result for perforin expression in different groups. B, results of uNK-cell cytotoxicity in different groups. C, data summary of flow cytometry results for perforin expression. The value is the percent of perforin-positive cells in the total number of uNK cells. Experiments were independently repeated 5 independent experiments. Data were analyzed using ANOVA and expressed as means ± SEM. *, <i>P</i><0.05.</p

Image_2.JPEG

<p>Previously, we reported the biocontrol effects of Saccharothrix yanglingensis strain Hhs.015 on Valsa mali. Here, we report a novel protein elicitor BAR11 from the biocontrol strain Hhs.015 and its functions in plant defense responses. Functional analysis showed that the elicitor BAR11 significantly stimulated plant systemic resistance in Arabidopsis thaliana to Pseudomonas syringae pv. tomato DC3000. In addition, systemic tissues accumulated reactive oxygen species and deposited callose in a short period post-treatment compared with the control. Quantitative RT-PCR results revealed that BAR11 can induce plant resistance through the salicylic acid and jasmonic acid signaling pathways. Further analysis indicated that BAR11 interacts with host catalases in plant cells. Taken together, we conclude that the elicitor BAR11 from the strain Hhs.015 can trigger defense responses in plants.</p

Effects of mifepristone on the ERK signaling pathway in uNK cells.

<p>Western blot of p-ERK and total ERK in uNK cells treated with mifepristone ± cortisol. To detect p-ERK and total ERK, total cell lysates were harvested after a 30 min treatment with mifepristone (1.0 µM) ± cortisol (1.0 µM). Uterine NK cells were isolated and cultured in the medium without IL-2 for 12 h as a negative control of MAPK/ERK activation. In the drug treatment groups, uNK cells were cultured in the medium supplemented with IL-2. Uterine NK cells were cultured in the medium with IL-2 and IL-15 for 15 min as a positive control. A typical blot (A) and densitometric scans of triplicate blots (B) are shown. Data were analyzed using ANOVA and expressed as means ± SEM. *, <i>P</i><0.05.</p

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<p>Previously, we reported the biocontrol effects of Saccharothrix yanglingensis strain Hhs.015 on Valsa mali. Here, we report a novel protein elicitor BAR11 from the biocontrol strain Hhs.015 and its functions in plant defense responses. Functional analysis showed that the elicitor BAR11 significantly stimulated plant systemic resistance in Arabidopsis thaliana to Pseudomonas syringae pv. tomato DC3000. In addition, systemic tissues accumulated reactive oxygen species and deposited callose in a short period post-treatment compared with the control. Quantitative RT-PCR results revealed that BAR11 can induce plant resistance through the salicylic acid and jasmonic acid signaling pathways. Further analysis indicated that BAR11 interacts with host catalases in plant cells. Taken together, we conclude that the elicitor BAR11 from the strain Hhs.015 can trigger defense responses in plants.</p

Effects of mifepristone on the phosphorylation level of MAPK in uNK cells.

<p>A, a representative immunoblot is shown after the treatment of uNK cells with mifepristone (1.0 µM) for 0, 15, 30, 60, and 120 min. Uterine NK cells were isolated and cultured in the medium without IL-2 for 12 h as a negative control of MAPK/ERK activation. In the drug treatment group, uNK cells were cultured in the medium supplemented with IL-2. Uterine NK cells were cultured in the medium with IL-2 and IL-15 for 15 min as a positive control. Immunodetection of MAPK members used specific antibodies for phosphorylated and total proteins of ERK1/2, P38 and JNK. B, densitometric scans of triplicate blots are shown. Experiments were independently repeated 3 times in each group. Data were analyzed using ANOVA and expressed as means ± SEM. *, <i>P</i><0.05.</p

Image_3.PDF

<p>Previously, we reported the biocontrol effects of Saccharothrix yanglingensis strain Hhs.015 on Valsa mali. Here, we report a novel protein elicitor BAR11 from the biocontrol strain Hhs.015 and its functions in plant defense responses. Functional analysis showed that the elicitor BAR11 significantly stimulated plant systemic resistance in Arabidopsis thaliana to Pseudomonas syringae pv. tomato DC3000. In addition, systemic tissues accumulated reactive oxygen species and deposited callose in a short period post-treatment compared with the control. Quantitative RT-PCR results revealed that BAR11 can induce plant resistance through the salicylic acid and jasmonic acid signaling pathways. Further analysis indicated that BAR11 interacts with host catalases in plant cells. Taken together, we conclude that the elicitor BAR11 from the strain Hhs.015 can trigger defense responses in plants.</p

Analysis of uNK-cell cytotoxicity and perforin expression after treatment with different concentrations of mifepristone.

<p>The purified uNK cells were incubated with 0, 65, 200, and 1000 nmol/L mifepristone for 24 h. Then, they were subjected to analysis of uNK-cell cytotoxicity and perforin expression. A, a representative flow cytometry profiles of perforin expression was shown. Uterine NK-cell cytotoxicity (B) and perforin (C) expression after treatment with different concentrations of mifepristone were evaluated. Values are expressed as means ± SEM. The influence of mifepristone was evaluated by 4 independent experiments. <i>P-</i>value<i>s</i> referred to One-way analysis of variance, n = 6, * <i>P</i><0.05 vs. control group.</p

Study on Interactions between the Major Apple Valsa Canker Pathogen <i>Valsa mali</i> and Its Biocontrol Agent <i>Saccharothrix yanglingensis</i> Hhs.015 Using RT-qPCR

<div><p>The mechanism of biocontrol agent <i>Saccharothrix yanglingensis</i> Hhs.015 action against <i>Valsa mali</i>, a major apple Valsa canker pathogen, was examined using a novel, sensitive (minimum detection limit 100 pg/μL) and reliably RT-qPCR technique. Prior to lesion formation, total concentration of <i>V</i>. <i>mali</i> in the bark showed a significant decrease (<i>p</i><0.05) after 24 h of Hhs.015 treatment. This was more pronounced at 48 and 96 h post treatment. After lesion formation, levels of <i>V</i>. <i>mali</i> remained constant at the boundary between infected and uninfected bark tissues, although the relative expansion rate of the lesion was significantly reduced (<i>p</i><0.05). Gene expression levels of endo-polygalacturonase, a marker for fungal pathogenicity, were sharply reduced while host induced resistance callose synthase levels increased significantly (<i>p</i><0.05) at the boundary bark at 9 d after Hhs.015 treatment. The results showed that biocontrol agent Hhs.015 prevented infection of <i>V</i>. <i>mali</i> by inhibiting pathogen growth, down-regulating pathogenicity factor expression and inducing a high level of host resistance.</p></div