20 research outputs found

    Subcellular localizations of P-bodies and stress granules in response to heat stress.

    No full text
    <p>AoDcp2-EGFP and AoPab1-mDsRed were used as markers of P-bodies and stress granules, respectively. Approximately 10<sup>4</sup> conidia of cells co-expressing AoDcp2-EGFP and AoPab1-mDsRed were grown in CD medium at 30°C for 18 h before being exposed to 45°C for 10 min. The lowest panels show magnified images of the apical region of the cell (within the boxed area in the above image). Scale bars  =  5 µm.</p

    Stress-induced formation of stress granules.

    No full text
    <p>Approximately 10<sup>4</sup> conidia of cells expressing AoPab1-EGFP were grown in CD+Met medium at 30°C for 18 h before being exposed to various types of stress. (A) Subcellular localization of AoPab1-EGFP. Accumulation of AoPab1-EGFP (indicated by the arrow) was induced when cells were exposed to 45°C for 10 min. (B) Subcellular localization of EGFP. Accumulation of EGFP was not observed in cells exposed to heat stress. (C) Accumulation of AoPab1-EGFP (indicated by the arrows) was induced in cells treated with cold stress (4°C, 30 min), glucose deprivation (10 min), osmotic stress (1.2 M sorbitol, 30 min), and ER stress (10 mM DTT, 60 min). Scale bars  =  5 µm.</p

    Effect of <i>Aoso</i> deletion on stress granules.

    No full text
    <p>(A) Stress granule formation in wild-type (WT) and <i>Aoso</i>-deletion mutant (▵<i>Aoso</i>) cells was detected using AoPab1-EGFP as a marker. Approximately 10<sup>4</sup> conidia of cells expressing AoPab1-EGFP were grown in CD+Met medium at 30°C for 18 h before being exposed to 45°C for 10 min. Scale bar  =  5 µm. (B) Distribution of stress granule localization. The distance of AoPab1-EGFP foci from the hyphal tip is displayed using a box plot where the top and bottom of the box represent limits of the upper and lower quartiles, with the median being indicated by the horizontal line within the box. The whiskers show the highest and lowest reading within 1.5 times the interquartile range. The outliers are indicated by dots. The data were derived from three independent experiments with a total of 260 measurements in the WT and ▵<i>Aoso</i> strains, respectively.</p

    Time-lapse observation of stress granule formation upon oxidative stress.

    No full text
    <p>Approximately 10<sup>4</sup> conidia of cells expressing AoPab1-EGFP were grown in CD+Met medium at 30°C for 18 h before being exposed to oxidative stress (2 mM H<sub>2</sub>O<sub>2</sub>). Accumulation of AoPab1-EGFP in cells was observed in a time-lapse manner. Scale bar  =  5 µm.</p

    Subcellular localizations of AoSO-EGFP and AoPab1-mDsRed in response to heat stress.

    No full text
    <p>(A) A wild-type strain co-expressing AoSO-EGFP and AoPab1-mDsRed was used to examine the relative localizations of AoSO and stress granules under normal growth conditions and heat stress. Approximately 10<sup>4</sup> conidia of cells were grown in CD medium at 30°C for 18 h before being exposed to 45°C for 10 min. Arrowheads indicate AoSO foci, and arrows indicate stress granules. Colocalization of a stress granule and an AoSO cytoplasmic focus is indicated by the asterisk. The effect of cycloheximide on the formation of mRNP granules was examined by pre-treating cells with 200 µg/ml cycloheximide for 30 min before being exposed to heat stress. Scale bar  =  5 µm. (B) Effect of cycloheximide on the formation of AoSO cytoplasmic foci at the hyphal tip. Cells were incubated with CD medium containing 200 µg/ml cycloheximide for 30 min before being exposed to heat stress. The percentage of cells displaying AoSO cytoplasmic foci at the hyphal tip was determined. Error bars represent the standard error. ***P < 0.0001. The presented data are from three independent experiments, each with n  =  50.</p

    <i>Aopub1</i> disruptant showed defects in the conidia formation and more severe growth retardation in stress conditions.

    No full text
    <p>(A) Stress-induced formation of stress granules (indicated by arrows) in an AoPub1-EGFP expressing wild-type strain. Approximately 10<sup>4</sup> conidia of cells were grown in CD+Met medium at 30°C for 18 h before being exposed to ER stress (10 mM DTT, 60 min), oxidative stress (2 mM H<sub>2</sub>O<sub>2</sub>, 30 min), and osmotic stress (1.2 M sorbitol, 30 min). Scale bar  =  5 µm. (B) Approximately 10<sup>3</sup> conidia of wild-type and <i>Aopub1</i> disruptant cells were spotted onto PD plates, and cultured at 30°C for 4 days. (C) Approximately 10<sup>3</sup> conidia of wild-type and <i>Aopub1</i> disruptant cells were spotted onto PD plates with or without 10 mM DTT, 2 mM H<sub>2</sub>O<sub>2</sub>, or 1.2 M sorbitol, and cultured at 30°C for 4 days. Colony diameters were compared to those of the wild-type strain under the control condition, which was set to 100%. Data represent the mean ± S.D. of three biological replicates. The asterisk denotes a statistically significant difference, as judged by the Student’s t-test with P < 0.005 (**) and P < 0.0005 (***).</p

    <i>A. oryzae</i> strains used in this study.

    No full text
    <p><i>A. oryzae</i> strains used in this study.</p

    Enhanced Production of Bovine Chymosin by Autophagy Deficiency in the Filamentous Fungus <i>Aspergillus oryzae</i>

    Get PDF
    <div><p><i>Aspergillus oryzae</i> has been utilized as a host for heterologous protein production because of its high protein secretory capacity and food-safety properties. However, <i>A. oryzae</i> often produces lower-than-expected yields of target heterologous proteins due to various underlying mechanisms, including degradation processes such as autophagy, which may be a significant bottleneck for protein production. In the present study, we examined the production of heterologous protein in several autophagy (<i>Aoatg</i>) gene disruptants of <i>A. oryzae</i>. We transformed <i>A. oryzae</i> gene disruptants of <i>Aoatg1</i>, <i>Aoatg13</i>, <i>Aoatg4</i>, <i>Aoatg8</i>, or <i>Aoatg15</i>, with a bovine chymosin (CHY) expression construct and found that the production levels of CHY increased up to three fold compared to the control strain. Notably, however, conidia formation by the <i>Aoatg</i> gene disruptants was significantly reduced. As large amounts of conidia are necessary for inoculating large-scale cultures, we also constructed <i>Aoatg</i> gene-conditional expression strains in which the promoter region of the <i>Aoatg</i> gene was replaced with the thiamine-controllable <i>thiA</i> promoter. Conidiation by the resultant transformants was clearly enhanced in the absence of thiamine, while autophagy remained repressed in the presence of thiamine. Moreover, these transformants displayed increased CHY productivity, which was comparable to that of the <i>Aoatg</i> gene disruptants. Consequently, we succeeded in the construction of <i>A. oryzae</i> strains capable of producing high levels of CHY due to defects in autophagy. Our finding suggests that the conditional regulation of autophagy is an effective method for increasing heterologous protein production in <i>A. oryzae</i>.</p></div

    AoAtg26, a putative sterol glucosyltransferase, is required for autophagic degradation of peroxisomes, mitochondria, and nuclei in the filamentous fungus <i>Aspergillus oryzae</i>

    No full text
    <p>Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus <i>Aspergillus oryzae</i> AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast <i>Pichia pastoris</i>. Deletion of <i>Aoatg26</i> caused a severe decrease in conidiation and aerial hyphae formation, which is typically observed in the autophagy-deficient <i>A. oryzae</i> strains. In addition, cup-shaped AoAtg8-positive membrane structures were accumulated in the <i>Aoatg26</i> deletion strain, indicating that autophagic process is impaired. Indeed, the <i>Aoatg26</i> deletion strain was defective in the degradation of peroxisomes, mitochondria, and nuclei. Taken together, AoAtg26 plays an important role for autophagic degradation of organelles in <i>A. oryzae</i>, which may physiologically contribute to the differentiation in filamentous fungi.</p> <p>Involvement of AoAtg26 in autophagic organelle degradation.</p

    Extracellular bovine chymosin (CHY) production by <i>A.oryzae</i> autophagy gene-conditional expression strains.

    No full text
    <p>(A) Approximately 2×10<sup>5</sup> conidia of the control (SlD-AKC1), SlD-PtA1-AKC, SlD-PtA4-AKC, SlD-PtA8-AKC, and SlDPtA15-AKC strains expressing CHY were inoculated into 20 ml 5×DPY medium (pH 5.5) supplemented with and without thiamine. CHY activities in the culture supernatant were measured after 4 days of growth at 30°C. Five experiments were performed, and the values of the average and standard deviations are represented (*<i>p</i><0.01, Student’s <i>t</i> test). (B) Western blot analysis of the culture supernatant of the CHY-expressing strains. Mature CHY bands of 35.4 kDa were detected using an anti-CHY antibody.</p
    corecore