An Amylase-Like Protein, AmyD, Is the Major Negative Regulator for α-Glucan Synthesis in Aspergillus nidulans during the Asexual Life Cycle
"> Figure 1
<p>Localization of AmyD associates with cell membrane; (<b>A</b>) Strategy of chimera-AmyD-GFP strain construction. The amylase-like domain of AmyD was replaced by a GFP, whereas the localization determining elements of AmyD were maintained. The localization of AmyD corresponded to a chimera-GFP signal; (<b>B</b>) PCR verification of AmyD-GFP strain. Primers targeting sites were labeled in (<b>A</b>). PCR using SE101 + SE102 primers confirmed the replacement of <span class="html-italic">amyD</span> by <span class="html-italic">gfp</span>. PCR using SE103 + AME16 and SE103 + SE106 primers confirmed the integration of the construct at the designed place. No ectopic insertion was found in the AmyD-GFP genome; (<b>C</b>) the GFP signal was examined using a Zeiss META501 confocal epifluorescence microscope at 63 × NA 1.2 or 25 × N.A. 1.0 objective lens. Confocal imaging used 488 nm excitation with emission controlled by a BP 505–530 nm filter; and (<b>D</b>) a magnified hypha image showed the GFP signal strongly associated with septa and the cell membrane.</p> "> Figure 2
<p>Overexpression of <span class="html-italic">a</span><span class="html-italic">myC</span> and <span class="html-italic">a</span><span class="html-italic">m</span><span class="html-italic">yE</span> does not affect significantly α-glucan accumulation. (<b>A</b>) 10<sup>5</sup> freshly harvested conidia of each strain were inoculated on complete medium and the plates were incubated at 30 °C for 48 h. All constructed strains showed the wild type colony phenotype on solid medium; (<b>B</b>) a large number (5 × 10<sup>7</sup>) of freshly harvested conidia were inoculated in flask with 20 mL complete medium, then the flask was incubated at 30 °C, 150 rpm overnight. Only <span class="html-italic">actA</span>(p)-<span class="html-italic">amyD</span> formed tiny colonies; (<b>C</b>) A large number (2 × 10<sup>7</sup>) of spores of each strain were inoculated in 100 mL complete medium. Samples were grown in flasks at 30 °C with 150 rpm for 24 h. α-glucan was extracted from 1 mg dry cell wall, and then digested to glucose and quantified by an anthrone assay. Results represent the mean of three independent quantification tests with duplicates each time ± standard deviation. The data for each strain were compared with wild type (column 1) individually by Mann Whitney <span class="html-italic">U</span> test. Significant difference (<span class="html-italic">p</span> < 0.05) is indicated by asterisks.</p> "> Figure 3
<p>AgnB and MutA are functional α-glucanases. (<b>A</b>) Freshly harvested conidia (10<sup>5</sup>) of each strain were inoculated on complete medium and the plates were incubated at 30 °C for 48 h. All constructed strains showed the wild-type colony phenotype on solid medium; (<b>B</b>) freshly harvested conidia (5 × 10<sup>7</sup>) were inoculated in a flask with 20 mL complete medium, then the flask was incubated at 30 °C, 150 rpm overnight. All strains behaved the same as the wild-type; (<b>C</b>,<b>D</b>) spores of the indicated strain (2 × 10<sup>7</sup>) were inoculated in 100 mL complete medium. Samples were grown in flasks at 30 °C with 150 rpm for 24 h. α-glucan was extracted from 1 mg of dry cell wall, and then digested to glucose and quantified using an anthrone assay [<a href="#B24-ijms-18-00695" class="html-bibr">24</a>]. Results represent the mean of three independent quantification tests with duplicates each time ± standard deviation. The data for each strain were compared with the wild-type (column 1) individually by a Mann Whitney <span class="html-italic">U</span> test. Significant difference (<span class="html-italic">p</span> < 0.05) was indicated by asterisks; (<b>E</b>) conidia of each strain were prepared and inoculated as in (<b>A</b>). Only <span class="html-italic">actA</span>(p)-<span class="html-italic">agnB</span> showed pigment deficiency; and (<b>F</b>) all strains behaved the same as the wild-type in shaken liquid medium. Growth condition was the same as described in (<b>B</b>).</p> "> Figure 4
<p>The absence of AmyD cannot suppress the increased α‑glucan production of <span class="html-italic">agnB</span>- or <span class="html-italic">mutA</span>-overexpression strains. (<b>A</b>) All constructed strains showed the wild-type colony phenotype on solid medium; (<b>B</b>) all strains behaved the same as the wild-type; and (<b>C</b>) α-glucan was extracted from 1 mg of dry cell wall, and then digested to glucose and quantified using an anthrone assay. Results represent the mean of three independent quantification tests with duplicates each time ± standard deviation. The data for each strain were compared with wild type (column 1) individually by a Mann Whitney <span class="html-italic">U</span> test. Significant difference (<span class="html-italic">p</span> < 0.05) was indicated by asterisks.</p> "> Figure 5
<p>Dynamics of α-glucan accumulation and its impact on CFW sensitivity. (<b>A</b>) Dynamics of α-glucan accumulation in each strain with time. 2 × 10<sup>7</sup> spores of each strain were inoculated in 100 mL complete medium. Samples were grown in flasks at 30 °C with 150 rpm for 16 h, 20 h, and 24 h, respectively. α-glucan was extracted from 1 mg of dry cell wall, and then digested to glucose and quantified by an anthrone assay. Wild-type and <span class="html-italic">actA</span>(p)-<span class="html-italic">amyD</span> showed the same increasing trend from 16 h to 24 h, except the glucose concentration in <span class="html-italic">actA</span>(p)-<span class="html-italic">amyD</span> was much lower than that of the wild-type at each time point. The <span class="html-italic">actA</span>(p)-<span class="html-italic">agnB</span> and <span class="html-italic">actA</span>(p)-<span class="html-italic">mutA</span> had the same decreasing trend from 16 h to 24 h; (<b>B</b>) freshly harvested conidia (10<sup>5</sup>) of each strain were inoculated on 50 μg/mL CFW plates and the plates were incubated at 30 °C for 48 h. Only <span class="html-italic">actA</span>(p)-<span class="html-italic">amyD</span> showed delayed germination and growth.</p> ">
Abstract
:1. Introduction
2. Results
2.1. AmyD Mainly Localizes at the Cell Membrane
2.2. AmyC and AmyE Do Not Affect α-Glucan Accumulation
2.3. MutA, as Well as AgnB, but Not AgnE, Can Repress α-Glucan Accumulation
2.4. Functions of AgnB and MutA Are Independent from AmyD
2.5. Dynamics of α-Glucan Accumulation Affects Colony Formation in Liquid Medium and Drug Sensitivity
3. Discussion
3.1. AmyD Is the Major Negative Regulator of α-Glucan Accumulation in the A. nidulans Asexual Life Cycle
3.2. Functions of α-Glucanases and AmyD Are Independent from Each Other
3.3. α-Glucan Content in Early Life Stage Is Critical for Colony Formation in Shaken Liquid as Well as Drug Sensitivity
4. Materials and Methods
4.1. Strains, Plasmids, and Medium
4.2. Quantification of Conidiation
4.3. α-Glucan Quantification
4.4. RT-PCR and qPCR
4.5. Drug Sensitivity Test
4.6. Confocal Imaging
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Shaken Growth | Static Growth | Overexpression by actA(p) | |||
---|---|---|---|---|---|
genes | 14 h | 24 h | 14 h | 24 h | 14 h |
amyC | 1 | 1.44 ± 0.44 | 1.28 ± 0.28 | 18.84 ± 6.36 | 794.13 ± 190.28 |
amyE | 1 | 1.72 ± 0.71 | 1.93 ± 0.49 | 27.82 ± 9.79 | 484.82 ± 140.61 |
agnB | 1 | 2.16 ± 0.63 | 2.39 ± 0.83 | 11.71 ± 3.93 | 871.00 ± 191.59 |
agnE | 1 | 1.92 ± 0.57 | 1.59 ± 0.53 | 274.90 ± 85.55 | 1268.84 ± 292.38 |
mutA | 1 | 2.97 ± 0.89 | 2.31 ± 0.96 | 6.96 ± 1.99 | 1753.59 ± 654.97 |
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He, X.; Li, S.; Kaminskyj, S. An Amylase-Like Protein, AmyD, Is the Major Negative Regulator for α-Glucan Synthesis in Aspergillus nidulans during the Asexual Life Cycle. Int. J. Mol. Sci. 2017, 18, 695. https://doi.org/10.3390/ijms18040695
He X, Li S, Kaminskyj S. An Amylase-Like Protein, AmyD, Is the Major Negative Regulator for α-Glucan Synthesis in Aspergillus nidulans during the Asexual Life Cycle. International Journal of Molecular Sciences. 2017; 18(4):695. https://doi.org/10.3390/ijms18040695
Chicago/Turabian StyleHe, Xiaoxiao, Shengnan Li, and Susan Kaminskyj. 2017. "An Amylase-Like Protein, AmyD, Is the Major Negative Regulator for α-Glucan Synthesis in Aspergillus nidulans during the Asexual Life Cycle" International Journal of Molecular Sciences 18, no. 4: 695. https://doi.org/10.3390/ijms18040695
APA StyleHe, X., Li, S., & Kaminskyj, S. (2017). An Amylase-Like Protein, AmyD, Is the Major Negative Regulator for α-Glucan Synthesis in Aspergillus nidulans during the Asexual Life Cycle. International Journal of Molecular Sciences, 18(4), 695. https://doi.org/10.3390/ijms18040695