MOESM2 of Aspergillus nidulans protein kinase A plays an important role in cellulase production

Additional file 2: Table S2. MIPS FunCat 8 h

MOESM1 of Aspergillus nidulans protein kinase A plays an important role in cellulase production

Additional file 1: Table S1. List of CAZymes

Functional Characterization of <em>Aspergillus nidulans ypkA,</em> a Homologue of the Mammalian Kinase SGK

<div><p>The serum- and glucocorticoid-regulated protein kinase (SGK) is an AGC kinase involved in signal cascades regulated by glucocorticoid hormones and serum in mammals. The <i>Saccharomyces cerevisiae ypk1</i> and <i>ypk2</i> genes were identified as SGK homologues and Ypk1 was shown to regulate the balance of sphingolipids between the inner and outer plasma membrane. This investigation characterized the <i>Aspergillus nidulans YPK1</i> homologue, YpkA, representing the first filamentous fungal <i>YPK1</i> homologue. Two conditional mutant strains were constructed by replacing the endogenous <i>ypk1</i> promoter with two different regulatable promoters, <i>alcA</i> (from the alcohol dehydrogenase gene) and <i>niiA</i> (from the nitrate reductase gene). Both constructs confirmed that <i>ypkA</i> was an essential gene in <i>A. nidulans</i>. Repression of <i>ypkA</i> caused decreased radial growth, a delay in conidial germination, deficient polar axis establishment, intense branching during late stages of growth, a lack of asexual spores, and a terminal phenotype. Membrane lipid polarization, endocytosis, eisosomes and vacuolar distribution were also affected by <i>ypkA</i> repression, suggesting that YpkA plays a role in hyphal morphogenesis via coordinating the delivery of cell membrane and wall constituents to the hyphal apex. The <i>A. nidulans</i> Pkh1 homologue <i>pkhA</i> was also shown to be an essential gene, and preliminary genetic analysis suggested that the ypkA gene is not directly downstream of <i>pkhA</i> or epistatic to <i>pkhA</i>, rather, <i>ypkA</i> and <i>pkhA</i> are genetically independent or in parallel. <i>BarA</i> is a homologue of the yeast <i>Lag1</i> acyl-CoA-dependent ceramide synthase, which catalyzes the condensation of phytosphingosine with a fatty acyl-CoA to form phytoceramide. When <i>barA</i> was absent, <i>ypkA</i> repression was lethal to the cell. Therefore, there appears to be a genetic interaction between <i>ypkA</i>, <i>barA</i>, and the sphingolipid synthesis. Transcriptional profiling of <i>ypkA</i> overexpression and down-regulation revealed several putative YpkA targets associated with the observed phenotypes.</p> </div

The <i>A. nidulans</i> YpkA interacts with BarA.

<p>The radial growth of the wild-type, <i>niiA::ypkA</i>, <i>barA1</i>, and <i>niiA::ypkA barA1</i> mutant strains were grown for 72 hours at 37°C on MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM (Wt = Wild-type).</p

polarized delivery of membrane lipids and cell wall deposition was not confined to the hyphal apex in the <i>niiA::ypkA</i> mutant upon repression.

<p>In all experiments, germlings were grown for 16 hours at 37°C on inducing (sodium nitrate) and repressing conditions (ammonium tartrate). Stains utilized: (A) Hoescht, (B) Filipin, (C) FITC-conjugated wheat germ, and (D) CFW. Bars, 5 and 10 µm.</p

<i>A. nidulans</i> YpkA is involved in polarized growth.

<p>The percentage of wild-type and <i>niiA::ypkA</i> mutant germlings that exhibited polar growth (defined here as the emergence of the germ tube) (A) and the number of nuclear per germling (B). Conidia were grown for 2 to 8 hours at 37°C. Averages (± standard deviation) represent 100 germlings from three independent experiments (Wt = Wild-type). (C) The germination pattern of the wild-type and <i>niiA::ypkA</i> conidiospores. Conidia were allowed to germinate on MM media for 8 to 16 hours. Conidia possessing germ tubes were classified as displaying (left to right) unipolar (1), bipolar (2), unipolar plus lateral branches (3) or bipolar plus lateral branches (4).</p

YpkA expression affects eisosomes distribution in <i>A. nidulans</i>.

<p>The <i>niiA::ypkA pilA::gfp</i> (A) and <i>niiA::ypkA pilB::gfp</i> (B) strains were grown for 16 hours at 37°C in MM+10 mM sodium nitrate or 50 mM ammonium tartrate. Bars, 5 µm.</p

The <i>pkhA</i> gene is essential to <i>A. nidulans</i> and interacts with <i>ypkA</i>.

<p>(A) The wild-type and <i>niiA::pkhA</i> mutant strains were grown for 72 hours at 37°C on MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM (Wt = Wild-type). (B) The <i>alcA::ypkA</i>, <i>niiA::pkhA</i>, and <i>alcA::ypkA niiA::pkhA</i> strains were grown for 72 hours at 37°C on different combinations of MM+glucose 2% or glycerol 2% plus threonine 100 mM plus sodium nitrate 10 mM or ammonium tartrate 50 mM (Wt = Wild-type).</p

Evaluation of the effect of YpkA depletion and overexpression on the growth of <i>A. nidulans</i> under different experimental conditions.

<p>Five µl of a ten-fold dilution series starting at a concentration of 2×10⁷ for the wild-type (A-D), <i>alcA::ypkA</i> (A-B), and <i>niiA::ypkA</i> (C–D) strains were spotted on different growth media and grown for 72 hours at 37°C, except for experiments where the temperature was evaluated (A and C). CFW = calcofluor white; CR = congo red; MR = myriocin; PHS = phytosphingosine; and SDS = sodium dodecyl sulphate.</p

The <i>ypkA</i> gene is to essential <i>A. nidulans</i>.

<p>(A) Wild-type and primary <i>ΔypkA</i> transformant were grown (left and center panel) or streaked (right panel) on YAG medium for 96 hours at 37°C. (B) The <i>alcA::ypkA</i> strain was grown for 6 hours in MM+4% glucose or MM+2% glycerol +threonine 100 mM (C) The <i>niiA::ypkA</i> strain was grown for 6 hours in MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM. The relative quantitation of <i>ypkA</i> and tubulin gene expression was determined by a standard curve (i.e., C_T –values plotted against a logarithm of the DNA copy number). The presented results are the means (± standard deviation) of four biological replicates. The growth phenotypes of the <i>alcA::ypkA</i> (D) and <i>niiA::ypkA</i> (E) mutant strains. The <i>A. nidulans</i> wild-type, <i>alcA::ypkA</i>, and <i>niiA::ypkA</i> mutant strains were grown for 72 hours at 37°C either on MM+4% glucose or MM+2% glycerol and MM+sodium nitrate 10 mM or MM+ammonium tartrate 50 mM.</p