Supplemental Material for Ries et al., 2018

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Overexpression of <i>pkcA</i> in a <i>ΔcnaA</i> background rescues aberrant septa formation in a Δ<i>cnaA</i> background.

<p>Wild-type, Δ<i>cnaA</i> and <i>alcA</i>::<i>pkcA</i> Δ<i>cnaA</i> strains were grown in minimal medium supplemented with 2% (w/v) glycerol plus 100 mM threonine for 16 hours at 30°C before being fixed for 30 minutes at room temperature (RT) and stained with calcofluor white (CFW) for 5 minutes at RT. Mycelial fluorescence was then assessed under the microscope (scale bars indicate 5 µm).</p

Overexpression of <i>pkcA</i> in a Δ<i>cnaA</i> background restores mitochondrial number.

<p>Wild-type, <i>ΔcnaA</i>, <i>alcA::pkcA</i> and <i>alcA::pkcA ΔcnaA</i> germlings were grown in minimal medium supplemented with 2% glycerol plus 100 mM threonine for 16 hours at 37 C and stained with nonyl acridine orange for 10 minutes at 37°C. Mycelial fluorescence was subsequently assessed under the microscope (scale bars indicate 5 µm).</p

Hierarchal and k-means (KM) clustering of the genes which were differentially (<i>t</i>-test <i>p</i><0.001) expressed between the Δ<i>cna</i> and <i>alcA</i>::<i>pkcA</i> Δ<i>cnaA</i> strains.

<p>Microarrays were performed on RNA extracted from both strains when grown in glucose or glycerol and threonine for 24 and 48 hours.</p

Overexpression of <i>pkcA</i> suppresses the phenotype defects caused by the <i>cnaA</i> deletion.

<p>(A) The wild-type, <i>alcA::pkcA</i>, <i>ΔcnaA</i> and <i>alcA::pkcA ΔcnaA</i> strains were grown for 72 hours at 37°C on agar plates containing either 4% glucose, 2% glycerol or 2% glycerol plus 100 mM threonine, or (B) in liquid media containing 2% glycerol plus 100 mM threonine for 12, 24 and 48 hours at 37°C, before their growth was assessed under the microscope (scale bars indicate 5 µm) and (C) mycelial dry weight measured for three biological replicates from each collected timepoint.</p

The <i>alcA::pkcA ΔcnaA</i> strain has increased <i>pkcA</i> mRNA accumulation.

<p>The wild−type, <i>alcA::pkcA</i>, <i>ΔcnaA</i>, and <i>alcA::pkcA ΔcnaA</i> strains were grown for 16 hours in minimal medium supplemented either with glucose 2% (G), glycerol 2% (Gly) or glycerol 2% plus 100 mM threonine (G+T), RNA extracted and RT-qPCR performed for <i>pkcA</i>.</p

Protein Kinase C Overexpression Suppresses Calcineurin-Associated Defects in <i>Aspergillus nidulans</i> and Is Involved in Mitochondrial Function

<div><p>In filamentous fungi, intracellular signaling pathways which are mediated by changing calcium levels and/or by activated protein kinase C (Pkc), control fungal adaptation to external stimuli. A rise in intracellular Ca²⁺ levels activates calcineurin subunit A (CnaA), which regulates cellular calcium homeostasis among other processes. Pkc is primarily involved in maintaining cell wall integrity (CWI) in response to different environmental stresses. Cross-talk between the Ca²⁺ and Pkc-mediated pathways has mainly been described in <i>Saccharomyces cerevisiae</i> and in a few other filamentous fungi. The presented study describes a genetic interaction between CnaA and PkcA in the filamentous fungus <i>Aspergillus nidulans</i>. Overexpression of <i>pkcA</i> partially rescues the phenotypes caused by a <i>cnaA</i> deletion. Furthermore, CnaA appears to affect the regulation of a mitogen-activated kinase, MpkA, involved in the CWI pathway. Reversely, PkcA is involved in controlling intracellular calcium homeostasis, as was confirmed by microarray analysis. Furthermore, overexpression of <i>pkcA</i> in a <i>cnaA</i> deletion background restores mitochondrial number and function. In conclusion, PkcA and CnaA-mediated signaling appear to share common targets, one of which appears to be MpkA of the CWI pathway. Both pathways also regulate components involved in mitochondrial biogenesis and function. This study describes targets for PkcA and CnaA-signaling pathways in an <i>A. nidulans</i> and identifies a novel interaction of both pathways in the regulation of cellular respiration.</p></div

Overexpression of <i>pkcA</i> in a <i>ΔcnaA</i> background rescues aberrant cell wall deposition in a Δ<i>cnaA</i> background.

<p>Wild-type, Δ<i>cnaA</i> and <i>alcA</i>::<i>pkcA</i> Δ<i>cnaA</i> strains were grown in minimal medium supplemented with 2% (w/v) glycerol plus 100 mM threonine for 16 hours at 30°C before being fixed for 30 minutes at room temperature (RT) and stained with fluorescein isothiocyanate wheat germ agglutinin (FITC-WGA) for 5 minutes at RT. Mycelial fluorescence was then assessed under the microscope (scale bars indicate 5 µm).</p

Overexpression of <i>pkcA</i> in a Δ<i>cnaA</i> background restores the levels of phosphorylated MpkA.

<p>Western blots (A) of the protein crude extract from the wild-type, <i>ΔcnaA</i>, <i>alcA::pkcA</i> and <i>alcA::pkcA ΔcnaA</i> strains, when grown in 2% glycerol plus 100 mM threonine for 16 hours at 30°C before being treated with Congo Red (CR) for 1 hour. Phosphorylated MpkA (49 kDa band) was probed for with anti-MpkA antibodies (B) and signal intensities were quantified using the Image J software by dividing the intensity of Western band by a the Coomassie stained protein bands. kDa, kilo Daltons. These results are from a single representative experiment from three different repetitions that provided comparable results.</p

Number of genes and the associated functions of the proteins they encode that had restored or elevated expression in the <i>alcA::pkcA ΔcnaA</i> strain when compared to the Δ<i>cnaA</i> strain.

<p>sP is number of genes in each category.</p