Western blot analysis of mitochondrial PaLON protease and the molecular chaperone HSP60.

<p><b>A</b> Mitochondrial proteins in WT and mutants grisea and <i>PaCox17</i>::ble were analysed with antibodies against HSP60 after transfer to a PVDF membrane. HSP60 levels are increased in the two mutants. <b>B</b> Protein levels of LON protease (PaLON) are moderately increased in the mutants compared to the WT. Below each immunodetection a densitometric analysis of signal intensities (x-fold level compared to the WT) is shown. Intensities of the PaPORIN signals were used for normalisation. UniProt accession numbers: PaLON: B2AZ54; PaHSP60: B2B270 and PaPORIN: B2B736.</p

Metabolic rates of live mycelia.

<p><b>A</b> Respirometry reveals that mutants grisea and <i>PaCox17</i>::ble are characterized by elevated oxygen consumption compared to the WT. <b>B</b> Assessment of heat production by calorimetry is also increased in the two mutants. <b>C</b> However, the mutant genotype does not influence the calorimetric/respirometric (CR) ratio. Data represent mean ± standard error. *: p<0.05; **: p<0.01; n. s.: not significant.</p

Total superoxide dismutase and catalase activity.

<p><b>A</b> The measurement of SOD activity shows a significant increase in <i>PaCox17</i>::ble compared to the WT. <b>B</b> Catalase activity is not significantly changed between the WT and mutants grisea and <i>PaCox17</i>::ble, respectively. Data represent mean ± standard error. **: p<0.01; n. s.: not significant.</p

Mycelial hydrogen peroxide production.

<p>H₂O₂ production in WT and mutants grisea and <i>PaCox17</i>::ble as measured by mycelial DAB precipitation. While the amount of H₂O₂ is slightly reduced in mutant grisea compared to the WT, it is strongly increased in <i>PaCox17</i>::ble.</p

Mitochondrial content.

<p><b>A</b> Mycelia were stained with Mitotracker Green and analysed by fluorescence microscopy. Representative hyphae are shown. Scale bar: 2 µm. <b>B</b> Protoplasts prepared from mutants grisea and <i>PaCox17</i>::ble contain significantly more mitochondria than the WT as revealed by NAO staining. Mitochondrial content in the WT was set to 100%. <b>C</b> Determination of the mtDNA/nuclear DNA ratio as a marker for mitochondrial quantity by PCR. <i>left</i>: representative 1% agarose gel showing separated <i>PaGpd</i> (nuclear DNA) and <i>PaLsu</i> (mtDNA) amplification products stained with ethidiumbromide, NC: negative control, pd: primer dimers. <i>right</i>: densitometric analysis of band intensities. The mtDNA/nuclear DNA ratio in the WT was set to 1. <b>D</b> Western blot analysis to detect PaPORIN levels in total protein extracts from the wild type strain and the two mutants. As a loading control the Coomassie-stained transfer membrane is shown. Data represent mean ± standard error. *: p<0.05; ***: p<0.001, Student's <i>t</i> test, two-tailed.</p

Determination of ATP content in mycelial homogenates.

<p>ATP levels were measured by a luminescence based assay. Mutants grisea and <i>PaCox17</i>::ble contain significantly less ATP than the WT. The age of the mycelia from which the homogenates were prepared is 10 d. Data represent mean ± standard error. *: p<0.05.</p