Lignin-degrading peroxidases in white-rot fungus <i>Trametes hirsuta</i> 072. Absolute expression quantification of full multigene family

<div><p>Ligninolytic heme peroxidases comprise an extensive family of enzymes, which production is characteristic for white-rot Basidiomycota. The majority of fungal heme peroxidases are encoded by multigene families that differentially express closely related proteins. Currently, there were very few attempts to characterize the complete multigene family of heme peroxidases in a single fungus. Here we are focusing on identification and characterization of peroxidase genes, which are transcribed and secreted by basidiomycete <i>Trametes hirsuta</i> 072, an efficient lignin degrader. The <i>T</i>. <i>hirsuta</i> genome contains 18 ligninolytic peroxidase genes encoding 9 putative lignin peroxidases (LiP), 7 putative short manganese peroxidases (MnP) and 2 putative versatile peroxidases (VP). Using ddPCR method we have quantified the absolute expression of the 18 peroxidase genes under different culture conditions and on different growth stages of basidiomycete. It was shown that only two genes (one MnP and one VP) were prevalently expressed as well as secreted into cultural broth under all conditions investigated. However their transcriptome and protein profiles differed in time depending on the effector used. The expression of other peroxidase genes revealed a significant variability, so one can propose the specific roles of these enzymes in fungal development and lifestyle.</p></div

Total relative expression of PODs complex at different growth stages and under different culture conditions of <i>T</i>. <i>hirsuta</i>.

<p>I, lag phase; II, exponential growth phase; III, stationary and lysis phase. The samples were taken in 12 h interval. Each dot represents arithmetic mean from three biological replicates on each cultivation media (GP, BR and AL). Polynomial regression line is superimposed on the mean scatter plot. Error bars represent standard deviations.</p

Relative expression of PODs genes under different culture conditions. Gene wise expression of PODs.

<p>Each doted square represents absolute expression normalized on internal control gene expression (RQ). The expression correlates both with the size of a dot and the color of a box. Samples in which expression was not detected depicted as small unsquared dots. GP, BR and AL stand for glucose-peptone medium and glucose-peptone medium with addition of bromocresol green and alkali lignin respectively.</p

Maximum likelihood phylogenetic tree of <i>T</i>. <i>hirsuta</i> PODs, characteristic PODs amino acid residues and genes’ intron positions.

<p>Numbers at nodes are bootstrap percentages from 100 replicates. The outgroup containing the three peroxidase genes of <i>Auricularia subglabra</i> (XM_007341671.1, XM_007341680.1 and XM_007347393.1) is highlighted in pink. Characteristic LiP residues are highlighted in violet; characteristic MnP residues–in green and red. Non-standard aa residues are highlighted in blue. The numeration of aa residues is corresponding to <i>P</i>. <i>chrysosporium</i> PODs (MnP1 and LiP -H8) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173813#pone.0173813.ref037" target="_blank">37</a>].</p

Properties of the predicted protein sequences for <i>T</i>. <i>hirsuta</i> peroxidases.

<p>Genes with high expression rate/RQ (according to ddPCR data, discussed below) are marked in dark grey.</p