Capturing an Early Gene Induction Event during Wood Decay by the Brown Rot Fungus Rhodonia placenta

Abstract

Brown rot fungi dominate wood decomposition in coniferous forests, and their carbohydrate-selective mechanisms are of commercial interest. Brown rot was recently described as a two-step, sequential mechanism orchestrated by fungi using differentially expressed genes (DEGs) and consisting of oxidation via reactive oxygen species (ROS) followed by enzymatic saccharification. There have been indications, however, that the initial oxidation step itself might require induction. To capture this early gene regulation event, here, we integrated fine-scale cryosectioning with whole-transcriptome sequencing to dissect gene expression at the single-hyphal-cell scale (tens of micrometers). This improved the spatial resolution 50-fold, relative to previous work, and we were able to capture the activity of the first 100 μm of hyphal front growth by Rhodonia placenta in aspen wood. This early decay period was dominated by delayed gene expression patterns as the fungus ramped up its mechanism. These delayed DEGs included many genes implicated in ROS pathways (lignocellulose oxidation [LOX]) that were previously and incorrectly assumed to be constitutively expressed. These delayed DEGs, which include those with and without predicted functions, also create a focused subset of target genes for functional genomics. However, this delayed pattern was not universal, with a few genes being upregulated immediately at the hyphal front. Most notably, this included a gene commonly implicated in hydroquinone and iron redox cycling: benzoquinone reductase. IMPORTANCE Earth's aboveground terrestrial biomass is primarily wood, and fungi dominate wood decomposition. Here, we studied these fungal pathways in a common "brown rot"-type fungus, Rhodonia placenta, that selectively extracts sugars from carbohydrates embedded within wood lignin. Using a space-for-time design to map fungal gene expression at the extreme hyphal front in wood, we made two discoveries. First, we found that many genes long assumed to be "on" (constitutively expressed) from the very beginning of decay were instead "off" before being upregulated, when mapped (via transcriptome sequencing [RNA-seq]) at a high resolution. Second, we found that the gene encoding benzoquinone reductase was "on" in incipient decay and quickly downregulated, implying a key role in "kick-starting" brown rot.

Keywords: RNA; biodegradation; decomposition; lignocellulose; transcriptomics.

FIG 1

Using space to parse time at a micrometer scale. Wood wafers colonized directionally by the brown rot fungus Rhodonia placenta were cryosectioned to a 100-μm thickness at the hyphal front and closely behind in order to map gene expression patterns using whole transcriptomic RNA (RNA-seq). Differentially expressed genes (DEGs) with a >4-fold difference (0 to 100 μm versus 4,900 to 5,000 μm) were grouped as being downregulated after high levels near the extreme front (which we label incipient) or upregulated after a delay (delayed). An additional section was included as a reference for later decay, as shown in Fig. 4.

FIG 2

A major induction event during early wood decay by R. placenta. Expression patterns within the first 5 mm (∼48 h) of decay show 21-fold more delayed genes than incipient genes (n = 1,176 versus n = 56) when examining significant DEGs with a >4-fold change (i.e., dotted lines at ±2). The fold change was calculated using the ratio of 0 to 100 μm to 4,900 to 5,000 μm. Genes were considered significant if the P_adjusted value was <0.05; low-count genes (n = 1,668) are not displayed.

FIG 3

Distribution of incipient versus delayed R. placenta DEGs involved in deconstructing lignocellulose. Total DEGs (>4-fold [P_adjusted < 0.05]) for incipient and delayed expression are organized by categories of interest. Here, functional assignments were adopted from those described previously by Zhang et al. (2), in which carbohydrate-active enzymes (CAZys) follow definitions from the Carbohydrate Active Enzymes database (http://www.cazy.org/) (14), including glycoside hydrolases (GHs), carbohydrate esterases, and glycosyltransferases, except for enzymes with auxiliary activities (e.g., peroxidases and oxidases, etc.), which are in the lignocellulose oxidation enzyme (LOX) categories. AKR, aldo-keto reductase.

FIG 4

How short-term expression dynamics at the hyphal front fit within longer-term decay trends. R. placenta transcript investments in lignocellulose decay genes vary across early decay and a reference section at 15 mm behind the hyphal front. CAZys, represented here by key GHs (GH5, -12, -27, -43, and -51), have delayed expression within the first 5 mm and continue to ramp up into later decay stages. Most LOX genes, while delayed at the onset, still peak in the early decay stage. Conversely, benzoquinone reductase is upregulated in the first 100 μm of decay (same pattern for both alleles, as shown) and may play a key role in initiating brown rot decay. CPM, counts per million. Note the different scales on the y axes.