Capturing early gene expression dynamics during wood decay by brown rot fungus Rhodonia placenta

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 cryo-sectioning with whole transcriptome sequencing to dissect gene expression at the single hyphal cell scale (tens of µm). We improved spatial resolution 50x, 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. By comparing the first 100-µm section with a 100-µm from a later decay stage, it was clear that the 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. However, this delayed pattern was not universal, with a handful of genes upregulated immediately at the hyphal front. Overall design: To compare gene expression at different stages of decay, we used a space-for-time experimental design, during which Rhodonia placenta grew directionally along a wood wafer. We sampled each wafer at four locations (0-100 µm, 4900-5000 µm, 15000-15100 µm, and 19900-20000 µm) yielding a 100-µm section for each sample from which RNA was extracted and sequenced. Two replicates from each wafer (n=6) were sampled for each location.