Oxidative Damage Control during Decay of Wood by Brown Rot Fungus Using Oxygen Radicals

Abstract

Brown rot wood-degrading fungi deploy reactive oxygen species (ROS) to loosen plant cell walls and enable selective polysaccharide extraction. These ROS, including Fenton-generated hydroxyl radicals (HO˙), react with little specificity and risk damaging hyphae and secreted enzymes. Recently, it was shown that brown rot fungi reduce this risk, in part, by differentially expressing genes involved in HO˙ generation ahead of those coding carbohydrate-active enzymes (CAZYs). However, there are notable exceptions to this pattern, and we hypothesized that brown rot fungi would require additional extracellular mechanisms to limit ROS damage. To assess this, we grew directionally on wood wafers to spatially segregate early from later decay stages. Extracellular HO˙ production (avoidance) and quenching (suppression) capacities among the stages were analyzed, along with the ability of secreted CAZYs to maintain activity postoxidation (tolerance). First, we found that HO and Fe concentrations in the extracellular environment were conducive to HO˙ production in early (HO:Fe ratio 2:1) but not later (ratio 1:131) stages of decay. Second, we found that ABTS radical cation quenching (antioxidant capacity) was higher in later decay stages, coincident with higher fungal phenolic concentrations. Third, by surveying enzyme activities before/after exposure to Fenton-generated HO˙, we found that CAZYs secreted early, amid HO˙, were more tolerant of oxidative stress than those expressed later and were more tolerant than homologs in the model CAZY producer Collectively, this indicates that uses avoidance, suppression, and tolerance mechanisms, extracellularly, to complement intracellular differential expression, enabling this brown rot fungus to use ROS to degrade wood. Wood is one of the largest pools of carbon on Earth, and its decomposition is dominated in most systems by fungi. Wood-degrading fungi specialize in extracting sugars bound within lignin, either by removing lignin first (white rot) or by using Fenton-generated reactive oxygen species (ROS) to "loosen" wood cell walls, enabling selective sugar extraction (brown rot). Although white rot lignin-degrading pathways are well characterized, there are many uncertainties in brown rot fungal mechanisms. Our study addressed a key uncertainty in how brown rot fungi deploy ROS without damaging themselves or the enzymes they secrete. In addition to revealing differentially expressed genes to promote ROS generation only in early decay, our study revealed three spatial control mechanisms to avoid/tolerate ROS: (i) constraining Fenton reactant concentrations (HO, Fe), (ii) quenching ROS via antioxidants, and (iii) secreting ROS-tolerant enzymes. These results not only offer insight into natural decomposition pathways but also generate targets for biotechnological development.

Gene Regulation Shifts Shed Light on Fungal Adaption in Plant Biomass Decomposers

“…The products of these genes may have evolved characteristics adapted to oxidatively reacted wood components such as carbonyl and carboxylic acid groups introduced on residual carbohydrates (21, 33). In line with this, cellulases and hemicellulases of brown rot fungi were recently shown to better tolerate Fenton-generated oxidative radicals, relative to those enzymes in Trichoderma reesei (34). Overall, these unique late-stage dynamics may involve genes inherited from white rot ancestors (1, 10) with functions adapted to substrate conditions that characterize brown rot, including many unexplored options relevant to industrial processes.…”

Cycling in degradation of organic polymers and uptake of nutrients by a litter‐degrading fungus

“…a role of Fenton chemistry and/or lipid peroxidation) during its cultivation process as well as during bursts. Recent insights in the decay strategy of brown rot fungi points to the production of hydroxyl radicals by their hyphal front while the bulk of (hemi-) cellulases is produced by older mycelium (Zhang et al, 2016(Zhang et al, , 2019Presley and Schilling, 2017;Castaño et al, 2018). H 2 O 2 and other radical species can damage proteins involved in polysaccharide deconstruction and spatial separation of radical generation and (hemi-) cellulases can protect these CAZYs from oxidative damage (Zhang et al, 2016;Castaño et al, 2018).…”

“…Recent insights in the decay strategy of brown rot fungi points to the production of hydroxyl radicals by their hyphal front while the bulk of (hemi-) cellulases is produced by older mycelium (Zhang et al, 2016(Zhang et al, , 2019Presley and Schilling, 2017;Castaño et al, 2018). H 2 O 2 and other radical species can damage proteins involved in polysaccharide deconstruction and spatial separation of radical generation and (hemi-) cellulases can protect these CAZYs from oxidative damage (Zhang et al, 2016;Castaño et al, 2018). In addition, the pre-treatment of plant cell walls with Fenton chemistry derived radicals or a ligninolytic system allows a more efficient (hemi-) cellulose deconstruction by CAZYs (ten Have et al, 2003;Arantes et al, 2012).…”

“…Recently, it was found that the mycelium of mushroom forming brown rot fungi spatially separate the radical generating Fenton chemistry from CAZYs (Zhang et al, 2016(Zhang et al, , 2019Presley and Schilling, 2017;Castaño et al, 2018). The outer 5 mm of the colony of brown rots like Postia placenta generate •OH radicals using the Fenton reaction, while the inner 15-35 mm regions of the colony secrete the bulk of (hemi-) cellulases.…”

“…This spatial separation would prevent inactivation of these enzymes by radical oxygen species generated by Fenton chemistry. In addition, the radical pre‐treatment of cell walls may facilitate the activity of CAZYs (Castaño et al ., 2018; Zhang et al ., 2019).…”