Project description:Wood-degrading brown rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and non-selective, these two steps are likely segregated. A common hypothesis has been that brown rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that lignocellulose-oxidation (LOX) components involved in ROS production are differentially expressed by brown rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole transcriptome sequencing (RNAseq) and assayed relevant enzyme activities. We found a marked pattern of LOX upregulation in a narrow (5-mm; 48-hr) zone at the hyphal front, which included many genes likely involved in ROS generation. Upregulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for brown rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization. We sequenced mRNA from 9 Postia placenta samples taken from 3 wood sections of wafer design, with 3 bioreplicates for each wood section, to compare the gene expression during brown rot processes. Three wood sections of the wafer are representing early to late decay stages.
Project description:Brown rot fungi evolved the unique strategy to efficiently decay wood structures and selectively extract carbohydrates, and this involved the sophistical regulation of functional genes (Zhang et al., PNAS, 2016, 113: 10968-). However, the regulatory mechanisms of brown rot genes were not well known, impeding the implication and application of brown rot machinery in biomass conversions. In this work, we systematically studied the roles of environmental carbon signals (e.g., aspen, cellobiose, glucose and no-carbon) in regulating gene expression in model brown rot fungus Postia placenta by RNA-seq. We found the complex substrate aspen (Populus sp.), but not the commonly recognized disaccharide cellobiose, was the universal inducer for Carbohydrate Active Enzymes (CAZYs) expression. Even though, cellobiose clearly induced the expression of cellulase (GH5 and GH12, endoglucanase) and xylanase (GH10, endoxylanase) (cellobiose vs. no-carbon, fold change > 4), as we reported previously (Zhang and Schilling, FGB, 2017, 106: 1-). When response to easy to use carbons, P. placenta lost the CCR effect on the main-chain cleaving CAZYs expression, but kept this repressing effect on side-chain cleaving CAZYs and AAs, which indicated a clear adaption relative to that in saprotrophic ascomycete ancestors. This “loss of CCR effect” was independent of the glucose concentrations. To explore the distinctive brown rot regulatory machinery, the gene modules subjected to inducing or CCR effects were then used to predict the regulatory motifs and transcriptional factors to build the regulatory network in P. placenta. Together, these findings will facilitate us to understand the adaptions of regulatory elements in brown rot fungi, as well as the efficient brown rot strategy.
Project description:Wood-degrading brown rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and non-selective, these two steps are likely segregated. A common hypothesis has been that brown rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that lignocellulose-oxidation (LOX) components involved in ROS production are differentially expressed by brown rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole transcriptome sequencing (RNAseq) and assayed relevant enzyme activities. We found a marked pattern of LOX upregulation in a narrow (5-mm; 48-hr) zone at the hyphal front, which included many genes likely involved in ROS generation. Upregulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for brown rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization.
Project description:Using whole genome microarrays based on the annotated genomes of Postia placenta, we monitored the changes in its transcriptomes relevant to cell wall degradation during growth on three chemically distinct Populus trichocarpa (poplar) wood substrates. The research goal is to identtify genes essential for cellulose depolymerization.
Project description:Mass spectrometry raw data for secreted wood-degrading enzymes. Sample IDs are named as follows. ppl: Postia placenta, treesi: Trichoderma reesei, tvers: Trametes versicolor. Control: 1, oxidative treatment: 2. Biological replicates: a, b, c. For example, ppl_1a is Postia placenta, control sample, biological replicate a.
Project description:Wood-degrading fungi vary in their strategies for deconstructing wood, and their competitive successes shape the rate and fate of carbon released from wood, Earth’s largest pool of aboveground terrestrial carbon. In this study, one-on-one interspecific interactions between two model brown rot (carbohydrate-selective) fungi, Gloeophyllum trabeum and Rhodonia (Postia) placenta, were studied on wood wafers where a clearly resolved interaction zone (IZ) could be generated, reproducibly. Comparative RNAseq and proteomics between the IZ and non-interacting hyphae of each species identified combative strategies for each fungus. Glycoside hydrolases were a relatively smaller portion of the interaction secretome compared to non-interacting hyphae. The interaction zone showed higher pectinase specific activity than all other sampling locations, and higher laminarinase specific activity (branched β‐glucan proxy) was seen in the IZ secretome relative to equivalent hyphae in single‐species cultures. Our efforts also identified two distinct competitive strategies in these two fungi with a shared nutritional mode (brown rot) but polyphyletic ancestral lineages. Gloeophyllum trabeum (Gloeophyllum clade) employed secondary metabolite (SM) synthesis in response to a competitor, as shown by the upregulation of several SM-synthesizing genes in the interaction. R. placenta (Antrodia clade) instead upregulated a larger variety of uncharacterized oxidoreductases in interacting hyphae, suggesting that an oxidative burst may be a response to competitors in this fungus. Both species produced several hypothetical proteins exclusively in the interaction zone, leaving abundant unknowns on the battlefield. This work supports the existence of multiple interaction strategies among brown rot fungi and highlights the functional diversity among wood decay fungi.