Most brown rot fungi are copper-tolerant, which makes them difficult to control with copper-based wood preservatives like MCQ. To better understand what biological processes are regulated, we used our model species, Antrodia radiculosa, to examine expression of genes on MCQ-treated wood. Our hypothesis was genes that decreased copper bioavailability would be up-regulated early, when wood showed no strength loss, while genes that degraded the structural polysaccharides would be up-regulated late, when wood exhibited high strength loss. We used a global profiling strategy called RNA-Seq to record all the genes that were actively being expressed at the two time points. We found 544 differentially expressed gene models. 52 of these gene models had putative functions directly related to oxalate production and polysaccharide degradation. Increased oxalate production at the early time point was caused by up-regulated expression of the following gene models: two pyruvate decarboxylases (3x), one citrate synthase (4x), one isocitrate lyase (8x), one oxaloacetate hydrolase (4x), and four mitochondrial carrier proteins (up to 9x). Up-regulation of oxalate is consistent with the theory that fungi remove copper toxicity by forming insoluble copper oxalate crystals. With respect to the late time point, we found sixteen gene models from at least six different glycoside hydrolase families (GH5, GH10, GH12, GH3, GH61, and GH53) that were highly up-regulated (as much as 23x), along with many sugar transporter genes. Function of the glycoside hydrolases involved cleavage of the -bonds typical of the hemicelluloses and cellulose, which explained the 52% strength loss observed at the late time point. Interestingly, two different sets of gene models for pectin hydrolysis were up-regulated at both early and late time points, suggesting that the pectic substances they targeted were slightly different. These results are significant for wood protection because we have identified the genes that are regulated to uptake sugars, control oxalate levels, and to enzymatically degrade pectin, cellulose, and the hemicelluloses. By knowing these control points, we can rationally develop the next generation of environmentally safe wood preservatives. We also hope to exploit novel brown rot biochemistries for new industries, like biological pretreatment of wood for cellulosic ethanol production.

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