Mechanisms of decay and interspecific interactions of white and brown rot fungi

Abstract

Wood is the largest source of biotic carbon on earth and the principle drivers of its decay are basidiomycete fungi. The biochemical mechanisms of wood decay by basidiomycete fungi are fundamental processes in forest ecosystems that dictate carbon evolution rates, soil organic matter deposition, and overall ecosystem function. These decay mechanisms are also unique in their ability to efficiently convert recalcitrant woody biomass to fermentable sugars and can serve as a biological template for industrial lignocellulose conversion to make renewable biofuels more economical. However, basidiomycete wood decay mechanisms are not fully understood and therefore not replicable in vitro, due in part to a lack of understanding of how decay mechanisms change throughout the progression of decay. In addition, gene transcripts and proteins used to facilitate decay are produced in concert with other biomolecules with non-degradative functions which makes resolution of degradative genes difficult. This dissertation contributes to resolving these problems by describing the temporal progression of decay among several species of wood-degrading basidiomycetes and functionally categorizing genes and secreted proteins involved in mediating interspecific interactions. This was done by first spatially resolving decay into a temporal sequence by growing model brown-rot fungi directionally on thin wood wafers. This system was used to co-localize changes in fungal physiology with chemical changes in wood substrates and the production of fungal metabolites to identify the functional significance of those physiological changes. The same wafer culture design was then used to resolve changes in fungal secretomes between two phylogenetically disparate brown-rot species over the course of decay using proteomics co-localized with lignocellulose-degrading enzyme assays. Interspecific differences were further investigated by comparing decay performance of the same two fungi on a Poales substrate, sorghum bagasse. Sorghum decay rates along with component removal and enzyme assays were monitored during decay to determine the genetic and biochemical basis of substrate preferences of the two species. Temporal alterations to fungal secretomes were compared among several model white and brown-rot fungi as well. Comparative proteomics concurrent with lignocellulose-degrading enzyme assays were used to identify common patterns among both rot types, as well as interspecific variability of decay mechanisms within species. Finally, changes in gene expression, protein secretion, and enzyme activity profiles in response to fungal competitors were described by modifying the thin wood wafer microcosms to incorporate two brown-rot species grown in opposition to one another. Resolution of decay into a sequence revealed a biphasic decay mechanism in brown-rot fungi delineated by early stage, non-hydrolytic pretreatment followed by later stage glycoside hydrolase-mediated saccharification. Proteomic investigation confirmed this pattern by showing later stage secretomes contain a greater proportion of glycoside hydrolases and their activities than earlier stages of decay. Brown-rot secretomes varied considerably by species as did their ability to degrade sorghum bagasse, likely due to a difference in the ability to hydrolyze ferulic acid esters present in sorghum biomass. Comparison of white and brown-rot secretomes identified a common segregation of decay into a biphasic decay mechanism characterized by high lignolysis, in white-rot fungi, upon wood colonization followed by later stage glycoside hydrolase secretion in both decay types. Considerable interspecific variability in decay mechanism within decay types was also detected, with the white-rot species producing different suites of ligninolytic enzymes and brown-rot species diverging in the types of glycoside hydrolases produced. Investigation of interspecific interactions identified several proteins exclusively produced during the interaction of two brown-rot species as well as identifying the general downregulation of lignocellulose-degrading genes during the interaction. In addition, comparative transcriptomics identified two different interaction strategies employed by species and implicates several secondary metabolite-synthesizing genes in facilitating interspecific interactions. Overall, this work contributes toward functional categorization of a wide range of basidiomycete proteins and provides a better understanding of decay mechanisms and interspecific interactions in these understudied organisms.