Our research, supported by a new DOE BER Systems Biology grant (as of Sept. 2018), focuses on "brown rot" fungi that selectively extract sugars embedded in lignin. These fungi, we discovered, loosen plant cell walls with reactive oxygen species (ROS) deployed ahead of most carbohydrate-active enzymes (CAZYs), including cellulases. Cellulases were long thought to be constitutively produced by brown rot fungi - we shifted the paradigm, showing that differential gene expression enabled an ROS pretreatment followed by inducible cellulases.
We hypothesize that there is another major deficiency in the brown rot paradigm that will be accessible to us only with tools and expertise at EMSL. Specifically, we have coarse-scale evidence that ROS is itself inducible, and that there are key"out-of-step" exceptions to the two-step ROS-CAZY sequence that, if teased apart at hyphal tips, will reveal genes enabling ROS-based deconstruction. This begs for expertise, high-resolution tools, and collaboration with EMSL.
With the goal of increasing resolution from mycelium scale (5 mm) to hyphal tip scale (30-100 micrometers) to discern ROS-specific brown rot pathways, our aims for an EMSL effort are as follows:
1) Use fine-scale cryo-toming and laser capture, coupled with fine-scale and/or single-cell RNA-seq, to increase resolution near hyphal tips in the model fungus Postia placenta.
2) Use cell-free expression, coupled with functionalization (including ETEM), to characterize key CAZYs produced at this hyphal front, with specific focus on ROS tolerance mechanisms.
3) Use super resolution microscopy and RNA-seq to disentangle sugar metabolism from detox and combat functions at hyphal tips by varying substrate extractives content and competitor presence.