LINKING STRUCTURE WITH FUNCTION AMONG PLANT-DECOMPOSING FUNGI

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: MCINTIRE-STENNIS
• Reporting Frequency: Annual
• Accession No.: 1006799
• Project No.: MIN-12-087
• Project Start Date: Oct 1, 2015
• Project End Date: Mar 31, 2018

Project Director
Schilling, J.

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Bioproducts & Biosystems Engineering

Non Technical Summary
The decomposition of plant biomass (lignocellulose) is achieved by a unique set oforganisms, primarily bacteria and fungi in temperate ecosystems. These organismsaccomplish something we strive to achieve - sustainable energy from plant biomass. InMinnesota and most of Earth's temperate northern forests, this is more specifically aprocess dominated by fungi. The dominance of certain decomposer fungi can have distinctconsequences depending on the species and their mechanisms for metabolizing planttissues such as wood. These mechanisms for deconstruction and metabolism, however,remain poorly characterized for the fungi that dominate our forests, and it is complicatedby ecological interactions that drive colonization dynamics. This project is geared toimprove both our biological baseline information for these fungi and our understanding ofthe consequences of these mechanisms as an emergent property in complex bacterialfungalcommunities. Specifically, our overall goal is to link the biology and ecology of thesedecomposer microbes with their functional consequences. Our approach couples modernmolecular analyses of both DNA and its products (mRNA and proteins) with thephysiochemical signatures that these organisms leave behind in lignocellulosic residues.There are three objectives thus proposed: 1) Assessing the variability among nutritionalmodes of lignocellulose?degrading fungi, 2) identifying unique evolutionary adaptationsthat enable dominance and thus have promise in biotechnology, and 3) contextualizingthese mechanisms among complex microbial communities, either to harness theseconsortia in production or to predict their emergent properties in nature.

Animal Health Component

0%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
123	4020	1060	100%

Knowledge Area
123 - Management and Sustainability of Forest Resources;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1060 - Biology (whole systems);

Keywords

microbe

wood

climate

carbon

fungi

mycology

Goals / Objectives
The overall goal is to link the biology and ecology of thesedecomposer microbes with their functional consequences. Our approach couples modernmolecular analyses of both DNA and its products (mRNA and proteins) with thephysiochemical signatures that these organisms leave behind in lignocellulosic residues.There are three objectives thus proposed: 1) Assessing the variability among nutritionalmodes of lignocellulose?degrading fungi, 2) identifying unique evolutionary adaptationsthat enable dominance and thus have promise in biotechnology, and 3) contextualizingthese mechanisms among complex microbial communities, either to harness theseconsortia in production or to predict their emergent properties in nature.

Project Methods
Objective 1: Assess variability along a spectrum of lignocellulose-degrading fungi.1.a. Isolates and MicrocosmsUtilizing the lineages of Floudas et al. (2012), distinguishing fungal rot types based on phylogeny, this objective will be approached using comparative transcriptomics and physiochemical changes in wood, using the wafer system outlined in Schilling et al. (2013) (Figure 2). The isolates will be representative of the 7+ clades of brown rot lineages that evolved from white rot ancestors, to be compared with those white rot ancestors, but remain undefined until growth strategies are assessed. Pint jars containing a peat:vermiculite:soil mix (Figure 2) will be inoculated with a 1-cm2 plug of a 14-d cultures of our test fungi. Birch feeder strips cut to fit the jars are laid flat on the soil. At the point of full mycelial coverage of the strips (2-4 weeks), aspen or spruce wafers will be added, propped vertically against the sides of the jars. Up to four wafers can be added per jar. Wafers (7-cm length) will be cut from untreated lumber into 7-mm thick wafers, the largest plane being transverse (23-mm width). Wafers will be oven-dried (100oC, 48 h) and weighed prior to steam sterilization and addition to microcosms, with the radial plane side resting on the mycelium. A second set of wafers (6-cm length) used for targeted testing are typically pre-soaked in distilled water under vacuum for three minutes prior to sterilization and will develop decay faster than those without a pre-soak. The wafer design will force hyphae to grow across the grain but not across annual rings, minimizing variability and maximizing cell-to-cell gradients.1.b. HarvestingWood sectioned can then be analyzed and re-assembled to create a composite image, similar to building a panorama from a progressive series of pictures. At harvest, the distance of visible fungal growth up the wafers will be recorded, and hyphae scraped from the surfaces. Strips (strips=vertical cuts) will be cut from fresh wafers over the length from the bottom to the top using a razor and a vice as a straight edge (Figure 2). Fresh strips will be sectioned (sections=horizontal cuts) at intervals, depending on analyses. Sections will be stored at -80oC until sectioning or microscopy, will be oven-dried and ground to 40 mesh in a Wiley mill for wood modification analyses, or will be sampled fresh.1.c. Hyphal localizationHyphal distance vertically up the wafers must be recorded for each sampled wafer and generally will be uniform as a horizontal front moving up the wafer. This must be an established relationship for each fungus tested. The visible hyphal front will be matched by analysis of hyphal progress within wood cells by fluorescent staining of chitin. Cryotomed radial sections (100 μm thick) will be made at intervals progressing up the wafers. Wood will be infiltrated with a non-crystal forming Tissue Freezing medium™ (Triangle Biomedical Sciences, Durham, NC, USA)and sectioned to approximately 100 µm with an OM 2488 MinotomeR microtome-crystat (International Equipment Company, Needham Heights, MA, USA) at−20°C. Sections will be mounted on glass slides, hydrated with pH 7.4 phosphate buffered saline (PBS) for 10 minutes and dyed for 10 minutes with wheat germ agglutinin, tetramethylrhodamine conjugate (10 mg/ml) (Invitrogen, USA). PBS-rinsed sections will be viewed on a Nikon C1 Spectral Imaging Confocal Laser Scanning Microscope (CLSM). The 488 and 567 laser bands will be used to excite and separate the wood tissue from fungal hyphae, and similar ranges will be used for detection.1.d. Colocalization analysesFor these samples (similar to bread slices), analyses will be performed prior to a colocalization effort (reassembling bread slices into a whole 'loaf'). This will allow us a spatial map of progress of reactions along an advancing mycelial growth of a fungus as it colonizes wood. Analyses of dried material will include DAS to determine lignin-hemicellulose decoupling and depolymerization (combined), lignin and carbohydrates (wt%), pH, and extractives contents, if desired. This material can also be used to test the concentrations of soluble sugars using high-performance liquid chromatography (HPLC) as for carbohydrate analyses, although this is better done with fresh material alongside any enzyme assays. Analyses for fresh material will involve an extraction procedure outlined in Schilling et al. (2013), and enzyme analyses will include cellulase and hemicellulases. Any hydrolytic enzyme activity can be analyzed using dye-linked substrates, with the chief limit being cost of individual substrates. For fresh material, depending on the level of resolution desired, we can use qPCR to assay the gene expression of target enzymes, or further use smFISH for highly-resolved expression colocalization. Overall, the design provides freedom in terms of isolate selection as well as analytical overlays, similar to the power of using GIS for map overlays at the landscape scale. This correlative power will allow us to connect response variables such as gene expression with cause (induction) or consequence.Objective 2: Identifying unique genetic adaptations of promiseUsing the data collected from the spatially-resolved systems, in concert with more expensive omics results ongoing from the DOE BER grant, this Objective involves comparative transcriptomic (mRNA quantification) analyses among isolates. Specifically, the qPCR targets from the Objective 1 will be placed in context with the RNAseq global analysis made possible with the collaborative efforts ongoing with the Minnesota Supercomputing Institute (MSI) at the University of Minnesota and with faculty (Figueroa and Bradeen) in the Department of Plant Pathology. A hire, to be named in Fall 2015, will be joining to contribute with Dr. Jiwei Zhang and Dr. Kevin Silverman on the bioinformatics component of that grant. This bioinformatics pipeline will be leveraged here to do comparative transcriptomics, extracting mRNA from wood samples for global transcript quantification. These large data sets will be analyzed in tandem with targeted qPCR mRNA gene sets, not only for the target genes (e.g. quinone reductase, glycosyl hydrolase family enzymes) but in context with those genes of putative function.Objective 3: Contextualize these mechanisms within more complex microbial communities.These biological efforts can be aided by adding context with other organisms present, and likewise, can reciprocally benefit ecological efforts trying to pin functional outcomes on the decomposer organisms responsible (as outlined in the Previous Work & Present Outlook section). An example of the former (using interactions to learn about biology) is at the advancing front of the fungus Gloeophyllum trabeum. We have found a number of exciting upregulated genes at this advancing front that may be critical to initial brown rot pretreatments of wood and that may be of great interest in cellulosic biofuel efforts. These genes, however, may instead be involved in combat upon colonizing wood, rather than metabolizing the wood substrate. Paired combative trials may help disentangle those genes of value in biomass-to-fuels from those used for competition, and offer a moderate-complexity system in which to bridge biology within isolates to ecology among many.

Progress 10/01/15 to 03/31/18

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project is being terminated, as the PI left the college

Publications

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:This project focuses on the basic biology and biochemistry of plant-decomposing fungi, but with contexts in both biotechnology and ecosystem science. Therefore, the target audiences are those studying basic microbiology, including the fungi, as well as those interested in harnessing plant-decomposing biology/biochemistry for biotechnological applications and those modeling carbon cycling dynamics from plants being decomposed in nature. Our work in the previous period has reached audiences in all three. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In terms of training, there are now 6 lab members who are advised by PI Schilling, a very engaged dynamic that involves a great deal of deliberate mentoring. We have bi-weekly lab meetings that are led by students and are informal but engaged. One of the overall training efforts has been bioinformatics, which has included several workshops and training exercises with the Minnesota Computing Institute. Schilling lab also hosted an Ethiopian student Tamirat Ali over the 2016 summer, and that relationship is ongoing as Tamirat is presenting his work for the second time in 2017 in Washington DC at an undergraduate STEM conference. Students have also been collaborating with research scientists and staff at the Pacific Northwest National Laboratory doing transcriptomics, super-resolution fluorescence microscopy, and several electron/Helium ion microscopy techniques. These are powerful learning experiences in using the cutting edge knowledge and tools at this National Lab in Richland, WA, and also seeing how they operate when networking. This training on instrumentation outside the University setting not only complements what they are learning here with non-redundant technology access but also in seeing how serious those scientists are about research, providing context that studying the little things is a National science motivation. For professional development, the PI has traveled and presented in various venues and formats in the past year, ranging from departmental talks at Oregon State University, Stellenbosch and the University of the Western Cape in South Africa, to non-technical talks at the ARC in South Africa and several 'bug club' talks to student groups interested in microbiology. These are more than reaching out with information, and more a two-way exchange of science and views. Our Professional developmetn- conferences seminars workshops (bioinformatics, mycoclub reading, etc. For the entire lab group. the bioinformatics efforts have been highly valuable, and also would count as training. In terms of networking, the Schilling lab has also created several fruitful relationships that have already spun off publications, including work with Yale that is in review for publication at Soil Biology and Biochemistry and a project with Peter Kennedy here at the University of Minnesota that was published in the journal Functional Ecology, with another in review at ISME Journal. How have the results been disseminated to communities of interest?Yes. As stated above, our work is of great interest to those trying to harness plant-decomposing metabolic machinery for biotechnology and those trying to do so to better predict carbon releases to the atmosphere. In line with this, we have published papers this year focusing on both the basic ecology and patterns of these fungi and the gene bases for these patterns, singling out key genes that might improve predictions of carbon and be harnessed to do so in a bioreactor, both. Publications are listed, previously, and we report here that PI Schilling gave 6 invited talks, including a talk in Paris in April at the European Fungal Genetics conference. The lab also presented several times at conferences this past year, including the Department of Energy's SPRUCE All-hands meeting in Saint Paul on the role of wood-degrading fungi in carbon allocations in a temperature/CO2 treatment design known as a 'free air carbon emission' design with deep peat heating, as well, to capture peatland methanogen/methanotrophic responses to changes in climate. What do you plan to do during the next reporting period to accomplish the goals?Our intended timeline indicated that this coming period we would be moving forward with microcosm trials and bioinformatics training. We are always setting and resetting microcosms, but the designs put forth in the proposal are done. The bioinformatics training is also now mature enough to complete our objectives, although it is ever-evolving and the tools/capacities are advancing rapidly. For co-localization assays, our goal is to expand the four-clade study results to match with proteomics data and carbon fraction analyses to allow something we have called 'connectomics.' Specifically, one target is to complete the four-clade synthesis and submit our results for publication as a transcriptomics-focused paper, including the bioinformatics that both inform our patterns and can be used to annotate the publically-available genomes. This will also be the intended goal for the comparative transcriptomics effort, but will be synergized with microscopy efforts in Richland intended to delineate hyphal localization during wood colonization. The other target is to then overlay these data with full proteomics along developed fungal wafers in parallel with wood porosity and wood chemistry, an effort in collaboration with Pacific Northwest National Lab and the Forest Products Laboratory in Madison, WI. We will also plan to increase the complexity of species-species interactions on wood wafers, with help in proteomics from Ellen Panisko and led by Gerrry Presley. Gerry will be looking to graduate and transition this coming year, so this effort is on the 'front burner.' This complexity in microcosm competition trials will be match by the progress in analyzing Cloquet field samples, a laborious process which we have only just begun. At present, an undergraduate student Emily Groenhoff is handling extractions and data/metadata efforts, and our goal is to determine the lignin loss:density loss ratios (an indicator of rot type, and a metric of nutritional mode) for the time zero and year 6 material in-hand. We have samples stored in the time series between those points, but if we can verify that by year 6, we have different rot type outcomes, we can both proceed with sampling in an informed way and utilize the preliminary findings to write grant proposals.

Impacts
What was accomplished under these goals? 1) Assessing the variability among nutritional modes of lignocellulose-degrading fungi. Our work on this Objective is ahead of schedule, both in terms of our hyphal localization and co-localization efforts. Our timeline was geared to have these experiments running and to be ready to handle data via bioinformatics, both of which are complete. We published several papers in 2016 focused on these spatially-motivated trials, specifically using a 'wafer' system of thin wood wafers to encourage directional growth and create space-for-time samples. We are also working with the Pacific Northwest National Lab on imaging and other spatially-explicit techniques that are enabling us to understand both the gene expression dynamics and proteomic aspects of wood-degrading fungi and the realistic context in which they are deployed in nature. Finally, we have an active relationship with Kevin Silverstein at the Minnesota Supercomputing Institute who is working with our postdoc Jiwei Zhang on the bioinformatics. These efforts have blossomed, and the transcriptomics information we have generated using RNA have been used both to reveal gene upregulation patterns but also to better annotate the genomes, themselves. This public service, by redepositing at the DOE Joint Genome Institute database, improves the ability for scientists to declare the functions for genes, a valuable asset for us and others in understanding why and how various genes are regulated. 2) Identifying unique evolutionary adaptations. We have now finished a trial we call the 'four clade' trial with two brown rot fungi and two white rot fungi, all from different evolutionary clades of wood-degrading fungi. In these trials, we have grown these fungi along our space-for-time design wood wafers to create a sequence along the wafer of hyphal ages. We have discovered, previously, that brown rot fungi in one of these clades staggers the expression of key oxidative genes at the hyphal front, while upregulating genes involved in glycoside hydrolysis later, in the older colonized sections of these wafers. Here, we are expanding on that work with four clades, being sure that one of these fungi is the brown rot fungus previously tested. At present, we have run these data through a rigorous bioinformatics pipeline that defines and clusters upregulated genes, and we have seen the same two-step oxidative-hydrolytic pattern that we showed before in the repeated brown rot fungal replicates. In the other brown rot fungi, we have seen a similar two-step pattern of gene regulation. I the other two white-rot fungi, we see some surprising similarities, however, in what we assumed was a brown rot-specific pattern, but the gene identies reveal some key differences related to lignin degradation. These patterns are being processed and synthesized, currently, to identify genes specific to either rot type; however, these early results promise another high-tier publication for several reasons that make the work of broad interest: 1) these genes in the hyphal front that are upregulated have clear biotechnological promise because the enzymes they code likely would be valuable in lignocellulose bioconversion schemes in industry, 2) the mechanisms of brown rot fungi, if better understood, will increase our ability to curb this destructive problem in lumber in service, and 3) these decay processes recycle nearly 80% of aboveground terrestrial biomass locked within wood. 3) Contextualizing these mechanisms among complex microbial communities, either to harness these consortia in production or to predict their emergent properties in nature. This work is on schedule, and has led to a grant proposal. Specifically, the work on proteomics in competitive environments as well as the fieldwork at Cloquet assessing patterns of wood-degrading fungal development are all active. An NSF-funded student, Gerry Presley, has been leading much of the proteomics efforts with the help of Ellen Panisko at the Pacific Northwest National Lab. In that work, we have identified a number of proteins being expressed at the hyphal front (using our wafer system design) that are likely not involved with metabolism, rather with combat. Assigning and colocalizing these proteins in our system offers several interesting payoffs in that we can match the protein profiles with gene expression patterns (described above) to verify a functional product, and in our quest for candidate genes of interest in bioconversion can winnow those targets by eliminating genes involved with combat rather than conversion. This is atop of the useful molecular ecology information of the genes and metabolites involved in combat when fungi and bacteria compete to colonize and metabolize wood.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Oliver, J.P., Schilling, J.S. (in press) Capture of methane by biofilter fungi  Evidence from lab-scale and chromatographic isotherm studies. Transactions of ASABE 59(6)
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Song, Z., Kennedy, P.G., Liew, F.J., Schilling, J.S. (online first) Fungal endophytes as priority colonizers during wood decomposition. Functional Ecology doi:10.1111/1365-2435.12735
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Oliver, J.P., Schilling, J.S.* (2016) Applying trait-function relationships for microbial plant decomposition to predict media longevity in engineered bioreactors. Applied Microbiology & Biotechnology 100: 2843-2853.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Oliver, J.P., Janni, K.A., and Schilling, J.S. (2016) Bait and scrape: An approach for assessing biofilm microbial communities on organic media used for gas-phase biofiltration. Ecological Engineering 91: 50-57.