Unlocking proteomic and ecological heterogeneity between Schizophyllum commune and Trametes versicolor as early sapwood colonizers on woodDissertation
Datum der mündl. Prüfung:2021-07-23
Betreuer:Prof. Dr. Carsten Mai
Gutachter:Prof. Dr. Andrea Polle
Gutachter:Dr. Markus PD. Euring
Gutachter:Prof. Dr. Oliver Gailing
EnglischFungi are an essential part of forest ecosystems and wood decay fungi play key roles in degradation of wood and in nutrient recycling. The complete degradation of wood by a single fungal species is a challenge due to the reluctant physical structure and chemical composition of wood. In nature, therefore, decaying wood is a harbor for multiple wood decaying fungi. These fungi, among others, are key players in the wood decay process. Certain wood-decay fungi colonize early the wood and start the decay process. In this context, two early sapwood colonizers Schizophyllum commune and Trametes versicolor are often found together in the form of mushrooms on branches of living trees, on freshly fallen dead wood and also on cut stems. In previously reported and in own observations, fruiting bodies of both fungi appeared on the decaying wood first during the early decay stage. However, S. commune is a weak degrader with features in between white and brown rot (grey rot), and may take advantages from co-resident fungi better able to degrade wood. T. versicolor in contrast is an aggressive white rot. Interactions between these two early sapwood colonizers exist which might be competitive or cooperative. In this thesis work, the central focus was to understand better the interactions between the early sapwood colonizers (S. commune and T. versicolor) in laboratory experiments in dual cultures on beech wood, and to isolate the secretomes/ proteomes from single and dual cultures grown on beech sapwood or artificial substrate to define functions in growth on wood, in detoxification of harmful wood compounds and in defense to other fungi. Beech sapwood and bark decay tests were performed to test and compare the decay capacity of two early sapwood colonizers, S. commune and T. versicolor, under different light schemes. Three strains of S. commune (dikaryon Sc-D; the two parental monokaryons Sc-M1 and Sc-M2) and two strains of T. versicolor (dikaryon Tv-D; monokaryon Tv-M) were inoculated on beech particles (ca. 4 x 1 mm) and incubated at 25 oC under different light conditions (in continuous dark - CD, in dark - D, in continuous light - CL, and in 12 h dark/12 h light cycles – 12L/12D) for 30 and 90 days. The aggressive behavior of T. versicolor has been verified by beech sapwood and bark decay tests. Overall, T. versicolor caused higher reduction in bark and sapwood weights than S. commune. In sapwood, T. versicolor reduced the weight at maximum 53% (Tv-D) and 24% (Tv-M) within 90 days, while S. commune achieved the weight loss in the same time only up to 6% (Sc-D: 4%; Sc-M1: 5%, and Sc-M2: 6%). All strains of S. commune reduced only a same minor amount of the weight under all light and dark conditions. Differences have been observed in the weight losses by both strains of T. versicolor between all tested light schemes. The maximum reduction in weight losses caused by the Tv-M strain were 19%, 9%, 24%, and 24% under CL, 12L/12D, D and CD conditions, respectively. Maximum 44%, 48%, 53%, and 52% sapwood weight losses by the Tv-D strain were recorded under CL, 12L/12D, D and CD, respectively. In bark, the weight loss by T. versicolor was maximum 33% with 60 days of incubation in dark. In contrast, 4% bark weight was reduced by S. commune in dark and in light. In dark, both monokaryotic and dikaryotic strains of T. versicolor showed higher reduction in bark weights (Tv-M: 10 %, Tv-D: 33 %) in comparison to light condition (Tv-M: 8 %, Tv-D: 28 %). In the tests of fungal growth on wood wash-out collected from European ash tree log, three S. commune (Sc-M1, Sc-M2, and Sc-D) and two T. versicolor (Tv-M, and Tv-D) strains were grown on different dilutions (1:1, 1:4, and 1:10) in dark/light at 25 oC. The growth of Sc-M2, Sc-D, Tv-M, and Tv-D were also tested on wood wash-out with the addition of thiamine, glucose and nitrogen. S. commune grew under all the conditions tested but T. versicolor could not grow on concentrated wood wash-out (1:1 dilution) with and without thiamine. With the presence of carbon, Sc-M2, Tv-M, and Tv-D produce dense mycelium except Sc-D. The tested strains (Sc-M2, Sc-D, Tv-M, and Tv-D) gave thin mycelial growth in the presence of nitrogen. The interactions between both early sapwood colonizers were studied on natural (beech sapwood particles) and artificial (S. commune minimal medium) substrates. All possible interactions between three strains of S. commune (Sc-M1, Sc-M2, and Sc-D) and two strains of T. versicolor (Tv-M, and Tv-D) were observed on both substrates under continuous light and continuous dark conditions at 25 oC for 16 days. The dikaryotic strain of T. versicolor (Tv-D) was encountered with the monokaryotic strains of S. commune (Sc-M1 and Sc-M2) on day 6 and with the dikaryotic strain (Sc-D) on day 8, and dense mycelia appeared in the interaction zone. After that, the Tv-D strain overgrew throughout the S. commune (all strains) colonized compartments while growing on wood. However, on S. commune minimal medium, the Tv-D strain overgrew the S. commune strains while forming barriers of dense mycelia. In contrast, deadlocks were formed between the monokaryotic strain of T. versicolor (Tv-M) and the three strains of S. commune which were sustained till the end of the experiment on both natural and artificial substrates. In the dark, T. versicolor grew faster over the wood with S. commune as compared to in light. Therefore, S. commune inhabited wood particles were invaded by T. versicolor over time in the interactional studies. S. commune also produced specialized hyphal structures i.e., spicules (small needle-like anatomical structures on hyphae) and hyphal coils in the interaction with T. versicolor. Deadlocks (mutual inhibition after mycelial contact), barrage formation (barrier zone of hyphal growth), and pigmentation of different colors and intensities were observed in the interactions on agar media. For production of medium-free pellets, two strains of T. versicolor (Tv-M and Tv-D) and three strains of S. commune (Sc-D, Sc-M1 and Sc-M2) were grown on liquid BSM and S. commune minimal medium, respectively in dark conditions at 25 oC. In submerged cultures, medium-free pellets of all strains of both fungi were produced successfully to inoculate the wood for the secretome/ proteome isolations. These pellets were stored at 4 oC in fridge. Four different buffers were tested for the isolation of the T. versicolor (Tv-D) secretome from fungus-wood samples which were incubated at 25 oC in dark. In the experiment performed, buffer 1 was the best to isolate the secretome as it extracted a low number of contaminating intracellular proteins. The T. versicolor secretomes from early (10 days) and late (28 days) stages of decay were isolated and compared. 206 and 217 secreted proteins with signal peptides (identified by using SignalP 4.1) were then identified from 10- and 28- days old T. versicolor wood samples, respectively. In the experiment, at the early decay stage more laccases were found than at the late stage of decay at which more chitinases were found. The secreted proteins (183) shared between early and late decay stages were grouped according to their putative functions. The potential function of an enzyme was identified from the databases of JGI, InterPro, UniProt, HMMER web server, Pfam, NCBI blastp and NCBI conserved domain database (CDD). Enzymes and proteins secreted by T. versicolor were grouped according to their potential functions, like; lignin-modifying enzymes (e.g., peroxidases, laccases), cellulose-degrading enzymes (e.g., β-glucosidasese, exo- and endo-glucanases), enzymes cleaving main and side chains of hemicellulose (e.g., endo-1,4-β-xylanases, β-galactosidaseses), pectinases (e.g., endo-polygalacturonase, pectinesterase), chitinases, auxiliary activity enzymes, proteases and peptidases, (e.g. aspartyl peptidases, and serine proteases), a few enzymes of other functions and some proteins of no known function. After extracting the T. versicolor (Tv-D) secretome from the fungus-wood samples, the remaining samples were processed for the intracellular proteins to identify the proteins involved in different metabolisms while growing on wood. The potential function of an enzyme was identified from the databases of JGI, InterPro, UniProt, HMMER web server, Pfam, NCBI blastp and NCBI conserved domain database (CDD). In the analysis of T. versicolor intracellular proteins, the identified proteins were involved in the following different pathways: amino acid metabolism, biodegradation of xenobiotics, biosynthesis of secondary metabolites, energy metabolism, metabolism of carbohydrates and complex carbohydrates, metabolism of lipids and complex lipids, metabolism of cofactors and vitamins, metabolism of other amino acids, nucleotide metabolism, sorting and degradation, transcription, and translation. For the secretomic analysis of S. commune, the Sc-M1 (monokaryotic) strain was grown on beech sapwood particles for 2 weeks in dark at 25 oC. In the secretome of S. commune, 197 proteins with signal peptides were identified, and lignin-modifying enzymes (laccases and other multi-copper oxidases) were not expressed. According to the potential functions of proteins, the identified proteins were involved in the degradation of cellulose (found all three-essential group of enzymes i.e., exoglucanases, endoglucanases and β-glucosidases), hemicellulose (identified enzymes involved in the cleavage of xylan, mannans, and others), and pectin (identified enzymes acting on main and side chains). Besides these, chitinases, amylases, lipases, proteases and peptidases, auxiliary activities enzymes (identified members of AA3 (glucose-methanol-choline family of oxidoreductases), AA6 (reduction of the quinones by 1,4-benzoquinone reductases), and AA9 (copper-dependent lytic polysaccharide monooxygenase - copper dependent cleavage of cellulose with oxidation of carbons)), enzymes of other functions, and some proteins of unknown function were found in the secretome. In the dual culture interaction of dikaryotic S. commune (Sc-D) and T. versicolor (Tv-D) strains on glass-fiber filters, the interaction was carried out on two layers of glass-fiber filters soaked in S. commune minimal medium which were placed in Petri-dish having solid S. commune minimal medium and incubated in dark for 10 days at 25 oC. From 453 identified proteins in the secretome, 284 and 169 proteins were from T. versicolor and S. commune respectively. The identified proteins from both fungi in the secretome were grouped as fungal cell wall degrading enzymes (glucanases and mannanases), chitinases, oxidases and peroxidases (e.g., laccases, peroxidases), proteases and peptidases (aspartic, metallo and serine peptidases), nucleases, and proteins with potential antimicrobial activities.
Keywords: Schizophyllum commune; Trametes versicolor; proteome; ecology; fungi; wood; early sapwood colonizers; beech; wood decay; grey rot; white rot; brown rot; bark decay test; sapwood decay test; fungal interactions; medium-free pellets; secretome; intracellular proteins; fungus-wood samples; monokaryon; JGI; InterPro; UniProt; HMMER; Pfam; NCBI blastp; S. commune minimal medium; Dikaryon