Unlocking the distinctive enzymatic functions of the early plant biomass deconstructive genes in a brown rot fungus by cell-free protein expression.

Saprotrophic fungi that cause brown rot of woody biomass evolved a distinctive mechanism that relies on reactive oxygen species (ROS) to kick-start lignocellulosic polymers' deconstruction. These ROS agents are generated at incipient decay stages through a series of redox relays that shuttle electrons from fungus's central metabolism to extracellular Fenton chemistry. A list of genes has been suggested encoding the enzyme catalysts of the redox processes involved in ROS's function. However, navigating the functions of the encoded enzymes has been challenging due to the lack of a rapid method for protein synthesis. Here, we employed cell-free expression system to synthesize four redox or degradative enzymes, which were identified, by transcriptomic data, as conserved players of the ROS oxidation phase across brown rot fungal species. All four enzymes were successfully expressed and showed activities that enable confident assignment of function, namely, benzoquinone reductase (BQR), ferric reductase, α-L-arabinofuranosidase (ABF), and heme-thiolate peroxidase (HTP). Detailed analysis of their catalytic features within the context of brown rot environments allowed us to interpret their roles during ROS-driven wood decomposition. Specifically, we validated the functions of BQR as the driver redox enzyme of Fenton cycles and reconstructed its interactions with the co-occurring HTP or laccase and ABF. Taken together, this research demonstrated that the cell-free expression platform is adequate for synthesizing functional fungal enzymes and provided an alternative route for the rapid characterization of fungal proteins, escalating our understanding of the distinctive biocatalyst system for plant biomass conversion.IMPORTANCEBrown rot fungi are efficient wood decomposers in nature, and their unique degradative systems harbor untapped catalysts pursued by the biorefinery and bioremediation industries. While the use of "omics" platforms has recently uncovered the key "oxidative-hydrolytic" mechanisms that allow these fungi to attack lignocellulose, individual protein characterization is lagging behind due to the lack of a robust method for rapid synthesis of crucial fungal enzymes. This work delves into the studies of biochemical functions of brown rot enzymes using a rapid, cell-free expression platform, which allowed the successful depictions of enzymes' catalytic features, their interactions with Fenton chemistry, and their roles played during the incipient stage of brown rot when fungus sets off the reactive oxygen species for oxidative degradation. We expect this research could illuminate cell-free protein expression system's use to fulfill the increasing need for functional studies of fungal enzymes, advancing the discoveries of novel biomass-converting catalysts.

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences.

Pleurotus ostreatus, also known as the oyster mushroom, is a popular edible mushroom cultivated worldwide. This review aims to survey recent progress in the molecular genetics of this fungus and demonstrate its potential as a model mushroom for future research. The development of modern molecular genetic techniques and genome sequencing technologies has resulted in breakthroughs in mushroom science. With efficient transformation protocols and multiple selection markers, a powerful toolbox, including techniques such as gene knockout and genome editing, has been developed, and numerous new findings are accumulating in P. ostreatus. These include molecular mechanisms of wood component degradation, sexual development, protein secretion systems, and cell wall structure. Furthermore, these techniques enable the identification of new horizons in enzymology, biochemistry, cell biology, and material science through protein engineering, fluorescence microscopy, and molecular breeding. KEY POINTS: • Various genetic techniques are available in Pleurotus ostreatus. • P. ostreatus can be used as an alternative model mushroom in genetic analyses. • New frontiers in mushroom science are being developed using the fungus.

Isolation of bacteria from Grifola frondosa cultivation on wood logs to find mycelial growth-promoting bacteria.

This study aimed to isolate bacteria that coexist with the edible mushroom Grifola frondosa when it is cultured on wood, and to determine their interactions; in turn, the aim was to find bacteria that stimulate mycelial growth so as to decrease the time required for spawn preparation on potato dextrose agar (PDA). Some Pseudomonas, Dyella, Bacillus, and Priestia spp. isolated from the cultivation surroundings of G. frondosa had a positive effect on the mycelial growth of the fungus in PDA. However, some isolated bacteria had a severe negative effect on the mycelial growth, especially Burkholderia spp. Thus, both mycelial-promoting bacteria and potentially pathogenic bacteria coexist with G. frondosa in cultivation. Enzyme activity assays indicated that some wood-degrading bacteria inhabit the cultivation surroundings of G. frondosa, and these bacteria probably help the fungus to degrade wood (especially cellulose).

Targeted disruption of hir1 alters the transcriptional expression pattern of putative lignocellulolytic genes in the white-rot fungus Pleurotus ostreatus.

Pleurotus ostreatus is frequently used in molecular genetics and genomic studies on white-rot fungi because various molecular genetic tools and relatively well-annotated genome databases are available. To explore the molecular mechanisms underlying wood lignin degradation by P. ostreatus, we performed mutational analysis of a newly isolated mutant UVRM28 that exhibits decreased lignin-degrading ability on the beech wood sawdust medium. We identified that a mutation in the hir1 gene encoding a putative histone chaperone, which probably plays an important role in DNA replication-independent nucleosome assembly, is responsible for the mutant phenotype. The expression pattern of ligninolytic genes was altered in hir1 disruptants. The most highly expressed gene vp2 was significantly inactivated, whereas the expression of vp1 was remarkably upregulated (300-400 fold) at the transcription level. Conversely, many cellulolytic and xylanolytic genes were upregulated in hir1 disruptants. Chromatin immunoprecipitation analysis suggested that the histone modification status was altered in the 5'-upstream regions of some of the up- and down-regulated lignocellulolytic genes in hir1 disruptants compared with that in the 20b strain. Hence, our data provide new insights into the regulatory mechanisms of lignocellulolytic genes in P. ostreatus.

Influence of carbon source on wood decay-associated gene expression in sequential hyphal zones of the brown rot fungus Gloeophyllum trabeum.

Brown rot fungi show a two-step wood degradation mechanism comprising oxidative radical-based and enzymatic saccharification systems. Recent studies have demonstrated that the brown rot fungus Rhodonia placenta expresses oxidoreductase genes ahead of glycoside hydrolase genes and spatially protects the saccharification enzymes from oxidative damage of the oxidoreductase reactions. This study aimed to assess the generality of the spatial gene regulation of these genes in other brown rot fungi and examine the effects of carbon source on the gene regulation. Gene expression analysis was performed on 14 oxidoreductase and glycoside hydrolase genes in the brown rot fungus Gloeophyllum trabeum, directionally grown on wood, sawdust-agar, and glucose-agar wafers. In G. trabeum, both oxidoreductase and glycoside hydrolase genes were expressed at higher levels in sections behind the wafers. The upregulation of glycoside hydrolase genes was significantly higher in woody substrates than in glucose, whereas the oxidoreductase gene expression was not affected by substrates.

Influence of light on lignin-degrading activities of fungal genus Polyporus s. str.

Six strains belonging to five species of Polyporus (P. arcularius, P. arcularioides, P. tricholoma, P. cfr. tricholoma, and P. varius), collected from an Atlantic Forest area in Misiones (Argentina), where species usually grow exposed to high temperatures and humidity, were identified by morphological and molecular analyses. P. tricholoma (BAFC 4536) and P. arcularioides (BAFC 4534) were selected by their lignin-degrading enzyme production, their ability to produce primordial of basidiomes under submerged fermentation, and the decrease in lignin content caused in Poplar wood (up to 29% after 45 days). Among several variables evaluated with a Plackett-Burman design (glucose, copper, vanillic acid and manganese concentration, incubation period, and light incidence), the most important factor affecting laccase and Mn-peroxidase (MnP) production by both strains, was light incidence. Light induced fruit body development but diminished laccase and MnP production. Moreover, a modified isoenzymatic laccase pattern was observed, showing additional isoenzymes when fungi were cultivated under darkness and differences in optimal temperature. Although the studied strains did not produce high laccase and MnP titers (uppermost detected 4230 and 90 U L-1 , respectively), their laccases showed thermal stability and optimal temperature above 70 °C, representing an interesting source in the search of thermo-tolerant enzymes for biotechnological applications.

RNA extraction from decaying wood for (meta)transcriptomic analyses.

Wood decomposition is a key step of the terrestrial carbon cycle and is of economic importance. It is essentially a microbiological process performed by fungi and to an unknown extent by bacteria. To gain access to the genes expressed by the diverse microbial communities participating in wood decay, we developed an RNA extraction protocol from this recalcitrant material rich in polysaccharides and phenolic compounds. This protocol was implemented on 22 wood samples representing as many tree species from 11 plant families in the Angiosperms and Gymnosperms. RNA was successfully extracted from all samples and converted into cDNAs from which were amplified both fungal and bacterial protein coding genes, including genes encoding hydrolytic enzymes participating in lignocellulose hydrolysis. This protocol applicable to a wide range of decomposing wood types represents a first step towards a metatranscriptomic analysis of wood degradation under natural conditions.

Competitive antagonistic action of laccase between Trichoderma species and the newly identified wood pathogenic Ganoderma camelum.

Ganoderma, a well-known genus in the Ganodermataceae family, has caused the extinction of several tree species due to its pathogenicity. This study explored the pathogenic effect of a newly identified Ganoderma species on trees and its competitive efficiency against Trichoderma species. Ganoderma camelum sp. nov. is characterized by small sessile basidiomata and a velvety, soft, camel-brown pileus. Phylogenetic analysis and ITS rDNA sequences indicated that the species were Trichoderma and Ganoderma camelum. Both fungal species competed antagonistically by secreting laccase. The laccase activity of G. camelum, with a value of 8.3 ± 4.0 U/mL, demonstrated the highest competitive activity against Trichoderma species. The laccase produced by T. atroviride (2.62 U/mL) was most effective in countering the pathogenic action of the novel G. camelum. The molecular weights of laccase were determined using SDS-PAGE (62.0 kDa for G. camelum and 57.0 kDa for T. atroviride). Due to the white rot induced by this Ganoderma species in the host tree, G. camelum showed the highest percentage inhibition of radial growth (76.3%) compared to T. atroviride (28.7%). This study aimed to evaluate the competitive antagonistic activity of Ganoderma and Trichoderma on malt extract agar media in the context of white rot disease in the host tree. This study concluded that the laccase from G. camelum caused weight loss in rubber wood blocks through laccase action, indicating tissue injury in the host species. Therefore, it was also concluded that G. camelum was more effective in pathogenic action of the host and resisted the biological action of T. atroviride. In principal components analysis (PCA), all the species associated with laccase exhibited a very strong influence on the variability of the system. The PIRG rate (percentage inhibition of radial growth) was strongly and positively correlated with laccase activity.

Genome-Wide Identification of the ABC Gene Family and Its Expression in Response to the Wood Degradation of Poplar in Trametes gibbosa.

Wood-rotting fungi's degradation of wood not only facilitates the eco-friendly treatment of organic materials, decreasing environmental pollution, but also supplies crucial components for producing biomass energy, thereby reducing dependence on fossil fuels. The ABC gene family, widely distributed in wood-rotting fungi, plays a crucial role in the metabolism of lignin, cellulose, and hemicellulose. Trametes gibbosa, as a representative species of wood-rotting fungi, exhibits robust capabilities in wood degradation. To investigate the function of the ABC gene family in wood degradation by T. gibbosa, we conducted a genome-wide analysis of T. gibbosa's ABC gene family. We identified a total of 12 Tg-ABCs classified into four subfamilies (ABCA, ABCB, ABCC, and ABCG). These subfamilies likely play significant roles in wood degradation. Scaffold localization and collinearity analysis results show that Tg-ABCs are dispersed on scaffolds and there is no duplication of gene sequences in the Tg-ABCs in the genome sequence of T. gibbosa. Phylogenetic and collinearity analyses of T. gibbosa along with four other wood-rotting fungi show that T. gibbosa shares a closer phylogenetic relationship with its same-genus fungus (Trametes versicolor), followed by Ganoderma leucocontextum, Laetiporus sulphureus, and Phlebia centrifuga in descending order of phylogenetic proximity. In addition, we conducted quantitative analyses of Tg-ABCs from T. gibbosa cultivated in both woody and non-woody environments for 10, 15, 20, 25, 30, and 35 days using an RT-qPCR analysis. The results reveal a significant difference in the expression levels of Tg-ABCs between woody and non-woody environments, suggesting an active involvement of the ABC gene family in wood degradation. During the wood degradation period of T. gibbosa, spanning from 10 to 35 days, the relative expression levels of most Tg-ABCs exhibited a trend of increasing, decreasing, and then increasing again. Additionally, at 20 and 35 days of wood degradation by T. gibbosa, the relative expression levels of Tg-ABCs peak, suggesting that at these time points, Tg-ABCs exert the most significant impact on the degradation of poplar wood by T. gibbosa. This study systematically reveals the biological characteristics of the ABC gene family in T. gibbosa and their response to woody environments. It establishes the foundation for a more profound comprehension of the wood-degradation mechanism of the ABC gene family and provides strong support for the development of more efficient wood-degradation strategies.

Evaluation of Chemical and Morphological Properties of Spruce Wood Stored in the Natural Environment.

This paper focuses on the changes in chemical structure and fiber morphological properties of spruce wood during 15 months of its storage in an open forest woodshed. From the chemical composition, the extractives, cellulose, holocellulose, and lignin content were determined. The pH value was measured on the wood surface using a contact electrode. Acetic and formic acid, saccharides (glucose, xylose, galactose, arabinose and mannose), and polymerization degree (PD) of cellulose were analyzed using the HPLC method. Fiber length and width were determined using a fiber tester analyzer. After 15 months of storage the content of both cellulose (determined by the Seifert method) and lignin did not change; the quantity of hemicelluloses decreased by 13.2%, due to its easier degradation and less stability compared to cellulose; and the pH value dropped by one degree. HPLC analyses showed a total decrease in the cellulose DP of 9.2% and in saccharides of 40.2%, while the largest decreases were recorded in the quantity of arabinose, by 72%, in the quantity of galactose, by 61%, and in the quantity of xylose, by 43%. Organic acids were not detected due to their high volatility during wood storage. The total decrease in average fiber length was 38.2% and in width was 4.8%. An increase in the proportion of shorter fibers, and a decrease in the proportion of longer fibers, was recorded. It can be concluded that fundamental changes occurred in the wood, which could affect the quality of further products (e.g., chips, pulp, paper, particleboards).

Evaluation and modeling of fungi towards wood degradation.

Fungi play a significant role in wood fiber degradation since they possess enzymatic tools for the degradation of recalcitrant plant polymers. The study aims to demonstrate the interactive fungal traits when they grow together and its development with total dead wood fiber degradation speed. A lab experiment was designed to describe decomposition rates and fungal properties using nonlinear fitting model and logistic equation from preliminary data sets. The degradation speed of five (A, B, C, D, and E) different types of fungi with different growth rates were calculated at various relative humidity's (35, 50, 65, 80, and 95 g.kg-). Results showed that the mycelium length of fungus A, has faster ideal growth rate than that of fungus B, with ecological niche width A < B. Besides this the growth rate of fungus 1 was vg1 = 0.12 and the environmental-holding capacity k1 = 3000; vg2 = 0.15 and k2 = 2000 for fungus 2. Comparing the results of fiber decomposition with a single fungus, we were able to find that the overall efficiency of the two-fungal system decomposition model was higher in a defined environment. Besides this the successfully simulated the competitive relationship between different species of fungi and the effect of different environments on the decomposition rate of fungi, with a good fit and in accordance with the biological laws. Our model is well generalizable and can be extended to multiple environmental variables (light, temperature, and heat) with good accuracy.

The impact of biochar on wood-inhabiting bacterial community and its function in a boreal pine forest.

Biochar is considered to be a possible means of carbon sequestration to alleviate climate change. However, the dynamics of the microbial community during wood decomposition after biochar application remain poorly understood. In this study, the wood-inhabiting bacterial community composition and its potential functions during a two-year decomposition period after the addition of different amounts of biochar (0.5 kg m-2 and 1.0 kg m-2), and at different biochar pyrolysis temperatures (500 °C and 650 °C), in a boreal Scots pine forest, were analyzed using Illumina NovaSeq sequencing combined with Functional Annotation of Prokaryotic Taxa (FAPROTAX). The results showed that the wood decomposition rates increased after biochar addition to the soil surface in the second year. Treatment with biochar produced at high temperatures increased the diversity of wood-inhabiting bacteria more than that produced at low temperatures (P < 0.05). The wood-inhabiting bacterial diversity and species richness decreased with decomposition time. The biochar treatments changed the wood-inhabiting bacterial community structure during the decomposition period. The pyrolysis temperature and the amount of applied biochar had no effect on the bacterial community structure but shifted the abundance of certain bacterial taxa. Similarly, biochar application shifted the wood-inhabiting bacterial community function in the first year, but not in the second year. The wood-inhabiting bacterial community and function were affected by soil pH, soil water content, and soil total nitrogen. The results provide useful information on biochar application for future forest management practices. Long-term monitoring is needed to better understand the effects of biochar application on nutrient cycling in boreal forests.

Gene expression of the white-rot fungus Lenzites gibbosa during wood degradation.

To determine the wood degradation mechanism and its key genes and biological processes of Lenzites gibbosa, we sequenced 15 transcriptomes of mycelial samples under woody environment at 3, 5, 7, and 11 d (D3, D5, D7, and D11) and nonwoody environment (control). All the transcripts were annotated as much as possible in eight databases to determine their function. The key genes and biological processes relating to wood degradation were predicted and screened. The expression of 11 key genes during wood degradation after 5 d of sawdust treatment was detected by quantitative polymerase chain reaction (PCR). A total of 2069 differentially expressed genes (DEGs) were obtained in 10 differential groups. Comparing wood with nonwood treatment condition, the key genes were those participating in oxidation-reduction process, they were oxidoreductase and peroxidase genes and their regulator genes; these genes mainly focused on the three biological processes of carbohydrate metabolism, lignin catabolism, and secondary metabolite biosynthesis, transport, and catabolism. The mostly enriched subcategories in molecular function were oxidoreductase activity, peroxidase activity, and heme binding in Gene Ontology (GO) annotation. One cellulose and hemicellulose degradation pathway and seven pathways related to lignin-derived aromatic compound degradation or the later degradation of lignin were found. In conclusion, during the process of L. gibbosa growing on wood, gene expression at the transcriptional level indicated that lignin catabolism and hyphal growth were promoted, but the metabolism of carbon and carbohydrates including cellulose in lignocellulose in overall trend was inhibited to some extent. The results have important reference value for the study of degradation mechanism of wood white rot.

Bio-based wood preservatives: Their efficiency, leaching and ecotoxicity compared to a commercial wood preservative.

Companies in the wood industry are constantly developing their outdoor products. The possibility of using bio-based chemicals as an alternative to traditional wood preservatives-regulated in Europe by The Biocidal Products Regulation No 528/2012-has been considered, but chemical leaching from the wood decreases its effectiveness and may negatively affect the environment. This study aims to compare the effectiveness of bio-based chemicals with potential use in wood preservation to commercially available preservatives, to investigate their fixation to wood and their ecotoxicity and to quantify the potentially toxic elements leached from the wood. Pyrolysis distillates of tree bark, organic acids found in distillates, Colatan GT10 tannin extract and log soaking liquid as a hardwood veneer process residue were tested and compared with commercial pine oil and a copper-based wood preservative. In the wood decay test of impregnated pine sapwood specimens, Colatan GT10 extract performed as well as the commercial wood preservatives. The same decay trial with leached specimens significantly reduced the performance of the bio-based chemicals. The results of the ecotoxicity test with photoluminescent Aliivibrio fischeri bacteria showed that many bio-based chemicals with potential use in wood preservation have markedly lower ecotoxicity than commercially available wood preservatives, but the ecotoxicity of some bio-based chemicals is higher, as in the case of some of the pyrolysis distillates. The wood preservation efficiency and the ecotoxicity of the studied chemicals had a poor correlation, implying that other factors besides treatment agent toxicity play a role in deterring fungal growth on treated wood. The amount of elemental toxins in the leachates was low. These results emphasize the importance of the chemical ecotoxicity of bio-based preservative compounds, as their detrimental effect on the environment can be higher than that of the traditional preservatives unless effectively linked to wood to prevent leaching.

Comparative Transcriptomics During Brown Rot Decay in Three Fungi Reveals Strain-Specific Degradative Strategies and Responses to Wood Acetylation.

Brown rot fungi degrade wood in a two-step process in which enzymatic hydrolysis is preceded by an oxidative degradation phase. While a detailed understanding of the molecular processes during brown rot decay is mandatory for being able to better protect wooden products from this type of degradation, the underlying mechanisms are still not fully understood. This is particularly true for wood that has been treated to increase its resistance against rot. In the present study, the two degradation phases were separated to study the impact of wood acetylation on the behavior of three brown rot fungi commonly used in wood durability testing. Transcriptomic data from two strains of Rhodonia placenta (FPRL280 and MAD-698) and Gloeophyllum trabeum were recorded to elucidate differences between the respective decay strategies. Clear differences were found between the two decay stages in all fungi. Moreover, strategies varied not only between species but also between the two strains of the same species. The responses to wood acetylation showed that decay is generally delayed and that parts of the process are attenuated. By hierarchical clustering, we could localize several transcription factors within gene clusters that were heavily affected by acetylation, especially in G. trabeum. The results suggest that regulatory circuits evolve rapidly and are probably the major cause behind the different decay strategies as observed even between the two strains of R. placenta. Identifying key genes in these processes can help in decay detection and identification of the fungi by biomarker selection, and also be informative for other fields, such as fiber modification by biocatalysts and the generation of biochemical platform chemicals for biorefinery applications.

Factors That Affect the Mechanical Strength of Archaeological Wood-A Case Study of 18th-Century Wooden Water Pipes from Bóznicza Street in Poznan, Poland.

Large amounts of archaeological wood are often excavated during groundworks in cities and towns. Part of the unearthed artefacts is usually saved, conserved and then presented in museums. However, if the finding contains several similar objects, some of them could potentially be further employed for some other practical purposes. The research aimed to determine the mechanical performance of the remains of wooden water mains excavated at Bóznicza street in Poznan, Poland and evaluate its potential usefulness for any practical purposes. First, wood density was determined along with its mechanical strength in compression. The density of archaeological wood identified as Scots pine was lower than contemporary pinewood (383 kg × m-3 vs. 572 kg × m-3); therefore, its mechanical properties in compression tests were also lower, as expected, making the wood unsuitable for any practical applications. However, the differences in modulus of elasticity and compressive strength were not justified by the differences in wood density. Further infrared spectroscopy and X-ray diffraction analyses revealed additional differences in chemical composition and cellulose crystallinity between archaeological and contemporary wood. The results indicated the decrease in carbohydrate content and cellulose crystallinity in degraded wood, which, in addition to wood density, apparently contribute to the deterioration in mechanical strength of archaeological wood. The case study of the excavated archaeological wooden pipes shows that they have historical value but are not useful for practical purposes. It also revealed that not only wood density but also its chemical composition and cellulose crystallinity level has a substantial impact on the wood mechanical properties, particularly in compression.

Transcriptional shifts in delignification-defective mutants of the white-rot fungus Pleurotus ostreatus.

White-rot fungi efficiently degrade lignin and, thus, play a pivotal role in the global carbon cycle. However, the mechanisms of lignin degradation are largely unknown. Recently, mutations in four genes, namely wtr1, chd1, pex1, and gat1, were shown to abrogate the wood lignin-degrading ability of Pleurotus ostreatus. In this study, we conducted a comparative transcriptome analysis to identify genes that are differentially expressed in ligninolysis-deficient mutant strains. Putative ligninolytic genes that are highly expressed in parental strains are significantly downregulated in the mutant strains. On the contrary, many putative cellulolytic and xylanolytic genes are upregulated in the chd1-1, Δpex1, and Δgat1 strains. Identifying transcriptional alterations in mutant strains could provide new insights into the regulatory mechanisms of lignocellulolytic genes in P. ostreatus.

Degradative Capacity of Two Strains of Rhodonia placenta: From Phenotype to Genotype.

Brown rot fungi, such as Rhodonia placenta (previously Postia placenta), occur naturally in northern coniferous forest ecosystems and are known to be the most destructive group of decay fungi, degrading wood faster and more effectively than other wood-degrading organisms. It has been shown that brown rot fungi not only rely on enzymatic degradation of lignocellulose, but also use low molecular weight oxidative agents in a non-enzymatic degradation step prior to the enzymatic degradation. R. placenta is used in standardized decay tests in both Europe and North America. However, two different strains are employed (FPRL280 and MAD-698, respectively) for which differences in colonization-rate, mass loss, as well as in gene expression have been observed, limiting the comparability of results. To elucidate the divergence between both strains, we investigated the phenotypes in more detail and compared their genomes. Significant phenotypic differences were found between the two strains, and no fusion was possible. MAD-698 degraded scots pine more aggressively, had a more constant growth rate and produced mycelia faster than FPRL280. After sequencing the genome of FPRL280 and comparing it with the published MAD-698 genome we found 660,566 SNPs, resulting in 98.4% genome identity. Specific analysis of the carbohydrate-active enzymes, encoded by the genome (CAZome) identified differences in many families related to plant biomass degradation, including SNPs, indels, gaps or insertions within structural domains. Four genes belonging to the AA3_2 family could not be found in or amplified from FPRL280 gDNA, suggesting the absence of these genes. Differences in other CAZy encoding genes that could potentially affect the lignocellulolytic activity of the strains were also predicted by comparison of genome assemblies (e.g., GH2, GH3, GH5, GH10, GH16, GH78, GT2, GT15, and CBM13). Overall, these mutations help to explain the phenotypic differences observed between both strains as they could interfere with the enzymatic activities, substrate binding ability or protein folding. The investigation of the molecular reasons that make these two strains distinct contributes to the understanding of the development of this important brown rot reference species and will help to put the data obtained from standardized decay tests across the globe into a better biological context.

Estimating the Fitness Effect of Deleterious Mutations During the Two Phases of the Life Cycle: A New Method Applied to the Root-Rot Fungus Heterobasidion parviporum.

Many eukaryote species, including taxa such as fungi or algae, have a lifecycle with substantial haploid and diploid phases. A recent theoretical model predicts that such haploid-diploid lifecycles are stable over long evolutionary time scales when segregating deleterious mutations have stronger effects in homozygous diploids than in haploids and when they are partially recessive in heterozygous diploids. The model predicts that effective dominance-a measure that accounts for these two effects-should be close to 0.5 in these species. It also predicts that diploids should have higher fitness than haploids on average. However, an appropriate statistical framework to conjointly investigate these predictions is currently lacking. In this study, we derive a new quantitative genetic model to test these predictions using fitness data of two haploid parents and their diploid offspring, and genome-wide genetic distance between haploid parents. We apply this model to the root-rot basidiomycete fungus Heterobasidion parviporum-a species where the heterokaryotic (equivalent to the diploid) phase is longer than the homokaryotic (haploid) phase. We measured two fitness-related traits (mycelium growth rate and the ability to degrade wood) in both homokaryons and heterokaryons, and we used whole-genome sequencing to estimate nuclear genetic distance between parents. Possibly due to a lack of power, we did not find that deleterious mutations were recessive or more deleterious when expressed during the heterokaryotic phase. Using this model to compare effective dominance among haploid-diploid species where the relative importance of the two phases varies should help better understand the evolution of haploid-diploid life cycles.

Transcriptome-based analysis of the saprophytic fungus Abortiporus biennis - response to oxalic acid.

In this study, the transcriptomic-based response of the white rot fungus Abortiporus biennis to oxalic acid induction was reported. The whole transcriptome of A. biennis was analysed using the RNA-based sequencing technology and Solid 5500 platform. De novo assembly of reads generated 37,719 contigs. A molecular function for 26,280 unique transcripts was assigned. The analysis of the A. biennis transcriptome predicted 635 hypothetical open reading frames encoding carbohydrate active enzymes distributed in 122 families. 82 genes were identified, whose expression level was significantly changed after oxalic acid addition. Among them, 18 genes were up-regulated and 64 genes were down-regulated. Genes coding for putative cellulose and hemicellulose degrading enzymes were predominantly up-regulated in the mycelium induced with oxalic acid; it was in the case of cellulases and xylanases (hemicellulases), in particular, ß-glucosidase and endo-1,4-ß-xylanases. On the contrary, several genes coding for lignolytic enzymes were down-regulated, with the significant repression level in the case of versatile peroxidase. Finally, we identified putative genes involved in oxalate metabolism. Among the transcripts detected in the A. biennis transcriptome, one was annotated as coding for putative oxalate decarboxylase (ODC) and nine transcripts were annotated as formate dehydrogenases (FDH). The addition of oxalic acid to the culture caused upregulation of the gene coding for ODC and three genes for FDH. Amongst the transcripts of putative FDH genes, one designated as NODE_36057, demonstrated the highest induction level recorded in this study after the oxalic acid addition.