Project description:In this study we performed RNA sequencing to determine and compare the transcriptome of regulatory T cells (Tregs) present in the thymus, placenta, spleen and white adipose tissue (FAT) of pregnant mice. We further assessed how the expression of the receptor Rank in the thymic medullary epithelia affects the transcriptional program of these Treg populations. For the isolation of viable and bona fide Tregs, we first generated RankWt (wild type control mice) and RankΔFoxn1 (which lack expression of Rank in the thymic epithelia) and further crossed them to mice that express GFP from the Foxp3 promoter. Neuropilin1 was used to mark the thymic origin of the Tregs. Equal numbers of Tregs (280 cells) were sorted as CD45-CD8-CD4+GFP+Neuropilin1High cells from the thymus and placenta of RankWtFoxp3GFP/GFP and RankΔFoxn1Foxp3GFP/GFP pregnant females at E17.5 (both samples from the same female; n=4 females per genotype). Thymus Tregs from non-pregnant littermate female mice for each genotype cohort were also studied. To increase robustness, the 280 placental Tregs were purified from 5 individual placentas per pregnant female (56 Tregs/placenta). Our study is the first one to determine the transcriptome of thymus and placenta Tregs during pregnancy and reveals that, partially dependent on Rank expression in the thymic epithelia, placental-resident Tregs are molecularly distinct from thymic Tregs. In a separate experiment with different pregnant mice than those used for thymus and placenta analysis, VAT Tregs (20-30 cells) and splenic Tregs (150 cells) were sorted (both samples from the same female) by using the same mouse lines (Rank Wt and RankΔFoxn1), embryological days of analysis (E17.5) and sorting strategies.
Project description:The bark represents the outer protective layer of trees. It contains high concentrations of antimicrobial extractives, in addition to regular wood polymers. It represents a huge underutilized side stream in forestry, but biotechnological valorization is hampered by a lack of knowledge on microbial bark degradation. Many fungi are efficient lignocellulose degraders, and here, spruce bark degradation by five species, Dichomitus squalens, Rhodonia placenta, Penicillium crustosum, Trichoderma sp. B1, and Trichoderma reesei, was mapped, by continuously analyzing chemical changes in the bark over six months. The study reveals how fungi from different phyla degrade bark using diverse strategies, regarding both wood polymers and extractives, where toxic resin acids were degraded by Basidiomycetes but unmodified/tolerated by Ascomycetes. Proteome analyses of the white-rot D. squalens revealed several proteins, with both known and unknown functions, that were specifically upregulated during growth on bark. This knowledge can accelerate improved utilization of an abundant renewable resource.
Project description:Wood-degrading fungi vary in their strategies for deconstructing wood, and their competitive successes shape the rate and fate of carbon released from wood, Earth’s largest pool of aboveground terrestrial carbon. In this study, one-on-one interspecific interactions between two model brown rot (carbohydrate-selective) fungi, Gloeophyllum trabeum and Rhodonia (Postia) placenta, were studied on wood wafers where a clearly resolved interaction zone (IZ) could be generated, reproducibly. Comparative RNAseq and proteomics between the IZ and non-interacting hyphae of each species identified combative strategies for each fungus. Glycoside hydrolases were a relatively smaller portion of the interaction secretome compared to non-interacting hyphae. The interaction zone showed higher pectinase specific activity than all other sampling locations, and higher laminarinase specific activity (branched β‐glucan proxy) was seen in the IZ secretome relative to equivalent hyphae in single‐species cultures. Our efforts also identified two distinct competitive strategies in these two fungi with a shared nutritional mode (brown rot) but polyphyletic ancestral lineages. Gloeophyllum trabeum (Gloeophyllum clade) employed secondary metabolite (SM) synthesis in response to a competitor, as shown by the upregulation of several SM-synthesizing genes in the interaction. R. placenta (Antrodia clade) instead upregulated a larger variety of uncharacterized oxidoreductases in interacting hyphae, suggesting that an oxidative burst may be a response to competitors in this fungus. Both species produced several hypothetical proteins exclusively in the interaction zone, leaving abundant unknowns on the battlefield. This work supports the existence of multiple interaction strategies among brown rot fungi and highlights the functional diversity among wood decay fungi.
Project description:Brown rot fungi Rhodonia (Postia) placenta grown on radiata pine modified with different levels of furfuryl alcohol - a wood protection system
Project description:Brown rot fungi dominate wood decomposition in coniferous forests, and their carbohydrate-selective mechanisms are of commercial interest. Brown rot was recently described as a two-step, sequential mechanism orchestrated by fungi using differentially expressed genes (DEGs) and consisting of oxidation via reactive oxygen species (ROS) followed by enzymatic saccharification. There have been indications, however, that the initial oxidation step, itself, might require induction. To capture this early gene regulation event, here we integrated fine-scale cryo-sectioning with whole transcriptome sequencing to dissect gene expression at the single hyphal cell scale (tens of μm). We improved spatial resolution 50x, relative to previous work, and we were able to capture the activity of the first 100 μm of hyphal front growth by Rhodonia placenta in aspen wood. By comparing the first 100-μm section with a 100-μm from a later decay stage, it was clear that the early decay period was dominated by delayed gene expression patterns as the fungus ramped up its mechanism. These delayed DEGs included many genes implicated in ROS pathways (lignocellulose oxidation, LOX) that were previously and incorrectly assumed to be constitutively expressed. However, this delayed pattern was not universal, with a handful of genes upregulated immediately at the hyphal front.
Project description:Over the last 20-80 million years the mammalian placenta has taken on a variety of morphologies through both divergent and convergent evolution. Recently we have shown that the human placenta genome has a unique epigenetic pattern of large partially methylated domains (PMDs) and highly methylation domains (HMDs) with gene body DNA methylation positively correlating with level of gene expression. In order to determine the evolutionary conservation of DNA methylation patterns and transcriptional regulatory programs in the placenta, we performed a genome-wide methylome (MethylC-seq) analysis of human, rhesus macaque, squirrel monkey, mouse, dog, horse, and cow placentas as well as opossum extraembryonic membrane. We found that, similar to human placenta, mammalian placentas and opossum extraembryonic membrane have globally lower levels of methylation compared to somatic tissues. However, not all species have clear PMD/HMDs in their placentas. Instead what is conserved is higher methylation over the bodies of genes involved in mitosis, vesicle-mediated transport, protein phosphorylation, and chromatin modification compared with the rest of the genome. As in human placenta, high gene body methylation is associated with higher gene expression across species. Analysis of DNA methylation in mouse and cow oocytes shows the same pattern of gene body methylation over many of the same genes as in the placenta, suggesting that this conserved pattern of active gene body methylation of the placenta may be established very early in development. MethylC-seq on placentas of 7 mammals, trophoblasts of rhesus, brains of 3 mammals, oocytes of cow, and human cordblood