Characterization of Neurospora crassa and Fusarium graminearum mutants defective in repeat-induced point mutation

Mutation of repetitive DNA by repeat-induced point mutation (RIP) is a process that occurs in many filamentous fungi of the Ascomycota during the sexual cycle. Concurrently, direct DNA repeats are often deleted by homologous recombination at high frequency during the sexual cycle. Thus, the processes of RIP and deletion compete to either mutate or remove repetitive DNA from the genome of filamentous fungi during sexual cycles. Both processes contribute to genome streamlining by controlling proliferation of transposable elements and by limiting expansion of gene families. While the genetic requirements for deletion by homologous recombination are well known, the mechanism behind the specific detection and mutation of repetitive DNA by RIP has yet to be elucidated as only a single gene essential for RIP, rid, has been identified. We have developed Fusarium graminearum as a model organism for the study of RIP by showing that it mutates repetitive DNA frequently during the sexual cycle and that the mutations due to RIP are dependent on rid. Further, we have sequenced a genetic mapping strain of F. graminearum (00-676-2) and identified 62,310 single nucleotide polymorphisms (SNPs) compared to the reference strain (PH-1). The SNP map will be useful for quickly mapping new mutants by bulk segregant analysis and high-throughput sequencing for which bioinformatic tools were specifically developed. The groundwork has thus been laid for identification of novel RIP mutants in F. graminearum, which being homothallic has a major advantage for identification of recessive mutations. We used a forward genetics approach to shed light on the mechanism of RIP in Neurospora crassa. Two rrr mutants that dominantly r̲educe R̲IP and r̲ecombination were characterized and identified as different mutated alleles of the same gene, rrr-1[superscript L496P] and rrr-1[superscript G325N] by bulk segregant analysis and high-throughput sequencing. Bioinformatic characterization suggests RRR-1 belongs to a previously uncharacterized group of dynamin-like proteins, which are generally involved in membrane fission and fusion. RRR-1-GFP localizes to the nuclear membrane, but not DNA, suggesting it affects RIP and recombination frequency indirectly by altering nuclear membrane dynamics during sexual development and thereby altering temporal aspects of RIP and recombination. We used a reverse genetics approach to determine whether high frequency RIP and homologous recombination of repetitive DNA during the sexual cycle are linked mechanistically or spatio-temporally. We tested strains where genes important for deletion by homologous recombination were knocked out and found all to be completely RIP competent except mre11, which, while sterile in homozygous deletion crosses, displayed lower RIP frequency in heterozygous crosses. This suggests that mre11 has roles in homologous recombination as well as non-homologous end joining may be important for RIP. Collectively, this work developed methods for efficiently mapping mutations and identified a novel protein that reduces RIP and recombination frequency but did not identify any mechanistic link between the two processes

Regulation of amino-acid metabolism controls flux to lipid accumulation in <i>Yarrowia lipolytica</i>

Yarrowia lipolytica is a promising microbial cell factory for the production of lipids to be used as fuels and chemicals, but there are few studies on regulation of its metabolism. Here we performed the first integrated data analysis of Y. lipolytica grown in carbon and nitrogen limited chemostat cultures. We first reconstructed a genome-scale metabolic model and used this for integrative analysis of multilevel omics data. Metabolite profiling and lipidomics was used to quantify the cellular physiology, while regulatory changes were measured using RNAseq. Analysis of the data showed that lipid accumulation in Y. lipolytica does not involve transcriptional regulation of lipid metabolism but is associated with regulation of amino-acid biosynthesis, resulting in redirection of carbon flux during nitrogen limitation from amino acids to lipids. Lipid accumulation in Y. lipolytica at nitrogen limitation is similar to the overflow metabolism observed in many other microorganisms, e.g. ethanol production by Sacchromyces cerevisiae at nitrogen limitation

A molecular genetic toolbox for Yarrowia lipolytica

Background: Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain. Results: We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the "Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. Conclusions: These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression

This is the publisher’s final pdf. The published article is copyrighted by BioMed Central Ltd and can be found at: http://www.biomedcentral.com/.Background: DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. \ud \ud Results: We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. \ud \ud Conclusions: We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation

Increase in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase function

Excess fatty acids and sterols are stored as triacylglycerols and sterol esters in specialized cellular organelles, called lipid droplets. Understanding what determines the cellular amount of neutral lipids and their packaging into lipid droplets is of fundamental and applied interest. Using two species of fission yeast, we show that cycling cells deficient in the function of the ER-resident CDP-DG synthase Cds1 exhibit markedly increased triacylglycerol content and assemble large lipid droplets closely associated with the ER membranes. We demonstrate that these unusual structures recruit the triacylglycerol synthesis machinery and grow by expansion rather than by fusion. Our results suggest that interfering with the CDPDG route of phosphatidic acid utilization rewires cellular metabolism to adopt a triacylglycerol-rich lifestyle reliant on the Kennedy pathway.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the American Society for Cell Biology. The published article can be found at: http://www.molbiolcell.org/

Genome resequencing reveals multiscale geographic structure and extensive linkage disequilibrium in the forest tree Populus trichocarpa

This is the publisher’s final pdf. The article is copyrighted by the New Phytologist Trust and published by John Wiley & Sons, Inc. It can be found at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291469-8137. To the best of our knowledge, one or more authors of this paper were federal employees when contributing to this work.•Plant population genomics informs evolutionary biology, breeding, conservation and bioenergy feedstock development. For example, the detection of reliable phenotype–genotype associations and molecular signatures of selection requires a detailed knowledge about genome-wide patterns of allele frequency variation, linkage disequilibrium and recombination.\ud •We resequenced 16 genomes of the model tree Populus trichocarpa and genotyped 120 trees from 10 subpopulations using 29 213 single-nucleotide polymorphisms.\ud •Significant geographic differentiation was present at multiple spatial scales, and range-wide latitudinal allele frequency gradients were strikingly common across the genome. The decay of linkage disequilibrium with physical distance was slower than expected from previous studies in Populus, with r² dropping below 0.2 within 3–6 kb. Consistent with this, estimates of recent effective population size from linkage disequilibrium (N[subscript e] ≈ 4000–6000) were remarkably low relative to the large census sizes of P. trichocarpa stands. Fine-scale rates of recombination varied widely across the genome, but were largely predictable on the basis of DNA sequence and methylation features.\ud •Our results suggest that genetic drift has played a significant role in the recent evolutionary history of P. trichocarpa. Most importantly, the extensive linkage disequilibrium detected suggests that genome-wide association studies and genomic selection in undomesticated populations may be more feasible in Populus than previously assumed

Methylome reorganization during in vitro dedifferentiation and regeneration of Populus trichocarpa

Background: Cytosine DNA methylation (5mC) is an epigenetic modification that is important to genome stability and regulation of gene expression. Perturbations of 5mC have been implicated as a cause of phenotypic variation among plants regenerated through in vitro culture systems. However, the pattern of change in 5mC and its functional role with respect to gene expression, are poorly understood at the genome scale. A fuller understanding of how 5mC changes during in vitro manipulation may aid the development of methods for reducing or amplifying the mutagenic and epigenetic effects of in vitro culture and plant transformation. Results: We investigated the in vitro methylome of the model tree species Populus trichocarpa in a system that mimics routine methods for regeneration and plant transformation in the genus Populus (poplar). Using methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq), we compared the methylomes of internode stem segments from micropropagated explants, dedifferentiated calli, and internodes from regenerated plants. We found that more than half (56%) of the methylated portion of the genome appeared to be differentially methylated among the three tissue types. Surprisingly, gene promoter methylation varied little among tissues, however, the percentage of body-methylated genes increased from 9% to 14% between explants and callus tissue, then decreased to 8% in regenerated internodes. Forty-five percent of differentially-methylated genes underwent transient methylation, becoming methylated in calli, and demethylated in regenerants. These genes were more frequent in chromosomal regions with higher gene density. Comparisons with an expression microarray dataset showed that genes methylated at both promoters and gene bodies had lower expression than genes that were unmethylated or only promoter-methylated in all three tissues. Four types of abundant transposable elements showed their highest levels of 5mC in regenerated internodes. Conclusions: DNA methylation varies in a highly gene-and chromosome-differential manner during in vitro differentiation and regeneration. 5mC in redifferentiated tissues was not reset to that in original explants during the study period. Hypermethylation of gene bodies in dedifferentiated cells did not interfere with transcription, and may serve a protective role against activation of abundant transposable elements.Keywords: Jacq, Palm Elaeis guineensis, Expression analysis, Genome, Cytosine methylation, Arabidopsis cells, Somaclonal variation, Plants, Tissue culture, Gene

Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression

Background: DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. Results: We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. Conclusions: We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.Keywords: Populus, DNA methylation, 5-methylcytosine, Epigenetics, Epigenomic

Engineering Rhodosporidium toruloides for production of 3-hydroxypropionic acid from lignocellulosic hydrolysate

Microbial production of valuable bioproducts is a promising route towards green and sustainable manufacturing. The oleaginous yeast, Rhodosporidium toruloides, has emerged as an attractive host for the production of biofuels and bioproducts from lignocellulosic hydrolysates. 3-hydroxypropionic acid (3HP) is an attractive platform molecule that can be used to produce a wide range of commodity chemicals. This study focuses on establishing and optimizing the production of 3HP in R. toruloides. As R. toruloides naturally has a high metabolic flux towards malonyl-CoA, we exploited this pathway to produce 3HP. Upon finding the yeast capable of catabolizing 3HP, we then implemented functional genomics and metabolomic analysis to identify the catabolic pathways. Deletion of a putative malonate semialdehyde dehydrogenase gene encoding an oxidative 3HP pathway was found to significantly reduce 3HP degradation. We further explored monocarboxylate transporters to promote 3HP transport and identified a novel 3HP transporter in Aspergillus pseudoterreus by RNA-seq and proteomics. Combining these engineering efforts with media optimization in a fed-batch fermentation resulted in 45.4&nbsp;g/L 3HP production. This represents one of the highest 3HP titers reported in yeast from lignocellulosic feedstocks. This work establishes R. toruloides as a host for 3HP production from lignocellulosic hydrolysate at high titers, and paves the way for further strain and process optimization towards enabling industrial production of 3HP in the future