Search

The morphological diversity of the inflorescence determines flower and seed production, which is critical for plant adaptation. Hall's panicgrass (Panicum hallii, P. hallii) is a wild perennial grass that has been developed as a model to study perennial grass biology and adaptive evolution. Highly divergent inflorescences have evolved between the 2 major ecotypes in P. hallii, the upland ecotype (P. hallii var hallii, HAL2 genotype) with compact inflorescence and large seed and the lowland ecotype (P. hallii var filipes, FIL2 genotype) with an open inflorescence and small seed. Here we conducted a comparative analysis of the transcriptome and DNA methylome, an epigenetic mark that influences gene expression regulation, across different stages of inflorescence development using genomic references for each ecotype. Global transcriptome analysis of differentially expressed genes (DEGs) and co-expression modules underlying the inflorescence divergence revealed the potential role of cytokinin signaling in heterochronic changes. Comparing DNA methylome profiles revealed a remarkable level of differential DNA methylation associated with the evolution of P. hallii inflorescence. We found that a large proportion of differentially methylated regions (DMRs) were located in the flanking regulatory regions of genes. Intriguingly, we observed a substantial bias of CHH hypermethylation in the promoters of FIL2 genes. The integration of DEGs, DMRs, and Ka/Ks ratio results characterized the evolutionary features of DMR-associated DEGs that contribute to the divergence of the P. hallii inflorescence. This study provides insights into the transcriptome and epigenetic landscape of inflorescence divergence in P. hallii and a genomic resource for perennial grass biology.

Different high temperatures adversely affect crop and algal yields with various responses in photosynthetic cells. The list of genes required for thermotolerance remains elusive. Additionally, it is unclear how carbon source availability affects heat responses in plants and algae. We utilized the insertional, indexed, genome-saturating mutant library of the unicellular, eukaryotic green alga Chlamydomonas reinhardtii to perform genome-wide, quantitative, pooled screens under moderate (35

Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. Finally, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.

Microbial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 712 full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night primarily in cyanobacteria, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts., Marc Van Goethem et al. combine short- and long-read sequencing from biocrust samples to report nearly 3,000 biosynthetic gene clusters (BGCs) encoding microbial secondary metabolites. Their results demonstrate the advantage of integrated metatranscriptomic and long-read metagenomic sequencing in analyzing BGCs and provides insight into the role of secondary metabolites in maintaining phylogenetic niches in biocrusts.

Chromatin modifications are epigenetic regulatory features with major roles in various cellular events, yet they remain understudied in algae. We interrogated the genome-wide distribution pattern of mono- and trimethylated histone H3 lysine 4 (H3K4) using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) during key phases of the Chlamydomonas cell cycle: early G

Different high temperatures adversely affect crop and algal yields with various responses in photosynthetic cells. The list of genes required for thermotolerance remains elusive. Additionally, it is unclear how carbon source availability affects heat responses in plants and algae. We utilized the insertional, indexed, genome-saturating mutant library of the unicellular, eukaryotic green algaChlamydomonas reinhardtiito perform genome-wide, quantitative, pooled screens under moderate (35°C) or acute (40°C) high temperatures with or without organic carbon sources. We identified heat-sensitive mutants based on quantitative growth rates and identified putative heat tolerance genes (HTGs). By triangulating HTGs with heat-induced transcripts or proteins in wildtype cultures and MapMan functional annotations, we present a high/medium-confidence list of 933Chlamydomonasgenes with putative roles in heat tolerance. Triangulated HTGs include those with known thermotolerance roles and novel genes with little or no functional annotation. About 50% of these high-confidence HTGs in Chlamydomonas have orthologs in green lineage organisms, including crop species.Arabidopsis thalianamutants deficient in the ortholog of a high-confidenceChlamydomonasHTG were also heat sensitive. This work expands our knowledge of heat responses in photosynthetic cells and provides engineering targets to improve thermotolerance in algae and crops.

The morphological diversity of the inflorescence determines flower and seed production, which is critical for plant adaptation and fitness. Cytosine methylation is an epigenetic mark that contributes to gene expression regulation during inflorescence development. Panicum hallii is a wild perennial grass in the subfamily Panicoideae that has been developed as a model to study perennial grass biology and adaptive evolution. Highly divergent inflorescences have evolved between the two major ecotypes in P. hallii, the upland ecotype with compact inflorescence and large seed and the lowland ecotype with an open inflorescence and small seed. Here we performed a comparative transcriptome and DNA methylome analysis across different stages of inflorescence between these two divergent ecotypes of P. hallii. Global transcriptome analysis identified differentially expressed genes (DEGs) involved in panicle divergence, stage-specific expression, and co-expression modules underlying inflorescence development. Comparing DNA methylome profiles revealed a remarkable level of differential DNA methylation associated with the evolution of the P. hallii inflorescence. We found that most differentially methylated regions (DMRs) occurred within the flanking regulatory regions of genes, especially the promoter. Integrative analysis of DEGs and DMRs characterized the global features of DMR-associated DEGs in the divergence of P. hallii inflorescence, which includes homologs of important inflorescence and seed developmental genes that have been previously identified in domesticated crops. Evolutionary analysis measured by Ka/Ks ratio suggested that most DMR-associated DEGs are under relatively strong purifying selection. This study provides insights into the transcriptome and epigenetic landscape of inflorescence divergence in P. hallii and a novel genomic resource for perennial grass biology.One sentence summary:A comparative transcriptome and DNA methylome analysis of inflorescence between upland and lowland ecotypes reveal gene expression and DNA methylation variation underlying inflorescence divergence in Panicum hallii.

ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

Significance Understanding the molecular response of plants to drought is critical to efforts to improve agricultural yields under increasingly frequent droughts. We grew 2 cultivars of the naturally drought-tolerant food crop sorghum in the field under drought stress. We sequenced the mRNA from weekly samples of these plants, resulting in a molecular profile of drought response over the growing season. We find molecular differences in the 2 cultivars that help explain their differing tolerances to drought and evidence of a disruption in the plant’s symbiosis with arbuscular mycorrhizal fungi. Our findings are of practical importance for agricultural breeding programs, while the resulting data are a resource for the plant and microbial communities for studying the dynamics of drought response., Drought is the most important environmental stress limiting crop yields. The C4 cereal sorghum [Sorghum bicolor (L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants’ roots.

Significance The plant-specific H3K27me1 methyltransferases ATXR5 and ATXR6 play integral roles connecting epigenetic silencing with genomic stability. However, how H3K27me1 relates to these processes is poorly understood. In this study, we performed a comprehensive transcriptome analysis of tissue- and ploidy-specific expression in a hypomorphic atxr5/6 mutant and revealed that the tissue-specific defects correlate with residual ATXR6 expression. We also determined that ATXR5/6 function is essential for female germline development. Furthermore, we provide a comprehensive analysis of H3K27me1 changes in relation to other epigenetic marks. We also determined that some previously reported suppressors of atxr5/6 may act by restoring the levels of H3K27me1, such as through up-regulation of the ATXR6 transcript in the atxr6 hypomorphic promoter allele., ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis. Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.

The eukaryotic green alga

The eukaryotic green alga Chromochloris zofingiensis is a reference organism for studying carbon partitioning and a promising candidate for the production of biofuel precursors. Recent transcriptome profiling transformed our understanding of its biology and generally algal biology, but epigenetic regulation remains understudied and represents a fundamental gap in our understanding of algal gene expression. Chromatin Immunoprecipitation followed by deep sequencing (ChIP-Seq) is a powerful tool for the discovery of such mechanisms, by identifying genome-wide histone modification patterns and transcription factor-binding sites alike. Here, we established a ChIP-Seq framework for Chr. zofingiensis yielding over 20 million high quality reads per sample. The most critical steps in a ChIP experiment were optimized, including DNA shearing to obtain an average DNA fragment size of 250 bp and assessment of the recommended formaldehyde concentration for optimal DNA-protein crosslinking. We used this ChIP-Seq framework to generate a genome-wide map of the H3K4me3 distribution pattern and to integrate these data with matching RNA-Seq data. In line with observations from other organisms, H3K4me3 marks predominantly transcription start sites of genes. Our H3K4me3 ChIP-Seq data will pave the way for improved genome structural annotation in the emerging reference alga Chr. zofingiensis.

Chromatin modifications are key epigenetic regulatory features with roles in various cellular events, yet histone mark identification, gene wide distribution and relationship to gene expression remains understudied in green algae. Histone lysine methylation is regarded as an active chromatin mark in many organisms, and is implicated in mediating active euchromatin. We interrogated the genome wide distribution pattern of mono- and trimethylated H3K4 using Chromatin-Immunoprecipitation followed by deep-sequencing (ChIP-Seq) during key phases of the Chlamydomonas cell cycle: early G1 phase (ZT1) when cells initiate biomass accumulation, S/M phase (ZT13) when cells are undergoing DNA replication and mitosis, and late G0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at TSSs of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at one or more time points during the cell cycle but not necessarily by continuous active transcription. This finding even applied to early zygotic genes whose expression may be dormant for hundreds or thousands of generations between sexual cycles; but core meiotic genes were completely missing H3K4me3 peaks at their TSS. In addition, bi-directional promoters regulating expression of replication dependent histone genes, had transient H3K4me3 peaks that were present only during S/M phase when their expression peaked. In agreement with biochemical studies, mono-methylated H3K4 was the default state for the vast majority of histones that were outside of TSS and terminator regions of genes. A small fraction of the genome which was depleted of any H3 lysine methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Genome wide H3K4me3 ChIP-Seq data will be a valuable resource, facilitating gene structural annotation, as exemplified by our validation of hundreds of long non-coding RNA genes.

A number of crop wild relatives can tolerate extreme stressed to a degree outside the range observed in their domesticated relatives. However, it is unclear whether or how the molecular mechanisms employed by these species can be translated to domesticated crops. PaspalumPaspalum vaginatumis a self-incompatible and multiply stress-tolerant wild relative of maize and sorghum. Here we describe the sequencing and pseudomolecule level assembly of a vegetatively propagated accession ofP. vaginatum. Phylogenetic analysis based on 6,151 single-copy syntenic orthologous conserved in 6 related grass species placed paspalum as an outgroup of the maize-sorghum clade demonstrating paspalum as their closest sequenced wild relative. In parallel metabolic experiments, paspalum, but neither maize nor sorghum, exhibited significant increases in trehalose when grown under nutrient-deficit conditions. Inducing trehalose accumulation in maize, imitating the metabolic phenotype of paspalum, resulting in autophagy dependent increases in biomass accumulation.

Progress in sequencing, microfluidics, and analysis strategies has revolutionized the granularity at which multicellular organisms can be studied. In particular, single-cell transcriptomics has led to fundamental new insights into animal biology, such as the discovery of new cell types and cell type-specific disease processes. However, the application of single-cell approaches to plants, fungi, algae, or bacteria (environmental organisms) has been far more limited, largely due to the challenges posed by polysaccharide walls surrounding these species’ cells. In this perspective, we discuss opportunities afforded by single-cell technologies for energy and environmental science and grand challenges that must be tackled to apply these approaches to plants, fungi and algae. We highlight the need to develop better and more comprehensive single-cell technologies, analysis and visualization tools, and tissue preparation methods. We advocate for the creation of a centralized, open-access database to house plant single-cell data. Finally, we consider how such efforts should balance the need for deep characterization of select model species while still capturing the diversity in the plant kingdom. Investments into the development of methods, their application to relevant species, and the creation of resources to support data dissemination will enable groundbreaking insights to propel energy and environmental science forward., In this Perspective, Diane Dickel and colleagues review recent progress and opportunities in applying single-cell sequencing and microfluidics methods to plants. The authors highlight the need for new tools developed with plants in mind, and advocate for the creation of a centralized, open-access database to house plant single-cell data.

Metagenomic sequence data from defined mock communities is crucial for the assessment of sequencing platform performance and downstream analyses, including assembly, binning and taxonomic assignment. We report a comparison of shotgun metagenome sequencing and assembly metrics of a defined microbial mock community using the Oxford Nanopore Technologies (ONT) MinION, PacBio and Illumina sequencing platforms. Our synthetic microbial community BMock12 consists of 12 bacterial strains with genome sizes spanning 3.2–7.2 Mbp, 40–73% GC content, and 1.5–7.3% repeats. Size selection of both PacBio and ONT sequencing libraries prior to sequencing was essential to yield comparable relative abundances of organisms among all sequencing technologies. While the Illumina-based metagenome assembly yielded good coverage with few misassemblies, contiguity was greatly improved by both, Illumina + ONT and Illumina + PacBio hybrid assemblies but increased misassemblies, most notably in genomes with high sequence similarity to each other. Our resulting datasets allow evaluation and benchmarking of bioinformatics software on Illumina, PacBio and ONT platforms in parallel., Measurement(s)metagenomic data • sequence_assemblyTechnology Type(s)ONT MinION • Illumina sequencing • PacBio RS IIFactor Type(s)sequencing platformSample Characteristic - OrganismBacteriaSample Characteristic - Environmentmock community Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.10260740

The DOE Joint Genome Institute (JGI) Metagenome Workflow performs metagenome data processing, including assembly; structural, functional, and taxonomic annotation; and binning of metagenomic data sets that are subsequently included into the Integrated Microbial Genomes and Microbiomes (IMG/M) (I.-M. A. Chen, K. Chu, K. Palaniappan, A. Ratner, et al., Nucleic Acids Res, 49:D751–D763, 2021, https://doi.org/10.1093/nar/gkaa939) comparative analysis system and provided for download via the JGI data portal (https://genome.jgi.doe.gov/portal/). This workflow scales to run on thousands of metagenome samples per year, which can vary by the complexity of microbial communities and sequencing depth. Here, we describe the different tools, databases, and parameters used at different steps of the workflow to help with the interpretation of metagenome data available in IMG and to enable researchers to apply this workflow to their own data. We use 20 publicly available sediment metagenomes to illustrate the computing requirements for the different steps and highlight the typical results of data processing. The workflow modules for read filtering and metagenome assembly are available as a workflow description language (WDL) file (https://code.jgi.doe.gov/BFoster/jgi_meta_wdl). The workflow modules for annotation and binning are provided as a service to the user community at https://img.jgi.doe.gov/submit and require filling out the project and associated metadata descriptions in the Genomes OnLine Database (GOLD) (S. Mukherjee, D. Stamatis, J. Bertsch, G. Ovchinnikova, et al., Nucleic Acids Res, 49:D723–D733, 2021, https://doi.org/10.1093/nar/gkaa983). IMPORTANCE The DOE JGI Metagenome Workflow is designed for processing metagenomic data sets starting from Illumina fastq files. It performs data preprocessing, error correction, assembly, structural and functional annotation, and binning. The results of processing are provided in several standard formats, such as fasta and gff, and can be used for subsequent integration into the Integrated Microbial Genomes and Microbiomes (IMG/M) system where they can be compared to a comprehensive set of publicly available metagenomes. As of 30 July 2020, 7,155 JGI metagenomes have been processed by the DOE JGI Metagenome Workflow. Here, we present a metagenome workflow developed at the JGI that generates rich data in standard formats and has been optimized for downstream analyses ranging from assessment of the functional and taxonomic composition of microbial communities to genome-resolved metagenomics and the identification and characterization of novel taxa. This workflow is currently being used to analyze thousands of metagenomic data sets in a consistent and standardized manner.

Organisms orchestrate cellular functions through transcription factor (TF) interactions with their target genes, although these regulatory relationships are largely unknown in most species. Here we report a high-throughput approach for characterizing TF-target gene interactions across species and its application to 354 TFs across 48 bacteria, generating 17,000 genome-wide binding maps. This dataset revealed themes of ancient conservation and rapid evolution of regulatory modules. We observed rewiring, where the TF sensing and regulatory role is maintained while the arrangement and identity of target genes diverges, in some cases encoding entirely new functions. We further integrated phenotypic information to define new functional regulatory modules and pathways. Finally, we identified 242 new TF DNA binding motifs, including a 70% increase of known Escherichia coli motifs and the first annotation in Pseudomonas simiae, revealing deep conservation in bacterial promoter architecture. Our method provides a versatile tool for functional characterization of genetic pathways in prokaryotes and eukaryotes.

Sensing available nutrients and efficiently utilizing them is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of inorganic and organic nitrogen sources. Nitrogen utilization in N. crassa is regulated by a network of pathway-specific transcription factors that activate genes necessary to utilize specific nitrogen sources in combination with nitrogen catabolite repression regulatory proteins. We identified an uncharacterized pathway-specific transcription factor, amn-1, that is required for utilization of the nonpreferred nitrogen sources proline, branched-chain amino acids, and aromatic amino acids. AMN-1 also plays a role in regulating genes involved in responding to the simple sugar mannose, suggesting an integration of nitrogen and carbon metabolism. The utilization of nonpreferred nitrogen sources, which require metabolic processing before being used as a nitrogen source, is also regulated by the nitrogen catabolite regulator NIT-2. Using RNA sequencing combined with DNA affinity purification sequencing, we performed a survey of the role of NIT-2 and the pathway-specific transcription factors NIT-4 and AMN-1 in directly regulating genes involved in nitrogen utilization. Although previous studies suggested promoter binding by both a pathway-specific transcription factor and NIT-2 may be necessary for activation of nitrogen-responsive genes, our data show that pathway-specific transcription factors regulate genes involved in the catabolism of specific nitrogen sources, while NIT-2 regulates genes involved in utilization of all nonpreferred nitrogen sources, such as nitrogen transporters. Together, these transcription factors form a nutrient sensing network that allows N. crassa cells to regulate nitrogen utilization.

Plant growth is mediated by the integration of internal and external cues, perceived by cells and transduced into a developmental program that gives rise to cell division, elongation, and wall thickening. Extra-, inter-, and intra- physical cellular forces contribute to this regulation. Across the plant kingdom thigmomorphogenesis is widely observed to alter plant morphology by reducing stem height and increasing stem diameter. The transcriptome is highly responsive to touch, including components of calcium signaling pathways and cell wall modification. One aspect of this cascade involves gibberellins (GA) and bZIP family transcription factors. Here, we present data connecting thigmomorphogenesis with secondary cell wall synthesis by GA inactivation and bZIP translocation into the nucleus. Brachypodium distachyon SECONDARY WALL INTERACTING bZIP (SWIZ) protein translocated into the nucleus in response to mechanical stimulation. This translocation was mitigated by supplementing with exogenous bioactive GA, and induced by chemical inhibition of GA synthesis. Misregulation of SWIZ expression resulted in plants with reduced stem and root elongation, and following mechanical stimulation, increased secondary wall thickness. Classical touch responsive genes were upregulated in B. distachyon following touch, and we observe significant induction of the glycoside hydrolase 17 family, which may be indicative of a facet of grass thigmomorphogenesis. SWIZ protein binding to an E-box variant in exons and introns was associated with immediate activation followed by repression of gene expression. Thus, SWIZ is a transcriptional regulatory component of thigmomorphogenesis, which includes the thickening of secondary cell walls.

We report here the draft genome sequence of Yokenella regensburgei strain WCD67, isolated from the boxelder bug (Boisea trivittata). The genome is 5,277,883 bp in size, has a GC content of 54.12%, and has 5,416 genes. A total of 17 mobile elements were discovered, 6 of which were predicted to be phages.

Significance Microorganisms have evolved signaling networks to identify and prioritize utilization of carbon sources. For fungi that degrade plant biomass, such as Neurospora crassa, signaling networks dictate the metabolic response to carbon sources present in plant cell walls, resulting in optimal utilization of nutrient sources. However, within a fungal colony, regulatory hierarchies associated with activation of transcription factors and temporal and spatial production of proteins for plant biomass utilization are unclear. Here, we perform expression profiling of N. crassa on simple sugars to complex carbohydrates to identify regulatory factors and direct targets of regulatory transcription factors using DNA affinity purification sequencing (DAP-seq). These findings will enable more precise tailoring of metabolic networks in filamentous fungi for the production of second-generation biofuels., Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.

The epigenome orchestrates genome accessibility, functionality, and three-dimensional structure. Because epigenetic variation can impact transcription and thus phenotypes, it may contribute to adaptation. Here, we report 1,107 high-quality single-base resolution methylomes and 1,203 transcriptomes from the 1001 Genomes collection of Arabidopsis thaliana. Although the genetic basis of methylation variation is highly complex, geographic origin is a major predictor of genome-wide DNA methylation levels and of altered gene expression caused by epialleles. Comparison to cistrome and epicistrome datasets identifies associations between transcription factor binding sites, methylation, nucleotide variation, and co-expression modules. Physical maps for nine of the most diverse genomes reveal how transposons and other structural variants shape the epigenome, with dramatic effects on immunity genes. The 1001 Epigenomes Project provides a comprehensive resource for understanding how variation in DNA methylation contributes to molecular and non-molecular phenotypes in natural populations of the most studied model plant.

Filamentous fungi depend on asexual cell-to-cell communication and cell fusion to establish and maintain an interconnected mycelial colony. In Neurospora crassa, genetically identical asexual spores undergo chemotropic interactions resulting in cell fusion and colony establishment... Maintenance of cell integrity and cell-to-cell communication are fundamental biological processes. Filamentous fungi, such as Neurospora crassa, depend on communication to locate compatible cells, coordinate cell fusion, and establish a robust hyphal network. Two MAP kinase (MAPK) pathways are essential for communication and cell fusion in N. crassa: the cell wall integrity/MAK-1 pathway and the MAK-2 (signal response) pathway. Previous studies have demonstrated several points of cross-talk between the MAK-1 and MAK-2 pathways, which is likely necessary for coordinating chemotropic growth toward an extracellular signal, and then mediating cell fusion. Canonical MAPK pathways begin with signal reception and end with a transcriptional response. Two transcription factors, ADV-1 and PP-1, are essential for communication and cell fusion. PP-1 is the conserved target of MAK-2, but it is unclear what targets ADV-1. We did RNA sequencing on Δadv-1, Δpp-1, and wild-type cells and found that ADV-1 and PP-1 have a shared regulon including many genes required for communication, cell fusion, growth, development, and stress response. We identified ADV-1 and PP-1 binding sites across the genome by adapting the in vitro method of DNA-affinity purification sequencing for N. crassa. To elucidate the regulatory network, we misexpressed each transcription factor in each upstream MAPK deletion mutant. Misexpression of adv-1 was sufficient to fully suppress the phenotype of the Δpp-1 mutant and partially suppress the phenotype of the Δmak-1 mutant. Collectively, our data demonstrate that the MAK-1/ADV-1 and MAK-2/PP-1 pathways form a tight regulatory network that maintains cell integrity and mediates communication and cell fusion.

To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX). The resulting DNA library is incubated with an affinity-tagged in vitro-expressed TF, and TF-DNA complexes are purified using magnetic separation of the affinity tag. Bound genomic DNA is eluted from the TF and sequenced using next-generation sequencing. Sequence reads are mapped to a reference genome, identifying genome-wide binding locations for each TF assayed, from which sequence motifs can then be derived. A researcher with molecular biology experience should be able to follow this protocol, processing up to 400 samples per week.

Due to a large and growing collection of genomic and experimental resources, Brachypodium distachyon has emerged as a powerful experimental model for the grasses. To add to these resources we sequenced 21 165 T-DNA lines, 15 569 of which were produced in this study. This increased the number of unique insertion sites in the T-DNA collection by 21 078, bringing the overall total to 26 112. Thirty-seven per cent (9754) of these insertion sites are within genes (including untranslated regions and introns) and 28% (7217) are within 500 bp of a gene. Approximately 31% of the genes in the v.2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized six T-DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies, demonstrating the utility of this collection for plant functional genomics. The Brachypodium T-DNA collection can be accessed at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/brachypodium-t-dna-collection/.

The complete genome sequence of the gammaproteobacterial isolate Serratia quinivorans 124R consists of 5 Mb over 2 scaffolds and a G+C content of 52.85%. Genes relating to aromatic metabolism reflect its isolation on organosolv lignin as a sole carbon source under anoxic conditions as well as the potential for lignin biorefinery applications.

Background Transcription factors (TFs) play a key role in regulating plant development and response to environmental stimuli. While most genes revert to single copy after whole genome duplication (WGD) event, transcription factors are retained at a significantly higher rate. Little is known about how TF duplicates have diverged in their expression and regulation, the answer to which may contribute to a better understanding of the elevated retention rate among TFs. Results Here we assessed what features may explain differences in the retention of TF duplicates and other genes using Arabidopsis thaliana as a model. We integrated 34 expression, sequence, and conservation features to build a linear model for predicting the extent of duplicate retention following WGD events among TFs and 19 groups of genes with other functions. We found that TFs was the least well predicted, demonstrating the features of TFs are substantially deviated from duplicate genes in other function groups. Consistent with this, the evolution of TF expression patterns and cis-regulatory cites favors the partitioning of ancestral states among the resulting duplicates: one “ancestral” TF duplicate retains most ancestral expression and cis-regulatory sites, while the “non-ancestral” duplicate is enriched for novel regulatory sites. By modeling the retention of ancestral expression and cis-regulatory states in duplicate pairs using a system of differential equations, we found that TF duplicate pairs in a partitioned state are preferentially maintained. Conclusions These TF duplicates with asymmetrically partitioned ancestral states are likely maintained because one copy retains ancestral functions while the other, at least in some cases, acquires novel cis-regulatory sites that may be important for novel, adaptive traits. Electronic supplementary material The online version of this article (10.1186/s12862-019-1398-z) contains supplementary material, which is available to authorized users.

While genome assembly projects have been successful in a number of haploid or inbred species, one of the current main challenges is assembling non-inbred or rearranged heterozygous genomes. To address this critical need, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble Single Molecule Real-Time (SMRT®) Sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We demonstrate the quality of this approach by assembling new reference sequences for three heterozygous samples, including an F1 hybrid of the model species Arabidopsis thaliana, the widely cultivated V. vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata that have challenged short-read assembly approaches. The FALCON-based assemblies were substantially more contiguous and complete than alternate short or long-read approaches. The phased diploid assembly enabled the study of haplotype structures and heterozygosities between the homologous chromosomes, including identifying widespread heterozygous structural variations within the coding sequences.

Draft genome sequences of 11 bacteria belonging to the family Microbacteriaceae were obtained using Illumina technology. The genomes of these strains have sizes from 3.14 to 4.30 Mb with their genomic DNA characterized as having high G+C contents (above 65%)., Draft genome sequences of 11 bacteria belonging to the family Microbacteriaceae were obtained using Illumina technology. The genomes of these strains have sizes from 3.14 to 4.30 Mb with their genomic DNA characterized as having high G+C contents (above 65%). These genomic data will be useful for natural taxonomy and comparative genomic studies of bacterial strains of the family Microbacteriaceae.

Grass biomass is comprised chiefly of secondary walls that surround fiber and xylem cells. A regulatory network of interacting transcription factors in part regulates cell wall thickening. We identified Brachypodium distachyon SECONDARY WALL ASSOCIATED MYB1 (SWAM1) as a potential regulator of secondary cell wall biosynthesis based on gene expression, phylogeny, and transgenic plant phenotypes. SWAM1 interacts with cellulose and lignin gene promoters with preferential binding to AC-rich sequence motifs commonly found in the promoters of cell wall-related genes. SWAM1 overexpression (SWAM-OE) lines had greater above-ground biomass with only a slight change in flowering time while SWAM1 dominant repressor (SWAM1-DR) plants were severely dwarfed with a striking reduction in lignin of sclerenchyma fibers and stem epidermal cell length. Cellulose, hemicellulose, and lignin genes were significantly down-regulated in SWAM1-DR plants and up-regulated in SWAM1-OE plants. There was no reduction in bioconversion yield in SWAM1-OE lines; however, it was significantly increased for SWAM1-DR samples. Phylogenetic and syntenic analyses strongly suggest that the SWAM1 clade was present in the last common ancestor between eudicots and grasses, but is not in the Brassicaceae. Collectively, these data suggest that SWAM1 is a transcriptional activator of secondary cell wall thickening and biomass accumulation in B. distachyon.

Maintenance of cell integrity and cell-to-cell communication are fundamental biological processes. Filamentous fungi, such asNeurospora crassa, depend on communication to locate compatible cells, coordinate cell fusion, and establish a robust hyphal network. Two MAP-Kinase pathways are essential for communication and cell fusion inN. crassa; the Cell Wall Integrity/MAK-1 pathway and the MAK-2 (signal response) pathway. Previous studies have demonstrated several points of cross talk between the MAK-1 and MAK-2 pathways, which is likely necessary for oordinating chemotropic growth toward an extracellular signal, and then mediating cell fusion. Canonical MAP-Kinase pathways begin with signal reception and end with a transcriptional response. Two transcription factors, ADV-1 and PP-1, are essential for communication and cell fusion. PP-1 is the conserved target of MAK-2, while it is unclear what targets ADV-1. We did RNAseq onΔadv-1, Δpp-1, and wild-type cells and found that ADV-1 and PP-1 have a shared regulon including many genes required for communication, cell fusion, growth, development, and stress response. We identified ADV-1 and PP-1 binding sites across the genome by adapting thein vitromethod of DNA-Affinity Purification sequencing (DAP-seq) forN. crassa. To elucidate the regulatory network, we misexpressed each transcription factor in each upstream MAPK deletion mutant. Misexpression ofadv-1was sufficient to fully suppress the phenotype of theΔpp-1mutant and partially suppress the phenotype of theΔmak-1mutant. Collectively, our data demonstrate that the MAK-1-ADV-1 and MAK-2- PP-1 pathways form a tight regulatory network that maintains cell integrity and mediates communication and cell fusion.

Maintenance of cell integrity and cell-to-cell communication are fundamental biological processes. Filamentous fungi, such as Neurospora crassa, depend on communication to locate compatible cells, coordinate cell fusion, and establish a robust hyphal network. Two MAP kinase (MAPK) pathways are essential for communication and cell fusion in N. crassa: the cell wall integrity/MAK-1 pathway and the MAK-2 (signal response) pathway. Previous studies have demonstrated several points of cross-talk between the MAK-1 and MAK-2 pathways, which is likely necessary for coordinating chemotropic growth toward an extracellular signal, and then mediating cell fusion. Canonical MAPK pathways begin with signal reception and end with a transcriptional response. Two transcription factors, ADV-1 and PP-1, are essential for communication and cell fusion. PP-1 is the conserved target of MAK-2, but it is unclear what targets ADV-1. We did RNA sequencing on Δadv-1, Δpp-1, and wild-type cells and found that ADV-1 and PP-1 have a shared regulon including many genes required for communication, cell fusion, growth, development, and stress response. We identified ADV-1 and PP-1 binding sites across the genome by adapting the in vitro method of DNA-affinity purification sequencing for N. crassa. To elucidate the regulatory network, we misexpressed each transcription factor in each upstream MAPK deletion mutant. Misexpression of adv-1 was sufficient to fully suppress the phenotype of the Δpp-1 mutant and partially suppress the phenotype of the Δmak-1 mutant. Collectively, our data demonstrate that the MAK-1/ADV-1 and MAK-2/PP-1 pathways form a tight regulatory network that maintains cell integrity and mediates communication and cell fusion.

Transcription factors (TFs) play a key role in regulating plant development and response to environmental stimuli. While most genes revert to single copy after a duplication event, transcription factors are retained at a significantly higher rate. However, it is unclear why TF duplicates have higher rates of retention relative to other genes. In this study, we compared three types of features (expression, sequence, and conservation) of retained TFs following whole genome duplication (WGD) events to genes with other functions, usingArabidopsis thalianaas a model. We found that gene function groups with higher maximum expression but lower mean expression tended to have higher duplicate retention rate post WGD, though TFs in particular are retained more often than would be expected based on the features examined. Conversely, expression of individual genes was not associated with duplication, but sequence conservation was. Furthermore, we found that the evolution of TF expression patterns and cis-regulatory cites favors the partitioning of ancestral states among the resulting duplicates. In particular, we found that one duplicate retains the majority of ancestral expression and cis-regulatory sites, while the “non-ancestral” duplicate was enriched for novel regulatory sites. To investigate how this pattern of partitioning pattern evolved, we modeled the retention of ancestral states in duplicate pairs using a system of differential equations. Our findings indicate that duplicate pairs evolve to a partitioned state more often than away from it, which in combination with accumulation of new regulatory sites in non-ancestral duplicates, suggest that selection favors partitioning via neofunctionalization.Author SummaryGene expression is controlled by regulatory proteins known as transcription factors. These factors control how an organism develops and responds to its environment. The evolution of transcription factor functions also contributes to the emergence of new species and crop domestication. In plants, new transcription factors mainly arise due to polyploidy, multiplication of the genome. Although most duplicated copies are lost following a genome duplication event, transcription factors are exceptional because they are often kept. Furthermore, we found that transcription factor duplicates that tend to diverge in how they are expressed and regulated in an unusual way where one copy mirrors the original, pre-duplication functional states of the ancestral gene, while the other loses the ancestral status and instead accumulates novel regulatory sites. Our results suggest these duplicate transcription factors may have been kept because one copy preserve ancestral function while the other has evolved new ones.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination.

SUMMARY Single-cell transcriptome profiling of heterogeneous tissues can provide high-resolution windows into developmental dynamics and environmental responses, but its application to plants has been limited. Here, we used the high-throughput Drop-seq approach to profile >12,000 cells from Arabidopsis roots. This identified numerous distinct cell types, covering all major root tissues and developmental stages, and illuminated specific marker genes for these populations. In addition, we demonstrate the utility of this approach to study the impact of environmental conditions on developmental processes. Analysis of roots grown with or without sucrose supplementation uncovers changes in the relative frequencies of cell types in response to sucrose. Finally, we characterize the transcriptome changes that occur across endodermis development and identify nearly 800 genes with dynamic expression as this tissue differentiates. Collectively, we demonstrate that single-cell RNA-seq can be used to profile developmental processes in plants and show how they can be altered by external stimuli., Graphical Abstract, In Brief The application of single-cell transcriptome profiling to plants has been limited. Shulse et al. performed Drop-seq on Arabidopsis roots, generating a transcriptional resource for >12,000 cells across major populations. This revealed marker genes for distinct cell types, cell frequency changes resulting from sucrose addition, and genes dynamically regulated during development.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6. Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors. In this report, we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that AFB4 and AFB5 are targets of the picloram family of auxinic herbicides in addition to indole-3-acetic acid. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

Genotype is the primary determinate of phenotype. During the past two decades, however, there has been an emergent recognition of the epigenotype, a separate layer of heredity distinct from the primary DNA sequence that can have profound effects on phenotype. The epigenotype is a collection of chemical modifications to the DNA and nucleosomes in conjunction with noncoding RNA transcripts, and together these epigenetic marks act as a potent and expansive regulatory system for controlling gene expression. In this review, we discuss our current understanding of variation in epigenotype in the model plant Arabidopsis and how allelic differences attributable to epigenetic changes, or epialleles, can affect phenotype. We discuss examples of epialleles that have been created in the laboratory and others that have been identified in natural populations, because these two models provide complementary information regarding the genetic pathways, mechanisms of transmission, and biological and evolutionary context for the role of the epigenotype in phenotypic variation.

The plant hormone auxin is perceived by a family of F-box proteins called the TIR1/AFBs. Phylogenetic studies reveal that these proteins fall into four clades in flowering plants called TIR1, AFB2, AFB4, and AFB6 (Parry et al. 2009). Genetic studies indicate that members of the TIR1 and AFB2 groups act as positive regulators of auxin signaling by promoting the degradation of the Aux/IAA transcriptional repressors (Dharmasiri et al. 2005; Parry et al. 2009). In this report we demonstrate that both AFB4 and AFB5 also function as auxin receptors based on in vitro assays. We also provide genetic evidence that both AFB4 and AFB5 are targets of the picloram family of auxinic herbicides. In contrast to previous studies we find that null afb4 alleles do not exhibit obvious defects in seedling morphology or auxin hypersensitivity. We conclude that AFB4 and AFB5 act in a similar fashion to other members of the family but exhibit a distinct auxin specificity.

Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.

SummaryDeciphering the multiple layers of epigenetic regulation that control transcription is critical to understanding how plants develop and respond to their environment. Using sequencing-by-synthesis technology we directly sequenced the cytosine methylome (methylC-seq), transcriptome (mRNA-seq), and small RNA transcriptome (smRNA-seq) to generate highly integrated epigenome maps for wild-type Arabidopsis thaliana and mutants defective in DNA methyltransferase or demethylase activity. At single-base resolution we discovered extensive, previously undetected DNA methylation, identified the context and level of methylation at each site, and observed local sequence effects upon methylation state. Deep sequencing of smRNAs revealed a direct relationship between the location of smRNAs and DNA methylation, perturbation of smRNA biogenesis upon loss of CpG DNA methylation, and a tendency for smRNAs to direct strand-specific DNA methylation in regions of RNA-DNA homology. Finally, strand-specific mRNA-seq revealed altered transcript abundance of hundreds of genes, transposons, and unannotated intergenic transcripts upon modification of the DNA methylation state.