Engineering topics

Lipzen, Anna

Publications and source records attributed to Lipzen, Anna.

At least 37 records · Page 2

DOE OSTI2024

Genome evolution and transcriptome plasticity is associated with adaptation to monocot and dicot plants in Colletotrichum fungi

Colletotrichum fungi infect a wide diversity of monocot and dicot hosts, causing diseases on almost all economically important plants worldwide. Colletotrichum is also a suitable model for studying gene family evolution on a fine scale to uncover events in the genome associated with biological changes. Here we present the genome sequences of 30 Colletotrichum species covering the diversity within the genus. Evolutionary analyses revealed that the Colletotrichum ancestor diverged in the late Cretaceous in parallel with the diversification of flowering plants. We provide evidence of independent host jumps from dicots to monocots during the evolution of Colletotrichum, coinciding with a progressive shrinking of the plant cell wall degradative arsenal and expansions in lineage-specific gene families. Comparative transcriptomics of 4 species adapted to different hosts revealed similarity in gene content but high diversity in the modulation of their transcription profiles on different plant substrates. Combining genomics and transcriptomics, we identified a set of core genes such as specific transcription factors, putatively involved in plant cell wall degradation. These results indicate that the ancestral Colletotrichum were associated with dicot plants and certain branches progressively adapted to different monocot hosts, reshaping the gene content and its regulation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Expression of dehydroshikimate dehydratase in poplar induces transcriptional and metabolic changes in the phenylpropanoid pathway

Abstract Modification of lignin in feedstocks via genetic engineering aims to reduce biomass recalcitrance to facilitate efficient conversion processes. These improvements can be achieved by expressing exogenous enzymes that interfere with native biosynthetic pathways responsible for the production of the lignin precursors. In planta expression of a bacterial 3-dehydroshikimate dehydratase in poplar trees reduced lignin content and altered the monomer composition, which enabled higher yields of sugars after cell wall polysaccharide hydrolysis. Understanding how plants respond to such genetic modifications at the transcriptional and metabolic levels is needed to facilitate further improvement and field deployment. In this work, we acquired fundamental knowledge on lignin-modified poplar expressing 3-dehydroshikimate dehydratase using RNA-seq and metabolomics. The data clearly demonstrate that changes in gene expression and metabolite abundance can occur in a strict spatiotemporal fashion, revealing tissue-specific responses in the xylem, phloem, or periderm. In the poplar line that exhibited the strongest reduction in lignin, we found that 3% of the transcripts had altered expression levels and ~19% of the detected metabolites had differential abundance in the xylem from older stems. The changes affected predominantly the shikimate and phenylpropanoid pathways as well as secondary cell wall metabolism, and resulted in significant accumulation of hydroxybenzoates derived from protocatechuate and salicylate.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

XA21-mediated resistance to Xanthomonas oryzae pv. oryzae is dose dependent

The rice receptor kinase XA21 confers broad-spectrum resistance to Xanthomonas oryzae pv. oryzae ( Xoo ), the causal agent of rice bacterial blight disease. To investigate the relationship between the expression level of XA21 and resulting resistance, we generated independent HA-XA21 transgenic rice lines accumulating the XA21 immune receptor fused with an HA epitope tag. Whole-genome sequence analysis identified the T-DNA insertion sites in sixteen independent T0 events. Further, through quantification of the HA-XA21 protein and assessment of the resistance to Xoo strain PXO99 in six independent transgenic lines, we observed that XA21-mediated resistance is dose dependent. In contrast, based on the four agronomic traits quantified in these experiments, yield is unlikely to be affected by the expression level of HA-XA21 . These findings extend our knowledge of XA21-mediated defense and contribute to the growing number of well-defined genomic landing pads in the rice genome that can be targeted for gene insertion without compromising yield.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Diverse fates of ancient horizontal gene transfers in extremophilic red algae

Abstract Horizontal genetic transfer (HGT) is a common phenomenon in eukaryotic genomes. However, the mechanisms by which HGT‐derived genes persist and integrate into other pathways remain unclear. This topic is of significant interest because, over time, the stressors that initially favoured the fixation of HGT may diminish or disappear. Despite this, the foreign genes may continue to exist if they become part of a broader stress response or other pathways. The conventional model suggests that the acquisition of HGT equates to adaptation. However, this model may evolve into more complex interactions between gene products, a concept we refer to as the ‘Integrated HGT Model’ (IHM). To explore this concept further, we studied specialized HGT‐derived genes that encode heavy metal detoxification functions. The recruitment of these genes into other pathways could provide clear examples of IHM. In our study, we exposed two anciently diverged species of polyextremophilic red algae from the Galdieria genus to arsenic and mercury stress in laboratory cultures. We then analysed the transcriptome data using differential and coexpression analysis. Our findings revealed that mercury detoxification follows a ‘one gene‐one function’ model, resulting in an indivisible response. In contrast, the ars H gene in the arsenite response pathway demonstrated a complex pattern of duplication, divergence and potential neofunctionalization, consistent with the IHM. Our research sheds light on the fate and integration of ancient HGTs, providing a novel perspective on the ecology of extremophiles.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Genome-scale model development and genomic sequencing of the oleaginous clade Lipomyces

The Lipomyces clade contains oleaginous yeast species with advantageous metabolic features for biochemical and biofuel production. Limited knowledge about the metabolic networks of the species and limited tools for genetic engineering have led to a relatively small amount of research on the microbes. Here, a genome-scale metabolic model (GSM) of Lipomyces starkeyi NRRL Y-11557 was built using orthologous protein mappings to model yeast species. Phenotypic growth assays were used to validate the GSM (66% accuracy) and indicated that NRRL Y-11557 utilized diverse carbohydrates but had more limited catabolism of organic acids. The final GSM contained 2,193 reactions, 1,909 metabolites, and 996 genes and was thus named iLst996. The model contained 96 of the annotated carbohydrate-active enzymes. iLst996 predicted a flux distribution in line with oleaginous yeast measurements and was utilized to predict theoretical lipid yields. Twenty-five other yeasts in the Lipomyces clade were then genome sequenced and annotated. Sixteen of the Lipomyces species had orthologs for more than 97% of the iLst996 genes, demonstrating the usefulness of iLst996 as a broad GSM for Lipomyces metabolism. Pathways that diverged from iLst996 mainly revolved around alternate carbon metabolism, with ortholog groups excluding NRRL Y-11557 annotated to be involved in transport, glycerolipid, and starch metabolism, among others. Overall, this study provides a useful modeling tool and data for analyzing and understanding Lipomyces species metabolism and will assist further engineering efforts in Lipomyces .

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Draft genome sequencing and assembly of Favolaschia claudopus CIRM-BRFM 2984 isolated from oak limbs

Favolaschia claudopus, a wood-inhabiting basidiomycete of the Mycenaceae family, is considered an invasive species that has recently spread from Oceania to Europe. The CIRM-BRFM 2984 strain of this fungus was originally isolated from a basidiome collected from the fallen limb of a decayed oak tree in Southwest France. The genome sequence of this strain shared characteristics with other Mycenaceae species, including a large genome size and enriched content of protein-coding genes. The genome sequence provided here will facilitate further investigation on the factors that contribute to the successful global dissemination of F. claudopus.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

A large sequenced mutant library – valuable reverse genetic resource that covers 98% of sorghum genes

SUMMARY Mutant populations are crucial for functional genomics and discovering novel traits for crop breeding. Sorghum , a drought and heat‐tolerant C4 species, requires a vast, large‐scale, annotated, and sequenced mutant resource to enhance crop improvement through functional genomics research. Here, we report a sorghum large‐scale sequenced mutant population with 9.5 million ethyl methane sulfonate (EMS)‐induced mutations that covered 98% of sorghum's annotated genes using inbred line BTx623. Remarkably, a total of 610 320 mutations within the promoter and enhancer regions of 18 000 and 11 790 genes, respectively, can be leveraged for novel research of cis ‐regulatory elements. A comparison of the distribution of mutations in the large‐scale mutant library and sorghum association panel (SAP) provides insights into the influence of selection. EMS‐induced mutations appeared to be random across different regions of the genome without significant enrichment in different sections of a gene, including the 5′ UTR, gene body, and 3′‐UTR. In contrast, there were low variation density in the coding and UTR regions in the SAP. Based on the K a / K s value, the mutant library (~1) experienced little selection, unlike the SAP (0.40), which has been strongly selected through breeding. All mutation data are publicly searchable through SorbMutDB ( https://www.depts.ttu.edu/igcast/sorbmutdb.php ) and SorghumBase ( https://sorghumbase.org/ ). This current large‐scale sequence‐indexed sorghum mutant population is a crucial resource that enriched the sorghum gene pool with novel diversity and a highly valuable tool for the Poaceae family, that will advance plant biology research and crop breeding.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Altered Expression of Two Small Secreted Proteins (ssp4 and ssp6) Affects the Degradation of a Natural Lignocellulosic Substrate by Pleurotus ostreatus

Pleurotus ostreatus is a white-rot fungus that can degrade lignin in a preferential manner using a variety of extracellular enzymes, including manganese and versatile peroxidases (encoded by the vp1-3 and mnp1-6 genes, respectively). This fungus also secretes a family of structurally related small secreted proteins (SSPs) encoded by the ssp1-6 genes. Using RNA sequencing (RNA-seq), we determined that ssp4 and ssp6 are the predominant members of this gene family that were expressed by P. ostreatus during the first three weeks of growth on wheat straw. Downregulation of ssp4 in a strain harboring an ssp RNAi construct (KDssp1) was then confirmed, which, along with an increase in ssp6 transcript levels, coincided with reduced lignin degradation and the downregulation of vp2 and mnp1. In contrast, we observed an increase in the expression of genes related to pectin and side-chain hemicellulose degradation, which was accompanied by an increase in extracellular pectin-degrading capacity. Genome-wide comparisons between the KDssp1 and the wild-type strains demonstrated that ssp silencing conferred accumulated changes in gene expression at the advanced cultivation stages in an adaptive rather than an inductive mode of transcriptional response. Based on co-expression networking, crucial gene modules were identified and linked to the ssp knockdown genotype at different cultivation times. Based on these data, as well as previous studies, we propose that P. ostreatus SSPs have potential roles in modulating the lignocellulolytic and pectinolytic systems, as well as a variety of fundamental biological processes related to fungal growth and development.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Near-complete genome sequence of Lipomyces tetrasporous NRRL Y-64009, an oleaginous yeast capable of growing on lignocellulosic hydrolysates

ABSTRACT Lipomyces tetrasporous is an oleaginous yeast that can utilize a variety of plant-based sugars. It accumulates lipids during growth on lignocellulosic biomass hydrolysates. We present the annotated genome sequence of L. tetrasporous NRRL Y-64009 to aid in its development as a platform organism for producing lipids and lipid-based bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Genome-scale phylogeny and comparative genomics of the fungal order Sordariales

The order Sordariales is taxonomically diverse, and harbours many species with different lifestyles and large economic importance. Despite its importance, a robust genome-scale phylogeny, and associated comparative genomic analysis of the order is lacking. In this study, we examined whole-genome data from 99 Sordariales, including 52 newly sequenced genomes, and seven outgroup taxa. We inferred a comprehensive phylogeny that resolved several contentious relationships amongst families in the order, and cleared-up intrafamily relationships within the Podosporaceae. Extensive comparative genomics showed that genomes from the three largest families in the dataset (Chaetomiaceae, Podosporaceae and Sordariaceae) differ greatly in GC content, genome size, gene number, repeat percentage, evolutionary rate, and genome content affected by repeat-induced point mutations (RIP). All genomic traits showed phylogenetic signal, and ancestral state reconstruction revealed that the variation of the properties stems primarily from within-family evolution. Together, the results provide a thorough framework for understanding genome evolution in this important group of fungi.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

IMA genome-F18

Sequencing fungal genomes has now become very common and the list of genomes in this manuscript reflects this. Particularly relevant is that the first announcement is a re-identification of Penicillium genomes available on NCBI. The fact that more than 100 of these genomes have been deposited without the correct species names speak volumes to the fact that we must continue training fungal taxonomists and the importance of the International Mycological Association (after which this journal is named). When we started the genome series in 2013, one of the essential aspects was the need to have a phylogenetic tree as part of the manuscript. This came about as the result of a discussion with colleagues in NCBI who were trying to deal with the very many incorrectly identified bacterial genomes (at the time) which had been submitted to NCBI. We are now in the same position with fungal genomes. Sequencing a fungal genome is all too easy but providing a correct species name and ensuring that the fungus has in fact been correctly identified seems to be more difficult. We know that there are thousands of fungi which have not yet been described. The availability of sequence data has made identification of fungi easier but also serves to highlight the need to have a fungal taxonomist in the project to make sure that mistakes are not made.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Convergent reductive evolution and host adaptation in Mycoavidus bacterial endosymbionts of Mortierellaceae fungi

Intimate associations between fungi and intracellular bacterial endosymbionts are becoming increasingly well understood. Phylogenetic analyses demonstrate that bacterial endosymbionts of Mucoromycota fungi are related either to free-living Burkholderia or Mollicutes species. The so-called Burkholderia-related endosymbionts or BRE comprise Mycoavidus, Mycetohabitans and Candidatus Glomeribacter gigasporarum. These endosymbionts are marked by genome contraction thought to be associated with intracellular selection. However, the conclusions drawn thus far are based on a very small subset of endosymbiont genomes, and the mechanisms leading to genome streamlining are not well understood. The purpose of this study was to better understand how intracellular existence shapes Mycoavidus and BRE functionally at the genome level. To this end we generated and analyzed 14 novel draft genomes for Mycoavidus living within the hyphae of Mortierellomycotina fungi. We found that our novel Mycoavidus genomes were significantly reduced compared to free-living Burkholderiales relatives. Using a genome-scale phylogenetic approach including the novel and available existing genomes of Mycoavidus, we show that the genus is an assemblage composed of two independently derived lineages including three well supported clades of Mycoavidus. Using a comparative genomic approach, we shed light on the functional implications of genome reduction, documenting shared and unique gene loss patterns between the three Mycoavidus clades. We found that many endosymbiont isolates demonstrate patterns of vertical transmission and host-specificity, but others are present in phylogenetically disparate hosts. We discuss how reductive evolution and host specificity reflect convergent adaptation to the intrahyphal selective landscape, and commonalities of eukaryotic endosymbiont genome evolution.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

A scaffolded and annotated reference genome of giant kelp (Macrocystis pyrifera)

Abstract Macrocystis pyrifera (giant kelp), is a brown macroalga of great ecological importance as a primary producer and structure-forming foundational species that provides habitat for hundreds of species. It has many commercial uses (e.g. source of alginate, fertilizer, cosmetics, feedstock). One of the limitations to exploiting giant kelp’s economic potential and assisting in giant kelp conservation efforts is a lack of genomic tools like a high quality, contiguous reference genome with accurate gene annotations. Reference genomes attempt to capture the complete genomic sequence of an individual or species, and importantly provide a universal structure for comparison across a multitude of genetic experiments, both within and between species. We assembled the giant kelp genome of a haploid female gametophyte de novo using PacBio reads, then ordered contigs into chromosome level scaffolds using Hi-C. We found the giant kelp genome to be 537 MB, with a total of 35 scaffolds and 188 contigs. The assembly N50 is 13,669,674 with GC content of 50.37%. We assessed the genome completeness using BUSCO, and found giant kelp contained 94% of the BUSCO genes from the stramenopile clade. Annotation of the giant kelp genome revealed 25,919 genes. Additionally, we present genetic variation data based on 48 diploid giant kelp sporophytes from three different Southern California populations that confirms the population structure found in other studies of these populations. This work resulted in a high-quality giant kelp genome that greatly increases the genetic knowledge of this ecologically and economically vital species.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

Comparative Genomics and Transcriptomics Analyses Reveal Divergent Plant Biomass-Degrading Strategies in Fungi

Plant biomass is one of the most abundant renewable carbon sources, which holds great potential for replacing current fossil-based production of fuels and chemicals. In nature, fungi can efficiently degrade plant polysaccharides by secreting a broad range of carbohydrate-active enzymes (CAZymes), such as cellulases, hemicellulases, and pectinases. Due to the crucial role of plant biomass-degrading (PBD) CAZymes in fungal growth and related biotechnology applications, investigation of their genomic diversity and transcriptional dynamics has attracted increasing attention. In this project, we systematically compared the genome content of PBD CAZymes in six taxonomically distant species, Aspergillus niger, Aspergillus nidulans, Penicillium subrubescens, Trichoderma reesei, Phanerochaete chrysosporium, and Dichomitus squalens, as well as their transcriptome profiles during growth on nine monosaccharides. Considerable genomic variation and remarkable transcriptomic diversity of CAZymes were identified, implying the preferred carbon source of these fungi and their different methods of transcription regulation. In addition, the specific carbon utilization ability inferred from genomics and transcriptomics was compared with fungal growth profiles on corresponding sugars, to improve our understanding of the conversion process. This study enhances our understanding of genomic and transcriptomic diversity of fungal plant polysaccharide-degrading enzymes and provides new insights into designing enzyme mixtures and metabolic engineering of fungi for related industrial applications.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Vertical and horizontal gene transfer shaped plant colonization and biomass degradation in the fungal genus Armillaria

The fungal genus Armillaria contains necrotrophic pathogens and some of the largest terrestrial organisms that cause tremendous losses in diverse ecosystems, yet how they evolved pathogenicity in a clade of dominantly non-pathogenic wood degraders remains elusive. Here we show that Armillaria species, in addition to gene duplications and de novo gene origins, acquired at least 1,025 genes via 124 horizontal gene transfer events, primarily from Ascomycota. Horizontal gene transfer might have affected plant biomass degrading and virulence abilities of Armillaria, and provides an explanation for their unusual, soft rot-like wood decay strategy. Furthermore, combined multi-species expression data revealed extensive regulation of horizontally acquired and wood-decay related genes, putative virulence factors and two novel conserved pathogenicity-induced small secreted proteins, which induced necrosis in planta. Overall, this study details how evolution knitted together horizontally and vertically inherited genes in complex adaptive traits of plant biomass degradation and pathogenicity in important fungal pathogens.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2023

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom

Abstract Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated. Here we describe the Joint Genome Institute (JGI) Plant Gene Atlas, an updateable data resource consisting of transcript abundance assays spanning 18 diverse species. To integrate across these diverse genotypes, we analyzed expression profiles, built gene clusters that exhibited tissue/condition specific expression, and tested for transcriptional response to environmental queues. We discovered extensive phylogenetically constrained and condition-specific expression profiles for genes without any previously documented functional annotation. Such conserved expression patterns and tightly co-expressed gene clusters let us assign expression derived additional biological information to 64 495 genes with otherwise unknown functions. The ever-expanding Gene Atlas resource is available at JGI Plant Gene Atlas (https://plantgeneatlas.jgi.doe.gov) and Phytozome (https://phytozome.jgi.doe.gov/), providing bulk access to data and user-specified queries of gene sets. Combined, these web interfaces let users access differentially expressed genes, track orthologs across the Gene Atlas plants, graphically represent co-expressed genes, and visualize gene ontology and pathway enrichments.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Expression quantitative trait loci mapping identified PtrXB38 as a key hub gene in adventitious root development in Populus

Summary Plant establishment requires the formation and development of an extensive root system with architecture modulated by complex genetic networks. Here, we report the identification of the PtrXB38 gene as an expression quantitative trait loci (eQTL) hotspot, mapped using 390 leaf and 444 xylem Populus trichocarpa transcriptomes. Among predicted targets of this trans ‐eQTL were genes involved in plant hormone responses and root development. Overexpression of PtrXB38 in Populus led to significant increases in callusing and formation of both stem‐born roots and base‐born adventitious roots. Omics studies revealed that genes and proteins controlling auxin transport and signaling were involved in PtrXB38‐mediated adventitious root formation. Protein–protein interaction assays indicated that PtrXB38 interacts with components of endosomal sorting complexes required for transport machinery, implying that PtrXB38‐regulated root development may be mediated by regulating endocytosis pathway. Taken together, this work identified a crucial root development regulator and sheds light on the discovery of other plant developmental regulators through combining eQTL mapping and omics approaches.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2023

Characterization of a novel polyextremotolerant fungus, Exophiala viscosa , with insights into its melanin regulation and ecological niche

Black yeasts are polyextremotolerant fungi that contain high amounts of melanin in their cell wall and maintain a primary yeast form. These fungi grow in xeric, nutrient depletes environments which implies that they require highly flexible metabolisms and have been suggested to contain the ability to form lichen-like mutualisms with nearby algae and bacteria. However, the exact ecological niche and interactions between these fungi and their surrounding community are not well understood. We have isolated 2 novel black yeasts from the genus Exophiala that were recovered from dryland biological soil crusts. Despite notable differences in colony and cellular morphology, both fungi appear to be members of the same species, which has been named Exophiala viscosa (i.e. E. viscosa JF 03-3 Goopy and E. viscosa JF 03-4F Slimy). A combination of whole genome sequencing, phenotypic experiments, and melanin regulation experiments have been performed on these isolates to fully characterize these fungi and help decipher their fundamental niche within the biological soil crust consortium. Our results reveal that E. viscosa is capable of utilizing a wide variety of carbon and nitrogen sources potentially derived from symbiotic microbes, can withstand many forms of abiotic stresses, and excretes melanin which can potentially provide ultraviolet resistance to the biological soil crust community. Besides the identification of a novel species within the genus Exophiala, our study also provides new insight into the regulation of melanin production in polyextremotolerant fungi.

60 APPLIED LIFE SCIENCES↗