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Abstract BackgroundGenetic and epigenetic perturbation of cis-regulatory sequences can shift patterns of gene expression and result in novel phenotypes. Phased genome assemblies now enable the local dissection of linkages between cis-regulatory sequences, including their epigenetic state, and allele-specific gene expression to further characterize gene regulation and resulting phenotypes in heterozygous genomes. ResultsWe assembled a locally phased genome for a mandarin hybrid named ‘Fairchild’ to explore the molecular signatures of allele-specific gene expression. With local genome phasing, genes with allele-specific expression were paired with haplotype-specific chromatin states, including levels of chromatin accessibility, histone modifications, and DNA methylation. We found that 30% of variation in allele-specific expression could be attributed to haplotype associated factors, with allelic levels of chromatin accessibility and three histone modifications in gene bodies having the most influence. Structural variants in promoter regions were also associated with allele-specific expression, including specific enrichments of hAT and MULE-MuDR DNA transposon sequences. Integration of haplotype-resolved genetic and epigenetic landscapes with high-throughput phenotypic analysis of fruit traits in a panel of 154 accessions with mandarin and pummelo ancestry revealed that trait-associated variants were enriched in regions of open chromatin. Mining of trait-associated variants uncovered a Gypsy retrotransposon insertion in a gene that regulates potassium transport and may contribute to the reduction in fruit size that is observed in mandarins. Conclusions​​Using a locally phased assembly of a heterozygous cultivar of citrus, we dissected the interplay between genetic variants and molecular phenotypes to reveal cis-regulatory sequences with potential functional effects on phenotypes relevant for genetic improvement. 

Abstract MotivationSingle-cell Hi-C (scHi-C) technologies have significantly advanced our understanding of the 3D genome organization. However, scHi-C data are often sparse and noisy, leading to substantial computational challenges in downstream analyses. ResultsIn this study, we introduce SHICEDO, a novel deep-learning model specifically designed to enhance scHi-C contact matrices by imputing missing or sparsely captured chromatin contacts through a generative adversarial framework. SHICEDO leverages the unique structural characteristics of scHi-C matrices to derive customized features that enable effective data enhancement. Additionally, the model incorporates a channel-wise attention mechanism to mitigate the over-smoothing issue commonly associated with scHi-C enhancement methods. Through simulations and real-data applications, we demonstrate that SHICEDO outperforms the state-of-the-art methods, achieving superior quantitative and qualitative results. Moreover, SHICEDO enhances key structural features in scHi-C data, thus enabling more precise delineation of chromatin structures such as A/B compartments, TAD-like domains, and chromatin loops. Availability and implementationSHICEDO is publicly available at https://github.com/wmalab/SHICEDO. 

ABSTRACT The fungusAspergillus melleusis an important biosynthesis host for varied commercial applications. Gene annotation of a previously published genome produced 12,841 protein-coding genes and identified 102 biosynthetic gene clusters. 

ABSTRACT Infections caused by the emerging pathogenic yeastClavispora (Candida) lusitaniaecan be difficult to manage due to multi-drug resistance. Resistance to the frontline antifungal fluconazole (FLZ) inCandidaspp. is commonly acquired through gain-of-function (GOF) mutations in the gene encoding the transcription factor Mrr1. These activated Mrr1 variants enhance FLZ efflux via upregulation of the multi-drug transporter geneMDR1. Recently, it was reported that, unlike in the well-studiedCandida albicansspecies,C. lusitaniaeandCandida parapsilosiswith activated Mrr1 also have high expression ofCDR1, which encodes another multi-drug transporter with overlapping but distinct transported substrate profiles and Cdr1-dependent FLZ resistance. To better understand the mechanisms of Mrr1 regulation ofMDR1andCDR1, and other co-regulated genes, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analysis of Mrr1 binding sites. Mrr1 bound the promoter regions ofMDR1andCDR1, as well asFLU1, which encodes another transporter capable of FLZ efflux. Mdr1 and Cdr1 independently contributed to the decreased susceptibility of theMRR1^GOFstrains against diverse clinical azoles and other antifungals, including 5-flucytosine. A consensus motif, CGGAGWTAR, enriched in Mrr1-boundC. lusitaniaeDNA was also conserved upstream ofMDR1andCDR1across species, includingC. albicans. CUT&RUN and RNA-seq data were used to define the Mrr1 regulon, which includes genes involved in transport, stress response, and metabolism. Activated and inducible Mrr1 bound similar regions in the promoters of Mrr1 regulon genes. Our studies provide new evolutionary insights into the coordinated regulation of multi-drug transporters and potential mechanism(s) that aid secondary resistance acquisition in emergingCandida. IMPORTANCEUnderstanding antifungal resistance in emergingCandidapathogens is essential to managing treatment failures and guiding the development of new therapeutic strategies. Like otherCandidaspecies, the environmental opportunistic fungal pathogenClavispora(Candida)lusitaniaecan acquire resistance to the antifungal fluconazole by overexpression of the multi-drug efflux pump Mdr1 through gain-of-function (GOF) mutations in the gene encoding the transcription factor Mrr1. Here, we show thatC. lusitaniaeMrr1 also directly regulatesCDR1, another major multi-drug transporter gene, along withMDR1. In strains with activated Mrr1, upregulation ofMDR1andCDR1protects against diverse antifungals, potentially aiding the rise of other resistance mutations. Mrr1 also regulates several stress response and metabolism genes, thereby providing new perspectives into the physiology of drug-resistant strains. The identification of an Mrr1 binding motif that is conserved across strains and species will advance future efforts to understand multi-drug resistance acrossCandidaspecies. 

ABSTRACT The fungusConoideocrella luteorostratais a recently discovered pathogen of invasive elongate hemlock scale insects (EHS;Fiorinia externa) in Christmas tree farms in the eastern U.S. Here, we report a scaffold-level genome and assembly along with an initial survey of biosynthetic gene clusters for strain ARSEF 14590 from EHS. 

Abstract Genomic clusters of immune genes, including those encoding nucleotide-binding leucine-rich repeat (NLR) proteins, are a model for exploring the dynamics of genomic regions in flux. Rapid sequence evolution of immune genes, including NLRs, and variation in their gene content, may enable long-lived plants, which lack adaptive immune systems, to keep pace with the fast evolution of pathogens. To explore the patterns and processes shaping the evolution of NLR gene content in a genus of long-lived tree species, we unified the annotation of NLR genes across 11 accessions (or 15 haplotypes) from the genusCitrusand its relatives, including three new diploid genome assemblies. A majority of NLRs were arranged in genomic clusters composed of paralogous genes, typically from a single gene family. Even larger clusters, with 10 or more NLRs, were limited to genes derived from one or few gene families. These patterns suggested that genomic clustering of NLRs arose through local expansion of phylogenetically related NLRs, but the mechanistic processes driving these patterns are not clear. Local gene duplication can be mediated by multiple processes, including transposon-mediated gene capture and subsequent proliferation, and non-allelic repair of double stranded breaks, including unequal recombination. Examples of retrotransposon-mediated duplication of NLRs were identified, but these were not sufficient to explain massive regional expansions. Signatures of unequal recombination are challenging to identify. Focusing on recent lineage-specific sequence duplications, at least one case of unequal recombination was identified, supporting a role for unequal recombination in shaping genomic variation in these regions. 

Abstract Reactivation of toxoplasmosis is a significant health threat to chronically infected individuals, especially those who are or become immunocompromised. An estimated one-third of the world population is infected withToxoplasma, placing millions at risk. TheToxoplasmacyst is the foundation of disease with its ingestion leading to infection and its reactivation, from slow replicating bradyzoites to fast replicating tachyzoites, leading to cell lysis in tissues such as the brain. There are no treatments that prevent or eliminate cysts in part due to our poor understanding of the mechanisms that underlie cyst formation and recrudescence. In this study, we aimed to understand the biology of bradyzoites prior to recrudescence and the developmental pathways they initiate. We have discovered ME49EW cysts from infected mice harbor multiple bradyzoite subtypes that can be identified by their expression of distinct proteins. Sorting of these subtypes revealed they initiate distinct developmental pathways in animals and in primary astrocyte cell cultures. Single bradyzoite RNA sequencing indicates 5 major bradyzoite subtypes occur within these cysts. We further show that a crucial subtype comprising the majority of bradyzoites in chronically infected mice is absent from conventional in vitro models of bradyzoite development. Altogether this work establishes new foundational principles ofToxoplasmacyst development and reactivation that operate during the intermediate life cycle ofToxoplasma. 

ABSTRACT Fungal infections are difficult to prevent and treat in large part due to strain heterogeneity, which confounds diagnostic predictability. Yet, the genetic mechanisms driving strain-to-strain variation remain poorly understood. Here, we determined the extent to whichStarships—giant transposons capable of mobilizing numerous fungal genes—generate genetic and phenotypic variability in the opportunistic human pathogenAspergillus fumigatus. We analyzed 519 diverse strains, including 11 newly sequenced with long-read technology and multiple isolates of the same reference strain, to reveal 20 distinctStarshipsthat are generating genomic heterogeneity over timescales relevant for experimental reproducibility.Starship-mobilized genes encode diverse functions, including known biofilm-related virulence factors and biosynthetic gene clusters, and many are differentially expressed during infection and antifungal exposure in a strain-specific manner. These findings support a new model of fungal evolution whereinStarshipshelp generate variation in genome structure, gene content, and expression among fungal strains. Together, our results demonstrate thatStarshipsare a previously hidden mechanism generating genotypic and, in turn, phenotypic heterogeneity in a major human fungal pathogen.IMPORTANCENo “one size fits all” option exists for treating fungal infections in large part due to genetic and phenotypic variability among strains. Accounting for strain heterogeneity is thus fundamental for developing efficacious treatments and strategies for safeguarding human health. Here, we report significant progress toward achieving this goal by uncovering a previously hidden mechanism generating heterogeneity in the human fungal pathogenAspergillus fumigatus: giant transposons, calledStarships, that span dozens of kilobases and mobilize fungal genes as cargo. By conducting a systematic investigation of these unusual transposons in a single fungal species, we demonstrate their contributions to population-level variation at the genome, pangenome, and transcriptome levels. TheStarshipcompendium we develop will not only help predict variation introduced by these elements in laboratory experiments but will serve as a foundational resource for determining howStarshipsimpact clinically relevant phenotypes, such as antifungal resistance and pathogenicity. 

ABSTRACT Astrocytes provide physical and metabolic support for neurons, regulate the blood–brain barrier, and react to injury, infection, and disease. When astrocytes become reactive, maintenance of the inflammatory state and its functional implications throughout chronic neuroinflammation are all poorly understood. Several models of acute inflammation have revealed astrocyte subpopulations that go beyond a two‐activation state model, instead encompassing distinct functional subsets. However, how reactive astrocyte (RA) subsets evolve over time during chronic inflammatory disease or infection has been difficult to address. Here we use a prolific human pathogen,Toxoplasma gondii, that causes lifelong infection in the brain alongside aLcn2CreERT2reporter mouse line to examine reactive astrocyte subsets during chronic neuroinflammation. Single‐cell RNA sequencing revealed diverse astrocyte populations including transcriptionally uniqueLcn2CreERT2+ RAs which change over the course of infection in a subset‐dependent manner. In addition to an immune‐regulatingLcn2CreERT2+ astrocyte population enriched with gene transcripts encoding chemokines CCL5, CXCL9, CXCL10, and receptors CCR7 and IL7R, a specific subset ofLcn2CreERT2+ astrocytes highly expressedtransthyretin(Ttr), a secreted carrier protein involved in glycolytic enzyme activation and potential vasculature regulation and angiogenesis. These findings provide novel information about the evolution and diversity of reactive astrocyte subtypes and functional signatures at different stages of infection, revealing an undocumented role for transthyretin‐expressing astrocytes in immune regulation at the central nervous system (CNS) vasculature. 

Abstract The Global Panzootic Lineage (GPL) of Batrachochytrium dendrobatidis (Bd) has been described as a main driver of amphibian extinctions. Pathogen studies have benefited from three Bd-GPL strain genomes, but identifying the genetic and molecular features that distinguish the B. dendrobatidis lineages requires additional high-quality genomes from diverse lineages. We sequenced and assembled genomes with Oxford Nanopore Technologies to produce assemblies of three Bd-BRAZIL isolates and one nonpathogen outgroup species Polyrhizophydium stewartii. The Bd-BRAZIL assembly sizes ranged between 22.0 and 26.1 Mb with 8,495 to 8,620 predicted protein-coding genes. We sought to categorize the pangenome of the species by identifying homologous genes across the sampled genomes as either being core and present in all strains, or accessory and shared among strains in a lineage, an analysis that has not yet been conducted on B. dendrobatidis and its lineages. We identified a core genome consisting of 6,278 gene families, and an accessory genome of 202 Bd-BRAZIL and 172 Bd-GPL specific gene families. We discovered copy number differences in pathogenicity gene families: M36 Peptidases, Crinkler Necrosis genes, Aspartyl Peptidases, Carbohydrate-Binding Module-18 genes, and S41 Proteases, between Bd-BRAZIL and Bd-GPL strains. Comparison of B. dendrobatidis and two closely related saprophytic species identified differences in protein sequence and domain counts for M36 and CBM18 families respectively. Our pangenome analysis of lineage-specific gene content led us to explore how the selection of the reference genome affects recovery of RNAseq transcripts when comparing different strains. We tested the hypothesis that genomic variation among Bd-GPL and Bd-BRAZIL lineages can impact transcript count data by comparing results with our new Bd-BRAZIL genomes as the reference genomes. Our analysis examines the genomic variation between strains in Bd-BRAZIL and Bd-GPL and offers insights into the application of these high-quality reference genomes resources for future studies.