PLoS Genetics最新文献

Non-crossover gene conversion is a type of meiotic recombination characterized by the non-reciprocal transfer of genetic material between homologous chromosomes. Gene conversions are thought to occur within relatively short tracts of DNA. In this study, we propose a statistical method to model the length distribution of gene conversion tracts in humans, using nearly one million gene conversion tracts detected from the UK Biobank whole autosome data. To handle the large number of tracts, we designed a computationally efficient inferential framework. Our method further accounts for regional variation in the density of variant sites and heterozygosity across the genome, which can influence the observed length of gene conversion tracts. We allow for multiple candidate tract length distributions and select the best fitting distribution using the Bayesian Information Criterion (BIC). Using a mixture of two geometric components for the tract length distribution, we estimate that the smaller component has a mean of 16.9 bp (95% CI: [16.4, 17.0]), and the larger component has a mean of 724.7 bp (95% CI: [720.1, 728.7]). We further estimate the proportion of tracts from the second component to be 0.00525 (95% CI: [0.005, 0.00525]). After stratifying by crossover-hotspot overlap, we infer that tracts whose midpoints lie within crossover hotspots are, on average, longer than the remaining tracts.

During meiosis, the programmed formation of DNA double-strand breaks (DSBs) by Spo11, a conserved topoisomerase VI family protein, initiates homologous recombination that leads to crossovers between homologous chromosomes, essential for accurate chromosome segregation and genome evolution. The DSB number, distribution and timing of formation are regulated during meiosis to ensure crossing over on all chromosomes and prevent genome instability. In S. cerevisiae, DSB interference suppresses the coincident formation of DSBs in neighboring hotspots through a Tel1/ATM dependent mechanism that remains unexplored. Here, we demonstrate that Tel1 is recruited to meiotic DSB hotspots and chromosomal axis sites in a DSB-dependent manner. This supports the tethered loop-axis complex (TLAC) model that postulates meiotic DSBs are formed within the chromosome axis environment. Tel1 recruitment to meiotic DSBs, DSB interference and the meiotic DNA damage checkpoint are all dependent on the C-terminal moiety of Xrs2, known to mediate Tel1-Xrs2 interaction in vegetative cells. However, mutation of the Xrs2 FxF/Y motif, known to stabilize Tel1 interaction with Xrs2, does not affect DSBs interference but abolishes the Tel1-dependent DNA damage checkpoint. Altogether, this work uncovers the dynamic association of Tel1 with meiotic chromosomes and highlights the critical role of its interaction with Xrs2 in fine-tuning both the meiotic DNA damage checkpoint and DSB interference.

The CRISPR/Cas9 gene-editing system is a powerful tool in plant genetic engineering; however, screening for Cas9-free edited plants remains complex and time-consuming. To address this limitation, we developed an RNA aptamer-assisted CRISPR/Cas9 system, termed 3WJ-4 × Bro/Cas9. In this system, the engineered RNA aptamer 3WJ-4 × Bro functions as a transcriptional reporter, serving as an alternative to traditional fluorescent proteins and thus avoiding their potential interference with Cas9 activity. Compared to the conventional GFP/Cas9 system, 3WJ-4 × Bro/Cas9 showed more efficient transformation and higher accuracy in fluorescence-based selection of positive T1 transformants, without significantly affecting plant growth. Furthermore, 3WJ-4 × Bro/Cas9 achieved a 78.6% increase in the T1 mutation rate compared to GFP/Cas9, with the homozygous mutation rate reaching 1.78%. In addition, 3WJ-4 × Bro/Cas9 enabled fluorescence-based visual screening in the T2 generation for rapid identification of Cas9-free mutants, improving sorting efficiency by 30.2% over the GFP-based method. Moreover, 3WJ-4 × Bro/Cas9 enabled more efficient generation of homozygous double-target mutants compared to GFP/Cas9. These results demonstrate that the 3WJ-4 × Bro/Cas9 system provides a non-transgenic, efficient, and broadly applicable strategy for plant genome editing and selection.

Obligate host-associated bacteria with reduced genomes, such as phytoplasmas, face strong evolutionary constraints, including metabolic dependence on hosts, limited opportunities for horizontal gene transfer (HGT), and frequent population bottlenecks. Despite these limitations, phytoplasmas, which are parasitic, insect-transmitted plant pathogens, maintain a diverse arsenal of secreted effectors that manipulate both plant and insect hosts to promote infection and transmission. These effectors can suppress immunity and reprogram plant development, inducing alterations such as witch's broom and leaf-like flowers, through ubiquitin-independent degradation of key transcription factors. However, how phytoplasmas diversify and maintain these effectors in the absence of frequent genetic exchange remains unclear. To address this, we analysed the effectoromes of 239 phytoplasma genomes and identified a diverse set of secreted proteins, which we designated as putative Phytoplasma Effectors (PhAMEs). We found that PhAMEs targeting evolutionarily conserved and structurally constrained surfaces of host proteins are widespread across phytoplasmas. These effectors adopt compact, efficient folds. They often function as molecular scaffolds with dual interaction surfaces capable of linking host proteins or integrating signalling pathways. Such scaffolding PhAMEs have evolved multiple times independently, providing clear evidence of convergent evolution. Despite severe genomic constrains imposed by genome reduction and limited HGT, gene duplications, interface variations, domain fusions, and repeat expansions have helped the shaping effector fold and diversity. While the overall effector repertoire of phytoplasmas appeared largely unique, some PhAME domains share similarities with proteins from other mollicutes and pathogens. Collectively, our findings shed light on how genome-reduced bacteria innovate molecular functions and offer insights into phytoplasma biology, effector evolution, and host-pathogen dynamics. They also lay the groundwork for protein engineering approaches aimed at discovering or designing novel biomolecules with biotechnological potential.

The fungal genus Cryptococcus includes several life-threatening human pathogens as well as diverse saprobic species whose genome architecture, ecology, and evolutionary history remain less well characterized. Understanding how some lineages evolved into major pathogens remains a central challenge and may be advanced by comparisons with their nonpathogenic counterparts. Integrative approaches have become essential for delimiting species and reconstructing evolutionary relationships, particularly in lineages with cryptic diversity or extensive chromosomal rearrangements. Here, we formally characterize six Cryptococcus species representing distinct evolutionary lineages, comprising both newly discovered and previously recognized but unnamed taxa, through a combination of phylogenomic analyses, divergence metrics, chromosomal comparisons, mating assays, and phenotypic profiling. Among pathogenic taxa, we formally name Cryptococcus hyracis sp. nov., corresponding to the previously characterized VGV lineage within the C. gattii complex. In parallel, we describe five saprobic, nonpathogenic species isolated from fruit, soil, and bark beetle galleries, spanning four phylogenetic clades. We identify a strong ecological association with bark beetles for Cryptococcus porticicola sp. nov., the only newly described nonpathogenic species with multiple sequenced strains from diverse sites. In this species, we detect strain-level chromosomal variation and evidence of sexual reproduction, along with population-level signatures of recombination. Across the genus, chromosome-level comparisons reveal extensive structural variation, including species- and strain-specific rearrangements that may restrict gene flow. We also identify multiple instances of chromosome number reduction, often accompanied by genomic signatures consistent with centromere inactivation or loss of centromeric identity. Comparative metabolic profiling with Biolog phenotype microarrays reveals clade-level differentiation and distinct substrate preferences, which may reflect metabolic divergence and habitat-specific diversification. Notably, we confirm that thermotolerance is restricted to clinically relevant taxa. These findings refine the species-level taxonomy of Cryptococcus, broaden its known genomic and ecological diversity, and strengthen the framework for investigating speciation, adaptation, and the emergence of pathogenicity within the genus.

Dysregulation of the cellular mechanisms that coordinate the interpretation and transduction of microenvironmental biophysical signals are a unifying feature of tissue remodeling pathologies such as fibrosis and cancer. While genomic regulation downstream of normal mechanotransduction (i.e., cases where cells sense soft and stiff appropriately) is well studied, significantly less is known about the consequences of abnormal mechanoperception and subsequent misinterpretation of the mechanical environment. Leveraging Thy-1 (CD90) loss as a model of impaired mechanoperception, we employed ATAC- and RNA-sequencing in parallel to characterize the changes in lung fibroblast genomic activity in response to a combination of substrate stiffness and culture time. Notably, we find perturbed mechanoperception elicits a near-complete shutdown of HOXA5, a transcription factor responsible for pattern specification and development in the nascent lung. In vitro investigation of HOXA5 expression reveals a potential mechanism connecting increased αv integrin signaling, cytoskeletal tension, and SRC kinase activity to HOXA5 silencing. These results establish novel links between integrin signaling and the expression dynamics of genes necessary for tissue formation and regeneration in the injured and/or developing lung, particularly HOXA5.

The predatory bacterium Bdellovibrio bacteriovorus kills and consumes other bacteria, thrives in diverse environments and holds great potential to address major challenges in medicine, agriculture, and biotechnology. As a bacterial predator it represents an alternative to traditional antimicrobial strategies to combat multidrug-resistant bacterial pathogens and prevent food waste, while the multitude of predatory enzymes it produces have potential for biotechnological applications. However, while a limited set of genetic tools exist, the lack of secretion assays and fine-tuning of secretion constrain both fundamental studies and bioengineering of B. bacteriovorus. Here, we present a molecular toolbox for B. bacteriovorus by systematically tuning gene expression and secretion of a reporter protein. Building on functional native and synthetic promoters from the Anderson library with varying expression levels of fluorescent reporter protein mScarletI3, we evaluated different ribosomal binding sites (RBS) to fine-tune gene expression. To examine secretion, we established a novel protocol to quantify extracellular release of a Nanoluc luciferase reporter protein in B. bacteriovorus using different native Sec-dependent signal sequences. We anticipate that the newly developed genetic toolkit and techniques will advance research on this fundamental predator-prey system, laying the foundation for its broader application and future bioengineering efforts. This work will pave the way for tailored applications of B. bacteriovorus in microbial ecology, agriculture, biotechnology, and medicine.

Background: Liver cirrhosis is a progressive chronic disease with high morbidity and mortality, thereby posing a major challenge to global health. Evidence suggests that thyroid dysfunction, particularly hypothyroidism, is linked to liver diseases. Hypothyroidism disrupts metabolism, immune homeostasis, and inflammatory pathways, processes central to cirrhosis pathophysiology. However, its causal role and molecular mechanisms remain unclear.

Methods: The study initiated by analyzing the association between thyroid dysfunction and cirrhosis through retrospective analysis of longitudinal data obtained from the Medical Information Mart for Intensive Care clinical database. To assess genetic correlation, we applied linkage disequilibrium score regression, followed by bidirectional Mendelian randomization to explore potential causal relationships. Through transcriptome-wide association studies, we identified candidate genes, which were then prioritized using a combination of weighted gene co-expression network analysis and differential gene expression data integration. To interpret the biological relevance of these genes, we conducted functional enrichment analyses. We further explored gene function at the cellular level by leveraging single-cell RNA sequencing (scRNA) to map cell-specific expression patterns, analyze intercellular communication, and simulate gene knockouts. Finally, we performed molecular docking and phenome-wide Mendelian randomization to identify potential therapeutic compounds targeting the prioritized genes.

Results: Through a combination of observational and genetic insights, we established a causal relationship between hypothyroidism and cirrhosis, identifying hypothyroidism as a risk factor for cirrhosis. Subsequent multi-omics analyses highlighted HLA-DQA1 and CD27 as potential therapeutic targets. ScRNA revealed key roles of these molecules in macrophages and CD8 ⁺ T cells, and simulated knockouts confirmed their importance in T cell activation and lymphocyte proliferation. Finally, molecular docking analysis identified glycyrrhizic acid and levothyroxine sodium as candidate drugs targeting HLA-DQA1 and CD27, while phenome-wide Mendelian randomization analysis revealed potential adverse effects associated with these targets.

Conclusions: This study is the first to reveal a causal relationship between hypothyroidism and cirrhosis, potentially driven by immune dysregulation mediated by HLA-DQA1 and CD27. These findings offer novel insights into disease progression and identify HLA-DQA1 and CD27 as potential therapeutic targets, with glycyrrhizic acid and levothyroxine sodium as promising candidate drugs.

The small GTPase CDC42 promotes axon growth through actin filament polymerization and this growth is driven by axonal localization of the mRNA encoding the prenylated CDC42 isoform (Prenyl-Cdc42). Here, we show that axonal Prenyl-Cdc42 mRNA levels and the mRNA's translation are decreased by growth-inhibiting stimulation and increased by growth-promoting stimulation. In contrast, axonal RhoA mRNA transport and translation are increased by growth-inhibiting but unaffected by growth-promoting stimuli. Localized increase in KHSRP in response to growth inhibitory stimulation, through elevation of intracellular Ca2+, promotes decrease in axonal levels of Prenyl-Cdc42 mRNA. Distinct 3'UTR motifs regulate transport and axonal levels of Prenyl-Cdc42 mRNA. KHSRP protein binds to a Prenyl-Cdc42 mRNA motif within nt 801-875 and the mRNA is remarkably increased in axons of Khsrp-/- mice. Depletion of the mRNA from sciatic nerve indicates that the increased axonal Prenyl-CDC42 contributes to the accelerated nerve regeneration when neuronal KHSRP is depleted.

Proper neural circuit organization requires individual neurons to project to their targets with high specificity. While several guidance molecules have been shown to mediate axonal fasciculation and pathfinding, less is understood about how neurons intracellularly interpret and integrate these cues. Here we provide genetic evidence that the Crk-Associated Substrate (Cas) family of intracellular adaptor proteins is required for proper fasciculation and guidance of two cortical white matter tracts: the Anterior Commissure (AC) and thalamocortical axons (TCAs). Using a Cas Triple Conditional Knock Out (Cas TcKO) mouse model, we show that Cas proteins are required for proper TCA projection by a non-neuronal cortical cell population. We also demonstrate a requirement of the β1-integrin receptor for TCA projection, similarly in a population of non-neuronal cortical cells. Additional analysis of Cas TcKO mutants reveals a role for Cas proteins in AC fasciculation, here within the neurons themselves. This AC fasciculation requirement is not phenocopied in β1-integrin deficient mutants, suggesting that Cas proteins might signal downstream of a different receptor during this axon pathfinding event. These findings implicate Cas proteins as key mediators of cortical axon tract fasciculation and guidance.