Biology subjects

Shao, M.

Publications and source records attributed to Shao, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

N1-methylpseudouridine mRNA modification enhances efficiency and specificity of gene overexpression by preventing Prkra-mediated global translation repression

In vitro transcribed messenger RNA (IVT mRNA) has emerged as a pivotal tool in mRNA-based therapies and has been extensively employed in gene function studies and genetic tool applications. However, the IVT process generates double-stranded RNA (dsRNA) by-products that are recognized by dsRNA sensors, triggering innate immune responses. In this study, we comprehensively analyzed the detrimental effects of dsRNA by-products on early zebrafish embryos, revealing that these by-products induce cell necrosis and delay maternal-zygotic transition by reducing global translation efficiency via Prkra, a recently identified dsRNA sensor in pluripotent cells. Importantly, we demonstrate that N1-methylpseudouridine (m1{varphi}) modification of IVT mRNAs effectively mitigates these adverse effects, as m1{varphi}-modified dsRNAs exhibit significantly lower binding affinity to the Prkra dimer. Our findings underscore a previously overlooked challenge in the use of IVT mRNA in early embryos and offer a robust solution to enhance the fidelity of mRNA applications. Furthermore, we elucidate that m1{varphi} modification minimizes the dsRNA-induced stress response in pluripotent cells through a distinct mechanism.

developmental biology↗

bioRxiv2024

An Exact and Fast SAT Formulation for the DCJ Distance

Reducing into a satisfiability (SAT) formulation has been proven effective in solving certain NP-hard problems. In this work, we extend this research by presenting a novel SAT formulation for computing the double-cut-and-join (DCJ) distance between two genomes with duplicate genes. The DCJ distance serves as a crucial metric in studying genome rearrangement. Previous work achieved an exact solution by transforming it into a maximum cycle decomposition (MCD) problem on the corresponding adjacency graph of two genomes, followed by reducing this problem into an integer linear programming (ILP) formulation. Using both simulated datasets and real genomic datasets, we firmly conclude that the SAT-based method scales much better and runs faster than using ILP, being able to solve a whole new range of instances which the ILP-based method cannot solve in a reasonable amount of time. This underscores the SAT-based approach as a versatile and more powerful alternative to ILP, with promising implications for broader applications in computational biology.

bioinformatics↗

bioRxiv2024

Accurate Detection of Tandem Repeats from Error-Prone Sequences with EquiRep

A tandem repeat is a sequence of nucleotides that occurs as multiple contiguous and near-identical copies positioned next to each other. These repeats play critical roles in genetic diversity, gene regulation, and are strongly linked to various neurological and developmental disorders. While several methods exist for detecting tandem repeats, they often exhibit low accuracy when the repeat unit length increases or the number of copies is low. Furthermore, methods capable of handling highly mutated sequences remain scarce, highlighting a significant opportunity for improvement. We introduce EquiRep, a tool for accurate detection of tandem repeats from erroneous sequences. EquiRep estimates the likelihood of positions originating from the same position in the unit by self-alignment followed by a novel approach that refines the estimation. The built equivalent classes and the consecutive position information will be then used to build a weighted graph, and the cycle in this graph with maximum bottleneck weight while covering most nucleotide positions will be identified to reconstruct the repeat unit. We test EquiRep on simulated and real HOR and RCA datasets where it consistently outperforms or is comparable to state-of-the-art methods. EquiRep is robust to sequencing errors, and is able to make better predictions for long units and low frequencies which underscores its broad usability for studying tandem repeats.

bioinformatics↗

bioRxiv2024

Augmenting Transcriptome Annotations through the Lens of Splicing Evolution

Alternative splicing (AS) is a ubiquitous mechanism in eukaryotes. It is estimated that 90% of human genes are alternatively spliced. Despite enormous efforts, transcriptome annotations remain, nevertheless, incomplete. Conventional means of annotation were largely driven by experimental data such as RNA-seq and protein sequences, while little insight was shed on understanding transcriptomes and alternative splicings from the perspective of evolution. This study addresses this critical gap by presenting TENNIS (Transcript EvolutioN for New Isoform Splicing), an evolution-based model to predict unannotated isoforms and refine existing annotations without requiring additional data. The model of TENNIS is based on two minimal premises-AS isoforms evolve sequentially from existing isoforms, and each evolutionary step involves a single AS event. We formulate the identification of missing transcripts as an optimization problem and parsimoniously find the minimal number of novel transcripts. Our analysis showed approximately 80% of multi-transcript groups from six transcriptome annotations satisfy our evolutionary model. At a high confidence level, 40% of isoforms predicted by TENNIS were validated by deep long-read RNA-seq. In a simulated incomplete annotation scenario, TENNIS dramatically outperforms two randomized baseline approaches by a 2.25-3 fold-change in precision or a 3.5-3.9 fold-change in recall, after controlling the same level of recall or precision of the baseline methods. These results demonstrate that TENNIS effectively identifies missing transcripts by complying with minimal propositions, offering a powerful approach for transcriptome augmentations through the lens of alternative splicing evolutions. TENNIS is freely available at https://github.com/Shao-Group/tennis.

bioinformatics↗

bioRxiv2024

Transcriptome Assembly at Single-Cell Resolution with Beaver

Emerging single-cell RNA sequencing techniques (scRNA-seq) has enabled the study of cellular transcriptome heterogeneity, yet accurate reconstruction of full-length transcripts at single-cell resolution remains challenging due to high dropout rates and sparse coverage. While meta-assembly approaches offer promising solutions by integrating information across multiple cells, current methods struggle to balance consensus assembly with cell-specific transcriptional signatures. Here, we present Beaver, a cell-specific transcript assembler designed for short-read scRNA-seq data. Beaver implements a transcript fragment graph to organize individual assemblies and designs an efficient dynamic programming algorithm that searches for candidate full-length transcripts from the graph. Beaver in-corporates two random forest models trained on 51 meticulously engineered features that accurately estimate the likelihood of each candidate transcript being expressed in individual cells. Our experiments, performed using both real and simulated Smart-seq3 scRNA-seq data, firmly show that Beaver substantially outperforms existing meta-assemblers and single-sample assemblers. At the same level of sensitivity, Beaver achieved 32.0%-64.6%, 13.5%-36.6%, and 9.8%-36.3% higher precision in average compared to meta-assemblers Aletsch, TransMeta, and PsiCLASS, respectively, with similar improvements over single-sample assemblers Scallop2 (10.1%-43.6%) and StringTie2 (24.3%-67.0%). Beaver is freely available at https://github.com/Shao-Group/beaver. Scripts that reproduce the experimental results of this manuscript are available at https://github.com/Shao-Group/beaver-test.

bioinformatics↗

bioRxiv2024

Prkra is the double-stranded RNA sensor to mediate an embryonic integrated stress response

Double-stranded RNAs (dsRNAs), known as conserved pathogen-associated molecular patterns, are recognized by interferon-induced protein kinase R (PKR) to trigger an integrated stress response, characterized by the inhibition of global translation. However, the interferon system is inactive in pluripotent cells, thus the mechanism underlying dsRNA sensing and translational control is unclear. In this study, we utilized early zebrafish embryos as a model of pluripotent cells and discovered a PKR-independent blockage of translation initiation induced by dsRNA stimulation. Prkra dimer was identified as the genuine dsRNA sensor. Upon binding to dsRNAs, the dimerized dsRNA binding domain 3 becomes activated to sequester the eIF2 complexes from the translation machinery, leading to a hindrance in global protein synthesis. This distinctive embryonic stress response restricts RNA virus SVCV replication in zebrafish embryos but is also conserved in early mouse embryos and embryonic stem cells. Therefore, the Prkra-mediated dsRNA sensing and translation blocking mechanism potentially represents a common strategy for reestablishing physiological homeostasis in response to environmental stresses.

immunology↗

bioRxiv2024

Transcriptome signatures of the medial prefrontal cortex underlying GABAergic control of resilience to chronic stress exposure

Analyses of postmortem human brains and preclinical studies of rodents have identified somatostatin (SST)-positive interneurons as key elements that regulate the vulnerability to stress-related psychiatric disorders. Conversely, genetically induced disinhibition of SST neurons or brain region-specific chemogenetic activation of SST neurons in mice results in stress resilience. Here, we used RNA sequencing of mice with disinhibited SST neurons to characterize the transcriptome changes underlying GABAergic control of stress resilience. We found that stress resilience of male but not female mice with disinhibited SST neurons is characterized by resilience to chronic stress-induced transcriptome changes in the medial prefrontal cortex. Interestingly, the transcriptome of non-stressed stress-resilient male mice resembled the transcriptome of chronic stress-exposed stress-vulnerable mice. However, the behavior and the serum corticosterone levels of non-stressed stress-resilient mice showed no signs of physiological stress. Most strikingly, chronic stress exposure of stress-resilient mice was associated with an almost complete reversal of their chronic stress-like transcriptome signature, along with pathway changes indicating stress-induced enhancement of mRNA translation. Behaviorally, the mice with disinhibited SST neurons were not only resilient to chronic stress-induced anhedonia -- they also showed an inversed anxiolytic-like response to chronic stress exposure that mirrored the chronic stress-induced reversal of the chronic stress-like transcriptome signature. We conclude that GABAergic dendritic inhibition by SST neurons exerts bidirectional control over behavioral vulnerability and resilience to chronic stress exposure that is mirrored in bidirectional changes in expression of putative stress resilience genes, through a sex-specific brain substrate.

neuroscience↗

bioRxiv2024

Phenotypically wild barley shows evidence of introgression from cultivated barley

Plant conservation hinges on preserving biodiversity, which is crucial for long-term adaptation. Multiple studies have reported genetic evidence of crop-to-wild introgression in phenotypically wild accessions of wild barley (Hordeum vulgare ssp. spontaneum). We examined 318 Wild Barley Diversity Collection (WBDC) accessions for evidence of introgression from cultivated barley. Using SNP genotype and exome capture data, we performed local ancestry inference between the 318 WBDC accessions and cultivated barley to identify genomic regions with evidence of introgression. Using the genomic intervals for well-characterized genes involved in domestication and improvement, we examined the evidence for introgression at genomic regions potentially important for maintaining a wild phenotype. Our analysis revealed that nearly 16% (48 of 318) of WBDC accessions showed evidence of introgression from cultivated barley, and up to 16.6% of the genome has been introgressed. All accessions identified as introgressed based on domestication-related phenotypes show clear genetic evidence of introgression. The size of runs of identity by state and local ancestry inference suggests that most introgression did not occur recently. This study suggests a long history of genetic exchange between wild and cultivated barley, highlighting the potential for introgression to influence the genetic makeup and future adaptation of wild populations, with implications for plant conservation strategies.

evolutionary biology↗

bioRxiv2024

Efficient Seeding for Error-Prone Sequences with SubseqHash2

Seeding is an essential preparatory step for large-scale sequence comparisons. Substring-based seeding methods such as kmers are ideal for sequences with low error rates but struggle to achieve high sensitivity while maintaining a reasonable precision for error-prone long reads. SubseqHash, a novel subsequence-based seeding method we recently developed, achieves superior accuracy to substring-based methods in seeding sequences with high mutation/error rates, while the only drawback is its computation speed. In this paper, we propose SubseqHash2, an improved algorithm that can compute multiple sets of seeds in one run by defining k orders over all length-k subsequences and identifying the optimal subsequence under each of the k orders in a single dynamic programming framework. The algorithm is further accelerated using SIMD instructions. SubseqHash2 achieves a 10-50x speedup over repeating SubseqHash while maintaining the high accuracy of seeds. We demonstrate that SubseqHash2 drastically outperforms popular substring-based methods including kmers, minimizers, syncmers, and Strobemers for three fundamental applications. In read mapping, SubseqHash2 can generate adequate seed-matches for aligning hard reads that minimap2 fails on. In sequence alignment, SubseqHash2 achieves high coverage of correct seeds and low coverage of incorrect seeds. In overlap detection, seeds produced by SubseqHash2 lead to more correct overlapping pairs at the same false-positive rate. With all the algorithmic breakthroughs of SubseqHash2, we clear the path for the wide adoption of subsequence-based seeds in long-read analysis. SubseqHash2 is available at https://github.com/Shao-Group/SubseqHash2.

bioinformatics↗

bioRxiv2024

Social state gates vision using three circuit mechanisms in Drosophila

Animals are often bombarded with visual information and must prioritize specific visual features based on their current needs. The neuronal circuits that detect and relay visual features have been well-studied. Yet, much less is known about how an animal adjusts its visual attention as its goals or environmental conditions change. During social behaviors, flies need to focus on nearby flies. Here, we study how the flow of visual information is altered when female Drosophila enter an aggressive state. From the connectome, we identified three state-dependent circuit motifs poised to selectively amplify the response of an aggressive female to fly-sized visual objects: convergence of excitatory inputs from neurons conveying select visual features and internal state; dendritic disinhibition of select visual feature detectors; and a switch that toggles between two visual feature detectors. Using cell-type-specific genetic tools, together with behavioral and neurophysiological analyses, we show that each of these circuit motifs function during female aggression. We reveal that features of this same switch operate in males during courtship pursuit, suggesting that disparate social behaviors may share circuit mechanisms. Our work provides a compelling example of using the connectome to infer circuit mechanisms that underlie dynamic processing of sensory signals.

neuroscience↗

bioRxiv2024

Accurate Assembly of Circular RNAs with TERRACE

Circular RNA (circRNA) is a class of RNA molecules that forms a closed loop with its 5 and 3 ends covalently bonded. Due to this specific structure circRNAs are more stable than linear RNAs, admit distinct biological properties and functions, and have been proven to be promising biomarkers. Circular RNAs were severely overlooked previously owing to the biases in the RNA-seq protocols and in the detection algorithms, but recently gained tremendous attentions in both aspects. However, most existing methods for assembling circRNAs heavily rely on the annotated transcriptomes, and hence exhibit unsatisfactory accuracy when a high-quality transcriptome is unavailable. Here we present TERRACE, a new algorithm for full-length assembly of circRNAs from paired-end total RNA-seq data. TERRACE uses the splice graph as the underlying data structure to organize the splicing and coverage information. We transform the problem of assembling circRNAs into finding two paths that "bridge" the three fragments in the splice graph induced by back-spliced reads. To solve this formulation, we adopted a definition for optimal bridging paths and a dynamic programming algorithm to calculate such paths, an approach that was proven useful for assembling linear RNAs. TERRACE features an efficient algorithm to detect back-spliced reads that are missed by RNA-seq aligners, contributing to its much improved sensitivity. It also incorporates a new machine-learning approach that is trained to assign a confidence score to each assembled circRNA, which is shown superior to using abundance for scoring. TERRACE is compared with leading circRNA detection methods on both simulations and biological datasets. Our method consistently outperforms by a large margin in sensitivity while maintaining better or comparable precision. In particular, when the annotations are not provided, TERRACE can assemble 123%-412% more correct circRNAs than state-of-the-art methods on human tissues. TERRACE presents a major leap on assembling full-length circRNAs from RNA-seq data, and we expect it to be widely used in the downstream research on circRNAs.

bioinformatics↗

bioRxiv2023

Castration Model Illuminates Sex Differences in Healthy Aging: Insights from Metabolome and Transcriptome Analyses

BackgroundSex, as a critical biological variable, has historically been underappreciated, despite the pervasive influence of sexual dimorphism across physiological and pathological processes. A significant obstacle to advancing sex-biased biological research is the absence of an effective animal model. In recent years, castration has emerged as a potential model for elucidating sex-based differences in the context of healthy aging, where it has been shown to equalize lifespan and growth trajectories in genetically diverse mice. However, the molecular shifts induced by castration in common laboratory models, such as C57BL/6 mice, and the broader applicability of this model to other sex-related biological contexts remain largely unexplored. MethodsWe employed multi-omics and observational analyses to investigate the molecular changes associated with sex and sex hormones following castration. We analyzed serum, kidney, and liver samples from 12-week-old and 18-month-old castrated male C57BL/6 mice, alongside intact male and female counterparts. The castration model was further applied to assess differences in cisplatin-induced toxicity and age-related cognitive decline in comparison to unaltered controls. ResultsLC-MS/MS metabolomics revealed that castrated males exhibited substantial alterations in steroid hormone levels and increased concentrations of antioxidant compounds, such as taurine, despite identical diets. Integrated metabolome-transcriptome analysis confirmed distinct patterns of lipid peroxidation and oxidative stress across sham-operated female, male, and castrated male mice. Histopathological evaluations following cisplatin treatment and aging-related behavioral tests demonstrated the models utility in investigating sex-dependent drug toxicity and cognitive decline. These findings underscored the critical role of sex hormones in modulating both toxicity defense mechanisms and cognitive performance. ConclusionThis study provides a systematic multi-omics spectrum on the castration model and demonstrates its capacity to feminize metabolic and transcriptomic profiles, establishing it as a valuable tool for exploring sex hormone-driven biological differences. Our findings lay the groundwork for further mechanistic studies and broaden the potential applications of the castration model in diverse biomedical research domains.

systems biology↗

bioRxiv2023

Utility of T1 mapping and T2 mapping for non-invasive assessment of liver fibrosis: preclinical results

ObjectiveTo explore the potential of T1 and T2 mappings in assessing liver fibrosis (LF) and investigate the relationships between MRI and liver fibrogenesis markers. Materials and methods A total of 39 male C57BL/6 mice were divided into the control group (n = 9) and the model group (n = 10 per subgroup) with carbon tetrachloride (CCl4) administration for 2, 4, and 6 weeks. The METAVIR system (F0-4) was performed to stage fibrosis. MRI T1 and T2 mappings were performed and T1, and T2 values were calculated. One-way analysis of variance (ANOVA), Spearmans rank correlation analysis, and receiver operating characteristic (ROC) curves were performed. ResultsT1 and T2 values increased with progressing severity of fibrosis induction (P < 0.01). T1 and T2 were significantly correlated with LF stages ({rho} = 0.854, 0.697, P < 0.001). The area under the curves (AUCs) range of T1 and T2 for predicting [≥]F1, [≥]F2, [≥]F3, and F4 were 0.842-0.994 and 0.808-0.883, respectively. T1 and T2 showed moderate to strong correlations with collagen-associated protein and inflammatory factors. DiscussionT1 and T2 mappings can evaluate and differentiate LF stages in the CCl4-induced model. T1 is better correlated with collagen deposition and inflammation of LF than T2.

bioengineering↗

bioRxiv2023

Transcript Assembly and Annotations: Bias and Adjustment

MotivationTranscript annotations play a critical role in gene expression analysis as they serve as a reference for quantifying isoform-level expression. The two main sources of annotations are RefSeq and Ensembl/GENCODE, but discrepancies between their methodologies and information resources can lead to significant differences. It has been demonstrated that the choice of annotation can have a significant impact on gene expression analysis. Furthermore, transcript assembly is closely linked to annotations, as assembling large-scale available RNA-seq data is an effective data-driven way to construct annotations, and annotations are often served as benchmarks to evaluate the accuracy of assembly methods. However, the influence of different annotations on transcript assembly is not yet fully understood. ResultsWe investigate the impact of annotations on transcript assembly. We observe that conflicting conclusions can arise when evaluating assemblers with different annotations. To understand this striking phenomenon, we compare the structural similarity of annotations at various levels and find that the primary structural difference across annotations occurs at the intron-chain level. Next, we examine the biotypes of annotated and assembled transcripts and uncover a significant bias towards annotating and assembling transcripts with intron retentions, which explains above the contradictory conclusions. We develop a standalone tool, available at https://github.com/Shao-Group/irtool, that can be combined with an assembler to generate an assembly without intron retentions. We evaluate the performance of such a pipeline and offer guidance to select appropriate assembling tools for different application scenarios.

bioinformatics↗

bioRxiv2022

PHYTOCHROME C regulation of PHOTOPERIOD1 is mediated by EARLY FLOWERING 3 in Brachypodium distachyon

Daylength sensing in many plants is critical for coinciding the timing of flowering with the appropriate season. Temperate-climate-adapted grasses such as Brachypodium distachyon flower during the spring when days are becoming longer. The photoreceptor PHYTOCHROME C is essential for long-day (LD) flowering in B. distachyon. PHYC is required for the LD activation of a suite of genes in the photoperiod pathway including PHOTOPERIOD1 (PPD1) that, in turn, result in the activation of FLOWERING LOCUS T (FT1)/FLORIGEN, which causes flowering. Thus, phyC mutants are extremely delayed in flowering. Here we show that PHYC-mediated activation of PPD1 occurs via EARLY FLOWERING 3 (ELF3), a component of the evening complex in the circadian clock. The extreme delay of flowering of the phyC mutant disappears when combined with an elf3 loss-of-function mutation. Moreover, the dampened PPD1 expression in phyC mutant plants is elevated in phyC/elf3 mutant plants consistent with the rapid flowering of the double mutant. We show that loss of PPD1 function also results in reduced FT1 expression levels and extremely delayed flowering consistent with reports from wheat and barley. Additionally, elf3 mutant plants have elevated expression levels of PPD1 and we show that overexpression of ELF3 results in delayed flowering, which is associated with a reduction of PPD1 and FT1, demonstrating ELF3 represses PPD1 transcription, consistent with previous studies showing that ELF3 binds to the PPD1 promoter. Indeed, PPD1 is the main target of ELF3-mediated flowering as elf3/ppd1 double mutant plants are delayed flowering. Our results indicate that ELF3 operates downstream from PHYC and acts as a repressor of PPD1 in the photoperiod flowering pathway of B. distachyon. AUTHOR SUMMARYDaylength is an important environmental cue that plants and animals use to coincide important life history events with a proper season. In plants, timing of flowering to a particular season is an essential adaptation to many ecological niches. Perceiving changes in daylength starts with the perception of light via specific photoreceptors such as phytochromes. In temperate grasses, how daylength perception is integrated into downstream pathways to trigger flowering is not fully understood. However, some of the components involved in the translation of daylength perception into the induction of flowering in temperate grasses have been identified from studies of natural variation. For example, specific alleles of two genes called EARLY FLOWERING 3 (ELF3) and PHOTOPERIOD1 (PPD1) have been selected during breeding of different wheat and barley varieties to modulate the photoperiodic response to maximize reproduction in different environments. Here, we show in the temperate grass model Brachypodium distachyon that the translation of the light signal perceived by phytochromes into a flowering response is mediated by ELF3, and that PPD1 is genetically downstream of ELF3 in the photoperiodic flowering pathway. These results provide a genetic framework for understanding the photoperiodic response in temperate grasses that include agronomically important crops such as wheat, oats, barley, and rye.

plant biology↗

bioRxiv2022

Genetic Analysis of Wild and Cultivated Populations of Northern Wild Rice (Zizania palustris L.) Reveal New Insights into Gene Flow and Domestication

Northern Wild Rice (NWR; Zizania palustris L.) is an aquatic, annual grass with significant ecological, cultural, and economic importance to the Great Lakes region of North America. In this study, we assembled and genotyped a diverse collection of 839 NWR individuals using genotyping-by-sequencing (GBS) and obtained 5,955 single-nucleotide polymorphisms (SNPs). Our collection consisted of samples from 12 wild NWR populations collected across Minnesota and Western Wisconsin, some of which were collected over two time points; a representative collection of cultivated NWR varieties and breeding populations; and a Zizania aquatica outgroup. Using these data, we characterized the genetic diversity, relatedness, and population structure of this broad collection of NWR genotypes. We found that wild populations of NWR clustered primarily by their geographical location, with some clustering patterns likely influenced by historical ecosystem management. Cultivated populations were genetically distinct from wild populations, suggesting limited gene flow between the semi-domesticated crop and its wild counterparts. The first genome-wide scans of putative selection events in cultivated NWR suggest that the crop is undergoing heavy selection pressure for traits conducive to irrigated paddy conditions. Overall, this study presents a large set of SNP markers for use in NWR genetic studies and provides new insights into the gene flow, history, and complexity of wild and cultivated populations of NWR.

genetics↗

bioRxiv2022

The response mechanism of oreorchis patens (Lindl.) Lindl. to cold stress

Cold stress, a major environmental factor, has an important impact on the production of landscape plants and crop yield, and its stress and resistance mechanisms have always been hot research issues. Oreorchis patens (Lindl.) Lindl., an important germplasm resource, has strong frost resistance and can resist low temperatures of -40 {degrees}C. However, the mechanism by which O. patens responds to cold stress remains poorly understood. Here, we examined the adaptation to the low-temperature environment of O. patens by changing the leaf tissue structure, while the synergistic effect of osmotic regulation, reactive oxygen scavenging and protein improved the resistance to cold stress. In addition, analysis of the photosynthetic system showed that cold stress changes the photosynthetic capacity of O. patens leaves to affect cold resistance. Analysis by nonparametric transcriptome sequencing revealed 2402 genes that were differentially expressed, most of which were related to resistance. Simultaneously, quantitative real-time polymerase chain reaction (qRT-PCR) analysis obtained results consistent with the transcriptome. These results indicated that O. patens could alter leaf structure and physiological and biochemical metabolic processes by initiating resistance-related molecular regulatory networks to improve the ability to resist cold stress. This study was the first to discuss the physiological, biochemical and molecular regulatory mechanisms of O. patens resistance to cold stress, which laid a foundation for revealing the biological and molecular mechanisms of overwintering of O. patens and breeding cold-resistant horticultural crops in northern China.

plant biology↗

bioRxiv2022

Developmental transitions coordinate assembly of the Coxiella burnetii Dot/Icm type IV secretion system

Coxiella burnetii is an obligate intracellular bacterial pathogen that has evolved a unique bi-phasic developmental cycle. The infectious form of C. burnetii is the dormant small cell variant (SCV) that transitions to a metabolically active large cell variant (LCV) that replicates inside the lysosome-derived host vacuole. A Dot/Icm type IV secretion system (T4SS), which can deliver over 100 effector proteins to host cells, is essential for the biogenesis of the vacuole and for intracellular replication. How the distinct C. burnetii life cycle impacts the assembly and function of the Dot/Icm T4SS has remained unknown. Here, we combine advanced cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET) imaging to visualize all developmental transitions and assembly of the Dot/Icm T4SS. Importantly, assembled Dot/Icm T4SSs were not present in the infectious SCV. The appearance of an assembled Dot/Icm machine correlated with the transition of the SCV to the LCV intracellularly. Furthermore, temporal characterization of C. burnetii morphological changes revealed regions of the inner membrane that invaginated to form tightly packed stacks during the LCV to SCV transition at late stages of infection, which could enable the SCV to LCV transition that occurs upon infection of a new host cell. Overall, these data establish how C. burnetii developmental transitions control critical bacterial processes to promote intracellular replication and transmission.

microbiology↗