Biology subjects

Zhuang, X.

Publications and source records attributed to Zhuang, X..

At least 91 records · Page 5Linked to original sources

bioRxiv2020

Reference-free Cell-type Annotation for Single-cell Transcriptomics using Deep Learning with a Weighted Graph Neural Network

Advances in single-cell RNA sequencing (scRNA-seq) have furthered the simultaneous classification of thousands of cells in a single assay based on transcriptome profiling. In most analysis protocols, single-cell type annotation relies on marker genes or RNA-seq profiles, resulting in poor extrapolation. Here, we introduce scDeepSort (https://github.com/ZJUFanLab/scDeepSort), a reference-free cell-type annotation tool for single-cell transcriptomics that uses a deep learning model with a weighted graph neural network. Using human and mouse scRNA-seq data resources, we demonstrate the feasibility of scDeepSort and its high accuracy in labeling 764,741 cells involving 56 human and 32 mouse tissues. Significantly, scDeepSort outperformed reference-dependent methods in annotating 76 external testing scRNA-seq datasets, including 126,384 cells (85.79%) from ten human tissues and 134,604 cells from 12 mouse tissues (81.30%). scDeepSort accurately revealed cell identities without prior reference knowledge, thus potentially providing new insights into mechanisms underlying biological processes, disease pathogenesis, and disease progression at a single-cell resolution.

bioinformatics↗

bioRxiv2020

The CCCTC-binding factor CTCF represses hepatitis B virus Enhancer I and regulates viral transcription

Hepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to the virus during their lifetime and at risk of progressive liver disease, cirrhosis and hepatocellular carcinoma. HBV is a member of the hepadnaviridae family that replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate viral transcription. The chromatin-organising transcriptional insulator protein CCCTC-binding factor (CTCF) has been reported to regulate transcription in a diverse range of viruses. We identified two conserved CTCF binding sites in the HBV genome within Enhancer I and chromatin immunoprecipitation (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or episomal copies of the viral genome. siRNA knockdown of CTCF results in a significant increase in pre-genomic RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF binding and increased pre-genomic RNA levels, providing evidence of a direct role for CTCF in repressing HBV transcription. IMPORTANCEHepatitis B virus (HBV) is a global cause of liver disease. At least 300 million individuals are chronically infected with HBV, frequently leading to life-threatening liver cirrhosis and cancer. Following viral entry, HBV DNA enters the nucleus and is bound by histones that are subject to epigenetic modification. The HBV genome contains two enhancer elements that stimulate viral transcription but the interplay between the viral enhancers and promoters is not fully understood. We have identified the host cell protein CCCTC binding factor (CTCF) as a repressor of HBV gene expression. CTCF binds to the HBV genome within Enhancer I and represses transcription of pre-genomic RNA. These findings provide new insights into how HBV transcription is regulated and show a new role for CTCF as a transcriptional insulator by associating with the viral genome between Enhancer I and the downstream basal core promoter.

microbiology↗

bioRxiv2020

Pharmacological activation of the circadian component REV-ERB inhibits HIV-1 replication

Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks a curative therapy or vaccine. Despite the reduction in AIDS-related deaths achieved by current antiretroviral therapies, drawbacks including drug resistance and the failure to eradicate infection highlight the need to identify new pathways to target the infection. Circadian rhythms are endogenous 24-hour oscillations which regulate physiological processes including immune responses to infection, and there is an emerging role for the circadian components participating viral replication. The molecular clock consists of transcriptional/translational feedback loops that generate rhythms. In mammals, CLOCK and BMAL1 activate rhythmic transcription of genes including the nuclear receptor REV-ERB, which represses BMAL1 and plays an essential role in sustaining a functional clock. We investigated whether REV-ERB activity regulates HIV-1 replication, and found REV-ERB agonists inhibited HIV-1 promoter activity in cell lines, primary human CD4 T cells and macrophages, whilst antagonism or genetic disruption of REV-ERB increased promoter activity. Furthermore, the REV-ERB agonist SR9009 inhibited promoter activity of different HIV-subtypes and HIV-1 replication in primary T cells. This study shows a role for REV-ERB synthetic ligands to inhibit HIV-1 LTR promoter activity and viral replication, supporting a role for circadian clock transcription factors in regulating HIV-1 replication.

microbiology↗

bioRxiv2020

Synchronized infection identifies early rate-limiting steps in the hepatitis B virus life cycle.

Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronized infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4{degrees}C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6h of infection and cccDNA was first detected at 24h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.

microbiology↗

bioRxiv2020

Multi-epitope vaccine design using an immunoinformatics approach for 2019 novel coronavirus in China (SARS-CoV-2)

A new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. An in silico approach based on the available virus genome was applied to identify 19 high immunogenic B-cell epitopes and 499 human-leukocyte-antigen (HLA) restricted T-cell epitopes. Thirty multi-epitope peptide vaccines were designed by iNeo Suite, and manufactured by solid-phase synthesis. Docking analysis showed stable hydrogen bonds of epitopes with their corresponding HLA alleles. When four vaccine peptide candidates from the spike protein of SARS-CoV-2 were selected to immunize mice, a significantly larger amount of IgG in serum as well as an increase of CD19+ cells in ILNs was observed in peptide-immunized mice compared to the control mice. The ratio of IFN-{gamma}-secreting lymphocytes in CD4+ or CD8+ cells in the peptides-immunized mice were higher than that in the control mice. There were also a larger number of IFN-{gamma}-secreting T cells in spleen in the peptides-immunized mice. This study screened antigenic B-cell and T-cell epitopes in all encoded proteins of SARS-CoV-2, and further designed multi-epitope based peptide vaccine against viral structural proteins. The obtained vaccine peptides successfully elicited specific humoral and cellular immune responses in mice. Primate experiments and clinical trial are urgently required to validate the efficacy and safety of these vaccine peptides. ImportanceSo far, a new coronavirus SARS-CoV-2 has caused over 9.2 million infection cases and 475758 deaths worldwide. Due to the rapid dissemination and the unavailability of specific therapy, there is a desperate need for vaccines to combat the epidemic of SARS-CoV-2. Different from the development approaches for traditional vaccines, the development of our peptide vaccine is faster and simpler. In this study, we performed an in silico approach to identify the antigenic B-cell epitopes and human-leukocyte-antigen (HLA) restricted T-cell epitopes, and designed a panel of multi-epitope peptide vaccines. The resulting SARS-CoV-2 multi-epitope peptide vaccine could elicit specific humoral and cellular immune responses in mice efficiently, displaying its great potential in our fight of COVID-19.

bioinformatics↗

bioRxiv2020

Role of RgsA in oxidative stress resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an extremely common opportunistic pathogen in clinical practice. Patients with metabolic disorders, hematologic diseases, malignancies, who have undergone surgery or who have received certain treatments are susceptible to this bacterium. In addition, P. aeruginosa is a multidrug-resistant that tends to form biofilms and is refractory to treatment. Small regulatory RNAs are RNA molecules that are 40-500 nucleotides long, possess regulatory function, are ubiquitous in bacteria, and are also known as small RNA (sRNA). sRNAs play important regulatory roles in various vital life processes in diverse bacteria and their quantity and diversity of regulatory functions exceeds that of proteins. In this study, we showed that deletion of the sRNA RgsA decreases the growth rate and ability to resist different concentrations and durations of peroxide in P. aeruginosa. These decreases occur not only in the planktonic state, but also in the biofilm state. Finally, protein mass spectrometry was employed to understand changes in the entire protein spectrum. The results presented herein provide a description of the role of RgsA in the life activities of P. aeruginosa at the molecular, phenotypic, and protein levels.

microbiology↗

bioRxiv2020

Nicotine exposure and neuronal activity regulate Golgi membrane dispersal and distribution

How nicotine exposure produces long-lasting changes that remodel neural circuits with addiction is unknown. Here, we report that long-term nicotine exposure alters the trafficking of 4{beta}2-type nicotinic acetylcholine receptors (4{beta}2Rs) by dispersing and redistributing the Golgi apparatus. In cultured neurons, dispersed Golgi membranes were distributed throughout somata, dendrites and axons. Small, mobile vesicles in dendrites and axons lacked standard Golgi markers and were identified by other Golgi enzymes that modify glycans. Nicotine exposure increased levels of dispersed Golgi membranes, which required 4{beta}2R expression. Similar nicotine-induced changes occurred in vivo at dopaminergic neurons at mouse nucleus accumbens terminals, consistent with these events contributing to nicotines addictive effects. Characterization in vitro demonstrated that dispersal was reversible, that dispersed Golgi membranes were functional, and that membranes were heterogenous in size, with smaller vesicles emerging from larger "ministacks", similar to Golgi dispersal induced by nocadazole. Protocols that increased cultured neuronal synaptic excitability also increased Golgi dispersal, without the requirement of 4{beta}2R expression. Our findings reveal novel activity- and nicotine-dependent changes in neuronal intracellular morphology. These changes regulate levels and location of dispersed Golgi membranes at dendrites and axons, which function in local trafficking at subdomains.

neuroscience↗

bioRxiv2020

RNA-binding protein Syncrip regulates Starvation-Induced Hyperactivity in adult Drosophila

How to respond to starvation determines fitness. One prominent behavioral response is increased locomotor activities upon starvation, also known as Starvation-Induced Hyperactivity (SIH). SIH is paradoxical as it promotes food seeking but also increases energy expenditure. Despite its importance in fitness, the genetic contributions to SIH as a behavioral trait remains unexplored. Here, we examined SIH in the Drosophila melanogaster Genetic Reference Panel (DGRP) and performed genome-wide association studies. We identified 23 significant loci, corresponding to 14 genes, significantly associated with SIH in adult Drosophila. Gene enrichment analyses indicated that genes encoding ion channels and mRNA binding proteins (RBPs) were most enriched in SIH. We are especially interested in RBPs because they provide a potential mechanism to quickly change protein expression in response to environmental challenges. Using RNA interference, we validated the role of syp in regulating SIH. syp encodes Syncrip (Syp), an RBP. While ubiquitous knockdown of syp led to semi-lethality in adult flies, adult flies with neuron-specific syp knockdown were viable and exhibited decreased SIH. Using the Temporal and Regional Gene Expression Targeting (TARGET) system, we further confirmed the role of Syp in adult neurons in regulating SIH. To determine how syp is regulated by starvation, we performed RNA-seq using the heads of flies maintained under either food or starvation conditions. RNA-seq analyses revealed that syp was alternatively spliced under starvation while its expression level was unchanged. We further generated an alternatively-spliced-exon-specific knockout (KO) line and found that KO flies showed reduced SIH. Together, this study demonstrates a significant genetic contribution to SIH as a behavioral trait, identifies syp as a SIH gene, and highlights the significance of RBPs and post-transcriptional processes in the brain in regulating behavioral responses to starvation. Author summaryAnimals living in the wild often face periods of starvation. How to physiologically and behaviorally respond to starvation is essential for survival. One behavioral response is Starvation-Induced Hyperactivity (SIH). We used the Drosophila melanogaster Genetic Reference Panel, derived from a wild population, to study the genetic basis of SIH. Our results show that there is a significant genetic contribution to SIH in this population, and that genes encoding RNA binding proteins (RBPs) are especially important. Using RNA interference and the TARGET system, we confirmed the role of an RBP Syp in adult neurons in SIH. Using RNA-seq and Western blotting, we found that syp was alternatively spliced under starvation while its expression level was unchanged. Further studies from syp exon-specific knockout flies showed that alternative splicing involving two exons in syp was important for SIH. Together, this study identifies syp as a SIH gene and highlights an essential role of post-transcriptional modification in regulating this behavior.

genomics↗

bioRxiv2019

Arginine-GlcNAcylation of death domain and NleB/SseK proteins is crucial for bacteria pathogenesis by regulating host cell death

Death receptor signaling is critical for cell death, inflammation, and immune homeostasis. Hijacking death receptors and their corresponding adaptors through type III secretion system (T3SS) effectors has been evolved to be a bacterial evasion strategy. NleB from enteropathogenic Escherichia coli (EPEC) and SseK1/2/3 from Salmonella enterica serovar Typhimurium (S. Typhimurium) can modify some death domains involved in death receptor signaling through arginine-GlcNAcylation. This study applied a limited substrate screen from 12 death domain proteins with conserved arginines during EPEC and Salmonella infection and found that NleB from EPEC hijacked death receptor signaling tumor necrosis factor receptor 1 (TNFR1)-associated death domain protein (TRADD), FAS-associated death domain protein (FADD), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1), whereas SseK1 and SseK3 disturbed TNFR signaling through the modification of TRADD Arg235/245 and TNFR1 Arg376, respectively. SseK1 and SseK3 delivered by Salmonella inhibited TNF-- but not TNF-related apoptosis-inducing ligand (TRAIL)-induced cell death, which was consistent with their host substrate recognition specificity. Taking advantage of the substrate specificity of SseK effectors, we found that only SseK1 fully rescued the bacteria colonization deficiency contributed by NleBc in Citrobacter rodentium infection animal model, indicating that TRADD was likely to be the preferred in vivo substrate corresponding to NleB/SseK1-induced bacterial virulence. Furthermore, novel auto-arginine-GlcNAcylation was observed in NleB and SseK1/3, which promoted the enzyme activity. These findings suggest that arginine-GlcNAcylation in death domains and auto-arginine-GlcNAcylation catalyzed by type III-translocated bacterial effector proteins NleB/SseKs are crucial for bacteria pathogenesis in regulating nuclear factor-{kappa}B (NF-{kappa}B) and death receptor signaling pathways. This study provides an insight into the mechanism by which EPEC and Salmonella manipulate death receptor signaling and evade host immune defense through T3SS effectors.\n\nAuthor SummaryEnteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are important food-borne pathogens infecting the intestine. They deliver type III secretion system effector NleB/SseKs to modify host death domain proteins by arginine GlcNAcylation. We screened the modification of 12 death domains containing conserved arginine in human genome by NleB, SseK1, SseK2, and SseK3 through ectopic co-expression and bacterial infection. Unlike multiple death receptor signaling inhibition by NleB, we found that SseK1 and SseK3 specifically hijacked tumor necrosis factor receptor 1 (TNFR1)-mediated death signaling through targeting TNFR1-associated death domain protein (TRADD) and receptor TNFR1, respectively. We identified the modification sites and suggested that TRADD was the in vivo target of NleB in mice infection model by utilizing the substrate specificity of SseK1 and SseK3, which highlighted anti-bacterial infection role of TRADD in death receptor signaling and non-death receptor signaling. In addition to the modification on host death domain substrates, we firstly elucidated the effect of auto-modification of the arginine GlcNAc transferases on the enzymatic activity, which widened our understanding of the newly discovered post translational modification in the process of pathogen-host interaction.

microbiology↗

bioRxiv2019

A robust deep neural network for denoising task-based fMRI data: An application to working memory and episodic memory

In this study, a deep neural network (DNN) is proposed to reduce the noise in task-based fMRI data without explicitly modeling noise. The DNN artificial neural network consists of one temporal convolutional layer, one long short-term memory (LSTM) layer, one time-distributed fully-connected layer, and one unconventional selection layer in sequential order. The LSTM layer takes not only the current time point but also what was perceived in a previous time point as its input to characterize the temporal autocorrelation of fMRI data. The fully-connected layer weights the output of the LSTM layer, and the output denoised fMRI time series is selected by the selection layer. Assuming that task-related neural response is limited to gray matter, the model parameters in the DNN network are optimized by maximizing the correlation difference between gray matter voxels and white matter or ventricular cerebrospinal fluid voxels. Instead of targeting a particular noise source, the proposed neural network takes advantage of the task design matrix to better extract task-related signal in fMRI data. The DNN network, along with other traditional denoising techniques, has been applied on simulated data, working memory task fMRI data acquired from a cohort of healthy subjects and episodic memory task fMRI data acquired from a small set of healthy elderly subjects. Qualitative and quantitative measurements were used to evaluate the performance of different denoising techniques. In the simulation, DNN improves fMRI activation detection and also adapts to varying hemodynamic response functions across different brain regions. DNN efficiently reduces physiological noise and generates more homogeneous task-response correlation maps in real data.

neuroscience↗

bioRxiv2019

m6A-binding YTHDF proteins promote stress granule formation by modulating phase separation of stress granule proteins

Diverse RNAs and RNA-binding proteins form phase-separated, membraneless granules in cells under stress conditions. However, the role of the prevalent mRNA methylation, m6A, and its binding proteins in stress granule (SG) assembly remain unclear. Here, we show that m6A-modified mRNAs are enriched in SGs, and that m6A-binding YTHDF proteins are critical for SG formation. Depletion of YTHDF1/3 inhibits SG formation and recruitment of m6A-modified mRNAs to SGs. Both the N-terminal intrinsically disordered region and the C-terminal m6A-binding YTH domain of YTHDF proteins are crucial for SG formation. Super-resolution imaging further reveals that YTHDF proteins are in a super-saturated state, forming clusters that reside in the periphery of and at the junctions between SG core clusters, and promote SG phase separation by reducing the activation energy barrier and critical size for condensate formation. Our results reveal a new function and mechanistic insights of the m6A-binding YTHDF proteins in regulating phase separation.

biochemistry↗

bioRxiv2019

LFMD: a new likelihood-based method to detect low-frequency mutations without molecular tags

As next-generation sequencing (NGS) and liquid biopsy become more prevalent in research and in the clinic, there is an increasing need for better methods to reduce cost and improve sensitivity and specificity of low-frequency mutation detection (where the Alternative Allele Frequency, or AAF, is less than 1%). Here we propose a likelihood-based approach, called Low-Frequency Mutation Detector (LFMD), which combines the advantages of duplex sequencing (DS) and the bottleneck sequencing system (BotSeqS) to maximize the utilization of duplicate reads. Compared with the existing state-of-the-art methods, DS, Du Novo, UMI-tools, and Unified Consensus Maker, our method achieves higher sensitivity, higher specificity (< 4 x 10-10 errors per base sequenced) and lower cost (reduced by ~70% at best) without involving additional experimental steps, customized adapters or molecular tags. LFMD is useful in areas where high precision is required, such as drug resistance prediction and cancer screening. As an example of LFMDs applications, mitochondrial heterogeneity analysis of 28 human brain samples across different stages of Alzheimers Disease (AD) showed that the canonical oxidative damage related mutations, C:G>A:T, are significantly increased in the mid-stage group. This is consistent with the Mitochondrial Free Radical Theory of Aging, suggesting that AD may be linked to the aging of brain cells induced by oxidative damage.

bioinformatics↗

bioRxiv2019

Pyridox(am)ine 5’-phosphate oxidase deficiency induces seizures in Drosophila melanogaster

Pyridox(am)ine 5-phosphate oxidase (PNPO) is a rate-limiting enzyme in converting dietary vitamin B6 (VB6) to pyridoxal 5-phosphate (PLP), the biologically active form of VB6, and involved in the synthesis of neuro-transmitters including GABA, dopamine, and serotonin. In humans, PNPO mutations have been increasingly identified in neonatal epileptic encephalopathy and more recently also in early-onset epilepsy. Till now, little is known about the neurobiological mechanisms underlying PNPO-deficiency-induced seizures due to the lack of animal models. Previously we identified a c.95 C > A missense mutation in sgll - the Drosophila homolog of human PNPO (hPNPO) and found mutant (sgll95) flies exhibiting a lethal phenotype on a diet devoid of VB6. Here we report the establishment of both sgll95 and ubiquitous sgll knockdown (KD) flies as valid animal models of PNPO-deficiency-induced epilepsy. Both sgll95 and sgll KD flies exhibit spontaneous seizures before they die. Electrophysiological recordings reveal that seizures caused by PNPO deficiency have characteristics similar to that in flies treated with GABA antagonist picrotoxin. Both seizures and lethality are associated with low PLP levels and can be rescued by ubiquitous expression of wild-type sgll or hPNPO, suggesting the functional conservation of the PNPO enzyme between humans and flies. Results from cell type-specific sgll KD further demonstrate that PNPO in the brain is necessary for seizure prevention and survival. Our establishment of the first animal model of PNPO deficiency will lead to better understanding of VB6 biology, the PNPO gene and its mutations discovered in patients, and can be a cost-effective system to test therapeutic strategies.

neuroscience↗

bioRxiv2018

Multiplexed detection of RNA using MERFISH and branched DNA amplification

Multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows simultaneous imaging of numerous RNA species in their native cellular environment and hence spatially resolved single-cell transcriptomic measurements. However, the relatively modest brightness of signals from single RNA molecules can become limiting in a number of important applications, such as increasing the imaging throughput, imaging shorter RNAs, and imaging samples with high degrees of background, such as some tissue samples. Here, we introduce a branched DNA (bDNA) amplification approach for MERFISH measurements. This approach produces a drastic signal increase in RNA FISH samples without increasing the fluorescent spot size for individual RNAs or increasing the variation in brightness from spot to spot. Using this approach in combination with MERFISH, we demonstrated RNA imaging and profiling with a near 100% detection efficiency. We further demonstrated that signal amplification improves MERFISH performance when fewer FISH probes are used for each RNA species, which should allow shorter RNAs to be imaged. We anticipate that the combination of bDNA amplification with MERFISH should facilitate many other applications and extend the range of biological questions that can be addressed by this technique in both cell culture and tissues.

cell biology↗

bioRxiv2017

Multiplexed imaging of high density libraries of RNAs with MERFISH and expansion microscopy

As an image-based single-cell transcriptomics approach, multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows hundreds to thousands of RNA species to be identified, counted and localized in individual cells while preserving the native spatial context of RNAs. In MERFISH, RNAs are identified via a combinatorial labeling approach that encodes RNA species with error-robust barcodes followed by sequential rounds of single-molecule FISH (smFISH) to read out these barcodes. The accuracy of RNA identification relies on spatially separated signals from individual RNA molecules, which limits the density of RNAs that can be measured and makes the multiplexed imaging of a large number of high-abundance RNAs challenging. Here we report an approach that combines MERFISH and expansion microscopy to substantially increase the total density of RNAs that can be measured. Using this approach, we demonstrate accurate identification and counting of RNAs, with a near 100% detection efficiency, in a ~130-RNA library composed of many high-abundance RNAs, the total density of which is more than 10 fold higher than previously reported. In parallel, we demonstrate the combination of MERFISH with immunofluorescence. These advances increase the versatility of MERFISH and will facilitate its application of a wide range of biological problems.

cell biology↗

bioRxiv2017

Exploring the Genetics of Lesion and Nodal Resistance in Pea (Pisum sativum L.) to Sclerotinia sclerotiorum Using Genome-wide Association Studies and RNA-Seq

The disease white mold caused by the fungus Sclerotinia sclerotiorum is a significant threat to pea production and improved resistance to this disease is needed. Nodal resistance in plants is a phenomenon where a fungal infection is prevented from passing through a node and the infection is limited to an internode region. Nodal resistance has been observed in some pathosystems such as the pea (Pisum sativum L.)-S. sclerotiorum pathosystem. Other than nodal resistance, different pea lines display different levels of stem lesion size restriction, referred to as lesion resistance. It is unclear whether the genetics of lesion resistance and nodal resistance are identical or different. This study applied genome-wide association studies (GWAS) and RNA-Seq to understand the genetic makeup of these two types of resistance. The time series RNA-Seq experiment consisted of two pea lines (the susceptible Lifter and the partially resistant PI 240515), two treatments (mock samples and S. sclerotiorum inoculated samples), and three time points (12, 24, and 48 hours post-inoculation). Integrated results from GWAS and RNA-Seq analyses identified different redox-related transcripts for lesion and nodal resistances. A transcript encoding a glutathione S-transferase was the only shared resistance source for both phenotypes. There were more leucine rich-repeat containing transcripts found for lesion resistance, while different candidate resistance transcripts such as a VQ motif-containing protein and a myo-inositol oxygenase were found for nodal resistance. This study demonstrated the robustness of combining GWAS and RNA-Seq for identifying white mold resistance in pea, and results suggest different genetics underlying lesion and nodal resistance.

plant biology↗

bioRxiv2017

Molecular Identification and Characterization of Two Rubber Dandelion Amalgaviruses

The Amalgaviridae family comprise persistent viruses that share the genome architecture of Totiviridae and gene evolutionary resemblance to Partitiviridae. Two genera have been assigned to this family, including genus Amalgavirus consisting in nine recognized species, corresponding to plant infecting viruses with dsRNA monosegmented genomes of ca. 3.4 kb. Here, we present the molecular characterization of two novel viruses detected in rubber dandelion (Taraxacum kok-saghyz). The sequenced viruses are 3,409 and 3,413 nt long, including two partially overlapping ORFs encoding a putative coat protein and an RNA-dependent RNA polymerase (RdRP). Phylogenetic insights based on the RdRP suggest them to be members of two new species within the Amalgavirus genus. Multiple independent RNAseq data suggest that the identified viruses have a dynamic distribution and low relative RNA levels in infected plants. Virus presence was not associated with any apparent symptoms on the plant hosts. We propose the names rubber dandelion latent virus 1 & 2 to the detected amalgaviruses; the first viruses to be associated to this emergent and sustainable natural rubber crop.

microbiology↗

bioRxiv2017

High-throughput, image-based screening of genetic variant libraries

Image-based, high-throughput, high-content screening of pooled libraries of genetic perturbations will greatly advance our understanding biological systems and facilitate many biotechnology applications. Here we introduce a high-throughput screening method that allows highly diverse genotypes and the corresponding phenotypes to be imaged in numerous individual cells. To facilitate genotyping by imaging, barcoded genetic variants are introduced into the cells, each cell carrying a single genetic variant connected to a unique, nucleic-acid barcode. To identify the genotype-phenotype correspondence, we perform live-cell imaging to determine the phenotype of each cell, and massively multiplexed FISH imaging to measure the barcode expressed in the same cell. We demonstrated the utility of this approach by screening for brighter and more photostable variants of the fluorescent protein YFAST. We imaged 20 million cells expressing ~60,000 YFAST mutants and identified novel YFAST variants that are substantially brighter and/or more photostable than the wild-type protein.

systems biology↗