Biology subjects

Hu, Q.

Publications and source records attributed to Hu, Q..

At least 109 records · Page 6Linked to original sources

bioRxiv2021

Structure and ion-release mechanism of PIB-4-type ATPases

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here we present structures and complementary functional analyses of an archetypal PIB-4-ATPases, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy metal binding domains, and provides fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release, and as an internal counter ion, of an invariant, central histidine. We also establish that the turn-over of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in e.g. drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.

biochemistry↗

bioRxiv2021

Single cell RNA Sequencing Identifies G-protein Coupled Receptor 87 as a Novel Basal Cell Marker of Distal Honeycomb Cysts in Idiopathic Pulmonary Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal lung disease with limited therapeutic options. Epithelial reprogramming and honeycomb cysts are key pathological features of IPF, however, the IPF distal bronchiole cell subtypes and their potential contribution to IPF development and progression still remain poorly characterized. Here, we utilized single-cell RNA sequencing on enriched EpCAM+ cells of the distal IPF and Donor lung. Using the 10x Genomics platform, we generated a dataset of 47,881 cells and found distinct cell clusters, including rare cell types, such as suprabasal cells recently reported in the healthy lung. We identified G-protein coupled receptor (GPR) 87 as a novel surface marker of distal Keratin (KRT)5+ basal cells. GPR87 expression was localized to distal bronchioles and honeycomb cysts in IPF in situ by RNA Scope and immunolabeling. Modulation of GPR87 in primary human bronchial epithelial cells cultures resulted in impaired airway differentiation and ciliogenesis. Thus, GPR87 is a novel marker and potentially druggable target of KRT5+ basal progenitor cells likely contributing to bronchiole remodeling and honeycomb cyst development in IPF.

cell biology↗

bioRxiv2021

Deep molecular characterization linked to drug response profiling of pancreatic ductal adenocarcinoma using patient-derived organoids.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by high drug resistance and poor prognosis. Novel therapeutic and stratification strategies are urgently needed. Here, we present an integration of in-depth genomic and transcriptomic characterization with drug screening and clinical outcome based on a catalogue of 51 patient-derived tumor organoids (PDOs) from resected PDAC. Known PDAC molecular subtypes and their prognostic value are conserved in organoids. Integration of transcriptomic and drug response profiles suggest a metabolism-mediated modulations of drug resistance. Copy number alterations on chromosome 13q and wild-type status of TP53 emerged as potential novel genomic biomarkers for sensitivity to 5-FU and oxaliplatin treatment, respectively. Functional testing of targeted drugs in PDOs revealed its additional value for genome-driven personalized oncology. Co-deletion of TP53/POLR2A increased vulnerability to RNA polymerase II inhibition, pointing to a promising target for personalized treatment in PDAC. SignificancePatient-derived PDAC organoids hold great promise as surrogate tumor models for personalized oncology. By integrating highly granular molecular, drug sensitivity and clinical data, we demonstrate that PDOs are valid models for molecular characterization and response prediction that also enable identification of novel drug sensitivity biomarkers and resistance mechanisms in PDAC.

cancer biology↗

bioRxiv2021

Directing an mRNA-LNP vaccine toward lymph nodes improves humoral and cellular immunity against SARS-CoV-2

The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic has captured the worlds attention and remains an ongoing issue in order to protect against emerging variants of concern (VoCs) while generating long lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid (mRNA) encoding the spike protein in a lipid nanoparticle formulation (LNP) (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In efforts to continually improve immunity, a lymphatic drug delivery device (MuVaxx) was engineered and tested to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) to elicit adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine producing CD8 T cells, and neutralizing antibodies against the Washington (Wild Type, WT) and South African (beta) variants. Remarkably, a 4-fold elevated T cell response was observed in MuVaxx administered vaccination as compared to that of IM administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic mediated delivery using MuVaxx for SARS-CoV-2 and VoCs vaccines.

immunology↗

bioRxiv2021

An atlas of healthy and injured cell states and niches in the human kidney

Understanding kidney disease relies upon defining the complexity of cell types and states, their associated molecular profiles, and interactions within tissue neighborhoods. We have applied multiple single-cell or -nucleus assays (>400,000 nuclei/cells) and spatial imaging technologies to a broad spectrum of healthy reference (n = 42) and disease (n = 42) kidneys. This has provided a high resolution cellular atlas of 100 cell types that include rare and novel cell populations. The multi-omic approach provides detailed transcriptomic profiles, epigenomic regulatory factors, and spatial localizations for major cell types spanning the entire kidney. We further identify and define cellular states altered in kidney injury, encompassing cycling, adaptive or maladaptive repair, transitioning and degenerative states affecting several segments. Molecular signatures of these states permitted their localization within injury neighborhoods using spatial transcriptomics, and large-scale 3D imaging analysis of [~]1.2 million neighborhoods provided linkages to active immune responses. These analyses further defined biological pathways relevant to injury niches, including signatures underlying the transition from reference to predicted maladaptive states that were associated with a decline in kidney function during chronic kidney disease. This human kidney cell atlas, including injury cell states and neighborhoods, will be a valuable resource for future studies.

genomics↗

bioRxiv2021

BRET-based self-cleaving biosensors for SARS-CoV-2 3CLpro Inhibitor Discovery

The 3C-like protease (3CLpro) of SARS-CoV-2 is an attractive drug target for developing antivirals against SARS-CoV-2. A few small molecule inhibitors of 3CLpro are in clinical trials for COVID-19 treatments and more inhibitors are being developed. One limiting factor for 3CLpro inhibitors development is that the cellular activities of such inhibitors have to be evaluated in a Biosafety Level 3 (BSL-3) or BSL-4 laboratory. Here, we design genetically encoded biosensors that can be used in BSL-2 laboratories to set up cell-based assays for 3CLpro inhibitor discovery. The biosensors were constructed by linking a green fluorescent protein (GFP2) to the N-terminus and a Renilla luciferase (RLuc8) to the C-terminus of SARS-CoV-2 3CLpro, with the linkers derived from the cleavage sequences of 3CLpro. After over-expression of the biosensors in HEK293 cells, 3CLpro can be released from GFP2 and RLuc by self-cleavage, resulting in a decrease of the bioluminescence resonance energy transfer (BRET) signal. Using one of these biosensors, pBRET-10, we evaluated the cellular activities of several 3CLpro inhibitors. These inhibitors restored the BRET signal by blocking the proteolysis of pBRET-10, and their relative activities measured using pBRET-10 were consistent with their anti-SARS-CoV-2 activities reported previously. We conclude that the biosensor pBRET-10 is a useful tool for SARS-CoV-2 3CLpro inhibitor discovery. Furthermore, our strategy can be used to design biosensors for other viral proteases that share the same activation mechanism as 3CLpro, such as HIV protease PR and HCV protease NS3. HighlightsO_LISensitive cell-based biosensors for 3CLpro inhibitor discovery in BSL-2 laboratories. C_LIO_LIThe BRET-based self-cleaving biosensors mimic the in vivo autoproteolytic activation of 3CLpro. C_LIO_LISimilar biosensors can be designed for other self-cleaving proteases, such as HIV protease PR and HCV protease NS3. C_LI

synthetic biology↗

bioRxiv2021

NNAL, a major metabolite of tobacco-specific carcinogen NNK, promotes lung cancer progression through deactivating LKB1 in an isomer-dependent manner

Smoking is associated with worse clinical outcomes for lung cancer patients. Cell-based studies suggest that NNK (a tobacco specific carcinogen) promotes lung cancer progression. Given its short half-life, the physiological relevance of these in vitro results remains elusive. NNAL, a major metabolite of NNK with a similar structure, a chiral center, and a longer half-life, has never been evaluated in cancer cells. In this study, we characterized the effect of NNAL and its enantiomers on cancer progression among a panel of NSCLC cell lines and explored the associated mechanisms. We found that (R)-NNAL promotes cell proliferation, enhances migration, and induces drug resistance while (S)-NNAL has much weaker effects. Mechanistically, (R)-NNAL phosphorylates and deactivates LKB1 via the {beta}-AR signaling in the LKB1 wild type NSCLC cell lines, contributing to the enhanced proliferation, migration, and drug resistance. Of note, NNK exposure also increases the phosphorylation of LKB1 in A/J mice. More importantly, human lung cancer tissues appear to have elevated LKB1 phosphorylation. Our results reveal, for the first time, that NNAL may promote lung cancer progression through LKB1 deactivation in an isomer-dependent manner.

cancer biology↗

bioRxiv2021

Role of the G-Protein Coupled Receptor 3-Salt Inducible Kinase 2 Pathway in Human β CellProliferation

Loss of pancreatic {beta} cells is the hallmark of type 1 diabetes (T1D) 1, for which provision of insulin is the standard of care. While regenerative and stem cell therapies hold the promise of generating single-source or host-matched tissue to obviate immune-mediated complications2-4, these will still require surgical intervention and immunosuppression. Thus, methods that harness the innate capacity of {beta}-cells to proliferate to increase {beta} cell mass in vivo are considered vital for future T1D treatment5, 6. However, early in life {beta} cells enter what appears to be a permanent state of quiescence 7-10, directed by an evolutionarily selected genetic program that establishes a {beta} cell mass setpoint to guard against development of fatal endocrine tumours. Here we report the development of a high-throughput RNAi screening approach to identify upstream pathways that regulate adult human {beta} cell quiescence and demonstrate in a screen of the GPCRome that silencing G-protein coupled receptor 3 (GPR3) leads to human pancreatic {beta} cell proliferation. Loss of GPR3 leads to activation of Salt Inducible Kinase 2 (SIK2), which is necessary and sufficient to drive cell cycle entry, increase {beta} cell mass, and enhance insulin secretion in mice. Taken together, targeting the GPR3-SIK2 pathway represents a novel avenue to stimulate the regeneration of {beta} cells.

cell biology↗

bioRxiv2021

Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B

Safe and effective vaccines are needed to end the COVID-19 pandemic caused by SARS-CoV-2. Here we report the preclinical development of a lipid nanoparticle (LNP) formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern (VOCs), including the B.1.1.7, B.1.351 and P.1 lineages. No adverse effects were induced by PTX-COVID19-B in both mice and hamsters. These preclinical results indicate that PTX-COVID19-B is safe and effective. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 1 clinical trial ongoing (ClinicalTrials.gov number: NCT04765436). One Sentence SummaryPTX-COVID19-B is a SARS-CoV-2 mRNA vaccine that is highly immunogenic, safe, and effective in preventing SARS-CoV-2 infection in mice and hamsters and is currently being evaluated in human clinical trials.

immunology↗

bioRxiv2021

Thermal Fogging with Disinfectant Didecyl Dimethyl Ammonium Bromide Effectively Kills A Coronavirus, An Influenza Virus and Two Indicator Bacteria in Subzero Cold-Chain Environment

BackgroundThe origin of several local COVID-19 outbreaks in China in 2020 were confirmed to be frozen food or packages contaminated with SARS-CoV-2, revealing the lack of effective disinfection measures in the frozen food chain. ObjectiveTo evaluate the disinfection efficacy at -20{degrees}C of a recently marketed thermal fogging disinfection product, with disinfectant-antifreeze combination being didecyl dimethyl ammonium bromide (DDAB) - propylene glycol (PPG). MethodCarriers with porcine epidemic diarrhea virus (PEDV) YN1, a coronavirus, and a swine influenza virus H1N1 (SIV-H1N1), and three indicator bacteria, E. coli, S. aureus, and B. subtilis endospores, were respectively placed in a -20{degrees}C freezer warehouse with or without DDAB-PPG fogging and activities of the microorganisms were tested. ResultsAt -20{degrees}C, DDAB-PPG fogging, which fully settles in 3.5-4.5 hours, fully inactivated PEDV of 10-3.5 TCID50/0.1ml and SIV-H1N1 of 26 hemagglutination titer within 15-30 min, and inactivated S. aureus and E. coli vegetative cells (106/ml) within 15 or 60 min, respectively, but had little effect on B. subtilis spores. The bactericidal effect lasted at least 3 hours for bacteria on carrier plates and for 6 hours for airborne bacteria. ConclusionsA practical subzero temperature disinfection technology was confirmed its efficacy in killing enveloped viruses and vegetative bacteria. It would help to meet the urgent public health need of environmental disinfection in frozen food logistics against pandemic and other potential pathogens and to enhance national and international biosecurity. HighlightsO_LIFirst report of efficacy of disinfectant didecyl dimethyl ammonium bromide (DDAB) at subzero temperatures C_LIO_LIThe customized thermal fogging machine makes fine droplets of DDAB-propylene glycol (PPG) fog which can suspend for ~4 hours at -20{degrees}C C_LIO_LIDDAB-PPG thermal fogging at -20{degrees}C effectively inactivated a coronavirus and an influenza virus C_LIO_LIDDAB-PPG thermal fogging at -20{degrees}C for effectively killed S. aureus and E. coli C_LIO_LIDDAB-PPG thermal fogging at -20{degrees}C inactivated airborne microorganism for up to 6 h C_LI

molecular biology↗

bioRxiv2021

A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration

The dramatically expanding COVID-19 needs multiple effective countermeasures. Neutralizing antibodies are a potential therapeutic strategy for treating COVID-19. A number of neutralizing nanobodies (Nbs) were reported for their in vitro activities. However, in vivo protection of these nanobodies was not reported in animal models. In the current report, we characterized several RBD-specific Nbs isolated from a screen of an Nb library derived from an alpaca immunized with SARS-CoV-2 spike glycoprotein (S); among them, three Nbs exhibited picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and 15 circulating SARS-CoV-2 variants. To improve the efficacy, various configurations of Nbs were engineered. Nb15-NbH-Nb15, a novel trimer constituted of three Nbs, was constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb15-NbH-Nb15 exhibited sub-ng/ml neutralization potency against the wild-type and currently circulating variants of SARS-CoV-2 with a long half-life in vivo. In addition, we showed that intranasal administration of Nb15-NbH-Nb15 provided 100% protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb15-NbH-Nb15 is a potential candidate for both prevention and treatment of SARS-CoV-2 through respiratory administration. One sentence summaryNb15-NbH-Nb15, with a novel heterotrimeric bispecific configuration, exhibited potent and broad neutralization potency against SARS-CoV-2 in vitro and provided in vivo protection against SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/429275v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@a30bc8org.highwire.dtl.DTLVardef@5a872eorg.highwire.dtl.DTLVardef@1610f74org.highwire.dtl.DTLVardef@13d9bd8_HPS_FORMAT_FIGEXP M_FIG Graphical abstract: C_FIG HighlightsO_LIWe described a novel heterotrimeric configuration of Nb-NbH-Nb (Nb15-NbH-Nb15) that exhibited improved viral inhibition and stability. C_LIO_LINb15-NbH-Nb15 provides ultrahigh neutralization potency against SARS-CoV-2 wild type and 18 mutant variants, including the current circulating variants of D614G and N501Y predominantly in the UK and South Africa. C_LIO_LIIt is the first to demonstrate the Nbs efficacy in preventing and treating SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery. C_LI

microbiology↗

bioRxiv2021

Restoration of deficient DNA Repair Genes Mitigates Genome Instability and Increases Productivity of Chinese Hamster Ovary Cells

Chinese Hamster Ovary (CHO) cells are the primary host used for manufacturing of therapeutic proteins. However, production instability of high-titer cell lines is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein titer. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide polymorphisms (SNPs) in DNA repair genes. Comparison with other mammalian cells confirmed DNA repair is compromised in CHO. Restoration of key DNA repair genes by SNP reversal or expression of intact cDNAs improved DNA repair and genome stability. Moreover, the restoration of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show for the first time that correction of key DNA repair genes yields considerable improvements in stability and protein expression in CHO, and provide new opportunities for cell line development and a more efficient and sustainable production of therapeutic proteins.

cell biology↗

bioRxiv2020

A Systematic and Consistent Assay for High-throughput Characterization of Stalk Quality in Sugarcane by Near-infrared Spectroscopy

Stalk quality improvement is deemed a promising strategy to enhance sugarcane production. However, the lack of efficient approaches for a systematic evaluation of sugarcane germplasm limited stalk quality improvement. In this study, 628 sugarcane samples were employed to take a high-throughput assay for determining the sugarcane stalk quality. Based on the high-performance anion chromatography method, large sugarcane stalk quality variations were detected in biomass composition and the corresponding fundamental ratio values. Online and offline Near-infrared Spectroscopy (NIRS) modeling strategies were applied for multiple purpose calibration. Consequently, 25 equations were generated with the excellent determination coefficient (R2) and ratio performance deviation (RPD) values. Notably, for some observations, RPD values as high as 6.3 were observed that indicated their exceptional performance potential and prediction capacity. Hence, this study provides a feasible way for high-throughput assessment of stalk quality, permitting large-scale screening of optimal sugarcane germplasm.

plant biology↗

bioRxiv2020

Selective vulnerability of supragranular layer neurons in schizophrenia

Schizophrenia is one of the most wide-spread mental brain disorders with complex and largely unknown etiology. To characterize the impact of schizophrenia at a cellular level, we performed single nucleus RNA sequencing of >190,000 neurons from the dorsolateral prefrontal cortex of patients with schizophrenia and matched controls (7 vs 11, respectively). In addition, to correlate data with cortical anatomy, >100,000 neurons were analyzed topographically by immunohistochemistry in an extended cohort of cases with schizophrenia and controls (10 vs 10). Compositional analysis of RNA sequencing data revealed reduction in relative abundance across all families of GABAergic neurons and a concomitant increase in principal neurons, which was most pronounced for supragranular subtypes (layers 2-3). Moreover, supragranular subtypes of GABAergic interneurons showed most dramatic transcriptomic changes. These results were substantiated by histological analysis, which revealed a reduction in the density of calretinin, calbindin and parvalbumin GABAergic interneurons particularly in layer 2. Common effect of schizophrenia on supragranular neuronal networks was underlined by downregulation of protein processing genes and upregulation of neuronal development/plasticity genes across supragranular subtypes of principal neurons and GABAergic interneurons. In situ hybridization and spatial transcriptomics further confirmed supragranular layer neuron vulnerability, revealing complexity of schizophrenia-affected cortical circuits. These point towards general network impairment within supragranular layers being a core substrate associated with schizophrenia symptomatology.

neuroscience↗

bioRxiv2020

Template-independent genome editing and repairing correct frameshift disease in vivo

Frameshift mutation caused by small insertions/deletions (indels) often generate truncated and non-functional proteins, which underlies 22% inherited Mendelian disorders in humans. However, there is no efficient in vivo gene therapy strategies available to date, especially in postmitotic systems. Here, we leveraged the non-homologous end joining (NHEJ) mediated non-random editing profiles to compensate the frameshift mutation in a USH1F mouse model - av3j. After treatment by the selected gRNA, about 50% editing products showed reading-frame restoration, and more than 70% targeted hair cells recovered mechanotransduction. In vivo treatment ameliorated the hearing and balance symptoms in homozygous mutant mice. Furthermore, a scale-up analysis of 114 gRNAs targeting 40 frameshift deafness mutations reveals that 65% loci have at least one gRNA with predicted therapeutic potential. Together, our study demonstrates that the NHEJ-mediated frame restoration is a simple and highly efficient therapeutic strategy for small-indel induced frameshift mutations.

neuroscience↗

bioRxiv2020

Piezo2 mediates ultrasonic hearing via cochlear outer hair cells in mice

Ultrasonic hearing is exploited for hunting and navigation as in echolocation by microbats and bottleneck dolphins, and for social communication like ultrasonic vocalization by mice and rats. However, the molecular and cellular basis for ultrasonic hearing is not known yet. Here we show that knockout of the mechanosensitive ion channel PIEZO2 in cochlea disrupts the ultrasonic hearing but not the low-frequency hearing in mice, as shown by audiometry and acoustically-associative freezing behavior. Deletion of Piezo2 in the outer hair cells specifically abolishes the associative learning of the mice upon hearing the ultrasonic frequency. Ex vivo cochlear Ca2+ imaging revealed that the ultrasonic transduction requires both PIEZO2 and the hair-cell mechanotransduction channel. Together, our study demonstrates that the outer hair cells are the effector cells with PIEZO2 as an essential molecule for ultrasonic hearing in mice. Significance StatementSome animals have evolved an incredible ability for vocalizing and hearing ultrasonic frequencies that is inaudible for humans (> 20 kHz). For many years, it has been considered that animals hear ultrasonic frequencies with their cochlear hair cells, using the identical set of mechanotransduction molecules in the hair bundles for hearing audible frequencies. Here, we show that the mice lacking the mechanosensitive ion channel PIEZO2 hardly hear ultrasonic frequencies, while can still be sensitive to audible frequencies. Thus, animals may use a partially different mechanism for sensing physiological ultrasound.

neuroscience↗

bioRxiv2020

Phytophthora sojae effector Avr1d functions as E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection

Oomycete pathogens such as Phytophthora secrete a repertoire of effectors to host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy-roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By co-immunoprecipitation, gel infiltration and ITC assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure of Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy-roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor, negatively regulating soybean resistance against P. sojae. Altogether, this study highlights a novel virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This is the first study to unravel the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding. Significance StatementUbiquitination acts as a crucial regulator in plant immunity. Accordingly, microbial pathogens secrete effectors to hijak host ubiquitination system. However, the molecular mechanisms by which microbial effectors modulate host ubiquitination system are not yet clear. Here, we found that the Phytophthora sojae effector Avr1d physically binds to the U-box type E3 ligase GmPUB13, a susceptibility factor in soybean. The crystal structure of Avr1d in complex with GmPUB13 revealed that Avr1d occupies the binding site in GmPUB13 for the E2 ubiquitin conjugating enzyme and competes with E2 for physical binding to GmPUB13. Avr1d stabilized GmPUB13 by suppressing the self-ubiquitination activity of GmPUB13 and thereby promoting Phytophthora infection. This study provides structural basis for modulation of host targets by Phytophthora effectors.

microbiology↗

bioRxiv2020

Upgraded CRISPR/Cas9 Tools for Tissue-Specific Mutagenesis in Drosophila

ABSTRACTCRISPR/Cas9 has emerged as a powerful technology for tissue-specific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available in Drosophila remain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here we tackle these deficiencies by building upon our previous CRISPR-mediated tissue restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissue-specific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissue-specific Cas9 lines based on homology-assisted CRISPR knock-in (HACK). In this way, we expand the choices of Cas9 for CRISPR-TRiM analysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective in Drosophila. These improvements may be also applied to other model systems.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genetics↗