Biology subjects

Cheng, Z.

Publications and source records attributed to Cheng, Z..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Hybrid Modeling of Engineered Biological Systems through Coupling Data-Driven Calibration of Kinetic Parameters with Mechanistic Prediction of System Performance

Mechanistic models can provide predictive insight into the design and optimization of engineered biological systems, but the kinetic parameters in the models need to be frequently calibrated and uniquely identified. This limitation can be addressed by integrating mechanistic models with data-driven approaches, a strategy known as hybrid modeling. Herein, we developed a hybrid modeling strategy using bioelectrochemical systems as a platform system. The data-driven component of the model consisted of artificial neural networks (ANNs) that were trained by using mechanistically derived parameter values (e.g., the maximum specific growth rate {micro}max and the maximum substrate utilization rate kmax for the fermentative, electroactive, and methanogenic populations, and the mediator yield for electroactive microbes YM) as outputs to compute error signals. The hybrid model was built using 148 samples collected from 25 publications. After ten-fold cross-validation, the model was tested with another 28 samples. Internal resistance was accurately predicted with a relative root-mean-square error (RMSE) of 3.9%. Microbial kinetic parameters were also calibrated using the data-driven component. They were fed into the mechanistic component to predict system performance. The R2 between the predicted and observed organic removal and current production for systems fed with a simple substrate were 0.90 and 0.94, respectively, significantly higher than those obtained with a standalone data-driven model (0.51 and 0) and a standalone mechanistic model (0.07 and 0.15). The hybrid modeling strategy can potentially be applied to a variety of engineered biological systems for in silico system design and optimization. SYNOPSISA hybrid modeling strategy was developed to predict the performance of engineered biological systems without the need for laborious experiment-based parameter calibration.

microbiology↗

bioRxiv2023

Bacterial Cytochrome P450-catalyzed Post-translational Macrocyclization

Bacterial cytochrome P450s represent an emerging enzyme family that can modify ribosomally synthesized peptides to generate structurally complex macrocyclic skeletons. However, the functional sequence space of this type of enzyme is largely unexplored. In this study, we conduct a systematic genome mining of small ribosomal peptide-tailoring P450s from genomes of actinobacteria via a precursor-centric, primary sequence-, and structure-guided strategy. We uncovered 1,957 putative P450s, prioritized two representative families for functional study, and characterized two P450 enzymes that can respectively catalyze Tyrosine-to-Tryptophan and Tryptophan-to-Tryptophan crosslinks to form 3-mer or 4-mer macrocycle. These two P450 enzymes exhibit broad substrate selectivity, suggesting a promising starting template for engineering unnatural cyclic peptide construction. Our work expanded the enzymatic catalysis of P450s and could inspire the community to discover hidden peptide-modifying enzymes.

biochemistry↗

bioRxiv2023

Pathogen protein modularity enables elaborate mimicry of a host phosphatase

Pathogens produce diverse effector proteins to manipulate host cellular processes. However, how functional diversity is generated in an effector repertoire is poorly understood. Many effectors in the devastating plant pathogen Phytophthora contain tandem repeats of the "(L)WY" motif, which are structurally conserved but variable in sequences. Here, we discovered a functional module formed by a specific (L)WY-LWY combination in multiple Phytophthora effectors, which efficiently recruit the Serine/Threonine protein phosphatase 2A (PP2A) core enzyme in plant hosts. Crystal structure of an effector-PP2A complex shows that the (L)WY-LWY module enables hijacking of the host PP2A core enzyme to form functional holoenzymes. While sharing the PP2A-interacting module at the amino terminus, these effectors possess divergent C-terminal LWY units and regulate distinct sets of phosphoproteins in the host. Our results highlight the appropriation of an essential host phosphatase through molecular mimicry by pathogens and diversification promoted by protein modularity in an effector repertoire.

plant biology↗

bioRxiv2023

Anti-CRISPR AcrIIC5 is a dsDNA mimic that inhibits type II-C Cas9 effectors by blocking PAM recognition

Anti-CRISPR proteins are encoded by phages to inhibit the CRISPR-Cas systems of the hosts. AcrIIC5 inhibits several naturally high-fidelity type II-C Cas9 enzymes, including orthologs from Neisseria meningitidis (Nme1Cas9) and Simonsiella muelleri (SmuCas9). Here, we solve the structure of AcrIIC5 in complex with Nme1Cas9 and sgRNA. We show that AcrIIC5 adopts a novel fold to mimic the size and charge distribution of double-stranded DNA, and uses its negatively charged grooves to bind and occlude the protospacer adjacent motif (PAM) binding site in the target DNA cleft of Cas9. AcrIIC5 is positioned into the crevice between the WED and PI domains of Cas9, and one end of the anti-CRISPR interacts with the phosphate lock loop and a linker between the RuvC and BH structural domains. We employ biochemical and mutational analyses to build a model for AcrIIC5s mechanism of action, and identify residues on both the anti-CRISPR and Cas9 that are important for their interaction and inhibition. Together, the structure and mechanism of AcrIIC5 reveal convergent evolution among disparate anti-CRISPR proteins that use a DNA-mimic strategy to inhibit diverse CRISPR-Cas surveillance complexes, and provide new insights into a tool for potent inhibition of type II-C Cas9 orthologs.

biochemistry↗

bioRxiv2022

Enhanced survival and low proliferation marks multifunctional virus specific memory CD4 T cells

Cytokine production by memory T cells is a key mechanism of T cell mediated protection. However, we have a limited understanding of the survival and secondary responses of memory T cells with cytokine producing capacities. We interrogate antigen-specific CD4 T cells using a mouse influenza A virus infection model. CD4 T cells with the capacity to produce cytokines survive better than non-cytokine+ cells, displaying a low fold contraction and expressing high levels of pro-survival molecules, CD127 and Bcl2. Transcriptomic analysis reveals a heterogenous population of memory CD4 T cells with three clusters of cytokine+ cells. These clusters match flow cytometry data revealing an enhanced survival signature in cells capable of producing multiple cytokines. These multifunctional cells are, however, less likely to proliferate during and following primary and secondary infections. Despite this, multifunctional memory T cells form a substantial fraction of the secondary memory pool, indicating that survival rather than proliferation may dictate which populations survive within the memory pool. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/520219v3_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@93e62forg.highwire.dtl.DTLVardef@1ed2444org.highwire.dtl.DTLVardef@1e7f79borg.highwire.dtl.DTLVardef@30e31d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

immunology↗

bioRxiv2022

Crowdsourcing Temporal Transcriptomic Coronavirus Host Infection Data: resources, guide, and novel insights.

The emergence of SARS-CoV-2 reawakened the need to rapidly understand the molecular etiologies, pandemic potential, and prospective treatments of infectious agents. The lack of existing data on SARS-CoV-2 hampered early attempts to treat severe forms of COVID-19 during the pandemic. This study coupled existing transcriptomic data from SARS-CoV-1 lung infection animal studies with crowdsourcing statistical approaches to derive temporal meta-signatures of host responses during early viral accumulation and subsequent clearance stages. Unsupervised and supervised machine learning approaches identified top dysregulated genes and potential biomarkers (e.g., CXCL10, BEX2, and ADM). Temporal meta-signatures revealed distinct gene expression programs with biological implications to a series of host responses underlying sustained Cxcl10 expression and Stat signaling. Cell cycle switched from G1/G0 phase genes, early in infection, to a G2/M gene signature during late infection that correlated with the enrichment of DNA Damage Response and Repair genes. The SARS-CoV-1 meta-signatures were shown to closely emulate human SARS-CoV-2 host responses from emerging RNAseq, single cell and proteomics data with early monocyte-macrophage activation followed by lymphocyte proliferation. The circulatory hormone adrenomedullin was observed as maximally elevated in elderly patients that died from COVID-19. Stage-specific correlations to compounds with potential to treat COVID-19 and future coronavirus infections were in part validated by a subset of twenty-four that are in clinical trials to treat COVID-19. This study represents a roadmap to leverage existing data in the public domain to derive novel molecular and biological insights and potential treatments to emerging human pathogens. The data from this study is available in an interactive portal (http://18.222.95.219:8047).

bioinformatics↗

bioRxiv2022

Neuron Navigator 1 Regulates Learning, Memory, and the Response to Multiple Potentially Addictive Drugs

Genetic variation accounts for much of the risk for developing a substance use disorder (SUD). Inbred mouse strains exhibit substantial and heritable differences in the extent of voluntary cocaine intravenous self-administration (IVSA). Computational genetic analysis of IVSA data obtained from an inbred strain panel identified Nav1, a member of the neuron navigator family that regulates dendrite formation and axonal guidance, as a candidate gene. To test this hypothesis, we generated and characterized Nav1 knockout (KO) mice. Nav1 KO mice exhibited increased cocaine intake during IVSA testing. Surprisingly, Nav1 KO mice also displayed a reduced susceptibility to become opioid dependent or develop opioid-induced hyperalgesia after chronic morphine administration, and had impaired spatial learning/memory. Immunohistochemistry and electrophysiology studies revealed that inhibitory synapse density in the cortex of Nav1 KO mice was reduced, and excitatory synaptic transmission was increased in the Nav1 KO cortex and hippocampus. Transcriptomic analysis revealed that Nav1 KO mice had a marked increase in excitatory neurons in a deep cortical layer. Collectively, our results indicate that Nav1 regulates learning, memory, and the response to multiple addictive drugs, and that changes in the excitatory and inhibitory synaptic balance in the cortex and hippocampus could possibly mediate these phenotypic effects.

genetics↗

bioRxiv2022

Using spatio-temporal information in weather radar data to detect and track communal bird roosts

O_LIThe exodus of swallows from communal nighttime roosts is often visible as an expanding ring-shaped pattern in weather radar data. The WSR-88D network operated by the National Weather Service archives more than 25 years of data across 143 stations in the contiguous US. However, access to information about the roosting behavior of swallows is limited by the cost of manual annotation of these scans. C_LIO_LIWe develop an AI system to detect and track swallow roosts in weather radar data. Our model is based on the Faster R-CNN architecture and is customized to incorporate multiple spatial and temporal channels in volumetric radar scans using novel adaptor layers. We systematically study the impact of network architecture and input representation for this task. We incorporate our detection outputs into an AI-assisted system with an interface for human screening to collect research-grade data about roosting behavior. We deploy the system to collect information from 12 radar stations in the Great Lakes region of the US spanning 21 years. C_LIO_LIThe addition of temporal information improves roost detection performance from 47.0% mean average precision to 54.7%. Temporal information helps the model recognize the expanding pattern of roosts and filter false positives due to rain and static structures. Our system allowed the annotation of 15,628 roost signatures with 64,620 single-frame detections in 612,786 radar scans with 183.6 total hours of human screening, or 1.08 seconds per radar scan. C_LIO_LIOur AI-assisted system provides research-quality roost data with far less human effort than manual annotation of radar scans. The data contains critical information about the phenology and population trends of swallows and martins, a declining group of aerial insectivores. Our successful deployment to collect historical data for 8% of the radar stations in the contiguous US lays the groundwork for continentscale analysis of swallow roosts, and provides a starting point for analysis of other family-specific phenomena in weather radar, such as bat roosts and mayfly hatches. C_LI

ecology↗

bioRxiv2022

Structural Analysis of Phosphorylation Proteoforms in a Dynamic Heterogeneous System Using Flash Oxidation Coupled In-Line with Ion Exchange Chromatography

Protein post-translational modifications (PTMs) are key modulators of protein structure and function that often change in a dynamic fashion in response to cellular stimuli. Dynamic post-translational modifications are very challenging to structurally characterize using modern techniques, including covalent labeling methods, due to the presence of multiple proteoforms and conformers together in solution. Here, we have coupled ion exchange HPLC with a flash oxidation system (IEX LC-FOX) to successfully elucidate structural changes among three phosphoproteoforms of ovalbumin (OVA) during dephosphorylation with alkaline phosphatase (AP). Real-time dosimetry indicates no difference in effective radical dose between peaks or across the peak, demonstrating both the lack of scavenging of the NaCl gradient and the lack of a concentration effect on radical dose between peaks of different intensities. The use of IEX LC-FOX allows us to structurally probe each phosphoproteoform as it elutes from the column, capturing structural data before the dynamics of the system reintroduce heterogeneity. We found significant differences in residue-level oxidation between the hydroxyl radical footprint of non-phosphorylated, mono-phosphorylated and di-phosphorylated ovalbumin. Not only were our data consistent with the previously reported stabilization of ovalbumin structure by phosphorylation, but local structural changes were also consistent with the measured order of dephosphorylation of Ser344 being removed first. These results demonstrate the utility of IEX LC-FOX for measuring the structural effects of PTMs, even in dynamic systems.

biochemistry↗

bioRxiv2022

Role of mPFC DUSP1 in regulating the sensitivity to cocaine in adolescent cocaine-exposed mice of adulthood

Adolescent cocaine abuse increases the risk for developing addiction in later life, which remains a big public health concern, but the underlying molecular mechanism is poorly understood. Here, adolescent cocaine-exposed (ACE) male mice models were established by administrating cocaine during adolescent period. When growing to adult age, mice were subjected to conditioned place preference (CPP) to evaluate the sensitivity to cocaine, then potential molecule of dual specificity phosphatase 1 (DUSP1) were screened out by transcriptomic sequencing. Subthreshold dose of cocaine (sdC), that is insufficient to produce CPP, was used to induce CPP in adulthood. The sdC treatment effectively induced CPP in ACE mice during adulthood, accompanied with the more triggered CaMKII-positive neurons, and induced higher Dusp1 gene, lower DUSP1 protein, lower DUSP1 activity and lower DUSP1 expression on CaMKII-positive neurons (DUSP1CaMKII) in medial prefrontal cortex (mPFC). Overexpressing DUSP1CaMKII suppressed CaMKII-positive neuronal activation, and ultimately blocked sdC-induced CPP in ACE mice during adulthood. While, knocking-down DUSP1CaMKII activated more CaMKII-positive neurons, and aggravated sdC-preferred behavior in ACE mice during adulthood. ERK1/2 might be potential subsequent signal for DUSP1 in the process. Collectively, our findings reveal a novel molecular mechanism underlying adolescent drug abuse-induced susceptibility to addiction during adulthood, and mPFC DUSP1CaMKII is a promising pharmacological target to predict or treat addiction, especially caused by adolescent substance use. SummaryAdolescent cocaine exposure causes higher cocaine-preferred behaviors during adulthood, along with evoked mPFC activity in response to cocaine challenge. Locally overexpressing but not knocking-down the dual specificity phosphatase 1 (DUSP1) on CaMKII-positive neurons (DUSP1CaMKII) suppresses mPFC activation, and ultimately rescues the higher sensitivity to cocaine during adulthood.

neuroscience↗

bioRxiv2022

β1-adrenergic receptor on pyramidal neurons of mPFC regulates METH-preferred behaviors in METH-sired male mice

Paternal abuse of drugs, such as methamphetamine (METH), elevates the risk of developing addiction in subsequent generations, however, its underlying molecular mechanism remains poorly understood. Male adult mice (F0) were exposed to METH for 30 days, followed by mating with naive female mice to create the first-generation mice (F1). When growing to adulthood, F1 were subjected to conditioned place preference (CPP) test. Subthreshold dose of METH, insufficient to induce CPP normally, were used in F1 (METHF1). Selective antagonist (betaxolol) for {beta}1-adrenergic receptor (ADRB1) or its knocking-down virus were administrated into mPFC to regulate ADRB1 function and expression on CaMKII-positive neurons. METH-sired male F1 acquired METHF1-induced CPP, indicating that paternal METH exposure induce higher sensitivity to METH in male F1. Compared with saline (SAL)-sired male F1, CaMKII-positive neuronal activity was normal without METHF1, but strongly evoked after METHF1 treatment in METH-sired male F1 during adulthood. METH-sired male F1 had higher ADRB1 levels without METHF1, which was kept at higher levels after METHF1 treatment in mPFC. Either inhibiting ADRB1 function with betaxolol, or knocking-down ADRB1 level on CaMKII-positive neurons (ADRB1CaMKII) with virus transfection efficiently suppressed METHF1-evoked mPFC acyivation, and ultimately blocked METHF1-induced CPP in METH-sired male F1. In the process, the p-ERK1/2 and {Delta}FosB may be potential subsequent signals of mPFC ADRB1CaMKII. The mPFC ADRB1CaMKII mediates paternal METH exposure-induced higher sensitivity to drug addiction in male offspring, raising a promising pharmacological target for predicting or treating transgenerational addiction. HIGHLIGHTSO_LIPaternal methamphetamine (METH) exposure induces higher sensitivity to METH in male F1 during adulthood, accompanied with higher ADRB1 level in mPFC. C_LIO_LIMETH use in F1 (METHF1) evokes more CaMKII-positive neurons in mPFC of METH-sired than saline-sired male F1. C_LIO_LIInhibiting ADRB1 function or knocking-down ADRB1 level on CaMKII-positive neurons (ADRB1CaMKII) efficiently suppresses METHF1-evoked mPFC activation, and ultimately rescues transgenerational susceptibility to addiction in male F1. C_LI

neuroscience↗

bioRxiv2022

Biofunctional matrix models reveal mineral-dependent mechanoregulation of bone metastatic breast cancer

Bone metastasis is a leading cause of breast cancer-related deaths and often initiated by tumor cell dissemination to osteogenic niches. During new bone formation, osteoblasts first deposit osteoid, the collagen I-rich, unmineralized component of bone ECM, within which carbonated hydroxyapatite nanoparticles subsequently form. However, it remains elusive how bone matrix mineralization dictates tumor cell phenotype due in part to the lack of relevant model systems. Using biofunctional, collagen I-based bone matrix models with physiological, intrafibrillar mineralization, we show that mineralization inhibits proliferation, while inducing a stem-like phenotype in tumor cells. These changes were due to reduced mechanosignaling contradicting the conventional assumption that increased rigidity caused by mineralization stimulates metastatic progression. Our findings are translationally relevant as the presence of mineral reduced tumor growth in vivo and upregulated a gene signature that correlated with decreased patient mortality. Our results could help explain why decreased bone mineral density increases the risk for bone metastasis in patients and highlight that bone metastasis models should integrate organic and inorganic matrix components in a manner that mimics physiological mineralization.

bioengineering↗

bioRxiv2022

Neutrophils reprograms the bone marrow to impair T-cell immunity during tuberculosis

Mycobacterium tuberculosis (Mtb) infection induces persistent influx of neutrophils that associates with poor bacterial control and clinical outcome from tuberculosis (TB). Although implicated in TB pathology, the mechanism by which these cells contribute to pathogenesis is poorly understood. Using Cell-DIVE multiplexed immunofluorescence imaging and spatial analysis of inflammatory TB lesions, we demonstrated that persistent neutrophil infiltration affects the spatiotemporal organization of T-lymphocytes and impairs their function. Instead of directly suppressing T-cells, neutrophils produce granulocyte colony stimulating factor (CSF3/G-CSF) that collaborates with type I interferon (IFN-I) to promote a granulocyte-skewed hematopoiesis impacting T-lymphocyte production. Importantly, neutrophil-intrinsic IFN-I receptor 1 (IFNAR1) is both necessary and sufficient to promote pathologic granulopoiesis. Finally, inhibition of IFNAR1-signaling alone mitigates immunopathogenesis by restoring hematopoietic equilibrium. Collectively, our work uncovers a potential immunevasion strategy by which virulent Mtb strains induce IFN-I to generate pathogen-permissive neutrophils that produce G-CSF and sustain pathogenic hematopoiesis to impair T-cell immunity during TB.

immunology↗

bioRxiv2022

Automated detection of extracellular action potentials from single neurons

Multi-electrode arrays (MEAs) non-invasively record extracellular action potentials (eAPs, also known as spikes) from hundreds of neurons simultaneously. However, because extracellular electrodes sample from the local electrical field, each electrode can detect eAPs from multiple nearby neurons. Interpreting spike trains at individual electrodes of high-density arrays requires spike sorting, a computational process which groups eAPs from single units based on assumptions of how spike waveforms correlate with different neuronal sources. Additionally, when experimental conditions result in changes to eAP waveforms, spike sorting routines may have difficulty correlating eAPs from multiple neurons at single electrodes before and after such waveform changes. We present here a novel, empirical method for unambiguously isolating eAPs from individual, uniquely identifiable neurons, based on automated multi- point detection of action potential propagation. This method is insensitive to changes in eAP waveform morphology because it makes no assumptions about the relationship between spike waveform and neuronal source. Our algorithm for automated detection of action potential propagation produces a fingerprint that uniquely identifies those spikes from each neuron. By unambiguously isolating eAPs from multiple neurons in each recording, on a range of platforms and experimental preparations, our method now enables high-content screening with contemporary MEAs. We outline the limitations and strengths of propagation-based isolation of eAPs from single neurons and propose how our automated method complements spike sorting and could be adapted to in vivo use.

neuroscience↗

bioRxiv2022

Probing neuronal functions with precise and targeted laser ablation in the living cortex: reply

In the comment on the article [Optica 8, 12 (2021)], the authors performed theoretical calculation to show that with a 1.1 NA objective and 140 fs laser pulses with single pulse energy of 2.2 nJ, and 80 MHz repetition rate, the focal point temperature rises 0.3 K and reaches equilibrium after 100 s in water. They suggest that the damage to brain tissue by laser could not be caused by thermal effects but rather by plasma-mediated chemical effects. To quantify the thermal accumulation due to the fs laser illumination in living animals, we used a thermocouple sensor to measure the temperature change in the vicinity of the laser focus at a depth of 300 m in the adult mouse cortex. Our results show that at 930 nm wavelength, 25 m from the laser focus, 19-300 mW laser can all lead to a brain tissue temperature rise of more than 0.3 K at 1 second and the maximum equilibrium temperature rise of more than 28 K. These experimental measurements are significantly higher than theoretically calculated values in the comment. These results suggest that the thermal accumulation effect of focused low-energy pulses from fs laser oscillators could contribute significantly to the collateral damage in the living brain.

neuroscience↗

bioRxiv2022

Topological supramolecular network enabled highly conductive and stretchable organic bioelectronics

Intrinsically stretchable bioelectronic devices based on soft and conducting organic materials have been regarded as the ideal interface for seamless and biocompatible integration with the human body. However, the grand challenge remains for the conducting polymer to possess both high mechanical ductility and good electrical conduction at cellular level feature sizes. This longstanding material limitation in organic bioelectronics has impeded the full exploitation of its unique benefits. Here, we introduce a new molecular engineering strategy based on rationally designed topological supramolecular networks, which allows effective decoupling of competing effects from multiple molecular building blocks to meet complex requirements. We achieve two orders of magnitude improvement in the conductivity under 100% strain in physiological environment, along with the capability for direct photopatterning down to 2 m. These unprecedented capabilities allow us to realize previously inaccessible bioelectronic applications including high-resolution monitoring of soft and malleable creatures, e.g., octopus, and localized neuromodulation down to single nucleus precision for controlling organ-specific activities through delicate tissues, e.g., brainstem.

bioengineering↗

bioRxiv2021

Super-resolution vibrational imaging using expansion stimulated Raman scattering microscopy

Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously due to spectral overlap. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, we describe a new technique called Molecule Anchorable Gel-enabled Nanoscale Imaging of Fluorescence and stImulatEd Raman Scattering microscopy (MAGNIFIERS), that integrates SRS microscopy with expansion microscopy (ExM). ExM is a powerful strategy providing significant improvement in imaging resolution by physical magnification of hydrogel-embedded preserved biological specimens. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. We used MAGNIFIERS to visualize nanoscale features in a label-free manner with C-H vibration of proteins, lipids and DNA in a broad range of biological specimens, from mouse brain, liver and kidney to human lung organoid. In addition, we applied MAGNIFIERS to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, we used MAGNIFIERS to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.

bioengineering↗

bioRxiv2021

Efficient transformation and genome editing of watermelon assisted by genes that encode developmental regulators

AbstructThe Cucurbitaceae contains multiple species of important food plants. But most of them are difficult to be genetically transformed. Watermelon is one of the most important fruit species of Cucurbitaceae, and it is a model horticulture crops. Its low transgenic efficiency is the major bottleneck in functional genome research and genome editing-based breeding. Here, with the help of genes that encode developmental regulators (DRs), in particular AtGRF5, and an appropriate Agrobacterium strain (GV3101), we have significantly increased the transformation efficiency of watermelon to about 25%, which is a 40-fold increase compared with a traditional vector. AtGRF5-mediated transformation increased the number of transformable watermelon genotypes without causing obvious side effects. Moreover, when applying this strategy to CRISPR/Cas9-mediated genome editing, clpds knockout mutants were generated in the T0 generation. Our results show that AtGRF5 is a powerful and robust tool to effectively create transgenic plants or knockout mutations in watermelon. Similar strategies using DRs might also be able to overcome the transformation barriers in many other Cucurbitaceae species.

plant biology↗