High-throughput Functional Characterization Research Articles

Transporter annotations are holding up progress in metabolic modeling

Mechanistic, constraint-based models of microbial isolates or communities are a staple in the metabolic analysis toolbox, but predictions about microbe-microbe and microbe-environment interactions are only as good as the accuracy of transporter annotations. A number of hurdles stand in the way of comprehensive functional assignments for membrane transporters. These include general or non-specific substrate assignments, ambiguity in the localization, directionality and reversibility of a transporter, and the many-to-many mapping of substrates, transporters and genes. In this perspective, we summarize progress in both experimental and computational approaches used to determine the function of transporters and consider paths forward that integrate both. Investment in accurate, high-throughput functional characterization is needed to train the next-generation of predictive tools toward genome-scale metabolic network reconstructions that better predict phenotypes and interactions. More reliable predictions in this domain will benefit fields ranging from personalized medicine to metabolic engineering to microbial ecology.

Disruption of KLHL6 Fuels Oncogenic Antigen Receptor Signaling in B-Cell Lymphoma.

Pathomechanisms that activate oncogenic B-cell receptor (BCR) signaling in diffuse large B-cell lymphoma (DLBCL) are largely unknown. Kelch-like family member 6 (KLHL6) encoding a substrate-adapter for Cullin-3-RING E3 ubiquitin ligase with poorly established targets is recurrently mutated in DLBCL. By applying high-throughput protein interactome screens and functional characterization, we discovered that KLHL6 regulates BCR by targeting its signaling subunits CD79A and CD79B. Loss of physiologic KLHL6 expression pattern was frequent among the MCD/C5-like activated B-cell DLBCLs and was associated with higher CD79B levels and dismal outcome. Mutations in the bric-a-brac tramtrack broad domain of KLHL6 disrupted its localization and heterodimerization and increased surface BCR levels and signaling, whereas Kelch domain mutants had the opposite effect. Malfunctions of KLHL6 mutants extended beyond proximal BCR signaling with distinct phenotypes from KLHL6 silencing. Collectively, our findings uncover how recurrent mutations in KLHL6 alter BCR signaling and induce actionable phenotypic characteristics in DLBCL. Significance: Oncogenic BCR signaling sustains DLBCL cells. We discovered that Cullin-3-RING E3 ubiquitin ligase substrate-adapter KLHL6 targets BCR heterodimer (CD79A/CD79B) for ubiquitin-mediated degradation. Recurrent somatic mutations in the KLHL6 gene cause corrupt BCR signaling by disrupting surface BCR homeostasis. Loss of KLHL6 expression and mutant-induced phenotypes associate with targetable disease characteristics in B-cell lymphoma. See related commentary by Leveille et al. See related commentary by Corcoran et al.

Genome-wide association study identifies human genetic variants associated with fatal outcome from Lassa fever

Infection with Lassa virus (LASV) can cause Lassa fever, a haemorrhagic illness with an estimated fatality rate of 29.7%, but causes no or mild symptoms in many individuals. Here, to investigate whether human genetic variation underlies the heterogeneity of LASV infection, we carried out genome-wide association studies (GWAS) as well as seroprevalence surveys, human leukocyte antigen typing and high-throughput variant functional characterization assays. We analysed Lassa fever susceptibility and fatal outcomes in 533 cases of Lassa fever and 1,986 population controls recruited over a 7 year period in Nigeria and Sierra Leone. We detected genome-wide significant variant associations with Lassa fever fatal outcomes near GRM7 and LIF in the Nigerian cohort. We also show that a haplotype bearing signatures of positive selection and overlapping LARGE1, a required LASV entry factor, is associated with decreased risk of Lassa fever in the Nigerian cohort but not in the Sierra Leone cohort. Overall, we identified variants and genes that may impact the risk of severe Lassa fever, demonstrating how GWAS can provide insight into viral pathogenesis.

PANDA: a self-driving lab for studying electrodeposited polymer films.

We introduce the polymer analysis and discovery array (PANDA), an automated system for high-throughput electrodeposition and functional characterization of polymer films. The PANDA is a custom, modular, and low-cost system based on a CNC gantry that we have modified to include a syringe pump, potentiostat, and camera with a telecentric lens. This system can perform fluid handling, electrochemistry, and transmission optical measurements on samples in custom 96-well plates that feature transparent and conducting bottoms. We begin by validating this platform through a series of control fluid handling and electrochemistry experiments to quantify the repeatability, lack of cross-contamination, and accuracy of the system. As a proof-of-concept experimental campaign to study the functional properties of a model polymer film, we optimize the electrochromic switching of electrodeposited poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) films. In particular, we explore the monomer concentration, deposition time, and deposition voltage using an array of experiments selected by Latin hypercube sampling. Subsequently, we run an active learning campaign based upon Bayesian optimization to find the processing conditions that lead to the highest electrochromic switching of PEDOT:PSS. This self-driving lab integrates optical and electrochemical characterization to constitute a novel, automated approach for studying functional polymer films.

An improved method for large scale generation and high-throughput functional characterization of human iPSC-derived microglia

Neuroscience drug discovery has faced significant challenges due to restricted access to relevant human cell models and limited translatability of existing preclinical findings to human pathophysiology. Induced pluripotent stem cells (iPSCs) have emerged as a promising solution, offering the potential to generate patient-specific cell types, including in the recent years, iPSC-derived microglia (iMGL). Current methods rely on complex and time-consuming differentiation procedures, leading to considerable batch-to-batch variability consequently hindering the establishment of standardized and reproducible high-throughput functional screening approaches. Addressing these challenges is critical in ensuring the generation of homogenous iMGL populations with consistent functional properties. In this study we describe an improved high-yield protocol for generating iMGL, which allows for increased reproducibility and flexibility in the execution of high-throughput functional screens. We introduce a two-step process in embryoid bodie (EB) maintenance and a stop point allowing for cryopreservation at the hematopoietic progenitor cell (iHPC) stages. Furthermore, we demonstrate inter-operator robustness of this modified protocol in a range of high-throughput functional assays including phagocytosis, lysosomal acidification, chemotaxis, and cytokine release. Our study underscores the importance of quality control checks at various stages of iPSC-differentiation and functional assay set up, highlighting novel workarounds to the existing challenges such as limited yield, flexibility, and reproducibility, all critical in drug discovery.

High-throughput functional characterization of combinations of transcriptional activators and repressors.

Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, whereas combining strong activators often results in weaker activation. In contrast, repressors combine linearly and produce full gene silencing, and repressor domains often overpower activation domains. We use this information to build a synthetic transcription factor whose function can be tuned between repression and activation independent of recruitment to target genes by using a small-molecule drug. Altogether, we outline the basic principles of how effector domains combine to regulate gene expression and demonstrate their value in building precise and flexible synthetic biology tools. A record of this paper's transparent peer review process is included in the supplemental information.

Abstract P1019: High-throughput Generation And Functional Characterization Of Myosin Heavy Chain 7 Variants In Genome-edited Cardiomyocytes

Hypertrophic cardiomyopathy (HCM), an inheritable cardiac condition characterized by idiopathic left ventricular hypertrophy, can cause sudden cardiac death or heart failure. Autosomal-dominant missense mutations in MYH7 (encoding the sarcomeric βMHC protein) account for 33% of genotype-positive HCM cases. While pathogenic MYH7 variants are clinically-actionable, 75% of MYH7 missense variants in ClinVar are variants of unknown significance (VUS). Gene-editing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can help determine the function of MYH7 VUS, however, the discovery of new variants by genetic testing outpaces the ability to gene-edit hiPSCs. Thus, we developed a novel method called CR ISPR a On- T arget E diting R etrieval (CRaTER) which increases the success of gene-editing by 25-fold compared to current methods. We leveraged CRaTER to enrich for hiPSCs edited with a library of MYH7 missense variants and recovered 113 different heterozygous variants. As deleterious mutations can reduce protein stability, we flow sorted MYH7 variant hiPSC-CMs into bins based on differing βMHC abundance followed by next generation sequencing of each bin to calculate variant abundance scores. This assay identified 31 MYH7 missense variants as functionally abnormal (lower abundance scores than synonymous controls), including all known pathogenic variants in the library. This βMHC depletion phenotype was validated in clonal hiPSC-CMs and supported by western blotting of myocardium from a patient with a pathogenic MYH7 missense mutation. Furthermore, we rescued βMHC protein levels by genetically ablating the gene encoding for Muscle RING-finger protein-1, an E3 protein ligase that targets βMHC for degradation, and found that the cardiomyocyte hypertrophy induced by the pathogenic MYH7 E848G HCM variant was significantly blunted. In summary, CRaTER enables the generation of gene-edited hiPSC lines at unprecedented scale. Functional analysis of MYH7 variant hiPSC-CMs reveals a novel correlation between loss of βMHC protein and clinical pathogenicity that can be leveraged for clinical variant interpretation. Interventions that prevent the loss of βMHC may potentially be therapeutic for MYH7 -associated HCM.

Single-Cell-Based High-Throughput Cultivation and Functional Characterization of Biosurfactant-Producing Bacteria from Soil and Oilfield-Produced Water.

Biosurfactants are a group of surface-active compounds that can be produced by diverse microorganisms. They have been widely used in various industrial fields. Reducing production costs, improving efficiency, and collecting more diverse producing strains have become major challenges in the biosurfactant industry. These challenges could be overcome by screening for more diverse and efficient biosurfactant-producing strains. The conventional methods for the isolation and functional characterization of microorganisms are laborious and biased toward fast-growing or strongly competitive microorganisms. Here, we established a high-throughput approach of single-cell-based cultivation and functional characterization of biosurfactant-producing bacteria (SCCBB). This approach combines single-cell cultivation with the detection of optical distortions. Using this approach, we isolated 431 strains with biosurfactant production potential from petroleum-contaminated soil and oilfield-produced water. The surfactant production capabilities of the strains were subsequently validated using surface tension measurements, TLC, and CMC measurements. To investigate the industrial production potential, we optimized the production conditions of a representative glycolipids-producing strain, Pseudomonas sp. L01, using response surface methodology (RSM). Optimal conditions yielded a crude biosurfactant yield of 8.43 g/L in a flask. Our work provides a high-throughput approach to the isolation and screening of biosurfactant-producing bacteria, as well as other functional bacteria in a wide range of fields.

Dysregulation and therapeutic targeting of RNA splicing in cancer.

High-throughput sequencing and functional characterization of the cancer transcriptome have uncovered cancer-specific dysregulation of RNA splicing across a variety of cancers. Alterations in the cancer genome and dysregulation of RNA splicing factors lead to missplicing, splicing alteration-dependent gene expression and, in some cases, generation of novel splicing-derived proteins. Here, we review recent advances in our understanding of aberrant splicing in cancer pathogenesis and present strategies to harness cancer-specific aberrant splicing for therapeutic intent.

Crowdsourced RNA design discovers diverse, reversible, efficient, self-contained molecular switches.

Internet-based scientific communities promise a means to apply distributed, diverse human intelligence toward previously intractable scientific problems. However, current implementations have not allowed communities to propose experiments to test all emerging hypotheses at scale or to modify hypotheses in response to experiments. We report high-throughput methods for molecular characterization of nucleic acids that enable the large-scale video game–based crowdsourcing of RNA sensor design, followed by high-throughput functional characterization. Iterative design testing of thousands of crowdsourced RNA sensor designs produced near–thermodynamically optimal and reversible RNA switches that act as self-contained molecular sensors and couple five distinct small molecule inputs to three distinct protein binding and fluorogenic outputs. This work suggests a paradigm for widely distributed experimental bioscience.

Generation of heterozygous (MRli003-A-5) and homozygous (MRli003-A-6) voltage-sensing knock-in human iPSC lines by CRISPR/Cas9 editing of the AAVS1 locus

Assessment of the electrophysiological properties of cardiomyocytes is necessary for phenotyping cardiac disorders and for drug screening. Optical action potential imaging using a genetically encoded voltage-sensing fluorescent protein (VSFP) allows for high-throughput functional characterization of cardiomyocytes, which offers an advantage over the traditional patch-clamp technique. Here, we knocked VSFP into the AAVS1 safe harbor locus of human iPSCs, generating two stable voltage indicator lines - one heterozygous (MRIi003-A-5) and the other homozygous (MRI003-A-6). Both lines can be used for optical membrane potential recordings and provide a powerful platform for a wide range of applications in cardiovascular biomedicine.

EP158: The utility of transcriptomics to increase diagnostic yield in cases of rare genetic disease

Rare diseases affect less than 1 in 200,000 individuals by definition, however, collectively are common, with approximately 30 million individuals in the United States impacted. Frequently individuals experience an arduous journey towards a rare disease diagnosis, often termed a “diagnostic odyssey”. Exome sequencing (ES) has become standard of care for patients with suspected rare genetic disease, with a diagnostic yield of 30%. Here we report on preliminary results of the largest study to date integrating transcriptome sequencing (RNAseq) with ES to increase diagnostic yield for rare disease patients. A cohort of 406 patients who were consented into the Mayo Clinic Diagnostic Odyssey study were selected for study. All patients had received clinical or research exome sequencing which failed to result in a definitive diagnosis. RNA was extracted from whole blood collected in PAXgene tubes, and sequencing libraries prepared using a hybridization capture protocol with the TruSeq RNA Access Library Prep Kit (Illumina, San Diego, CA). Paired-end 101-basepair reads were sequenced using Illumina instruments. Resulting RNA sequences were analyzed for fusion using two algorithms, LeafCutterMD and Fraser, with results integrated for review. Allele specific expression was determined using AnevaDot. Gene expression was analyzed for outlier expression patterns in individual patients compared to the entire cohort using Outrider. Fusion analysis was performed using an internal workflow previously described. The major advantages RNAseq in addition to ES are the high-throughput functional characterization of DNA variation and the detection of aberrations not identifiable by ES sequencing alone. Recent work has shown a 7.5-35% yield using RNAseq in unsolved rare disease cases, dependent on tissue and methodology. In this study RNAseq was performed on blood from 406 individuals with suspected rare genetic disease for whom clinical ES was non-diagnostic. The transcriptome data was analyzed to interrogate four different effects: genes with outlier expression, aberrant RNA-splicing, gene fusions, and allele specific expression. Splicing analysis utilized two complementary algorithms, FRASER and LeafcutterMD, to identify modified intron usage, shifted splice donor and acceptor sites, intron retention events, and creation of cryptic exons. All data were studied for ultra-rare modifications with potential disease ramifications, using the remaining patient cohort as a control RNASeq population. Our preliminary results include gene fusion events, splice site variants with confirmed splicing alterations, and significantly altered outlier gene expression which were integral to the patient’s genetic diagnosis. RNA sequencing is complementary to DNA sequencing in the search for genetic diagnoses in Mendelian disease. When analyzing RNA data, care must be taken to evaluate all forms of RNA mediated events to obtain a thorough representation of the underlying genetics. Here we discuss a 406 patient cohort study using RNA and DNA sequencing to profile patient seen at the Mayo Clinic. Complete results of this study will provide valuable information regarding the clinical utility of RNAseq in tandem with ES for identifying a genetic diagnosis for rare disease patients.

Saturation variant interpretation using CRISPR prime editing.

High-throughput functional characterization of genetic variants in their endogenous locus has so far been possible only with methods that rely on homology-directed repair, which are limited by low editing efficiencies. Here, we adapted CRISPR prime editing for high-throughput variant classification and combined it with a strategy that allows for haploidization of any locus, which simplifies variant interpretation. We demonstrate the utility of saturation prime editing (SPE) by applying it to the NPC intracellular cholesterol transporter 1 gene (NPC1), mutations in which cause the lysosomal storage disorder Niemann-Pick disease type C. Our data suggest that NPC1 is very sensitive to genetic perturbation, with 410 of 706 assayed missense mutations being classified as deleterious, and that the derived function score of variants is reflective of diverse molecular defects. We further applied our approach to the BRCA2 gene, demonstrating that SPE is translatable to other genes with an appropriate cellular assay. In sum, we show that SPE allows for efficient, accurate functional characterization of genetic variants.

Construction of a high-density mutant population of Chinese cabbage facilitates the genetic dissection of agronomic traits

Chinese cabbage (Brassica rapa ssp. pekinensis) is an economically important vegetable crop throughout the world, especially in Asia. High-quality genome sequences are available for Chinese cabbage, but gene functional studies remain challenging. To promote functional genomic studies of Chinese cabbage, we generated an ethyl methane sulfonate (EMS) mutant population of ∼8000 M2 plants using the double haploid inbred line A03 as the parent. The genome of A03 was sequenced and used as a reference for high-throughput functional characterization of gene mutations at the whole-genome level. A total of 300 M2 to M5 EMS mutants were phenotypically screened and then sequenced, revealing 750 629 SNPs and 46 272 InDel mutations that cover 98.27% of all predicted genes in the A03 genome. A forward-genetics approach was successfully used to identify two genes with chloroplast-related functions that are responsible for the yellow leaf mutant trait. A reverse-genetics approach was also used to identify associations between mutations in five genes of the glucosinolate biosynthetic pathway and variations in glucosinolate content of the mutant plants. In addition, we built the Chinese cabbage EMS mutation database (CCEMD, www.bioinformaticslab.cn/EMSmutation/home) to increase the usability of this mutant population resource. In summary, we performed large-scale screening of a heading Chinese cabbage EMS mutant collection at the phenotypic and genotypic levels, which will facilitate gene mining of Chinese cabbage and might also be useful for the study of other Brassica crops.

High-throughput functional characterization of protein phosphorylation sites in yeast.

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, less than 5% of thousands of recently discovered phosphosites have been functionally annotated. Here, we devised a chemical genetic approach to study the functional relevance of phosphosites in S. cerevisiae. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions, and validated a subset by thermal proteome profiling and lipidomics. A high fraction exhibited phenotypes not seen in the corresponding gene deletion suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This high-throughput approach allows for functionally characterizing individual phosphosites at scale.

A high-efficiency Agrobacterium-mediated transient expression system in the leaves of Artemisia annua L.

BackgroundThe Agrobacterium-mediated transient transformation, which proved effective in diverse plant species, has been widely applied for high-throughput gene function studies due to its simplicity, rapidity, and high efficiency. Despite the efforts have made on Artemisia annua transient expression, achieving high-throughput gene functional characterization basing on a fast and easy-manipulated transient transformation system in A. annua remains challenging.ResultsThe first pair of true leaves of A. annua is an ideal candidate for Agrobacterium injection. EHA105 was the optimal strain that can be used for the development of the transient expression system. The supplementation of Triton X-100 at a concentration of 0.005% greatly improved the transient expression frequency. According to the histochemical β-Glucuronidase (GUS) staining assay, high transient expression level of the reporter gene (GUS) maintained at least a week. Dual-luciferase (Dual-LUC) transient assays showed that the activity of cauliflower mosaic virus 35S (CaMV35S) promoter and its derivates varied between A. annua and tobacco. In A. annua, the CaMV35S promoter had comparable activity with double CaMV35S promoter, while in tobacco, CaMV35S exhibited approximately 50% activity of double CaMV35S promoter. Otherwise, despite the CaMV35S promoter and double CaMV35S promoter from GoldenBraid Kit 2.0 displayed high activity strength in tobacco, they demonstrated a very low activity in transiently expressed A. annua. The activity of UBQ10 promoter and endogenous UBQb promoter was investigated as well. Additionally, using our transient expression system, the transactivation of AaGSW1 and AaORA on AaCYP71AV1 promoter was confirmed. Dual-LUC assays demonstrated that AaHD8 activated the expression of two glandular secreting trichomes-specific lipid transfer protein genes AaLTP1 and AaLTP2, indicating that AaLTP1 and AaLTP2 might serve as downstream components of AaHD8-involved glandular trichome initiation and cuticle formation, as well as artemisinin secretion in A. annua.ConclusionsA simple, rapid, good-reproducibility, high-efficiency and low-cost transient transformation system in A. annua was developed. Our method offered a new way for gene functional characterization studies such as gene subcellular localization, promoter activity and transcription activation assays in A. annua, avoiding the aberrant phenotypes resulting from gene expression in a heterologous system.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation for gene functional and gene editing analysis in soybean

BackgroundAgrobacterium-mediated genetic transformation is a widely used and efficient technique for gene functional research in crop breeding and plant biology. While in some plant species, including soybean, genetic transformation is still recalcitrant and time-consuming, hampering the high-throughput functional analysis of soybean genes. Thus we pursue to develop a rapid, simple, and highly efficient hairy root system induced by Agrobacterium rhizogenes (A. rhizogenes) to analyze soybean gene function.ResultsIn this report, a rapid, simple, and highly efficient hairy root transformation system for soybean was described. Only sixteen days were required for the whole workflow and the system was suitable for various soybean genotypes, with an average transformation frequency of 58–64%. Higher transformation frequency was observed when wounded cotyledons from 1-day-germination seeds were inoculated and co-cultivated with A. rhizogenes in 1/2 B5 (Gamborg’ B-5) medium. The addition of herbicide selection to root production medium increased the transformation frequency to 69%. To test the applicability of the hairy root system for gene functional analysis, we evaluated the protein expression and subcellular localization in transformed hairy roots. Transgenic hairy roots exhibited significantly increased GFP fluorescence and appropriate protein subcellular localization. Protein–protein interactions by BiFC (Bimolecular Fluorescent Complimentary) were also explored using the hairy root system. Fluorescence observations showed that protein interactions could be observed in the root cells. Additionally, hairy root transformation allowed soybean target sgRNA screening for CRISPR/Cas9 gene editing. Therefore, the protocol here enables high-throughput functional characterization of candidate genes in soybean.ConclusionA rapid, simple, and highly efficient A. rhizogenes-mediated hairy root transformation system was established for soybean gene functional analysis, including protein expression, subcellular localization, protein–protein interactions and gene editing system evaluation.

High-Throughput Functional Characterization of Visceral Afferents by Optical Recordings From Thoracolumbar and Lumbosacral Dorsal Root Ganglia.

Functional understanding of visceral afferents is important for developing the new treatment to visceral hypersensitivity and pain. The sparse distribution of visceral afferents in dorsal root ganglia (DRGs) has challenged conventional electrophysiological recordings. Alternatively, Ca2+ indicators like GCaMP6f allow functional characterization by optical recordings. Here we report a turnkey microscopy system that enables simultaneous Ca2+ imaging at two parallel focal planes from intact DRG. By using consumer-grade optical components, the microscopy system is cost-effective and can be made broadly available without loss of capacity. It records low-intensity fluorescent signals at a wide field of view (1.9 × 1.3 mm) to cover a whole mouse DRG, with a high pixel resolution of 0.7 micron/pixel, a fast frame rate of 50 frames/sec, and the capability of remote focusing without perturbing the sample. The wide scanning range (100 mm) of the motorized sample stage allows convenient recordings of multiple DRGs in thoracic, lumbar, and sacral vertebrae. As a demonstration, we characterized mechanical neural encoding of visceral afferents innervating distal colon and rectum (colorectum) in GCaMP6f mice driven by VGLUT2 promotor. A post-processing routine is developed for conducting unsupervised detection of visceral afferent responses from GCaMP6f recordings, which also compensates the motion artifacts caused by mechanical stimulation of the colorectum. The reported system offers a cost-effective solution for high-throughput recordings of visceral afferent activities from a large volume of DRG tissues. We anticipate a wide application of this microscopy system to expedite our functional understanding of visceral innervations.

An improved and efficient method of Agrobacterium syringe infiltration for transient transformation and its application in the elucidation of gene function in poplar

BackgroundForest trees have important economic and ecological value. As a model tree, poplar has played a significant role in elucidating the molecular mechanisms underlying tree biology. However, a lack of mutant libraries and time-consuming stable genetic transformation processes severely limit progress into the functional characterization of poplar genes. A convenient and fast transient transformation method is therefore needed to enhance progress on functional genomics in poplar.MethodsA total of 11 poplar clones were screened for amenability to syringe infiltration. Syringe infiltration was performed on the lower side of the leaves of young soil-grown plants. Transient expression was evaluated by visualizing the reporters β-glucuronidase (GUS) and green fluorescent protein (GFP). The experimental parameters of the syringe agroinfiltration were optimized based on the expression levels of the reporter luciferase (LUC). Stably transformed plants were regenerated from transiently transformed leaf explants through callus-induced organogenesis. The functions of Populus genes in secondary cell wall-thickening were characterized by visualizing lignin deposition therein after staining with basic fuchsin.ResultsWe greatly improved the transient transformation efficiency of syringe Agrobacterium infiltration in poplar through screening for a suitable poplar clone from a variety of clones and optimizing the syringe infiltration procedure. The selected poplar clone, Populus davidiana × P. bolleana, is amenable to Agrobacterium syringe infiltration, as indicated by the easy diffusion of the bacterial suspension inside the leaf tissues. Using this technique, we localized a variety of poplar proteins in specific intracellular organelles and illustrated the protein–protein and protein–DNA interactions. The transiently transformed leaves could be used to generate stably transformed plants with high efficiency through callus induction and differentiation processes. Furthermore, transdifferentiation of the protoxylem-like vessel element and ectopic secondary wall thickening were induced in the agroinfiltrated leaves via the transient overexpression of genes associated with secondary wall formation.ConclusionsThe application of P. davidiana × P. bolleana in Agrobacterium syringe infiltration provides a foundation for the rapid and high-throughput functional characterization of Populus genes in intact poplar plants, including those involved in wood formation, and provides an effective alternative to Populus stable genetic transformation.

LentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements.

Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.