Knockout Lines Research Articles

Generation and validation of a myoglobin knockout zebrafish model

Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.

Loss of function of male-specific lethal 3 (Msl3) does not affect spermatogenesis in rodents.

Male-specific lethal 3 (Msl3) is a member of the chromatin-associated male-specific lethal MSL complex, which is responsible for the transcriptional upregulation of genes on the X chromosome in males of Drosophila. Although the dosage complex operates differently in mammals, the Msl3 gene is conserved from flies to humans. Msl3 is required for meiotic entry during Drosophila oogenesis. Recent reports indicate that also in primates, Msl3 is expressed in undifferentiated germline cells before meiotic entry. However, if Msl3 plays a role in the meiotic entry of mammals has yet to be explored. To understand, if Msl3a plays a role in the meiotic entry of mammals, we used mouse spermatogenesis as a study model. Analyses of single-cell RNA-seq data revealed that, in mice, Msl3 is mostly expressed in meiotic cells. To test the role of Msl3 in meiosis, we used a male germline-specific Stra8-iCre driver and a newly generated Msl3flox conditional knock-out mouse line. Msl3 conditional loss-of-function in spermatogonia did not cause spermatogenesis defects or changes in the expression of genes related to meiosis. Our data suggest that, in mice, Msl3 exhibits delayed expression compared to Drosophila and primates, and loss-of-function mutations disrupting the chromodomain of Msl3 alone do not impede meiotic entry in rodents.

The R2R3-MYB transcription factor EVER controls the emission of petunia floral volatiles by regulating epicuticular wax biosynthesis in the petal epidermis.

The epidermal cells of petunia (Petunia × hybrida) flowers are the main site of volatile emission. However, the mechanisms underlying the release of volatiles into the environment are still being explored. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by virus-induced gene silencing and clustered regularly interspaced short palindromic repeat (CRISPR), and metabolomics, we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)-a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. To generate ever knockout lines, we developed a viral-based CRISPR/Cas9 system for efficient gene editing in plants. These knockout lines, together with transient-suppression assays, revealed EVER's involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in volatile production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. Liquid chromatography/gas chromatography-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. We reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a regulatory layer in the scent-emission machinery in petunia flowers.

Functional dissection and assembly of a small, newly evolved, W chromosome-specific genomic region of the African clawed frog Xenopus laevis.

Genetic triggers for sex determination are frequently co-inherited with other linked genes that may also influence one or more sex-specific phenotypes. To better understand how sex-limited regions evolve and function, we studied a small W chromosome-specific region of the frog Xenopus laevis that contains only three genes (dm-w, scan-w, ccdc69-w) and that drives female differentiation. Using gene editing, we found that the sex-determining function of this region requires dm-w but that scan-w and ccdc69-w are not essential for viability, female development, or fertility. Analysis of mesonephros+gonad transcriptomes during sexual differentiation illustrates masculinization of the dm-w knockout transcriptome, and identifies mostly non-overlapping sets of differentially expressed genes in separate knockout lines for each of these three W-specific gene compared to wildtype sisters. Capture sequencing of almost all Xenopus species and PCR surveys indicate that the female-determining function of dm-w is present in only a subset of species that carry this gene. These findings map out a dynamic evolutionary history of a newly evolved W chromosome-specific genomic region, whose components have distinctive functions that frequently degraded during Xenopus diversification, and evidence the evolutionary consequences of recombination suppression.

C-Cbl Regulates Murine Subventricular Zone-Derived Neural Progenitor Cells in Dependence of the Epidermal Growth Factor Receptor.

The localization, expression, and physiological role of regulatory proteins in the neurogenic niches of the brain is fundamental to our understanding of adult neurogenesis. This study explores the expression and role of the E3-ubiquitin ligase, c-Cbl, in neurogenesis within the subventricular zone (SVZ) of mice. In vitro neurosphere assays and in vivo analyses were performed in specific c-Cbl knock-out lines to unravel c-Cbl's role in receptor tyrosine kinase signaling, including the epidermal growth factor receptor (EGFR) pathway. Our findings suggest that c-Cbl is significantly expressed within EGFR-expressing cells, playing a pivotal role in neural stem cell proliferation and differentiation. However, c-Cbl's function extends beyond EGFR signaling, as its loss upon knock-out stimulated progenitor cell proliferation in neurosphere cultures. Yet, this effect was not detected in hippocampal progenitor cells, reflecting the lack of the EGFR in the hippocampus. In vivo, c-Cbl exerted only a minor proneurogenic influence with no measurable impact on the formation of adult-born neurons. In conclusion, c-Cbl regulates neural stem cells in the subventricular zone via the EGFR pathway but, likely, its loss is compensated by other signaling modules in vivo.

Loss of ATG4B and ATG4A results in two-stage cell cycle defects in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) exhibits elevated levels of autophagy, which promote tumor progression and treatment resistance. ATG4B is an autophagy-related cysteine protease under consideration as a potential therapeutic target, but it is largely unexplored in PDAC. Here, we investigated the clinical and functional relevance of ATG4B expression in PDAC. Using two PDAC patient cohorts, we found that low ATG4B mRNA or protein expression is associated with worse patient survival outcomes, poorly differentiated PDAC tumors and a lack of survival benefit from adjuvant chemotherapy. In PDAC cell lines, ATG4B knockout reduced proliferation, abolished processing of LC3B (also known as MAP1LC3B), and reduced GABARAP and GABARAPL1 levels, but increased ATG4A levels. ATG4B and ATG4A double knockout lines displayed a further reduction in proliferation, characterized by delays in G1-S phase transition and mitosis. Pro-LC3B accumulated aberrantly at the centrosome with a concomitant increase in centrosomal proteins PCM1 and CEP131, which was rescued by exogenous ATG4B. The two-stage cell cycle defects following ATG4B and ATG4A loss have important therapeutic implications for PDAC.

Molecular mechanisms of the stress-induced regulation of the inflammatory response in fish

Stressors in the environment of aquatic organisms can profoundly affect their immune system. The stress response in fish involves the activation of the hypothalamus-pituitary-interrenal (HPI) axis, leading to the release of several stress hormones, among them glucocorticoids, such as cortisol, which bind and activate corticosteroid receptors, namely the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). These receptors are highly expressed on immune cells, thereby allowing stress to have a potent effect that is classically considered to suppress immune function. In this review, we highlight the conserved structure and function of GR and MR among vertebrates and describe their role in modulating inflammation by regulating the expression of pro-inflammatory and anti-inflammatory genes. In particular, the involvement of MR during inflammation is reviewed, which in many studies has been shown to be immune-enhancing. In recent years, the use of zebrafish as a model organism has opened up new possibilities to study the effects of stress on inflammation, making it possible to investigate knockout lines for MR and/or GR, in combination with transgenic models with fluorescently labeled leukocyte subpopulations that enable the visualization and manipulation of these immune cells. The potential roles of other hormones of the HPI axis, such as corticotrophin-releasing hormone (Crh) and adrenocorticotropic hormone (Acth), in immune modulation are also discussed. Overall, this review highlights the need for further research to elucidate the specific roles of GR, MR and other stress hormones in regulating immune function in fish. Understanding these mechanisms will contribute to improving fish health and advancing our knowledge of stress signalling.

The Effects of Glypican-3 Deficiency on Radiosensitivity in Liver Cancer Cells

Glypican-3 (GPC-3), a heparan sulfate proteoglycan involved in cellular proliferation, modulates signaling of FGF/FGFR, IGF/IGFR, HGF/Met, Wnt/Frizzled, among others and correlates with survival. GPC-3 is overexpressed in the majority of hepatocellular carcinoma and hepatoblastoma, but not in normal hepatocytes. Accordingly, it is being investigated as a liver cancer-selective target for radiopharmaceutical imaging and therapy. However, the potential linkage between GPC-3 expression and radiosensitivity has not yet been defined. In this study, we investigated the effects of GPC-3 deficiency on radiosensitivity in liver cancer cell lines. CRISPR/Cas9 system was used to engineer GPC-3 knockout variants of liver cancer cell lines, HepG2 & Hep3B, both of which natively express GPC-3. Confirmation of knockout of GPC-3 was evaluated by RT-PCR, western blotting, flow cytometry, immunocytochemistry, and gDNA sequencing. Cell growth and migration were evaluated by BrdU insertion and wound-healing assays, respectively. In vitro radiosensitivity was examined by radiation-induced apoptosis/necrosis (Annexin V-APC and PI staining), cell cycle modification, γH2AX foci formation, and clonogenic assays (6 Gy). Wildtype and knockout lines were engrafted into athymic mice to assess tumor growth kinetics. RT-PCR, western blotting, flow cytometry, and immunocytochemistry all confirmed GPC-3 knockout in both HepG2 and Hep3B cell lines. Nucleotide deletion at exon 3 of the GPC-3 gene was confirmed by gDNA sequencing in HepG2ΔGPC3 and Hep3BΔGPC3. GPC-3 deficiency reduced liver cancer cell proliferation (HepG2ΔGPC3, p = 0.027, and Hep3BΔGPC3, p = 0.031) and migration (HepG2ΔGPC3: 1.5-fold, p<0.001, and Hep3BΔGPC3: 2.3-fold, p<0.001) significantly when compared with wild type. GPC-3 deficiency reduced cell survival and clonogenicity (HepG2ΔGPC3: DEF = 1.23, Hep3BΔGPC3: DEF = 1.23) in liver cancer cells exposed to irradiation (6 Gy). The delayed repair of double-stranded DNA damage was observed in irradiated GPC-3 deficient liver cancer cells. Tumor growth was dramatically delayed by GPC-3 deficiency. Tumor weight measured at 50 (Hep3B) and 60 (HepG2) days after liver cancer cell inoculation corroborated these effects. Knockout lines of HepG2 and Hep3B exhibited decreased cell proliferation, migration, and in vivo tumor growth compared to wildtype. GPC-3 deficiency was associated with increased sensitivity to radiation therapy. Studies identifying the pathways through which this radiosensitivity is mediated are ongoing.

Mitfa deficiency promotes immune vigor and potentiates antitumor effects in zebrafish

The mitfa gene is a well-known transcription factor associated with microphthalmia and is essential for early melanophore development. However, little is known about how mitfa affects the immune system. Here, we generated a novel mitfa knock-out zebrafish line using the CRISPR/Cas9 system. The mitfa−/− zebrafish exhibited reduced melanin levels compared to the nacre mutant. We investigated the impact on the immune system after exposure to Edwardsiella tarda and bifenazate in zebrafish larvae, and observed that the macrophage numbers were reduced in both treated groups. Remarkably, the expression levels of immune-related genes exhibited significant increases after bacterial challenge or bifenazate exposure in the mitfa−/− zebrafish, except for tlr4 and rela. Furthermore, we conducted xenograft experiments using mouse B16 melanoma cells. Notably, the cancer cells didn't show a high cell migration ratio, implying that the immune system was highly activated after the loss of mifta. Taken together, our findings suggest that mitfa−/− zebrafish serve as a valuable model for investigating the relationship between the immune system and melanocytes, providing new insights into the role of mitfa in immune responses.

Del(8p) and TNFRSF10B loss are associated with a poor prognosis and resistance to fludarabine in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, the prognosis of which varies according to the cytogenetic group. We characterized a rare chromosomal abnormality (del(8p), deletion of the short arm of chromosome 8) in the context of CLL. By comparing the largest cohort of del(8p) CLL to date (n = 57) with a non-del(8p) cohort (n = 155), del(8p) was significantly associated with a poor prognosis, a shorter time to first treatment, worse overall survival (OS), and a higher risk of Richter transformation. For patients treated with fludarabine-based regimens, the next-treatment-free survival and the OS were shorter in del(8p) cases (including those with mutated IGHV). One copy of the TNFRSF10B gene (coding a pro-apoptotic receptor activated by TRAIL) was lost in 91% of del(8p) CLL. TNFRSF10B was haploinsufficient in del(8p) CLL, and was involved in the modulation of fludarabine-induced cell death - as confirmed by our experiments in primary cells and in CRISPR-edited TNFRSF10B knock-out CLL cell lines. Lastly, del(8p) abrogated the synergy between fludarabine and TRAIL-induced apoptosis. Our results highlight del(8p)'s value as a prognostic marker and suggest that fit CLL patients (i.e. with mutated IGHV and no TP53 disruption) should be screened for del(8p) before the initiation of fludarabine-based treatment.

Genetic Deletion of DNAJB3 Using CRISPR-Cas9, Produced Discordant Phenotypes.

Several pathways and/or genes have been shown to be dysregulated in obesity-induced insulin resistance (IR) and type 2 diabetes (T2D). We previously showed, for the first time, impaired expression of DNAJB3 mRNA and protein in subjects with obesity, which was concomitant with increased metabolic stress. Restoring the normal expression of DNAJB3 attenuated metabolic stress and improved insulin signaling both in vivo and in vitro, suggesting a protective role of DNAJB3 against obesity and T2D. The precise underlying mechanisms remained, however, unclear. This study was designed to confirm the human studies in a mouse model of dietary obesity-induced insulin resistance, and, if validated, to understand the underlying mechanisms. We hypothesized that mice lacking DNAJB3 would be more prone to high-fat (HF)-diet-induced increase in body weight and body fat, inflammation, glucose intolerance and insulin resistance as compared with wild-type (WT) littermates. Three DNAJB3 knockout (KO) lines were generated (KO 30, 44 and 47), using CRISPR-Cas9. Male and female KO and WT mice were fed a HF diet (45% kcal fat) for 16 weeks. Body weight was measured biweekly, and a glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at week 13 and 14, respectively. Body composition was determined monthly by nuclear magnetic resonance (NMR). Following euthanasia, white adipose tissue (WAT) and skeletal muscle were harvested for further analyses. Compared with WT mice, male and female KO 47 mice demonstrated higher body weight and fat mass. Similarly, KO 47 mice also showed a slower rate of glucose clearance in GTT that was consistent with decreased mRNA expression of the GLUT4 gene in WAT but not in the muscle. Both male and female KO 47 mice exhibited higher mRNA levels of the pro-inflammatory marker TNF-a in WAT only, whereas increased mRNA levels of MCP1 chemokine and the ER stress marker BiP/Grp78 were observed in male but not in female KO 47 mice. However, we did not observe the same changes in the other KO lines. Taken together, the phenotype of the DNAJB3 KO 47 mice was consistent with the metabolic changes and low levels of DNAJB3 reported in human subjects. These findings suggest that DNAJB3 may play an important role in metabolic functions and glucose homeostasis, which warrants further phenotyping and intervention studies in other KO 47 and other KO mice, as well as investigating this protein as a potential therapeutic target for obesity and T2D.

EXTRACELLULAR VESICLES TRANSFER RESISTANCE TO VINCRISTINE IN SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract AIMS • To determine the expression patterns of specific ATP-binding cassette (ABC) transporters in medulloblastoma • To assess whether ABC transporters play a role in acquired drug resistance in SHH medulloblastoma using drug-tolerant cell line models • To assess whether extracellular vesicles transfer drug-tolerance in SHH medulloblastoma and determine the mechanism by which they do this METHOD Drug-tolerant cell lines were generated by continuous culture in drug-containing culture medium. ABC trans- porter gene and protein expression was assessed via qPCR and western blot. EVs were isolated from EV-depleted medium using size-exclusion chromatography. Nanoparticle tracking analysis was used to quantify and deter- mine size. Cytotoxicity analysis was used to determine EV-mediated transfer of resistance in the presence or absence of an ABC transporter inhibitor. RNA sequencing of EVs from matched vehicle and drug tolerant cell lines was carried out. RESULTS Analysis of drug-tolerant cell lines revealed significant increases in specific drug transporter gene expression across medulloblastoma subgroups. ABCB1 protein expression was significantly higher in drug-tolerant sonic- hedgehog (SHH) lines and inhibition of ABCB1 in a drug-tolerant SHH line abolished acquired resistance to vincristine. Cytotoxicity assays on sensitive cells, after pre-treatment with drug-tolerant EVs, showed a significant increase in vincristine resistance, implicating EVs in the transfer of resistance. CONCLUSIONS Data to date supports the hypothesis that multidrug transporter carrying extracellular vesicles may transfer their multidrug resistant phenotype to surrounding cells in medulloblastoma. We are currently testing this using inhibitors and knockout lines, in addition to RNA sequencing, with the hopes of providing new therapeutic targets to patients with medulloblastoma.

A citrate efflux transporter important for manganese distribution and phosphorus uptake in rice.

The plant citrate transporters, functional in mineral nutrient uptake and homeostasis, usually belong to the multidrug and toxic compound extrusion transporter family. We identified and functionally characterized a rice (Oryza sativa) citrate transporter, OsCT1, which differs from known plant citrate transporters and is structurally close to rice silicon transporters. Domain analysis depicted that OsCT1 carries a bacterial citrate-metal transporter domain, CitMHS. OsCT1 showed citrate efflux activity when expressed in Xenopus laevis oocytes and is localized to the cell plasma membrane. It is highly expressed in the shoot and reproductive tissues of rice, and its promoter activity was visible in cells surrounding the vasculature. The OsCT1 knockout (KO) lines showed a reduced citrate content in the shoots and the root exudates, whereas overexpression (OE) line showed higher citrate exudation from their roots. Further, the KO and OE lines showed variations in the manganese (Mn) distribution leading to changes in their agronomical traits. Under deficient conditions (Mn-sufficient conditions followed by 8 days of 0 μm MnCl2 · 4H2 O treatment), the supply of manganese towards the newer leaf was found to be obstructed in the KO line. There were no significant differences in phosphorus (P) distribution; however, P uptake was reduced in the KO and increased in OE lines at the vegetative stage. Further, experiments in Xenopus oocytes revealed that OsCT1 could efflux citrate with Mn. In this way, we provide insights into a mechanism of citrate-metal transport in plants and its role in mineral homeostasis, which remains conserved with their bacterial counterparts.

Approaches for timeline reductions in pathogenesis studies using genetically modified mice.

Although genetically modified mouse models have long been a powerful tool for microbiology research, the manipulation of the mouse genome is expensive, time consuming, and has historically remained the domain of dedicated animal facilities. The recent use of in vivo clustered regularly interspaced short palindromic repeats (CRISPR)-based editing technology has been reported to reduce the expertise, cost, and time required to generate novel mouse lines; it has remained unclear, however, if this new technology could meaningfully alter experimental timelines. Here, we report the optimization of an in oviduct murine genetic manipulation technique for use by microbiologists. We use this approach to generate a series of knockout mice and detail a protocol using an influenza A virus infection model to test the preliminary importance of a host factor in as short as 11 weeks (with a fully backcrossed knockout line in ~22 weeks) from initiation of the study. Broader use of this approach by the microbiology community will allow for more efficient, and rapid, definition of novel pathogenic mechanisms in vivo. IMPORTANCE Clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies have already begun to revolutionize biomedical science. An emerging application of this technology is in the development of genetically modified model organisms to study the mechanisms underlying infectious disease. Here, we describe a protocol using an in vivo CRISPR-based approach that can be used to test the importance of a candidate host factor for microbial pathogenesis in less than 3 months and before complete establishment of a new mouse line. Adoption of this approach by the broader microbiology community will help to decrease the resources and time required to understand how pathogens cause disease which will ultimately speed up the development of new clinical interventions and therapies.

The role of WRKY transcription factors, FaWRKY29 and FaWRKY64, for regulating Botrytis fruit rot resistance in strawberry (Fragaria × ananassa Duch.)

BackgroundThe cultivated strawberry (Fragaria × ananassa Duch.) is one of the most economically important horticultural crops worldwide. Botrytis fruit rot (BFR) caused by the necrotrophic fungal pathogen Botrytis cinerea is the most devasting disease of cultivated strawberries. Most commercially grown strawberry varieties are susceptible to BFR, and controlling BFR relies on repeated applications of various fungicides. Despite extensive efforts, breeding for BFR resistance has been unsuccessful, primarily due to lack of information regarding the mechanisms of disease resistance and genetic resources available in strawberry.ResultsUsing a reverse genetics approach, we identified candidate genes associated with BFR resistance and screened Arabidopsis mutants using strawberry isolates of B. cinerea. Among the five Arabidopsis T-DNA knockout lines tested, the mutant line with AtWRKY53 showed the greatest reduction in disease symptoms of BFR against the pathogen. Two genes, FaWRKY29 and FaWRKY64, were identified as orthologs in the latest octoploid strawberry genome, ‘Florida Brilliance’. We performed RNAi-mediated transient assay and found that the disease frequencies were significantly decreased in both FaWRKY29- and FaWRKY64-RNAi fruits of the strawberry cultivar, ‘Florida Brilliance’. Furthermore, our transcriptomic data analysis revealed significant regulation of genes associated with ABA and JA signaling, plant cell wall composition, and ROS in FaWRKY29 or FaWRKY64 knockdown strawberry fruits in response to the pathogen.ConclusionOur study uncovered the foundational role of WRKY transcription factor genes, FaWRKY29 and FaWRKY64, in conferring resistance against B. cinerea. The discovery of susceptibility genes involved in BFR presents significant potential for developing resistance breeding strategies in cultivated strawberries, potentially leveraging CRISPR-based gene editing techniques.

Generation and characterization of a zebrafish knockout model of abcb4, a homolog of the human multidrug efflux transporter P-glycoprotein

The ATP-binding cassette subfamily B member 1 (ABCB1), encoding a multidrug transporter referred to as P-glycoprotein (Pgp), plays a critical role in the efflux of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. Therefore, developing high-throughput animal models to screen for Pgp function and bioavailability of substrates and inhibitors is paramount. Here, we generated and validated a zebrafish knockout line of abcb4, a human Pgp transporter homolog. CRISPR/Cas9 genome editing technology was deployed to generate a frameshift mutation in exon 4 of zebrafish abcb4. The zebrafish abcb4 homozygous mutant exhibited elevated accumulation of fluorescent rhodamine 123, a substrate of human Pgp, in the intestine and brain area of embryos. Moreover, abcb4 knockout embryos were sensitized toward toxic compounds such as doxorubicin and vinblastine compared to the WT zebrafish. Immunostaining for zebrafish Abcb4 colocalized in the endothelial brain cells of adult zebrafish. Transcriptome profiling using Gene Set Enrichment Analysis uncovered that the 'cell cycle process,' 'mitotic cell cycles,' and 'microtubule-based process' were significantly downregulated in the abcb4 knockout brain with age. This study establishes and validates the abcb4 knockout zebrafish as an animal model to study Pgp function in vivo. Unexpectedly it reveals a potentially novel role for zebrafish abcb4 in age-related changes in the brain. The zebrafish lines generated here will provide a platform to aid in the discovery of modulators of Pgp function as well as the characterization of human mutants thereof.

Validation and in vivo characterization of research antibodies for Moesin, CD44, Midkine, and sFRP-1.

Background: A major goal of the Target Enablement to Accelerate Therapy Development for Alzheimer’s disease (TREAT-AD) program is to develop and identify high-quality tools to test target or mechanistic hypotheses. As part of this initiative, it is important that commercial reagents including research antibodies being used to interrogate drug targets have confirmed validation data in knock-out cell lines. Ideally, these antibodies should also have utility for both in vitro and in vivo studies such that the levels of target proteins in target tissues can be quantified. Methods: We evaluated commercial antibodies against TREAT-AD protein targets Moesin (Uniprot ID: P26038), CD44 (Uniprot ID: P16070), Midkine (Uniprot ID: P21741) and Secreted frizzled-related protein 1, referred to as “sFRP-1” (sFRP-1; Uniprot ID: Q8N474). Moesin, Midkine and sFRP-1, that were confirmed as selective based on data in knock-out cell lines. Western blot analysis was used to compare protein levels in brain homogenates from a mouse model with AD-relevant pathology (5XFAD) versus age-matched C57BL/6J control mice. Results: Anti-Moesin ab52490 reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa. Moesin protein expression was 2.8 times higher in 5xFAD compared to WT. Anti-CD44 ab189524 reacted with a band at the predicted size of 82 kDa. CD44 protein expression was 1.9 times higher in 5xFAD compared to WT. Anti-Midkine AF7769 reacted with a band ~16 kDa and a 17.8 times greater expression in 5xFAD compared to WT. Anti-sFRP-1 ab267466 reacted with a band at 35 kDa as predicted. sFRP-1 protein expression was 11.9 times greater in 5xFAD compared to WT. Conclusions: These data confirm the utility of these selective commercially available antibodies against Moesin, CD44, Midkine, and sFRP-1 for in vivo studies in mice and provide insight into the use of 5XFAD mice for in vivo target engagement studies for these target proteins.

Natural variation in Arabidopsis responses to Plasmodiophora brassicae reveals an essential role for Resistance to Plasmodiophora brasssicae 1 (RPB1).

Despite the identification of clubroot resistance genes in various Brassica crops our understanding of the genetic basis of immunity to Plasmodiophora brassicae infection in the model plant Arabidopsis thaliana remains limited. To address this issue, we performed a screen of 142 natural accessions and identified 11 clubroot-resistant Arabidopsis lines. Genome-wide association analysis identified several genetic loci significantly linked with resistance. Three genes from two of these loci were targeted for deletion by CRISPR/Cas9 mutation in resistant accessions Est-1 and Uod-1. Deletion of Resistance to Plasmodiophora brassicae 1 (RPB1) rendered both lines susceptible to the P. brassicae pathotype P1+. Further analysis of rpb1 knock-out Est-1 and Uod-1 lines showed that the RPB1 protein is required for activation of downstream defence responses, such as the expression of phytoalexin biosynthesis gene CYP71A13. RPB1 has recently been shown to encode a cation channel localised in the endoplasmic reticulum. The clubroot susceptible Arabidopsis accession Col-0 lacks a functional RPB1 gene; when Col-0 is transformed with RPB1 expression driven by its native promoter it is capable of activating RPB1 transcription in response to infection, but this is not sufficient to confer resistance. Transient expression of RPB1 in Nicotiana tabacum induced programmed cell death in leaves. We conclude that RPB1 is a critical component of the defence response to P. brassicae infection in Arabidopsis, acting downstream of pathogen recognition but required for the elaboration of effective resistance.

Activated Expression of Rice DMR6-like Gene OsS3H Partially Explores the Susceptibility to Bacterial Leaf Streak Mediated by Knock-Out OsF3H04g.

Downy Mildew Resistance 6-like (DMR6-like) genes are identified as salicylic acid (SA) hydroxylases and negative regulators of plant immunity. Previously, we identified two rice DMR6-like genes, OsF3H03g, and OsF3H04g, that act as susceptible targets of transcription activator-like effectors (TALEs) from Xanthomonas oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak (BLS) in rice. Furthermore, all four homologs of rice DMR6-like proteins were identified to predominantly carry the enzyme activity of SA 5-hydroxylase (S5H), negatively regulate rice broad-spectrum resistance, and cause the loss of function of these OsDMR6s, leading to increased resistance to rice blast and bacterial blight (BB). Here, we curiously found that an OsF3H04g knock-out mutant created by T-DNA insertion, osf3h04g, was remarkedly susceptible to BLS and BB and showed an extreme reduction in SA content. OsF3H04g knock-out rice lines produced by gene-editing were mildly susceptible to BLS and reduced content of SA. To explore the susceptibility mechanism in OsF3H04g loss-of-function rice lines, transcriptome sequencing revealed that another homolog, OsS3H, had induced expression in the loss-of-function OsF3H04g rice lines. Furthermore, we confirmed that a great induction of OsS3H downstream and genomically adjacent to OsF3H04g in osf3h04g was primarily related to the inserted T-DNA carrying quadruple enhancer elements of 35S, while a slight induction was caused by an unknown mechanism in gene-editing lines. Then, we found that the overexpression of OsS3H increased rice susceptibility to BLS, while gene-editing mediated the loss-of-function OsS3H enhanced rice resistance to BLS. However, the knock-out of both OsF3H04g and OsS3H by gene-editing only neutralized rice resistance to BLS. Thus, we concluded that the knock-out of OsF3H04g activated the expression of the OsS3H, partially participating in the susceptibility to BLS in rice.

GRAS family member LATERAL SUPPRESSOR regulates the initiation and morphogenesis of watermelon lateral organs.

The lateral organs of watermelon (Citrullus lanatus), including lobed leaves, branches, flowers, and tendrils, together determine plant architecture and yield. However, the genetic controls underlying lateral organ initiation and morphogenesis remain unclear. Here, we found that knocking out the homologous gene of shoot branching regulator LATERAL SUPPRESSOR in watermelon (ClLs) repressed the initiation of branches, flowers, and tendrils and led to developing round leaves, indicating that ClLs undergoes functional expansion compared with its homologs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and tomato (Solanum lycopersicum). Using ClLs as the bait to screen against the cDNA library of watermelon, we identified several ClLs-interacting candidate proteins, including TENDRIL (ClTEN), PINOID (ClPID), and APETALA1 (ClAP1). Protein-protein interaction assays further demonstrated that ClLs could directly interact with ClTEN, ClPID, and ClAP1. The mRNA in situ hybridization assay revealed that the transcriptional patterns of ClLs overlapped with those of ClTEN, ClPID, and ClAP1 in the axillary meristems and leaf primordia. Mutants of ClTEN, ClPID, and ClAP1 generated by the CRISPR/Cas9 gene editing system lacked tendrils, developed round leaves, and displayed floral diapause, respectively, and all these phenotypes could be observed in ClLs knockout lines. Our findings indicate that ClLs acts as lateral organ identity protein by forming complexes with ClTEN, ClPID, and ClAP1, providing several gene targets for transforming the architecture of watermelon.