Collectively, our data underscore the role of IR-mediated signaling in maintaining pluripotency, the extracellular matrix necessary for the stem cell niche, and regulating cell fate specification including the neural lineage.Human embryonic stem cells are a type of pluripotent stem cells (hPSCs) that are used to investigate their differentiation into diverse mature cell types for molecular studies. The mechanisms underlying insulin receptor (IR)-mediated signaling in the maintenance of human pluripotent stem cell (hPSC) identity and cell fate specification are not fully understood. Here, we used two independent shRNAs to stably knock down IRs in two hPSC lines that represent pluripotent stem cells and explored the consequences on expression of key proteins in pathways linked to proliferation and differentiation. We consistently observed lowered pAKT in contrast to increased pERK1/2 and a concordant elevation in pluripotency gene expression. ERK2 chromatin immunoprecipitation, luciferase assays, and ERK1/2 inhibitors established direct causality between ERK1/2 and OCT4 expression. Of importance, RNA sequencing analyses indicated a dysregulation of genes involved in cell differentiation and organismal development. Mass spectrometry–based proteomic analyses further confirmed a global downregulation of extracellular matrix proteins. Subsequent differentiation toward the neural lineage reflected alterations in SOX1 Human embryonic stem cells (hESCs) are prototypical human pluripotent stem cells (hPSCs) widely used for studying pluripotency and differentiation . PluripoThese hPSC media that contain supraphysiological levels of insulin and insulin-like growth factors (IGFs) suggest a requirement for insulin and/or IGF-I receptors in regulating hPSCs . In geneTo directly address the significance of IR in pluripotency and hPSC identity, we used two independent shRNAs to stably knock down IR in two different hPSC lines and interrogated the consequences on insulin/IGF-I signaling pathways . Knock dConsistent with previous reports , 13, we INSR) resulted in a reproducible knockdown of IR transcript and protein expression. Of a total of 52 clones (35 for CHB8 and 17 for H9) that were screened both at the transcript and protein levels, we obtained two independent IR knocked down clones in CHB8 (shIR-CHB8) and three in H9 hESCs (shIR-H9). QPCR and Western blot analyses confirmed the knock down of IR in CHB8  and H9 hESCs . Of interest, knock down of IR resulted in a concomitant decrease in IGF1R protein expression (A and B).To directly investigate the role of IR signaling in mediating human pluripotency, we stably knocked down IR in two different hESC lines (CHB8 and H9). Among the four shRNA constructs (Sigma) we tested, the short hairpins that target the CACTGATTACTTGCTGCTCT sequence in exon 2 and GTGCTGTATGAAGTGAGTTA sequence in exon 13 of IR gene (C–E). Notwithstanding the mild changes in expression of the proximal adaptor proteins, we observed a drastic decrease in pAKTS473 and a surprising reciprocal increase in pERK1/2T202/Y204 signaling (C and D). Western blot analyses on additional independent IR knocked down clones in H9 hESCs, generated using different shRNA constructs, reflected similar changes in protein expression (E). Together, these data indicate that IR/AKT/ERK1/2 signaling is active in hPSCs and the loss of IR modulates PI3K/AKT and MEK/ERK signaling pathways in a reciprocal manner, which may, in turn, impact the hPSC state.Next, upon IR knockdown, we observed that IRS-1 and p85α protein expression was marginally decreased, whereas IRS-2 protein expression either increased or did not change C–E. Notignaling , C and Dpression E. TogethS473 upon insulin stimulation (E), indicating primary activation of the PI3K/AKT signaling pathway in the maintenance of self-renewal and pluripotency. In contrast, shIR-hPSCs lost their responsiveness to exogenous insulin stimulation (E).To determine if the shIR-hPSCs continued to respond normally to ligand, we starved the hPSCs for 24 h in DMEM/F-12 + 0.5% bovine serum albumin (BSA) + 10 ng/ml Activin + 12 ng/ml FGF2  . We also differentiated them into the three germ layers in vivo via teratoma assays (A) and performed in vitro directed differentiation (B) to definitively demonstrate their pluripotency. We next undertook a systems biology approach to gain further insights into the impact of IR knock down on hPSCs. RNA-Seq performed on shIR-CHB8 hESCs indicated that INSR transcript was knocked down without affecting IGF1R transcripts  is secondary to the loss of IR protein expression. Proteomics analyses further confirmed that IR protein was knocked down in shIR-CHB8 hESCs  and H9 (E and F) hESCs (D) and H9 (G and H) hESCs.RNA-Seq analyses performed on shIR-CHB8 hESCs revealed an interesting trend of upregulation of numerous pluripotency genes Fig. 2) and H9 E and F F) hESCs  and S3.  and H9 ) and H9 G and H Y204 and ERK2Y187 . These data gain significance since ERK2 has been reported to bind to promoter/enhancer regions of pluripotency genes OCT4, SOX2, DPPA4, LIN28A, SALL4, and DNMT3B  (OCT4 enhancer region (E). To confirm that the OCT4 enhancer region bound by ERK2 is transcriptionally active, we cloned the promoter/enhancer regions of OCT4, SOX2, and DPPA4 into luciferase assay vectors. Among these three pluripotency genes, indeed we found the OCT4 enhancer region to be transcriptionally active (F). To further demonstrate that the direct interaction between ERK1/2 and OCT4 genomic loci is functionally active, we used two different ERK1/2 inhibitors (instead of MEK inhibitors) on shScr- and shIR-hPSCs and observed that the inhibition of ERK1/2 reproducibly abolished the protein expression of OCT4 but not that of SOX2 (G and H). The knockdown of ERK1/2 via shRNAs was unfortunately unsuccessful across multiple attempts.To establish direct causality between ERK1/2 and pluripotency, we first performed ERK2 chromatin immunoprecipitation (ChIP) qPCR analyses on promoter/enhancer regions of pluripotency genes . (2013) . Among tr region E. To cony active F. To fur of SOX2 , G and Hp < 0.05) in shIR-CHB8 hESCs (p < 0.05) in shIR-H9 hESCs (p < 0.05) in both hESCs  via Gene Ontology (GO) analyses indicated an upregulation of genes linked to nervous system development and neurogenesis (A–C). This correlated with the increased expression of all Hox genes (majorly expressed in the vertebrate nervous system) detected in the RNA-Seq data  (B). In addition, a majority of the most differentially upregulated genes in shIR-hPSCs were related to the neural lineage  B. In add lineage .Figure 3C and D). Among the early germ layer specification genes, we observed that indeed numerous neuroectodermal genes such as PAX2, GBX2, CHRD, and NES and numerous HOX genes were upregulated, whereas most of the mesodermal and endodermal genes were strikingly downregulated in shIR-hPSCs (A and B). GO analyses performed on downregulated proteins in the proteomics data from both shIR-CHB8 and shIR-H9 hESC lines (fold change > 1.5) (E) corroborated the RNA-Seq data (D). QPCR analyses then confirmed that some of the most downregulated genes  such as EOMES, SOX17, and FOXA2 were severely suppressed in shIR-hPSCs (F and G). Thus, knockdown of IR in hPSCs causes an imbalance in germ layer specification genes, likely owing to an increase in phosphorylation coupled with total protein expression of numerous pluripotency genes.GO analyses indicated that multiple biological processes were both up- and downregulated in the RNA-Seq data , C and DIR-hPSCs , A and Be > 1.5) E corroboSeq data D. QPCR aIR-hPSCs , F and GCOL), keratins (KRT), vitronectin (VTN), fibronectin (FN1), laminins (LAM), and prolyl 4-hydroxylase (P4H) genes were dramatically downregulated in shIR-hPSCs (A). Proteomics data in shIR-CHB8 and shIR-H9 hESCs further reflected this global downregulation of ECM proteins (B and C). QPCR analyses on FN1, COL1A1, and KRT19 genes in both shIR-CHB8 and shIR-H9 hESCs (D) and Western blot analyses on FN1, COL1, and P4HB (E) provided corroborative independent validations.The knock down of IR in hPSCs also distinctly resulted in a global downregulation of ECM gene expression. RNA-Seq data indicated that a majority of the collagen (IR-hPSCs A. Proteoproteins B and C.H9 hESCs D and Wesand P4HB E provideA–C), we then differentiated both shScr- and shIR-hPSCs into neuroectoderm and cerebral organoids to evaluate the effects of IR knockdown on neural lineage differentiation. QPCR analyses revealed an upregulation of neuroectodermal markers SOX2, SOX1, PAX6, and SIP1 in shIR-hPSCs differentiated into day 7 neuroectoderm (A). Immunostaining then revealed that SOX1 and PAX6 proteins colocalized in shScr-hPSC–derived neuroectoderm but not in shIR-hPSC–derived neuroectoderm . However, we consistently found the forebrain marker FOXG1 and Cajal–Retzius marker REELIN to be significantly downregulated at the end of the cerebral differentiation (C). Immunostaining for FOXG1 in these cerebral organoids then showed that shScr-hPSC–derived cerebral organoids formed FOXG1+ cortical membrane-like structures (see white arrows) but shIR-hPSC–derived cerebral organoids did not (D). This suggests that the knockdown of IR in hPSCs led to aberrant neuroectoderm development, giving rise to disrupted neural gene expression and the proper formation of mature cortical structures in this cerebral organoid model.Moving forward, we differentiated both shScr- and shIR-hPSCs into cerebral organoids using the protocol published by Lancaster et al. . We contprotocol C. Howeventiation C. Immuno did not D. This sE) decreased pAKT  and resulted in a global decrease in ECM gene expression necessary for the hPSC niche . The knockdown also resulted in a compensatory increase in pERK1/2 , an inherent compensatory mechanism, leading to increased pluripotency marker gene expression , and perturbations in cell fate specification such as that of neural development .Collectively, our data demonstrate that the knock down of IR in hPSCs 1; E decreas pAKT 2; E and resniche 3; E. The knRK1/2 4; E, an inhssion 5; E, and pepment 6; E.via ligand binding to IR/IGF1R.Insulin/IGF-I signaling pathways regulate the growth and specialized functions of most mammalian tissues including pluripotent stem cells. Although a role for IGF1R has been reported , 7, virtAmong the nodes downstream of insulin/IGF-I signaling, both the AKT and ERK1/2 pathways  are consOCT4, SOX2, DPPA4, LIN28A, SALL4, and DNMT3B and is necessary for the maintenance of pluripotency . Overall, this study links IR-mediated signaling to the pluripotent function of hPSCs and the proper regulation of cell fate specification, including that of the neural lineage. Future experiments are warranted to elucidate ERK1/2-bound targets in shIR-hPSCs to potentially reveal direct regulatory relationships with germ layer–specific genes.In summary, our follow-up study  using gemmitment E. OveralCHB8  and H9 (WiCell) hESCs stably transfected with shIR plasmids were cultured in mTeSR1 media supplemented with 1 μg/ml puromycin . Two difet al.  was performed by the Joslin Specialized Assay Core.https://massive.ucsd.edu/) with accession number MSV000085298. See Supplementary Information for details.RNA-Seq was performed at the Broad Institute. Raw data have been uploaded to GEO with accession number GSE60328. MS was performed in the Environmental Molecular Science Laboratory at Pacific Northwest National Laboratory. Raw data have been deposited in MassIVE (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


E2F/DP proteins have been shown to regulate genes implicated in cell cycle control and DNA repair. However, to date, research into the potential role of the Moso bamboo E2F/DP family has been limited.E2F/DP regulated the expression of cell cycle-related genes. A yeast two-hybrid assay and expression analysis based on transcriptome data and in situ hybridization indicated that the PheE2F-PheDP complex played important roles in winter Moso bamboo shoot growth. The qRT-PCR results showed that the PheE2F/DPs exhibited diverse expression patterns in response to drought and salt treatment and diurnal cycles.Here, we identified 23 E2F/DPs in the Moso bamboo genome, including nine E2F genes, six DP genes, eight DEL genes and one gene with a partial E2F domain. An estimation of the divergence time of the paralogous gene pairs suggested that the E2F/DP family expansion primarily occurred through a whole-genome duplication event. A regulatory element and coexpression network analysis indicated that Our findings provide novel insights into the Moso bamboo E2F/DP family and partial experimental evidence for further functional verification of the PheE2F/DPs.The online version contains supplementary material available at 10.1186/s12870-021-02924-8. Phyllostachys edulis) generates nearly 5 billion US dollars annually in economic value. It is the most important non-timber product used for commercial purposes in East Asia due to its fast growth rate [Moso bamboo  groups based on their conserved domains . The E2FArabidopsis. During cell proliferation, the canonical AtE2Fs reportedly play an antagonistic role, because E2Fc is a negative regulator, whereas E2Fa and E2Fb are positive regulators [The E2F/DP family has eight members in gulators –7. E2Fa gulators .Arabidopsis have indicated that plants also have all the core regulators in the E2F signaling pathway, such as cyclin-dependent kinase inhibitors (CKIs), retinoblastoma (RBs), cyclins, and cyclin-dependent kinases (CDKs). In plants, E2F/DP binds to the E2FAT (TTTCCCGCC) motif in its target genes, playing a role in promoting transcription [The E2F member might perform distinct roles in controlling the cell fate determination . In mammcription .PhE2F/DPs were still missing annotations. In addition, the expression changes and potential roles of the PheE2F/DP genes during bamboo shoot growth and in response to stress stimuli and diurnal cycles were still absent. In this study, a total of 23 E2F/DPs were identified based on the new version of the Moso bamboo genome annotation project, which provided chromosome-level de novo genome assembly. The phylogenetic relationships, gene structures, and conserved motifs of 23 PheE2F/DP genes were analyzed. The promoter analysis indicated that various cis-acting elements, including light response and hormone signaling as well as many transcription factor binding sites, were present in the promoter region of the PheE2F genes. We surveyed potential gene promoters containing E2F/DP binding sites within the Moso bamboo genome and identified 580 E2F/DP target genes. Based on bioinformatic predictions, we further studied the expression dynamics of the PheE2F genes during bamboo shoot growth and in response to stress stimuli and diurnal cycles. A yeast two-hybrid assay and an expression analysis were conducted to investigate the potential roles of PheE2F/DPs involved in Moso bamboo shoot growth. Our study provided new insights and some valuable information for the further functional verification of E2F/DPs in Moso bamboo.Previous studies identified 12 PhE2F/DP transcription factors from the first version of the Moso bamboo genome database . HoweverPH02Gene25981.t1 sequence, including exons, introns, 5′-UTRs and 3′-UTRs, but no complete E2F_DD or DP domains were identified in the PH02Gene25981.t1 sequence. Second, we searched the expression information and potential splicing variants of PH02Gene25981.t1 in many previously published and unpublished transcriptome sequencing data, including single molecule sequencing data [PH02Gene25981.t1 from any of the tissues. Thus, we speculated that PH02Gene25981.t1 is just a pseudogene.A total of 24 potential PheE2F/DP genes were identified in the Moso bamboo genome, including a PH02Gene25981.t1 with an incomplete E2F domain. To find whether the incomplete E2F domain in PH02Gene25981.t1 was caused by incorrect gene structure annotation in the bamboo genome or PH02Gene25981.t1 was merely just a pseudogene, we used three different methods to provide bioinformatic and experimental evidence. First, we used the CD-Search program from the NCBI to identified conserved domains involved in the whole ing data ; howeverOryza sativa, Brachypodium and Arabidopsis was constructed. For the 23 identified PheE2F/DP genes from Moso bamboo, the proteins could be classified into three functional groups: E2F, DP, and DEL [Brachypodium, rice, and Moso bamboo.To determine the evolutionary relationship of E2F/DPs in Moso bamboo and other model plants, a maximum likelihood tree of E2F/DP proteins from Moso bamboo, /DP family revealed that the genes within the same group shared similar intron lengths and numbers  of all the PheE2F/DP genes using the PlantCARE database. A large number of cis-elements including light response sites, hormone response sites, transcription factor binding sites and meristem-related motifs were found  site was abundant in the PheE2F/DP promoters, and this element was found in 21 promoters. In addition, the TGACG motif and TCA element, which are responsive to methyl jasmonate (MeJA) and salicylic acid stimulation, appeared in most of the PheE2F/DP promoter regions. Nineteen promoters contained CAT box, a meristem expression-related cis-element, indicating the importance of the PheE2Fs involved in cell division and proliferation. In addition, no significant difference was observed between different groups following a comparison of cis-acting elements.The  stimuli . Thus, wund Fig. . All 23 A previous study reported that E2F/DP binds to the E2FAT (TTTCCCGCC) motif of its target genes and regulates their transcription . Thus, wPheE2F/DPs. Most E2F/DPs showed relatively high expression levels in winter bamboo shoots (S1) and spring bamboo shoots, especially during the early growth period (S2-S5) compared with the other growing culms, such as the rhizome (R) and seedling stems (SS1 and SS2). Of the 18 genes that were expressed in at least one tissue, 11 showed the highest expression level in winter bamboo shoots (S1) , while PH02Gene46765.t1 exhibited the highest accumulation level in the outward rhizome (O). PH02Gene05248.t2 and PH02Gene10996.t1 were highly expressed in S4  and S5 , respectively. The expression analysis results revealed that the PheE2F/DPs played important roles in the growth of the bamboo shoots, especially during the winter period.The transcriptome data generated from 13 different culm tissue samples were used to investigate the expression patterns of the riod S2-S comparedPH02Gene35099.t1 (LSD1), PH02Gene44324.t1 (ATXR3), PH02Gene09249.t1 (HOP), etc. In addition, PH02Gene05248.t2 and PH02Gene01086.t1 had the second and third-highest connecting times. All three hub genes reached the highest accumulation level in winter bamboo shoots, and all of them were regulated by PH02Gene08546.t1 (MYB), PH02Gene20517.t1 (MYB) and PH02Gene25898.t2 (MYB). In addition, all three genes regulated the expression of cell cycle-associated genes, including PH02Gene32333.t1 (CDKF), PH02Gene29974.t1 (MRB1), PH02Gene36158.t1 (SPR2), and PH02Gene33706.t1 (MCM2) as well as DNA replication-associated genes, including PH02Gene32448.t3 (POLD3) and PH02Gene37503.t2 (POLA2). Furthermore, many genes involved in RNA processing or environmental stress response showed high correlations with many E2F/DP members. The in situ hybridization data showed that the mRNAs of the hub genes PH02Gene01086.t1 and PH02Gene26414.t1 were highly expressed in ground tissues of winter bamboo shoots but showed low expression levels in vascular bundles  screening, a total of 58 genes had a strong correlation with at least one PheE2F/DP gene Fig. . In the Arabidopsis, E2F can interact with DP to activate the cell cycle and cell proliferation-associated gene expression [PH02Gene34520.t1 (E2F), PH02Gene26414.t1 (DP), and PH02Gene34005.t1 (DP), which showed the highest accumulation level in winter bamboo shoots, were selected and further tested. The combinations of pGBKT7-PH02Gene26414.t1 + pGADT7-PH02Gene34520.t1 and pGBKT7-PH02Gene34005.t1 + pGADT7-PH02Gene34520.t1 were co-transformed into yeast strain AH109. The Y2H results showed that the positive control, negative control, and both test transformants grew well on SD/−Leu/−Trp medium  and salt treatments .To understand the transcriptional changes in iod Fig. . The qRTArabidopsis (7), Zea mays (19), wheat (18) and Brachypodium (11) [Zea mays (2300 Mb) [Triticeae) (17 Gb) [Brachypodium (300 Mb), Arabidopsis (164 Mb) and Oryza sativa (441 Mb) [PheE2F/DPs in Moso bamboo indicated that the abundance of PheE2F/DPs may be associated with genome duplication events. Previous reports suggested that Moso bamboo was tetraploid in origin, it went through a long evolutionary progression from tetraploidy to diploidy, and it carried two duplicates, similar to Oryza sativa gene model sets [E2F/DP controls the spatial and temporal expression of genes that are essential for multiple biological processes during the cell cycle, and their transcription level determined the cell proliferation . In a geium (11) –17. The ), wheat  and BracZea mays , wheat ((441 Mb) –22. Rathdel sets , 23. A sPheE2F/DPs, including five E2Fs and four DPs, showed the highest expression in winter bamboo shoots , PH02Gene20517.t1 (MYB) and PH02Gene25898.t2 (MYB). In addition, several MYB genes, such as PH02Gene26882.t1 and PH02Gene09101.t1, repressed the expression of E2F/DP genes , which showed a high correlation with PH02Gene41928.t1 (E2F/DP) and PH02Gene26414.t1 (E2F/DP), is involved in stress responsiveness. In Arabidopsis, AtRGGA is induced by salt and osmotic stress, and atrgga mutants exhibit increased sensitivity to abiotic stress [PH02Gene25752.t1 (an ABA-responsive gene) and PH02Gene37465.t1  were also regulated by E2F/DPs in the network [PheE2F/DPs under abiotic stress might be essential for plant resistance to numerous adverse environments.The identification of regulatory elements in the ess Fig. . Coexprec stress . In addi network . The higE2F expression. However, there is a great deal of new evidence that suggests that drought and salt stress can stimulate the expression of cell cycle genes. For the Medicago truncatula E2Fb gene, an upregulation trend was reported under high salt concentration treatment [Oryza CDKC1, which is involved in cell proliferation and differentiation, was triggered by NaCl treatment through the ABA signaling pathway [Arabidopsis, CDC2aAt, a cyclin-dependent kinase-coding gene and two mitotic cyclin genes, namely, Arath;CycB1;1 and Arath;CycA2;1, were monitored during salt stress [PheE2F/DPs participated in different biological processes in Moso bamboo.The regulation of the cell cycle might be subject to interference by drought and salt stress at the transcriptional level through repression of reatment , 27. In  pathway . In Arabt stress . In mammt stress . Many Phcis-acting elements involved in the photoperiodic response, hormone signaling, meristem growth, and many MYB and MYC2 binding sites were present in the PheE2F/DP promoters. Recent whole-genome duplication played important roles in the E2F/DP family expansion. Transcriptome sequencing and in situ hybridization analysis revealed that E2F/DPs play important roles in bamboo shoot growth, especially during the winter period. Expression profiles derived from qRT-PCR indicated that E2F/DP expression is stimulated by various forms of abiotic stress and diurnal cycles. These results provide comprehensive insights into the Moso bamboo E2F/DP gene family and lay a solid foundation for further functional verification of the PheE2F/DP family.In conclusion, a total of 23 PheE2F/DPs were identified in the Moso bamboo genome, including nine E2F, six DP, and eight DEL genes. The E2F/DP sequences of Arabidopsis thaliana (Arabidopsis), Oryza sativa (rice), and Brachypodium distachyon (Brachypodium) were downloaded from The Arabidopsis Information Resource (TAIR) [Brachypodium distachyon genome database [AtE2F/DP and OsE2F/DP sequences were then used as queries in BLASTP searches against the local bamboo protein database using the default parameters. The sequences were considered as predicted E2F/DPs if they had E-values of ≤ − 10. Lastly, the protein families database (Pfam) was further used to confirm each candidate E2F/DP sequence.The e (TAIR) , the rice (TAIR) , and thedatabase , respectMoso bamboo shoots as well as other culm samples were collected by Long Li in Guangde County , Anhui Province, from January to August 2018 . A drought stress test was conducted by irrigating the two-month-old seedlings with 18% PEG6000 medium. A salt stress test was conducted by irrigating the two-month-old seedlings with medium containing 250 mM NaCl. The leaves were harvested by Long Li at 1, 3, 12 and 24 h after abiotic stress treatment. Seedlings irrigated with tap water were used as the control (CK). The plant samples were frozen in liquid nitrogen and stored at − 80 °C prior to use. Five individuals that showed similar growth patterns were pooled at each time point and three biological repeats were employed for each sample. The voucher specimens were deposited in the herbarium at the International Center for Bamboo and Rattan, Beijing, catalog numbers ICBR-B-18101 (bamboo shoot), ICBR-B-18201 (rhizome), ICBR-B-18301(outward-rhizome), ICBR-B-18401 (seedling stem), ICBR-B-18501 (seed), ICBR-B-18601 (seedling leaf).The Gene Structure Display Server program (GSDS) was used to investigate the gene structure of E2F/DP based on the GFF annotation file with the default parameters . The conPheE2F/DPs as well as the other 51,050 Moso bamboo genes (from − 2000 bp to + 1 bp) were analyzed using the PlantCARE online program [− 6 and a minimum similarity of 55% were annotated with GO terms. The promoters of genes that contained E2F/DP binding sites were further used for Gene Ontology (GO) enrichment analysis via TBtools [The transcription start sites (mRNA start sites) were designated as + 1 and obtained by GFF file as downloaded from the GigaDB, the new version of the Moso bamboo genome annotation project . The reg program , 42. A G TBtools .E2F/DP genes, homology data derived from protein-protein comparisons made using BLASTN, with an E-value cutoff of 1e-6, and the other parameters were the defaults or recommended ones. The BLASTP result was analyzed using MCScanX to find the collinear regions between PheE2F/DP genes as well as other synteny blocks across the whole genome. The minimum block size was 5 and the other parameters were the default or recommended ones. The synteny blocks were extracted to draw a collinearity map within PheE2F/DPs using TBtools software.To investigate the synteny of blocks containing − 9 in monocotyledons and 1.5 × 10− 8 in dicotyledons [The Ka (nonsynonymous substitution), Ks (synonymous substitution) and the Ka/Ks ratio between the paralogous pairs were calculated using DNA Sequence Polymorphism software (DnaSP). The formula T = Ks/2λ was used to estimate the divergence time of the duplication event, with a divergence rate λ = 6.5 × 10tyledons .The ClustalW program was executed for multiple sequence alignments using the full-length E2F/DP amino acids with the default settings. The parameters used to run the CLUSTALW program were as follows: Scoring matrix for amino acid sequences: BLOSUM62, gap open: 10, gap extension: 0.2, iteration: none. The Maximum Likelihood tree was built using the IQ-Tree software with the best fit tree model, and the JTT + R3 was found to be the best model according to the Bayesian Information Criterion. The bootstrap method was applied in phylogeny test with 1000 replications. After that, the results were imported to FigTree v1.4.2 software for processing.-△△Ct method.The total RNA from each sample was isolated using the TRIzol reagent . First-strand cDNA was synthesized with 2 μg RNA using a PrimeScriptTM RT Reagent Kit  according to the manufacturer’s instructions. qRT-PCR experiments were performed using SYBR Green chemistry  on a Light Cycler 480 instrument  according to the manufacturer’s directions. The Primer 3 online program was used for gene-specific primer design  [PheE2F/DPs was measured as fragments per kilobase of exon model per million mapped reads (FPKM). The heatmap was generated using R.The transcriptome data for A706151) . These tThe transcription factor-target gene (TF-TG) interactions were predicted based on the regulatory elements in the promoters of the target genes. The gene expression correlation was calculated by Pearson correlation coefficient (PCC) using FPKM through the R program. Only the transcription factor (TF) and target relationships that displayed PCC values of ≥ + 0.90 or < − 0.90 were retained for coexpression network construction. The network was ultimately visualized using Cytoscape 3.7.0 software.PH02Gene01086.t1 and PH02Gene26414.t1 were amplified by PCR using gene-specific primers with T7 and SP6 RNA polymerase-binding sites. The images were captured using an Olympus Nikon E600.Bamboo shoot tissues were fixed in 4% paraformaldehyde for one day at 4 °C and then hybridized as described previously . The speTo test whether E2F can interact with DP in Moso bamboo, the Matchmaker GAL4 two-hybrid system  was applied. Based on the in-fusion cloning method , the full length of the PH02Gene26414.t1 and PH02Gene34005.t1 cDNAs were independently cloned into pGBKT7, and the full length PH02Gene34520.t1 cDNAs were cloned into pGADT7 (Additional file Additional file 1: Figure S1. Sequence logo of the different motifs identified in the PheE2F proteins. Figure S2. Moso bamboo tissues used for expression analysis. Table S1. Estimated divergence period of E2F/DP gene pairs in four species. Table S2. The primer sequences used for qRT-PCR. Table S3. The primer sequences used for gene cloning


The outbreaks of H5N2 avian influenza viruses have occasionally caused the death of thousands of birds in poultry farms. Surveillance during the 2018 winter season in South Korea revealed three H5N2 isolates in feces samples collected from wild birds . Phylogenetic tree analysis revealed that these viruses arose from reassortment events among various virus subtypes circulating in South Korea and other countries in the East Asia–Australasian Flyway. The NS gene of the KNU18-28 and KNU18-86 isolates was closely related to that of China’s H10N3 strain, whereas the KNU18-93 strain originated from the H12N2 strain in Japan, showing two different reassortment events and different from a low pathogenic H5N3 (KNU18-91) virus which was isolated at the same day and same place with KNU18-86 and KNU18-93. These H5N2 isolates were characterized as low pathogenic avian influenza viruses. However, many amino acid changes in eight gene segments were identified to enhance polymerase activity and increase adaptation and virulence in mice and mammals. Experiments reveal that viral replication in MDCK cells was quite high after 12 hpi, showing the ability to replicate in mouse lungs. The hematoxylin and eosin-stained (H&E) lung sections indicated different degrees of pathogenicity of the three H5N2 isolates in mice compared with that of the control H1N1 strain. The continuing circulation of these H5N2 viruses may represent a potential threat to mammals and humans. Our findings highlight the need for intensive surveillance of avian influenza virus circulation in South Korea to prevent the risks posed by these reassortment viruses to animal and public health. Influenza viruses infect many animal species in addition to humans. Avian influenza viruses (AIVs) have a very wide host range, including mallard ducks, wild ducks, chickens, bean gooses, shorebirds, pigs, and swans. There are 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes that were classified according to the basic surface proteins (H17-18 and N10-11 were identified in bats) . VirusesH5 and H7 subtypes LPAIVs evolve into HPAI viruses through a change in the HA cleavage site and cause an infectious disease in humans. The outbreak of avian influenza viruses significantly affects both the human health and the economy. The outbreak of H5N1 in Hong Kong in 1997 reported six cases of death . In 1983Yeo et al. have reported the low-pathogenic avian influenza H5N3 reassortment virus  during AIV surveillance in South Korea in the 2018-winter season. This H5N3 virus has resulted from the reassortment event of multiple virus strains coming from China and Mongolia via migratory birds . In thisFresh wild bird’s feces samples were collected in South Korea environment by sterile swabs in the 2018-winter season. The feces samples were maintained at 2–8 °C and then doing further analysis within 12 h in the laboratory.Phosphate buffered saline  containing 100 U/mL penicillin and 100 mg/mL streptomycin was used to resuspend the feces samples. The mixed samples were centrifuged 3000 rpm at 4 °C for 10 min to collect the supernatant, which was then filtered by a 0.45 µm pore size GVS Syringe Filter . Next, virus samples were inoculated in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (ECEs)  for virus amplification at 37 °C for 72 h under humidified conditions. Allantoic fluid was collected from the inoculated ECEs, and the hemagglutination assay was performed to verify the presence of the influenza virus.® RNA extraction kit  according to the manufacturer’s instructions. RNA was finally eluted in 60 µL of RNase-free water, supplied with 20 units of RNase inhibitor for storage at −80 °C for further analysis. A reverse transcription polymerase chain reaction (RT-PCR) was performed to verify the presence of the influenza virus in the sample. The matrix gene was amplified using universal primers and probes according to the World Health Organization guidelines [Viral RNA was extracted directly from the allantoic fluid of ECEs using the NucleoSpinidelines . Host ididelines .Next generation sequencing was conducted by GnCBio  on the Illumina Hiseq X platform , as previously reported . Brieflyhttp://www.gisaid.org, accessed on 1 September 2021) and the National Center for Biotechnology Information . The blasted sequence of all three isolates was combined, and the replicated sequences were deleted. Next, phylogenetic trees were generated by the maximum likelihood method with 1000 bootstrap replicates using MEGA-X software version 10.2.6 .Nucleotide blast analysis was used to identify the closest relatives of the viral genes. All reference sequences were downloaded from Global Initiative on Sharing All Influenza Data  for detecting the viral nucleoprotein (NP) antigen.Three isolated H5N2 viruses  together with two reference strains  and H5N3 (A/spot-billed duck/Korea/KNU SYG06/2006)) were infected into the Madin–Darby canine kidney (MDCK) cell line (American Type Culture Collection) to examine the viral replication capacity in mammalian cells. Briefly, five kinds of viruses were inoculated with 0.01 multiplicity of infection (MOI) into MDCK cell monolayers in DMEM containing 1 µg/mL TPCK-treated trypsin  and were incubated at 37 °C. The virus supernatant was collected every 12 h until 72 h post-infection (hpi). Viral replication titers were determined by the TCID50) titers as reported previously [2. When MDCK cells reached 80–90% confluence, cells were washed with PBS. The cells were then inoculated with 10-fold serial dilutions of viral suspensions. The medium was removed; the cells were washed with PBS once and fixed with 80% cold acetone after 3 days of cell incubation. The plates were blocked with 5% skim milk to remove the non-specific binding for 2 h at 37 °C. Next, the anti-influenza nucleoprotein antibody (0.1 µg/well) was applied to capture the nucleoprotein fixed on the well bottom surface for 1 h at 37 °C. After washing 3 times with PBS-T, the rabbit polyclonal antibody to mouse IgG (HRP)  was added at 1:5000 dilution. The TMB substrate was applied after stringent washing with PBS-T five times and the signal was measured at wavelength 450 nm with a SpectraMax® iD3 microplate reader . The TCID50 titers were determined using the Reed and Muench method [The enzyme-linked immunosorbent assay (ELISA) was used to measure the 50% tissue culture infectious dose , the chorioallantoic cavities of 10-day-old SPF ECEs were inoculated with 100 μL serial 10-fold dilutions of the viruses, using five ECEs for each dilution. The ECEs were incubated at 37 °C for 3 days, after which the allantoic fluid was harvested and tested for hemagglutination activity. Calculation of EIDn = 12) that were intranasally inoculated with 105 EID50/50 µL of the virus. Mice were anesthetized using 1% isoflurane following the manufacturer’s instructions  and the guidelines of Vertebrate Animal Research, University of Iowa. The body weight and survival rate of mice were observed for 14 days. Mice were euthanized and their lungs were collected on days 3, 6, and 14 post-infection (n = 3). The lung tissue was homogenized, and the TCID50 was determined to test the viral titers of homogenate supernatant. This study was conducted following the relevant guidelines and regulations of the Animal Ethics Committee of the Wonkwang University . The lungs from three mice collected at day 6 post-infection for each group were fixed in 10% formalin/saline, and the tissue was embedded in paraffin. By using standard hematoxylin and eosin (H&E) staining, histological characterization of the lung sections was observed under microscopy at 40× magnification.The pathogenic potential of the new isolate was determined in 6-week-old female BALB/c mice purchased from Orient   of biological replicates.All results were statistically analyzed using GraphPad Prism 5.0. One-way and two-way analyses of variance (ANOVA) were used to analyze the bodyweight change, the lung weight and the TCIDAmong the 30 positive samples out of the collected 500 fecal samples from various wild birds, such as mallard duck, big goose, spot-billed duck, rump pigeon, and bean goose, in 2018, three isolated viruses were determined as H5 subtype AIVs based on HA and NA gene sequencing results from NGS. The A/Wild duck/South Korea/KNU18-28/2018 (H5N2) (KNU18-28) strain was collected from Han River Estuary, Paju, Gyeonggi-do  on 16 October 2018. The A/Bean Goose/South Korea/KNU18-86/2018 (H5N2) (KNU18-86) and A/Wild duck/South Korea/KNU18-93/2018 (H5N2) (KNU18-93) strains were collected from Hwaseong Sihwa Lake, Gyeonggi-do  on 2 December 2018. The viruses were inoculated in ECEs and identified using hemagglutination assay. The full genome sequences of the three H5N2 isolates were submitted to the NCBI GenBank database, with accession numbers MT477766-MT477773, MT477790-MT477797, and MT477798-MT477805 corresponding to eight gene segments of KNU18-28, KNU18-86, and KNU18-96, respectively.The highest nucleotide identities for the three isolates obtained from the GenBank database are shown in The putative original gene segments of the KNU18-28, KNU18-86, and KNU18-93 isolates are illustrated in Phylogenetic tree analysis was performed for eight genes of the three novel H5N2 isolates to evaluate their genetic relationship with those of wild birds and domestic poultry in Korea and neighboring countries. All eight genes  in our three selected H5N2 strains were mainly distributed in East Asian and Eurasian lineages, far from highly pathogenic H5N2 strains as well as the H5N3 isolate (KNU18-91) . The thrWe proposed a hypothesis identifying the ancestor of each gene segment of the three H5N2 isolates in two different reassortment events as shown in The ancestors of 5/8 gene segments  were identified in various strains  in Korea during the migratory season 2016–2017. A/mallard/Korea/H50-4/2016 (H5N3) may donate HA gene to the A/spot-billed duck/Korea/H10-1/2017 (H5N3), and A/bean goose/Korea/F54-8/2017 (H6N1) may donate M gene to the A/mallard/Korea/A21-2/2017 (H5N2) before the reassortment resulting in KNU18-28 and KNU18-86 strains A. This iWhile lacking key mutations that support the virus become highly pathogenic; many amino acid mutation sites of internal genes, involved in enhancing the virulence of virus in mammalian cells, birds, and mice, of all three H5N2 isolates are shown in The viral replication kinetics of three H5N2 isolates in mammalian cells was investigated compared to that of the H1N1  and H5N3 (A/spot-billed duck/Korea/KNU SYG06/2006) control virus strains.50 assay raw data for growth kinetics are presented in 5 EID50/50 µL. The control H1N1 and H5N3 strains were included for comparison. The body weight of the mice infected with the H1N1 strain was found to decrease regularly, with the lowest weight (84.14% ± 2.29%) observed at 7 days post-infection (dpi). In contrast, the three novel H5N2 isolates and H5N3 control strain maintained a stable body weight for 14 days (10 TCID50/mL at 3 dpi and 5.25 ± 0.31 log10 TCID50/mL at 6 dpi), whereas H5N3 control strain in the lung with the lowest titer (2.84 ± 0.21 log10 TCID50/mL at 3 dpi and 3.58 ± 0.12 log10 TCID50/mL at 6 dpi). The three novel H5N2 isolates  were observed with high viral replication titer at 3 dpi  and at 6 dpi . Among them, the KNU18-93 strain replicated with a low titer at 3 dpi, which then became higher than that of the KNU18-86 strain at 6 dpi. Raw data from the TCID50 assay are shown in p < 0.01) and KNU18-93 (p < 0.001) strains, compared with control strain H5N3.To determine the pathogenic potential in mammals, the six-week-old female BALB/c mice were used to inoculate the three novel H5N2 isolates with a 10 14 days A. All in 14 days B. The vin = 3 in each group). Except for the normal and H5N3 mice groups, the H&E-stained sections of the KNU18-28, KNU18-86, KNU18-93, and H1N1-infected lungs revealed that neutrophils had penetrated the alveolar air spaces at 6 dpi  and KNU18-93 (p < 0.001) at 6 dpi  isolate did not replicate well in mice, showing a similar pattern and significantly lower virus titer in the lung at 3 dpi compared with the H5N3 control strain (A/spot-billed duck/Korea/KNU SYG06/2006) . In our at 6 dpi C. Taken Apart from the cleavage site, the mutation sites also support the adaptation of avian influenza viruses to mammals, especially the E627K and D701N substitutions in the polymerase basic protein 2 (PB2). Both the key mutations, E627K plays a critical role in mammalian adaptation ,59 and DHost adaptation for virulence is generally performed through 5 to 20 lung-to-lung passages in mouse models. In 2017, Nam et al. reported that a low pathogenic avian influenza H5N2 virus, A/Aquatic Bird/Korea/CN2/2009, shifted rapid virulence during a single passage in mice lungs. All mice infected with the passage 1 virus died within 8 dpi, indicating the potential of the virus to become virulent with 100% lethality after a single passage in mice. The molecular analysis indicated PB2 E627K and PA T97I mutations that are responsible for the acquisition of mouse virulence in a common LPAI H5N2 isolate after a single passage [Our present study indicates that H5 AIVs circulating in Korea have undergone reassortment with other AIVs in Korea, as well as in other countries, via bird migration, resulting in two genetic groups of novel reassorted H5N2 AIVs causing virulence in mice. Thus, continuously monitoring AIVs in wild migratory birds has become a critical task in the prevention and control of the AIV in South Korea.


The length and width of the natural mature pod in spm were only 71.34% and 73.36% of those in WT, respectively. We performed comparative analyses for morphological characteristics, anatomy, physiology, and global transcriptome between spm and WT pods. Samples were collected at 10, 20, and 30 days after peg elongation into the soil, representing stages S1, S2, and S3, respectively. The differences in pod size between WT and spm were seen at stage S1 and became even more striking at stages S2 and S3. The cell sizes of the pods were significantly smaller in spm than in WT at stages S1, S2, and S3. These results suggested that reduced cell size may be one of the important contributors for the small pod in spm. The contents of indole-3-acetic acid (IAA), gibberellin (GA), and brassinosteroid (BR) were also significantly lower in spm pods than those in WT pods at all three stages. RNA-Seq analyses showed that 1,373, 8,053, and 3,358 differently expressed genes (DEGs) were identified at stages S1, S2, and S3, respectively. Functional analyses revealed that a set of DEGs was related to plant hormone biosynthesis, plant hormone signal transduction pathway, and cell wall biosynthesis and metabolism. Furthermore, several hub genes associated with plant hormone biosynthesis and signal transduction pathways were identified through weighted gene co-expression network analysis. Our results revealed that IAA, GA, and BR may be important regulators in controlling pod size by regulating cell size in peanuts. This study provides helpful information for the understanding of the complex mechanisms of plant hormones in controlling pod size by regulating the cell size in peanuts and will facilitate the improvement of peanut breeding.Pod size is an important yield-influencing trait in peanuts. It is affected by plant hormones and identifying the genes related to these hormones may contribute to pod-related trait improvements in peanut breeding programs. However, there is limited information on the molecular mechanisms of plant hormones that regulate pod size in peanuts. We identified a mutant with an extremely small pod ( Arachis hypogaea L., 2n = 4x = 40, AABB) is one of the most important oil crops for the human diets worldwide  in Arabidopsis (BnDA1 in rapeseed (Grain width 2 (GW2) in rice  in wheat (KERNEL NUMBER PER ROW 6 (KNR6) in maize (AUXIN-REGULATED GENE INVOLVEDIN ORGAN SIZE (ARGOS) gene increased cell size and cell number, resulting in enlarged leaves and siliques (AUXIN RESPONSE FACTOR 18 (ARF18) encodes an auxin-response factor, and disfunction of the ARF18 gene can induce longer siliques by accelerating cell expansion in the silique wall of Brassica napus L. (GA 20-oxidases (GA20ox) genes, GA20ox1 and GA20ox2, involved in gibberellin (GA) biosynthesis, regulate hypocotyl, internode, and siliques elongation in Arabidopsis  is allelic to GROWTH REGULATION FACTOR4 (OsGRF4) and positively regulates brassinosteroid (BR) responses to promote rice grain development  related to peanut pod size have been reported in recent years . Luo et Peanut has an unique characteristic of fruit production. It accomplishes flowering and fertilization aerially, but forms the fruits underground . After fspm) from Yuanza 9102 (WT) using 60Co γ-radiation mutagenesis, and performed morphological, anatomical, physiological, and transcriptome analyses for pods in WT and spm at three key pod developmental stages. The comparative analyses revealed significant differences in the pod size, cell size, contents of plant hormones, and dynamic gene expression between WT and spm. Pairwise comparisons and WGCNA analyses identified several plant hormone-related genes, which were involved in determining peanut pod sizes. Our study contributes to the understanding of the transcriptome dynamics of peanut pod development and are helpful for elucidating the complex regulatory mechanism of pod size in peanuts.In the present study, we identified a mutant with an extremely small pod (spm mutant was selected from an 60Co γ-radiation-induced population originating from Yuanza 9102 (WT). The spm mutant was self-pollinated for 10 generations to ensure a stabilized line for this study. Both WT and spm mutant were grown in 2019 in the Yuanyang experiment station of the Henan Academy of Agricultural Sciences. Days to first flower, we marked the flowers and tied the elongating aerial pegs with colored tags  for 24 h. Pericarps were dehydrated in an ethanol series . Pericarps were then processed in xylene for 10 min and embedded in paraffin . The perspm and WT used for indole-3-acetic acid (IAA), BR, and GA quantification were sampled from the same plants used for RNA-seq at stages S1, S2, and S3. A detailed method for pod collection and store was described in the RNA sample collection. The method for extraction, purification, and quantification of the IAA, BR, and GA by enzyme-linked immunosorbent assay kits was modified following the protocol described in spm and WT pods were tested using ANOVA.All the pods of PRJCA006624 . Low-quality bases and adaptor sequences were eliminated through fastp  using HISAT  < 0.05 were considered to be significantly enriched. The MapMan categories with significance value < 0.05 were used for performing pathway enrichment analysis of DEGs.Total RNA from the marked pod harvested at stages S1, S2, and S3 was extracted using the TRIzol reagent kit . RNA integrity and quality were tested by using an Agilent 2100 bioanalyzer . Subsequently, a total of 18 libraries were constructed, and the transcriptome sequencing was performed on Illumina HiSeq2500 platform by the Genedenovo Biotechnology Company . The RNA-seq sequencing data in this study have been uploaded to China National Center for Bioinformation under BioProjects, v0.18.0; . The remv0.18.0; , and the v2.0.0; . Transcr v2.0.0; . Princip v2.0.0; . The difWGCNA was performed for analyzing gene correlation patterns among different samples using the WGCNA software package as described in −ΔΔCt method , which differ significantly in pod size/weight, were selected to investigate the pod differences (P < 0.001) than those of spm  and its small pod mutant  and S2 (P = 0.002). Similarly, the mesocarp cell areas in spm were significantly smaller than those in WT at stages S1 (P < 0.001) and S2 (P = 0.002). The cell areas of spm in the endocarp were always significantly smaller (P < 0.001) than those of WT from stages S1 to S2  in WT and e stages , indicatspm. A total of 18 samples were sequenced, and 35.80–67.65 million high-quality reads were obtained for each sample  accounted for approximately 4% in different samples. Approximately 20%, 40%, and 36% of genes with high (10 ≤ FPKM < 50), moderate (2 ≤ FPKM < 10), and low (0.1 ≤ FPKM < 2) expression levels, respectively, were identified in different samples  were detected in spm pods compared with WT pods , xyloglucan glycosyltransferase , xyloglucan xylosyltransferase (XXT), cellulose synthase , and plant invertase/pectin methylesterase inhibitor (PMEI) had significantly lower expression levels in stage S2 . These gstage S2 . In addie length . The CLAxpansion . The LNGbidopsis .spm pods at stage S2 for IAA (spm than in WT at stages S1–S3. The genes encoding protein PIN-LIKES 2 (PILS2) displayed significantly higher expression levels in spm than in WT at stages S1 and S2, however, PILS1 genes exhibited significantly lower transcriptional activity in spm than in WT at stages S1 and S2. The expression level of gene encoding protein TRANSPORT INHIBITOR RESPONSE 1 (TIR) was significantly higher in spm at stages S2. The genes encoding ARF9 and auxin-responsive proteins (IAA26) exhibited significantly higher transcriptional activity in spm at stages S2 and S3, whereas other genes encoding ARF10 and Aux/IAA family members  showed significantly lower expression levels in spm at stages S2 and S3. The expression levels of genes encoding tryptophan aminotransferase-related protein 2 (TAR2), ATP-binding cassette subfamily proteins (ABCB1 and ABCB19), auxin-induced in root cultures protein 12 (AIR12), IAA-amido conjugating enzyme family members (GH3.1 and GH3.6), auxin efflux carrier family proteins , and auxin transporter-like proteins (LAX1 and LAX5) were significantly higher in WT than in spm at stage S2.The expression of DEGs related to IAA, GA, and BR showed striking differences at stages S1–S3, especially at stage S2 . A total for IAA . The genspm pods at stage S2 for GA (spm compared with WT at stages S2 and S3. The genes encoding GIBBERELLIN INSENSITIVE DWARF1 (GID1B and GID1C) had higher expression levels in spm than in WT at three stages. The genes encoding gibberellin-regulated proteins  showed significantly lower expression levels in spm than in WT at stage S2.A total of 20 genes showed strikingly different expression levels between WT and 2 for GA . The genspm pod at stage S2 in BR  showed significantly lower expression levels in spm than in WT at three stages, whereas, BES1/BZR1 homolog protein 2 (BEH2) showed significantly higher expression levels in spm than in WT at stages S2 and S3. The transcriptional activity of genes encoding BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1), BRI1 kinase inhibitor 1 (BKI1), Serine/threonine-protein kinase BSK1 (BSK1), and Brassinosteroid LRR receptor kinase (CURL3) was significantly lower in spm than in WT at stages S2 and S3. The genes encoding steroid 5-alpha-reductase DET2 (DET2), 3-hydroxy-3-methylglutaryl-coenzyme A reductase-like proteins (HMG1), sterol methyltransferases (SMT1 and SMT2), serine/threonine-protein kinase BSK3 (BSK3), and CYCLIN D3 (CYCD3-3) exhibited significantly lower expression levels in spm than in WT at stage S2.A total of 30 genes exhibited significantly different expression levels between WT and S2 in BR . Eleven spm compared with WT at stages S1, S2, and S3, respectively. These TFs were classified into 47 gene families , the remaining 30,233 genes were used to perform WGCNA. According to pairwise correlations of gene expression across all samples, a hierarchical clustering tree with 23 co-expression modules was constructed . The cor WT pods . The exp WT pods . Therefo WT pods . Forteen WT pods  and 10F.l stages . These rNine hub genes were selected to validate the accuracy of RNA-seq results. These included four genes related to auxin signal transduction pathways, two genes related to GA biosynthesis or signal transduction pathways, and three genes related to BR biosynthesis or signal transduction pathway. The results revealed that the overall expression trend of these nine genes, as quantified by the qRT-PCR, were consistent with those obtained from RNA-seq analysis , thus despm) at three stages. The differences in pod size appeared at stage S1, expanded rapidly at stage S2, and peaked at stage S3  than in WT  decreased seed size and seeds per fruit in cucumber  were detected in Click here for additional data file.


Permanent coronary ligation was performed to induce MI in a murine model. BM activation, inflammatory cell composition and cardiac function were assessed using global and platelet-specific gene knockout and pharmacological inhibition models for P2Y12. Complementary in vitro studies allowed for investigation of ADP-dependent effects on LSK cells. We found that ADP acts as a danger signal for the hematopoietic BM and fosters emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. We were able to detect P2Y12 in LSK, implicating a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 knockout and P2Y12 inhibitor treatment with prasugrel reduced emergency hematopoiesis and the excessive inflammatory response to MI, translating to lower numbers of downstream progeny and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition preserved cardiac function and reduced chronic adverse cardiac remodeling after MI. P2Y12-dependent signaling is involved in emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel, non-canonical value for P2Y12 antagonists beyond inhibition of platelet-mediated atherothrombosis.Emergency hematopoiesis is the driving force of the inflammatory response to myocardial infarction (MI). Increased proliferation of hematopoietic stem and progenitor cells (LSK) after MI enhances cell production in the bone marrow (BM) and replenishes leukocyte supply for local cell recruitment to the infarct. Decoding the regulation of the inflammatory cascade after MI may provide new avenues to improve post-MI remodeling. In this study, we describe the influence of adenosine diphosphate (ADP)-dependent P2YThe online version contains supplementary material available at 10.1007/s00395-022-00927-6. Inflammation is an essential prerequisite for wound healing and cardiac remodeling after myocardial infarction (MI) . Acute iInjured cardiac cells secrete various chemoattractants that have been appreciated to locally regulate post-MI inflammation , 15. In Lineage neg, Sca-1+, c-Kit+)  . Mean ± S.E.M., *p < 0.05. (PNG 132 KB)Supplementary file2 Proliferation analysis performed with BrdU incorporation assay in C57BL/6 mice treated with prasugrel compared to vehicle control. Bar graph shows relative cycling LSK rates in % on day 3 after MI. (A IL-1β levels in plasma on day 1 after MI in prasugrel-treated C57BL/6 mice compared to vehicle control and wildtype (WT), assessed by ELISA . B Bone marrow niche factors CXCL12, VCAM-1, Angpt-1 and SCF on day 3 after MI in C57BL/6 mice under prasugrel treatment compared to vehicle control and WT, evaluated by qPCR . C LSK cell and hematopoietic progenitor mobilization into peripheral blood on day 3 after MI under prasugrel treatment in comparison to vehicle control, evaluated by FACS . Mean ± S.E.M., **p < 0.01. (PNG 222 KB)Supplementary file3 A Schematic illustration of the experimental setup. After establishing cyclooxygenase-mediated platelet inhibition by a loading dose of ASA, LAD was ligated for MI and analysis was performed on day 2 after MI as shown. B Bleeding time under oral treatment with ASA versus vehicle control . C Flow cytometric gating for LSK cells and cell cycle analysis performed with Ki67/DAPI assay in ASA-treated C57BL/6 mice compared to vehicle control. Bar graphs show cycling LSK rates and LSK in non G0-phase in % and cell counts of the progenitor populations GMP and MDP per femur on day 2 after MI . Mean ± S.E.M., *p < 0.05. (PNG 449 KB)Supplementary file4 Below is the link to the electronic supplementary material.


In the originally published version of this manuscript, two errors were noted and are listed in this erratum.The following sentences appear in the first paragraph of the introduction and should have appeared as a footnote:The nucleotide sequences reported herein have been submitted to DDBJ under accession numbers LC189561 (p35S-Cas9), LC189562 (pX2-Cas9), LC189563 (pKIR1.0), LC189564 (pKIR1.1), LC189565 (pKI1.1) and LC189566 (pKI1.1R). Patent: a patent has been applied for on the method to drive Cas9 using the RPS5A promoter for plant genome editing (patent application number: 2016–171590).In Figure 4F, one point of data is lacking a label. This label should read .The Publisher apologises for these errors.


Stylosanthes guianensis is an excellent forage legume in subtropical and tropical regions with drought tolerance, but little is known about its drought tolerance mechanism. Dehydration responsive element binding proteins (DREBs) are responsive to abiotic stresses. A SgDREB2C was cloned from S. guianensis, while SgDREB2C protein was localized at nucleus. SgDREB2C transcript was induced by dehydration treatment. Transgenic Arabidopsis overexpressing SgDREB2C showed enhanced osmotic and drought tolerance with higher levels of relative germination rate, seedlings survival rate and Fv/Fm and lower levels of ion leakage compared with WT after osmotic and drought stress treatments. In addition, higher levels of superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities and stress responsive gene  transcripts were observed in transgenic Arabidopsis than in WT under drought stress. These results suggest that SgDREB2C regulated drought tolerance, which was associated with increased SOD and APX activities and stress-responsive gene expression under drought stress. DREB members are classified into six subgroups, named A-1, A-2, A-3, A-4, A-5 and A-6 in Arabidopsis [DREB2A and DREB2B from Arabidopsis plants is induced by dehydration and salt stress [AtDREB2A and AtDREB2B have functional redundancy in response to heat stress [Arabidopsis plants overexpressing AtDREB2A show increased drought tolerance and heat tolerance [OsDREB2A from rice results in enhanced drought and salt tolerance [OsDREB2B expression leads to improved drought tolerance in transgenic rice [ZmDREB2A from Zea mays improves heat and drought tolerance in transgenic Arabidopsis [PcDREB2A-overexpressing Arabidopsis plants show increased tolerance to drought and osmotic stress [EsDREB2B from Eremosparton songoricum leads to increased tolerance to osmotic stress, salt, cold and heat in transgenic tobacco [Drought is one of the major abiotic stresses reducing crop productivity . Thousanbidopsis , while Dbidopsis ,9. Expret stress . In addit stress . Transgeolerance , but onlolerance . Overexpolerance ,15, whilnic rice . Overexpbidopsis . PcDREB2c stress . Overexp tobacco .AtDREB2C is induced by oxidative stress [AtDREB2C results in improved thermotolerance and freezing tolerance in transgenic Arabidopsis plants [Arabidopsis [DREB2C enhances tolerance to oxidative stress in transgenic Arabidopsis by regulating HsfA3, which promotes APX2 expression [Arabidopsis plants overexpressing AtDREB2C perform delayed germination and increase abscisic acid (ABA) content [OsDREB2C expression in rice is induced by drought and salinity [MsDREB2C from Malus sieversii is induced by salicylic acid (SA), jasmonic acid (JA), ABA, drought, salt, cold and heat [DREB2C confers tolerance to multiple abiotic stresses in plants. Transcription of e stress , heat [2e stress  and salte stress . Constits plants ,21. It sbidopsis ,23. Overpression . In addipression ,25. Transalinity . MsDREB2and heat . Its oveand heat . CompareMn-SOD than those in drought-susceptible ones [MsAPX is shown in the drought-tolerant variety [The antioxidant defense system protects plants against drought-induced oxidative damage . It consble ones . APX act variety .Stylosanthes guianensis (Aublet) Sw. is a tropical forage legume with drought tolerance that is commonly cultivated in tropical and subtropical areas, but it is sensitive to chilling [9-cis-EPOXYCAROTENOID DIOXYGENASE gene (SgNCED1) expression is induced in response to drought, salt and low temperature, which is associated with ABA accumulation under abiotic stress in S. guianensis [S. guianensis plants are limited. An SgDREB2C showing greatly induced expression by drought was observed in our unpublished transcriptome analysis, implying that it may play an important role in regulation of drought tolerance in S. guianensis. The objectives of this study were to identify the role of SgDREB2C in drought tolerance and provide a candidate gene for improvement of drought tolerance in forage legumes. Transgenic Arabidopsis overexpressing SgDREB2C was obtained and used for evaluation of drought tolerance. In addition, antioxidant enzyme activities and several stress-responsible genes’ expression in response to drought were detected.chilling ,34. ABA chilling . Higher chilling . 9-cis-Eianensis . Its oveianensis ,34. HoweDREB2C gene from S. guianensis (SgDREB2C) was cloned. It contains an open reading frame (ORF) of 1227 bp (GeneBank accession number OK663529) and encodes a deduced polypeptide of 408 amino acids with MW of 44.8 kDa and an isoelectric point at 4.94. Sequence alignment analysis showed that SgDREB2C was most homologous (85.7%) to AipDREB2C (XP_016167062.1) from peanut (Arachis ipaensis) in amino acids  with higher levels of SgDREB2C transcript were selected for evaluation of drought tolerance . Drought causes the production of reactive oxygen species (ROS) that can damage PSII and further lead to decreased photosynthesis capability under drought stress [Fv/Fm than the sensitive one under drought stress [Arabidopsis plants maintained a survival rate that was associated with higher photosynthesis capability compared with that in WT under drought conditions.Transgenic plants overexpressing bidopsis . Photosybidopsis . Fv/Fm rt stress . In chict stress . In the PcDREB2A-overexpressing lines have lower ROS levels in transgenic plants under drought stress [Arabidopsis, and higher CAT activity was maintained in two lines of SgDREB2C-overexpressing transgenic plants compared with WT under drought. Nevertheless, the higher antioxidant enzyme activities are associated with increased drought tolerance in transgenic plants overexpressing SgDREB2C.The antioxidant system protects plants against oxidative damage by scavenging ROS under abiotic stress ,43. Hight stress . Higher cis-acting dehydration-responsive element/C-repeat (DRE/CRT) element in their promoter regions [RD29A, COR47 and COR15A genes are responsive to drought in Arabidopsis [VvNAC08-OE [VyP5CR-OE transgenic lines [ataf1-1, ataf1-2 mutants of A. thaliana [RD29A, COR47 and COR15A. Higher transcript levels of COR15A and COR47 were maintained in three SgDREB2C-overexpressing lines and higher levels of RD29A transcript were maintained in two lines compared with WT plants under drought stress. Similarly, the COR15A transcript was higher in transgenic Arabidopsis overexpressing AtDREB2C compared with its level in WT plants, although COR47 and RD29A levels were not affected [SgDREB2C transgenic lines.Stress-responsive genes are regulated by DREBs through binding to the  regions ,21,28. Rbidopsis . The incNAC08-OE  and VyP5ic lines  and atafSgDREB2C in the regulation of drought tolerance was identified. The SgDREB2C transcript was induced by drought, and its constitutive expression led to enhanced drought tolerance in transgenic plants. The increased drought tolerance was associated with the higher levels of stress-responsive gene transcripts and antioxidant enzyme activities compared with the wild type under drought stress.In summary, the role of S. guianensis cv CIAT 184 were sown in 11 cm diameter plastic pots with a mixture of peat and perlite . The seedlings were grown in a greenhouse under natural light with temperatures from 25 to 30 °C for 3 weeks as previously described [The germinated seeds of escribed . Roots, S. guianensis leaves using TRI-Gene RNA Extraction Kit . Two ug of total RNA was used for reverse transcription by using Evo M-MLV RT Kit with gDNA Clean  in the presence of oligo (dT)18. The cDNA was used as a template to amplify the coding sequence of SgDREB2C using two primers (forward: 5′-ATGGGTGCTTATGATCAAGGTTCTA-3′ and reverse: 5′-CTCTAATCATCCTTCAACGTTTGCATTATTTCCCTTTTGGAT-3′), and 2×TSINGKE® Master Mix . The conserved domain of SgDREB2C protein was analyzed using SMART (http://smart.embl-heidelberg.de/), which was accessed on 20 April 2018. SgDREB2C and its homologous DREB2C sequences from other plants obtained from National Center for Biotechnology Information (NCBI) were aligned using DNAMAN software version 6.0. The phylogenic tree was generated using MEGA version 7.0.26 with the best model JTT+F+I+G4.Total RNA was isolated from 0.1 g of SgDREB2C without termination codon was cloned into pYL322-eGFP-N and fused with eGFP. The recombinant vector (35S-SgDREB2C-GFP) and control vector of nucleic localization sequence (35S-NLS-mCherry) were transiently co-transformed into protoplast isolated from 2-week-old rice sheath using PEG-mediated transformation as previously described [The coding sequence of escribed . The fluSgDREB2C was cloned into pCAMBIA3301 driven by CaMV35S promoter. The recombinant vector was introduced into Agrobacterium tumefaciens strain EHA105, which was used to transform Arabidopsis by the floral dip method [Arabidopsis were placed on MS medium containing 50 mg/L basta to obtain positive transformants. Homozygous transgenic lines (T3) were identified.The coding sequence of p method . The steFifty sterilized seeds of transgenic lines and wild type were placed on 1/2 MS medium containing 200 mM mannitol or without mannitol as control as previously described . After 4−2 s−1. After the seedlings were fully irrigated, plants were withheld from irrigation so that the soil gradually became dry. When the wild type plants showed serious wilting at 21 d after withholding irrigation, ion leakage and maximal photochemical efficiency (Fv/Fm) of photosystem II were measured as previously described [Seven-day-old seedlings grown on 1/2 MS medium were transferred to a 50-hole tray to grow for 7 d in a growth chamber at 22 °C under light of 200 μmol mescribed . The plaw/v) PVP and 2 mM EDTA, followed by centrifugation at 14,000 rpm for 30 min at 4 °C. The supernatants were collected for measurements of superoxide dismutase (SOD) and catalase (CAT) activities as previously described [2O2 (extinction coefficient 0.0394 mM/cm) or ascorbic acid (AsA) (extinction coefficient 2.8 mM/cm) within 1 min [Fresh leaves (0.3 g) were ground in 2 mL of 50 mM ice-cold phosphate buffer (pH 7.8) containing 2%  on the Bio-Rad CFX Manager version 1.6 . ACTIN was used as reference gene to normalized gene expression. qPCR primers are listed in Total RNA was extracted using TRI Gene reagent , followed by synthesis of the first strand cDNA using Evo M-MLV Mix Kit . PCR was performed using 2 × SYBRp < 0.05).All data for these experiments were processed using Excel version 2016 and SPSS software Version 19.0 . The data were tested by one-way analysis of variance (ANOVA) based on least significant difference (LSD) (


The COPII component SEC23A overlaps functionally with its paralogous protein and rescues the SEC23B-deficient CDAII defect. SEC23B. In contrast to humans, SEC23B-deficient mice deletion do not exhibit CDAII but die perinatally with pancreatic degeneration. Here, we demonstrate that expression of the full SEC23A protein  from the endogenous regulatory elements of Sec23b completely rescues the SEC23B-deficient mouse phenotype. Consistent with these data, while mice with erythroid-specific deletion of either Sec23a or Sec23b do not exhibit CDAII, we now show that mice with erythroid-specific deletion of all four Sec23 alleles die in mid-embryogenesis with features of CDAII and that mice with deletion of three Sec23 alleles exhibit a milder erythroid defect. To test whether the functional overlap between the SEC23 paralogs is conserved in human erythroid cells, we generated SEC23B-deficient HUDEP-2 cells. Upon differentiation, these cells exhibited features of CDAII, which were rescued by increased expression of SEC23A, suggesting a novel therapeutic strategy for CDAII.Congenital dyserythropoietic anemia type II (CDAII) results from loss-of-function mutations in  SEC23B  are a group of hereditary disorders resulting from defects in late erythroid maturation  vesicles, which traffic ~7000 mammalian proteins from the ER to the Golgi apparatus  cell lines that are deficient in SEC23B; these cell lines exhibited features of human CDAII, which were equally rescued by expression of either SEC23A or SEC23B, demonstrating that the functional equivalence of SEC23A and SEC23B is also reflected in human erythroid cells. Moreover, we showed that increasing SEC23A levels using CRISPRa rescues the SEC23B-deficient erythroid defect observed in HUDEP-2 cells. Together, these results suggest that increasing the expression of the unaffected SEC23A gene may prove a novel therapeutic strategy for CDAII.Here, we address several outstanding questions in the field. We first generated mice expressing full-length Sec23a under the control of Sec23b regulatory elements. To generate these mice, we obtained a ~170-kb mouse bacterial artificial chromosome (BAC) that spans the Sec23b gene and that contains the key regulatory sequences for Sec23b, as we previously described  were crossed to +/−Sec23b mice. The latter cross generated −/− Sec23b-aSec23b BAC+ mice at the expected Mendelian ratio at weaning  stain  stain . Similar tissues . FurtherWe have previously demonstrated that in contrast to humans, mice with erythroid-specific SEC23B deficiency do not exhibit anemia or a defect in erythroid maturation (−/−Sec23a) has been previously shown to result in embryonic lethality at E11.5 , in which exon 3 is flanked by loxP sites, to a mouse expressing Cre recombinase in erythroid cells under the control of the EpoR promoter (EpoR-Cre). Mice with erythroid-specific Sec23a deletion (−/fl EpoRSec23a-Cre+ or fl/fl EpoRSec23a-Cre+ mice) were generated using various mating schemes as demonstrated in fl/fl EpoRSec23a-Cre+ mice and demonstrated no detectable fl allele by polymerase chain reaction (PCR), consistent with complete (or near complete) excision of the flSec23a allele in the erythroid cells  are not viable at weaning. Mice with combined erythroid-specific SEC23A/B deficiency , using various mouse crosses as described in het Sec23bkoSec23a mice die at ~E14.5 to 16.5, exhibiting pale appearance compared to WT littermate controls .We next generated mice with combined controls . Livers aluation  and flowaluation . Sec23ahSec23a deletion and Sec23b haploinsufficiency in the erythroid compartment (erythroid-specific ko Sec23bhetSec23a) using several mating schemes as described in ko Sec23bhetSec23a mice exhibited a mild reduction in hemoglobin levels (P < 0.01)   and a blSec23 alleles, we conclude that (i) SEC23A functionally overlaps with SEC23B in murine erythroid cells, and (ii) the severity of the murine erythroid defect inversely correlates with the total SEC23 protein (SEC23A + SEC23B) level in erythroid cells . Notably, the SEC23B-targeting single-guide RNA (sgRNA) used in this manuscript did not target SEC23A (fig. S4B). Compared to control WT K562 clonal cell lines, which were generated using a nontargeting sgRNA, SEC23B-deficient K562 cells exhibited no increased percentage of binucleated cells either before or following differentiation into erythroid cells with hemin . In addition, we generated SEC23A-deficient K562 cell lines (fig. S4B) and similarly demonstrated no increased percentage of binucleated cells at baseline or following hemin-mediated erythroid differentiation . K562 cells with combined deficiency for both SEC23A and SEC23B could not be generated, suggesting that combined SEC23A/SEC23B deletion is not tolerated in these cells.We have shown that SEC23A compensates for SEC23B deficiency in murine erythroid cells. To determine whether SEC23A also functionally compensates for SEC23B deficiency in human erythroid cells, we first generated clonal human erythroleukemia K562 cell lines that are deficient in SEC23B using CRISPR-Cas9 genome editing. We confirmed biallelic frameshift insertions/deletions (indels) in exon 2 of SEC23B in the HUDEP-2 cell line using CRISPR-Cas9–mediated genome editing. The HUDEP-2 immortalized cell line can be induced to differentiate into mature erythroid cells and is a good model of human terminal erythroid maturation , resulting in a stop codon in exon 2 of SEC23B and undetectable SEC23B protein by immunoblotting  –tagged SEC23A (SEC23A-HA) or HA-tagged SEC23B (SEC23B-HA), or with an empty vector as control. As expected, relatively similar levels of SEC23A and SEC23B proteins were achieved by lentiviral expression of SEC23A-HA or SEC23B-HA, as demonstrated by immunoblotting for the HA tag (fig. S7A). Notably, the reduced cell viability , likely explaining the incomplete rescue achieved with this vector. The differentiation defects resulting from SEC23B deficiency were also equally rescued by expression of SEC23A cDNA from the same lentiviral construct . Similar to CD34-differentiated erythroid cells (fig. S6B), SEC23A expression in this reporter cell line decreases with erythroid differentiation (fig. S6C). Using this reporter cell line, we show that two independent SEC23A-targeting sgRNAs, CRISPRa SEC23A sgRNA-1 and sgRNA-2, respectively increased SEC23A level by ~30 and ~300% in undifferentiated HUDEP-2 cells and by ~5 and ~80% at day 11 of differentiation, as determined by flow cytometry –tagged SEC23A from the endogenous ytometry . Increasytometry , reducedytometry , increasytometry , impaireytometry , and binytometry  observedSEC23 duplication, it is estimated to have occurred ~615 million years ago  293T cells and complementation of yeast Sec23 by both SEC23 paralogs  of the duplicated gene become fixed in the genome, either because one paralog acquires a new function  or because both paralogs split the function of the ancestral gene . Regarding the SEC23 paralogs.We previously showed that in contrast to humans, mice with hematopoietic SEC23B deficiency do not exhibit anemia or other CDAII characteristics ] was previously reported to result in increased binucleated cells  in which exons 5 and 6 are flanked by LoxP sites and a Sec23b null allele (−Sec23b) with excision of Sec23b exons 5 and 6 were generated as previously described  in which exon 3 is flanked by loxP sites and a Sec23a null allele (−Sec23a) with deletion of exon 3 were also generated as previously described ] was purchased from the BACPAC Resources Center. We have previously shown that the key regulatory sequences required for Sec23b expression reside in a 127-kb region that is contained within the 170-kb RP24 BAC (Sec23a cDNA synthesized de novo (by GenScript) without the stop codon, a sequence encoding three glycines followed by an HA tag and three stop codons, a bovine growth hormone polyadenylation transcriptional termination sequence, a kanamycin resistance cassette with its PGK promoter flanked by two FRT sites, and a 3′ homology sequence with RP24  was purified using the Macherey-Nagel NucleoBond Xtra Maxi EF kit .A ~170-kb mouse BAC containing ith RP24 . This veb-aSec23 BAC that expresses Sec23a under the control of the Sec23b regulatory elements was microinjected into the pronucleus of zygotes generated from the in vitro fertilization of C57BL/6J oocytes with sperm from +/−Sec23b male mice. Zygotes were subsequently implanted in pseudopregnant foster mothers. Pups were genotyped at 2 weeks of age, and germline transmission of the BAC transgene was confirmed in one mouse line by genotyping.The recombineered flSec23a and the −Sec23a alleles were crossed to mice expressing Cre recombinase under the control of the erythropoietin receptor promoter (EpoR-Cre mice)  for 45 to 60 min on an end-over-end rotator at 4°C. Following centrifugation at 20,000SEC23B exon 2 (GGAACGTGTGGCCTTCCAGC) or an sgRNA-targeting SEC23A exon 2 (TCAAGTCGACTGGAAGCTAC), respectively, as previously described . Clonal K562 cell lines without SEC23B or SEC23A mutations were used as controls. K562 cells were differentiated into erythroid cells with hemin, as previously described , followed by Sanger sequencing.HUDEP-2 cells were coelectroporated (Lonza 4D nucleofector) with a PX459 plasmid expressing an sgRNA (ATTAGCACTTCAAGCAGCAC) targeting the SEC23A locus immediately upstream of the stop codon and with a donor template allowing insertion of the GFP coding sequence by homology-directed repair. The donor template was assembled as previously described , 2% penicillin-streptomycin , human holo-transferrin , recombinant human insulin , 5% inactivated human AB pooled plasma (Rhode Island Blood Center X00004), Epogen (3 U/ml), doxycycline (1 μg/ml), and rhSCF (100 ng/ml). The supplementation of rhSCF and doxycycline to the differentiation media was stopped at days 5 and 8, respectively.HUDEP-2 cells were cultured as previously described  of ~0.3. Twenty-four hours following spinfection, transduced cells were selected with puromycin (Sigma-Aldrich P8833) at a concentration of 1 μg/ml for 96 hours.LV1-5 lentiviral vectors (Addgene #68411) expressing either SEC23A or SEC23B fused to a C-terminal HA tag were assembled using the NEBuilder HiFi DNA assembly cloning kit (NEB). The integrity of the assembly was confirmed by Sanger sequencing. The same LV1-5 lentiviral vector expressing no protein was used as a negative control. To package the constructs into lentiviral particles, 3.56 μg of each lentiviral vector was individually cotransfected with 2.67 μg of psPAX2  and 1.78 μg of pCMV-VSV-G   at an MOI of ~0.3. Twenty-four hours after transduction, cells were selected with hygromycin (Invitrogen #10687010) at 300 μg/ml for 15 days. Subsequently, and after a recovery period of 2 to 4 days, cells were transduced with lentiSAMv2  that expresses a SEC23A-targeting sgRNA or a nontargeting (NT) control sgRNA, again at an MOI of 0.3. Transduced cells were selected with blasticidin (Gibco R21001) at 8 μg/ml for 10 days and subsequently analyzed. Two independent sgRNAs targeting SEC23A (CRISPRa SEC23A sgRNA-1: AGAGGAGGGGACGGGGCGTG and CRISPRa SEC23A sgRNA-2: AAGCAAGCTCAGGGGTCCGG) and an NT sgRNA (CRISPRa NT sgRNA: CTGAAAAAGGAAGGAGTTGA) were used and cloned into lentiSAMv2 as previously described (To activate the expression of All experiments using mice were approved by and performed in accordance with the regulations of the University Committee on Use and Care of Animals.


The recently reported “UK variant” (B.1.1.7) of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-Å resolution cryo-electron microscopy (cryo-EM) structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to 2 representative potent neutralizing antibody fragments. The rapid international spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is associated with numerous mutations that alter viral fitness. Mutations have been documented in all 4 structural proteins encoded by the viral genome including the small envelope glycoprotein (E), membrane glycoprotein (M), nucleocapsid (N) protein, and the spike (S) protein. The most prominent mutations are in the spike protein, which mediates entry of the virus into cells by engaging with the angiotensin converting enzyme 2 (ACE2) receptor. Several structures of SARS-CoV-2 spike protein variants in pre- and post-fusion conformations have been reported, including complexes with ACE2 and a variety of antibodies . MutatioIn December 2020, new variants of SARS-CoV-2 carrying several mutations in the spike protein were documented in the UK (SARS-CoV-2 VOC202012/01) and South Africa 501Y.V2) ,15. Earl01Y.V2 ,1To understand the structural effects of the N501Y mutation on ACE2 binding, we expressed and purified spike (S) protein ectodomains with and without the N501Y mutation in Expi293F cells , and conCryo-EM structural analysis of the complex formed between the N501Y spike protein ectodomain and the ACE2 receptor ectodomain provides a detailed glimpse of both the overall structure of the receptor and the binding interface between the RBD and ACE2 ; S4 Fig.H Fc ab8 and IgG ab1, both derived from a large human library of antibody sequences [Potent neutralization of SARS-CoV-2 has been achieved with a number of antibodies, including 2 recently reported examples, Vequences ,21. We ckoff)  . Notablykoff) . The com reports –28, thesH Fc ab8 and IgG ab1  and Fab ab1 (the antigen binding fragment of IgG ab1) with the N501Y mutant spike protein ectodomain.Next, we tested the effect of the N501Y mutation on the relative strengths of binding and neutralization potency of V IgG ab1 . ELISA a IgG ab1 . Second, IgG ab1 , while V extents . This isY spikes . Consistted form . OverallH ab8 and the N501Y spike protein ectodomain shows a single dominant conformation with 2 VH ab8 fragments bound to RBDs in the down conformation and weak density for the other RBD, which is flexible and primarily in the up position . The N501Y mutation was introduced by site-directed mutagenesis . Successful subcloning and mutation were confirmed by Sanger sequencing (Genewiz). Expi293F cells (Thermo Fisher) were grown in suspension culture using Expi293 Expression Medium (Thermo Fisher) at 37°C and 8% CO2. Cells were transiently transfected at a density of 3 × 106 cells/ml using linear polyethylenimine (Polysciences). Twenty-four hours following transfection, the medium was supplemented with 2.2 mM valproic acid, and expression carried out for 5 d at 37°C and 8% CO2. The supernatant was harvested by centrifugation and filtered through a 0.22-μm filter before loading it onto a 5-ml HisTrap excel column (Cytiva). The column was washed with 20 column volumes (CVs) of wash buffer , followed by 5 CVs of wash buffer supplemented with 20 mM imidazole. The protein was eluted with elution buffer . Elution fractions containing the protein were pooled and concentrated  for gel filtration (GF). GF was conducted using a Superose 6 10/300 GL column (Cytiva) pre-equilibrated with GF buffer . Peak fractions corresponding to soluble protein were pooled and concentrated to 4.5–5.5 mg/ml . Protein purity was estimated as >95% by SDS-PAGE, and protein concentration was measured spectrophotometrically .The wild-type SARS-CoV-2 S HexaPro expression plasmid was a gift from Jason McLellan  and was H ab8 (0.02 mg/ml) for 30 min, followed by addition of ACE2 (0.05 mg/ml) for another 30 min. Grids (copper 200 or 300 mesh coated with continuous ultrathin carbon) were plasma cleaned using an H2/O2 gas mixture for 15 s in a Solarus II Plasma Cleaner (Gatan) or 10 s in a PELCO easiGlow Glow Discharge Cleaning System (Ted Pella). The protein mixtures (4.8 μl) were applied to the grid and allowed to adsorb for 30 s before blotting away excess liquid, followed by a brief wash with Milli-Q H2O. Grids were stained by 3 successive applications of 2% (w/v) uranyl formate . Negative stain grids were imaged using a 200-kV Glacios (Thermo Fisher Scientific) transmission electron microscope (TEM) equipped with a Falcon3 camera operated in linear mode. Micrographs were collected using EPU at nominal 92,000× magnification  over a defocus range of −2.0 μm to −1.0 μm with a total accumulated dose of 40 e–/Å2.For negative stain, purified S protein (0.05 mg/ml) was mixed with soluble ACE2 (0.05 mg/ml) and incubated on ice for 15 min. For the competition experiment, the S protein (0.05 mg/ml) was first incubated on ice with VH ab8, or Fab ab1 at molar ratios of 1:1.25, 1:9, and 1:8 (spike trimer to binding partner), respectively. Incubations were performed for 20 min on ice prior to centrifugation at 14,000g for 10 min. Grids were plasma cleaned using an H2/O2 gas mixture for 15 s in a Solarus II Plasma Cleaner (Gatan) before 1.8 μl of protein suspension was applied to the surface of the grid. Using a Vitrobot Mark IV (Thermo Fisher Scientific), the sample was applied to either Quantifoil Holey Carbon R1.2/1.3 copper 300 mesh grids  or UltrAuFoil Holey Gold 300 mesh grids (N501Y spike in complex with VH ab8 or Fab ab1) at a chamber temperature of 10°C with a relative humidity level of 100%, and then vitrified in liquid ethane after blotting for 12 s with a blot force of −10. All cryo-EM grids were screened using a 200-kV Glacios (Thermo Fisher Scientific) TEM equipped with a Falcon4 direct electron detector followed by high-resolution data collection on a 300-kV Titan Krios G4 (Thermo Fisher Scientific) TEM equipped with a Falcon4 direct electron detector in electron event registration (EER) mode. Movies were collected at 155,000× magnification  over a defocus range of −3 μm to −0.5 μm with a total dose of 40 e–/Å2 using EPU automated acquisition software (Thermo Fisher).For cryo-EM, both N501Y and unmutated SARS-CoV-2 spike ectodomain preparations were deposited on grids at a concentration of 2.25 mg/ml. Complexes were prepared by incubating spike ectodomain preparations with either ACE2 , VIn general, all data processing was performed in cryoSPARC v.2.15 or v.3.0.1  unless sftp://ftp.gwdg.de/pub/misc/sphire/crYOLO-GENERAL-MODELS/gmodel_phosnet_negstain_20190226.h5). After extraction, particles were imported into cryoSPARC and subjected to 2D classification and 3D heterogeneous classification. Final density maps were obtained by 3D homogeneous refinement.For negative stain data, motion correction and contrast transfer function (CTF) estimation were performed in RELION v.3.1.1 . ParticlH ab8 or ACE2 resulted in improvement of the binding interfaces. C3 symmetry expanded particles were used for local refinement of RBD and its bound Fab ab1. Overall resolution and locally refined resolutions were according to the gold-standard Fourier shell correlation (FSC) [For cryo-EM data, motion correction in patch mode , CTF estimation in patch mode, reference-free particle picking, and particle extraction  were performed on-the-fly in cryoSPARC. After preprocessing, particles were subjected to 2D classification and 3D heterogeneous classification. The initial consensus maps were obtained by 3D homogeneous refinement. Then particles were re-extracted with box size 800 and then binned to 400. Final 3D refinement was done with per particle CTF estimation and aberration correction. Local refinements with a soft mask covering a single RBD and its bound Von (FSC) .H ab8 and Fab ab1, respectively. Individual domains of SARS-CoV-2 HexaPro S trimer (PDB ID 6XKL) were docked into cryo-EM density using UCSF Chimera v.1.15 [Coordinates of PDB 6WGJ and 7CH5 were used as initial models to build the Va v.1.15 . Initiala v.1.15  and Phena v.1.15 . Glycansa v.1.15 . Figuresa v.1.15 , and PyM50 values were calculated using a 4-parameter dose–response  curve in GraphPad Prism . This function provides the 95% confidence interval (95% CI) and standard error of the mean (SEM).SARS-CoV-2 S N501Y plasmid was obtained from SARS-CoV-2 S plasmid (HDM-IDTSpike-fixK) by site-directed mutagenesis . SARS-CoV-2 S and SARS-CoV-2 S N501Y pseudotyped retroviral particles were produced in HEK293T cells as described previously [H Fc ab8 or ACE2-mFc  in PBS-T + 0.5% BSA buffer for 1 h at room temperature. After washing, wells were incubated with either Goat Anti-Human IgG (Jackson ImmunoResearch) or Goat Anti-Mouse IgG Fc Secondary Antibody, HRP (Invitrogen) at a 1:8,000 dilution in PBS-T + 0.5% BSA buffer for 1 h at room temperature. After washing, the substrate solution (Pierce 1-Step) was used for color development according to the manufacturer’s specifications. Optical density at 450 nm was read on a Varioskan Lux plate reader (Thermo Fisher Scientific). For ACE2 competition assays, experiments were conducted as described above with amendments. Serial dilutions of VH Fc ab8 were incubated for 30 min at room temperature prior to the addition of 2.5 nM ACE2-mFc . Wells were then further incubated for 45 min at room temperature. After washing, wells were incubated at a 1:8,000 dilution with Goat Anti-Mouse IgG Fc Secondary Antibody, HRP (Invitrogen) in PBS-T + 0.5% BSA buffer for 1 h at room temperature. After washing, the substrate solution (Pierce 1-Step) was used for color development according to the manufacturer’s specifications. Optical density at 450 nm was read on a Varioskan Lux plate reader (Thermo Fisher Scientific). For all experiments, controls for antibody–BSA interactions were performed. For competition assays, controls for Goat Anti-Mouse IgG Fc Secondary Antibody recognition of VH Fc ab8 were performed. The EC50 values were calculated using a 4-parameter dose–response  curve in GraphPad Prism.One hundred microliters of wild-type or N501Y SARS-CoV-2 S protein preparation was coated onto 96-well MaxiSorp plates at 2 μg/ml in PBS overnight at 4°C. All washing steps were performed 5 times with PBS + 0.05% Tween 20 (PBS-T). After washing, wells were incubated with blocking buffer (PBS-T + 2% BSA) for 1 h at room temperature. After washing, wells were incubated with dilutions of Vkon) and dissociation (koff) rates were derived from the sensorgram fitting and used to calculate the binding equilibrium constant (KD).The binding kinetics of SARS-CoV-2 trimers and human ACE2 was analyzed with the biolayer interferometer BLItz (ForteBio). Protein-A biosensors  were coated with ACE2-mFc (40 μg/ml) for 2 min and incubated in DPBS (pH = 7.4) to establish baselines. Concentrations of 100 nM, 200 nM, and 400 nM spike trimers were used for association for 2 min followed by dissociation in DPBS for 5 min. The association  Size exclusion chromatography profile of the indicated affinity-purified HexaPro constructs. Fractions pooled for structural and biochemical studies are indicated by gray shading. (B) SDS-PAGE analysis of pooled and concentrated HexaPro constructs.(TIF)Click here for additional data file.S2 Fig(A) Representative micrograph. (B) Representative 2D class averages. (C) Cryo-EM data processing workflow. (D) Fourier shell correlation (FSC) between 2 half maps (red) and FSC between the refined map and model (blue). (E) Local resolution estimation. (F) Viewing direction distribution.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(A) Representative micrograph. (B) Representative 2D class averages. (C) Cryo-EM data processing workflow. (D) Fourier shell correlation (FSC) between 2 half maps (red) and FSC between the refined map and model (blue). (E) Local resolution estimation of the global map. (F) Viewing direction distribution. (G) FSC between 2 half maps of local refinement (red) and FSC between the locally refined map and model (blue). (H) Local resolution estimation of the locally refined map.(TIF)Click here for additional data file.S5 Fig50 of soluble ACE2-mFC neutralization is 0.066 μg/ml (95% CI 0.026–0.17 μg/ml) for unmutated pseudotyped virus, and 0.0074 μg/ml  for N501Y pseudotyped virus. The IC50 for wild-type is greater than that for N501Y, as demonstrated by a 1-tailed Welch test (p = 3 × 10−5). (C and D) Biolayer interferometry analysis of immobilized ACE2 binding by increasing concentrations of either N501Y (A) or unmutated (B) spike ectodomain. Shown is the extent of binding as determined by shift in wavelength (nm: nanometers). Biophysical parameters  are shown as mean ± standard deviation.(A) Analysis of cell entry of N501Y or unmutated SARS-CoV-2 S pseudotyped viral particles. N501Y or unmutated SARS-CoV-2 S pseudotyped virus was normalized for p24 levels and incubated with HEK293T-ACE2 cells for 48 h prior to cell lysis and luciferase activity quantification. (RLU: relative luminescent units). (B) Analysis of N501Y or unmutated SARS-CoV-2 S pseudotyped virus neutralization by soluble ACE2-mFC. The IC(TIF)Click here for additional data file.S6 FigH ab8 antibody fragment, then with soluble ACE2. The density corresponding to bound VH ab8 is colored in red. The VH ab8 antibody fragment competes with ACE2 binding, as demonstrated by the reduced ACE2 occupancy in both (I) the unmutated spike (1 RBD bound) and (J) the N501Y spike (2 RBDs bound).(A and B) Representative micrograph selected from the total dataset for the unmutated (A) or N501Y (B) spike ectodomains in complex with ACE2. The concentrations of spike proteins and soluble ACE2 are the same for both unmutated and N501Y preparations. (C and D) 2D class averages corresponding to (C) the unmutated dataset  and (D) the N501Y dataset , covering the same range of stain thickness. (E and F) Processing workflow. (E) For unmutated spikes, 3D classification reveals an occupancy of 2 or fewer RBDs bound for the 2 most populated initial classes . (F) For N501Y spikes, the most populated initial class (63%) has 3 RBDs bound. (G and H) Final refinement of (G) unmutated spikes and (H) N501Y spikes. The density corresponding to bound soluble ACE2 is colored in green. The higher occupancy of ACE2 for N501Y spikes reflects a shift in the equilibrium stoichiometry, consistent with the higher affinity of N501Y spikes for ACE2. (I and J) Competition experiments. Spike ectodomains were first incubated with the V(TIF)Click here for additional data file.S7 Fig(A) Representative micrograph. (B) Representative 2D class averages. (C) Cryo-EM data processing workflow. (D) Fourier shell correlation (FSC) between 2 half maps (red) and FSC between the refined map and model (blue). (E) Local resolution estimation of the global map. (F) Viewing direction distribution. (G) FSC between 2 half maps of local refinement (red) and FSC between the locally refined map and model (blue). (H) Local resolution estimation of the locally refined map.(TIF)Click here for additional data file.S8 Fig(A) Representative micrograph. (B) Representative 2D class averages. (C) Cryo-EM data processing workflow. (D–I) Fourier shell correlation (FSC) between 2 half maps (red) and between the refined map and model (blue), with local resolution estimation and viewing direction distribution, for class 1 (D–F) and class 2 (G–I). (J) FSC between 2 half maps of local refinement (red) and between the locally refined map and the model (blue). (K) Local resolution estimation of the locally refined map.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.