Interferon-induced transmembrane protein 3 (IFITM3) is a potent antiviral protein that enhances cellular resistance to a variety of pathogens, including influenza virus. Classically defined as an interferon-stimulated gene, expression of IFITM3 on cells is rapidly up-regulated in response to type I and II interferon. Here we found that IFITM3 is rapidly up-regulated by T cells following their activation and this occurred independently of type I and II interferon and the interferon regulatory factors 3 and 7. Up-regulation of IFITM3 on effector T cells protected these cells from virus infection and imparted a survival advantage at sites of virus infection. Our results show that IFITM3 expression on effector T cells is crucial for these cells to mediate their effector function and highlights an interferon independent pathway for the induction of IFITM3 which, if targeted, could be an effective approach to harness the activity of IFITM3 for infection prevention. Cells are equipped with a variety of mechanisms to protect themselves from virus infection. The early detection of a viral infection by innate receptors triggers the production of type I interferon (IFN), which in turn signals expression of interferon-stimulated genes (ISG) within the host cell. The proteins encoded by these genes interfere with viral replication and enhance the ability of uninfected cells to resist infection. Interferon-induced transmembrane 3 (IFITM3) is a potent anti-viral protein that exhibits protection against a broad range of viruses including orthomyxoviruses, flaviviruses, filoviruses, and coronaviruses –3. IFITMWhile some cell types, including respiratory epithelial cells  and tissWhether IFITM3 expression is regulated differently in distinct cell types remains unclear. As immune cells often carry out effector functions at sites of infection and are therefore directly exposed to virus-infected cells, these cells are likely to benefit greatly from regulated expression of antiviral proteins. We have previously reported that dendritic cells stationed within the lung upregulate IFITM3 following influenza virus infection, a process driven by exposure to type I interferon, and this was crucial for these cells to successfully traffic influenza viral antigen from the lung to draining lymph node (LN) without becoming infected and perishing en route . Tissue Here we extend this research to investigate the expression pattern and drivers of IFITM3 expression in T cells during their activation and infiltration into influenza virus-infected lung tissue. We find that IFITM3 is rapidly up-regulated on T cells following their activation in the draining LN and expression is maintained as these cells infiltrate sites of virus infection. Up-regulation of IFITM3 on effector T cells protected these cells from virus infection and imparted a survival advantage at sites of virus infection. Interestingly, the expression of IFITM3 on activated T cells was not driven by type I or II interferon and did not rely on the interferon regulatory factors 3 and 7. Our results show that IFITM3 expression on effector T cells is crucial for these cells to mediate their effector function and highlights an interferon independent pathway for the induction of IFITM3, which if targeted, could be an approach to harness the activity of IFITM3 for infection prevention.tm1(RFP)Zhang, The Jackson Laboratory), IFITM3 KO x OT-I.CD45.1, gBT-I.GFP, IFNAR KO, IFNAR x IFNγR KO, IFNγKO, IRF3 KO, IRF7 KO (provided by T. Taniguchi), and IRF3 x IRF7 KO mice were bred in-house and housed in specific pathogen-free conditions in the animal facility at the Peter Doherty Institute of Infection and Immunity, the University of Melbourne. All experiments were approved and performed in accordance with the Institutional Animal Care and Use Committee guidelines of the University of Melbourne. Mice were infected intranasally with X31-OVA [257–264 epitope within the neuraminidase stalk) generously provided by Prof. S. Turner, Monash University, Melbourne, Australia. For total respiratory tract (TRT) infection, mice were anesthetized with inhalation anaesthetic and infected with 104 PFU of X31-OVA in a volume of 30μl.C57BL/6, OT-I.CD45.1, IFITM3 KO  and transferred onto nitrocellulose membrane. Membranes were stained with antibodies against mouse IFITM3 (Abcam) and Actin (Sigma). Horseradish peroxidase (HRP)–conjugated anti-rabbit IgG antibody (Sigma) was used for protein detection.+ or CD8+ T cells were plated at 106 cells per well in 2-ml 24-well flat-bottomed tissue culture plates  coated with anti-CD3(145-2C11) and anti-CD28 (37.51). Alternatively, OT-I cells were plated at 106 cells per well in 2-ml 24-well flat-bottomed tissue culture plates  with 1 μg/ml SIINFEKL peptide.T cells were purified from the LN and spleen of mice. Cells were purified after a depletion step using antibodies against CD11b (M1/70), F4/80, Ter-119, Gr-1 (RB6), MHC class II (M5/114), and CD4 (GK 1.5) or CD8 (YTS 169.4), followed by incubation with anti-rat IgG-coupled magnetic beads  following the manufacturer’s protocols. T cell preparations were 90–95% pure as determined by flow cytometry. Purified naïve CD4Influenza A virus X31 (H3N2) and Phil82X (H3N2) are high-yielding reassortants of A/Aichi/2/1968 (H3N2) and A/Philippines/2/1982 (H3N2), respectively, with PR8 that express the H3N2 surface glycoproteins. In addition, we have used the seasonal H1N1 influenza virus strains A/Brazil/11/78 (Braz78) and A/New Caledonia/20/1999 , as well as the H3N2 strains A/Udorn/2/1972 (Udorn72), A/Port Chalmers/1973  A/Beijing/353/89 (Beij89). Viruses were grown in 10-day embryonated chicken’s eggs by standard procedures. Titres of infectious virus were determined by plaque assay on Madin-Darby canine kidney (MDCK) cells .+ or CD8+ T cells magnetically enriched as described above, or sorted purified from the LN and spleen of mice, were in some experiments labelled with cell trace violet and cultured (105 cells/well) in serum free media with influenza virus (moi = 1–10) for 2 hours. Cells were then washed to remove free virus and cultured at 37°C in RPMI with 10% FCS for 12–48 hours. Susceptibility to infection was measured by intracellular staining for the nucleoprotein (NP) of influenza A viruses using anti-influenza NP-FITC .Naïve CD47 transgenic splenocytes from OT-I mice were cultured for 4 days with 5 × 107 C57BL/6 splenocytes pulsed with 0.1 μg of OVA257–264 peptide, in 40 ml of RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 5 × 10−5 M 2-ME, and antibiotics. The cultures were diluted 1 in 2 on days 2 and 3 with fresh medium containing 10 U/ml IL-2. On day 4, cells were collected, and 5 × 106 T cells were transferred into recipient mice via intravenous (i.v.) injections in a total volume of 200 μl of PBSA total of 5 × 10Perfused lung tissue, inflated with OCT were immediately embedded in OCT. Frozen sections (14 μm) were cut using a cryostat. Tissue sections were fixed in acetone, blocked in serum-free protein block and then stained for CD8 (53–6.7), CD45.1 (A20) and DAPI. Images were acquired on a Zeiss LSM700 microscope with a 20x objective. Acquired images were processed using ImageJ software.b-NP-tetramer was made in house. The levels of IFN-γ, IFN-ß, and interlukin-6 (IL-6) were analysed using a BD Cytometric Bead Array (CBA) Mouse Inflammation Kit  according to the manufacturer’s instructions.Single cell suspensions were prepared from spleens by mechanical disruption. Mice were perfused prior to the harvest of the lung tissue which was then incubated for 1 h at 37°C in 3 mL of collagenase type 3 (Worthington), (3 mg/mL in RPMI 1640 supplemented with 2% FCS) and DNAse I (0.5mg/ml) (Roche). Cells were stained for 25 min on ice with the appropriate mixture of monoclonal antibodies and washed with PBS with 1% BSA. The conjugated monoclonal antibodies were obtained from BD Pharmingen or Biolegend include; anti-CD8 (53–6.7), anti-CD45-1 (A20), anti-CD45.2(104). H2-DData were analysed with GraphPad Prism and the indicated statistical tests. * P<0.05, ** P<0.01, *** P<0.001.+ T cells do not express IFITM3 however following crosslinking of the TCR which triggers activation, T-cells transiently upregulate expression of this antiviral protein (+ and CD8+ T cells purified from wild type (WT) mice, IFNAR KO mice (which lack the IFNα receptor and therefore are unable to respond to type I interferon), or IFNγ KO mice (which are unable to synthesize interferon gamma), were activated in vitro using plate bound anti-CD3/CD28 and 3 days later the expression of IFITM3 in naïve (day 0) and activated T cells was measured (+ and CD4+ T cells that lacked the IFNα receptor upregulated IFITM3 expression following TCR engagement (in vitro activation of these cells (as described above) we observed upregulation of IFITM3 expression, albeit less effectively  whose expression is known to be up-regulated in response to type I and II interferons . We havemeasured . Both CDgagement , as did gagement . To detegagement . Followiectively  indicati+ T cells with influenza virus  and cultured these cells in vitro for 48 hrs. This infection protocol resulted in roughly 3% of the total WT and KO CD8+ T cells being infected with influenza virus, as measured by intracellular staining for the virus nuclear protein (NP)  . While bein (NP) , T-cellsein (NP) . Collect+ T cells from OT-I mice and co-cultured these cells with naïve polyclonal CD8+ T cells from B6 mice. The SIINFEKL peptide was added to the culture to activate only the transgenic OT-I T cells and on day 3 post co-culture, activated OT-I cells were separated from naïve B6 bystander CD8+ T cells, and IFITM3 expression was determined in each of the T cell populations  are known inducers of IFITM3 expression [ are knowulations . Consistulations . In cont T cells .+ T cells isolated from mice deficient in either IRF3 or IRF7, or both, showed equivalent levels of activation and expansion in response to TCR crosslinking. To do this, naïve CD8+ T cells purified from either WT, IRF3 KO, IRF7 KO or IRF3 x IRF7 KO mice were labelled with carboxyfluorescein succinimidyl ester (CFSE) and activated in vitro with plate bound anti-CD3/28 for 3–5 days. We observed similar proliferation profiles  . We nextprofiles  and coulprofiles  from thepression .+ T cells with violet trace membrane dye, activated these cells in vitro with plate-bound anti-CD3/28 for 3 days and then sort purified T cells into 4 groups according to the number of divisions the cells had undergone . We assessed each group of cells for expression of IFITM3 and found that only after cells had undergone 2–3 rounds of division could upregulation of IFITM3 be detected (+ T cells were activated in vitro (as described above) for 3 days, infected with a panel of different influenza A virus strains, and 12 hrs later the proportion of infected cells (Flu-NP+) undergoing 1–4 divisions was determined (+ T cells (with infection rates ranging from 5–28%) in general, we observed less infection of T cells that had undergone 4 rounds of division compared to T cells that had undergone 1–2 rounds of division. Thus, as T cells undergo division, expression of the antiviral IFITM3 protein increases, as does resistance to influenza virus infection.To gain insight into the kinetics of TCR engagement-induced expression of IFITM3, we labelled naïve B6 CD8detected . We nexttermined . While d+ T cells to influenza A virus infection was mediated by IFITM3, transgenic WT OT-I CD8+ T cells and IFITM3 KO OT-I CD8+ T cells were activated in vitro for 3 days and then infected with different influenza A viruses  and, 12 hrs later, the percentage of virus-infected cells was determined  were seeded with congenically marked (CD45.1) OT-I CD8+ T cells and, as a control, green fluorescent protein (GFP)-expressing TCR transgenic CD8+ T cell specific for the glycoprotein B of herpes simplex virus (gBT-I), prior to intranasal infection with a recombinant influenza virus expressing the model antigen OVA (X31-OVA). On days 4 and 10 post infection (p.i.), both transgenic CD8+ T cell populations were sort purified from the lung-draining lymph node (LN) and levels of IFITM3 expression was determined. At both 4 and 10 days p.i., effector OT-I T cells isolated from the LN of X31-OVA-infected mice expressed IFITM3 while the naïve gBT-I T cells did not (-) and activated (CD44+) CD8+ T cells recovered from lung draining LN on day 4 and 10 p.i. displayed a similar pattern of IFITM3 expression  and on day 7 p.i. influenza virus nuclear protein (NP) specific CD8+ T cells (NP-tetramer+) were sort purified from the lung draining LN and spleen  and the ratio and absolute numbers of WT and KO OT-I cells in the spleen and lung was measured 48 hrs later. Equivalent proportions of WT and IFITM3 KO OT-I cells were observed in the spleen and in the lung following transfer into mice that received the inflammatory agent LPS, indicating that both WT and KO OT-I cells can infiltrate these tissues effectively Click here for additional data file.S2 Fig-) and activated (CD44+) CD8+ T cells recovered from the LN of mice on day 4 and 10 p.i. with 104 PFU of X31-OVA. Data are representative of 2 experiments. Actin was included as a loading control.Western blot analysis of IFITM3 expression by endogenous (endo) naïve (CD44(PDF)Click here for additional data file.S3 Fig4 PFU of X31 and on day 7 p.i influenza specific (NP-tetramer+) cells were sort purified from the lung draining LN. (A) Representative flow cytometry profiles depicting the gating strategy for sorting the NP-tetramer+ cells. (B) Western blot analysis of IFITM3 expression by endogenous naïve (CD44-) and NP-tetramer+ CD8+ T cells recovered from the LN of WT mice on day 7 p.i. Data are representative of 2 experiments. Actin was included as a loading control. (C) WT and IFITM3 KO NP-tetramer+ cells sort purified from the spleen and LN and infected in vitro with different influenza A viruses (moi = 5) and 12 hrs later the absolute number of influenza virus-infected cells was measured by intracellular staining for influenza A virus nucleoprotein (NP-FITC). Data are pooled from 2 experiments, bars represent the mean ± SEM.(A-B) Mice, WT or IFITM3 KO were infected with 10(PDF)Click here for additional data file.S4 Fig6in vitro activated WT and IFITM3 KO OT-I T cells. The absolute number of WT and IFITM3 KO OT-I T cells in the (A) spleen and (B) lung was then determined 48 hrs later. Data are pooled from 3 independent experiments, dots represent individual mice.Mice were infected i.n. with X31-OVA (Influenza) or treated i.n. with LPS and 2 days later received 5 x 10(PDF)Click here for additional data file.


The anti-influenza therapeutic baloxavir targets cap-dependent endonuclease activity of polymerase acidic (PA) protein. We monitored baloxavir susceptibility in the United States with next generation sequencing analysis supplemented by phenotypic one-cycle infection assay. Analysis of PA sequences of 6,891 influenza A and B viruses collected during 2016/17 and 2017/18 seasons showed amino acid substitutions: I38L (two A(H1N1)pdm09 viruses), E23G (two A(H1N1)pdm09 viruses) and I38M (one A(H3N2) virus); conferring 4–10-fold reduced susceptibility to baloxavir. On 24 October 2018, the United States (US) Food and Drug Administration (FDA) approved a new anti-influenza therapeutic, baloxavir marboxil, following its approval in Japan earlier in the same year . In clinEarly detection of emerging resistance is essential for timely modification of public health policies and recommendations on the use of antiviral therapeutics. Therefore, our aim was to determine baloxavir susceptibility of seasonal viruses before the drug entered the US market and establish a methodology to conduct baloxavir surveillance.50) was used to list PA AAS conferring reduced susceptibility to baloxavir  guidance, public health laboratories (PHLs) submit up to two viruses of each subtype (type A) and lineage (type B) to national surveillance, twice a month. Since the implementation of the Sequence First Initiative, all submitted viruses have been subjected to codon-complete next generation sequencing (NGS) of viral genomes  Table 2Table 2. Sequences were analysed for changes in the PA protein previously associated with reduced baloxavir susceptibility . Of 6,89Supplemental Table S1). Notably, the heterogeneous subpopulation E119K/E was found in a virus isolate, but could not be detected in the respective clinical specimen, suggesting emergence of AAS during virus propagation.Other PA substitutions were detected either as pure populations (I38V) or heterogeneous subpopulations   [As molecular mechanisms of reduced susceptibility to baloxavir are not yet fully elucidated, it is necessary to supplement NGS analysis with phenotypic testing. A phenotypic reduction in susceptibility is expressed as a fold change compared with a reference ECay (FRA) .Supplemental Figure S1). Virus-infected cells are detected by immunofluorescence. HINT was used in this study to establish type-/subtype-specific baseline EC50 values.Previously, Stevaert et al. suggested using a one-cycle infection assay to test PA inhibitors . Recentl50 values were similar to those reported for viruses tested using a plaque reduction assay [50 compared with type A viruses. The consistency between HINT and plaque reduction assay was encouraging as EC50 values determined using a different assay, ViroSpot, appeared to be elevated [To this end, 116 viruses representing different subtypes (type A) and lineages (type B) circulating in the US during 2016/17 and 2017/18 seasons were tested . EC50 vat values ranged 19.06–22.23). The EC50 values ranged from 2.5–6.7 nM, which was within the established baseline for type B virus isolates (data not shown).Next, we tested 11 nasal washes from patients positive for influenza B . A/Louisiana/49/2017, containing I38M, displayed a 10-fold change in baloxavir susceptibility compared with the control virus A/Louisiana/50/2017, which is in agreement with a previous report for another A(H3N2) virus using a plaque reduction assay [Viruses flagged by NGS analysis were tested alongside their PA-matching counterpart (i.e. control viruses) ; nevertheless, their relevance cannot be ruled out at this time. As expected, I38V conferred one–two-fold change compared to control viruses, which was previously reported [The new substitution, I38L, conferred an eight- and seven-fold change compared with the control virus in the HINT and FRA assays, respectively. Two A(H1N1)pdm09 viruses carrying E23G displayed a four–five-fold change by HINT . When fo0 values , the rese median  and 4. TCombining NGS and HINT data from two seasons, the frequency of viruses displaying reduced baloxavir susceptibility (˃ three-fold change) was low: 0%, 0.032% and 0.3%, for type B, A(H3N2) and A(H1N1)pdm09 viruses, respectively. Similar to a previous report, A(H1N1)pdm09 viruses exhibited a higher frequency of PA amino acid polymorphism .Out of 26 A(H1N1)pdm09 viruses submitted from Illinois, two contained I38L, A/Illinois/37/2018 and A/Illinois/38/2018, which were identical on a nt level. Both were collected on 8 February 2018 from school age males residing in the same county. Because natural polymorphism at this residue is rare, we investigated for potential transmission.Supplemental Figure S2). Results were obtained for 30 specimens, all of which lacked I38L. The combined results from NGS and pyrosequencing estimated the rate of detection of I38L to be 3.6% (2/56) for Illinois and 4.3% (2/47) for this particular county.The CDC requested additional A(H1N1)pdm09 viruses collected in this state during the 2017/18 season. Forty-one additional A(H1N1)pdm09 positive specimens were available for evaluation. To expedite testing, the pyrosequencing assay  was usedIn this study, we established the methodology for monitoring baloxavir susceptibility in the US ahead of the drug entering the national market. As NGS analysis has become the cornerstone of influenza surveillance, it created a platform for monitoring resistance for all FDA-approved anti-influenza therapeutics. The frequency of AAS associated with reduced baloxavir susceptibility should be determined by type and subtype. This will allow for accurate inter-seasonal comparisons, as different types and subtypes predominate season to season. In the event of suspected emergence of resistance, either NGS or pyrosequencing could support epidemiological investigations in a timely manner. Pyrosequencing has been employed by many US PHLs to conduct enhanced antiviral surveillance, so this assay can readily be implemented for detecting baloxavir resistant viruses. Phenotypic assays detect viruses with reduced drug susceptibility regardless of underlying mutations. Here, HINT was successfully applied to establish the baseline susceptibility of seasonal viruses. Moreover, we have shown that clinical specimens could be tested directly by HINT, which could expedite surveillance.50 values were within their respective type/subtype baseline (data not shown).Overall, in the 2016/17 and 2017/18 seasons the frequency of AAS associated with reduced susceptibility to baloxavir was low. For the current 2018/19 season, we analysed PA sequences of 384 US viruses (accessed from GISAID on 11 January 2019) and found no AAS at positions of concern. Additionally, we tested 73 representative viruses by HINT and all ECThe antiviral testing algorithm described above will be applied to both foreign and domestic viruses. In collaboration with the Association of Public Health Laboratories, CDC aims to advance antiviral surveillance by implementing HINT at designated National Influenza Reference Centers. To assist in baloxavir susceptibility testing at the WHO National Influenza Centers and other laboratories, a CDC panel of reference viruses with reduced susceptibility to baloxavir will be made available from the International Reagent Resource . As balo


However, the contribution of TLR2 activation to adaptive immunity and HBV clearance remains to be clarified. In this study, we explored the hydrodynamic injection (HI) mouse models for HBV infection and examined how the TLR2 agonist Pam3CSK (P3C) influences HBV control and modulates HBV-specific T-cell response if applied in vivo. We found that TLR2 activation by P3C injection leads to the rapid but transient production of serum proinflammatory factors interleukin-6 and tumor necrosis factor-α and activation of CD8+ T cells in vivo. Then, the anti-HBV effect and HBV-specific T-cell immunity were investigated by TLR2 activation in the mouse models for persistent or acute HBV infections using HBV plasmids pAAV-HBV1.2 and pSM2, respectively. Both P3C application at early stage and pre-activation promoted HBV clearance, while only TLR2 pre-activation enhanced HBV-specific T-cell response in the liver. In the mouse model for acute HBV infection, P3C application had no significant effect on HBV clearance though P3C significantly enhanced the HBV-specific T-cell response. Collectively, TLR2 pre-activation enhances HBV-specific T-cell responses and accelerates HBV clearance in HI mouse models. Thus, the modulation of host immune status by TLR2 agonists may be explored for immunotherapeutic strategies to control HBV infection.Toll-like receptors (TLRs) play a crucial role in activation of innate immunity, which is essential for inducing effective adaptive immune responses. Our previous studies have shown that toll-like receptor 2 (TLR2) is required to induce effective virus-specific T-cell responses against hepatitis B virus (HBV)  CD8+ T cells kill infected cells and secrete antiviral cytokines [mainly interferon gamma (IFN-γ)] that inhibit HBV replication non-cytopathically (Hepatitis B virus (HBV) is a hepatotropic and non-cytopathic virus that causes more than one million deaths annually from liver cirrhosis and hepatocellular carcinoma , 2. The thically , 4. Neutthically .Hepatitis B virus was at one time considered to be a “stealth virus” that induces negligible innate immune responses during the early phase of infection . Gene ar+ T cells  is widely expressed by antigen-presenting cells, endothelial and epithelial cells, and lymphocyte subsets, including activated and memory T cells , 14. TLR T cells . The dow T cells .+ and CD8+ T cells, it may serve as a co-stimulatory molecule to enhance T-cell proliferation, survival, and effector functions  and were maintained under specific pathogen-free conditions in the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology. Animals were treated according to the Guidelines of the National Institutes of Health for Animal Care and Use.Plasmid pAAV/HBV1.2  contains a terminally redundant 1.2× copy of genotype A HBV genome . PlasmidThe peptide of Kb-restricted HBV Cor93–100 epitope (MGLKFRQL) was synthesized and purchased from Chinese Peptide Company . The synthetic lipopeptide Pam3CSK4  was purchased from EMC Microcollections .Briefly, 10 µg of plasmid DNA (pAAV/HBV1.2 or pSM2) was hydrodynamically injected into the tail vein of C57BL/6 mice in a volume of phosphate-buffered saline (PBS) solution equivalent to 10% body weight within 5–8 s , 26, 27.Serum samples were collected from mice at the indicated time points after HI and diluted 1:10 with Diluent Universal . The levels of serum HBsAg, Hepatitis B e antigen (HBeAg), anti-HBc, anti-HBs, and HBV DNA were determined as described previously . NucleocSerum samples were collected from mice at the indicated time points after injection of P3C or PBS. The levels of serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured by specific Mouse cytokine ELISA kits  according to the manufacturer’s instructions. Splenocytes were isolated from mice and cultured with αCD3/CD28 for 48 h. The IFN-γ levels in the supernatant were also measured by Mouse cytokine ELISA kits (eBioscience) in accordance with the manufacturer’s instructions.Total RNA was isolated from collected mouse liver tissue samples with Total RNA Kit (Omega) following the manufacturer’s protocol. One-step RT-PCR with real-time detection was carried out with the SYBR Green Real-time RT-PCR Master Mix (TaKaRa). The mRNA levels of IFN-β, IL-6, TNF-α, IL-10, and β-actin were detected by commercial QuantiTec Primer Assays (Qiagen) listed in Table S1.g without break. After removing the debris and hepatocytes on the top layer, IHLs in the pellet were collected, washed, and subjected to further analysis. CD4+, CD8+, CD11b+, CD11c+, and F4/80+ cells in mouse spleen were isolated by appropriate microbeads purchased from Miltenyi Biotec GmbH.Murine splenocytes (SPLs) and intrahepatic lymphocytes (IHLs) were isolated as described previously . In brieSurface and intracellular staining were performed as described previously . The ant+ T cells specific to the Kb-restricted HBV Cor93–100 epitope, Recombinant Soluble Dimeric Mouse H-2K[b]: Ig Fusion Protein  was loaded with Cor93–100 overnight, and then used to stain mouse lymphocytes according to the technical instructions . Data were analyzed using nonparametric one-way ANOVA and Dunn’s multiple comparison test or Student’s in vivo by application of TLR2 ligands P3C in C57BL/6 mice without and with HBV replication. C57BL/6 mice were treated with 50 and 100 µg of P3C by subcutaneous (SC) injection or PBS as control, respectively. The serum level of IL-6 and TNF-α was detected by specific ELISA at the indicated time points. The production of IL-6 and TNF-α was transient after P3C injection, with the levels peaking at 3 h, in a dose-dependent manner, and disappearing at 12 h after injection . The kinetics of serum HBsAg, HBeAg, and HBV DNA indicated that P3C treatment at the late time points had no effect on HBV replication in mice , a TLR3 ligand, demonstrated that direct application of TLR ligands into the liver may effectively recruit specific T cells and control HBV replication . Thus, w+, CD8+, CD11b+, CD11c+, and F4/80+ cells in the liver were analyzed by flow cytometry. We observed that the frequency of resident F4/80+ Kupffer cells in the liver was decreased. In contrast, the frequency of CD11c+ cells (DC) in the liver was slightly increased by P3C injection. However, the frequencies of CD4+ and CD8+ T cells in the liver and spleen were unchanged  from either control or P3C mice. Then, IFN-γ production in the supernatant was determined after 48 h stimulation with αCD3/CD28. Interestingly, we found that higher levels of IFN-γ were produced by CD4+, CD8+, CD11c+, and F4/80+ cells after TLR2 activation Figures First, we treated C57BL/6 mice with 20 µg of P3C or PBS by intravenous (IV) injection. Liver NPCs and splenocytes were separated at day 7 after IV. The frequencies of CD4ex vivo. The IHLs produced higher levels of IFN-γ  before HI of pAAV-HBV1.2. HI with pAAV-HBV1.2 established prolonged HBV replication and gene expression in C57BL/6 mice, as described in the previous experiment  before HI of plasmid pSM2. Again, the serum HBsAg, HBeAg, and HBV DNA disappeared in mice at day 21 after HI   . We prevoly(I:C) . Howeveroly(I:C) . Thus, toly(I:C) . In thisoly(I:C) .+ T cells were recruited in the dependence of CXCR3 into the liver. The peak of CD8+ cell infiltration into the liver was measured between days 20 and 30 after poly(I:C) application, which coincided with significant reduction of HBV loads .ML and YX conceived and designed the study. YL, XH, JW, JL, MC, ZM, YL, SH, and QL performed the experiments and analyzed the data. YL, ML, and YX wrote the manuscript. EZ, XZ, JH, and DY provided materials and technical support and contributed to helpful discussions and review of the final manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nonstructural protein 1 (NS1) is a virulence factor encoded by influenza A virus (IAV) that is expressed in the nucleus and cytoplasm of host cells during the earliest stages of infection. NS1 is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. However, to date, the phosphorylation sites of NS1 have not been identified, and the relationship between phosphorylation and protein function has not been thoroughly elucidated.In this study, potential phosphorylation sites in the swine influenza virus (SIV) NS1 protein were bioinformatically predicted and determined by Phos-tag SDS-PAGE analysis. To study the role of NS1 phosphorylation sites, we rescued NS1 mutants (Y73F and S83A) of A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability, cytokine production as well as the intracellular localization in cultured cells. Additionally, we used small interfering RNA (siRNA) assay to explore whether changes in the type I IFN response with dephosphorylation at positions 73 and 83 were mediated by the RIG-I pathway.We checked 18 predicted sites in 30 SIV NS1 genes to exclude strain-specific sites, covering H1N1, H1N2 and H3N2 subtypes and identified two phosphorylation sites Y73 and S83 in the H1N1 SIV protein by Phos-tag SDS-PAGE analysis. We found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and reduced the ability of NS1 to antagonize IFN-β expression but had no effect on nuclear localization. Knockdown of RIG-I dramatically impaired the induction of IFN-β and ISG56 in NS1 Y73F or S83A mutant-infected cells, indicating that RIG-I plays a role in the IFN-β response upon rSIV NS1 Y73F and rSIV NS1 S83A infection.We first identified two functional phosphorylation sites in the H1N1 SIV protein: Y73 and S83. We found that dephosphorylation at positions 73 and 83 of the NS1 protein affected the antiviral state in the host cells, partly through the RIG-I pathway. Swine influenza (SI) is a highly contagious respiratory disease that is characterized by fever, weight loss and acute respiratory problems. It is caused by influenza A virus (IAV), which belongs to the Orthomyxoviridae family. Outbreaks of swine flu cause significant morbidity and growth retardation in pigs, leading to a considerable economic loss to the infected farms , 2. The IAV possesses a segmented genome with eight single stranded negative-sense RNA molecules. The surface glycoproteins hemagglutinin (HA) and neuraminidase (NA) are the key antigens and can be used to classify IAV into different subtypes . NonstruThe ability of host cells to recognize viral pathogens relies on pattern recognition receptors (PRRs) which trigger an antiviral response. Retinoic acid-inducible gene I product (RIG-I) has been identified as one of the key cellular sensors of RNA viruses. Once activated, it binds to cytosolic double-stranded RNA and initiates a signaling cascade that depends on the E3 ligase activity of tripartite motif family 25 (TRIM25), resulting in IFN and inflammatory cytokine induction . Goffic Protein phosphorylation, a kind of post-translational modification, commonly occurs on threonine (T), serine (S), or tyrosine (Y) residues, and it regulates distinct activities of multifunctional proteins . During Here, we bioinformatically analyzed potential phosphorylation sites of the SIV NS1 protein. Upon generating recombinant influenza A/swine/Shanghai/3/2014(H1N1) viruses that encode mutant NS1 proteins (Y73F and S83A), we found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and diminished the capability of NS1 to antagonize IFN-β expression. The function of NS1 protein phosphorylation is complicated, and this work will help to elucidate how phosphorylation mutations influence the function of the NS1 protein.2. Rabbit polyclonal antibodies against RIG-I and horseradish peroxidase (HRP) conjugated goat anti-rabbit or anti-mouse secondary antibodies were purchased from Cell Signaling Technology . A mouse monoclonal antibody against β-actin was purchased from Sigma . Alexa Fluor 488-labeled goat anti-mouse IgG was purchased from Beyotime Biotechnology . A mouse polyclonal antibody against the NS1 protein was generously provided by Professor Ying Fang at Kansas State University, USA.The H1N1 SIV A/swine/Shanghai/3/2014(SH/2014) strain was isolated during routine surveillance and sequenced by our laboratory. Madin-Darby canine kidney (MDCK) cells and the human epithelial kidney cell line (293 T) were obtained from the American Type Culture Collection  and grown in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  at 37 °C and 5% COhttps://www.ncbi.nlm.nih.gov/). The amino acid sequences were aligned using the MegAlign program with DNASTAR software , and conserved S, T and Y phosphorylation sites were obtained. To exclude false negatives and false positives, we used two websites to predict the possible phosphorylation sites of the NS1 protein. We uploaded amino acid sequences of NS1 to NetPhos 3.1 Server (http://www.cbs.dtu.dk/services/NetPhos/) and Scansite Motif Scan (https://scansite4.mit.edu/4.0/) [The full-length sequences of 30 SIV NS1 genes from H1N1, H1N2 and H3N2 subtype strains were obtained from GenBank (du/4.0/) , 23. Scadu/4.0/) , 25.2, which were added to the separating gel before polymerization [The pCAGGS-(SH/2014)-NS1 and pCAGGS-(SH/2014)-NS1 mutants Y73F, S76A, S83A, S151A, S161A and S195A were constructed by inserting the corresponding cDNAs into the EcoI/NheI sites of the pCAGGS vector. Then, 293 T cells grown in six-well plates were cotransfected with 2 μg of NS1 plasmid by using Lipofectamine 2000 transfection reagent. At 48 h posttransfection, we collected the cells and analyzed the phosphorylation state of the different mutants via western blotting using Phos-tag SDS-PAGE. The gel consisted of a separating and a stacking gel containing 100 μM Phos-tag and 10 mM MnClrization . The gelrization . The pho50) using the Reed–Münch method.The eight gene segments of SH/2014 were cloned into the vRNA-mRNA bidirectional transcription vector pBD to rescue SH/2014, as described previously . Mutatio2. At 1 h post infection (hpi), the virus suspensions were removed and replaced with serum-free DMEM containing 2 μg/ml TPCK-treated trypsin. Supernatants were collected at 12, 24, 48, and 72 hpi and titrated on MDCK cells.To evaluate virus growth kinetics, confluent monolayers of MDCK cells were infected with rSIV or NS1-mutant viruses at a multiplicity of infection (MOI) of 0.001 and incubated at 37 °C in 5% COImmunofluorescence analysis was processed as described previously . MDCK ce293 T cells were infected with wt or NS1-mutant viruses at an MOI of 1 for different times, and total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s recommendations. qRT-PCR was performed using specific primers to measure the mRNA levels of IFN-β, ISG56 and RIG-I, and β–actin was used as a control. The sequences of the primers are listed in Table RNA interference was used to knock down endogenous RIG-I. 293 T cells grown to 40 to 50% confluence in 6-well plates were transfected with 100 nmol of siRNA using 3 μl of Lipofectamine 2000 (Invitrogen) in Opti-MEM (Invitrogen). Twenty-four hours after transfection, the medium was removed. The cells were then infected with wt or mutant viruses at an MOI of 1 for 24 h and then harvested for analysis by western blot analysis or real-time PCR. Scrambled siRNA was used as a negative control. The siRNAs were designed and synthesized by Gene Pharma Company  with the following sequences: siRIG-I, 5′-GCCCAUUGAAACCAAGAAAUU-3′; nontargeting control siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3’ [p-value of < 0.05 was considered to be statistically significant.The data are expressed as the means ± standard deviations (SD). The significance was determined with a two-tailed independent Student’s t-test. A To identify possible NS1 phosphorylation sites, the NetPhos 3.1 and Kinase Scansite 4.0 databases were used. As shown in Table 293 T cells were transfected with the different NS1 variants and we assayed the level of NS1 phosphorylation by western blotting using SDS-PAGE gels containing Phos-tag, a ligand that shifts the mobility of phosphorylated proteins. After electrophoresis, NS1 proteins could be separated into several bands due to the decreased mobility of phosphorylated NS1 proteins. As shown in Fig. 50 assay at the indicated time points. The result showed that two mutant viruses grew similarly in MDCK cells, and their titers peaked at 48 h, with values of 5.87 and 5.47 TICD50/ml, respectively ) and its NS1 protein mutants via reverse genetics and compared their replication ability in cultured cells. The growth curves revealed that dephosphorylation of Y73 and S83 caused a reduction in the amount of SIV particles in the cell culture supernatant, as well as viral protein synthesis, compared to wild-type virus at the same time point after infection. It has been shown earlier that in human IAV, phosphorylation of S42 and T49 attenuated virus replication, while T215 phosphorylation does not affect virus replication . We hypoIt is well known that type I IFN antagonism is one of the main biological properties of NS1 protein on host cells. Therefore, we explored whether the attenuated replication caused by dephosphorylation at Y73 and S83 in NS1 of the SH/2014 virus might be due to elevated induction of IFN-β. Indeed, both of the NS1 mutants resulted in strong IFN gene induction, indicating that dephosphorylation at Y73 and S83 is less capable of suppressing the cellular IFN response. RIG-I has been identified as a cellular sensor of RNA virus infection resulting in type I IFN induction, and IAV can inhibit type I IFN production through its NS1 protein by inhibiting RIG-I , 37. In In conclusion, we first identified two functional phosphorylation sites in the H1N1 SIV protein: Y73 and S83. We found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and reduced the ability of NS1 to antagonize IFN-β expression. The present study helps to clarify the mechanism by which NS1 protein phosphorylation affects SIV propagation and interferon-antagonistic activity.


Momordica charantia L.). A total of 2013 high quality SNP markers binned to 20 linkage groups (LG) spanning a cumulative distance of 2329.2 cM were developed. Each LG ranging from 185.2 cM (LG-12) to 46.2 cM (LG-17) and average LG span of 116.46 cM. The number of SNP markers mapped in each LG varied from 23 markers in LG-20 to 146 markers in LG-1 with an average of 100.65 SNPs per LG. The average distance between markers was 1.16 cM across 20 LGs and average distance between the markers ranged from 0.70 (LG-4) to 2.92 (LG-20). A total of 22 QTLs for four traits  were identified and mapped on 20 LGs. The gynoecious (gy-1) locus is flanked by markers TP_54865 and TP_54890 on LG 12 at a distance of 3.04 cM to TP_54890 and the major QTLs identified for the earliness traits will be extremely useful in marker development and MAS for rapid development of various gynoecious lines with different genetic background of best combiner for development of early and high yielding hybrids in bitter gourd.A high-density, high-resolution genetic map was constructed for bitter gourd ( Momordica charantia L.; 2n = 22) is an economically important vegetable crop belonging to the subtribe Thalidianthinae, tribe Joliffieae, subfamily Cucurbitoideae and family Cucurbitaceae   , whereasgy-1) . Since glination .Momordica species through sequencing of small insert genomic library enriched for 10 different repeat motifs  draft genome sequence . Draft genome sequence of bitter gourd revealed that, the MOMC3_649 in bitter gourd was presumed to be an ortholog of CmAcs11  and two proteins  were found in bitter gourd similar to CmAcs-7  grouped in the same clade in the phylogenetic tree sequence (The first genetic map and positions of major fruit trait loci of bitter melon were worked out by ed lines . Matsumusequence  of a monsequence . Cui et gy-1) gene, sex ratio, and earliness related traits in bitter gourd for further utilization in bitter gourd crop improvement.2 segregated population and 65 F2:3 families  derived from a cross between DBGy-201 (PVGy-201) and Pusa Do Mousami (PDM). It is very difficult in self-fertilizing and seed production of gynoecious lines to F2:3 population. The female parent PVGy-201 is a gynoecious line  and first pistillate flower appear at 7th node on 33 days after planting; whereas male parent Pusa Do Mousami is a monoecious plant (♂:♀ is 17:1) and first pistillate flower appear at 13th node on 60 days after planting. The F2 plants along with the two parents were planted at vegetable research farm of Indian Agricultural Research Institute (IARI), New Delhi, India during spring summer (February–May) 2015 for phenotyping the qualitative traits like gynoeciousim and sex ratio. A total of 65 F2:3 families along with two parents were planted during spring summer (February–May) 2016 to study the quantitative traits like node and days to first pistillate flower appearance. About 20 F3 seeds from each F2 plant were sown in single row with three replications and recommended agronomic practices were undertaken for the healthy crop.The genetic material used for mapping of gynoecious and earliness involved 90 F1, back crosses, F2 and F2:3 populations was performed to study the inheritance and mapping of traits related to sex ratio . The sequences of the two oligonucleotides comprising the barcode adapter are:Two different types of adapters were used in this protocol . The “ba5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTxxxx and5′-CWGyyyyAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTwhere “xxxx” and “yyyy” denote the barcode and barcode complement and sequences.ApeKI-compatible sticky end:The second, or “common,” adapter has only an 5′-CWGAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG and5′-CTCGGCATTCCTGCTGAACCGCTCTTCCGATCTFor preparing each library we have used 94 different barcodes for tagging samples, which have variable length from 5 to 10 nucleotides.2 and each one from two parents and F1) were sequenced using Illumina True Seq Version 3 single end sequencing chemistry with read lengths of 150 bp on HiSEQ 2000 Platform. Ninety four samples (plus a blank negative control) were sequenced per lane. The library was prepared for GBS by following the PRJNA493717).The final 93 libraries (90 FApeKI cut site (CWGC), no adapter dimers and reads with no “NS” (minimum Qscore of 10) across the first 72 bases. The final size of raw data from all libraries was 18.4 GB. As bitter gourd was not having reference genome, sequence reads from raw data FASTQ file were processed through de novo GBS analysis pipeline as implemented in UNEAK (Supplementary Data Sheet 1 (Supplementary Data Sheet The reads were filtered following ® 4.1 program (2 test was performed for identification of markers with aberrant segregation (p < 0.05) by calculating the locus genotyping frequencies in JOINMAP®4.1. Linkage groups (LGs) were constructed by grouping of markers at a minimum independence LOD threshold of 3.0 and a maximum of 10.0 with a step up of 0.5. The groups were converted to maps at LOD using regression algorithm with the following settings: linkages with recombination frequency (<0.49), LOD (>0.01) threshold for removal of loci with respect to jumps in goodness-of-fit (5.0) and performing a ripple after adding 2 loci. Distance was calculated using Kosambi’s mapping function and LGs were drawn with help of Map Chart.The genotypic data matrix was generated based on scoring pattern observed with all polymorphic SNP markers. The generated matrix data was integrated with all polymorphic SNP markers used as an input file in JOINMAP program  for cons2 individuals with phenotypic data for sex ratio and earliness (both F2 and F3 populations). The genotypic data consisted of marker loci. QTLs were detected with the WinQTL Cartographer v2.5 (p = 0.05)  . The 95% = 0.05) . Additiv2 individuals, F1 and two parents) have generated 93,926 SNP sites. After excluding SNPs that were monomorphic in the F2 population, those non-biallelic, with more than 40% of missing data, with minimum allele frequency (MAF) 20%, 4003 SNPs remained. The SNPs were again filtered to remove heterozygote SNPs in both parents and finally high quality 2013 SNPs were identified and used for linkage map construction.Raw data of Illumina sequence of all 93 libraries was 18.4 GB. The total 93 pooled, barcoded samples  generation all plants were monoecious which indicated it as dominant trait (Table 2 population (5–100% segregation for pistillate flower frequency), 72 plants were monoecious and 18 plants gynoecious. The observed frequency of F2 plants fitted well in the expected ratio of 3 monoecious: 1 gynoecious as evident from the non-significant χ2 values of 1.20 (P = 0.27). The BC1P1 population segregated into 16 monoecious and 14 gynoecious plants. The observed frequency of BC1P1 plants fitted well in the expected ratio of 1 monoecious: 1 gynoecious plants with non-significant χ2 values of 0.13 (P = 0.72).Inheritance pattern of gynoecy was investigated in bitter gourd in cross DBGy-201 (100% pistillate flower frequency) × Pusa Do Mousami (6% pistillate flower frequency), the Fit Table . Out of 1 (3) and segregated populations F2 and F2:3 ranged from 0 to 20 and 0.5 to 21, respectively. The transgressive segregants for sex ratio in F2:3 population were 13.85%. The bimodal distribution of F2 and F2:3 populations  is also an extremely important trait in cucurbits for getting higher fruit yields. The sex ratio most likely genetically controlled, but it is often affected by environmental and nutritional factors. The sex ratio of parents DBGy-201 , Pusa Do Mousami (17), F Figures , 5 revea1 had first pistillate flower appeared at 6th node after 35 days of sowing. The range of segregation for node at first pistillate flower appear was 3–16 and 2.5–15.75 in F2 and F2:3 populations, respectively. For trait, days to first pistillate appearance ranged from 35 to 47 and 32.56 to 46.63 in F2 and F2:3 populations, respectively.For getting higher price in the market, early picking is most important. Node and days to first pistillate flower appearance are directly contributing toward early harvesting of horticultural matured fruits. The first pistillate flower appeared for parent DBGy-201 on 7th node after 33 days of sowing and parent Pusa Do Mousami at 15th node, after 60 days of sowing. The F2 individuals and F2:3 families indicated that, this trait was governed by quantitative genes. For trait, node at first pistillate flower appearance, out of 90 plants in F2, 73 plants and in F2:3 out of 65 families, 55 families had shown lower than female parent PVGy-201 , which indicated predominance of major genes toward lower node for first pistillate appearance. The segregation pattern  and F2:3 families (45 families out 65 families had in the range of 35–40 days) were located between the two parents, but very close to female parent (PVGy-201), this indicated predominance of major genes toward earliness for pistillate flower appearance. The transgressive segregants for node at first pistillate flower appearance in F2:3 population were 86.15% and for days to first pistillate flower appearance were only 1.54%.The segregation pattern Figures , 7 of tr Figures , 9 of tr2 and F2:3 mapping populations with 2013 SNP markers over 20 LGs to construct high quality genetic map. High-resolution mapping of QTLs may be used to develop reliable markers for MAS  and LOD score of above 2.0 or 3.0 (most commonly 3.0) was usually chosen as the significance threshold for detecting QTL  . The marker TP_54923 on right side of gy-1 gene at a distance of 4.14 cM and marker TP_55091 was on right side at distance of 10.04 cM. The markers TP_54865 and TP_54827 were on left side at distance of 7.06 and 10.36 cM, respectively.The gynoecious  Figure  gene was2 and F2:3 populations. Detailed information about all these QTLs  is shown in Table 2 and F2:3 populations. The phenotypic variation (R2%) explained by these QTLs ranged from 0.05 to 58.75% and total 11 major QTLs (R2 = >10%) were identified. Most of the QTLs identified in this study were located in the same or adjacent regions on LG-14 and LG-16, this may be due to high correlation among the traits under study . The QTLs 3qSR14a-F and 3qSR14b-F were located between flanking markers TP_68232 and TP_68500 on LG-14, had shown LOD value of 6.30 and 6.40, respectively . The 2qSR14b-F was located between flanking markers TP_68643 and TP_67968 on LG-14 with LOD value of 6.30 (R2 = 20.95%). All four major QTLs together explained 65.60% of phenotyping variation. The QTLs 3, qSR14b-F3qSR14a-F, and 2qSR14b-F showed positive additive effect, indicating allele for increasing sex ratio was contributed by DBGy-201, whereas QTL 3qSR13-F showed negative additive effect, indicating allele for increasing sex ratio was contributed by Pusa Do Mousami.Four major QTLs were identified for sex ratio (♂: ♀), one on LG-13 and three on the linkage group LG-14. The QTL qSR13-F3qNPF9-F was located between flanking markers TP_46756 and TP_46766 on LG-9, showing LOD value of 4.00 (R2 = 13.94%). The 2qNPF14-F was located between flanking markers TP_67968 and TP_68232 on LG14 (R2 = 12.03%). Both the QTLs together contributed 25.97% of phenotyping variation and showed positive additive effect indicating positive allele for increasing node at first pistillate flower appearance was contributed by female parent PVGy-201.Two major QTLs were identified for node at first pistillate flower appearance one each on the linkage group LG-9 and LG-14. The 3qDPF5a-F and 3qDPF5b-F were located between flanking markers TP_11213 and TP_11334 on LG-5, showed LOD value of 3.20 and 3.30 explaining 15.13 and 15.33% of phenotyping variation (R2%), respectively. The QTLs 3qDPF16a-F and 3qDPF16b-F were located between flanking markers TP_74581 and TP_74591 on LG-16 had shown LOD value of 35.80 and 35.90, respectively . The 3qDPF14-F was located between flanking markers TP_67682 and TP_67839 on LG-14 with LOD value of 4.40 and explaining 17.34% of phenotyping variation (R2%) and both QTLs explained 25.97% of phenotyping variation. The QTL 3qDPF14-F showed positive additive effect, indicating allele for increasing days to first pistillate flower appearance was contributed by PVGy-201, whereas QTLs 3, qDPF5b-F3, qDPF16a-F3qDPF5a-F, and 3qDPF16b-F showed negative additive effect, indicating allele for increasing days to first pistillate flower appearance was contributed by Pusa Do Mousami.Economically most important horticultural traits in bitter gourd for getting early and high yield are early flowering and sex ratio which are highly variable, affected by genetic, environmental and hormonal factors. Five major additive QTLs were identified for days to first pistillate flower appearance, two each on the linkage groups LG-5 and LG-16; and one on LG-14. The QTLs ApeKI (GCWGC)    to identpipeline  and a to(n = 11)  even thoion size  and type2 population revealed that, the gynoecious trait (gy-1) single recessive gene (gy-1), marker TP_54890 was very close to gy-1 gene, with a distance of 3.04 cM. Gynoecy (gy-1) trait useful for low cost and quality hybrid seed production, early and high yielding hybrids. The gy-1 gene flanked to markers TP_54865 and TP_54890 on LG 12 are extremely useful in marker development and MAS for rapid development of various gynoecious lines with different genetic background of best combiner for development of early and high yielding hybrids. Based on a RAD-seq analysis of F2 progeny gy1.1 and gy1.2 at distal end of linkage map by RAD-based genetic map for anchoring scaffold sequences in bitter gourd. We identified two SNPs within 5 cM distance from gy-1 gene, TP_54890 (3.04 cM) and TP_54923 (4.14 cM). In this study we have taken highly diversified parents with highly segregated mapping population and less base recognition restriction endonuclease ApeKI (GCWGC) and constructed linkage map with high number (2013) quality SNPs.Inheritance pattern of qualitative traits were investigated in bitter gourd cross DBGy-201 × Pusa Do Mousami of Five gene , in cont2 and F2:3 populations in bitter gourd was bimodal distribution of sex ratio (♂: ♀) which revealed that sex ratio is governed by semi-quantitative genes and some major genes may be modified by other minor genes  and cultivars with better sex ratio. In the present study we identified QTLs that explain significant portions of the observed phenotypic variation for plant architecture. The appearance of the first pistillate flower at a lower node number is an indication of the earliness of the variety . On the 3qNPF9-F and 2qNPF14-F showed positive additive effects contributed from PVGy-201 alleles increased the nodes for first pistillate flower appearance at 1.24 and 0.91 node number, respectively. Similarly, Luffa  together explained 47.80% of phenotyping variation and other two major QTLs (3qDPF16a-F and 3qDPF16b-F) explained 58.75% of phenotyping variation individually for days to first pistillate flower appearance. The QTL 3qDPF14-F showed positive additive effect, thus PVGy-201 alleles for increased 7.92 days to first pistillate flower appearance, whereas QTLs 3, qDPF5b-F3, qDPF16a-F3qDPF5a-F, and 3qDPF16b-F showed negative additive effect, thus Pusa Do Mousami alleles increased days to first pistillate flower appearance by 7.25, 7.30, 19.22, and 19.22 days, respectively. Similarly four QTLs mapped for days to anthesis in cucumber  was mapped with SNP marker TP_54890 with a distance of 3.04 cM on LG-12. Major QTLs were mapped in present study has the potential to significantly improve the efficiency of MAS for gynoecious gene, sex ratio and earliness for development of best hybrids or varieties.A total of 22 QTLs for three traits were identified and mapped on 20 LGs and the gynoecy (PGR and TKB performed and conceived the experiments, collected data, writer and analyst of results. TKB, AG, and ADM supervised the project. GSJ and GB management and technical support. All authors reviewed and approved this submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arginine methylation widens the mechanism of control by E2F1 from a transcription factor to a regulator of alternative RNA splicing. E2F is a family of master transcription regulators involved in mediating diverse cell fates. Here, we show that residue-specific arginine methylation (meR) by PRMT5 enables E2F1 to regulate many genes at the level of alternative RNA splicing, rather than through its classical transcription-based mechanism. The p100/TSN tudor domain protein reads the meR mark on chromatin-bound E2F1, allowing snRNA components of the splicing machinery to assemble with E2F1. A large set of RNAs including spliced variants associate with E2F1 by virtue of the methyl mark. By focusing on the deSUMOylase SENP7 gene, which we identified as an E2F target gene, we establish that alternative splicing is functionally important for E2F1 activity. Our results reveal an unexpected consequence of arginine methylation, where reader-writer interplay widens the mechanism of control by E2F1, from transcription factor to regulator of alternative RNA splicing, thereby extending the genomic landscape under E2F1 control. E2F is a family of master transcription regulators involved in mediating diverse cell fates, which frequently becomes deregulated in cancer. The retinoblastoma protein (pRb)–E2F pathway is a central player in the control of cell cycle progression in diverse cell types and its deregulation of primary importance in proliferative disease such as cancer, where aberrant pRb activity occurs through a variety of oncogenic mechanisms  protein influenced E2F target gene activity through regulating chromatin SUMOylation and heterochromatin protein 1 (HP1) binding. Our results reveal an unexpected role for E2F1 in regulating the alternative RNA splicing machinery, which occurs through a meR mark–dependent reader-writer interplay, enabling E2F1 to broaden its influence to genes that otherwise are poor transcription targets. The methyl mark, therefore, confers a new mechanism of control and extends the genomic landscape under E2F1 control.Here, we show that methylation by PRMT5 enables E2F1 to regulate a diverse group of genes at the level of alternative RNA splicing, rather than through the classical transcription-based mechanism widely ascribed to E2F1. The impact of E2F1 on alternative RNA splicing requires the tudor domain protein p100/TSN to read the meR mark, allowing components of the splicing machinery such as small nuclear RNA (snRNA) to associate with the p100/TSN-E2F1 complex. Consistent with its role in RNA splicing, a large group of RNAs, including spliced intermediates, bind to the E2F1 complex. Most genes subject to alternative splicing are poor transcription targets for E2F1. We identified To clarify the role of the meR mark in regulating E2F1 activity, we developed a panel of Tet-On inducible cell lines . Each ceWe used RNA sequencing (RNA-seq) to assess the global transcript profile in each stable cell line. Mining the RNA-seq dataset for transcripts regulated twofold or more upon E2F1 expression (compared to the empty pTRE vector cell line) identified a large number, the majority (around 50% for each cell line) being derived from E2F target genes (fig. S1E and table S1), where an E2F target gene was defined by the presence of one or more E2F binding site consensus motifs in the proximal promoter region (−900 to +100) . Further, although a high proportion of up- and down-regulated transcripts were shared between the WT and KK E2F1 cell lines  , some ofWe performed a similar analysis on the R109K expression condition. In contrast to WT or KK E2F1, the R109K derivative was less able to influence transcription . We obseWe assessed the gene sets that were present in the RNA-seq by gene set analysis. There were a number of shared gene sets enriched in each condition, including E2F targets (as expected), whereas gene sets connected with the epithelial-mesenchymal transition and hypoxia were generally down-regulated in each condition (fig. S2A).LRRC4, ETV1, and FGF4 transcripts were expressed at high levels in the KK cell line, with reduced expression in the R109K cell line, and a similar pattern of expression was evident when transcription from each gene was individually measured in each cell line (fig. S1F). Conversely, at the global level, KCNIP showed higher expression in R109K compared to KK, and a similar expression pattern was apparent when gene expression was individually measured (fig. S1F). Moreover, we confirmed that the expression of each candidate gene was dependent on E2F1, as silencing endogenous E2F1 with siRNA caused reduced expression of each gene (fig. S1G).It was important to validate the results from the RNA-seq. We therefore measured the expression of a number of E2F target candidate genes identified in the RNA-seq dataset, where there was evidence for differential expression patterns. For example, It is noteworthy that the R109K derivative exhibits a reduced ability to affect transcription  (17). OfWe observed alternative splicing events in E2F gene transcripts, which included skipped exons, alternative 3′ (A3SS) or 5′ (A5SS) splice sites, mutually exclusive exons, and retained introns . Only 42When each set of alternatively spliced transcripts derived from E2F target genes was compared under the WT, KK, and R109K E2F1 expression conditions, qualitative and quantitative differences in the alternatively spliced RNA were apparent, with events that were both shared and unique . WT and We performed gene ontology (GO) analysis on the E2F gene sets from which the alternatively spliced transcripts were derived (fig. S3). Although there was considerable overlap in the GO terms enriched in each condition, such as cellular processes linked to cell cycle, there were a number of marked differences. For example, DNA damage–related terms were prevalent under KK expression conditions, while catabolic and biosynthetic terms were enriched upon R109K expression.We also studied the expression level of a variety of E2F target genes connected with splicing, many encoding components of the splicing machinery (table S3). From an analysis of the RNA-seq data, none of the genes were expressed at a notably different level under the WT, KK, or R109K expression conditions (table S3). The increased level of alternative splicing identified by rMATS, therefore, cannot be easily attributed to coincident changes in the expression of splicing components.We reasoned that the impact of E2F1 on alternative RNA splicing could be mediated by meR E2F1 interacting with components of the splicing machinery, since the meR reader protein p100/TSN functions in spliceosome assembly and enhances splicing activity . SignifiKAT2B) (ΔΨ = 0.171 to 0.232), the lysine methyl-transferase SET domain containing 2 (SETD2) (ΔΨ = −0.888), and max gene associated protein (MGA) (ΔΨ = 0.436) were identified as alternatively spliced transcripts by rMATS (table S2).Having established that arginine methylation and its reader p100/TSN enable E2F1 to influence alternative splicing  and furtSENP7, MDS1 and EVI1 complex locus (MECOM), P3H2, and SPG21 genes were identified in the E2F1 RIP-seq (SENP7 and MECOM shown as Sashimi plots in fig. S4E).We further mined the E2F1 RIP-seq dataset to identify peak sequencing reads that span exon junctions across the different RNAs, which were then related to genomic organization of the parent gene, enabling us to identify spliced RNA variants. We identified a subgroup of the 384 RNA species where the sequencing reads spanned 27 exon junctions, which correspond to 26 different transcripts derived from 18 genes (table S6). For example, multiple alternatively spliced transcripts derived from SENP7 and MECOM as the representative examples and characterized them in greater detail. SENP7 is a deSUMOylase that is involved with the control of protein stability, chromatin, and transcription  in cells (U2OS and HCT116) treated with EPZ015666  and identified an intronic E2F DNA binding site motif within 450 base pairs (bp) of the transcription start site, after the first exon  (https://cancergenome.nih.gov/) and thereafter whether there was any correlation with E2F1 and PRMT5 expression. The MECOM V7 transcript variant was present at increased levels in cervical, colon, and ovarian cancer compared to the normal tissue control where, importantly, its level coincided with the expression of E2F1 and PRMT5 (fig. S5). These results highlight the role of PRMT5 and p100/TSN-E2F1 in regulating alternative splicing of SENP7 and MECOM RNA and further suggest that it is relevant to clinical disease.We performed a similar analysis of  variant . We subs variant . By ChIP variant , and althibition . Most siSENP7 as previous studies had highlighted the role of SENP7 deSUMOylase in the control of HP1, an established repressor of E2F transcriptional activity  found that it was an efficient regulator of E2F target gene transcription, most likely through altering the repressive effect of HP1α on E2F target gene activity. It appears, therefore, that the ability of E2F1 to affect alternative RNA splicing has significant functional consequences on E2F1 activity.The analysis of alternative RNA splicing of the In conclusion, our study has revealed an unexpected mechanism whereby arginine methylation widens the regulatory impact of E2F1, from its classical mechanism of transcriptional control to one where alternative RNA splicing is the predominate level of regulation. The reader-writer interplay, which is dependent on the meR mark, endows E2F1 with a new regulatory RNA splicing mechanism that extends its genomic influence. The meR mark thus expands the repertoire of genomic landscape under E2F control.Hemagglutinin (HA)–tagged WT, the arginine to lysine 111/113 mutant E2F1 (KK), and the arginine to lysine 109 (R109K) constructs have been described previously , 150 mM NaCl, 1% (w/v) Igepal CA-630, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.2 mM sodium orthovanadate, and protease inhibitor cocktails] and incubated on ice for 30 min before SDS–polyacrylamide gel electrophoresis. The following antibodies were used in immunoblots: p100/TSN , E2F1 , E2F1 , HA , FLAG , β-actin , H4R3me2s , histone H4 , and SENP7 .20 (Invitrogen) was added. For splice variant analysis, RNA was deoxyribonuclease (DNase)–treated (Sigma-Aldrich) before cDNA synthesis using random hexamers (Invitrogen). Moloney Murine Leukemia virus (M-MLV) reverse transcriptase (Promega) was used as per the manufacturer’s instructions. Quantitative reverse transcription PCR (qRT-PCR) was carried out in triplicate using the indicated primer pairs and the Brilliant III SYBR Green qPCR Master Mix (Stratagene) on an MX3005P (Agilent) qPCR instrument. Results were expressed as average (mean) fold change compared to control treatments using the ΔΔCt method from three biological repeat samples. Glyceraldehyde-phosphate dehydrogenase or actin primer sets were used as an internal calibrator. Error bars represent SE unless otherwise indicated.RNA was isolated from cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. One microgram of total RNA was used for complementary DNA (cDNA) synthesis. For standard mRNA analysis, oligo(dT)Ct method from triplicate biological repeat samples. Alternatively, a standard curve was generated to calculate ChIP/input signals that were subsequently used to generate fold change values compared to IgG control. Error bars represent SE unless otherwise indicated.ChIP was performed as described previously . RIP lysis buffer  was added directly to the plate, on ice. The lysate was agitated at 4°C for 10 min before sample clarification at 13,000 rpm. For protein samples, 5% of inputs were taken and boiled in an SDS-loading buffer. For RNA samples, 10% of inputs were taken, and 10 μg of proteinase K was added for 30 min at 37°C before addition of TRIzol and RNA isolation. The rest of the lysate was precleared using preblocked protein A/G agarose beads, 1 μg of nonspecific IgG (Jackson ImmunoResearch), and heparin (0.1 mg/ml) for 1 hour at 4°C. The precleared lysate was added to a fresh tube with 1 μg of nonspecific IgG or a specific antibody  for 1 hour with rotation. Protein A/G beads were then added for a further hour. The beads were washed four times in RIP lysis buffer and resuspended in 400 μl of RIP lysis buffer. This was separated into two fractions—one for protein isolation and the other for RNA extraction. For protein isolation, beads were dried and resuspended in SDS-loading buffer before boiling. For RNA extraction, an equal amount of RIP extraction buffer  was added to the fraction, along with 15 μg of proteinase K, and incubated at 37°C for 30 min before RNA purification using TRIzol. RNA was DNase-treated before first-strand cDNA synthesis using random hexamers and M-MLV reverse transcriptase.Cells were washed once with phosphate-buffered saline (PBS) before ultraviolet cross-linking at 900 mJ/cmWT, E2F1-KK, or E2F1-R109K expression was induced in U2OS-Tet-ON cells for 24 hours before isolating the RNA using TRIzol. mRNA was subsequently enriched from three biological replicates using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), as per the manufacturer’s instructions. cDNA libraries were made using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs). Sequencing was carried out on an Illumina NextSeq platform.www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The trimmed reads were aligned to the human reference genome (build hg19) with STAR aligner v.2.5.1 , was less than 0.01, and the change in expression level was greater than twofold. Differential splicing analysis, Ψ calculation, and splicing events statistics were performed with rMATS turbo package v4.0.1 , and annotations were provided in org.Hs.eg.db (v.3.5.0) and GO.db (v.3.5) packages. Gene expression data have been deposited in the National Center for Biotechnology Information’s (NCBI) Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE111961.FASTQ files for pTRE, WT, KK, and R109K samples in three biological replicates were trimmed to remove adapters and low-quality bases with TrimGalore v.0.4.3 . Libraries were prepared using a NEBNext Ultra Directional RNA library Prep kit for Illumina (New England Biolabs). The library was sequenced on an Illumina NextSeq, and bioinformatics analysis was carried out (see below).P value threshold < 0.05) above the siE2F1 control RIP are recorded in table S4. RIP sequencing data have been deposited in NCBI’s GEO and are accessible through GEO Series accession number GSE111961.FASTQ files for two biological replicates in each condition were trimmed as described above. The reads were aligned to the human genome build hg19 by gsnap aligner v.2017-04-21 with two mismatches allowed  using the Mean method (https://xena.ucsc.edu/). The TCGA TARGET GTEx dataset was selected, which contained transcript expression data from TCGA (cancer tissue) and Genotype-Tissue Expression  samples. Cervical, colon, and ovarian cancers were selected alongside their respective healthy tissue and were categorized according to their E2F1 gene expression. Information on PRMT5 and MECOM gene expression was also displayed. MECOM V7 transcript was identified using the Ensembl transcript ID.For the analysis of E2F1, PRMT5, MECOM V7, and total MECOM expression levels in human cancers, Xena browser  was used  were plated on coverslips and transfected for 48 hours with the indicated plasmids, or U2OS-Tet-ON cells were induced to express WT E2F1, E2F1-KK, or E2F1-R109K for 24 hours as appropriate. Cells were fixed for 15 min with 4% paraformaldehyde in PBS and permeabilized for 15 min with 0.5% Triton X-100 in PBS. Coverslips were incubated with primary antibody for 1 hour, washed five times, and then incubated with Alexa Fluor 488–conjugated secondary antibody  for 1 hour. Coverslips were washed again before mounting on glass slides using VECTASHIELD mounting medium with DAPI . Proteins were visualized on a BX60 fluorescence microscope (Olympus) fitted with a Hamamatsu C4742-95 camera and analyzed with Openlab 5 software (Improvision).WT, E2F1-KK, or E2F1-R109K mutant U2OS-Tet-ON cells were induced with doxycycline for 24 hours before addition of fresh medium containing 20 μM etoposide and doxycycline for 48 hours. Then, cells were fixed and stained with propidium iodide for cell cycle analysis, as described previously , 2 mM MgCl2, 100 mM β-mercaptoethanol, and ortho-nitrophenyl-galactosidase (1.33 mg/ml)] and incubated at 37°C before absorbance monitoring (415 nm) on a Sunrise microplate reader (Tecan). Reporter activity was determined from triplicate technical repeats as luciferase/β-galactosidase reading and expressed as fold induction compared to empty vector–expressing cells. Average (mean) fold changes with SE from two biological repeat experiments are shown.U2OS cells were transfected with 500 ng of p73-luciferase or CDC6-luciferase plasmids, along with 500 ng of β-galactosidase and 2 μg of p3xFlag-CMV SENP7 V5 or empty vector for 48 hours. Cell extracts were then prepared in Reporter Lysis Buffer (Promega) and combined with luciferase reagent (Promega) for signal detection on a Microlumat Plus LB 96 V luminometer (Berthold Technologies). Alternatively, extract was mixed with β-galactosidase buffer [200 mM Nat test with Excel software (Microsoft). Data are shown as means with SE displayed. P values are indicated as *P < 0.05 or **P < 0.005.Statistical analyses were performed using two-tailed, unpaired Student’s


RCC1 genes within the same group shared similar exon/intron patterns and conserved motifs in their encoded proteins. Most genes of the RCC1 family are expressed differently under various hormone treatments and are negatively controlled by salt stress. Gh_A05G3028 and Gh_D10G2310, which encode two proteins located in the nucleus, were strongly induced under salt treatment, while mutants of their homoeologous gene (UVR8) in Arabidopsis and VIGS (virus induced gene silencing) lines of the two genes above in G. hirsutum exhibited a salt-sensitive phenotype indicating their potential role in salt resistance in cotton. These results provide valuable reference data for further study of RCC1 genes in cotton.The regulator of chromosome condensation 1 (RCC1) is the nucleotide exchange factor for a GTPase called the Ras-related nuclear protein, and it is important for nucleo-plasmic transport, mitosis, nuclear membrane assembly, and control of chromatin agglutination during the S phase of mitosis in animals. In plants, RCC1 molecules act mainly as regulating factors for a series of downstream genes during biological processes such as the ultraviolet-B radiation (UV-B) response and cold tolerance. In this study, 56 genes were identified in upland cotton by searching the associated reference genomes. The genes were found to be unevenly distributed on 26 chromosomes, except A06, A12, D03, and D12. Phylogenetic analysis by maximum-likelihood revealed that the genes were divided into five subgroups. The  Gossypium spp.) is an important economic crop worldwide. The demand for cotton production in China is high and the arable land area available to these crops decreases each year. Due to insufficient farm land for food crops, cotton is most often planted on saline-alkali and dry land. Abiotic stress, especially high salinity, restricts the productivity and quality of cotton. Although cotton is moderately salt-tolerant, seedlings are severely inhibited under high salt stress and the yield is seriously affected [Cotton , damage to membrane systems, and ultimately, apoptosis [High concentrations of Kpoptosis ,5,6. Celpoptosis .Plants harbor a series of mechanisms to cope with adverse effects under salt stress, such as osmotic adjustment, osmo-protection, sodium exclusion, sodium sequestration, and potassium retention ,6. Solubregulator of the chromosome condensation 1 (RCC1) gene family is important to the cell cycle. Although these genes have been studied extensively in mammals, no studies in cotton have yet been reported. The RCC1 is the nucleotide exchange factor for a GTPase called the Ras-related nuclear protein (Ran). The RCC1 was first discovered in 1987 and is considered to be a regulator of chromosome concentration. It encodes the 45 kDa nuclear protein whose sequence is highly conserved among all eukaryotes and consists of seven beta helices, also known as seven RCC1 repeat units [RCC1 is associated with cancer. For example, RCC1 expression is inhibited in gastric cancer cells [The at units ,11. Due er cells .Arabidopsis [Arabidopsis has made some progress. Normally, UVR8 is evenly distributed in the cytoplasm and nucleus; however, under UV-B treatment, it tends to accumulate in the nucleus through interaction with constitutive photomorphogenic 1 (COP1), thus triggering a UV-B cascade [In plants, the first RCC1 family protein to be identified was UVR8 (UVB-resistance protein 8) in bidopsis . UVB-res cascade ,16,17,18Arabidopsis also belongs to the RCC1 protein family and is involved in the splicing of nad2 messenger RNA in mitochondria and the formation of nicotinamide adenine dinucleotide (NADH) complexes. RCC1/UVR8/GEF-like3 is a member of the RCC1 family of seven basic structural units. Unlike UVR8, RUG3 localizes in mitochondria and has no homologous genes in aquatic plants, animals, or fungi. The RUG3 is a terrestrial plant-specific RCC1 structural protein [PRAF (PH (pleckstrin homology domain), RCC1 and FYVE  zinc-finger domain)) gene family is a subfamily of the plant RCC1 gene family. It is also a unique gene family in plants containing special domains and specific sequences [The mitochondrial protein RUG3 (RCC1/UVR8/GEF-like3) in  protein . Toleran protein . The PRAequences . The MtZequences . The expequences .RCC1 gene family in four species of cotton, we identified 174 RCC1 genes from the cotton genome [G. arboretum; 29 from G. raimondi; 59 from G. barbadense; and 56 from G. hirsutum. Phylogenetic analysis was carried out for the four species and the genetic structure and conserved motifs of G. hirsutum genes were studied. Tissue specificity was analyzed by real-time quantitative reverse transcription (qRT) PCR and the expression of 56 GhRCC1 genes under abiotic and hormone stress was investigated.To fully understand the possible function and evolution of the n genome , includiRCC1 genes in G. arboreum, G. raimondii, G. barbadense, and G. hirsutum, HMMER and BLAST searches were performed using 25 RCC1 genes from Arabidopsis as the query. A total of 174 RCC1 gene sequences were identified, of which 30 were G. arboreum sequences, 29 were G. raimondii sequences, 59 were G. barbadense sequences, and 56 were G. hirsutum sequences, and the number of G. hirsutum is almost the accumulation of G. arboreum and G. raimondii, and the same is true for G. barbadense  and the highest number were in Group V (69 genes) . Each subgroup includes the RCC1 family genes of 9 genes) B.Gh_D09G1044, Gh_A09G1023, and Gh_D08G2751 all have nine exons, and the fragment length of the sixth exon is the longest. Gh_A13G1460 has only three exons. Gh_A07G0164, Gh_D07G0221, Gh_D11G1503, and Gh_A11G1355 all contain four exons, but exon length is clearly divided into two groups.The analysis of the gene structure including intron–exon size and number indicated that different members of the RCC1 family  contain Gh_A08G0100, Gh_D08G2751, Gh_A09G1023, Gh_D09G1044, Gh_D10G1907, Gh_A10G1652, Gh_A13G0645, Gh_D13G0764, Gh_A03G0868, Gh_D02G1249, Gh_D11G0331, and Gh_A11G0277). These genes all contain the same motif , whereas Gh_D09G0059, Gh_A09G0062, Gh_D10G1050, and Gh_A10G0603 contain the same motif , which proves that the RCC1 gene family sequence is highly conserved. Although the motifs of closely related genes are similar, the size of the gene fragments varies widely , such that the Gh_A08G0100 gene fragment is much smaller than Gh_D08G2751.To further investigate the conservation of motifs in RCC1 in upland cotton, protein sequences of 56 GhRCC1 genes were identified and 20 conserved motifs were identified . CloselyGh_D08G2751 and Gh_A11G3194 were not mapped to chromosomes but to scaffolds. The largest number of genes were located on chromosome D11 (seven genes), followed by chromosome A11 (six genes), chromosome D05 (five genes), and chromosome A09 (four genes), while A06, A12, D03, and D12 contained none of the genes. Other chromosomes contained between one and three of the genes.Based on the complete genome sequence of upland cotton, we used the MG2C website to map all 56 genes to chromosomes . Among tGhRCC1 genes, qRT-PCR was used to determine the spatial specificity expression pattern of 56 GhRCC1 genes in fourteen cotton organs. As indicated in GhRCC1 genes were differently expressed in the fourteen tissues tested, while other GhRCC1 genes showed similar expression patterns in diverse tissues, which may manifest from functional differentiation of GhRCC1 genes during plant development. For example, Gh_D01G0233, Gh_D13G0764, and Gh_D07G0221 were preferentially expressed in terminal buds, implying that these genes may play regulatory roles at flower bud differentiation stages, while Gh_D13G1736, Gh_A04G0246, Gh_A11G1355, and Gh_D08G0551 were highly expressed in 0 DPA tissue, so these genes may be involved in fiber initiation. In general, the GhRCC1 genes that are highly expressed in specific tissues may be involved in the regulation of plant development. Gh_D11G2263, Gh_A11G0277, and Gh_A11G2084, for instance, were highly expressed in the ovule 30 DPA, suggesting that they might be related to the elongation of the developing fibers in the seed coat. Conversely, some genes are not expressed specifically. Furthermore, Gh_A05G3028 and Gh_A10G0603 were relatively highly expressed at each stage, suggesting that they may be involved in the development of every tissue.To explore the biological function of Phytohormones play a critical role in various biological processes throughout all the stages of vegetative growth and reproductive growth. Research focusing on the development of cotton fiber has revealed the important roles of auxin, gibberellic acid, and ethylene in fiber initiation and elongation ,29,30,31GhRCC1 genes responsive to auxin, gibberellic acid, and ethylene, we analyzed the expression profiles under IAA (indole-3-acetic acid), GA (gibberellic acid) and ACC (1-aminocyclopropane-1-carboxylic acid) treatment , a precursor of ethylene, was used for ethylene due to its relative stability. To identify the reatment . Under tGh_A05G3028, Gh_D10G2310, Gh_A10G2003, Gh_D04G0616, Gh_D13G1736, Gh_A11G2084, Gh_A07G0164, Gh_D11G2399, Gh_A03G1146, Gh_A10G0603, Gh_D05G2447, Gh_A02G0670, and Gh_D02G0718 were increased in both shoots and roots; Gh_A09G2009, Gh_D09G2222, Gh_A13G0645, and Gh_D13G0764 were downregulated in both shoots and roots; and Gh_D11G2263, Gh_A09G0062, and Gh_D10G1050 were upregulated in shoots and downregulated in roots.To probe their potential roles in the cotton response to various abiotic stresses, expression levels of GhRCC1 genes under 200 mM NaCl and 17% PEG6000 stress treatments were determined by qRT-PCR . Under tGh_A05G3028 and Gh_D10G2310 were dramatically upregulated under salt treatment, mutants of their homologous genes in Arabidopsis were used to determine their role in the control of salt resistance of cotton. Although the amino acid sequences of Gh_A05G3028 and Gh_D10G2310 are best matched to TCF1 in Arabidopsis, TCF1 was minimally expressed unless under low temperatures. Thus, T-DNA mutants of another homologous gene, UVR8, were identified  system. Seven days after infection, TRV2: GhCLA cotton exhibited an albino phenotype (Gh_A05G3028 and Gh_D10G2310 in negative control (TRV1 + TRV2) and their VIGS lines (TRV1 + TRV2: Gh_A05G3028 and TRV1 + TRV2: Gh_A05G3028) were detected by RT-qPCR. As the results showed, the expression level of the two genes in VIGS interfering lines was sharply decreasing in comparison to the negative control  Heynh Col-0 (wild type), uvr8-1 (SALK_072594C), and uvr8-2 (SALK_072594C) from the Nottingham Arabidopsis Stock Centre (NASC) (http://arabidopsis.info/BasicForm) were germinated and grown on 1/2 MS medium (pH 5.8) for 7 days under the following conditions: 12,000 Lx light for 16 h at 25 °C/dark 8 h at 23 °C, 80% humidity, and then transferred into 1/2 MS medium and 1/2 MS medium containing 150 μm NaCl, pH 5.8 under the same condition for 3 days. Leaves of Arabidopsis thaliana above were collected and used for determination of chlorophyll content.Seeds of TM-1  were germinated and cultivated under the conditions above in wet vermiculite, and then soaked in 1/2 MS liquid medium containing 50 μm indole-3-acetic acid (IAA), 5 μm gibberellic acid (GA), and 50 μm aminocyclopropane-1-carboxylic acid (ACC) for 3 h, respectively. For salt and PEG6000 treatment, ten-day-old seedlings of TM-1 were soaked in 1/2 MS liquid medium with 200 μm NaCl for 3, 6, and 12 h; and 17% PEG6000 for 3 h. The TM-1 soaked in normal 1/2 Murashige and Skoog (MS) liquid medium was used as the control. Samples of 7-, 14-, and 30-day-old seedling shoots and roots, 60-day-old leaves, stems, and buds, 0, 10, and 30 DPA (day post-anthesis) ovules, and 30 DPA fibers were harvested from the TM-1 growth in soil at an average temperature of 30 °C.For phytohormone treatment, seeds of Gh_A05G3028 and Gh_D10G231 and their potential downstream genes under salt treatment, seeds of TM-1 were germinated in soil under the conditions: 12,000 Lx light 12 h at 23 °C/dark 12 h at 23 °C, 80% humidity for 14 days. Then part of the plants were watered with 1/2 MS nutrient solution as the control and the others were watered with 200 mM salt solution regularly every 3 days until the phenotypes appeared. Cotton leaves of TM-1 under control and salt treatment were collected for fluorescence quantitative PCR at 0, 1, and 3 h after treatment, respectively.To further confirm the expression pattern of Gossypium spp., Arabidopsis RCC1 sequences were obtained from the TAIR database (http://www.arabidopsis.org) [Gossypium spp. genome database (https://www.cottongen.org) [GhRCC1 gene are listed in To identify members of the RCC1 gene family in sis.org)  and usedgen.org) . Genes wArabidopsis RCC1 genes were extracted using Clustal Omega  [http://itol.embl.de/) [Arabidopsis, G. raimondii, G. arboreum, G. barbadense, and G. hirsutum were used. The neighbor-joining method with amino acid p-distance was applied to construct the tree and reliability was obtained by bootstrapping with 1000 replicates.The amino acid sequences of the cotton and ustalo/)  for multmbl.de/) , RCC1 prhttps://www.ebi.ac.uk/Tools/msa/clustalo/).To better understand the evolutionary relationships between different members of the RCC1 gene family, the genomic DNA and cDNA sequences of predicted genes were obtained from the four cotton genomes. Multiple sequence alignment of all identified RCC1 proteins was performed using Clustal Omega with default parameters (http://gsds.cbi.pku.edu.cn/) mapped the exon–intron structure diagrams of individual genes in the GhRCC1 family based on the full-length coding sequences and genomes in the cotton genomics database. The exon–intron structure maps of all upland cotton RCC1 genes were drawn using TBtools software  based on the published Upland Cotton Genome Document and the ID of the GhRCC1 gene family.The Gene Structure Display Server (RCC1 were identified by the MEME suite program (http://meme-suite.org/tools/meme). The parameters were set as follows: the maximum number of motifs = 20; the minimum width of the pattern = 8; the maximum width of the pattern = 50. Other conditions were set to default values.Conservative motifs in cotton GhRCC1 genes was derived from the annotation files downloaded from the CottonGen website. The MG2C website was used to distribute the GhRCC1 genes along the chromosome from the top to the bottom (http://mg2c.iask.in/mg2c_v2.0/).The chromosomal location information of all Total RNA of plant materials was extracted from 14 different spatio-temporal organs using the RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich)  according to the manufacturer’s instructions.2O 38 μL, total RNA 8 μL, after mixing, put in a PCR instrument at 68 °C for 10 min, ice bath for 10 min, then add RRI (RNase Inhibitor) 2 μL, 5 × MLV buffer 16 μL, M-MLV 4 μL to the centrifuge tube, mix and mix, and put into the PCR instrument . The qRT-PCR was then performed using a 7300 Real-Time PCR System  according to the supplier’s protocols. Each reaction mixture contained 6 μL DNase/RNase-free water, 11 μL TB Green Real-Time PCR master mix, 2 μL diluted cDNA product from the reverse transcription PCR reaction, and 1 μL gene-specific primer. Three biological replicates were conducted for each tissue and each biological replicate was technically repeated three times. All primers are listed in UBQ. The relative expression levels were calculated using the 2−∆∆Ct method. A heat map for gene expression patterns was generated using OmicShare tools, a free online platform for data analysis (http://www.omicshare.com/tools).The RNA quantity and purity were assessed using a NanoDrop One spectrophotometer . Total RNA was reverse-transcribed by Reverse transcriptase M-MLV . The reaction system and procedures were as follows: Oligo dT 8 μL, dNTPs 4 μL, RNase-Free ddHArabidopsis rosette leaves was extracted using the TPS method. Primers for identification of uvr8-1 (SALK_072594C) and uvr8-2 (SALK_072594C) were designed from the website  and are listed in The DNA of Gh_A05G3028 and Gh_D10G2310, including upstream and downstream adaptors (21 bp matching to flanking sequences of multiple cloning sites in an over-expression vector), were amplified from cDNA of TM-1 and integrated into the linear pCAMBIA1300 vector containing a superfolder green fluorescent protein (sGFP) gene , thus constructing the CaMV35S driving and GENE-sGFP-fused expression vector. Gh_A05G3028 and Gh_D10G2310 fused GFP genes were transiently expressed in epidermal cells of tobacco via Agrobacterium with expressed nuclear localization sequence fused to a reporter gene mCherry as a nuclear marker. The GFP and red fluorescent protein fluorescence in epidermal cells of tobacco was detected and photographed using a fluorescence microscope Imager A2 . The primers Gh_A05G3028-CDS-U/L and Gh_D10G2310-CDS-U/L are listed in Full length open reading frames of http://structuralbiology.cau.edu.cn/gossypium/) was used for co-expression analysis. Meanwhile, the cotton FGD  website was used for gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.To better understand the regulatory mechanisms of RCC1 family genes and salt tolerance, ccNET  was used as a positive control to monitor the silencing efficiency. Then the recombinant plasmids of TRV: Gh_A05G3028, TRV: Gh_D10G2310 vectors, and TRV: GhCLA were transferred into Agrobacterium tumefaciens (GV3101). The seeds of TM-1 were germinated in soil under the conditions: 12,000 Lx light 12 h at 23 °C/dark 12 h at 23 °C, 80% humidity for 7 days. The cotyledons of cotton were injected with an infective solution containing Agrobacterium tumefaciens of pTVR1 and TRV2: GhCLA or pTRV1 and TRV2: target gene. Cotton infected by Agrobacterium tumefaciens was cultured under the conditions above until the white-striped leaf or albino phenotype appeared in the positive control. The negative control (pTRV1 coupled with pTRV2) and experimental group (pTRV1 coupled with TRV: Gh_A05G3028 or TRV: Gh_D10G2310). Some of the plants were watered with 1/2 MS nutrient solution as the control and the others were watered with 200 mM salt solution regularly every 3 days until the phenotypes appeared. The primers for the VIGS assay are listed in The TRV (tobacco rattle virus) constructs f Fradin . The fra−1) was calculated as the following formula, and the content of each pigment (mg·g−1) was calculated according to the calculated concentration:Fifty milligrams leaves of Arabidopsis thaliana or cotton were weighed and added into a 10 mL centrifugal tube, sealed with 2.5 mL 95% ethanol, and extracted at room temperature for 36 h under dark conditions. The extract solution was diluted two times, and then colorimetric analysis was carried out by the SYNERGY H1 microplate reader  at 665 and 649 nm, respectively. The concentration of chlorophyll a and b (mg·LThe content of pigments in leaves (mg/g) = concentration of pigments (m g/L) × total volume of extracts (ml) × dilution multiple/sample quality (g).RCC1 family genes in G. hirsutum were identified and comprehensively analyzed in terms of gene structure, chromosome distribution, conserved domains, phylogenetic relationship and expression patterns in different tissues. Besides, the expression patterns of 56 RCC1 family genes in G. hirsutum under various hormones and abiotic stresses were further analyzed and revealed that Gh_A05G3028 and Gh_D10G2310 were significantly induced by salt. The phenotypic analysis of their homologous mutants in Arabidopsis and VIGS lines in G. hirsutum under salt treatment further proved their crucial role in salt tolerance of cotton. Additionally, the co-expression network analysis of the two genes revealed their possible downstream genes in the regulation of salt tolerance in cotton. The results above provide valuable reference data for further study of RCC1 genes in cotton.In Conclusion, 56