The programmed death- (PD-) 1/PD-1 ligand (PD-L) pathway plays an important role in regulating T cell activation and maintaining peripheral tolerance. Accumulated studies showed that PD-1/PD-L1 pathway was involved in the development of type 1 diabetes (T1DM). Since the genetic background of type 1 diabetes differs greatly among the different population, we aim to investigate the association of genetic polymorphisms in PD-1 and PD-L1 with T1DM susceptibility in Chinese population.  In total, 166 T1DM patients and 100 healthy controls were enrolled into the study. Genomic DNA was extracted from 4 mL peripheral blood samples collected from each subject. Genotyping of 8 selected SNPs of PD-1 and PD-L1 was carried out by the pyrosequencing PSQ 24 System using PyroMark Gold reagents (QIAGEN).  SNP rs4143815 in PD-L1 was significantly associated with T1DM. People carrying the C allele of rs4143815 suffering less risk of T1DM and T1DM patients with G/G genotype showed higher levels of autoantibody (AAB) positive incidence compared with C allele carriers. No significant associations were found in other SNPs.  Our results indicate that rs4143815 of PD-L1 is significantly associated with T1DM and may serve as a new biomarker to predict the T1DM susceptibility. The increased prevalence of diabetes mellitus is considered one of the greatest public health challenges nowadays. Type 1 diabetes mellitus (T1DM), a polygenic autoimmune disease, is resulted from both genetic and environmental factors [Programmed cell death 1 (PD-1) is an immunoinhibitory factor belonging to the CD28/B7 family. It plays a vital role in regulating T cell activation and maintaining peripheral tolerance as a core costimulatory molecule . RecentlIncreasing studies had been committed to the association with PD-1/PD-L1 and autoimmune disease, including systemic lupus erythematosus, ankylosing spondylitis, allergic bronchial asthma, and autoimmune diabetes –17. The However, there is only a few studies focus on the associations between PD-1/PD-L1 gene polymorphisms and T1DM in Chinese population. Therefore, in the present study we investigated 8 selected SNPs of PD-1 and PD-L1 to ascertain whether PD-1 and PD-L1 SNPs influence the T1DM susceptibility.A total of 166 T1DM patients and 100 healthy controls were recruited for the study . Blood sEight SNPs in 2 genes (PD-1 and PD-L1) were selected from the National Center for Biotechnology Information (NCBI) database . All of 4 mL peripheral blood samples was collected in EDTA anticoagulant tubes for each patient. Genomic DNA was extracted using Genomic DNA Purification Kit according to the standard protocols. Genotyping of each polymorphism in the PD-1 and PD-L1 genes was carried out by the pyrosequencing PSQ 24 System using PyroMark Gold reagents (QIAGEN). Primers were designed using Pyrosequencing™ Assay Design Software and Primer Premier 5. The genomic region of interest was amplified by polymerase chain reaction (PCR) using one regular primer and one biotinylated primer; PCR products were sequenced using sequencing primer on PSQ 24 System. All the primers used in the present study were shown in P = 0.05.The chi-squared test was used to evaluate the Hardy-Weinberg equilibrium for allele frequencies of all SNPs. Unconditional logistic regression was conducted to calculate the adjusted odds ratio (OR) and 95% confidence intervals (95% CI). The associations between genotypes and T1DM susceptibility were tested using logistic regression analysis with Plink under additive, dominant, and recessive models. The test of significance was set at two-tailed http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi), which is a recognized software providing structure prediction service [The secondary structure of the mRNA was simulated using online webserver RNAfold ( service .P > 0.05)  age of 34.1 ± 15.9 at the time of recruitment. 8 SNPs were genotyped in these subjects, only five of which conformed to Hardy-Weinberg equilibrium , dominant model , and the recessive model . No significant association was observed between other SNPs and T1DM susceptibility. Considering multiple-testing correction, we calculated the P value for the SNPs by false detection rate (FDR) for correction. According to these results, rs4143815 remained significant in the additive model (P = 0.045).There was a significant association between the PD-L1 SNP rs4143815 and T1DM susceptibility in the additive model .In an exploratory analysis, clinical characteristics related to T1DM were compared among different genotypes of PD-L1 rs4143815 shown in . Age, geWe also simulated the secondary structure of partial PD-L1 3′UTR mRNA with different allele carrier using RNAfold . DiffereP = 0.045). No significant associations were observed with other SNPs. Furthermore, T1DM patients homozygous for the rs4143815 G/G genotype showed higher levels of AAB positive incidence compared with C allele carriers (P = 0.033).For the past few years, increasing studies had been committed to the associations with gene polymorphisms and T1DM susceptibility. The present study was made to investigate whether genetic polymorphisms in immune checkpoints could predict the onset of T1DM. In total, 266 subjects were included in the present study. With 8 selected SNPs genotyped, PD-L1 SNP rs4143815 remained significantly associated with T1DM susceptibility after FDR correction (additive model: PD-1 is a core costimulatory molecule expressed by T cells that interact with its ligands PD-L1 and PD-L2 on antigen-presenting cells. PD-L1 was shown to be overexpressed in human gastrointestinal tumors . PD-L1 nIncreasing studies had been committed to the association with PD-1/PD-L1 and type 1 diabetes; low serum level of PD-1 and PD-L1 was reported being associated with T1DM in Japanese and Chilean patients , 21. AssIn the present study, rs4143815 was found being associated with T1DM susceptibility. People carrying the C allele of rs4143815 suffering less risk of T1DM and T1DM patients with G/G genotype showed higher levels of AAB positive incidence compared with C allele carriers. Since the level of AABs has high associations of T1DM, the results of AABs reinforce the importance of rs4143815. People carrying the C allele of rs4143815 suffer lower AAB level and less risk of T1DM. However, HbA1c did not vary considerably among genotypes of the rs4143815 SNP. Since HbA1c is an important indicator for glycemic control and has little relevance to the susceptibility of the disease, this may be induced by the different treatment stage of T1DM patients.It had been reported that rs4143815 was associated with the increased risk of gastric cancer , 27 and The rs4143815 is located in the 3′UTR of PD-L1 and binds to miR-570 . DiffereSeveral studies had been committed to the association with PD-1 and autoimmune disease. PD 1.2, PD1.3, PD1.5, and PD1.9 were found being associated with systemic lupus erythematosus, ankylosing spondylitis, and allergic bronchial asthma –17. HoweOur study has some limitations. With limited SNPs and small sample size, the final result may have a certain deviation. We will select more typical SNPs and increase the sample size in the next step, and we will use gene chip and conduct multicenter studies to revalidate our results in the future.In this study, our results indicate that rs4143815 of PD-L1 is significantly associated with T1DM susceptibility. Importantly, this SNP remained significantly after FDR correction. Furthermore, the presence of AABs is higher in T1DM patients homozygous for the rs4143815 G/G genotype compared with C allele carriers.


Flaviviridae family and causes severe human neuroinfections. In this study, protective efficacy of the chimeric antibody chFVN145 was examined in mice infected with strains belonging to the Far-Eastern, European, and Siberian subtypes of TBEV, and the antibody showed clear therapeutic efficacy when it was administered once one, two, or three days after infection. The efficacy was independent of the TBEV strain used to infect the mice; however, the survival rate of the mice was dependent on the dose of TBEV and of the antibody. No enhancement of TBEV infection was observed when the mice were treated with non-protective doses of chFVN145. Using a panel of recombinant fragments of the TBEV glycoprotein E, the neutralizing epitope for chFVN145 was localized in domain III of the TBEV glycoprotein E, in a region between amino acid residues 301 and 359. In addition, three potential sites responsible for binding with chFVN145 were determined using peptide phage display libraries, and 3D modeling demonstrated that the sites do not contact the fusion loop and, hence, their binding with chFVN145 does not result in increased attachment of TBEV to target cells.Tick-borne encephalitis virus (TBEV) is the most important tick-transmitted pathogen. It belongs to the  Flaviviridae family, is a causative agent of one of the most severe human neuroinfections [Ixodes ticks that inhabit the forested areas of Eurasia from Western Europe to the Far East and from the Scandinavian peninsula to the Mediterranean [Tick-borne encephalitis virus (TBEV), a positive-sense single-stranded RNA virus from the fections –3. TBEV erranean . Three merranean –7. TBEV-erranean ,8–10. Tierranean . Althougerranean ,12. Seveerranean –15.In most endemic countries, there are no specific preparations for the treatment of TBE. Previously, specific anti-TBE serum immune globulin Encegam (FSME-Bulin) was used in European countries; however, the use of Encegam was suspended due to concerns regarding possible enhancement of TBE after its administration. In Russia, where the most severe and lethal cases of TBE have been recorded, specific anti-TBE serum immunoglobulin (anti-TBE-Ig) is applied for post-exposure prophylaxis and treatment of TBE ,16. Antiin vitro and one of them protected mice that were infected with TBEV-Eu [Previously, several anti-TBEV chimeric antibodies were constructed ,19 based TBEV-Eu . In thishttp://www.ckp-rf.ru, Irkutsk, Russia); the TBEV strain Absettarov (TBEV-Eu) was obtained from the repository at the Tomsk branch of the Federal State Unitary Company “Microgen” . All experiments with live TBEV were conducted under BSL-3 conditions.TBEV strains Sofjin and Vasilchenko, prototype Russian strains of TBEV-FE and TBEV-Sib, respectively, were obtained from the repository at the Federal State Public Scientific Institution «Scientific Centre for Family Health and Human Reproduction Problems»  were co-transfected into suspension CHO-S/FRT cells [To develop chFVN145, DNA fragments encoding variable domains of heavy and light chains (VH and VL) of a mouse monoclonal antibody against glycoprotein E of TBEV were amplified by RT-PCR using previously designed primers  and totaeviously  to generRT cells  using PED = kd/ka.The kinetics for chFVN145 binding with TBEV glycoprotein E was determined by a surface plasmon resonance (SPR) method using a ProteOn XPR36 System . Recombinant protein rE  was immoad libitum. Before the experiments, LD50 was determined for each viral stock. Groups of three-week old mice (n = 10 in each group) were infected intraperitoneally with serial ten-fold dilutions of a viral stock in 0.9% NaCl and LD50 for each stock was calculated by the Reed–Muench method [BALB/c mice were purchased from the animal care facility in the Federal State Research Center of Virology and Biotechnology “Vector” . Animals were housed under normal light-dark cycle; water and food were provided h method . Then, v50) was additionally determined in each experiment based on the method described previously [To examine the protective efficacy of antibody preparations, three-week old BALB/c mice (9–10 g) were infected intraperitoneally with TBEV at the appropriate dose in 0.2 mL. One, two, or three days after infection, the mice were treated once intramuscularly with chFVN145, a commercial preparation of human anti-TBE immunoglobulin (anti-TBE-Ig) produced from donor blood , or 0.9% NaCl in a volume of 100 μL. Preparations of chFVN145 and anti-TBE-Ig were diluted in 0.9% NaCl. Each experimental group of mice included 8–10 animals. Mice were monitored at least twice a day for clinical signs of the disease , which resulted in euthanasia using an overdose of isoflurane. The mice were observed for 21 days after infection, and the survival rate was recorded. In addition, the mean survival time (MST) was determined as the period between the infection and animal death; the MST of mice that survived until the end of the experiment was 21 days. To consider the effect of storage and freezing-thawing on the viral stock, actual infectious dose . All experiments with animals were approved by the local bioethics committee of the Federal State Public Scientific Institution «Scientific Centre for Family Health and Human Reproduction Problems».Escherichia coli HB2151 cells.Recombinant variants of TBEV glycoprotein E, rED1+2  and rED3_301  were designed previously . Breifly5′-GCGCCATGGCCGGCGGTGGCTCGGGTCTTACATACACAATGTGCG-3′ and 5′-TTAGCGGCCGCTGTTATCAACATGGCCACGTTCACATCCG-3′ for rED3delA+ and 5′-GCGCCATGGCCGGCGGTGGCTCGATGTTGATAACACCCAACCCC-3′ and 5′-TTAGCGGCCGCTTAGTGATGGTGATGATGATGACTCCCTTTTTGGAACCATTG-3′ for rED3delD+, respectively. The resulting PCR fragments were cleaved with NcoI and NotI and ligated independently into NcoI/NotI-digested pHEN2. E. coli HB2151 cells were transformed with the resulting plasmids, plated onto agar with 50 μg/mL ampicillin and 20 μg/mL isopropyl-β-D-1-thiogalactopyranoside and cultivated. Individual colonies of E. coli producing rED3delA+ and rED3delD+ were screened by PCR using the same primers, and their ability to produce the recombinant proteins was confirmed using 12.5% SDS-PAGE.The recombinant proteins rED3delA+ and rED3delD+, which included fragments of domain III of glycoprotein E , were obtained in this study using the expression vector pHEN2. DNA fragments encoding rED3delA+ and rED3delD+ were amplified by PCR using cDNA of the same strain (Sofjin-Ru) as a template and two pairs of the primers: E. coli cells producing the recombinant proteins rED1+2, rED3_301, rED3delA+, and rED3delD+ were harvested by centrifugation and lysed with a buffer . The lysates were fractioned using 12.5% SDS-PAGE and transferred onto a nitrocellulose membrane . After blocking with 5% skimmed milk in phosphate-buffered saline (PBS) with 0.05% Tween 20 (PBST), the membrane was incubated with 5 μg/mL chFVN145 at 37°C for 1 h. Immune complexes were detected using alkaline phosphatase-conjugated goat anti-human IgG  and visualized by a mixture of nitro blue tetrazolium  and 5-bromo-4-chloro-3-indolylphosphate  for 20 min.11 phage particles from each library were pre-incubated with a non-specific mock mouse antibody and added to the wells of 96-well microtiter plates coated with 200 ng chFVN145 in PBS, pH 7.4, and incubated for one hour at 37°C. Then, unbound phage particles were washed away with PBST, and bound phages were eluted with 100 μg/mL chFVN145. The eluted phages were used for the second round of biopanning, which was carried out using plates coated with 20 ng chFVN145. Phage particles that were eluted after the second round were used to transfect E. coli ER2738 cells. Phages with the selected exposed peptides were isolated from individual plaques and assayed for their binding to chFVN145 by ELISA. For indirect ELISA, the wells of 96-well microtiter plates were coated with 20 ng of chFVN145 or 3% bovine serum albumin in PBS, pH 7.4. After blocking, individual selected phages were diluted in PBST to yield ~1010 colony forming units (CFU) and added to the wells. Indirect ELISA was performed with anti-M13 polyclonal rabbit antibodies followed by alkaline phosphatase-conjugated anti-rabbit IgG (ICN) and stained with p-nitrophenyl phosphate.Selection of specific peptides exposed on the surface of bacteriophages from the phage display libraries PhD-12 and PhD-C7C  was carried out as described previously . Brieflyhttps://www.evanmiller.org/ab-testing/survival-curves.html. P < 0.05 was considered to indicate statistical significance.The data were analyzed using Microsoft Excel software and are expressed as mean values ± standard error of the mean (SEM). Comparisons were performed using the log-rank test. Data were analyzed using the on-line service available at D = (1.5 ± 0.2) × 10−9 M  and the second group at a dose of 10 μg/mouse (low dose), whereas two other groups of animals were treated with anti-TBE-Ig at doses of 100 μg and 10 μg per mouse, respectively. The fifth group of mice was administered 0.9% NaCl, while a group of non-treated mice was used as the control group. Post-exposure administration of chFVN145 at target doses of 100 μg and 10 μg per mouse resulted in 100% (8/8) and 50% (4/8) survival rates, respectively , anti-TBE-Ig at a dose of 100 μg/mouse, or 0.9% NaCl. Clear protection against TBEV infection in the treated mice was observed  one, two, or three days after infection . Administration of 100 μg of chFVN145 showed good therapeutic effect and resulted in 100% (8/8), 85.7% (7/8), and 62.5% (5/8) survival rates in infected mice that received the antibody at days +1, +2, and +3, respectively . Even tr50 TBEV-FE were administered chFVN145 at high and low doses at days +1, +2, and +3. Good therapeutic effect was observed at days +1 and +2 for both high and low doses, and only weak improvement in the survival rate was recorded on day +3 for the high dose , rED3_301 (aa 301–397), rED3delA+ (aa 301–359), and rED3delD+ (aa 356–397), were developed and produced in ED3delD+ .To localize the neutralizing epitope, two random peptide libraries were screened using chFVN145. The PhD-12 library contains phages each with a random 12-mer peptide inserted into the N-terminus of the phage minor protein p3 and exposed on the phage surface. In the PhD-C7C library, an exposed 7-mer random peptide is flanked by cysteine residues, which form a peptide loop. Three different peptides with DIII-like sequences were selected from the PhD-12 library along with six peptides from the PhD-C7C library . All ideIn this study, the therapeutic efficacy of the chimeric antibody chFVN145 was investigated in mice infected with various subtypes of TBEV. In previous experiments, ch14D5a purified from culture medium after transient expression in CHO-K1 cells showed protective efficacy against lethal infection of BALB/c mice with the TBEV strain Absettarov (TBEV-Eu) when ch14D5a was administered one day after infection . A similAs different TBEV subtypes occur in Eurasia, the protective efficacy of chFVN145 against virus strains belonging to TBEV-FE and TBEV-Sib was examined. The results of these experiments were similar to those obtained in mice infected with TBEV-Eu: chFVN145 demonstrated dose-dependent efficacy against both TBEV-FE and TBEV-Sib and was significantly more effective than anti-TBE-Ig. Importantly, despite the different pathogenicity of TBEV-Eu, TBEV-Sib, and TBEV-FE strains for humans, no substantial difference in the protective efficacy of chFVN145 against all three subtypes was observed in the mouse model.50 (TBEV-FE strain), good results were observed in mice that received chFVN145 on days +1 or +2. The survival rate of mice challenged with TBEV-Sib strain at a dose of approximately 4000 LD50 was substantially improved only when mice were treated one day after infection. However, even in the case of such a high infection dose, an improvement in the survival rate or the MST was observed when mice were given chFVN145 at days +2 or +3.Given this, the therapeutic efficacy of chFVN145 was assessed when mice were challenged with variable doses of target TBEV strains. The post-exposure window was expanded and mice were treated one, two, or three days after infection. The results indicated that survival rates were TBEV dose-dependent. When mice were infected at a dose of 20 LD50 (TBEV-Eu strain), the survival rate of mice administered chFVN145 at +1, +2, or +3 days substantially increased compared to that of non-treated mice and mice received 0.9% NaCl. When mice were infected at a dose of approximately 500 LDin vitro and in vivo experiments [The ability of chFVN145 to augment TBEV infection in mice was specifically investigated due to concerns that some flavivirus-specific antibodies can induce antibody-dependent enhancement (ADE) of infection. This phenomenon was repeatedly observed for flaviviruses in eriments –30. Moreeriments –33. ADE eriments ,35. In tRecently, a novel mechanism of antibody-induced enhancement of flavivirus infection, which is based on the antibody-promoted conformational changes in glycoprotein E that result in an increased availability of the usually buried fusion loop (FL), has been described . In thisin vivo efficacy studies using multiple administrations of the antibody are required.In conclusion, chFVN145 demonstrated clear therapeutic efficacy, which was TBEV dose-dependent. ChFVN145 protected mice infected with TBEV-Eu, TBEV-Sib, and TBEV-FE strains. Importantly, administration of chFVN145 did not enhance TBEV infection in the mouse model. The neutralizing epitope was localized in domain III of glycoprotein E; sites responsible for binding with chFVN145 are distant from the FL, and their interaction with chFVN145 would not result in rearrangement of glycoprotein E and exposing the FL, which would lead to enhanced TBEV infection. The obtained results indicate that chFVN145 would be of value in designing potential anti-TBE preparations; however, further


R-gene-mediated resistance that rapidly induces a symptomless resistance phenotype, which is different from the phenotypical R-resistance manifested by the programmed cell death, accumulation of reactive oxygen species, and hypersensitive response. The Rsv3 gene in soybean cultivar L29 is responsible for ER against the avirulent strain G5H of soybean mosaic virus (SMV), but is ineffective against the virulent strain G7H. Rsv3-mediated ER is achieved through the rapid accumulation of callose, which arrests SMV-G5H at the point of infection. Callose accumulation, however, may not be the lone mechanism of this ER. Analyses of RNA-seq data obtained from infected soybean plants revealed a rapid induction of the abscisic acid pathway at 8 h post infection (hpi) in response to G5H but not to G7H, which resulted in the down-regulation of transcripts encoding β-1,3 glucanases that degrade callose in G5H-infected but not G7H-infected plants. In addition, parts of the autophagy and the small interfering (si) RNA pathways were temporally up-regulated at 24 hpi in response to G5H but not in response to G7H. The jasmonic acid (JA) pathway and many WRKY factors were clearly up-regulated only in G7H-infected plants. These results suggest that ER against SMV-G5H is achieved through the quick and temporary induction of ABA, autophagy, and the siRNA pathways, which rapidly eliminate G5H. The results also suggest that suppression of the JA pathway in the case of G5H is important for the Rsv3-mediated ER.Extreme resistance (ER) is a type of  R-gene resistance, whose product can directly or indirectly recognize specific viral effectors  [Plants are equipped with several antiviral defense arrays that provide varying levels of resistance/tolerance depending on the specificity of the infecting virus ,2,3,4. Oroteins) . R-genes. The products of one type inhibit virus infection by targeting one or more stages in the infection cycle. One example of this type is the protein encoded by the tomato gene TM-1, which binds to the replicase of tomato mosaic virus (ToMV) and inhibits its replication [R-gene acts earlier than the first by initiating effector-triggered immunity (ETI) upon the recognition of viral effectors. ETI leads to the suppression of viral replication/spread at the site of infection by inducing programmed cell death (PCD), which is manifested by necrotic lesions that indicate a hypersensitive response (HR). This second type of resistance is positively regulated by the stress hormone salicylic acid (SA) and may also involve the antiviral small interfering (si) RNA pathway [There are two types of lication ,7. The o pathway ,9,10,11.Arabidopsis [R-gene Rsv3 in cultivar L29, indirectly recognizes the cylindrical inclusions (CI) of the virus. The triggered ER is partially regulated by the PP2C3a gene, which controls the rapid accumulation of callose at the points of infection, leading to a swift arrest of GH5 spread [PP2C3a is involved in the abscisic acid (ABA) signaling pathway. However, other reported examples of ER involve the SA pathway, such as the ER against CMV-Y in A. thaliana, or the ER against tomato bushy stunt virus (TBSV) in TM-22-trasgenic tobacco [Extreme resistance (ER) is a unique type of dominant resistance that results in early and rapid arrest of virus replication and spread, but seldom generates defensive symptoms such as PCD, HR, or accumulation of reactive oxygen species (ROS) ,5,12. ERbidopsis . With rebidopsis . ER agai5 spread . This ER tobacco . The ER Although callose accumulation arrests virus spread at the site of infection and is thus important in the ER against SMV-G5H, this mechanism may not explain the entire resistance response, because PCR failed to detect G5H in healthy-looking “G5H-infected” leaves . The latRsv3 resistance gene. We also compared the responses of L29 to SMV-G5H, which fails to infect L29, and SMV-G7H, which rapidly infects L29. This comparison enabled us to identify genes/networks in L29 that are specifically regulated in response to SMV-G5H. As noted in the previous paragraphs, ER has been documented against several viruses in different hosts. A detailed understanding of how ER is regulated, however, is lacking. More specifically, there is little information on the downstream signaling involved in ER. A better understanding of how plants deploy ER will require studies that determine the changes at the transcriptome level at an early stage of infection. In the current study, we used RNA-seq data to analyze defense responses regulated by ER in soybean cultivar L29, which expresses ER against SMV-G5H through the Rsv3) were obtained previously [RNA-seq data from soybean resistance cultivar L29 (carrying eviously . In brieGlycine max) annotation v1.0 on assembly v2.0 was used from the Phytozome v.12.1.5 release of December 2017 https://phytozome.jgi.doe.gov/pz/portal.html# and from EXPath databases [Soybean (atabases . Informap ≤ 0.01) [p > 0.05 were excluded. RNA-seq data were subjected to bioinformatics analyses using EXPath tool in order to obtain lists of deferentially expressed genes (DEGs) (fold-change ≥2 or ≤0.5 and  ≤ 0.01) . DEGs unhttp://bioinformatics.psb.ugent.be/webtools/Venn/). Heatmaps for genes involved in hormone pathways were generated by Heatmapper [Venn diagrams of DEGs were generated using the online tool (atmapper .The first trifoliate leaves of L29 line (age ~ 16 days) were sprayed with ABA (100 µM) or Mock (0.1% MeOH) 24 h prior to SMV-G7H-GFP inoculation, plants received another ABA treatment two days post infection (dpi), and samples were collected 5 dpi for analysis.The two unifoliate leaves from L29 plants were infected with 10 µg/leaf pSMV-G7H-GFP plasmid . A pool Total RNA was extracted using RNA-extraction kit  following manufacturer’s instruction. One µg of total RNA was used for cDNA synthesis using GoScript kit . Real-time quantitative RTq-PCR was carried out with SYBR-Green (Promega) to measure the relative expression of target genes using ΔΔCT method. Actin11 was used as internal control, and primer used in this study were listed in Total protein was extracted from 0.1 g of inoculated leaf pool from three plants as described previously . DetectiThe RNAseq data was first validated by RTqPCR using eight different genes . ExpressAs noted earlier, soybean cultivar L29 exhibits ER against the SMV avirulent strain G5H, but is highly susceptible to the virulent strain G7H. To determine which genes or defense mechanism/pathways are regulated upon infection with the avirulent strain G5H, we generated lists of DEGs by comparing expression in mock-treated, G5H-treated, and G7H-treated plants with the expression in healthy plants using EXPath Tool, a web-based platform that integrates gene expression data with metabolic pathways . DEGs ofGlyma03g28850.1 was temporarily and slightly increased at 24 hpi, but the expression of the other two glucan genes was drastically reduced at 8 hpi in response to G5H infection, and remained relatively down-regulated at 24 and 54 hpi  gene family, which is responsible for the synthesis of the callose deposited at different tissues and organelles [PDCBs) [Gsls and PDBPs were down-regulated at 8 hpi  . In respat 8 hpi C, but set 54 hpi C. Gsl-like-7 genes that were up-regulated in response to G5H at 24 hpi, the expression profiles of Gsl-like-7 genes were generally similar in response to G5H and G7H at 8 and 24 hpi , purple acid phosphatase 12 (PAP12), and glutamine-dependent asparagine synthase 1 (ASN1) were up-regulated by several fold at 24 hpi, and only PAP12 remained up-regulated at 8 to 54 hpi in response to G5H, but not in response to G7H  can increase plant tolerance to viruses by acting upstream of the SA pathway . There wBrassionsteroids (BRs) may also increase plant resistance to viruses in an SA-independent mechanism that is yet to be discovered . In the PP2C3a, which stops G5H spread. [PP2C3a by ABA on L29 leaves was able to reduce G7H levels to ~50%  make up the backbone of the vsiRNA, and several reports have highlighted their antiviral functions [Specific genes in the virus-derived (v)siRNA pathway were clearly induced in response to G5H infection at 24 hpi B. Ortholunctions ,50,51,52unctions ,35. Most of the genes involved in the ABA and siRNA pathways were down- or un-regulated in response to G7H infection B, indicaN-tobacco resistant to TMV, is negative [The JA pathway was reported to have positive roles in compatible interactions against few viruses such potato viruses X and Y ,54, and negative . In the Several autophagy and apoptosis genes were up-regulated in response to G5H infection at 8 hpi A; the reIn summary, Rsv3-mediated ER against SMV-G5H is evidently achieved in three steps: (1) the quick arresting of the virus at the site of infection by enhanced callose deposition; (2) the activation of the antiviral vsiRNA machinery to cleave SMV viral genes; and (3) the induction of autophagy to eliminate remaining viral components from the cell. In the susceptible interaction involving G7H, on the other hand, the virulent strain avoids RSV3-recognition (and thus downstream defenses) as a consequence of changes in a few amino acids in the CI region . This alOur understanding of Rsv3-mediated ER in soybean L29 plants will be increased by the identification of factors that are associated with Rsv3 and that help trigger the ER response.


Drosophila eye development, AJ reorganization includes the formation of isolated N-Cadherin AJ between photoreceptors R3/R4. Little is known about how these N-Cadherin AJ are established and maintained. This study focuses on the kinases Mbt/PAK4 and SRC, both known to alter E-Cadherin AJ across phyla. Drosophila p21-activated kinase Mbt and the non-receptor tyrosine kinases Src64 and Src42 regulate proper N-Cadherin AJ. N-Cadherin AJ elongation depends on SRC kinase activity. Cell culture experiments demonstrate binding of both Drosophila SRC isoforms to N-Cadherin and its subsequent tyrosine phosphorylation. In contrast, Mbt stabilizes but does not bind N-Cadherin in vitro. Mbt is required in R3/R4 for zipping the N-Cadherin AJ between these cells, independent of its kinase activity and Cdc42-binding. The mbt phenotype can be reverted by mutations in Src64 and Src42. Because Mbt neither directly binds to SRC proteins nor has a reproducible influence on their kinase activity, the conclusion is that Mbt and SRC signaling converge on N-Cadherin. N-Cadherin AJ formation during eye development requires a proper balance between the promoting effects of Mbt and the inhibiting influences of SRC kinases.Tissue morphogenesis is accompanied by changes of adherens junctions (AJ). During  Summary: N-Cadherin adherens junction formation in the Drosophila larval eye imaginal disc is controlled by the combined functions of the p21-activated kinase Mbt/PAK4 and the kinases Src64 and Src42. Drosophila compound eye development  allowing dynamic intercellular interactions. In developmental processes, ZA becomes manifold reorganized e.g. during epithelial-to-mesenchymal transition , or durielopment .Adherens junctions (AJ) are a main adhesive complex of the ZA. The central molecules of AJ, the classical cadherins such as E-Cadherin (E-Cad) and N-Cadherin (N-Cad), establish intercellular homophilic interactions. The intracellular domain of Cadherin binds to Catenins, thereby linking the AJ to the actin cytoskeleton . CadheriDrosophila eye imaginal discs are an easily accessible tissue to analyze AJ formation and reorganization necessary to finally establish the highly ordered arrangement of the 800 single eye units (ommatidia) in the adult compound eye. Eye discs are epithelia, from which all cell types of the eye are determined. Briefly, at the beginning of third larval stage, the morphogenetic furrow starts posterior and crosses the epithelium. Anterior to the morphogenetic furrow, cells proliferate and stick together by E-Cad AJ. Cells enter a G1 arrest and become determined posterior to the furrow in a stepwise fashion to build up ommatidia row by row 4, termed Mushroom bodies tiny (Mbt) in osophila . PAKs arosophila . Group Iosophila . Full stosophila . Dependiosophila . PAK4/Mbosophila  and Mbt osophila . PAK4/Mbosophila . One mecosophila . On one osophila . On the osophila . Par3/Baosophila . These oThe expression of N-Cad during eye development raises the question of whether Mbt in addition to E-Cad also influences N-Cad AJ. In human glioma xenograft cells, strong overexpression of PAK4 increases N-Cad levels . InformaBased on a previous report demonstrating the presence of N-Cad AJ at the cell boundary between photoreceptor R3 and R4 , we analFig. S1A). Mbt localization pattern is similar to E-Cad  larvae and compared to wild type and animals expressing a genomic rescue construct in the P1mbt background   of ommatidia with a loss of Mbt only in R2, R5 and/or R8 showed wild-type-like N-Cad AJ (n=29). Importantly, five out of six P1mbt clones only affecting R3/R4 but not R2/R5/R8 had a short N-Cad AJ  A. Postercohesion  and N-Cacohesion A. More atructure A. Phenottructure B. Quantihenotype A,B. Thishenotype . HomozygN-Cad AJ A,B. In cN-Cad AJ C. IntereN-Cad AJ D. As desN-Cad AJ . WhetherN-Cad AJ  and synaN-Cad AJ .Fig. 2.P1mbt eye discs, accumulation of E-Cad at the R2 and R5 AJ was impaired , a phenotype that could be reverted by expression of P[gen-mbt] . Impaired E-Cad accumulation in P1mbt might explain the loss of ommatidial integrity posterior row 10 , which is in line with the observation that E-Cad is not upregulated when N-Cad is absent . Following row 5 ommatidia from apical to more basal sections, Baz staining is first visible mainly in R4, followed by Baz localization at all photoreceptor boundaries and elevated Baz signal at the R3/R4 interface. Baz staining overlaps with N-Cad signals only in more basal sections , showing apical to basal polarity of apical determinants and ZA material. In P1mbt ommatidia, Baz showed weak, more diffuse, and uneven membrane localization . Comparable results were found in P1mbt pupal eye discs . Since the loss of Mbt, specifically in R4, leads to ommatidial rotation defects (Another important AJ modulator is Bazooka (Baz), which was shown in pupal eye discs to link Mbt to E-Cad AJ stability . In larvye discs . Despite defects D and api defects , it remaUAS-mbt transgenes encoding a PBD-defective , a kinase-deficient (MbtK397M) or a wild-type (Mbtwt) variant were expressed in the P1mbt background in photoreceptor R3 and R4 using sev-Gal4. Animals of the genotype P1;sev-Gal4/+mbt served as a control. N-Cad phenotypes were analyzed in row 5 ommatidia and quantified. Transgenic expressed Mbtwt localizes to the cytosol and cell boundaries of the cells . To verify this result a second independent H19,22LUAS-mbt fly line was tested, showing again a N-Cad phenotype between P1mbt and wild type (data not shown).To assess whether membrane localization of Mbt is needed for normal N-Cad AJ, the Cdc42-binding-deficient Mbtwas used . MbtH19,lization A. Besidearrow in A. Surpricomplete A,B. CompThe observation that Mbt staining is minimal at the R3/R4 cell–cell boundary E, but neDrosophila SRC isoforms (Src42 and Src64) are expressed in larval eye discs .A second kinase relevant for the R3/R4 AJ is the tyrosine kinase SRC . Both Drye discs . Studiesye discs  and induye discs . Y654 anye discs . The comDrosophila R3/R4 cell boundary , larvae were grown at 18°C to reduce expression levels. Also at 18°C, expression of Src42CA or Src64 interferes with eye development. However, the majority of row 5 ommatidia were intact but showed significantly truncated N-Cad AJ . Src42 did not exhibit such a strong phenotype at 18°C . Over-expression of Src42 at 25°C resulted in a fewer number of intact ommatidia and again in significantly truncated N-Cad AJ between R3 and R4 . In contrast, expression of Src42KD did not alter the N-Cad AJ , demonstrating the requirement of a functional kinase domain to induce this phenotype. To sum up, SRC proteins impede N-Cad AJ maturation during Drosophila eye development. The mechanism by which Src42 affects N-Cad AJ is most likely kinase dependent, because Src42CA had a much stronger influence compared to Src42, whereas Src42KD had no effect. This is consistent with known SRC functions towards N-Cad in other organisms . This finding argues against a function of SRC proteins upstream of Mbt in this cellular context. To substantiate the genetic interaction between SRC and Mbt, the reverse experiment was performed. Src42 or Src64 were overexpressed with gmr-Gal4 in combination with the P[gen-mbt] transgene. Increased levels of Mbt significantly reverted the N-Cad phenotype caused by Src42 or Src64 overexpression to a more wild-type appearance . The genetic data suggest a function of Drosophila SRC either downstream of Mbt in the same pathway or in a parallel pathway that converges with Mbt signaling on N-Cad.Both loss of Mbt and elevated SRC levels lead to truncated N-Cad AJ. This observation raises the question as to whether PAK4/Mbt and SRC act in concert or independently. To investigate a possible genetic interaction, N-Cad AJ in animals heterozygous for pearance A. Rescuee , raising the question of whether Mbt is able to bind Src64 and Src42. To test this, Myc-tagged Mbt and His-tagged full length Src64 or Src42 were expressed in Drosophila S2 cells and co-immunoprecipitations were performed. No interaction was detected (data not shown). However, binding could be very transient or dependent on unknown signaling events influencing localization or accessibility of interaction domains. It remains to be verified whether PAK4/Mbt binds to full length SRC in any organism.Following the hypothesis that both kinases act in the same pathway, Mbt should somehow act on SRC function. So far it is not clear whether Mbt/PAK4 and SRC undergo a direct protein–protein binding. However, evidence for bi-directional PAK4-SRC signaling comes from studies in vertebrates . SpecifiDrosophila SRC proteins: the N-terminal SRC sequences containing S17 and S12 are highly conserved in vertebrates but absent in Drosophila Src64 and Src42 . To sum up, the relevant protein sequences with respect to the PAK4-SRC signaling in vertebrates are different or absent in Drosophila. Nevertheless, the genetic interaction between mbt and Src64 or Src42 under loss of function and overexpression conditions raises the question of whether Mbt regulates SRC activity. To examine this possibility, SRC activity in lysates from S2 cells was monitored by western blot with a phospho-Y416-Src antibody under normal and Mbt overexpression conditions. No reproducible influence was observed, neither on Src64 nor Src42 . To sum up, a direct molecular link between Mbt and SRC in Drosophila is unlikely.A second hint for direct PAK4-SRC signaling comes from an integrated analysis of phosphoproteomic data and prediction of kinase activities in a mouse skin carcinogenesis model . This stmbt and Src on the one hand and the missing biochemical link between both proteins on the other hand, supported the idea that both kinases act in parallel pathways, which finally converge on N-Cad. To test for a potential effect of Mbt on N-Cad, stably transfected S2 cells expressing the GST-tagged intracellular domain of N-Cad [GST-N-Cad(intra)] alone or in combination with Myc-tagged Mbt were used. Western blot analysis of cell lysates detected two bands for GST-N-Cad(intra), which might be due to post-translational modifications or cleavage. Interestingly, in lysates from cells co-expressing GST-N-Cad(intra) and Mbt the upper band was consistently stronger in seven independent experiments  in S2 cells had no effect on GST-N-Cad(intra) expression levels  whereas SRC does not promote destabilization, although SRC binds and phosphorylates GST-N-Cad(intra). However, SRC's inability to destabilize N-Cad(intra) mbt rescue fly line, a genomic EcoRI fragment of the complete mbt sequence including 1410 bp upstream and 3526 bp downstream of the open reading frame was cloned into pW8 vector. The final vector was injected in 1118w embryos. The insertion was mapped on the third chromosome and crossed into the P1mbt background .To generate a genomic wt was used , 16-2UAS-Src42  for 30 min at room temperature. Eye discs were incubated at 4°C overnight with the following primary antibodies: rat anti-N-Cad ; mouse anti-Armadillo , rat anti-E-Cad , rabbit anti-Baz , rabbit anti-GFP  or rabbit anti-Mbt  supplemented with 10% fetal bovine serum (Biochrom AG), 2 mM L-glutamine (PAA) and 1% penicillin/streptomycin (PAA). Transfections of S2 cells were done with Cellfectin II Reagent (Invitrogen) according to the manufacturer's protocol. For stable transfection pCo-Hygro was added to the transfection mixture and afterwards cells were selected with 200 µg/ml Hygromycin (InvivoGen). Protein expression was induced by addition of 0.7 mM CuSO4 for about 20 h followed by cell lysis.S2 cells were maintained at 25°C in Schneider's g. Cell pellets were resuspended in lysis buffer  supplemented with Complete (Roche) and PhosSTOP (Roche). The lysate was incubated at 4°C for 40 min with gentle shaking, centrifuged for 30 min at 16,000× g and 4°C, and the supernatant was used for further experiments.S2 cells were harvested by centrifugation at 1000× To carry out GST-pulldown assay, lysates were incubated with Glutathione-Agarose (Machery-Nagel) for 4 h at 4°C with gentle shaking. Before elution, beads were washed three times with lysis buffer at 4°C. To elute proteins, beads were boiled in SDS-PAGE sample buffer.Protein samples were separated using 10% SDS polyacrylamide gels and transferred to nitrocellulose, membranes were blocked with 5% dry milk in TBST (0.1% Tween20 in TBS). Primary antibodies were diluted in blocking solution: anti-Myc , anti-His , anti-GST , anti-pTyr . Protein bands were detected with a ChemoCam (Intas) using HRP-coupled secondary antibodies  and ECL Prime Western Blotting Detection Reagent .


Enterovirus A species (EV-A) through a series of intertypic recombinations. Therefore, locally enriched outbreaks of cocirculating viruses from different genotypes/serotypes may facilitate recombination. Most recombination breakpoints we found had nonrandom distributions and were located within the region spanning from the boundary of P1  and P2  to the cis-acting replication element at P2, indicating that specific genome reassembly of structural and nonstructural genes may be subject to natural selection. Through intensive recombination, 11 EV-A71-like signatures  were found to be present in a variety of recently cocirculating EV-A viruses worldwide, suggesting that these viruses may be targets for wide-spectrum antiviral development.Enterovirus A71 (EV-A71) is an important nonpolio enterovirus that causes severe neurological complications. In 1998, Taiwan experienced an EV-A71 outbreak that caused 78 deaths. Since then, periodic epidemics of EV-A71 associated with newly emerging strains have occurred. Several of these strains are known to be recombinant; however, how these strains arose within such a short period of time remains unknown. Here, we sequenced 64 full-length genomes from clinical isolates collected from 2005 to 2016 and incorporated all 91 Taiwanese genomes downloaded from the Virus Pathogen Resource to extensively analyze EV-A71 recombination in Taiwan. We found that the B3 subgenotype was a potential recombinant parent of the EV-A71 C2-like and C4 strains by intratypic recombination. Such B3-similar regions were also found in many cocirculating coxsackieviruses belonging to  Enterovirus A species (EV-A) of Picornaviridae1. Notably, infection with EV-A71 in children under 5 years of age sometimes leads to severe neurological complications  and even death owing to cardiopulmonary failure3. EV-A71 is a nonenveloped small RNA virus with a positive, single-stranded RNA genome1. The viral genome can be directly translated into a polyprotein consisting of the structural (P1) and nonstructural (P2 and P3) regions; this polyprotein is subjected to a series of proteolytic cleavages to generate functional proteins, including structural proteins (VP1–4) functioning in capsid assembly and nonstructural proteins  required for viral replication1. Based on the nucleotide sequence of the VP1 protein, EV-A71 is classified into seven genotypes (A–G)6. Genotypes B and C can be further divided into subgenotypes from B0 to B5 and from C1 to C5, respectively7. Outbreaks caused by these variable genotypes have been reported8.Enterovirus A71 (EV-A71), a member of the nonpolio enterovirus family, belongs to the 8. From the 1970s to the late 1980s, genotype B dominated and led to outbreaks in the USA, Japan, Australia, and Europe. In the late 1980s, the prevalent genotype changed to genotype C, which is currently active outside the Asia-Pacific region. However, after the 1975 Bulgaria and 1978 Hungary outbreaks, no severe outbreaks occurred until 19978; since then, several EV-A71 outbreaks have accompanied fatal hand–foot–mouth disease (HFMD) cases in various countries in the Western Pacific region, and the threat continues8. Thus, EV-A71 has been selected by the World Health Organization as one of the top five viruses in the post-polio eradication era9. These recent outbreaks were associated with newly emerging strains, including the recombinant B3, B4, C2, and C4 subgenotypes8. Notably, recombination is believed to play a more important role than that of mutations in the evolution of EV-A7110. Although clinical trials of the EV-A71 vaccine are currently ongoing11, novel vaccine-resistant strains might appear as a result of recombination. Therefore, characterization of the mechanisms of viral recombination remains essential.In 1969 and the early 1970s, EV-A71 genotype A caused outbreaks in the United States of America (USA). However, genotype A did not recur until 2008, and the intervening worldwide epidemics were found to be caused by other genotypes3; however, the first severe outbreak occurred in 1998, and Taiwan experienced the most severe EV-A71 outbreak on record. In a subsequent outbreak in 2000–2002, 846 severe cases and 129 deaths were reported3. Thus, enterovirus infection has been evaluated as an important infectious disease in Taiwan. From 1989 to 2009, physicians and hospitals used sentinel surveillance systems to monitor highly infectious diseases; now, computerized systems carry this responsibility. Additionally, a laboratory-based virological surveillance system was established in 2000 to focus on influenza virus and enterovirus infections3, and reporting of severe cases is now mandatory in Taiwan.In Taiwan, EV-A71 has become a long-term pathogen and can be traced back to as early as 1980Here, we evaluated the evolution and recombination of different genotypes of EV-A71 and several cocirculating EV-A viruses in Taiwan based on full-genome sequence analyses. Our results provide insights into the crucial role of the similar nonstructural regions via a series of recombination events associated with various serotypes, which may be triggered by the emergence of the temporal EV-A71 B3 strain. These results may facilitate the development of wide-spectrum antivirals against cocirculating EV-A strains.13.We summarized epidemiological reports of enterovirus infections from the Taiwan Centre for Disease Control in Fig. Sixty-four clinical isolates collected from 2005 to 2016 were sequenced to investigate EV-A71 recombination. Yearly counts are presented in Table 13, genotype switching in Taiwan may be related to the frequent transmission of different strains outside the Taiwan region.Interestingly, a periodic switching between the EV-A71 C4 and B5 strains occurred in Taiwan during the years from 2005 to 2016 Table . The det8. Both the C2 and C4 strains are recombinogenic and carry partial genomes derived through intertypic recombination with coxsackievirus (CV)-A8 and CV-A16, respectively14. Furthermore, the B4 strain is an example of intratypic recombination within EV-A71 genotype B13. The emergence of various EV-A71 strains within a decade may be attributable to regionally enriched, large-scale outbreaks that can increase the risk of coinfection, a key requirement for recombination15. To examine this possibility, we evaluated Taiwanese full-genome sequences that consisted of a variety of genotypes/subgenotypes , some differences may still exist between the published full-length genomes and the actual viral population.In the phylogenetic tree constructed via the P1 region and rooted with the oldest strain (CVA2-Fleetwood), distinct clades representative of each genotype/subgenotype were observed, and all sequences of prototype CV appeared as an outgroup Fig. . In contion Fig. . The cluion Fig. . Given tsis Fig. . When cosis Fig. . The shient Fig. . Since t16. Thus, we next examined whether EV-A71 may be involved in the recombination of non-EV-A71 viruses of EV-A. To prevent sampling bias, we collected all of the historical EV-A full-genome sequences worldwide for the following analyses. Since most recombinations in other EV-A viruses also occur outside the P1 region16, we evaluated EV-A71 and CV sequences in the P2/P3 coding region. We utilized Bayesian evolutionary analysis to specify the spatial-temporal relationships among these sequences. When sequences spanning from P2 to the 3′ end of the viral genome were analyzed, several clusters were observed, most of which contained clades corresponding to different serotypes of EV-A and subgenotypes of EV-A71 . The overall identity of amino acid sequences among these viruses still reached 94%19. However, considering the requirement of coinfection for recombination, it is possible to obtain the “non-self” genome from cocirculating viruses15. In addition, as one of the countries having cocirculation of multiple EV-A71 subgenotypes and several EV-A viruses, Taiwan represents a good niche for clarifying the relationships among cocirculating viruses8. In this study, we proposed that the emergence of the EV-A71 C2-like and C4 strains may be explained by intratypic recombination with the B3 strain. Although the presence of the B3 strain in Taiwan has not been previously documented, it was involved in the recombination of the B4 strain, which caused severe outbreaks in the early 2000s13. The intensive recombinations were not EV-A71-specific and could also be found in several currently circulating EV-A viruses through intertypic recombinations. Among these viruses, only CV-A2, CV-A4, and CV-A6 have been documented in Taiwan. However, all these viruses have been reported to be common causes of HFMD in China and are recombinogenic with EV-A7124. Interestingly, a novel EV-A71 genotype C strain with a mosaic genome structure has been identified in Germany and Denmark26. This new strain had a C1-like VP1 region; however, the 5′ untranslated region and the P2/P3 region showed higher similarity to EV-A71 B3/C2-like and C4, respectively. This new EV-A71 strain may have been generated by recombination of the locally circulating C1 strain with the imported C4 strain that became dominant recently27. Although intensive recombination of cocirculating EV-A viruses with EV-A71 has been observed, there are some exceptions. Both CV-A5 and CV-A10 are commonly detected by the Taiwan enterovirus surveillance system; however, no recombination with EV-A71 has been observed. Instead, a close relationship in the nonstructural region, possibly caused by intertypic recombination between circulating CV-A5 and CV-A10, has been reported28. Therefore, recombination between cocirculating viruses may be more common than expected, and full-genome sequencing rather than sequencing of VP1 only should be considered when encountering a new epidemic.Sequences with high similarity to those of the prototype strains CV-A8 and CV-A16 were found in the EV-A71 C2 and B3/C4 strains, respectively29. Because the genome of EV-A can be directly translated into a polyprotein and then undergo proteolytic cleavage, changes in the functional entities by recombination could be deleterious to the virus. For example, viral 3Dpol is required for viral replication and recognizes several cis-elements throughout the genome29. In the case of EV-A71 B3, due to the presence of the CV-A16-like 3Dpol, the virulence was decreased when compared with those of EV-A71 B4 and CV-A16 in mouse model infections21. Therefore, the EV-A71-like signatures that consist of EV-A71 genotype B-like P2 and CV-A16-like P3 regions may have been less favored and discarded in the evolution of EV-A71 genotype B -containing viral cDNA using a ReverTra Ace -α- kit . Overlapping amplicons covering the entire viral genome were amplified by different sets of primers31, and genome assembly was carried out using SeqMan software . The five samples collected after 2014 were sequenced using the Illumina HiSeq platform for next-generation sequencing (NGS). NGS data preprocessing included the removal of low-quality and host reads. Using the Taiwanese B5 and C4 strains as an initial template, the viral genomes were assembled by an iterative mapping approach32. A total of 64 genomes obtained in this study were deposited in GenBank with accession numbers MG756691–MG756754.All of the 64 EV-A71 clinical specimens isolated from 2005 to 2016 were provided by the Linkou Chang Gung Memorial Hospital, Taiwan. Regardless of the illness diagnosed, we randomly picked clinical samples from epidemics in this time span. To prevent contamination, amplified viral stocks from human rhabdomyosarcoma cells were used for full-genome sequencing. Viral genomes were recovered using TRIzol LS reagent  according to the manufacturer’s instructions. The 59 samples collected before 2014 were sequenced by Sanger sequencing. Oligo-(dT)33. To identify genomic signatures associated with detected recombination in this study, a consensus sequence for each of the serotypes/genotypes was generated by using the Cons tool with the default setting from EMBOSS31.Eight hundred thirty-one EV-A71 genomes worldwide were initially retrieved from ViPR in September 2017. Sequences with ambiguous nucleotides or without known sampling dates and countries were removed. To reduce redundancy, we randomly selected 10 sequences with the same genotype and isolation year from each country. We then collected 427 EV-A71 sequences including all Taiwanese strains for analysis  tree under the HKY model. Based on our collected sequences, we generated 50 million Markov chain Monte Carlo (MCMC) chains with 10% burn-in. One MCC tree was constructed for every 25,000 chains, and a single consensus tree was summarized from these MCC trees. MCMC was also used in BEAST to estimate the time of divergence for each alignment.The ML method based on the Hasegawa–Kishino–Yano (HKY) model was performed to infer the evolutionary historySupplementary Table 1Supplementary Table 2


Scientific Reports 10.1038/s41598-019-42529-8, published online 17 April 2019Correction to: The Acknowledgements section in this Article is incomplete.“Authors would like to thank Dr Edorta Santos for assistance on hydrogel degradation conditions, Inés Mármol for enriching discussions on the qPCR experiments and Jacobo Ayensa for valuable advice on statistical analysis. This work has been supported by the Spanish national research program (DPI2015-65401-C3-1-R and BIO2016-79092-R grants) and the Aragon Government (T24_17R grant). The Spanish national government provided M.V. studentship. Authors would like to acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza. ORCHID and CISTEM projects have received funding from H2020 programme under grant agreement 766884 and 778354 respectively.”should read:“Authors would like to thank Dr Edorta Santos for assistance on hydrogel degradation conditions, Inés Mármol for enriching discussions on the qPCR experiments and Jacobo Ayensa for valuable advice on statistical analysis. This work has been supported by the Spanish national research program (DPI2015-65401-C3-1-R and BIO2016-79092-R grants) and the Aragon Government (T24_17R grant). The Spanish national government provided M.V. studentship. Authors would like to acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza. ORCHID and CISTEM projects have received funding from H2020 programme under grant agreement 766884 and 778354 respectively. M.V., D.J.B and M.K.L. acknowledge University of Wisconsin Carbone Cancer Center Support Grant P30 CA014520, Environmental Protection Agency 83573701-Human Models for Analysis of Pathways (H-MAPs) Center and NIH NCI Cancer Moonshot Grant R33CA225281.”


In vivo evidence supports a role for L-selectin in regulating TEM and chemotaxis, but the intracellular mechanism is poorly understood. The ezrin-radixin-moesin (ERM) proteins anchor transmembrane proteins to the cortical actin-based cytoskeleton and additionally act as signaling adaptors. During TEM, the L-selectin tail within transmigrating pseudopods interacts first with ezrin to transduce signals for protrusion, followed by moesin to drive ectodomain shedding of L-selectin to limit protrusion. Collectively, interaction of L-selectin with ezrin and moesin fine-tunes monocyte protrusive behavior in TEM. Using FLIM/FRET approaches, we show that ERM binding is absolutely required for outside-in L-selectin clustering. The cytoplasmic tail of human L-selectin contains two serine (S) residues at positions 364 and 367, and here we show that they play divergent roles in regulating ERM binding. Phospho-S364 blocks direct interaction with ERM, whereas molecular modeling suggests phospho-S367 likely drives desorption of the L-selectin tail from the inner leaflet of the plasma membrane to potentiate ERM binding. Serine-to-alanine mutagenesis of S367, but not S364, significantly reduced monocyte protrusive behavior in TEM under flow conditions. Our data propose a model whereby L-selectin tail desorption from the inner leaflet of the plasma membrane and ERM binding are two separable steps that collectively regulate protrusive behavior in TEM.The migration of circulating leukocytes toward damaged tissue is absolutely fundamental to the inflammatory response, and transendothelial migration (TEM) describes the first cellular barrier that is breached in this process. Human CD14 L-selectin shedding in primary human CD14+ monocytes has been recently shown to be triggered exclusively during TEM, and not before  describes the first physical barrier that is breached in this process . Chemokit before . MoreoveRolling leukocytes sense chemokines deposited on the apical aspect of the endothelium, triggering integrin activation and arrest from flow. Upon firm adhesion, leukocytes spread and polarize to establish front-back polarity on the apical aspect of the endothelium. Luminal crawling describes the coordinated protrusion and retraction behavior of leukocytes, sampling and identifying a suitable site to execute TEM. During TEM, leukocytes will protrude a leading edge, most commonly between inter-endothelial junctions, and organize their movement across inflamed endothelial monolayers to successfully enter the subendothelial space. A large amount of the intracellular molecular mechanisms governing TEM has been defined more in endothelial cells than in leukocytes . To datein vivo  isozymes α, τ and θ  promote phosphorylation of Ser τ and θ , 38. In selectin . Whetherselectin . In “resselectin . As TEM out mice , 41. Whaout mice . It has out mice .in silico simulation models  suggesting an important role for this residue in ERM binding and pseudopod protrusive behavior.Clustering of L-selectin is known to activate numerous effector responses in different immune cell subtypes. To better understand how L-selectin clustering  is regulated during TEM, we engineered the monocyte-like THP-1 cell line to co-express WT or mutant forms of L-selectin that were C-terminally tagged to green or red fluorescent proteins (GFP/RFP). Fluorescence lifetime imaging microscopy (FLIM) was used to quantify Förster resonance energy transfer (FRET) between the GFP and RFP tags, as a direct readout for L-selectin clustering during TEM. As published previously, WT L-selectin reproducibly clustered within transmigrating pseudopods of THP-1 cells captured in mid-TEM. Surprisingly, pharmacologic or genetic blockade of L-selectin shedding completely reversed the distribution of clustered L-selectin to non-transmigrated uropods. Serine-to-alanine mutagenesis of Ser364 and Ser367 within the L-selectin tail contribute to AMC, we mutated them both to alanines (SSAA) in the open reading frames of WT human L-selectin, or a “sheddase-resistant” mutant of L-selectin  led to a modest but significant drop in FRET efficiency compared to WT L-selectin (9.25% [SSAA] and 9.76% [ΔM-NSSAA] vs. 12.39%[WT]). However, no significant difference in the FRET efficiency of ΔM-N and ΔM-NSSAA. An underlying reason for this observation could be that the serine residues in ΔM-N are predominantly dephosphorylated. Indirect assessment of L-selectin serine phosphorylation by phos-tag Western blots revealed that serine phosphorylation was detected only when cells were robustly stimulated with the phorbol ester, PMA  of L-selectin has been shown to drive a multitude of responses in different leukocyte subsets. Examples include: the formation of a supramolecular complex between adjoining L-selectin tails, β1 and β2 integrin activation, chemokine receptor expression from intracellular stores, chemokine responsiveness of T-cells and reactive oxygen species production , 46–48. ter, PMA , which id Ser367 . These rd Ser367 ] is held364 and Ser367 contribute to L-selectin clustering in TEM. Our recent work showed that WT L-selectin clusters exclusively within transmigrated pseudopods of THP-1 cells before it is cleaved  allowed us to interrogate the clustering of this mutant during TEM. FRET/FLIM analysis revealed that the ΔM-NSSDD-GFP/RFP mutant faithfully phenocopied the clustering distribution of ΔM-N L-selectin, strongly suggesting that serine phosphorylation is driving L-selectin clustering in to non-transmigrated uropods. Taken together, the pool of ΔM-N L-selectin within the non-transmigrated uropod is likely to exist in a predominantly serine phosphorylated form. Although the extent of AMC in cells expressing ΔM-N or ΔM-NSSAA was completely indistinguishable , failed to compete-off the chip-immobilized moesin/FERM complex (367 (p-S367) peptide strongly competed chip-immobilized moesin FERM to the same degree as the NPP and suggested phospho-Ser367 would not interfere with FERM binding in cells  interacts with peptides corresponding to the tail of L-selectin , 43, 49. complex . In contin cells . The como-Ser364 , suggesthis site . Indeed, complex . Taken t367 didn't block the binding of either calmodulin  bilayer containing 6% PIP2 lipids randomly distributed in the lower leaflet showed agreement with previous MD simulations . Specifioups see . Engineeoups see . By trac367 regulates desorption of the L-selectin tail from the inner leaflet of the plasma membrane  monocytes disturbs front-back polarity in cells that have entered the subendothelial space, post-TEM post-TEM . CD14+ in humans –54. If bn humans .WT and ΔM-N L-selectin bind differently to ezrin and moesin during TEM, suggesting that altered signal transduction could precipitate as a direct consequence of skewed ERM binding behavior. Specifically, ezrin remains bound to ΔM-N L-selectin over a 25 min period of analysis during TEM . In contBased on recent data  and dataSSDD phenocopies that of ΔM-N L-selectin strongly implies that ΔM-N within non-transmigrated uropods is phosphorylated at positions Ser364 and Ser367. In support of this view, the ΔM-NSSAA mutant reverses the distribution of clustering from non-transmigrated uropods back to transmigrated pseudopods. Moreover, these data show that Ser364 and Ser367 play important roles in orchestrating the subcellular organization of L-selectin clustering of non-cleavable L-selectin during TEM. Given that WT L-selectin is phenocopied by SSAA L-selectin suggests that clustering within transmigrating pseudopods exists in a predominantly non-phosphorylated form. Indeed, no clustering is observed in cells expressing the SSDD mutant of L-selectin. We can now build on our previous findings  were used as secondary antibodies for LI-COR imaging of Western blots.2 under humidifying conditions. The THP-1 monocytic cell line was purchased from the American Type Culture Collection (LGC Standards) and passaged in RPMI medium containing 10% heat-denatured fetal calf serum (FCS), 1% antibiotics (penicillin/streptomycin) and 50 μM β-mercaptoethanol. Cells were tested negative for mycoplasma. HUVECs were purchased from Lonza and maintained in endothelial cell growth medium (EGM-2) supplemented with growth factors and antibiotics provided within their “bullet kits.” Cells were initially expanded for six or fewer passages, and were harvested and stored in liquid nitrogen for final use in flow assays or western blotting. Confluent HUVECs were disaggregated with trypsin/EDTA solution and seeded onto 10 μg/ml bovine-derived fibronectin. HEK 293T cells were used for lentiviral production and were a kind gift from Yolanda Calle, University of Roehampton, London, UK. Cells were routinely passaged at a 1:3 ratio on the third day. This “splitting” activity maintained an optimal cell density at 0.5 ×106 cells per mL.The generation of WT and mutant L-selectin lines have been described previously , includiThe open reading frame for WT and ΔM-N L-selectin was cloned into lentiviral vectors as previously described . The pHRMutagenesis of the serine to alanine or aspartate residues was conducted using a QuikChange Site-Directed Mutagenesis Kit (Agilent). The following forward (Fwd) and reverse (Rev) primers were used:S364AFwd GATTAAAAAAAGGCAAGAAAGCCAAGAGAAGTATGAATGACC.Rev GGTCATTCATACTTCTCTTGGCTTTCTTGCCTTTTTTTAATC.S367AFwd GGCAAGAAATCCAAGAGAGCTATGAATGACCCATATCAC.Rev GTGATATGGGTCATTCATAGCTCTCTTGGATTTCTTGCC.SSAAFwd GGCAAGAAAGCCAAGAGAGCTATGAATGACCCATATCAC.Rev GTGATATGGGTCATTCATAGCTCTCTTGGCTTTCTTGCC.SSDDFwd GGCAAGAAAGACAAGAGAGATATGAATGACCCATATCAC.Rev GTGATATGGGTCATTCATATCTCTCTTGTCTTTCTTGCC.R357AFwd GGCATTTATCATTTGGCTGGCAAGGGCATTAAAAAAAGGCA AGAAATCCAAG.Rev CTTGGATTTCTTGCCTTTTTTTAATGCCCTTGCCAGCCAAATGATAAATGCC.6 cells per mL. On the same day, 13 mm diameter glass coverslips (thickness “1”) were placed into the base of a 24 well dish, spotted with 100 μL of poly-L-lysine (PLL) to immobilize according to manufacturer's instruction. On the day of the assay, THP-1 cells were counted and adjusted to a density of 1 ×106 per mL in 500 μL containing antibody labeling buffer (RPMI culture medium containing FcR block [Miltenyi Biotec]). Cells were labeled with 2 μg per mL DREG56 for 30 min at 4°C, followed by washing (by centrifugation at 300 g and resuspension) in ice cold culture media to remove excess unbound antibody and then incubated back in ice cold labeling buffer containing secondary antibody conjugated to Alexa Fluor 663 (Thermo Fisher) for a further 30 min at 4°C. Cells were then washed twice in ice cold neat RPMI and resuspended to 100 μL of neat ice cold RPMI before 80 μL of the cell suspension was seeded onto 13 mm diameter glass coverslips (thickness = “1”), which were pre-coated with PLL-coated the night before. The seeded cells were placed into a humidified cell culture incubator at 37°C and 5% CO2 for 10 min to drive clustering. Adhered cells were flooded in excess 4% paraformaldehyde and fixed for 15 min at room temperature. Fixed coverslips were washed 3 times in PBS and then treated in 10 mg/mL of Sodium Borohydride dissolved in phosphate buffered saline for 10 min at room temperature . Coverslips were subsequently washed in PBS to remove sodium borohydride and mounted using DAKO mounting medium.THP-1 cells were adjusted the night before AMC to a density of 0.5 ×102 and according to manufacturer's instructions. After electrophoresis, Phos-tag™ acrylamide gels were transferred in SDS-containing transfer buffer  ethanol, 0.1% (w/v) SDS). Membranes were blocked in 5% (w/v) non-fat dried milk and incubated with the indicated primary antibodies, followed by incubation with LI-COR near infrared secondary antibodies. Immunodetection was carried out with a LI-COR Odyssey® CLx imaging system.SDS-PAGE was performed with 5% polyacrylamide gels. Proteins were transferred to 0.45 μm nitrocellulose membranes (Amersham™) using a wet blotting apparatus. Phos-tag™ SDS-PAGE was performed with 5% polyacrylamide gels containing 50 μM Phos-tag™ acrylamide  and 100 μM MnClFLIM measurement of FRET was performed with a multiphoton microscope system as described previously , 23. A NηFRET = (R06/(R06 + r6)) = 1–τFRET τd,where ηFRET is the FRET efficiency, R0 is the Förster radius, r is the molecular separation, τFRET is the lifetime of the interacting fraction and τd is the lifetime of the donor in the absence of an acceptor. The donor- only control is used as the reference against which all of other lifetimes are calculated in each experiment. τFRET and τd can also be taken to be the lifetime of the interacting fraction and non-interacting fraction, respectively. Quantification of FRET was made from all pixels within each cell that was analyzed. All image collection and data analysis were performed using TRI2 software .Surface plasmon resonance measurements of L-selectin/FERM competition studies have been published previously . Human FAll Molecular Dynamics (MD) simulations presented in this work are based on the Martini force field , 68. Folhttp://www.ccp4.ac.uk/newsletters/newsletter40.pdf#page=44) was used to generate atomistic models of all L-selectin variants, which were subsequently transferred to CG level using the martinize script (The studied L-selectin models consist of a transmembrane (TM) and the cytoplasmic tail of 23 and 17 residues, respectively. The sequences of the human and mouse L-selectin tails are shown in e script . In accoe script , the sec3). L-selectin was inserted into the bilayer parallel to the membrane normal. All systems were solvated with standard CG water beads and neutralized with sodium counterions.For the lipid bilayers, two types of lipids were used: zwitterionic POPC  and anionic PIP2 , q = −4e). Parameters for POPC were obtained from Wassenaar et al. . A model−4 bar-1 that was coupled to the system in a semiisotropic way. During both equilibration procedures the backbone beads of L-selectin were restrained to their initial positions by a harmonic potential with a force constant of 1,000 kJ mol-1 nm-2. After equilibration, three independent productions runs, 5 μs each, were conducted without any positions restraints; here constant pressure was achieved by using the Parrinello-Rahman barostat  were seeded onto 35 mm diameter glass coverslips  that were pre-coated with 10 μg/mL fibronectin (37°C for at least 1 h). Before each perfusion assay, HUVEC were stimulated overnight (16 h) with 10 ng/mL carrier-free recombinant human TNF-α (R&D Systems). Each perfusion assay was performed by injecting a bolus of cells for 6 min, followed by just perfusion media (without cells) for the remaining 25 min. THP-1 cells were perfused at a density of 0.5 ×106 cells per mL. THP-1 cells treated with 10 μM TAPI-0 required a preincubation time of 10 min at 37°C before perfusion over TNF-α-activated HUVEC. Note that 10 μM TAPI-0 was also supplemented in the perfusate. Stills were acquired once every 10 seconds using 10 × objective lens.All flow experiments were performed using a 35 mm diameter Glycotech parallel plate flow chamber. Perfusion experiments were performed at 1.5 dyn/cmFor FRET/FLIM analysis, coverslips were detached from the flow chamber after 15 min of flow, which is a period when protrusive activity is optimal, but ectodomain shedding is minimal , 23. CovAll datasets generated for this study are included in the manuscript/AI conceived the project, performed experiments, and wrote the paper. AN performed flow experiments and analyzed data. KR performed flow experiments and analyzed data. MK, CS, and SM performed experiments and analyzed all the data involving Molecular Dynamics. JJ performed experiments, contributed to data analysis, and compiled artwork for manuscript. AR-G contributed to experimental design. JD performed experiments related to surface plasmon resonance. MP performed analysis of FRET/FLIM data.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.


Bunyavirales cause serious diseases in animals, humans and plants. Family Tospoviridae in this order contains only one genus Orthotospovirus, and members in this genus exclusively infect plants. Tomato spotted wilt tospovirus (TSWV) is considered one of the most economically important plants viruses. Little is known about the regulatory elements in the TSWV genome. Here we show that, when in the cDNA form, the 5′-upstream region of the TSWV-coded GN/GC gene (pGN/GC) possesses putative cis-regulatory elements, including an auxin responsive element (AuxRE) for binding of auxin response factors (ARFs), as well as a circadian clock-associated 1 (CCA1) protein binding site (CBS). Due to the lack of a reverse genetics system, we verified the functionality of these elements in Arabidopsis. pGN/GC showed light-suppressive promoter activity in transgenic Arabidopsis, and mutation in the CBS was sufficient to switch the activity to light inducible. Additionally, exogenous auxin treatments repressed the promoter activity of both wild type and CBS-mutated pGN/GC. Mutation in AuxRE in both promoters abolished their sensitivity to auxin. As transcriptional repressors, both CCA1 and ARF2 were able to bind to pGN/GC directly. To our knowledge, this is the first report that a 5′-terminal sequence of an RNA virus has light-and hormone-responsive promoter activities when expressed as cDNA in host plant’s nuclear background. Our findings suggest new clues on the possible origin, evolution and function of the TSWV genomic sequence and its non-coding regions.Members of the virus order  Bunyavirales are characterized by segmented RNA genome with three RNAs packaged in enveloped virus particles (Tospoviridae contains only one genus Orthotospovirus (omato spotted wilt tospovirus (TSWV), a member of the genus Orthotospovirus, is considered one of the world’s most important plant viruses (Viruses in the order articles . Family spovirus . Virusesspovirus . Tomato  viruses . Transmi viruses . The thr viruses . In rece viruses .cis-regulatory elements (either outside or inside the coding regions) may play an important role in virus replication and RdRp-RNA interactions  that recognizes specific RNA elements (promoter sequences) in either the positive (+) or negative (-) strands of the viral RNA genomes, and viral ractions . During ractions . Both toractions .A major bottleneck in studying structure-function relationships of tospoviruses is the lack of a reverse genetics system. The ambisense genome organization of tospoviruses makes it very difficult to produce an infectious cDNA clone of the RNA genome. Despite years of effort by several groups, an infectious clone for any tospovirus remains elusive. Additionally, the roles of non-translated terminal regions of the genomic RNAs in tospovirus gene expression are largely unknown.N/GC gene (pGN/GC) possesses putative cis-regulatory elements, including an auxin response element (AuxRE) for binding of auxin response factors (ARFs)  , as wells (ARFs) . Since tArabidopsis thaliana ecotype Col-0 was used for Agrobacterium-mediated transformation. The Agrobacterium tumefaciens strain GV3101 was used for Arabidopsis transformation. The TSWV isolate T (M segment GenBank no. AY870389) was used. pCAMBIA1381Z was used as the plant expression binary vector.cis-regulatory elements. The list of known transcription factor (TF)-binding sites in plants was obtained from the Arabidopsis cis-regulatory element database (AtcisDB) in the Arabidopsis Gene Regulatory Information Server (AGRIS)1. Except for the transcription initiation site, both the original and the reverse-complement sequences of the TF-binding sites were used for scanning.The 5′-upstream sequences of the TSWV-coded genes were scanned for putative plant Nicotiana benthamiana leaves for cDNA synthesis. The pGN/GC fragment was amplified from cDNA using primers 5′-CGGAATTCAGAGCAATCAGTGCAAACAAA -3′ and 5′- CGGGATCCTTATTTTCCACTTGATAATAAACATTA -3′, and then cloned into EcoRI/BamHI sites of pCAMBIA1381Z. The pN fragment was amplified using primers 5′- CGGAATTCAGAGCAATCGTGTCAATTTTGTGTT -3′ and 5′- CGGGATCCGTATTGAGATTCTCAGAATTCCC -3′, and then cloned into EcoRI/BamHI sites of pCAMBIA1381Z. The pRdRp fragment was amplified using primers 5′- CGGAATTCAGAGCAATCAGGTAACAACGA -3′ and 5′- CGGGATCCTTATTTATTCTCTCAAACTCATCATC -3′, and then cloned into EcoRI/BamHI sites of pCAMBIA1381Z. All the PCR amplicons were verified by sequencing.Total RNA was extracted from TSWV infected Agrobacterium by electroporation and then used to transform Arabidopsis using the floral dip method . Homozygous transgenic seeds were obtained in the T3 generation. Two independent transgenic lines with single-locus T-DNA insertions were used for every promoter activity and GUS staining assay. To prepare material for PCR and GUS assays, seeds were placed on half-strength MS medium plates. After stratification at 4°C for 4 days, the seeds were treated by red light for 5 h to stimulate germination, followed by growing at 25°C for 4 days, either in dark or under continuous 80 μmol m-2 s-1 white light . The Arabidopsis UBQ10 gene was used as reference. The qRT-PCR primers for the detection of UBQ10 transcripts were 5′-TCTTCGTGGTGGTTTCTAAATCTCG-3′ and 5′-AAAGAGATAACAGGAACGGAAACATAGT-3′. Each data point had three biological replicates. Unpaired Student’s t-test was used to test the significance of difference in gene expression. The GUS staining was performed using the protocol previously described , Sigma RNase-free DNase I  was added during the RNA extraction to reduce gDNA contamination. Reverse transcription was done using the iScript Reverse Transcription Supermix . Quantitative RT-PCR (qRT-PCR) was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and the Applied Biosystems 7500 fast real-time PCR system . The qRT-PCR primers for the detection of escribed .N/GC-CBS in this case) was cloned into pDONR P4-P1R by BP reaction. The fragment was then subcloned into pMW#2 by LR reaction and integrated into the genome of yeast strain YM4271. pGN/GC-CBS was fused with the reporter gene HIS3 in the yeast genome. After verifying for self-activation, the bait yeast strain was transformed with CCA1 or ARF2 cloned in the Gateway prey vector pACT-GW and the empty vector as control. The activation of HIS3 was tested by yeast tolerance to 3-aminotriazole . The primers used for the initial amplification of pGN/GC-CBS were 5′- GGGGACAACTTTGTATAGAAAAGTTGGACTAATCTGATGCTAGAATCTC-3′ and 5′- GGGGACTGCTTTTTTGTACAAACTTGGAAGCATTCAAGCAGTTGTTAGG-3′. The sequence of pGN/GC-CBS is GACTAATCTGATGCTAGAATCTCAGACTCCTGGAACCCGTCAGATACGAGAAGAAGAATCAACCATCCCTATTTTTGCTGAGTCAACTACGGAAAAAACAATCTTTGTCTCGGATCTTCCTAACAACTGCTTGAATGCTTC. All the PCR amplicons were verified by sequencing. All the Gateway cloning reagents came from Invitrogen.The Gateway-compatible system was adopted for the Y1H assay . The DNAN/GC:GUS construct. The primers used to mutate CBS (AACAATCT) to CBSm (AACGGTCT) are 5′-CTGAGTCAACTACGGAAAAAACGGTCTTTGTCTCGGATCTTCCTAA-3′ and 5′-TTAGGAAGATCCGAGACAAAGACCGTTTTTTCCGTAGTTGACTCAG-3′. The primers used to mutate AuxRE (TGTCTC) to AuxREm (TGGATC) are 5′-AAGCAGTTGTTAGGAAGATCCGATCCAAAGATTGTTTTTTCCGTAGTTG-3′ and 5′-CAACTACGGAAAAAACAATCTTTGGATCGGATCTTCCTAACAACTGCTT-3′. The primers used to mutate CBS and AuxRE simultaneously are 5′-CAGTTGTTAGGAAGATCCGATCCAAAGACCGTTTTTTCCGTAG-3′ and 5′-CTACGGAAAAAACGGTCTTTGGATCGGATCTTCCTAACAACTG-3′. All the introduced mutations were verified by sequencing.The QuickChange site-directed mutagenesis kit (Agilent Technologies) was used for mutating the CBS and AuxRE elements in the pGN/GC  binding elements. As it is hard to define the precise boundaries for promoters, we analyzed the 500 bp in the 5′-upstream regions  of the five TSWV genes. No putative transcript initiation sites (TATA box or its variants) were found in the 5′-upstream regions of RdRp, NSm and NSs , respectively. The TATA-absent pRdRp fragment was also cloned into pCAMBIA1381Z (pRdRp:GUS) for use as a negative control. GUS driven by the constitutive CaMV 35S promoter (p35S:GUS) was used as the positive control. As a compatible host for TSWV, Arabidopsis ecotype Col-0 was used for pGN/GC:GUS transformation (GUS transcript accumulation levels in seedlings grown under continuous white light (80 μmol m-2 s-1). Two independent transgenic lines were tested for each construct. Same transgenic plant selection criteria, seedling age and growth conditions applied to all the assays in this research. The promoter strengths of both pRdRp (negative control) and pN were negligible when compared to that of pGN/GC  assay showed that CCA1 directly bound pGN/GC (N/GC (named as pGN/GC-CBS) was used as the bait in the assay. CBS was the only light-responsive element located in pGN/GC-CBS.Interactions between light-responsive TF-binding elements and their acting TFs may positively or negatively regulate the transcription of downstream genes. The SORLIP1 element, the T-box motif and the GATA binding motif are all light-activating elements , while Cby light , CBS coud pGN/GC . A 141-bN/GC:GUS with CBS mutated from AACAATCT to AACGGTCT (named as pGN/GC-CBSm:GUS) was used for stable transformation of Arabidopsis. Homozygous lines with single-locus T-DNA insertions were selected for subsequent experiments. qRT-PCR analysis of two independent transgenic lines (pGN/GC-CBSm:GUS/Col-0 -1 and pGN/GC-CBSm:GUS/Col-0 -2) showed that the promoter activity of pGN/GC-CBSm became light-inducible  can abolish the suppression effect of exogenous IAA on both pGN/GC  is the principal contributor to the light inducible characteristic of pGN/GC-CBSm. It is also possible that these elements simply have additive effect on light response.Our results suggest that the TSWV has regulatory elements that are found to be responsive to light and auxin when expressed as cDNA in the plant’s nuclear background. Following our finding that certain promoter-like elements are present in the cDNA of TSWV M RNA, we used Arabidopsis to test if these elements might be functional in a plant host. Our results showed that the putative promoter elements we identified, indeed, were light and auxin responsive when expressed as cDNA in the host nuclear background. A mutation in CBS was sufficient to switch the pGN/GC fragment exists in the native TSWV genome as RNA, and thus cannot act as a traditional promoter for the initiation of transcription. Our unpublished data also indicated that no DNA form of pGN/GC could be amplified by PCR from TSWV-infected plant tissues. The replication process of TSWV is not fully understood. It could be that the putative cis-regulatory elements in the M RNA may regulate the replication of M RNA or the expression of GN/GC mediated by TSWV RdRp. Unlike ARFs which are localized in the nucleus, CCA1 can be detected in both nucleus and cytoplasm (cis-RNA elements are indispensable for RdRp-mediated viral RNA synthesis (cis-elements in pGN/GC have a regulatory function. However, due to the lack of tospoviral infectious clones, it is difficult to test potential protein interactions with the RNA genome of TSWV in vivo. The majority of CCA protein molecules enter plant nucleus rapidly after their biosynthesis (Since TSWV is an RNA virus that completes its life cycle in the cell cytoplasm, the pGytoplasm . Therefoynthesis . Thus, iynthesis , which icis-acting elements from the host plants during viral evolution. There is evidence that RNA viruses may acquire host-derived sequences during reverse transcription, illegitimate recombination with retrotransposons, and host genome integration-excision processes (Another hypothesis is that TSWV may have obtained these rocesses . It is pTo our knowledge, this is the first report that the 5′ region of a viral RNA genome contains motifs suggestive of promoter elements. These elements were found to be light- and hormone-responsive when expressed as cDNA in a plant. It remains to be seen if these elements have a role in the virus lifecycle. Overall, we provide new clues on the origin and evolution of a bunyavirid genome sequence, and serves as a starting point to dissect the functions of end-genome or non-translational regions in RNA viruses.All datasets for this study are included in the manuscript and the All authors designed the project, wrote the manuscript, read and approved the final manuscript. YZ and HP performed the experiments and analyzed the data. HRP and MMN guided the experimental work.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.