Escherichia coli (STEC) O121 is among the top six non-O157 serogroups that are most frequently associated with severe disease in humans. While O121:H19 is predominant, other O121 serotypes have been frequently isolated from environmental samples, but their virulence repertoire is poorly characterized. Here, we sequenced the complete genomes of two animal isolates belonging to O121:H7 and O121:H10 and performed comparative genomic analysis with O121:H19 to assess their virulence potential. Both O121:H7 and O121:H10 strains carry a genome comparable in size with the O121:H19 genomes and belong to phylogroup B1. However, both strains appear to have evolved from a different lineage than the O121:H19 strains according to the core genes-based phylogeny and Multi Locus Sequence Typing. A systematic search of over 300 E. coli virulence genes listed in the Virulence Factor DataBase revealed a total of 73 and 71 in O121:H7 and O121:H10 strains, respectively, in comparison with an average of 135 in the O121:H19 strains. This variation in the virulence genes repertoire was mainly attributed to the reduction in the number of genes related to the Type III Secretion System in the O121:H7 and O121:H10 strains. Compared to the O121:H19 strains, the O121:H7 strain carries more adherence and toxin genes while the O121:H10 strain carries more genes related to the Type VI Secretion System. Although both O121:H7 and O121:H10 strains carry the large virulence plasmid pEHEC, they do not harbor all pEHEC virulence genes in O121:H19. Furthermore, unlike the O121:H19 strains, neither the O121:H7 nor O121:H10 strain carried the Locus of Enterocyte Effacement, OI-122, nor the tellurite resistance island. Although an incomplete Locus of Adhesion and Autoaggregation (LAA) was identified in the O121:H7 and O121:H10 strains, a limited number of virulence genes were present. Consistently, both O121:H7 and O121:H10 strains displayed significant reduced cytotoxicity than either the O157:H7 strain EDL933 or the O121:H19 strain RM8352. In fact, the O121:H7 strain RM8082 appeared to cause minimal cytotoxicity to Vero cells. Our study demonstrated distinct evolutionary lineages among the strains of serotypes O121:H19, O121:H10, and O121:H7 and suggested reduced virulence potentials in STEC strains of O121:H10 and O121:H7.Shiga toxin-producing  Such variation is attributed in part to the differences in their genetic makeup, especially the repertoire of virulence genes. The genomes of E. coli are extremely flexible, consisting of a core genome that is conserved among the E. coli strains, and an accessory genome containing strain, lineage, or pathovar-specific genes (Escherichia coli (EHEC) refer to a subset of STEC associated with severe human illnesses including bloody diarrhea and hemolytic uremic syndrome (HUS). The classical characteristics of EHEC include the production of Shiga toxin (Stx), formation of attaching-and-effacing (A/E) lesions on intestinal epithelial cells, and production of enterohemolysin , first reported in E. coli K-12 strain MG1655 (E. coli food isolate (terZABCDEF that is co-located with the gene encoding the Iha adhesin on a large GI known as tellurite resistance- and adherence-conferring island (TRI)  and #48 (OI-48)  and pathogenicity islands (PAIs) in n MG1655 , is a 14 isolate , confers isolate . Tellurind (TRI) . Great gnd (TRI) . In stra (OI-48) . Both OI (OI-48) . Other Eerotypes . In facterotypes . The OI-erotypes . The OI-erotypes .tia) encoding an invasion determinant that is primarily present in enterotoxigenic E. coli  is involved in iron storage and uptake and was originally identified in the Yersinia species  was revealed in a subset of LEE negative STEC strains, and some of these strains could cause hemorrhagic colitis or HUS . The com E. coli . Once th E. coli . The loc E. coli . This PA species . The HPI species .STEC O121 is among the top six non-O157 serogroups that are most frequently associated with severe disease in humans . Outbrea22.1Bacterial strains and their sources are listed in 2.2Escherichia coli #1 configuration   . The gennd recA) .2.3Escherichia Virulence Factors (VFs) listed in VFDB as of April 2022, which includes genes identified in 37 E. coli strains belonging to adherent invasive E. coli (AIEC), avian pathogenic E. coli (APEC), enteroaggregative E. coli (EAEC), Shiga toxin-producing enteroaggregative E. coli (StxEAEC), EHEC, enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), neonatal meningitis-associated E. coli (NMEC), or uropathogenic E. coli (UPEC). The identified virulence genes were further verified by a BLASTn search of a database containing all STEC strains examined in this study in Geneious Prime® with a threshold of 80% for gene coverage and 70% for sequence identity. Genes carrying a loss-of-function mutation in the coding region were marked as being absent.The putative virulence genes were identified using VFanalyzer at The Virulence Factor DataBase (VFDB) using pre-annotated genomes in GenBank format. Each pre-annotated genome was uploaded to VFanalyzer to scan for all 2.4E. coli K-12 strain MG1655 genome as a reference. A GI was called when a prediction was made by at least one of the three methods . The GIs and PAIs known to contribute to stress resistance and/or pathogenicity in enteric pathogens  of the enhanced green fluorescent protein (EGFP) as described previously  and were grown at 5% CO2 and 37°C under humidified conditions in Ham’s F-12 Nutrient medium (Life Technologies Corp.), supplemented with 10% fetal bovine serum (American Type Culture Collection) and 1% penicillin-streptomycin (Life Technologies). The Vero-d2EGFP cells were then exposed to Ham´s F-12 medium containing tenfold dilutions of cell-free culture supernatants from each bacterial strain and were incubated at 37°C for 20 hr in a 5% CO2 humidified incubator. Following intoxication, the Vero-d2EGFP cells were briefly rinsed three times with 1X phosphate-buffered saline, and the EGFP fluorescence from the Vero-d2EGFP cells was measured using a BioTek Synergy HT Multi-Detection Microplate Reader (Agilent Technologies) with the 485/20 nm excitation filter and the 528/20 nm emission filter. All experiments were performed in triplicate and each experimental condition consisted of six replicates. The results were expressed as percentages of the GFP fluorescence values for culture supernatant-treated Vero-d2EGFP cells when compared to the fluorescence values from control Vero-d2EGFP cells incubated without bacterial supernatants. The significant differences in the cytotoxicity levels among the STEC strains were determined using the two-tailed test function  in R version 4.2  carried a Stx1d-prophage inserted in the tRNA gene serU. All three O121:H19 strains carried a Stx2a-prophage inserted in the tRNA gene argW. Both the Stx2e-prophage (~ 48 kb) and the Stx1d-prophage (~ 47 kb) were smaller in size than any of the Stx2a-prophages (~ 66-70 kb) in the O121:H19 strains . Although homologs of the Stx1d-prophage late region were identified in multiple E. coli strains isolated worldwide, the region containing the stx1d coding sequence and its regulatory elements were only identified in a few strains, including C311, a cattle isolate, that originated from Hong Kong in 2018 , 2014C-3075, a clinical isolate that originated from the U.S. in 2013 , and FHI40, a clinical isolate that originated from Norway in 2009 .Strain RM10740 (O121:H10) carried a Stx2e-prophage inserted within the gene  strains Table 2.stx2a in the three O121:H19 strains were nearly identical and highly similar to the stx2a and stx1a regulatory regions in strain EDL933 , curli fimbriae (csgDEFG and csgBAC), E. coli common pilus (ecpRABCDE), E. coli laminin-binding fimbriae (elfADCG), and hemorrhagic E. coli pilus (hcpCBA) (eae) or the effector ToxB (toxB). Among the five O121 strains examined, only the O121:H7 strain RM8082 carried homologs of K88 fimbriae genes (faeCDEFHI) and P fimbriae genes (papC and papH)  and the chromosome-borne hemolysin gene (hlyE) were detected in the O121:H7 and O121:H10 strains, as observed for the O121:H19 strains. Interestingly, more toxin genes were detected in the O121:H7 strain RM8082, including cytolethal distending toxin genes cdtABC and enterotoxin 1 gene pic. Although a homolog of heat-labile enterotoxin gene eltA was detected, no homolog of eltB was identified in the genome of RM8082  Table 3.A total of 34 genes, related to biosynthesis of the T3SS apparatus, were detected in all three O121:H19 strains. In contrast, none of the T3SS apparatus genes were identified in either O121:H7 strain RM8082 or O121:H10 strain RM10740  contributing to the pathogenicity of enteric pathogens and three GIs , conferring eviously  and are 3.4.1selC. A LEE region was identified in all O121:H19 strains, but not in O121:H7 or O121:H10 strains (pheV. This integration site in the O121:H7 strain RM8082 was found occupied by a large Integrative and Conjugative Element (ICE) whereas unoccupied in the O121:H10 strain RM10740 , sheep (e.g. strain RHB10-C09), pig (e.g. strain W25K), deer (e.g. strain W49-2), and monkeys (e.g. strain A6). The 15-kb DNA fragment containing the genes related to integrating conjugative function is also present in the ETEC strain UMNK88 .The LEE region in O157:H7 strain EDL933 corresponds to the O-island #148 (OI-148) and is inserted downstream of tRNA gene  strains Table 4.3.4.2sisA and hes on module I, iha and lesP on module II, pagC, tpsA and tpsB on module III, and ag43 on module IV   and ckf. Additionally, there are two putative virulence genes, one (Locus tag: Z2112) encoding a Clp-like protease and the other one (Locus tag: Z2046) encoding a regulator of cell division .A 48-kb OI-57 like island was identified in the O121:H7 strain RM8082, overlapping with the genome of prophage 5 Table 4.3.4.5serW (OI-43), and serX (OI-48) (ileX (The two TRIs in strain EDL933 are located adjacent to tRNA gene  (OI-48) Table 5. (OI-48)   and the urease genes (ureDABCEFG). Noteworthy, the terF gene was missing in all O121:H19 strains examined and was replaced with a hypothetical gene and an IS3 transposase gene. The O121:H19 TRIs exhibited limited sequence similarity with the EDL933 TRIs  and for an efflux pump (mdtEF). Similar to both MG1655 and EDL933, the AFI in all O121 strains was inserted between a hypothetical gene and the gene encoding a cytochrome C peroxidase (yhiA). However, unlike the AFI in strain EDL933, the iron acquisition genes chuSA and chuTWXYU were not detected in any of the O121 genomes examined in this study.The 37-kb LPA often inserts downstream of tRNA gene n MG1655 Table 5,3.5hlyCABD), the type II secretion system (T2SS) (etpCDEFGHIJKLMNO), Toxin B (toxB), an EHEC-catalase (katP), and a serine protease autotransporter (espP). While the enterohemolysin genes were conserved in all O121 pEHEC plasmids, the espP and toxB genes were only detected in pEHEC of the O121:H19 strains and the katP was only detected in pEHEC of the O121:H7 strain. No homologs of any T2SS genes were detected in any of O121 pEHECs examined. The RM8082 pEHEC carried a large gene cluster  encoding a type IV conjugative transfer system and a F-type conjugative transfer system. This gene cluster was also present in the RM10740 pEHEC. Some unique genes located on the RM8082 pEHEC included several autotransporter genes, two colicin genes, a hydrolase gene, a sulfite reductase gene, a methyltransferase gene, two transcriptional regulator genes, and the toxin-antitoxin VapBC system. The unique genes carried by the RM10740 pEHEC included the accessory colonization factor AcfC gene, an EamA family transporter gene, a macrolide transporter gene, the gene cluster (merR and merTPCADE) conferring resistance to mercury , and the tetracycline resistance genes tetA and tetR.The main virulence factors encoded by the plasmid pEHEC in strain EDL933 are enterohemolysin , RM8082 (O121:H7), and RM10740 (O121:H10) was assessed in the mammalian host Vero cells by using bacterial cell-free culture supernatants and compared with the supernatants from the Stx-negative strain MG1655 and the Stx-positive EHEC prototype strain EDL933. Under the high concentration of the expressed Stx, resulting in low levels of GFP fluorescence in the host Vero cells  of ST641 thus are closely related based on this phylogenetic context to the ST641 strains as suggested previously , the main ST reported in the O121:H19 population . The O12eviously . The maieviously . The O12E. coli virulence genes in O121:H7 strain RM8082 and O121:H10 strain RM10740 than any of the O121:H19 strains examined. Additionally, both strains RM8082 and RM10740 carry a Stx subtype that has lower toxicity compared to Stx2a, which is predominantly present in the O121:H19 population  and the serine protease autotransporter (pic), which both are known important virulence factors in enteric pathogens (eltA) but not the gene for subunit B (eltB) was detected in the genome of RM8082.  and highly similar to the i T6SS-2 , which ii T6SS-1 . This geand APEC . Besidesand APEC .stx2a gene. A recent study that investigated the virulence evolution in the STEC O121:H19 population, and among the examined 639 strains, over 95% of the strains carried stx2a only, whereas the rest of the population (5%) carried either stx1a or both stx1a and stx2a , about 13%, 12%, and 8% of total CDSs are unique to the strain belonging to O121:H19, O121:H7, and O121:H10, respectively. As expected, this variation is mainly attributed to the difference in the content of MGEs, including prophages, plasmids, GIs/PAIs, and transposable elements. Both O121:H7 strain RM8082 and O121:H10 strain RM10740 carry more plasmids but a smaller number of prophages than the O121:H19 strains. Based on a BLASTn genome search with the ISfinder database, the O121:H10 strain RM10740 contained the greatest number of IS elements with a total of 170, followed by the O121:H19 strains with 136 IS elements, and the O121:H7 strain RM8082 harbored the lowest number of 104 IS elements. Functions encoded by strain-specific genes include, but are not limited to virulence, carbon metabolism, DNA restriction and modification, DNA transfer, and multidrug/heavy metals resistances. It is reasonable to hypothesize that, if not all, some of these functions are niche specific and reflect recent evolution of a particular strain. Although serotype O121:H7 or O121:H10 are not frequently associated with human disease, horizontal gene transfer of thogenic . Thus, mThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.Conceptualization: MC. Methodology: AP, BQ, and MC. Software: AP. Data curation: AP, BQ, and NL. Writing—original draft preparation: NL and MC. Writing—review and editing: BQ and MC. Funding acquisition: BQ and MC. Project administration: MC. All authors have read and agreed to the published version of the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by USDA-ARS CRIS projects 2030-42000-052-00D and 2030-42000-055-00D.We thank Bertram Lee for providing technical assistance with statistical analyses and for critical reading of the 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Gamma-glutamyl transferase (GGT) and high-density lipoprotein cholesterol (HDL-C) have been proven to be valuable predictors of type 2 diabetes mellitus (T2DM). The aim of this study was to investigate the association between GGT/HDL-C ratio and incident T2DM.The study retrospectively analyzed 15453 participants from 2004 to 2015. Cox proportional hazards regression models and Kaplan-Meier curves were used to elucidate the effect of GGT/HDL-C ratio on T2DM. Restricted cubic spline (RCS) analysis was performed to explore any non-linear correlation between GGT/HDL-C ratio and the risk of T2DM. The predictive performance of GGT, HDL-C and GGT/HDL-C ratio for T2DM was evaluated utilizing receiver-operating-characteristic (ROC) curves.During a median follow-up of 5.39 years, 373 cases of incident T2DM were observed. Kaplan-Meier curves showed that the cumulative probabilities of T2DM increased in the participants with higher GGT/HDL-C ratio significantly (P < 0.001). Cox models further clarified that high GGT/HDL-C ratio was an independent risk factor for T2DM . Linear positive correlation between GGT/HDL-C ratio and the risk of T2DM was demonstrated through RCS analysis. In the ROC analysis, GGT/HDL-C ratio  showed competitive role in the prediction of T2DM compared with single GGT and HDL-C.The GGT/HDL-C ratio could serve as a valuable predictor of T2DM, and the risk of T2DM increases in the condition of higher GGT/HDL-C ratio. With the improvement of living standards, diabetes mellitus has become an increasingly common disease, causing a serious public burden . AccordiThe pathogenesis of T2DM is complex, with insulin resistance and β-cell dysfunction being the most important . Liver iOn the other hand, metabolic syndrome is recognized as an important risk factor for T2DM and is characterized by the decline of high-density lipoprotein cholesterol (HDL-C) concentrations . HDL-C hGGT/HDL-C ratio was a new indicator proposed by a recent study .The studhttps://doi.org/10.5061/dryad.8q0p192)  . The dat8q0p192) . In addi8q0p192) Figure 1The study of Okamur et al. was approved by the Murakami Memorial Hospital Ethics Committee and all participants signed an informed consent form . This stAll participants completed a standardized self-administration questionnaire to provide medical history and lifestyle factors including alcohol consumption, smoking status, and habit of exercise. Additionally, body mass index (BMI), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were obtained by physical examination, as well as FPG, glycated hemoglobin (HbA1c), alanine aminotransferase (ALT), aspartate transaminase (AST), GGT, HDL-C, cholesterol and triglycerides by analyzing fasting blood samples.Meanwhile, Okamur et al. provided clear definitions for the following indicators . AlcoholContinuous variables would be described as mean ± standard deviation if they follow a normal distribution and compared using the Student’s t-test. Otherwise they would be reported as median (quartiles) and compared utilizing the Mann-Whitney U test. Categorical variables were expressed as frequency (percentage) and evaluated with chi-square test. Then, GGT/HDL-C ratio was divided into 4 groups based on quartiles, namely Q1 (<7.20), Q2 (7.20-10.78), Q3 (10.78-17.96) and Q4 (>17.96). Kaplan-Meier curves were applied to visualize the cumulative probability of T2DM in each group. Next, four Cox proportional hazards regression models were developed to investigate the independent effect of GGT/HDL-C ratio on T2DM, among which three adjusted models contributed to reducing bias. Model 1 was adjusted for age and gender. Model 2 was further adjusted for BMI, alcohol consumption, smoking status and fatty liver on the basis of model 1. Model 3 was further adjusted for habit of exercise, FPG, HbA1c, ALT, AST, cholesterol, triglycerides, SBP and DBP on the basis of model 2. Based on model 3, the restricted cubic spline (RCS) model with 4 knots was employed to explore any non-linear correlation between GGT/HDL-C ratio and the risk of T2DM. Finally, in order to assess the efficiency of GGT/HDL-C ratio in predicting T2DM, receiver-operating-characteristic (ROC) curves were performed and the best cut-off value were further obtained.All statistics and analyses were performed by SPSS (version 26.0) and R software (version 4.0.5). P values (two-sided) below 0.05 were considered to indicate statistical significance.A total of 15453 participants were recruited into the final retrospective cohort study. 373 of them (2.41%) developed incident T2DM after a median follow-up period of 5.39 years. It was clear that participants with T2DM were older and higher in the percentage of male than those without T2DM. Meanwhile, T2DM group involved in more participants with characteristics of heavy alcohol consumption, current smoking, fatty liver and no exercise habit. In addition, the baseline BMI, FPG, HbA1c, ALT, AST, GGT, cholesterol, triglycerides, SBP and DBP were higher but HDL-C was lower in T2DM group. The GGT/HDL-C ratio was further calculated. Its median was only 10.62 in non-T2DM group, whereas it reached 21.53 in T2DM group, showing a dramatically significance (P < 0.001). Table 1Subsequently, GGT/HDL-C ratio was divided into four groups based on quartiles. It was apparent that the distribution of GGT/HDL-C ratio in non-T2DM group was balanced, with each group accounting for approximately 25%. However, 63.27% of participants with T2DM were in the highest Q4 group, while only 5.36% were in Q1 group. Table 1 Next, univariate and multivariate Cox proportional hazards regression models were constructed to investigate the independent effect of GGT/HDL-C ratio on incident T2DM. In the univariate Cox model, elevated GGT/HDL-C ratio would increase the risk of T2DM . Similarly, the risk of T2DM was significant higher in groups Q2, Q3 and Q4 with higher GGT/HDL-C ratios compared to Q1 group, with Q4 group being 11.60 times higher than Q1 group . Model 1 and model 2, which were further adjusted for some covariates, showed similar results. The final model 3 adjusting for all covariates illustrated that GGT/HDL-C ratio was still an independent risk factor for T2DM as a continuous variable . Interestingly, when GGT/HDL-C ratio was considered as a categorical variable, no statistical difference was shown between Q2 and Q3 groups and Q1 group, but the risk of T2DM in Q4 group still was demonstrated to be 1.97 times higher than Q1 group . Table 2RCS model was established based on model 3 to examine whether there was a non-linear correlation between GGT/HDL-C ratio and the risk of T2DM. Obviously, GGT/HDL-C ratio was linearly correlated with the risk of T2DM due to a P for non-linear of 0.333. And the risk of T2DM increased with elevated GGT/HDL-C ratio. Figure 3Finally, ROC analysis was utilized to elucidate the predictive performance of GGT/HDL-C ratio in incident T2DM. Apparently, GGT/HDL-C ratio  was a better predictor when compared with single GGT  or single HDL-C . Figure 4In brief, this was the first study to explore the association between GGT/HDL-C ratio and the risk of T2DM. Furthermore, the large population-based retrospective cohort study demonstrated that GGT/HDL-C ratio was linearly correlated with the risk of T2DM and that the risk of T2DM increased in the condition of higher GGT/HDL-C ratio. GGT/HDL-C ratio could serve as a valuable predictor of T2DM with better predictive performance compared to single GGT and HDL-C.T2DM and its complications have become a serious threat to the quality of life of patients. Recent studies on the epidemiology of T2DM have revealed numerous risk factors, such as genetics, obesity, metabolic syndrome, unhealthy diet, unhealthy lifestyle habits, smoking and alcohol consumption, and these factors interact in the development and progression of T2DM . Given tde novo lipogenesis (DNL) pathway and decreasing the export of very low density lipoproteins , Data from: Ectopic fat obesity presents the greatest risk for incident type 2 diabetes: a population-based longitudinal study, Dryad, Dataset, The studies involving human participants were reviewed and approved by Ethics Committee of the Murakami Memorial Hospital. The patients/participants provided their written informed consent to participate in this study.XWC, LB and TYS analyzed the data and drafted the manuscript. XWC collected and assembled the data. SZS and YTS contributed to design and critically revised the manuscript for important intellectual content. All authors read and approved the final version of the manuscript.We appreciate Okamura et al. for sharing their scientific 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Together, these papers show a balance among mutations that enhance stability, those that increase binding to the human receptor ACE2, and those that confer resistance to neutralizing antibodies. —VVAs battles to contain the COVID-19 pandemic continue, attention is focused on emerging variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that have been deemed variants of concern because they are resistant to antibodies elicited by infection or vaccination or they increase transmissibility or disease severity. Three papers used functional and structural studies to explore how mutations in the viral spike protein affect its ability to infect host cells and to evade host immunity. Gobeil  How SARS-CoV-2 variants gain enhanced infectivity and evade host immune responses. A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429), which was originally detected in California, carries spike glycoprotein mutations S13I in the signal peptide, W152C in the N-terminal domain (NTD), and L452R in the receptor-binding domain (RBD). Plasma from individuals vaccinated with a Wuhan-1 isolate–based messenger RNA vaccine or from convalescent individuals exhibited neutralizing titers that were reduced 2- to 3.5-fold against the B.1.427/B.1.429 variant relative to wild-type pseudoviruses. The L452R mutation reduced neutralizing activity in 14 of 34 RBD-specific monoclonal antibodies (mAbs). The S13I and W152C mutations resulted in total loss of neutralization for 10 of 10 NTD-specific mAbs because the NTD antigenic supersite was remodeled by a shift of the signal peptide cleavage site and the formation of a new disulfide bond, as revealed by mass spectrometry and structural studies. SARS-CoV-2 infects target cells using the spike glycoprotein (S), which is organized as a homotrimer with each monomer comprising an SThe ongoing global spread of SARS-CoV-2 led to the fixation of the D614G substitution (https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html).The SARS-CoV-2 B.1.427/B.1.429 variant was reported for the first time at the beginning of 2021 in California, and as of May 2021 had been detected in 34 additional countries  for the B.1.427 and B.1.429 lineages, respectively. This VOC was detected in California and in other US states, and more recently in 34 additional countries worldwide . CollectTo assess the impact of the three mutations present in the B.1.427/B.1.429 S glycoprotein on neutralization, we first compared side by side the neutralization potency of mRNA vaccine–elicited Abs against the G614 S and B.1.427/B.1.429 S pseudoviruses. We used plasma from 15 individuals who received two doses of the Moderna mRNA-1273 vaccine and from 15 individuals who received two doses of Pfizer/BioNtech BNT162b2 vaccine collected between 7 and 27 days after booster immunization (table S2). All vaccinees had substantial plasma-neutralizing activity against G614 SARS-CoV-2 S pseudotyped viruses. Using a murine leukemia virus (MLV) pseudotyping system, geometric mean titers (GMTs) showed that the average neutralization potency of the Moderna mRNA1273–elicited plasma was reduced 2.4-fold for B.1.427/B.1.429 S (GMT: 178) compared with G614 S (GMT: 424)  collected 15 to 28 days after symptom onset (table S2). The neutralization potency of plasma from the nine convalescent donors was reduced 3.4-fold for B.1.427/B.1.429 S (GMT: 70) compared with G614 S (GMT: 240), similar to what we observed with B.1.351  and P.1  pseudotyped viruses, whereas neutralization of B.1.1.7 was less affected   recognizes distinct antigenic sites, as previously characterized  structure of the variant S ectodomain trimer [carrying the HexaPro mutations , and S2LWe subsequently used local refinement to account for the conformational dynamics of the NTD and S2L20 relative to the rest of S and obtained a cryoEM reconstruction of the NTD bound to S2L20 at 3.0-Å resolution . These sOverlaying an ACE2-bound SARS-CoV-2 RBD structure with the B.1.427/B.1.429 variant S structure showed that the R452 residue points away from and does not contact ACE2, suggesting that this substitution would not affect receptor engagement . We nextTo investigate further the molecular basis for the loss of NTD-directed mAb-neutralizing activity and structural changes in the NTD, we analyzed the binding of a panel of NTD-specific mAbs to recombinant SARS-CoV-2 NTD variants using ELISA. The S13I signal peptide mutation dampened binding of five mAbs and abrogated binding of five additional mAbs of the 11 neutralizing mAbs evaluated  . As a reAlthough the S13I and W152C NTD variants were respectively cysteinylated at positions C136 and W152C, the double mutant S13I/W152C was not cysteinylated, suggesting that C136 and W152C had formed a new disulfide bond . Tandem Collectively, these findings demonstrate that the S13I and W152C mutations found in the B.1.427/B.1.429 S variant are jointly responsible for escape from NTD-specific mAbs because of deletion of the two SARS-CoV-2 S N-terminal residues and overall rearrangement of the NTD antigenic supersite. Our data support that the SARS-CoV-2 NTD evolved a compensatory mechanism to form an alternative disulfide bond and that mutations of the S signal peptide occur in vivo in a clinical setting to promote immune evasion. The SARS-CoV-2 B.1.427/B.1.429 S variant therefore relies on an indirect and unusual neutralization escape strategy.Serum- or plasma-neutralizing activity is a correlate of protection against SARS-CoV-2 challenge in nonhuman primates (The SARS-CoV-2 NTD undergoes rapid antigenic drift and accumulates a larger number of mutations and deletions relative to other regions of the S glycoprotein (Similar to how the S13I/W152C mutations facilitate evasion of all tested NTD-specific mAbs, E484K causes broad resistance to many RBD-specific mAbs. The independent acquisition of the E484K mutation in the B.1.351, P.1, and B.1.526 variants and, more recently, in the B.1.1.7 variant (20210701-1Click here for additional data file.


ERG) fusion affects prostate cancer depending on the degree of expression of ERG. Solute Carrier Family 45 Member 3 (SLC45A3) is the second-most common 5′ partner gene of ERG rearrangement. However, the molecular pathological features of SLC45A3:ERG (S:E) fusion and therapeutic methods have not been studied at all. S:E fusion-positive cancers (n = 10) were selected from the Tumor Fusion Gene Data Portal website. Fusion-negative cancers (n = 50) were selected by sorting ERG expression level in descending order and selecting the bottom to 50th sample. Totally, 1325 ERG correlated genes were identified by a Pearson correlation test using over 0.3 of absolute correlation coefficiency (|R| > 0.3). Pathway analysis was performed using over-representation analysis of correlated genes, and seven cancer-related pathways /PI3K-Akt, JAK-STAT, Notch, receptor tyrosine kinase/PDGF, TGF-β, VEGFA, and Wnt signaling) were identified. In particular, focal adhesion kinase (FAK)/PI3K-Akt signaling and JAK-STAT signaling were significantly enriched in S:E fusion-positive prostate cancer. We further identified therapeutic targets and candidate drugs for S:E fusion-positive prostate cancer using gene–drug network analysis. Interestingly, PDGFRA and PDGFRB were the most frequently predicted therapeutic targets, and imatinib targeted both genes. In this study, we provide extensive information on cellular signaling pathways involved in S:E fusion-positive prostate cancer and also suggest therapeutic methods.ETS-related gene ( ERG), a member of the E-26 transformation-specific (ETS) family, is an oncogene regulating cell proliferation, differentiation, and metastasis [ERG overexpression driven by fusion of the ERG gene with androgen response genes such as TMPRSS2 [TMPRSS2:ERG (T:E) fusion is most frequently found in prostate cancer, and its oncogenic role and regulating molecular mechanisms have been well studied [T:E fusion have been suggested but no drugs have been FDA approved to date [T:E fusion-positive prostate cancer were investigated and actionable drugs with therapeutic targets were suggested [Prostate cancer is a common diagnosis in males and is the second most common cancer causing mortality in males. More than 190,000 cases of prostate cancer were reported in the United States in 2020 [ TMPRSS2 . TMPRSS2 studied ,5. Inhibuggested .SLC45A3 is the second-most common 5′ partner gene in ERG rearrangement [SLC45A3:ERG (S:E) fusion is found in approximately 6% of ERG fusion prostate cancer, and concurrent TMPRSS2 and SLC45A3 fusions to ERG are found in 11% of ERG fusion-positive cancer [S:E fusion-positive prostate cancers is very limited.angement . SLC45A3e cancer . HoweverS:E fusion-positive prostate cancer and provide potential therapeutic genes and candidate drugs. The study used The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) data from the Broad GDAC Firebrowse website. We analyzed cellular pathways through over-representation analysis (ORA), and presented actionable drugs for the patients through the Drug-Target database from Clinical Interpretation of Variants in Cancer (CIViC).In this study, we aimed to investigate the molecular pathological features of http://gdac.broadinstitute.org/ accessed on 21 August 2021). Additionally, TCGA PRAD patient characteristics including molecular and pathological information such as Age, TNM stage, Gleason Score, PSA, and vital status  was used to select fusion-positive samples. S:E fusion-positive cancers (n = 10) were selected after excluding two cases  which have concurrent T:E and S:E fusions. A fusion of exon 1 of TMPRSS2 with exon 4/5 of ERG were selected as T:E fusion-positive cancers (n = 86). Fusion-negative cancers (n = 50) were selected from a sample of TCGA PRAD data by sorting ERG expression level in descending order and selecting the bottom to 50th sample.In this study, ERG and other genes. Correlated genes were obtained by considering genes with an absolute correlation coefficient of over 0.3, based on our previous study [S:E fusion and T:E fusion, respectively. The Pearson correlation test was applied to measure the statistical correlations between gene expression levels of us study . As a reS:E fusion and T:E fusion was used as an input for Over-Representation Analysis (ORA) via ConsensusPathDB . Pathways were selected with this criteria: a minimum overlap input list (n = 2) and p-value cutoff , and resulted from databases described by a previous study [Each selected gene in us study .S:E fusion, and identified therapeutic target genes and their targeting drugs.A clinical drug database was obtained from Clinical Interpretation of Variants in Cancer (CIViC). We performed gene–drug network analysis with each selected gene (|R| > 0.3) in R statistical software (version 3.6.3) was used for analysis in this study. ERG. Ten cases were identified as S:E fusion-positive prostate cancer after removing two cases  with concurrent T:E and S:E fusions. T:E fusion-positive prostate cancer was obtained by considering fusions of exon 1 of TMPRSS2 to exon 4 or exon 5 of ERG (n = 86) because exon 4 or exon 5 of ERG are the most abundant breaking sites, generating T:E fusions [S:E fusion and T:E fusion result in ERG overexpression, and thus the ERG fusion-negative group was selected by a low expression of ERG (n = 50). First, we obtained RNA-seq data and clinical data of prostate cancer patients from TCGA . The Tum fusions . S:E fusS:E fusion-positive vs. ERG fusion-negative patients, S:E fusion-positive vs. T:E fusion-positive patients, and T:E fusion-positive vs. ERG fusion-negative patients, and there were no significant differences in PSA, tumor stages, and Gleason score as well as age and race  and FAF1 and INPP4B in the VEGFA signaling pathway , showed a negative correlation to ERG /Pl3k-Akt signaling pathway, JAK-STAT pathway, receptor tyrosine kinase/PDGF signaling pathway, and Wnt signaling pathway /Pl3K-Akt signaling pathway and JAK-STAT signaling pathway were significantly enriched in S:E fusion-positive prostate cancer.To identify unique cellular pathways in  cancers . Two patS:E fusion-positive prostate cancer by performing gene–drug network analysis. In total, there were 1325 genes serving as the input, and the CIViC database and Cytoscape software program were used; 24 drugs targeting 14 genes were found in the S:E fusion-positive group /Pl3K-Akt signaling pathway and JAK-STAT signaling pathway were uniquely identified in S:E fusion-positive prostate cancer  and AR to the specific sites [TOP2B-expressing cells inhibit TMPRSS2 gene expression. Considering that SLC45A3 is transcribed in an AR-dependent manner and shTOP2B-expressing cells inhibit SLC45A3 gene expression, TOP2B may be one of mechanisms to cause a break in SLC45A3 and ERG and induce S:E fusion. In addition, androgen and genotoxic stress recruit cytidine deaminase (AID) and LINE1-encoded ORF2 endonuclease to the specific sites and induce DNA cleavage of TMPRSS2, SLC45A3, ERG, and ETV1 in prostate cancer [SPOP increases topoisomerase 2 alpha (TOP2A) in DNA, and in turn removes tyrosyl-DNA phosphodiesterase 1(TDP1), tyrosyl-DNA phosphodiesterase 2(TDP2), and MRE11 from DNA, which triggers DNA damage and break [S:E fusion mechanism based on this concept, gene expression of SPOP, TOP2A, TDP1, TDP2, and MRE11 was examined, but no significant differences were found between the S:E fusion positive- and negative- groups /PI3K-Akt signaling pathway, JAK-STAT pathway, and receptor tyrosine kinase/PDGF signaling pathway in S:E fusion-positive prostate cancer. This suggests that PDGFRA and PDGFRB might be effective therapeutic targets, and imatinib could be a potential candidate drug for S:E fusion-positive prostate cancer. In a previous study, imatinib did not have beneficial effects on prostate cancer [S:E fusion-positive prostate cancer. In addition, imatinib was proposed as an anti-cancer targeted drug by targeting and suppressing PDGFR in breast cancer [PDGFRs are expressed for the bone metastasis of prostate cancer cells [FZD8, the highest ERG-correlated gene (R = 0.761) in S:E fusion-positive prostate cancer /PI3K-Akt signaling pathway and JAK-STAT pathway were characterized as cellular signaling pathways unique to S:E fusion-positive prostate cancer. In addition, imatinib, targeting PDGFRA and PDGFRB, was suggested as a potent actionable drug specific for S:E fusion-positive prostate cancer. Despite the obvious limitations of in silico analysis, as a first step, our study provides a rigorous method to understand the molecular characteristics of a certain type of cancer, and suggests possible therapeutic targets and candidate drugs. Further biochemical experiments will certainly necessary to validate our in silico analysis, and moreover investigate the underlying molecular mechanism and function of S:E fusion in prostate cancer. To conclude, we investigated the molecular features defined by different fusion partners of


N gene, an NLR that confers immunity to tobacco mosaic virus (TMV). This SR protein showed an interaction with a 3′ genomic regulatory sequence (GRS) and has a potential role in regulating the alternative splicing of N. Thus, it was named SR regulator for N, abbreviated SR4N. Further study showed that SR4N plays a positive role in N-mediated cell death but a negative role in N protein accumulation. SR4N also promotes multiple virus replications in co-expression experiments, and this enhancement may not function through RNA silencing suppression, as it did not enhance 35S-GFP expression in co-infiltration experiments. Bioinformatic and molecular studies revealed that SR4N belongs to the SR2Z subtype of the SR protein family, which was conserved in both dicots and monocots, and its roles in repressing viral immunity and triggering cell death were also conserved. Our study revealed new roles for SR2Z family proteins in plant immunity against viruses.Plant nucleotide-binding, leucine-rich, repeat-containing proteins (NLRs) play important roles in plant immunity. NLR expression and function are tightly regulated by multiple mechanisms. In this study, a conserved serine/arginine-rich protein (SR protein) was identified through the yeast one-hybrid screening of a tobacco cDNA library using DNA fragments from the  N gene that confers immunity against tobacco mosaic virus (TMV) was the first NLR to recognize plant viruses and is defined as one of the major types of NLR which has the N-terminal Toll/interleukin-1 receptor-like (TIR) domain and is called TNL [Plant immunity against viruses consists of multiple mechanisms. Plant Dicer-like (DCL) enzymes cut viral double-stranded RNA into 21- to 24-nucleotide (nt) small interfering RNAs (siRNA) to activate siRNA-based antiviral immunity. These virus-derived siRNAs (vsiRNAs) are loaded into Argonaute (AGO) proteins to form an RNA-induced silencing complex (RISC) with one strand retained. Then, vsiRNAs guide RISCs to silence viruses based on the complementarity between vsiRNA and viral RNA ,4. The plled TNL . Recentllled TNL ,10,11.Although NLRs confer immunity against devastating pathogens and are important for plant success in evolution, there is a fitness cost for plants carrying NLRs in their genomes, as their expression is harmful to plant growth and reproduction in the absence of cognate pathogens . To counSolanaceae family, many NLR genes have been found colocalized with various transposable elements, which generate abundant 24 nt siRNAs that may transcriptionally silence nearby NLR genes through the RNA-directed DNA methylation (RdDM) pathway [In the  pathway . Recentl pathway . Consist pathway . Thus, RSolanaceae-specific MIR6019/6020 family was derived from the inverted duplication of TIR-coding sequences from N gene ancestors [CPR1, MUSE3, and AtCDC48A, resulted in the overaccumulation of the TNL SNC1 protein and an enhanced disease resistance phenotype or autoimmunity [At the post-transcriptional level, numerous miRNAs have been identified targeting a major portion of the NLR transcripts in various plant species, and many of these NLR-targeting miRNA genes evolved through the inverted duplication of NLR gene fragments; for example, the ncestors ,16,17. Sncestors ,19. At timmunity .N gene requires full-length N and truncated N (Ntr) proteins that are translated from NS and NL, respectively, which contain an alternatively spliced exon (AE) encoding a premature stop codon [Besides the abovementioned regulatory mechanisms, many NLRs are also subjected to alternative splicing (AS), and many NLRs require multiple isoforms of transcripts to confer full resistance . For exaop codon . Mechaniop codon . HoweverN gene DNA fragments as bait. This SR protein interacts with N gene terminator sequences. It significantly reduced overall N/Ntr protein levels when co-expressed with N, and it has a moderate effect on the SN/LN transcript ratio, suggesting it may also affect the AS of N transcripts. Thus, we named this protein SR regulator for the N gene, abbreviated as SR4N. The overexpression of SR4N promoted TMV replication in both wild-type and N-transgenic plants, suggesting it is a negative regulator of plant immunity. However, knocking-down SR4N via virus-induced gene silencing (VIGS) inhibited N-p50-triggered cell death, and the overexpression of SR4N alone was sufficient to trigger cell death, suggesting SR4N is a positive regulator of cell death. Bioinformatics analysis showed that SR4N belongs to a conserved SR2Z subfamily of SR proteins. SR4N homologs in tomato, Arabidopsis, and rice also play negative roles in plant immunity against TMV and N protein accumulation and a positive role in cell death. Our results uncovered the opposite roles of RS2Z-type SR proteins: repressing plant immunity against viruses and promoting cell death.In this study, we identified a tobacco serine/arginine-rich (SR) protein in a yeast one-hybrid (Y1H) screening of a tobacco cDNA library using N. benthamiana was used for transient expression assay and VIGS assay. N. tabacum (SR1), S. lycopersicum (A57), A. thaliana (Col0), and Oryza sativa L. leaves were used to clone the SR4N gene. TRV2-GFP [N. tabacum transgenic line TG34 was used for SR4N expression analysis, which was transformed with the tobacco mosaic virus resistance gene N and described earlier [Wild-type TRV2-GFP , TMV-GFPTRV2-GFP , TBSV-GFTRV2-GFP , and pMSTRV2-GFP  vectors  earlier .N. benthamiana. After reverse transcription using M-MLV Reverse Transcriptase , the tobacco, tomato, Arabidopsis, and rice SR4N coding sequences were amplified and cloned into plant expression vector pH7LIC8.1 digested by StuI  through homologous recombination, which expressed the SR4N protein with N-terminal 3 × HA tags  through homologous recombination.SacI  and XhoI  through T4 DNA ligase. The SR4N were amplified from pH7LIC8.1-SR4N vector and cloned into yeast expression vector pGADT7LIC2.0 via homologous recombination.The N promoter containing 5′-UTR was divided into 11 fragments (NP1 to NP11), N intron III was divided into 5 fragments (NI1 to NI5), and N 3′-GS terminator containing 3′-UTR was divided into 3 fragments (NT1 to NT3). These fragments were amplificated from SPDK450 vector and cloned into pABAi vector digested by Primers used for constructing these vectors are listed in Total RNA was extracted from leaves collected from 1-, 3-, and 6-week-old tobacco plants and used for the construction of a prey cDNA library using Matchmaker Gold Yeast One-Hybrid Library Screening System Kit , according to the manufacturer’s instructions.N gene fragments  were individually transformed into yeast strain Y1HGold. The bait strain was grown on yeast nitrogen base (YNB) plates with different ABA concentrations, and 400 ng/mL was found to provide optimal selection pressure. Before mating, the fourteen bait yeast colonies, except for NT1, NI5, NP1, NP17, and NP19 bait yeast with autoactivation, were picked, resuspended in YNB liquid medium to OD600 of 0.5, 100 μL of each strain was mixed, and 50 mL of fresh YNB culture was seeded into a 250 mL flask. The mixed bait culture was grown at 28 °C for 16–20 h until OD600 reached 0.8; then, it was spun down and resuspended in 4–5 mL YNB medium. A tobacco cDNA library yeast culture of 1 mL was added to 45 mL 2× YPDA medium together with bait culture in a 2 L flask for 20–24 h while gently shaking at 30–50 rpm. After mating, the yeast culture was spun down, washed three times with 50 mL 2× YPDA, and resuspended in 10 mL 2× YPDA. Finally, the yeast culture was plated on YNB media  and cultivated at 30 °C for 3–5 days. The well-grown colonies were picked up and subjected to a colony PCR using primer pair ZH1010/1011 . Primers are listed in To detect SR4N expression in different timepoints after TMV infection, 6-week-old TG34 leaves were infected with TMV-U1, total RNA was extracted from the infected leaves at a different timepoint, and 2 µg total RNA after genomic DNA digesting by DNaseI  was used to obtain plant cDNA using oligo-dT and M-MLV reverse transcription enzyme . The N. benthamiana seed SRR21782212, N. benthamiana flower SRR696938, N. benthamiana leaf SRR696940, N. benthamiana root SRR696961, tomato root SRR19075381, tomato flower SRR14102833, tomato seeds SRR13787019, Arabidopsis root SRR656215, Arabidopsis flower SRR656217, Arabidopsis seeds ERR6351727, Arabidopsis leaf SRR656216, rice root SRR8051560, rice flower SRR8051546, rice seeds SRR7974062, rice leaf SRR8051557). The NGS transcriptome data were filtered and trimmed using Trimmomatic [SR4N gene expressions from different species were extracted from a merged expression matrix. After normalizing using log 10, a heatmap of gene expressions was drawn using the R package pheatmap .RNA-seq data from tobacco and tomato leaf samples from unpublished laboratory data were used. High-throughput RNA sequencing data from tobacco, tomato, Arabidopsis, and rice tissue samples were downloaded from NCBI . The MCT was placed in a Qiagen TissueLyser II  and ground for 15 s at 30 rpm. Then, 200 μL of extraction buffer  was added and mixed well. The MCT was then subjected to centrifugation at 12,000 rpm for 10 min at 4 °C. The upper clear lysate was kept at 4 ℃ for immediate use or kept at −80 °C for long-term storage.w/v) SDS, 20% (v/v) glycerol, 0.3 M β–thio-ethanol, 0.05% (w/v) Bromophenoblue), heated in 100 °C water bath for 5 min, and 20 μL of each was loaded onto the gel, together with a protein marker . The gel was first run at a constant voltage of 80 V for 30 min until the protein sample ran into the resolving gel and then ran at a constant 120 V for 45 min. N and actin were detected on one membrane via sequential hybridization using different antibodies and stripping. Ntr was detected on another membrane run at the same time.The TGX stain-free FastCast Acrylamide Kit  was used to prepare SDS PAGE gel for protein separation. Before loading, the prepared total protein sample was mixed with an equal volume of 2× SDS sample buffer (0.125 M Tris-HCl (pH 6.8), 4%  using Trans-Blot Turbo  according to the manufacture’s protocol. The membrane was then blocked in 1 × TBS buffer with 5% fat-free milk in a horizontal shaker  for 1–2 h, agitated at 100 rpm. Primary antibodies were added with the following dilution times: 1:5000 for anti-FLAG , anti-MYC , anti-HA , and anti-actin . They were incubated for 2 h. Next, buffer with primary antibody was removed, and the membrane was washed thrice with 1× TBST buffer, 10 min each time. Secondary antibody HRP-conjugated Affinipure Goat Anti-Mouse IgG (H+L)  with 1:5000 dilution was added and incubated for 1 h. Finally, the membrane was washed three times with 1× TBST again and developed using Clarity Western ECL Substrate  and imaged using ChemiDoc XRS +  according to the manufacturer’s instructions.N. benthamiana SR protein sequences were identified from protein databases [Arabidopsis and rice SR protein names were downloaded from a previous report , and SR atabases  through atabases ,35  caught our attention and was further tested in a point-to-point Y1H assay with each N gene fragment. The results showed that yeast containing SR4N and each prey DNA fragment grew well on the medium without Ura and Leu; similar results were observed for yeast containing the corresponding prey DNA and negative control bait, indicating all yeast strains are active under nonselective conditions  primers resulted in a clearly stronger SR4N DNA band in the p50-U1 and N-co-infiltrated sample than in the p50-Ob and N-co-infiltrated sample or the un-infiltrated control sample, while the tubulin bands were of similar intensity in all samples . Semi-quantitative RT-PCR was conducted to analyze the expression levels of NtaSR4N in different samples, and the results showed that the SR4N expression level clearly increased from 6 to 12 HPI A, indica samples B. An Ima samples C. These N. tabacum leaves [To gain more insights into the SR4N expression pattern, we analyzed its expression in our previously published RNA sequencing data from 3-week and 6-week-old m leaves . The FPKN-mediated resistance signaling, a virus-induced gene-silencing experiment was conducted. For this purpose, about 200 nt of SR4N cDNA was cloned into the TRV2-GFP vector, which expresses the GFP protein and enables the visual tracking of virus movements in plants. Three-week-old N. benthamiana plants were co-infiltrated with TRV1 and TRV2-GFP-SR4N or a TRV2-GFP control. Two weeks later, the upper leaves, which showed a GFP signal before, were co-infiltrated with agrobacterium containing N and p50 constructs. Five days after the second infiltration, cell death appeared in about 70% and 40% of the infiltrated patches in TRV2-GFP and TRV2-GFP-SR4N, respectively , and TMV-GFP mixture; an N, TMV-GFP, and SR4N mixture; and a TMV-GFP and SR4N mixture were infiltrated on the same leaf , one member in tomato (SlySR4N), and two members in Arabidopsis (AthSR4Na and AthSR4Nb). The latter was lost in Arabidopsis and has two members in tobacco (NtaSR4NLa and NtaSR4NLb), two members in N. benthamiana (NbeSR4NLa and NbeSR4NLb), and one member in tomato (SlySR4NL). The two rice members that shared higher identities with SR4N were named OsaSR4Na and OsaSR4Nb , followed by two CxxCxxxxHxxxxC zinc finger motifs, a serine/arginine-rich region, and a serine/proline-rich region at the C-terminal . A proteNtaSR4N in plant immunity, SlySR4N, AthSR4Na and b, and OsaSR4Na and b were cloned from tomato, Arabidopsis, and rice plants into expression vectors (see N gene expression and N protein accumulation were tested in co-infiltration experiments. A Western blot analysis showed that all these SR4N homologs significantly reduce N and Ntr protein accumulation and have stronger effects on N than Ntr (L and NS transcript levels, and the results showed that overall transcript levels were not significantly changed by the co-expression of these SR4Ns. The rice SR4Na and b expressions slightly increased the ratio of NL/NS, but the other ones did not (In order to investigate whether SR4N genes from other plant species have similar functions to tors see . First, than Ntr A. RT-PCR did not A. These N (N, plus various SR4Ns (AthSR4Na and OsaSR4Na.Next, the roles of various SR4Ns on N-mediated TMV resistance were tested with co-infiltration experiments. TMV-GFP was co-expressed with us SR4Ns B, indicaN. benthamiana leaves. The results showed that the overexpression of all tested SR4N homologs resulted in clear cell death in the infiltrated patches (NtaSR4Na and b.The overexpression of SR4Ns was also analyzed in transient assays in  patches C, indicaIn summary, the above experiments showed that SR4N gene functions on N expression, N-mediated immunity, and cell death regulation are conserved among both dicots and monocots, suggesting that SR4N is a highly conserved regulator of plant immunity.The promotion of TMV-GFP replication by NtaSR4Na and b raises the questions of whether they have a general role in other virus replications, whether this role is conserved in other plant species, and whether it is related to the suppression of siRNA-mediated silencing. To answer these questions, all SR4N homologs were tested with TBSV-GFP, TMV-GFP, and TRV2-GFP in a half-leaf assay. An agrobacterium containing each virus was co-infiltrated with EV control on one side of the leaf and with different SR4Ns on the other side . GFP imaN gene 3′GRS fragments (L/NS and Ntr/N (N. This is consistent with the role of splicing regulation. In the classic model, SR protein regulates splicing by binding to splicing enhancer or repressor elements in pre-mRNA, so it seemed contradictory for SR4N to play a role in the AS of N by binding to its 3′GRS DNA. Interestingly, it was shown in a previous study that the production of NL transcript is dependent on 3′GRS of N [N by interacting with 3′GRS to enhance AS. This is consistent with the “chromatin–adaptor” model of alternative splicing [N would be worthwhile for further investigation.NLRs play important roles in plant immunity yet they also impose potential threats to plant development; thus, the proper regulation of NLR expressions and functions is critical for the success of plants in evolution and for breeding crops to achieve a balance between disease resistance and optimal yields. In this work, using the N-TMV interaction as a model system, we studied the role of a class of RS2Z subtypes in SR proteins, which we named SR4N, in N regulation and plant immunity in general. SR proteins have been identified as being involved in RNA processing, such as transcription; splicing; polyadenylation; mRNP packing in the nucleus for mRNA export, translation, and decay in the cytoplasm; and non-coding RNA processing ,43,44,45ragments . The co-nd Ntr/N A. These GRS of N . The AraGRS of N . Thus, isplicing . The rolN, SR4N from all tested plant species clearly reduced overall N protein levels and promoted TMV-GFP replication when co-expressed with N and TMV-GFP (L/NS transcript levels did not significantly change when SR4N was co-expressed (N transcripts or promote N protein stability. Several mechanisms are possible for SR4N’s effect on N expression. SR4N may affect N mRNA translation by interfering in its mRNA polyadenylation and export considering it binds to its 3′GRS. It may reduce N translation or protein stability indirectly by regulating other factors in translation or protein degradation. It would be important to determine whether this effect on N is dependent on its interaction with its 3′GRS and specific for N or general for NLR.Besides potential roles in regulating the AS of  TMV-GFP A,B, suggxpressed C, SR4N mN. benthamiana plants (N is repressed by miRNAs [In this study, we also found that NbeSR4N was required for N-p50-triggered cell death via the VIGS knockdown of NbeSR4N, and further experiments showed that the overexpression of SR4N alone can trigger cell death C. These a plants  but did y miRNAs . These cThe functions of the SR4N family genes are understudied in plants. The overexpression of Arabidopsis AthSR4Na causes pleiotropic developmental phenotypes, affecting cell shape, stomata development, the formation of meristems, auxin signaling, in vitro regeneration, etc. . The exp