Also, the L753 mutation linked to the Y756 hydrogen bond prevents the S protein from being cleaved. Y756 and L753 mutations alter S protein subcellular localization. Importantly, Y756 and L753 mutations are demonstrated to reduce the infectivity of the SARS-CoV-2 pseudoviruses by interfering with the incorporation of S protein into pseudovirus particles and causing the pseudoviruses to lose their sensitivity to neutralizing antibodies. Furthermore, both mutations affect the assembly and production of SARS-CoV-2 virus-like particles in cell culture. Together, our findings reveal for the first time a critical role for the conserved L753-LQ-Y756 motif between S1/S2 and S2′ cleavage sites in S protein synthesis and processing as well as virus assembly and infection.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein mediates viral entry and membrane fusion. Its cleavage at S1/S2 and S2′ sites during the biosynthesis in virus producer cells and viral entry are critical for viral infection and transmission. In contrast, the biological significance of the junction region between both cleavage sites for S protein synthesis and function is less understood. By analyzing the conservation and structure of S protein, we found that intrachain contacts formed by the conserved tyrosine (Y) residue 756 (Y756) with three α-helices contribute to the spike’s conformational stability. When Y756 is mutated to an amino acid residue that can provide hydrogen bonds, S protein could be expressed as a cleaved form, but not IMPORTANCE The continuous emergence of SARS-CoV-2 variants such as the delta or lambda lineage caused the continuation of the COVID-19 epidemic and challenged the effectiveness of the existing vaccines. Logically, the spike (S) protein mutation has attracted much concern. However, the key amino acids in S protein for its structure and function are still not very clear. In this study, we discovered for the first time that the conserved residues Y756 and L753 at the junction between the S1/S2 and S2′ sites are very important, like the S2′ cleavage site R815, for the synthesis and processing of S protein such as protease cleavage, and that the mutations severely interfered with the incorporation of S protein into pseudotyped virus particles and SARS-CoV-2 virus-like particles. Consequently, we delineate the novel potential target for the design of broad-spectrum antiviral drugs in the future, especially in the emergence of SARS-CoV-2 variants. The spike (S) protein of coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a single-pass type I membrane protein and the major determinant of virus entry, host range, tissue tropism, pathogenesis, and virulence , 2. The 9–For SARS-CoV-2 and SARS-CoV entry, the proteolytic cleavage of coronavirus S protein by one or more host proteases, such as cysteine proteases cathepsin B and L or the transmembrane serine protease 2 (TMPRSS2), is essential to trigger membrane fusion , 13. TMP16–In structural analyses of the SARS-CoV-2, the S proteins were found to freely exist on the viral envelope, and the S head was connected to the viral membrane with the stalk domains with three flexible hinges in the S2 subunit , 20. Pre6VSB [The conservation analysis of S protein showed that the amino acid similarity among 27 SARS-CoV-2 isolates and SARS-CoV (CUHK-W1 strain) is 99.7 to 99.9%, and these SARS-CoV-2 strains share amino acid sequence similarities of only 74% with MERS virus (HCoV-EMC/2012) . Especially the region between the S1/S2 and S2′ cleavage sites of S protein , the ami6VSB ), which To investigate the influence of these amino acid residues between the S1/S2 and S2′ cleavage sites on the synthesis and function of S protein, a series of the S mutants was generated through site-directed mutagenesis , includiTo explore the regular pattern, we mutated the Y756 to other amino acid residues: tryptophan (W), alanine (A), glycine (G), histidine (H), or glutamate (E). We found that the mutation of Y756A, Y756G, or Y756E caused the S proteins to be produced as uncleaved forms, but the mutated S proteins with Y756W or Y756H were partially cleaved . OverallWe next evaluate the consequences of mutation of tyrosine 756 on SARS-CoV-2 infection using a lentivirus (HIV-1 NL4-3 strain) pseudotyping system . First, To further confirm pseudovirus infection, a HEK293T cell line stably expressing human ACE2 (293T-hACE2) was prepared through the lentiviral transduction system and blasticidin pressure selection. The ACE2-positive proportion of the 293T-ACE2 cell line was quantified as above 98% by flow cytometry, and the expression of ACE2 was detected by Western blotting . Then, tIn addition, we investigated the effect of L753, F759, or T998 mutation on SARS-CoV-2 spike-mediated virus infection. The three mutations significantly reduced the pseudovirus infection, especially the L753G mutation , consistSince these mutations, including R685, L753, Y756, or R815, affect the expression and processing of S protein, the subcellular localization of S protein may also be affected. For visual analysis of subcellular localization, the fusion expression plasmids of S or its mutants with a C-terminal enhanced green fluorescent protein (EGFP) tag and tyrosine protein kinase Lck with a C-terminal red fluorescent protein (RFP) tag were constructed and cotransfected into HeLa cells, and Lck was used as a subcellular localization marker; Lck localizes to the plasma membrane and pericentrosomal vesicles . As specBased on the results showing the effects of L753 and Y756 mutations on spike synthesis and processing in our experiments, we have boldly speculated that these mutations may affect the yield or quality of the SARS-CoV-2 pseudotyped viruses. During the packaging of S-enveloped pseudotyped lentiviruses in cell culture, the expression of S and its mutations was confirmed to be consistent with the data shown in In addition, except that no pseudovirus particles bearing Y756G S were observed, the characteristics of the particles formed by the WT S or its other mutants were observed under a negative-staining electron microscope. The virions embedded by WT S or its mutants with Y756F, R685A, D614G, Y756A, or Y756H exhibited similar morphologies and a diameter of ∼200 nm, but the virions embedded by the Y756C, Y756W, Y756E, and R815A S mutants were irregular in shape . AmazingWe next evaluated the impact of the mutation of residue L753, F759, or T998, structurally related to Y756, on pseudovirus production with SARS-CoV-2 S protein. Pseudoviruses with each mutant S protein were produced, concentrated, and detected by Western blotting as described above. Similarly, the quantification of pseudovirus particles was performed using p24 protein as a reference. The expression of WT or mutant S protein was detected by anti-spike RBD or S2 antibody in the producer cells . For theThe next question is whether the above-mentioned locus mutations affect the production of authentic SARS-CoV-2 virions. The SARS-CoV-2 virion, as is well established, is composed of four structural proteins: spike, membrane, envelope, and nucleocapsid protein. Recent studies have shown that the coexpression of four structural proteins in HEK293T cells could result in self-assembly into virus-like particles (VLPs) , 28, 29.For the VLPs, the WT or D614G mutated S proteins incorporated into SARS-CoV-2 VLPs composed of M, E, and N . HoweverFurthermore, in the pellets, compared to VLPs with WT S protein, F759G or T998G mutant VLPs only contained lower levels of cleaved S1 or S2 products, especially F759G mutant, whereas L753G spike did not appear in the precipitation . These rThe above-described results confirmed that although the R685A mutation causes the S protein to be incompletely cleaved, it can be assembled into pseudovirions with the typical spike structure of the SARS-CoV-2 prototypic virus. So then, an important question is raised: does this mutation affect antiviral therapy targeting the S protein? The neutralization potency of the monoclonal antibodies and horse or rhesus monkey antiserum targeting the S protein receptor-binding domain was assessed using the pseudotyped viruses bearing the WT S or D614G variant and vesicular stomatitis virus G protein (VSV G)-enveloped pseudotyped virus as a nonspecific control. As expected, the anti-RBD monoclonal antibodies efficiently neutralized both viruses pseudotyped with the WT S and D614G variant but not VSV G-enveloped pseudotyped viruses , which wAt the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a novel betacoronavirus broke into people’s lives; this virus had highly transmissible and pathogenic characteristics and caused a pandemic of acute respiratory disease named coronavirus disease 2019 (COVID-19) , 32. As 39–The SARS-CoV-2 virion mainly consists of four structural proteins, including the spike, envelope (E), membrane (M), and nucleocapsid (N) proteins. For SARS-related coronaviruses, the S protein is synthesized and processed, including posttranslational modification and cleavage, in the endoplasmic reticulum (ER) and Golgi, respectively , 45. MorMoreover, other proteases’ S1/S2 cleavage is processed beyond the furin proteolytic enzyme . For SAR6VSB) (The S2 subunit consists of the fusion peptide FP), a second proteolytic site (S2′), an internal fusion peptide (IFP), and two heptad-repeat domains (HR1 and HR2) preceding the transmembrane domain (TM) , 45. Fur6VSB)  and F. FP, a secoIt is noteworthy that the changes in the expression and subcellular localization of the S protein affect the assembly of virus particles and viral transduction to a certain extent. Indeed, we observed very low or even no infectivity of Y756-mutated S-based pseudovirus compared to that with WT S protein under the conditions of our experiments , which mTaken together, our results show that the S protein of SARS-CoV-2 as the primary receptor binding and membrane fusion mediator mediates virus entry into host cells and is also one of the main structural proteins for virus assembly. Therefore, the correct expression, structural stability, and precise subcellular localization of the S protein are critical to the correct assembly of the virus and indirectly affect the infection of the progeny virus. In this work, we confirm for the first time that the tyrosine residue at position 756 and the associated L753 and F759 are critical to the correct expression and subcellular localization of the S protein, regulate virion assembly and production, and, in turn, affect the S protein-mediated virus entry, using a pseudotyped virus or virus-like particle model. In addition to the resolved cryo-EM structure of the S protein, we found that the hydrogen bond formed by Y756 is also important for the stability of the S protein structure. In terms of significance, this study provides a new potential target for novel interventions, including antiviral drugs or CRISPR-mediated gene mutation therapy, by analyzing the key amino acid sites of the S protein. In the future, we will cooperate with other teams to carry out authentic SARS-CoV-2 verification and small-molecule drug screening research due to biosafety factors.NC_045512.2) and then inserted into the eukaryotic expression vector pCMV-3×Flag-14 and fused with 3×Flag for expression. The S mutants, including D614G, R685A, R815A, and series mutation of Y756, L753, F759, or T998, were engineered and constructed by PCR with a Q5 site-directed mutagenesis kit , using the S gene of the Wuhan-Hu-1 strain as the parent and WT sequence. The WT S gene and its mutants fused with EGFP at the C terminus were cloned into the pEGFP-N3 vector for subcellular localization assays. Human ACE2 was amplified using pcDNA3.1-ACE2-Flag  as a template and cloned into pLenti 6.3/V5-DEST  via homologous recombination to construct a cell line stably expressing human angiotensin-converting enzyme 2 (ACE2). The lentiviral backbone plasmid pNL4-3.Luc.R-E- was provided by Ningyi Jin. For the coexpression of SARS-CoV-2 M and E proteins, the M-IRES-E fusion fragment was composed of the M and E genes fused with Flag tag in the N and C termini, respectively, and M and E were linked by the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV). The pcDNA3.1-SARS-CoV-2-N-Myc was purchased from Beyotime (China). All plasmids were verified by DNA sequencing.The full-length SARS-CoV-2 spike (S) gene was synthesized  according to the sequence of SARS-CoV-2 Wuhan-Hu-1 strain  (GenBankDYKDDDDK) tag (no. 20543-1-AP), ACE2 (no. 21115-1-AP), GAPDH (no. 10494-1-AP), and β-actin (no. 20536-1-AP), and CoraLite 488-conjugated ACE2 monoclonal antibody (no. CL488-66699) were purchased from Proteintech (China). Horse anti-RBD/SARS-CoV-2 spike serum and cynomolgus monkey anti-RBD serum were gifts from Ningyi Jin.Rabbit monoclonal antibody against the SARS-CoV-2 receptor-binding domain (RBD) (no. 40592-R001), rabbit polyclonal antibody against SARS-CoV-2 nucleocapsid (no. 40588-T62) and spike S2 (no. 40590-T62), and HIV-1 p24 protein  antibody (no. 11695-T48) were purchased from Sino Biological (China). Rabbit polyclonal antibodies against Flag . The expression of hACE2 was examined by Western blotting with anti-ACE2 antibody, and the hACE2+ cell percentage was identified by flow cytometry.Human lung epithelial (A549) cells were maintained in Ham’s F-12K (Kaighn’s) medium  or supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin . HEK293T, HEK293, and HeLa cells were maintained in Dulbecco’s modified Eagle medium  containing 10% FBS and 1% penicillin-streptomycin. Human hepatoma (Huh7) cells were cultured in DMEM supplemented with 10% FBS, 1% GlutaMAX , and 1 mM sodium pyruvate . These cells were cultured at 37°C in a 5% COCells were lysed with a buffer of 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, and EDTA-free protease inhibitor cocktail  and phenylmethylsulfonyl fluoride . The extracted proteins were subjected to SDS-PAGE and then electrophoretically transferred to a nitrocellulose membrane , and protein bands were probed with the indicated primary antibodies.6 HEK293T cells in a 10-cm dish were cotransfected with 8 μg of S and its mutant expression plasmids pCMV-S-3×Flag including the WT, D614G, R685A, R815A, or Y756 mutant or L753G, F759G, or T998G and 8 μg of pNL4-3.Luc.R-E- using Lipofectamine 3000  to prepare various SARS-CoV-2 S-pseudotyped lentiviral luciferase reporter viruses. Six hours posttransfection, the supernatants were replaced with fresh DMEM supplemented with 2% FBS. The supernatants containing the pseudoviruses were harvested 48 h after transfection and purified by filtration with a 0.45-μm filter. In accordance with the manufacturer’s protocol, the pseudoviruses were titrated with the Lenti-X reverse transcription-quantitative PCR (qRT-PCR) titration kit . For SARS-CoV-2 S-pseudotyped virus entry assays, Huh7 or 293T-hACE2 cells were infected with a fixed dose of pseudoviruses for 48 h and then washed and lysed to detect luciferase signal with the ONE-Glo luciferase assay system  according to the instructions.A total of 5 × 10The supernatants containing the pseudoviruses with SARS-CoV-2 S protein or its mutants were obtained as described above and then layered onto 20% (wt/vol) sucrose cushions and purified and concentrated by ultracentrifugation  using an Optima XPN-100 ultracentrifuge (Beckman Coulter). Furthermore, the pellets were resuspended in sterilized 1× phosphate-buffered saline (PBS) and prepared for SDS-PAGE and Western blot analysis using the corresponding primary antibodies against RBD, Flag, or HIV p24.The resuspended pellets containing the pseudovirions obtained as described above were added to a glow-discharged 230-mesh copper grid covered with carbon-coated collodion film. After standing for several minutes, the excess liquid was absorbed with a filter paper from the edge of the copper mesh. Then, the copper mesh was stained with phosphotungstic acid  for 5 min and then sucked dry with a filter paper. Samples were analyzed with a JEM-1200EXII transmission electron microscope (JEOL).Analysis of the subcellular localization of C-terminally EGFP-tagged S or its mutants in HeLa cells was done using a laser scanning confocal microscope . The tyrosine protein kinase Lck was used as a subcellular localization marker. Briefly, HeLa cells were cultured on glass coverslips to cotransfect with S or its mutant expression plasmids and Lck marker for 48 h, then fixed in 4% paraformaldehyde and PBS for 30 min, and then permeabilized with 0.25% Triton X-100 according to the specific requirement. The nuclei were stained with 4′,6-diamidino-2-phenylindole . Images were taken under a fluorescence microscope or a Leica TCS SP8 confocal microscope.HEK293T cells in a 10-cm dish were cotransfected with the plasmids encoding the SARS-CoV-2 M, E, N, and S or its mutants. In the transfection mixtures, 6 μg of each plasmid M-IRES-E, pcDNA3.1-SARS-CoV-2-N-Myc, and pCMV-3×Flag-S or the plasmid with S mutant were added into 1 mL of Opti-MEM and then mixed with 18 μL of PEIpro transfection reagent . The transfection mixtures were incubated for 15 min at room temperature and dropped into HEK293T cells. At 72 h posttransfection, the culture medium was collected, filtered through a 0.45-μm filter, layered onto 20% (wt/vol) sucrose cushions, and purified and concentrated by ultracentrifugation  using an Optima XPN-100 ultracentrifuge (Beckman Coulter) as described above. Moreover, the pellets containing VLPs were resuspended in sterilized 1× PBS, aliquoted, and stored at −80°C freezer.The VLP pellets and the producer cell lysates were boiled with 5× SDS-PAGE loading buffer for 10 min at 100°C to identify the components of VLPs. The cell lysates were used to normalize loading conditions for SARS-CoV-2 structure proteins. These samples were resolved by a 12.5% SDS-PAGE separating gel and subsequently transferred to a nitrocellulose membrane for Western blot analysis with the indicated antibodies against SARS-CoV-2 S RBD S2 or N Flag tag. GAPDH was the loading control.4/well overnight. Threefold serial dilutions of a commercially available rabbit anti-RBD monoclonal antibody or anti-RBD serum samples were prepared and mixed with an equal volume of pseudoviruses. The mixture was incubated at 37°C for 1 h before adding 293T-hACE2 cells. At 48 h after infection, cells were lysed to detect luciferase signal with the ONE-Glo luciferase assay system  to determine the neutralization levels.The SARS-CoV-2 pseudovirus neutralization assay was used to test the effect of mutation on S antigenicity. The horse or rhesus monkey anti-RBD serum samples collected after two immunizations were gifts from Ningyi Jin and were inactivated at 56°C for 30 min. 293T-hACE2 cells were seeded in 96-well tissue culture plates at a density of 1 × 10t test or one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test.Data are presented as means ± standard deviations (SD) from 3 to 6 replicates. Data are representative of those from three independent experiments. Statistical analyses of the data were performed by using GraphPad Prism version 8.4.3. Statistical comparisons were made using a two-tailed paired or unpaired Student


We, hence, investigated whether oral administration of sodium thiosulfate (STS), a clinically applicable H2S-based therapy, can exert a protective effect against hypertension in an adenine-induced CKD rat model. Eight-week-old male Sprague–Dawley rats were fed with 0.5% adenine chow for 3 weeks to induce CKD. After 1 week, the rats were divided into two groups: one without and one with STS (2 g/kg body weight/day) in drinking water for 2 weeks. Treatment with STS lowered systolic and diastolic blood pressure by 7 and 9 mm Hg, respectively. Renal H2S-generating enzyme expression was inhibited by CKD, while STS therapy increased plasma levels of H2S and thiosulfate. Additionally, restoration of nitric oxide bioavailability and rebalance of the renin–angiotensin system may contribute to the protective effects of STS. Our data suggest that the oral administration of STS improves hypertension in an adenine-induced CKD model, which brings us closer to the clinical translation of H2S-targeting therapy in CKD-induced hypertension.Hypertension is highly prevalent in chronic kidney disease (CKD). Hydrogen sulfide (H Hyperure (BP) , being c disease ,2,4. In rtension ,6. Conve disease .2S from the substrate L-Cysteine occurs through three enzymes: cystathionine-lyase (CSE), cystathionine-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3MST) [2S is produced by cysteine amino transferase (CAT) and D-amino acid oxidase (DAO). Our prior work showed that dietary supplementation with L-Cysteine (the precursor of H2S) protects spontaneously hypertensive rats (SHRs) against high-salt-induced hypertension and kidney damage via the reduction of oxidative stress, mediation of the H2S-generating pathway, and restoration of the renin–angiotensin system (RAS) [Enzymatic production of He (3MST) . In an aem (RAS) .2S can be non-enzymatically produced from organic thiol. Thiosulfate, an intermediate in oxidative H2S metabolism, can alternatively be reduced and regenerate H2S [In addition, Hrate H2S . Althougrate H2S , limitedThe adenine diet model results in progressive kidney injury and several complications, truly mimicking the pathophysiology of human CKD . Our prin = 32) were obtained from BioLASCO Taiwan Co., Ltd. . On arrival, the rats were maintained in our animal facility awarded full accreditation from AAALAC International. The procedures used in this study were according to the rules of the Care and Use of Laboratory Animals of the National Institutes of Health and the IACUC of Chang Gung Memorial Hospital (Permit # 2020073101).Male Sprague–Dawley (SD) rats (n = 8 per group): normal diet (ND), diet supplemented with 0.5% adenine from 8 weeks of age for 3 weeks (CKD), normal diet with STS (2 g/kg body weight/day) in drinking water from 9 weeks of age for 2 weeks (ND + STS), and diet supplemented with 0.5% adenine from weeks 8 to 11 and STS from weeks 9 to 11 (CKD + STS). The doses of adenine and STS used here are based on previous studies conducted on rats [Afterward, each rat was randomly assigned to one of four treatments  system connected to an Agilent 6470 Triple Quadrupole LC/Mass Spectrometry (MS) . We added internal standard phenyl 4-hydroxybenzoate (PHB) to the extraction solvent. We detected thiosulfate derivative pentafluorobenzyl (PFB)-S2O3H and H2S derivative sulfide dibimane (SDB). Selected reaction monitoring mode was used to detect target compounds with a targeted m/z 212.99→93, m/z 415→223, and m/z 292.99→81 for PHB, SDB, and PFB-S2O3H, respectively.According to our validated protocol , concent2S production were determined: Cbs (encoding for protein CBS), Cth (CSE), Mpst (3MST), and Dao (DAO). We also determined the renal expression of the genes belonging to the RAS, including Ren (renin), Ace (angiotensin converting enzyme-1), Agtr1a (angiotensin II type 1 receptor), Ace2 (angiotensin converting enzyme-2), Agtr2 (angiotensin II type 2 receptor), and Mas1 (angiotensin (1–7) receptor MAS). Rn18s was used as the internal control as it was expressed at a constant level across the sample set. The comparative threshold cycle (Ct) method (2−ΔΔCt), represented as x-fold expression, was used to compare the mRNA levels. Rat kidney cortex tissue was homogenized in lysis buffer, and total RNA was extracted using the TRIZOL method , as described earlier . Two-stew/v) in 1% (v/v) acetic acid; Sigma-Aldrich, St. Louis, MO, USA) for 10 min on the rocker, followed by a brief rinse in distilled water so that the lanes and bands were clearly visible. The membranes were then scanned and the TIFF file saved for later quantification. After that, the membranes were incubated in distilled water for two washes of 5 min each and we proceeded with the blocking. The membranes were blocked in phosphate-buffered saline-Tween (PBS-T) containing 5% nonfat milk. For the detection of H2S-generating enzymes, the membranes were incubated with the following antibodies: a rabbit monoclonal anti-rat 3MST antibody , a mouse monoclonal anti-rat CBS antibody , or a rabbit polyclonal anti-rat CSE antibody . The bands were detected using an enhanced chemiluminescence reagent . The abundance of protein expression was quantified by densitometry  and calculated as the integrated optical density (IOD) in relation to the background value. The relative protein abundance was expressed as IOD/PonS to correct the variations in total protein loading.As previously described , kidney We used the HP Agilent 1100 HPLC System  with fluorescence detection of O-phthalaldehyde/3-mercaptopropionic acid (OPA/3-MPA) derivatives to measure NO-related parameters in the plasma. These parameters include L-Arginine, L-Citrulline (the precursor of L-Arginine), and NO synthase inhibitor asymmetric and symmetric dimethylarginine (ADMA and SDMA). The standards contained 1–100 mM L-Citrulline, 1–100 mM L-Arginine, 0.5–5 mM ADMA, and 0.5–5 mM SDMA. L-Arginine was divided by ADMA plasma levels for calculating the L-Arginine-to-ADMA ratio, a determinant of NO bioavailability .p < 0.05 was considered significant. Statistical analyses were performed using SPSS software .All data are presented as the mean ± the standard error of the mean. Statistical analyses were performed using one-way ANOVA or two-way ANOVA where appropriate. Tukey’s post hoc test was applied where multiple comparisons were made. The body weight (BW) of the CKD and CKD + STS groups was lower, while the kidney weight (KW) and the KW-to-BW ratio were higher compared to those of the control . Baselin2S pathway are shown in 2S and thiosulfate levels and the renal mRNA expression of H2S-generating enzymes. Compared to ND, the plasma H2S level was higher in the other three groups  STS treatment attenuated the elevation of BP in rats with adenine-induced CKD, (2) CKD caused a reduction in renal H2S-generating enzyme expression, while STS treatment had a negligible effect on their levels, (3) STS therapy increased plasma levels of H2S and thiosulfate, (4) the beneficial effect of STS was associated with the reduction of ADMA and the restoration of L-Arginine-to-ADMA ratio in the plasma, and (5) STS treatment enhanced RAS gene Ace2 and Mas1 but decreased Agtr1a.This work casts new light on the interaction between H2S-generating enzyme protein levels and/or activity is involved in the pathogenesis of hypertension and CKD [2S exhibits a wide range of biological functions [2S and thiosulfate while it had little effect on renal H2S-generating enzymes.The findings of this research are in line with prior studies indicating that decreased renal H and CKD ,16. Althunctions , limited2S [2S can be endogenously produced or exogenously delivered. We have previously shown that the administration of precursors of H2S, such as L-Cysteine or N-Acetylcysteine, could enhance endogenous H2S production and provide protection against hypertension [2S-generating enzymes are inhibited by CKD in our model, this might not be an appropriate approach for CKD-induced hypertension. H2S donors have been evaluated as having the therapeutic potential of exogenous H2S [2S donors might be toxic whenever they cause a short-term increase in H2S to supraphysiological concentrations, which limit their clinical translation.The reduction in BP observed in this study is consistent with previous findings displaying the vasorelaxant properties of H2S ,8,13. H2rtension . Considenous H2S . However2S [2S in our body. Treatment with STS for 2 weeks in our study reduced SBP and DBP by 7 and 9 mmHg in rats with adenine-induced CKD, respectively. The BP lowering effect of STS in our work is similar to that previously reported in an NO deficiency model [2S. As STS has been used for the treatment of calciphylaxis and has a proven safety profile [The current study provides another possibility in the use of sodium thiosulfate. As dynamic conversion exists between thiosulfate and H2S , thiosulcy model . In the  profile , our dat2S is to enhance NO signaling [2S, NO, and carbon monoxide (CO) against hypertension [2S can modify cysteine residues on key signaling molecules such as nuclear factor erythroid 2-related factor 2 (NRF2) and nuclear factor κB (NFκB), thereby promoting antioxidant and anti-inflammatory effects [A key vasorelaxant action of Hignaling ,17. In ortension ,6. H2S c effects . NO can  effects . Because2S was reported to inhibit the activity of renin [Another probable beneficial effect of STS is related to its ability to rebalance the RAS. Consistent with prior research indicating that the aberrant activation of the classic RAS is associated with CKD , our datof renin , we foun2S-generating enzymes by immunohistochemistry. Another limitation is that we did not test other treatment doses or duration of STS. Whether STS treatment displays a dose- or time-dependent effect deserves further clarification. Although the use of STS has been approved for the treatment of calciphylaxis in patients with CKD [2S [There are still some limitations. One limitation of this study is that we did not analyze other organs that control BP. The protective effects of STS against hypertension may be attributed to other organ systems, such as the vasculature, the heart, or the brain. For future research, it would be interesting to conduct a thorough examination of aforementioned protective mechanisms of STS in an organ-specific manner. Second, we did not determine which cells in the kidney are involved in the expression of Hwith CKD , its saf CKD [2S , additio2S pathway might have therapeutic potential in CKD-induced hypertension.In conclusion, we demonstrated that the oral administration of STS improves hypertension in an adenine-induced CKD model, which brings us closer to clinical translation. Approaches targeting the H


In this study, Prussian blue@Carbon-dot (PB@C-dot) hybrids have been developed by one-step hydrothermal method. The incorporation of C-dots into Prussian blue thin film as a way of improving its electrochromic performance was investigated. The structure of the PB@C-dot hybrid was characterized through X-ray diffraction, Raman spectroscopy and scanning electron microscopy. The electrochromic properties showed that incorporation of 10 mL C-dots into the film showed higher optical contrast of 1.6 and superior coloration/bleaching response of 10 and 3 s. It is proposed that the C-dots component used in the construction of the PB@C-dot hybrid plays a key role to achieve superior electrochromic performance. Electrochromic materials have attracted much attention because of their potential applications in antiglare mirrors, smart windows and sunglasses ,2,3. Wid2) electrochromic film, Br−/Br3− with Li+ as electrolyte was fabricated, and it shows advantages of high transparency in a bleached state when compared with conventional PECD. Tina et al. ) and 0.25 g trisodium citrate (C6H5Na3O7) were dissolved in the mixed solution of C-dots and distilled water; among them, the six different volume ratios of C-dots/H2O were chosen, i.e., 0/60, 2/58, 5/55, 10/50, 20/40 and 30/30, respectively. Afterward, 0.4 mL 36% hydrochloric acid (HCl) was dropped into the solution. After stirring for 20 min, the resulting solution was transferred into a 50 mL Teflon-lined stainless-steel autoclave containing a FTO substrate (1 × 5 cm2 in size) with the conducting side facing down. Then, the sealed autoclave was maintained at 120 °C for 1 h. After cooling naturally to room temperature, the product was washed with distilled water and dried at 60 °C in a vacuum oven. The final products were denoted as PB@C-dot-0, PB@C-dot-2, PB@C-dot-5, PB@C-dot-10, PB@C-dot-20 and PB@C-dot-30, respectively.All the chemicals are of analytical grade and used without any further purification. Firstly, the C-dots were prepared according to the reported method . BrieflyThe crystal structure and surface morphology of the PB@C-dot films were investigated by X-ray diffraction  with Cu Kα radiation and scanning electron microscopy . Raman spectra were recorded on an Invia Renishaw spectrometer using an excitation light wavelength of 532 nm. Electrochromic properties of these films were measured via UV-Vis spectrophotometer , by applying constant potentials and square-wave potentials using an Autolab PGSTAT302N workstation. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were conducted on a CHI660E electrochemical workstation using a three-electrode system with 1 M KCl as the electrolyte, PB@C-dot film as the working electrode, saturated calomel electrode (SCE) as the reference electrode and Pt sheet as the counter electrode, respectively.−1 and 2141 cm−1 correspond to the stretching vibration of the C≡N bond from the cyanide ligands in PB. The bands at 266 cm−1 and 530 cm−1 are assigned to Fe-C, Fe-CN and Fe-N modes [−1 (D-band) and 1590 cm−1 (G-band), suggesting that the C-dots were involved in the PB growth [XRD was applied to characterize the crystal phase of the PB@C-dot films. As shown in -N modes ,28. MeanB growth .The surface morphology of the PB@C-dot films were investigated by SEM, as presented in −1, as shown in 2+→Fe3+) and reduction reaction (Fe3+→Fe2+), respectively [In order to understand the electrochemical performance of PB@C-dot films, the CV curves were estimated in the potential range from −0.6 to 1.6 V at a scan rate of 50 mV sectively ,31. Compectively ,33. Whenectively ,35. Howect) and bleaching time (bt) of the PB@C-dot films can be evaluated according to the time to reach 90% of the entire change in absorbance. The results of the study are summarized in c/tbt of electrode containing no C-dots was 15/12; the addition of 5 mL and 10 mL C-dots quickened this to 12/6 and 10/3, respectively. The c/tbt for the electrode containing 30 mL C-dots was 15/10. Notably, PB@C-dot-10 hybrid had a slower coloration time (10 s) compared to its bleaching time (3 s). This result may be explained by a slower interaction of K+ ions and electrons, leading to a slower coloring speed. Furthermore, the accumulated K+ ions had a higher diffusion ability; therefore, they required the shortest bleaching time [+ ions and electrons within the PB@C-dot film. In order to further demonstrate the electrochemical behaviors of the PB@C-dot films, EIS was carried out within the frequency range from 10−2 to 105 Hz. As shown in ing time . This reThis study investigated the effects of the addition of C-dots on the electrochromic performance of PB film. The accelerated optical modulation and fast coloration/bleaching efficiency show that the addition of C-dots into the PB film enhances the electrochromic performance of the electrode, with 10 mL C-dots additions accelerating the coloration/bleaching time of the electrode notably. The excellent electrochromic performance is mainly attributed to the efficient charge transfer ability of C-dots. In this integrated system, the merits of both C-dots and the PB may create synergistic effects and lead to very high electrochromic efficiency and superior optical modulation. Thus, the incorporation of C-dots into the electrochromic materials maybe useful to improve both electrochromic performance and electrochemical behavior.


Verticillium dahliae and inhibiting miRm0002 lines were more sensitive, compared to the wild-type (WT) control. Some physiological indicators were selected and the results showed that SOD, POD, and CAT activities were significantly increased in Verticillium wilt-infected overexpressing miRm0002 lines, indicating that the expression of miRm0002 activates the antioxidant system. QRT-PCR assay showed that the transcript expression of miRm0002 candidate target ARF8, a gene encoding auxin response factor was negatively related to miRm0002 in WT as well as transgenic eggplants. However, RLM-RACE mapping and degradome sequencing showed miRm0002 could not cleave the sequence of ARF8. Taken together, these data suggest that miRm0002 plays a positive role in the defense response of eggplant against Verticillium wilt. Verticillium wilt is a major disease that severely affects eggplant production, and a new eggplant miRNA named miRm0002 identified through high-throughput sequencing was highly induced by Verticillium wilt infection. However, the miRm0002 function was still elusive. In this study, the sequence of the miRm0002 precursor was cloned and transgenic eggplants were constructed. In vivo inoculation test and in vitro fungistatic test showed that overexpressing miRm0002 lines were more resistant to  Verticillium dahliae, reduces up to 34.1–42.5% of eggplant production worldwide , or, or30], Total RNA was extracted from leaves of eggplant Suqi seedlings in four-leaf stage using the Trizol reagent , then it was directly ligated to the RNA 5′ adaptor (5′-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGCCCCCCCCCCCCCCC-3′) and 3′ and reverse-transcribed using M-MuLV First Strand cDNA Synthesis Kit (B532435) with oligo (dT) primer. The cDNA samples were amplified by nested PCR according to invitrogen 5′ race system manual. Initial PCR was carried out using the 5′ outer primer (5′-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGCCCCCCCCCCCCCCC-3′) and gene-specific outer primer (5′-TTCTAGCCCAAACATGCGAGCAAGC-3′). Nested PCR was carried out using 1 μL of the initial PCR reaction, and 5′ nested primer (5′-GCTGTCAACGATACGCTACGTAAC-3′) and gene-specific inner primer (5′-GAGTGACCGTCCAAAGGACCCTGATT-3′). After amplification, 5′-RACE products were gel purified and cloned into the pMD180T vector and the clones were sequenced.Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method  × 100%. In vitro anti-fungal efficacy of crude protein extracts from transgenic and control eggplants was carried out according to the improved mycelium growth inhibition method . The ant−1 cm−1) at 470 nm [2O2 into oxygen and water, and the enzyme activity was determined by the H2O2 decomposition rate at 240 nm [Enzyme solution for antioxidant assay was extracted using the method of Wan et al. , and prot 470 nm . Catalast 240 nm .p < 0.05; **, p < 0.01).The data were analyzed by one-way ANOVA using IBM SPSS Version 19.0. Displayed results are means ± SD (error bar) for two biological replicates and three technical replicates. Asterisks indicate the significance level in one-way ANOVA (*, In summary, this study reveals the positive regulation of miRm0002 in the defense response of eggplant V. wilt by in vivo inoculation test, in vitro fungistatic test, measurement of some physiological indicators in mO, mI lines, and controls. All these results provide not only important insights into the function and mechanism of miRm0002 in eggplant defense against V. wilt, but also theoretical basis for eggplant V. wilt resistance breeding.


CRISPR-Cas systems are immune systems that protect bacteria and archaea against their viruses, bacteriophages. Immunity is achieved through the acquisition of short DNA fragments from the viral invader’s genome. Flavobacterium columnare that carries an acquisition-deficient CRISPR-Cas subtype VI-B system and a complete subtype II-C system. We show that the VI-B locus acquires spacers from both the bacterial and phage genomes, while the newly acquired II-C spacers mainly target the viral genome. Both loci preferably target the terminal end of the phage genome, with priming-like patterns around a preexisting II-C protospacer. Through gene deletion, we show that the RNA-cleaving VI-B system acquires spacers in trans using acquisition machinery from the DNA-cleaving II-C system. Our observations support the concept of cross talk between CRISPR-Cas systems and raise further questions regarding the plasticity of adaptation modules.CRISPR-Cas immune systems adapt to new threats by acquiring new spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition despite variation in spacer content between microbial strains. It has been suggested that such loci may use acquisition machinery from cooccurring CRISPR-Cas systems within the same strain. Here, following infection by a virulent phage with a double-stranded DNA (dsDNA) genome, we observed spacer acquisition in the native host  The repposition . Immunitposition , is inseposition , 3, 5, 7position –10. Mostosition –. Upon reAdaptation is almost universally mediated by a complex consisting of Cas1 and Cas2 that may recruit additional proteins depending on the subtype. For example, type I-D requires Cas4 for correct adaptation , while t13–Spacer acquisition generally occurs through either naive or primed adaptation. Naive adaptation occurs when no preexisting spacers target the incoming genome , while p19–Streptococcus thermophilus, the conservation of the first 10 nucleotides on both sides of the leader-repeat junction is essential for spacer integration  is not optimal, as only half of the potential spacers may functionally target mRNAs. Adaptation in type VI systems has yet to be experimentally demonstrated, and it is unknown if new spacers are acquired through the in trans adaptation model and, if so, how this affects their capacity to target RNA.The recently discovered type VI CRISPR-Cas systems , 37, 39 trans 3941. HowevFlavobacterium columnare has subtype II-C and VI-B CRISPR-Cas loci , while this was the case for only half of the II-C protospacers  with its virulent dsDNA phage FCL-2 (accession no. NC_027125.1)  . We also27125.1) . The natCL-2 see . Only baCL-2 see  and S2.10.1128/mBio.03338-20.7TABLE S1A, either bacteria alone (B), bacteria plus phages (B+P), bacteria plus UV-treated phages (B+UVP), or bacteria plus phages and UV-treated phages (B+P+UVP). B, number of mutants found after the first plating on agar. C, number of colonies after multiple rounds of serially plating the initial colony. (B) Colonies with additional spacers and their sequences obtained after serial plating. ORF numbers indicate phage genes, and “self” indicates bacterial chromosomal genes. Download Table S1, DOCX file, 0.02 MB.(A) Results from colony screening. “Mutants” refers to isolates with an expanded II-C or VI-B array. Copyright © 2021 Hoikkala et al.2021Hoikkala et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03338-20.8TABLE S2Table S2, DOCX file, 0.01 MB.(A) Primers used in the study. (B) Ion Torrent reads and spacer counts from different samples. B+P refers to the treatment with bacteria plus phages. Download Copyright © 2021 Hoikkala et al.2021Hoikkala et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03338-20.9TABLE S3F. columnare strain B185. Array positions indicate distances from the variable end (with 1 being the most recently acquired spacer). Target strand indicates the coding (plus [+]) or template (minus [−]) strand: any crRNA complementary to predicted ORF mRNA is targeting the plus strand. Underlined sequences in the crRNA column and * in the target column indicate mismatches in the corresponding protospacer regions. (B) Preexisting type VI-B spacers in F. columnare strain B185. Array positions indicate distances from the variable end (with 1 being the most recently acquired spacer). Target strand indicates the coding (plus) or template (minus) strand: any crRNA complementary to predicted ORF mRNA is targeting the plus strand. (C) Preexisting type II-C spacers in F. columnare strain B245. Array positions indicate distances from the variable end (with 1 being the most recently acquired spacer). Target strand indicates the coding (plus) or template (minus) strand: any crRNA complementary to predicted ORF mRNA is targeting the plus strand. Underlined sequences in the crRNA column and * in the target column indicate mismatches in the corresponding protospacer regions. (D) Preexisting type VI-B spacers in F. columnare strain B245. Array positions indicate distances from the variable end (with 1 being the most recently acquired spacer). Target strand indicates the coding (plus) or template (minus) strand: any crRNA complementary to predicted ORF mRNA is targeting the plus strand. Underlined sequences in the crRNA column and * in the target column indicate mismatches in the corresponding protospacer regions. Download Table S3, DOCX file, 0.03 MB.(A) Preexisting type II-C spacers in Copyright © 2021 Hoikkala et al.2021Hoikkala et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Next, we focused on CRISPR adaptation at the bacterial population level by amplifying by PCR the variable ends of both arrays directly from the same liquid cultures. In contrast to individual colony screening, we observed spacer acquisition in both the phage mixture treatment (B+P+UVP) (data not shown) and the phage-only treatment (B+P) . The dif10.1128/mBio.03338-20.1FIG S1Flavobacterium columnare B185. The variable ends of the arrays were amplified from population-level DNA samples of five replicate cultures (a to e) in the presence or absence of phage FCL-2. (A) II-C array with phages; (B) VI-B array with phages; (C) II-C array without phages; (D) VI-B array without phages. Download FIG S1, TIF file, 1.8 MB.Gel electrophoresis of type II-C and VI-B CRISPR arrays in Copyright © 2021 Hoikkala et al.2021Hoikkala et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The lengths of new spacers varied between 28 and 32 bp, with the majority at 30 bp in both loci . We divi10.1128/mBio.03338-20.2FIG S2FIG S2, TIF file, 1.0 MB.PAM sequences from the unique spacer data set. (A) The 15-bp regions upstream and downstream of each protospacer displayed as WebLogos from replicates b and e for type II-C and replicates b, d, and e for type VI-B. PAMs were determined using the guide-oriented approach. (B) Variants of SAMs in both loci. Stacked bars show the proportions of different variants of SAMs in both loci and target genomes. Download Copyright © 2021 Hoikkala et al.2021Hoikkala et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the F. columnare strain B245. As a control plasmid, we replaced the matching protospacer sequence with a nonmatching sequence. Using Escherichia coli as the donor, the efficiency of conjugation was roughly three times lower in the matching protospacer treatment than in the control treatment under antibiotic selection  . As speculated to occur with Listeria monocytogenes, small colonies may result from low Cas9 expression, allowing the persistence of a small number of plasmids in the cell  . Survivit test]) . The II-the cell .NNNNNTAAA-3′)  (described in detail below). Both simulations were run 1,000 times, with each run sampling 430 spacer positions (the size of the VI-B spacer pool). We calculated the proportions of shared spacers between each run and the II-C spacers and found that on average, 6.3%  of the random spacers and 29.6%  of the PAM-adjacent spacers had a match in the subtype II-C spacer set (P < 10−10 by a one-tailed test), which suggests that new spacers are sampled from a limited protospacer pool that is common to both loci.To calculate the proportions of identical new spacers between the loci, we pooled unique phage-targeting spacers from both loci from all replicates. Remarkably, 58% of VI-B spacers were also found in the larger II-C spacer pool, suggesting that the loci share a predisposition toward a set of spacers. For statistical comparison, we simulated spacer acquisition across the phage genome by sampling random positions either freely or next to predicted PAM sites . The su10.1128/mBio.03338-20.3FIG S3FIG S3, TIF file, 0.2 MB.Transcription directions of subtype II-C and VI-B arrays. The RNA samples were taken in the absence of phages to provide enough RNA for sequencing, leading to minimal yet sufficient expression of the arrays to determine the transcription direction. (a) Eight reads revealed type II-C transcription from within the array toward the leader (167.8 reads per million). (b) Forty-seven reads mapped on the type VI-B array revealed the transcription direction starting from the leader end (986.6 reads per million). The VI-B locus was reverse complemented from its genomic orientation for easier comparison. Download Copyright © 2021 Hoikkala et al.2021Hoikkala et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the NNNNNTAAA was observed downstream of most (∼63%) phage-targeting spacers (F. columnare (TTTANNNNN) and located upstream  of the loci is seemingly different, the spacer acquisition motif (SAM) is shared by the loci. The term TIM is not strictly applicable to the studied RNA-targeting type VI systems, and their interference efficiency has instead been shown to be affected by short protospacer-flanking sites (PFSs) , 39. HerNNNNNTAAA-3′) across the phage genome  phage genomes , we examined the ability of each spacer crRNA to bind mRNA by assessing their complementarity to predicted open reading frames (ORFs) from both target genomes. Spacers from all replicates within a locus were also pooled for an overall estimate. For statistical context, we calculated the probabilities of mRNA-targeting proportions in both loci with a binomial distribution that uses sample sizes matching those of the pooled spacers and assumed an equal probability of targeting each strand. Deviation from these expected distributions signals possible bias toward or away from mRNA targeting.P = 0.074 by a one-tailed test), suggesting a weak or nonexistent bias toward non-mRNA-targeting spacers. VI-B mRNA targeting on the bacterial genome was rarer and significantly below expectation at 0.359 (P < 10−9 by a one-tailed test) (P = 0.0059 by a one-tailed test) (P = 10−10 by a one-tailed test)  . When coed test) , suggested test) .P < 10−10 by a one-tailed test), while self-targets were not clearly biased (P = 0.052 by a one-tailed test) (P = 0.124 by a one-tailed test). The proportions of absolute phage-targeting II-C spacers were slightly lower than in unique counts, at 0.319, and deviated strongly from expectation (P < 10−10) (Subtype II-C mRNA-targeting proportions in the unique spacer set were the same for both target genomes (0.37 and 0.37). Given their vastly different sample sizes, the proportion of mRNA-targeting spacers on the phage genome was significantly below expectation (ed test) . In the ed test)  but stem< 10−10) .n = 81,490), and the proportions are 51.6%/48.4% for FCL-2 . Another possibility is that competition between RNA and DNA targets for Cas9 association would impact subtype II-C immunity negatively.The minimal phage mRNA targeting in subtype II-C spacers was unexpected, as Cas9 functionality is indifferent to which strand is being targeted. One potential explanation would be a strongly uneven distribution of PAM sequences on the coding versus template strands, but this is not the case in either genome: the proportions of PAM sequences (5′-TAAA-3′) in annotated coding sequences from B185 are 51.2%/48.8% (coding/template) (F. columnare B185 (n = 7) (The lack of new VI-B self-mRNA-targeting spacers is expected due to their possible dormancy-inducing effects . This is (n = 7) . However (n = 7)  and C, w (n = 7) . If the F. columnare isolates, there was a saturation of phage mRNA-targeting VI-B spacers  . Our neF. columnare subtype II-C and VI-B CRISPR-Cas loci share spacer acquisition machinery. To verify this, we deleted cas1 from the subtype II-C locus from F. columnare strain B245. Using the resulting B245 Δcas1 strain, we performed a prolonged adaptation experiment with 10 phage-exposed (phage V156) and 2 bacterium-only replicates. As a control, we used a “reversion wild-type” (rev-wt) strain that resulted from an alternative outcome of the mutation process and contains an intact cas1 . We did not observe spacer acquisition in either CRISPR array in any of the 12 Δcas1 cultures  Subtype II-C array. (B) Subtype VI-B array. Ten replicates of Δcas1 and rev-wt cultures (annotated with numbers) were grown with phages along with two replicates without phages. Of the 12 rev-wt cultures, only 2 (replicates 1 and 5) had grown at the end of the 3-week experiment, both having acquired new spacers in the II-C and VI-B CRISPR-Cas loci. All Δcas1 replicates had grown by the end of the experiment, but none had acquired new spacers in either locus. The ladder used on gels is a 1-kb Plus DNA ladder (Thermo Fisher Scientific). Download FIG S4, TIF file, 0.7 MB.Spacer acquisition experiment using the ΔCopyright © 2021 Hoikkala et al.2021Hoikkala et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the cas1 is surprising as the inability to acquire new spacers should not abrogate interference using preexisting ones. These strains also belong to different genetic groups, and thus, their differences are not limited to spacer content. Further experiments to explore the effect of spacer diversity and the role of different cas genes and other host defenses in F. columnare are required.The inability of B245 rev-wt to survive in the presence of phages is in contrast with the previous experiment performed with strain B185 and phage FCL-2. This difference may partly stem from the experimental setups, as the bacterial strains B245 and B185 were cultivated in different media and for 3 weeks and 1 week, respectively . Also, strain B245 has four preexisting, fully matching phage mRNA-targeting spacers in the variable end of the VI-B arrays C and D, F. columnare, subtypes VI-B and II-C have similarly sized repeats (36 bp), with sequence similarity mostly in the leader-distal ends (5′-GTTGT-3′)  and poly(T) regions up to 100 bp from the junction (cas1 and cas2 in their VI-B locus). II-C and VI-B repeats shared the same leader-distal motifs across the species, but F. columnare was the only species with similarly sized II-C and VI-B repeats (II-C repeats in the other species were on average 11 bp longer)  did not acquire new spacers (In TTGT-3′) . The lacA system . The F. junction . To see  longer) . Leadersolumnare , which molumnare , 27, 61, spacers .10.1128/mBio.03338-20.5FIG S5FIG S5, TIF file, 0.2 MB.Alignments of II-C and VI-B repeats from species that carry both loci. The 3′ ends are leader adjacent. Download Copyright © 2021 Hoikkala et al.2021Hoikkala et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03338-20.6FIG S6FIG S6, PDF file, 0.9 MB.Alignments of the II-C and VI-B leaders from species that carry both loci. The 5′ end is repeat adjacent, and open reading frames downstream of the leader are annotated with yellow rectangles. (A) Alignment of type II-C leaders (200 bp). (B) Alignment of type VI-B leaders (200 bp). (C to H) Alignments of both leaders within clusters. (I) Phylogenetic tree of Cas1 and the derived clusters. (J) Nucleotide similarity of leaders in the clusters. Download Copyright © 2021 Hoikkala et al.2021Hoikkala et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the F. columnare spacer acquisition and interference. During acquisition, new spacers are captured by the Cas1/2 acquisition complex (possibly aided by Cas9) in a specific conformation relative to the PAM  for testing interference. Another possibility for low efficiency against phages may be phage-encoded anti-CRISPR proteins  at day 7 see . Colonie2 (4 mM) was required for amplification. To minimize PCR bias, four separate PCRs were performed for each of the five replicates, which were then pooled and cleaned using a Qiagen MinElute reaction cleanup kit. The resulting 10-μl samples were run on a 2% agarose gel (4.6 V/cm) for 2 h 45 min in Tris-acetate-EDTA (TAE) buffer. The expected PCR product sizes for the wild-type array  as well as for the expanded arrays with one new repeat-spacer unit  were extracted from the phage treatment using X-tracta (Sigma) extraction tools and a gel extraction kit (Qiagen) using MinElute columns (Qiagen). In the bacterium-only treatments, the wild-type PCR products from both loci were extracted as controls. All extractions underwent two gel purification rounds to reduce contamination.Total DNA was extracted from 1-ml liquid samples using a DNeasy blood and tissue kit (Qiagen). The variable ends of subtype II-C and VI-B CRISPR loci  from the phage and bacterium-only treatments were amplified with DreamTaq (Thermo Fisher Scientific), with one primer binding to the leader sequence (C2_B185_F and C1_B185_R) and one binding to the second (C1_B185_F) or third (C2_B185_R) spacer . The PCRTaq DNA polymerase (Thermo Fisher Scientific), the primers M13-B185_223bp_C2F and P1-B185_223bp_C2R, as well as the IonA_bc_M13 primer that contained multiplexing barcodes and an M13 adaptor (2 (4 mM) using the following program: 95°C for 5 min; 31 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 1 min; and 72°C for 15 min. The reaction mixture was then purified with Agencourt AMPureXP (Beckman Coulter) and reamplified using primers IonA-bc_M13 . Th. ThTaq DNontrimmed reads were obtained from PGM and sorted by their barcodes. Trimmomatic 0.36  was usedF. columnare was determined using DoriC 10.0.0  regions due to the large proportion of only-DNA-targeting type VI-B spacers. Similarly, we follow the guide-oriented approach for both loci, as is common with DNA-targeting systems  PAM is .NNNNNTAAA-3′). Sampling was repeated 1,000 times, and similarities of the simulated spacers were compared with the type II-C spacer pool using CD-hit-est-2D as described above. A normal distribution was fit onto the simulated distributions using the R function fitdistr from the MASS package. The probability of the observed similarity, given the null hypothesis, between subtype II-C and VI-B spacers (0.58) was measured using the function pnorm from the upper tails of the distributions.To calculate the proportion of subtype VI-B spacers that are identical to II-C spacers, unique VI-B spacers from all replicates  were first pooled. CD-hit-est was used to extract unique spacers from the pooled spacer set (clustering threshold of 0.8). The process was repeated for the type II-C spacers from replicates b and e. Next, the pooled unique spacer sets from both loci were compared to each other with CD-hit-est-2D using a similarity threshold of 0.9. The number of resulting clusters was then used to calculate the proportion of VI-B spacers that had a match in the type II-C spacer pool. Simulated sets of 430 spacers  were generated by sampling the phage genome either at random positions or from the 1,672 predicted PAM sites (5′-The ability of each spacer to target an ORF’s transcript was determined by two rules: (i) the crRNA of the spacer must be complementary to the coding strand, and (ii) the protospacer must be fully contained within an ORF. Intergenic spacers were excluded from the analysis. The number of mRNA-targeting spacers was divided by the total number of ORF-targeting spacers in the sample to obtain the proportion of mRNA-targeting spacers for each replicate separately. We also performed the analysis for pooled spacers from the replicates. Pools were made on the basis of locus and target, resulting in four pools .P values on their probability given the null hypothesis described above (one-tailed test using the pbinom function in R). This analysis was done separately for the unique and absolute spacer counts.The mRNA proportions from pooled spacers were compared to a null hypothesis that assumed an equal chance of a spacer targeting both strands. Since intergenic spacers were excluded from the analysis, a binomial distribution could be used to construct the model. Separate distributions were created for each spacer pool to account for different numbers of spacers in each. The observed values in both loci were then compared to their respective distributions to yield direct F. columnare B185 was grown without phages in 10 ml of Shieh medium at 24°C with constant shaking at 150 rpm. Next, 24-h-grown cultures  were centrifuged  and stored in RNAlater (Qiagen), until RNA was extracted with an Ambion MicrobExpress mRNA purification kit. RNA quality was verified using an Agilent Bioanalyzer 2100 RNA nanochip, and samples with RNA integrity values of >9.5 were selected for library preparation . The cDNA was sequenced with Ion Torrent using a 318 chip (v2) and an internal ERCC  spike-in control, after ensuring cDNA quality . Our results support previous reports showing type VI-B crRNA transcription from the leader end .The direction of CRISPR array transcription was determined from bacterial RNA sequencing (RNA-seq) data. ader end  and typeader end   and primers 2322 (adding a KpnI site) and 2323 (adding a BamHI site). The PCR product was digested with KpnI and BamHI and ligated into the plasmid pMS75 (cas1 was PCR amplified using primers 2324 (adding a BamHI site) and 2325  (E. coli S17-1λpir into F. columnare strain B245 by conjugation. One milliliter of a culture of the recombinant E. coli strain grown overnight was inoculated into 9 ml of LB containing 100 μg/ml ampicillin and incubated with shaking at 37°C until the OD at 600 nm (OD600) reached 0.6. Similarly, 5 ml of a culture of F. columnare B245 grown overnight was inoculated into 25 ml fresh TYES  broth  agar medium  . The PCRs) broth  and incur medium . After icas1 and B245_rev (rev-wt). The latter is a reversion mutant where the integrated plasmid was lost by recombination in a manner that regenerated the wild-type sequence. Phage V156 (cas1 and rev-wt) was measured with a standard double-layer method, where 300 μl of a turbid bacterial culture was mixed with 3 ml of TYES medium with 0.1% mucin and 1% agar. The mixture was poured onto a TYES agar plate, and 10 μl of phage V156 dilutions (10−1 to 10−7) were spotted in duplicates onto the solidified agar. The number of plaques was counted after 2 days from the 10−7 dilutions. Phage titers (PFU per milliliter) were 1.9 × 1010 and 3.7 × 1010 on the Δcas1 and rev-wt strains, respectively.Strains used in the new spacer acquisition experiment were Δage V156  was used600 of 0.45. Ten phage-infected cultures in 0.1× TYES medium as well as two bacterium-only cultures were started from these cultures grown overnight. The spacer acquisition protocol differed from the one used with B185, as follows: medium TYES (versus Shieh medium), volume of 1 ml (versus 5 ml), and duration of growth in 0.1× medium of 3 weeks (versus 5 days). The protocol was prolonged because follow-up experiments showed that longer incubation in diluted medium resulted in more efficient spacer acquisition. The medium or its volume has no detectable effect on the acquisition efficiency (data not shown) and was changed for practical reasons. After 3 weeks in diluted medium (0.1×), the cultures were transferred to regular TYES medium (1×), and the CRISPR arrays were PCR amplified after 2 days. All 12 phage-containing cultures of the Δcas1 strain showed growth in 1× medium, in contrast to only 2 of 12 replicates of the rev-wt cultures. The variable ends of both CRISPR arrays in all surviving cultures were PCR amplified using primers B245_C1_F and F_col_C1_R (B245 II-C array) and primers F_col_C2_F and B245_C2_R (B245 VI-B array)  .NNNNNTAAA-3′. Two plasmids were constructed: one with a protospacer matching the most recent II-C spacer (next to the leader sequence) of F. columnare strain B245 (ACTGCTAAAGGTAATTTTAAAACAAATGAGTATGTACGA [the PAM sequence is underlined]) and one with a nonmatching control sequence (ACTGCTAAAATCAGATCTAATCTCTATGTCAATGTATGA [the PAM sequence is underlined]). The insertions were added to the plasmid at an intergenic multiple-cloning-site region using the Q5 site-directed mutagenesis kit (New England BioLabs) and the oligonucleotide pair pAS43_protoSDM_F/R (matching protospacer) or pAS43_protoSDM_neg_F/R (nonmatching protospacer) (E. coli strain S17-1λpir. Successful transformation and insertion were verified by colony PCR and Sanger sequencing (primer pair pAS43_MCS2_seq_F/R) (A derivative of plasmid pAS43  (with a ospacer) . The iniseq_F/R) .F. columnare strain B245. Both the donor and the recipient cells were first grown overnight from a freezer stock. The turbid cultures were then transferred to fresh medium (LB for E. coli and TYES medium for F. columnare). E. coli was grown to an OD600 of 0.95, and the recipient F. columnare strain was grown to an OD600 of 0.414. The conjugation protocol was performed similarly to the cas1 deletion (described above), and the final donor/recipient mixtures were spotted on 0.45-μm filters resting on FCGM plates. After 24 h of growth at 30°C, the cells were scraped from the filters and resuspended in 2 ml TYES liquid medium. Next, 150 μl of the mixed cultures was plated on double-antibiotic selection plates without dilution (cefoxitin for conjugant selection and tobramycin for eliminating E. coli) as well as on tobramycin-only plates with 10−5 and 10−6 dilutions for determining the number of potential recipient cells in each replicate . Colonies were counted and plates were photographed after 72 h at 25°C. The efficiency of conjugation was determined by dividing the number of F. columnare conjugants (double-selection plate) by the number of potential recipients (tobramycin plates) in each replicate.In the quantitative conjugation assay, the plasmids were transferred into cas1, cas2, cas9, and a CRISPR array) and VI-B (containing cas13b and a CRISPR array) loci were identified using CRISPRCasdb . From each strain, repeat sequences from both loci were aligned with Geneious aligner . Next, 200-bp leader sequences were extracted downstream of the expected variable end of the array. The variable ends were primarily determined by the repeats’ direction compared to F. columnare strain B185. These initial predictions were supported by the presence of possible degenerate terminal repeats and alignment scores of both flanking regions compared to the leader of F. columnare B185. To allow the comparison of the highly divergent leaders, the strains were first clustered based on their Cas1 protein . The leaders from both loci within the resulting six clusters (most of which contained single species) were aligned with Geneious aligner . We used strict alignment rules to emphasize the importance of the distance of possible motifs from the repeat-leader junction. Analyses and alignments were performed with Geneious 9.1.8.Microbial species that carry intact subtype II-C  and phage FCL-2 (accession no. NC_027125.1) are available in GenBank. Custom code and instructions for analyzing the raw data and reproducing all figures in the manuscript are in GitHub at https://github.com/vihoikka/spacerAQ_vh.The B245 genome and raw reads from the spacer acquisition experiment have been submitted to GenBank under accession numbers CP071008 and SAMN18022999, respectively. The genomes of


The enteric human adenoviruses of species F (HAdVs-F), which comprise HAdV-F40 and HAdV-F41, are significant pathogens that cause acute gastroenteritis in children worldwide. The early transcription unit 3 (E3) of HAdVs-F is markedly different from that of all other HAdV species. To date, the E3 proteins unique to HAdVs-F have not been characterized and the mechanism by which HAdVs-F evade immune defenses in the gastrointestinal (GI) tract is poorly understood. Here, we show that HAdV-F41 infection of human intestinal HCT116 cells upregulated the expression of MHC class I-related chain A (MIC A) and MIC B relative to uninfected cells. Our results also showed that, for MIC B, this response did not however result in a significant increase of MIC B on the cell surface. Instead, MIC B was largely sequestered intracellularly. Thus, although HAdV-F41 infection of HCT116 cells upregulated MIC B expression, the ligand remained inside infected cells. A similar observation could not be made for MIC A in these cells. Our preliminary findings represent a novel function of HAdVs-F that may enable these viruses to evade immune surveillance by natural killer (NK) cells in the infected gut, thereby paving the way for the future investigation of their unique E3 proteins. HAdVs cause partially overlapping, species-specific diseases associated with infections of the respiratory , urinary (species B), gastrointestinal (species A and F), and ocular (species D) systems [HAdVs represents a large family of genetically diverse pathogens. To date, more than 100 different HAdVs have been identified and classified into seven species, A to G  was grown in 50–60% confluent HEK-293 cells (ATCC® CRL-1573™) in DMEM (ATCC® 30-2002) supplemented with 1–2% FBS (ATCC® 30-2020™). Infection was done with virus at passage five at an MOI = 1. After infection, when cells show clear cytopathic effect (round up with increased nucleus size), cultures were harvested with a cell scraper and transferred to falcon tubes. Cell suspensions were centrifuged at 700× g, 4 °C for 10 min, and cells were resuspended in culture medium discharging the supernatant. Samples were subjected to three freeze/thaw cycles (−80 °C and 37 °C), then centrifuged at 1500× g, 4 °C for 10 min. Supernatants were aliquoted in small volumes and kept at −80 °C until use. To determine viral titers, an aliquot of the virus preparation was used for titration in HEK-293 cells via immunohistochemistry using the QuickTiter™ Adenovirus Quantitation Kit , following instructions by the manufacturer. Two cell types were used for the in vitro infection models: human colorectal carcinoma HCT116 cells . HCT116 cells were grown in McCoy’s 5A medium  supplemented with 10% FBS.HAdV-F41 (ATCCFor immunofluorescence (IF) staining, cells were grown on sterile glass coverslips placed on 12-well plates prior to infection with HAdV-F41 (MOI 0.5). After 2 days, cells were fixed in 4% PFA for 10 min, permeabilized with 0.1% triton x-100 for 20 min, and blocked with 1% PBS/BSA for 30 min. For virus staining, a rabbit anti-pVIII polyclonal Ab  was used. Cells were washed and stained for 1 h with a mixture of donkey anti-rabbit secondary Ab conjugated with rhodamine , and Phalloidin-iFluor 488  to stain actin fibers. MIC A and MIC B staining were done using primary mouse anti-MIC A and mouse anti-MIC B Abs. A goat anti-mouse-FITC was used as the secondary Ab. Coverslips were mounted on slides using ProLong™ Diamond Antifade with DAPI  and cured at 4 °C for 24 h in the dark. Samples were analyzed under an Olympus BX51 IF microscope coupled with a CCD camera to acquire individual channels of DAPI, alexa fluor 488 or rhodamine. Acquired channels were merged using ImageJ software v1.53a. Uninfected cells, and secondary Abs alone, produced no relevant signals in the rhodamine channel.g for 10 min, incubated with Zombie Violet Fixable Viability Kit  at 1:500 for 30 min in the dark for discriminating live versus dead cells, washed, and fixed in 4% PFA for 20 min on ice. Cells were then washed and incubated with a mixture of anti-MIC A-phycoerythrin (PE)  and anti-MIC B-allophycocyanin (APC)  Abs for 40 min on ice. Isotype Abs recommended by the manufacturer, as well as uninfected HCT116 cells, were used as negative controls. In the case of samples prepared for extra- and intra-cellular staining, cells were incubated with Ab cocktail for surface staining prior to permeabilization with 0.1% triton x-100 for 10 min at RT. Hexon staining was carried out using a 2Hx-2 monoclonal anti-hexon Ab  [HCT116 cells were infected with HAdV-F41 (MOI 0.5) and expression levels of MIC A and MIC B were determined on the cell surface and intracellularly by flow cytometry on days 2 and 4 post-infection. Infection was assessed based on the expression of intracellular hexon protein. At the harvest time, cells were scraped, washed in PBS by centrifugation at 700× MO, USA)  with furThe GenBank accession number for HAdV-F41 E3 region is M85254 . BioinfoE3 is one of the most divergent gene regions between species : while sThat HAdVs-F are one of the least characterized species may be due to the difficulty of propagating these viruses in most common human cell culture systems that permit replication of all other HAdVs. We established an in vitro culture system for infection of human intestinal HCT116 with HAdV-F41 . To charWe examined if HAdV-F41 impairs the cell surface expression of MIC A and MIC B in HCT116 cells by flow cytometry and IF. We first characterized the basal expression levels of MIC ligands in uninfected HCT116 cells over four days. Results show that for both MIC A and MIC B, expression levels are higher intracellularly than on the cell surface a. Furthe+ cells than hexon- cells  (- cells)  b relativ- cells) a.Given the uniqueness of E3-19.4K and E3-31.6K to HAdV-F, and the strong possibility that these proteins participate in immune evasion functions in the gut, we characterized their basic properties. HAdV-F41 19.4K has 173 residues  and its 2 indicated that E3-31.6K has 17% α-helix, 50% β-strand, and 11% disordered regions, with the putative transmembrane domain correctly identified as an α-helix. A BLAST analysis indicated that the amino acid sequence of HAdV-F40 E3-31.6K shows no homology to E3-19K proteins. No other significant sequence homology was identified.HAdV-F41 E3-31.6K has 276 residues  and its HAdVs-F replicate preferentially in the GI tract and cause severe gastroenteritis in children. The selective pressure on HAdVs-F by the microenvironment of the gut has led these viruses to eliminate the E3-19K gene, and presumably the MHC I-binding function, and to evolve two new E3 genes that are conspicuously absent in all other species. This raises intriguing questions on the underlying mechanisms by which HAdVs-F are exquisitely adapted to the GI tract. Given that E3 proteins modulate various host immune functions ,7, it isHAdVs-F are notoriously difficult to grow in most cell culture systems ,46,47 anOur results raise important questions concerning the mechanism by which HAdV-F sequesters MIC B inside cells, and the viral factor responsible for this effect. The E3-19.4K and E3-31.6K proteins are highly conserved in HAdVs-F, 99% amino acid sequence identity between HAdV-F40 and HAdV-F41, which suggests that these proteins are critical for viral tropism or virulence in the gut. Interestingly, it was shown previously that infection of human fibroblasts by HAdV-C2 and HAdV-C5 led to the sequestration of MIC A and MIC B inside infected cells , an effeIn conclusion, we showed that HAdV-F41 sequesters MIC B inside infected cells. This represents a novel function of HAdVs-F. HAdVs-F have received considerably less attention than the other HAdVs, despite being significant pathogens, and our findings cast a new light on how these viruses, under immune pressure in the GI tract, have remarkably adapted to this site. Future investigations will provide more details about this function and the viral protein responsible for it, and will reveal if the suppression of MIC ligands impairs the recognition of HAdV-F-infected cells by NK cells.


With the application of fungicide in agriculture, resistance to fungicide has become a serious problem. It is important to assess the evolution of resistance for development of disease prevention and control. We confirmed, by site-directed mutagenesis, that single mutations conferring moderate or low resistance are more likely to evolve into double mutations conferring higher resistance under the selective pressure of SDHI. However, the double mutations suffer large of fitness penalty than single mutation. We recommend that the use of SDHI in agriculture should be appropriately reduced or that other types of fungicides should be used to control plant diseases, such as dicarboximide fungicides (DCFs), to avoid the emergence of very resistant plant pathogens.Corynespora cassiicola and analysis the evolution of double mutations. We confirmed, by site-directed mutagenesis, that all double mutations  conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation. Analyses of fitness showed that all double mutations had lower fitness than the wild type; most of double mutations suffered more fitness penalties than the corresponding single mutants. We also further found that double mutations (B-I280V+D-D95E/D-G109V/D-H105R) containing low SDHI-resistant single point mutation (B-I280V) exhibited higher resistance to SDHI and low fitness penalty than double mutations (B-H278Y+D-D95E/D-G109V) containing high SDHI-resistant single mutations (B-H278Y). Therefore, we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI. Taken together, our results provide some important reference for resistance management.With the further application of succinate dehydrogenase inhibitors (SDHI), the resistance caused by double mutations in target gene is gradually becoming a serious problem, leading to a decrease of control efficacy. It is important to assess the sensitivity and fitness of double mutations to SDHI in  Corynespora cassiicola, has become one of the most important plant diseases. The plant pathogenic fungus C. cassiicola can infect more than 530 species of plants, including Hevea brasiliensis, cucumber, tomato, cowpea, and other vegetables, causing substantial economic losses  × 100, where C is the colony diameter of the untreated control and N is the colony diameter after treatment.To test susceptibility to various environmental stresses , mycelial plugs (6 mm in diameter) obtained from the periphery of 5-day-old colony of each isolate were incubated with several compounds , and the single-copy insertion of the cassette.To obtain DHB gene A was repDHB gene B to geneDHB gene C. FinallTransformants with double mutations connected into the locus yielded a single 2575- or 2791-bp band with primers P3/P4 or P5/P6, respectively, whereas those in which integration was ‘random’ yielded no band. These primers did not amplify any fragment from the wild-type (WT) isolate , with EC50 values ranging from 2.472~4.062 µg/mL. However, three double mutations, including the B-H278Y+D-D95E, B-I280V+D-G109V, and D-B-H278R+D-G109V genotypes, were identical to high resistance . The EC50 values of B-I280V+D-H105R were >25 µg/mL and were identified as conferring very high resistance (VHR).Sensitivity to SDHI fungicides  was tested for all double mutations and parental isolates. All the data was shown in We also assessed these mutants for their sensitivity to fluopyram. All of the mutants were resistant to fluopyram, but the resistance level varied among the double mutations, with RF values ranging from 1.357 to 130.30. In isolates carrying only B-H278Y/R mutations, negative cross-resistance was observed between boscalid and fluopyram, whereas no negative cross-resistance was found in the double mutations carrying the B-H278Y/R single point mutation, with RF values ranging from 1.357 to 39.29 for fluopyram and from 16.48 to 113.40 for boscalid. Further evaluation revealed that most double mutations showed either low to moderate resistance to carboxin (5 < RFs < 25) or moderate to high resistance to carboxin (19 < RFs < 216). Only one double mutation was classified as conferring low resistance to penthiopyrad, with an RF value of 3.86.To further analyze the effect of double mutations on SDHI resistance compared with single mutations, synergistic coefficients (SCs) were calculated. However, the effect of double mutations varied among the different SDHI and mutants. All of the double mutations showed that the increased resistance to at least one fungicide . The eff50) values. Regression analyses and Spearman’s correlation tests revealed no significant cross-resistance between fluopyram and boscalid , fluopyram and carboxin , or carboxin and boscalid    , while s= 0.021) .The fitness of fungi, including the production and germination of conidia, pathogenicity, mycelial growth, and the sensitivity to various environmental stresses, plays an essential role in the ability of the fungus to spread and colonize in new environments .In the evaluation of pathogenicity, we found that the B-H278R+D-D95E and H-H278Y+D-D9E double mutations produced significantly smaller lesions than the wild type, but were not found in the other double mutations E. In terWhen assessed for sensitivity to various environmental stresses, we found that the B-H278Y+D-D95E mutant exhibited a lower ability to cope with environmental stress, as evidenced by inhibition of osmotic stress, cell wall damage, and alternative respiration. A similar phenomenon was found in the B-I280V+D-D95E and B-I280V+D-G109V mutants, which showed increased osmotic stress sensitivity. However, three double mutations  showed significantly reduced sensitivity to paraquat D.To accurately analyze the fitness of the double mutations, we scored their fitness. All double mutations had lower fitness scores than the wild type, and most double mutations suffered more fitness penalties than the corresponding single mutants (−18.75–68.75). Interestingly, the B-I280V+D-D95E double mutation showed higher fitness scores than the single mutant, but lower than the wild type .SDH gene expression levels, the expression of these genes in double mutations was quantified. Of the four SDH genes, only the expression of SDHB significantly differed in the double mutations compared to that of the wild type. The expression of the SDHB gene was 28.17-, 43.13-, and 22.96-fold higher in the double mutations B-H278R+D-D95E, B-H278R+D-G109V, and B-I280V+D-G109V, respectively, than that of the wild type. In contrast, the expression of the SDHB gene in the double mutations B-H278Y+D-D95E, B-H278Y+D-G109V, and B-I280V+D-H105R was not significantly different from that of the wild-type strain -reduction assay in the presence of SDHI. Our results showed that SDH activity was reduced to some extent in all of the double mutations except for the isolate carrying the B-I280V+D-D95E double mutation. The SDH enzyme activity of wild type was 18.90 U/mg protein. Of all the double mutations tested, the B-I280V+D-D95E mutant showed the highest SDH activity, with 20.09 U/mg of protein. The other six double mutations  had significantly lower SDH enzymatic activity than the wild-type isolate, and these six double mutations also exhibited lower activity than the corresponding single mutant .p = 0.033)  . HoweverSDH gene further reduces susceptibility to SDHI than single point mutation, leading to a reduction of control efficacy of SDHI fungicides [C. cassiicola. In this research, site-directed mutagenesis was performed to create double mutations in the SDH gene, and these double mutations were analyzed for their resistance to SDHI and fitness, and to further understand the evolution of resistance.SDHI-resistance has become a serious problem in agriculture, leading to a decrease of control efficacy ,26,27. Sngicides ,21. HoweSDH, CYP51, and β-tubulin genes significantly decreased the sensitivity to corresponding fungicides compared with the single mutation [SDH gene occur in field by a combination of pre-existing single point mutations, interestingly, increased sensitivity to fluopyram and penthiopyrad  were found in double mutations (B-H278Y+D-D95E/G109V and B-H278R+D-D95E). These results are in agreement with a previous study that showed that the B-H277Y+C-H134R double mutation in Alternaria alternata improved susceptibility to fluopyram and penthiopyrad compared to the single amino acid substitutions C-H134R and B-H277Y [Previous studies have shown that multiplex amino acid substitution in target genes of pathogens such as mutation ,21,22,28mutation ,30. This B-H277Y ,22. The  B-H277Y  and chan B-H277Y ,30.Zymoseptoria tritici [A previous study reported that fungicide applications are major determinants of the change of resistance genotype in  tritici . This re tritici ,23. In o50 values) of carboxin compared to the other SDHI [Interestingly, the synergistic coefficients of all the double mutations for carboxin were approximately 1.2, indicating that the structural changes in the ubiquinone domain caused by double mutations in the SDH enzyme had little effect on carboxin resistance. In contrast, changes in the structure of the ubiquinone domain had a greater impact on the resistance to boscalid, fluopyram and penthiopyrad. A possible hypothesis to explain this phenomenon is that the affinity between carboxin and the SDH enzyme is lower than that of the other SDHI, such as boscalid and fluopyram, as evidenced by the significantly lower activity   . A similt strain .C. cassiicola. All double mutations exhibited that the increased resistance to at least one fungicide and fitness penalty than single amino acid substitution in succinate dehydrogenase. Furthermore, single mutations conferring moderate or low resistance are more likely to evolve into double mutations conferring higher resistance under the selective pressure of SDHI. Therefore, we should reduce the application of SDHI used in controlling leaf spot in greenhouse-cultivated cucumber, and we recommend using other types of fungicides for controlling this disease, such as dicarboximide fungicides (DCFs).In summary, we first reported the sensitivity to SDHI of double mutations in