The authors show that TBSV co-opts the endosomal SNX-BAR (sorting nexin with Bin/Amphiphysin/Rvs- BAR domain) proteins, which bind to PI(3)P and have membrane-reshaping function during retromer tubular vesicle formation, directly into the VRCs to boost progeny viral RNA synthesis. We find that the viral replication protein-guided recruitment and pro-viral function of the SNX-BAR proteins depends on enrichment of PI(3)P at the site of viral replication. Depletion of SNX-BAR proteins or PI(3)P renders the viral double-stranded (ds)RNA replication intermediate RNAi-sensitive within the VRCs in the surrogate host yeast and in planta and ribonuclease-sensitive in cell-free replicase reconstitution assays in yeast cell extracts or giant unilamellar vesicles (GUVs). Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to promote VRC formation and to play structural roles and stabilize the VRCs during viral replication. Altogether, the interplay between the co-opted SNX-BAR membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable VRCs, which provide the essential protection of the viral RNAs against the host antiviral responses.Positive-strand RNA viruses replicate in host cells by forming large viral replication organelles, which harbor numerous membrane-bound viral replicase complexes (VRCs). In spite of its essential role in viral replication, the biogenesis of the VRCs is not fully understood. The authors identified critical roles of cellular membrane-shaping proteins and PI(3)P (phosphatidylinositol 3-phosphate) phosphoinositide, a minor lipid with key functions in endosomal vesicle trafficking and autophagosome biogenesis, in VRC formation for  Positive-stranded RNA viruses are major pathogens of plants, humans and animals. These viruses hijack and deform intracellular membranes to build viral replication compartments, which support virus replication. In this paper, the authors have identified the critical roles of cellular membrane-shaping proteins and a unique lipid in the formation of the replicase complex for tomato bushy stunt virus (TBSV). TBSV co-opts and retargets the endosomal sorting nexin-BAR proteins into the large TBSV-induced replication compartment. TBSV also promotes the enrichment of PI(3)P phosphoinositide within the subverted membranes. The interplay between the membrane-shaping proteins, PI(3)P and the viral replication proteins leads to stable membranous structures around the viral replicase, which provide the essential protection of the viral RNAs against the host antiviral responses. RNA virus replication depends on the formation of large intracellular viral replication compartments or organelles (VROs), which represent the sites of intensive viral RNA replication. Biogenesis of VROs for positive-strand (+)RNA viruses requires major membrane remodeling and proliferation, retargeting of trafficking vesicles, and recruitment of numerous host proteins. –5. The fMany (+)RNA viruses induce the formation of numerous small spherules, which are vesicle-like structures with narrow opening towards the cytosol –8. The spol are essential for viral replication. p92pol is the RdRp protein and translated from the genomic gRNA via readthrough of the translational stop codon in p33 ORF. The auxiliary p33 replication protein is an RNA chaperone involved in recruitment of the viral (+)RNA for replication and is the master regulator of the VRC assembly process [pol [Among plant viruses, tombusviruses are intensively studied to unravel host-virus interactions ,10. Tomb process –13. The ess [pol .Intriguingly, tombusviruses take advantage of various cellular compartments for the biogenesis of large VROs, including the formation and assembly of spherule-like VRCs . Tomato PI(3)P is a critical signaling and a minor structural lipid molecule, which is a key player in endosomal vesicle trafficking by conferring identity to endosomes . MoreoveIn this work, we show key evidence that a family of the PI(3)P effectors, namely the endosomal SNX-BAR (sorting nexin containing Bin/Amphiphysin/Rvs domain) proteins are recruited by TBSV and this is required for complete VRC formation. SNX-BAR proteins, such as Vps5p in yeast and SNX1 and SNX2a/b in plants and SNX1 in mammals, are recruited to specific subdomains of endosomes through binding to PI(3)P via their Phox-homology (PX) domain and sensing positive membrane-curvature through their banana-shaped BAR domains ,30. SNX-in vitro in replicase reconstitution assays. Based on our results, we propose that PI(3)P and the co-opted SNX-BAR proteins are coordinately exploited by tombusviruses to stabilize the VRCs during viral replication.We demonstrate that the yeast Vps5p SNX-BAR protein is required for TBSV replication in yeast. The p33 replication protein of TBSV re-localizes Vps5p into VROs as a permanent component of the viral replicase complex. The PI(3)P binding of Vps5p is required for its recruitment and pro-viral function in yeast. Depletion of SNX-BAR proteins renders the viral double-stranded (ds)RNA replication intermediate ribonuclease-sensitive within the VRCs in yeast and plants and also VPS5 in haploid yeast showed the largest inhibitory effect on the accumulation of TBSV among the genes tested (vps5Δ yeast (vps5Δ yeast (To test the putative role of the yeast PI(3)P-binding proteins in TBSV replication, we analyzed the accumulation of TBSV replicon (rep)RNA in yeast lacking one of fourteen known PI(3)P-effectors . We founs tested . The absvps5Δ yeast restored efficient replication of both TBSV and CIRV  and Snx2b in N. benthamiana plants. Knock-down of the SNX-BAR proteins led to ~4-fold reduced accumulation of TBSV genomic (g)RNA and CIRV gRNA, respectively  and Flock house virus (FHV)  in bidopsis  Tables. thamiana . Similara plants . The repa plants , suggestus (FHV) .N. benthamiana leaves. RT-PCR analysis showed the up-regulation of both Snx1 and Snx2b mRNA levels in tombusvirus-infected leaves  assay. This assay demonstrated that the p33 and p92ion Figs . The BiFion Figs . Confocaion Figs , or simuion Figs  to the lion Figs . Super-rion Figs . Based oNext, we tested if Vps5p is a permanent component of the viral replicase complex by purification of the viral replicase from yeast cells at various time points. New VRC formation was halted via blocking yeast translation through cycloheximide. Interestingly, the co-purified Vps5p level has not changed in comparison with Flag-p33 through 2.5 hours . This fiConfocal microscopy of plant protoplasts infected with TBSV revealed the efficient co-localization of PI(3)P (detected with anti-PI(3)P antibody or by using a RFP-2xFYVE biosensor, which specifically binds to PI(3)P in cells) ,41, and To test if PI(3)P affects the recruitment of SNX-BAR proteins into VROs, we performed BiFC assay with plant cells treated with Wortmannin, an inhibitor of Vps34 PI3K. The recruitment of both Snx1  and Snx2Y321A,R360A mutant in vps5Δ yeast poorly complemented TBSV replication P binding site in the plant Snx1 (Snx1rry) inhibited the recruitment into VROs by the TBSV p33 and the CIRV p36 replication proteins in a BiFC assay P, also interfered with the p33-driven recruitment of Snx1 into VROs, whereas nonphosphorylation mimicking mutant Snx1A, which has high binding affinity to PI(3)P, was recruited by TBSV similar to the case with wt Snx1 (in vitro (A mutagenesis approach was used to test if PI(3)P binding by SNX-BAR is important for TBSV replication. The conserved YR amino acids in the PI(3)P binding region (PX-domain) of the yeast Vps5p were mutated to AA to block PI(3)P binding . Expresslication , whereassay Figs  and S5D. wt Snx1 . Pull-doin vitro . TherefoLegionella as a fusion partner to p33 replication protein. RavZCT is known to bind to PI(3)P and senses positive-membrane curvature [CT-p33, however, strongly inhibited TBSV replication in yeast P and sense positive-membrane curvature, we wondered if it is possible to substitute the SNX-BAR proteins in the VRCs. We used the C-terminal domain of RavZ effector protein from urvature , similarin yeast . Deletio protein . These dN. benthamiana cells. We found co-localization of Snx2b and the TBSV p33 replication protein with Vps26, Vps29, and Vps35 retromer complex proteins in the large TBSV VROs -based replicase reconstitution assays, which can efficiently probe the exposure of the replicating viral RNAs to ribonucleases [pol replication proteins without the TBSV repRNA to allow the replicase pre-assembly step in vps5Δ yeast and in WT yeast as a control repRNA transcripts as schematically shown in in vitro assembled VRC based on the CFE prepared from vps5Δvps17Δ yeast provided 3-times less protection at the 60 min time point in comparison with the CFE from WT yeast. In the third assay, we isolated the VRCs replicating TBSV repRNA from vps5Δ and WT yeasts, followed by in vitro replication assay in the presence or absence of MNase [vps5Δ yeast was inhibited by the MNase by 2-fold more efficiently than the corresponding preparation from WT yeast P interaction helps stabilizing the VRCs and protection of the viral dsRNA during TBSV replication.Additional assay to test the level of protection provided by the VRCs included of MNase . The actWT yeast . AltogetTo obtain additional evidence for the protective role of Vps5p SNX-BAR protein in TBSV replication, we used a replicase reconstitution assay based on giant unilamellar vesicles (GUVs) . The artin vitro replicase reconstitution using CFE preparations obtained from vps34Δ yeast, lacking PI3K, provided lesser protection of dsRNA against MNase than WT CFE did P phosphatase, increased dsRNA protection against MNase in comparison with the WT CFE P phosphoinositide in the protection of the TBSV dsRNA replication intermediate, we measured vsiRNA abundance in yeast treated with Vps34p PI3K inhibitors in comparison with the DMSO-treated wt yeast expressing Dcr1 . Interes20Kstop missing the p20 silencing suppressor to allow for efficient RNAi activity during infection. CNV20Kstop accumulation was greatly suppressed in the inoculated leaves and CNV20Kstop was close to undetectable in systemic leaves of N. benthamiana repRNA and (-)repRNA of TBSV and purified yeast Vps5p, Snx1 RNA, while the RNA passes through the tight neck structure during exit from the spherule/VRC. Therefore, we tested if SNX-BAR proteins could bind to the viral RNA. In the first test, we used 5p, Snx1  and Snx25p, Snx1  in a gelpol replication proteins leads to replication of the repRNA. If nYFP-Snx1 could bind to the repRNA, whereas MS2-CP-cYFP could bind to the MS2-hairpins present in the repRNA, then we expect TriFC signal as shown schematically in To demonstrate if similar interaction could occur in plant cells, we cross-linked plant samples replicating the TBSV gRNA, followed by purification of Flag-tagged Snx1 and/or Snx2b. RT-PCR analysis of the RNAs isolated from purified Snx1, Snx2b and Snx1/Snx2b preparations with strand-specific primers showed the presence of the co-purified TBSV (+)gRNA, but not the (-)RNA . In the in vivo, we co-expressed repRNA(+)MS2hp with p33 and p92pol replication proteins (to support replication) and HA-tagged Vps5p in yeast. This was followed by capturing repRNA(+)MS2hp from yeast with the GST-tagged MS2-CP RNA viruses, including TBSV, induce the formation of large VROs harboring numerous membranous spherules, small vesicle-like structures during their replication –8. TheseIn this paper, we have identified the critical roles of endosomal SNX-BAR proteins and PI(3)P phosphoinositide in the formation and function of TBSV VRCs. The yeast Vps5p and the plant Snx1 and Snx2b SNX-BAR proteins are efficiently co-opted and retargeted into the large TBSV-induced VROs consisting of aggregated peroxisomes or the CIRV-induced aggregated mitochondria. The recruitment of SNX-BAR proteins to the sites of viral replication required PI(3)P, which is produced by the co-opted Vps34p PI3K in yeast and plants, as we have shown previously . Vps5p SCT-p33, but not RavZCT-P-p33 lacking the PI(3)P-binding domain. The dominant inhibitory effect of RavZCT-p33 is likely due to competition with Vps5p SNX-BAR to bind to PI(3)P within VRCs.The spherules consist of two different membranous subdomains: the body, which harbors the VRC and shows negative curvature, and likely enriched with PE (a negative membrane curvature-inducing lipid) and sterols . The secin vitro and in vivo data based on probing the extent of viral RNA protection by VRCs. For example, in vitro reconstitution of the TBSV replicase led to protective structure for the viral dsRNA against ribonucleases if wt yeast CFE was used. The reconstituted TBSV replicase was less protective to the dsRNA (i) in the absence of Vps5p SNX-BAR protein; (ii) when the amount of PI(3)P in the CFE was reduced by the addition of Ymr1 PI(3)P phosphatase; (iii) or in the absence of Vps34p PI3K. In contrast, the reconstituted TBSV replicase was more protective to the dsRNA in the absence of Ymr1 PI(3)P phosphatase. Moreover, yeast lacking Vps5p SNX-BAR protein provided lesser protection to the TBSV repRNA than wt yeast when a reconstituted RNAi pathway was activated. In comparison with the repRNA, the relative amount of vsiRNAs, produced by Dcr1 on dsRNA template, was increased when Vps34p PI3K was inhibited via chemical inhibitors. Moreover, knock down of the Snx1/Snx2b levels in N. benthamiana had a more dramatic inhibitory effect on a tombusvirus lacking the suppressor of gene silencing P interaction might stabilize VRCs, possibly the neck structure, which could be important to maintain continuous viral (+)RNA synthesis for several hours. This model is supported by the ilencing  than on ilencing . All theilencing .in vitro. All these observations support the model that the co-opted SNX-BAR proteins are in close association with the viral (+)RNA, possibly during the temporal passing of the viral (+)RNA through the tight neck structure in VRCs. Future, ultrahigh-resolution structural studies will be needed to confirm this model.The neck structure within the TBSV-induced spherules is rather narrow, ~10 nm in diameter ,21. The The advantage to co-opt the endosomal SNX-BAR proteins to the replicase complex is that TBSV co-opts the endosomal Rab5-containing vesicles, which are PE-rich, and the endosomal Vps34 PI3K into VROs ,25,28. TThe use of membrane curvature-sensing proteins during viral infections might be common among (+)RNA viruses. Accordingly, alphaviruses, semliki forest virus and Sindbis virus, co-opt the cellular amphiphysins for replication . Brome mIn conclusion, we have determined the critical roles of endosomal SNX-BAR proteins and PI(3)P phosphoinositide in the formation and function of TBSV VRCs. By co-opting the SNX-BAR proteins and enrichment of PI(3)P in VRCs, tombusviruses create a nuclease- and RNAi-protective microenvironment for viral replication.Some of the Materials and Methods are presented in the Parental yeast strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0), and single knock out strains vps5Δ, vps17Δ, vps34Δ, vps26Δ, vps29Δ, vps35Δ and ymr1Δ were purchased from Open Biosystems. SC1 (MATa his3Δ1 leu2Δ trp1Δ289 uraΔ52) yeast strain was purchased from Invitrogen. vps5Δvps17Δ double deletion strain and yeast strain (BY4741: Vps5-3xHA) with chromosomal HA tagging of Vps5 was created by using yeast toolbox plasmids .Plasmids and primers used in this study are described in -) supplemented with 2% glucose at 29°C for overnight, then tombusviral repRNA replication was induced by transferring the yeast to synthetic complete medium (ULH-) supplemented with 2% galactose at 23°C for 24 h for TBSV or 30 h for CIRV. Yeast total RNA and total protein were isolated and analyzed by Northern blotting and Western blotting, respectively [To identify the function of yeast Vps5 and Vps17 SNX-BAR proteins in the replication of TBSV and CIRV, yeast strains BY4741, vps5Δ and vps17Δ were transformed with plasmids pESC-His-p33/DI72, pYES-His-p92 and pRS315-cFlag for TBSV replication, pESC-Strep-p36/DI72, pYES-Strep-p95 and pRS315-cFlag for CIRV replication. The transformed yeast cells were pre-grown in synthetic complete medium lacking uracil, leucine and histidine . Plant leaf discs from inoculated leaves and systemic leaves were collected for total RNA extraction and viral RNA detection by Northern blotting [To measure the effect of plant SNX-BAR proteins on virus replication, NbSnx1 and NbSnx2b gene expression were silenced using tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) method in thamiana . The NbSblotting .N. benthamiana leaves [5 protoplasts were transformed with in vitro transcribed full-length TBSV, CIRV and TCV genomic RNAs, incubated in 35 x 10 mm petri dishes in dark at room temperature for 24 h. Total RNAs were extracted from these protoplasts, viral RNA accumulation was detected by Northern blotting [Plant protoplasts were isolated from NbSnx1/2b-silenced or cGFP-control a leaves . About 5blotting .N. benthamiana leaves were co-infiltrated with agrobacterium carrying plasmids pGD-AtSnx1-GFP (0.3 OD600) or pGD-AtSnx2b-GFP (0.3 OD600), pGD-RFP-SKL (0.2 OD600) together with pGD-p33-BFP (0.3 OD600) and p19 (0.1 OD600). Alternatively, pGD-p33-BFP, pGD-AtSnx1-GFP and pGD-AtSnx2b-RFP were co-agroinfiltrated into N. benthamiana. The agroinfiltrated leaves were inoculated with TBSV sap at 14 h post agroinfiltration. After 2 dpi, the agroinfiltrated leaves were subjected to confocal microscopy  using 405 nm laser for BFP, 488 nm laser for GFP and 559 nm for RFP. Images were captured successively and merged using the FLUOVIEW software [To examine the subcellular localization of plant SNX-BAR proteins upon virus infection, software .N. benthamiana leaves were co-infiltrated with agrobacterium carrying plasmids pGD-AtSnx2b-RFP (0.3 OD600), pGD-p33-BFP (0.3 OD600) and p19 (0.1 OD600) together with pGD-AtVps26-GFP(0.3 OD600) or pGD-AtVps29-GFP(0.3 OD600) or pGD-AtVps35-GFP(0.3 OD600). The agroinfiltrated leaves were subjected to confocal microscopy as above.To examine the co-localization of plant sorting nexins with the retromer complex upon virus infection, -) supplemented with 2% glucose at 29°C overnight. Tombusviral repRNA replication was induced by changing the yeasts to synthetic complete medium (ULH-) supplemented with 2% galactose for 21 h at 23°C. Yeast cells were collected to isolate spheroplasts for immunofluorescence [To analyze the subcellular localization of Vps5 in yeast cells upon tombusviral replication, pYes-His-p92, pEsc-GFP-p33/DI72 and pRS315-Vps5-Flag plasmids were transformed into BY4741 yeast. The transformed yeast cells were pre-grown in synthetic complete medium . Anti-p33 monoclonal mouse antibody and anti-Flag rabbit antibody  were used to visualize p33 and Vps5, respectively .in vivo, bimolecular fluorescence complementation (BiFC) assay was performed in N. benthamiana [Agrobacterium strain C58C1, as well as pGD-nYFP-AtSnx1, pGD-nYFP-AtSnx2b and SNX-BAR protein mutants. The obtained Agrobacterium transformants were co-infiltrated  into the leaves of four weeks-old N. benthamiana plants. Agroinfiltrated leaves were inoculated with TBSV or CIRV 14 h after agroinfiltration. Plant samples were subjected to confocal laser microscopy at 48 h post virus inoculation.To identify the interaction between SNX-BAR proteins and tombusviral replication proteins thamiana . The pla600) or pGD-AtSnx2b-GFP (0.3 OD600), together with pGD-p33-BFP (0.3 OD600) were agroinfiltrated into N. benthamiana leaves. The agroinfiltrated leaves were inoculated with TBSV sap at 12 h post agroinfiltration. Protoplasts were isolated from the infiltrated leaves with enzyme solution containing 1.5% (wt/vol) Cellulase R10  and 0.4% (wt/vol) Macerozyme R10  48 h post virus inoculation for immunofluorescence detection of PI(3)P [To observe the distribution and association between cellular PI(3)P and SNX-BAR proteins upon tombusvirus replication in plant cells, pGD-AtSnx1-GFP (0.3 ODf PI(3)P . The perf PI(3)P .Agrobacterium strain C58C1. The obtained Agrobacterium transformants were co-infiltrated (OD600 0.3 for each) into the leaves of four week-old N. benthamiana plants. Agroinfiltrated leaves were treated with 66 μM Wortmannin (an Vps34 PI3K inhibitor) 15 h after agroinfiltration. Plant samples were subjected to confocal laser microscopy at 31 h post inhibitor treatment.To investigate the role of PI(3)P in the recruitment of plant SNX-BAR proteins into viral replication compartments, the plasmids pGD-nYFP-AtSnx1, pGD-nYFP-AtSnx2b, pGD-p33-cYFP and pGD-RFP-SKL were separately transformed into 600, each), followed by TBSV sap inoculation 12 h post agroinfiltration. Membrane fraction was isolated from the leaves 2 dpi and solubilized with 1% Triton X-100 followed by purification of AtSnx1-Flag or AtSnx2b-Flag with anti-Flag M2 agarose . The co-purified p33-myc protein was detected by anti-myc antibody [To test the interaction between p33 and SNX-BAR proteins in plant, TBSV p33-myc was co-expressed with AtSnx1-Flag or AtSnx2b-Flag or GFP-Flag in plant by agroinfiltration (0.3 ODantibody .-) supplemented with 2% glucose and 100 μM BCS at 29°C overnight, followed by transferring the yeasts to synthetic complete medium (ULH-) supplemented with 2% galactose and 100 μM BCS at 29°C for 24 h. Tombusviral repRNA replication was induced by changing the media to synthetic complete medium (ULH-) supplemented with 2% galactose and 50 μM CuSO4 for 3 h at 23°C, 100 μg/ml cycloheximide was added to the culture to inhibit protein translation [To dissect whether Vps5 SNX-BAR protein is a permanent component of the tombusvirus replicase complex, pEsc-URA-Vps5-3xHA was transformed into yeasts with pGAD-Cup1-Flag-p92 and pGBK-Cup1-Flag-p33-Gal1-DI72. pGAD-Cup1-His-p92 and pGBK-Cup1-His-p33-Gal1-DI72 were transformed into yeast as the negative control. The transformed yeast cells were pre-grown in synthetic complete medium lacking uracil, leucine and histidine  [To investigate whether the association between p33 and SNX-BAR proteins is direct or indirect, MBP-p33, GST-Vps5, GST-AtSnx1 and GST-AtSnx2b were obtained from s cells) . First, s cells) .S. castellii as an intracellular RNAi probe in yeast as described [To measure if Vps5 contributes to the protection of the viral RNA, we co-expressed Dcr1 and Ago1 proteins from escribed . Detailsin vitro replication contained 2 μl of CFE, 0.15 μg DI-72 (+)RNA, 400 ng affinity-purified MBP-p33, 400 ng affinity-purified MBP-p92pol in 20 μl total volume. 0.1 U/μl micrococcal nuclease  was added to the reactions at different time points as shown in Figures, followed by incubation for 15 min at 25°C, then, 2.5 mM EGTA was added to the samples to inactivate the MNase. The CFE reactions were further incubated for a total of 3 h at 25°C.CFEs were prepared from untransformed BY4741, vps5Δ, vps5Δvps17Δ, vps34Δ and ymr1Δ yeast strains as described previously ,22,49. Rin vitro replicase reconstitution assay utilized the CFE from wild-type yeast was pre-treated with purified Ymr1 PI(3)P phosphatase at 25°C for 50 min [pol were added to the reaction mixture in 20 μl total volume, 0.1 U/μl MNase was added to the reactions at different time points as shown in Figures, the reaction mixtures were incubated for 15 min at 25°C, then, 2.5 mM EGTA was added to the samples to inactivate the MNase. The CFE reactions were further incubated for a total of 3 h at 25°C.The second in vitro replicase reconstitution assay utilized BY4741, vps5Δ and vps5Δvps17Δ yeast strains, which were transformed with pGAD-CUP1-His-p92 and pGBK-CUP1-His-p33. Expression of p33 and p92pol was induced by adding 50 μM CuSO4 for 40 min to the medium. CFEs were separately prepared from the above yeasts. The CFE-based reaction mixtures were programmed with 0.5 μg DI-72 (+)RNA transcripts as described previously [The third eviously ,49. 0.1 in vitro assay was based on yeast strains BY4741 and vps5Δ, which were transformed with pGAD-CUP1-His-p92 and pGBK-CUP1-His-p33-GAL1-DI72. CFEs were separately prepared from the above yeasts. MNase treatment was performed as describe above. The CFE-based replication mixtures were incubated at 25°C for a total of 3 h.The fourth 32P-labeled RNA products were separated in 5% semi-denaturing polyacrylamide gel containing 8 M urea [The total RNAs were isolated from the CFE-based reactions, the 8 M urea ,49.-) supplemented with 2% glucose and 100 μM BCS at 29°C overnight. This was followed by transferring the yeasts to synthetic complete medium (ULHT-) supplemented with 2% galactose and 100 μM BCS at 29°C for 24 h. Tombusviral repRNA replication was induced by changing the media to synthetic complete medium (ULHT-) supplemented with 2% galactose and 50 μM CuSO4 for 12 h at 23°C. The tombusviral repRNA replication in yeasts was confirmed by Northern blotting. To do the RNA-based purification, yeast cells were broken with glass beads, membrane fraction was solubilized with 1% Triton X-100 followed by incubation with GST resin coated with GST-MS2-CP (purified from E. coli), and then, the GST resins were washed five times. The co-purified proteins were detected by anti-HA and anti-Flag antibody for Vps5-3xHA and Flag-p33, respectively.To investigate the interaction between Vps5 SNX-BAR protein and viral RNA within viral replicase complex in yeast, MS2-tagged RNA affinity purification assay was performed . pGAD-CuN. benthamiana [Agrobacteria C58C1 strain. pGD-MS2-CP-cYFP (0.1 OD600), pGD-nYFP-Snx1 (0.1 OD600), pGD-p33-BFP (0.3 OD600), pGD-RFP-SKL (0.2 OD600), pGD-p33 (0.3 OD600), pGD-p92 (0.3 OD600) and pGD-repRNA (0.3 OD600) or pGD-35S-repRNA(+)MS2hp (0.3 OD600) or pGD-35S-repRNA(-)MS2hp (0.3 OD600) were co-agroinfiltrated into N. benthamiana. The plant leaves were studied by confocal microscopy 60 h post agroinfiltration, using confocal laser scanning microscope.To test if SNX-BAR proteins associate with viral RNAs within the viral replicase complex in plants, trimolecular fluorescence complementation (TriFC) was conducted in thamiana ,74. MS2-thamiana . MS2-CP in planta [600) or pGD-AtSnx2b-Flag (0.3 OD600) were infiltrated into in N. benthamiana leaves, and in parallel, pGD-AtSnx1-Flag (0.3 OD600) and pGD-AtSnx2b-Flag (0.3 OD600) were co-agroinfiltrated into plant leaves. pGD empty vector served as the negative control. TBSV sap inoculation was done 12 h post agroinfiltration. The leaf strips from the agroinfiltrated leaves (2 dpi) were cross-linked with 1% formaldehyde at 23°C for 1 h, then formaldehyde was quenched by adding 0.1 M glycine. Afterwards, the plant leaf strips were washed in 1xPBS buffer. The membrane fraction was isolated and solubilized with 1% Triton X-100. Flag-based purification of AtSnx1-Flag or AtSnx2b-Flag was performed with anti-Flag M2 agarose. The purified protein was detected with Western blot using anti-Flag antibody. Total RNAs were extracted from the purified protein-RNA complexes by phenol/chloroform, and then, cDNAs were synthesized for the detection of the co-purified viral genomic RNAs by RT-PCR. The PCR reactions were conducted with the same amount of samples and using the same number of PCR cycles for detecting the viral (+)gRNA and (-)gRNA, respectively [An RNA-based immuno-precipitation method was used to confirm the interaction between SNX-BAR proteins and viral RNA within the viral replicase complex n planta ,77. Agroectively .S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 Figvps5Δ yeast strain. Vps5p and its deletion mutants were expressed from the constitutive TEF1 promoter from a plasmid. The accumulation level of repRNA was normalized based on 18S rRNA levels (second panel). The accumulation of Strep-p36, Strep-p95 and Vps5-Flag is measured by western blotting and anti-Strep or anti-Flag antibodies. See further details in vps17Δ yeast strain. Vps17p SNX-BAR protein was expressed from the TEF1 promoter from a plasmid. The accumulation level of repRNA was normalized based on 18S rRNA levels (second panel). (C-D-E) VIGS-based knock-down of both SNX1 and SNX2b mRNA levels inhibits the accumulation of TBSV, CIRV and the related TCV RNAs in N. benthamiana protoplasts. Top panel: The accumulation of TBSV, CIRV and TCV gRNA and sgRNAs was measured using Northern blot analysis of total RNA samples obtained from N. benthamiana protoplasts. Second panel: ethidium-bromide stained gels show ribosomal RNA level. Protoplasts were isolated from the upper leaves of SNX1/SNX2b-silenced N. benthamiana on the 12th day, followed by transformation of TBSV, CIRV and TCV RNA, respectively. Twenty-four hours later, total RNA was analyzed by Northern blotting. (F) Up-regulation of Snx1 and Snx2b expression in TBSV and CIRV-infected N. benthamiana leaves. The mRNA levels for the SNX-BAR proteins were estimated by semi-quantitative RT-PCR in total RNA samples obtained from TBSV or CIRV-infected versus mock-infected N. benthamiana leaves. Tubulin mRNA and ribosomal RNA were used as controls (bottom panels). (G-H) The endosomal Vps5p SNX-BAR protein is not required for nodavirus replication in yeast. Top image: Northern blot analysis shows the accumulation level of NoV and FHV (+)RNA1 and the subgenomic RNA3 in vps5Δ yeast strain. The accumulation level of repRNA was normalized based on 18S rRNA levels (second panel). Bottom panel: The flag-tagged Protein A replication protein expression was measured with western blotting using anti-Flag antibody.(A) Top image: Northern blot analysis shows decreased CIRV (+)repRNA accumulation in (TIF)Click here for additional data file.S2 Figpol replication protein and the SNX-BAR proteins was detected by BiFC assay in planta. TBSV p92-cYFP and the nYFP-Snx2b, nYFP-Snx2a or nYFP-Snx1 proteins were co-expressed from the 35S promoter after co-agroinfiltration into N. benthamiana leaves. Bottom three panels: negative control was GST-cYFP and the SNX-BAR proteins analyzed by BiFC assay in planta. Note that the plants were infected with TBSV to induce the viral replication compartments in cells. Co-localization of RFP-SKL peroxisomal luminar marker with the BiFC signals demonstrates that the interaction between p92pol and SNX-BAR proteins occurs in the viral replication compartments. Scale bars represent 10 μm. (B) Western blots show the accumulation levels of the proteins used in the BiFC assays. Top panel for in planta. CIRV p36-cYFP and nYFP-Snx2b, nYFP-Snx2a or nYFP-Snx1 proteins were co-expressed from the 35S promoter after co-agroinfiltration into N. benthamiana leaves. nYFP-GST was co-expressed with p36-cYFP to serve as the negative control for BiFC assay. Note that the plants were infected with CIRV to induce the viral replication compartments in cells. Scale bars represent 10 μm.(A) Interaction between TBSV p92(TIF)Click here for additional data file.S3 FigN. benthamiana cells is detected by confocal laser microscopy. Scale bars represent 5 μm. (C) Localization pattern of Snx1-GFP in the absence of viral components in N. benthamiana cells is detected by confocal laser microscopy. (D) Co-localization of TBSV GFP-p33 and CIRV GFP-p36, respectively, with Vps5-Flag SNX-BAR protein in yeast cells replicating repRNA. Vps5-Flag was detected with anti-Flag antibody. Each experiment was repeated three times. (E) Absent of excessive co-localization of TBSV GFP-p33 with Vps5yr-AA-Flag mutant protein in yeast cells replicating repRNA.(A-B) Co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and GFP-tagged Snx2b in (TIF)Click here for additional data file.S4 FigN. benthamiana leaf tissues and protoplasts is detected by confocal laser microscopy. RFP-2xFYVE protein binds PI(3)P selectively. Top two panels: Co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and with RFP-2xFYVE protein in N. benthamiana leaves. Middle panel represents images in the absence of viral components. Scale bars represent 10 μm. Bottom panel shows the co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and with RFP-2xFYVE protein in N. benthamiana protoplasts. Scale bars represent 20 μm. (B) Co-localization of TBSV p33-BFP with the GFP-tagged Snx2b and with RFP-2xFYVE protein in N. benthamiana leaf tissues and protoplasts is detected by confocal laser microscopy. Top panel: Co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and with RFP-2xFYVE protein in N. benthamiana leaves. Second panel represents images in the absence of viral components. Scale bars represent 10 μm. Third panel shows the co-localization of TBSV p33-BFP with the GFP-tagged Snx2b and with RFP-2xFYVE protein in N. benthamiana protoplasts. Bottom panel represents images in the absence of viral components. Scale bars represent 20 μm.(A) Co-localization of TBSV p33-BFP with the GFP-tagged Snx1 and with RFP-2xFYVE protein in (TIF)Click here for additional data file.S5 FigN. benthamiana treated with Wortmannin, a Vps34 PI3K inhibitor, or with DMSO as a negative control. TBSV p33-cYFP and nYFP-Snx1 proteins were co-expressed from the 35S promoter after co-agroinfiltration into N. benthamiana leaves. RFP-SKL was expressed as a peroxisomal marker to identify the viral replication compartments. Scale bars represent 10 μm. The BiFC signals were quantified via Image J. Each experiment was repeated. (C) Western blot analysis shows the accumulation level of nYFP-Snx1 and nYFP-Snx2b proteins and p33-cYFP in Wortmannin or DMSO-treated N. benthamiana leaves. (D) BiFC assay shows that nYFP-Snx1rry protein was not recruited by CIRV p36-cYFP replication protein into the viral replication compartment in N. benthamiana infected with CIRV. The CIRV replication compartment was decorated with CoxIV-RFP mitochondria marker protein. Scale bars represent 10 μm. (E) Pull-down assay shows direct interaction of TBSV p33 replication protein with the shown Snx1 mutants in vitro. Top panel: Western blot analysis of the captured GST-Snx1 mutants with the MBP-affinity purified p33 . The negative control was MBP . Bottom panel: The captured MBP-p33 and MBP were detected with anti-MBP antibody. Note that equal amount of each GST fusion protein was incubated with MBP-p33 or MBP. (F) Expression of RavZCT-p33 fusion protein inhibits TBSV replication in yeast. Reduced repRNA accumulation by expression of RavZCT-p33 fusion protein in comparison with p33 replication protein in wt yeast replicating TBSV repRNA. RavZCT-p33 fusion protein lacking the PI(3)P-binding domain  loses the strong inhibitory effect on TBSV replication. Top panel shows the northern blot analysis of (+)repRNA accumulation. Middle panel: the 18S rRNA level. Accumulation of p33 and the fusion proteins was shown in bottom panel. (G) Co-expression of RavZCT or RavZCT-P with viral p33, p92 and repRNA does not affect TBSV replication in wt yeast. The accumulation of repRNA was normalized based on 18S rRNA levels (second panel). The protein level of RavZCT or RavZCT-P was shown in bottom panel.(A-B) BiFC assay shows the reduced level of interaction between p33 replication protein and the Snx1 protein in (TIF)Click here for additional data file.S6 FigN. benthamiana cells is detected by confocal laser microscopy. The plants were infected with TBSV. Scale bars represent 10 μm. (B) Different co-localization pattern of the GFP-tagged retromer proteins  and RFP-tagged Snx2b in the absence of viral components in N. benthamiana cells is detected by confocal laser microscopy. Scale bars represent 10 μm.(A) Co-localization of TBSV p33-BFP with the GFP-tagged retromer proteins  and RFP-tagged Snx2b in (TIF)Click here for additional data file.S7 Figin vps5Δ yeast strain. The yeast strains expressed the p33 and p92pol replication proteins and (+)repRNA, allowing for the assembly of the viral replicase complex in yeast peroxisomal membranes. The CFEs were treated with MNase 20 min latter, followed by the inactivation of MNase after 15 min with EGTA. Non-denaturing PAGE analysis shows the 32P-labeled TBSV repRNA products from the in vitro assay. (C-D) In vitro reconstitution of the TBSV replicase using purified recombinant p33 and p92pol replication proteins and TBSV (+)repRNA. The CFEs were prepared from wt yeast and were pre-incubated with purified recombinant Flag-tagged Ymr1 PI(3)P phosphatase to reduce the PI(3)P level in the CFE. The MNase treatments, which lasted for 15 min, were done at three different time points as shown. Non-denaturing PAGE analysis was done as in 32P-labeled (+)repRNA and (-)repRNA, respectively. Purified GST-Vps5, GST-Snx1 or GST was added in increasing amounts  to the assays. The Vps5-repRNA or Snx1-repRNA complex was analyzed on non-denaturing 5% polyacrylamide gels. Each experiment was repeated. (G) Scheme of the MS2-CP-based RNA purification assay. Flag-p33, Flag-p92 and Vps5-3xHA and the repRNA derivatives were expressed in yeast to allow the formation of RNA-protein complexes. This scheme is to explain the experiments presented in (A-B) Reduced repRNA production by the tombusvirus replicase assembled (TIF)Click here for additional data file.


In human macrophages and monocytes, lipopolysaccharide (LPS) induces nuclear factor kappa B (NFκB) activation and pro-inflammatory cytokines production. We tested the possible involvement of Ku70 and Ku80 in the process. In THP-1 macrophages and primary human peripheral blood mononuclear cells (PBMCs), shRNA-induced double knockdown of Ku70 and Ku80 potently inhibited LPS-induced production of pro-inflammatory cytokines . Additionally, we developed CRISPR/Cas-9 gene-editing methods to knockout both Ku70 and Ku80 in THP-1 cells and PBMCs. Double knockout (DKO) largely inhibited LPS-induced pro-inflammatory cytokines production. Conversely, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 enhanced the pro-inflammatory cytokines production by LPS. Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in the nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced NFκB activation was inhibited by Ku70 plus Ku80 double knockdown or DKO. It was however enhanced with Ku70 and Ku80 overexpression. Together, Ku70 and Ku80 promote LPS-induced NFκB activation and pro-inflammatory response in THP-1 cells and human PBMCs. Lipopolysaccharide (LPS) is one common pathogen-associated molecular pattern (PAMP) , 2. It iThe heterodimeric protein Ku is composed of two subunits: Ku70 and Ku80. The two were originally identified as possible auto-antigens associated with multiple autoimmune diseases, including systemic lupus erythematosus (SLE), scleroderma, polymyositis, and possible others , 12. Ku7Ku70 mRNA and Ku80 mRNA -induced mRNA expression of multiple pro-inflammatory cytokines, including TNF-α , IL-1β . Therefore, Ku70 or Ku80 SKO failed to inhibit LPS-induced pro-inflammatory response in THP-1 cells.To achieve single knockout (SKO) cells, THP-1 human macrophages were transduced with the lenti-CRISPR/Cas9-Ku70 KO construct or the lenti-CRISPR/Cas9-Ku80 KO construct. As shown, Ku70 SKO  or Ku80 Ku70 mRNA and Ku80 mRNA levels increased over 6-8 folds in the stable cells  plus the Ku80-expressing AAV were co-transfected to THP-1 macrophages. Stable cells were established by puromycin selection. Analyzing mRNA expression, by qPCR, confirmed that s, TNF-α , IL-1β   did not TNF-α mRNA expression  potently inhibited LPS-induced production of pro-inflammatory cytokines . While each single knockdown of Ku70 or Ku80 was complete ineffective. Further, CRISPR/Cas-9-mediated double knockout (DKO) of Ku70 and Ku80 largely inhibited LPS-induced pro-inflammatory cytokines production in THP-1 cells and human PBMCs. While single knockout failed to attenuate LPS-induced actions. On the contrary, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 (D-OE) enhanced pro-inflammatory cytokines production by LPS, with Ku70 or Ku80 single OE completely ineffective. These results convincingly showed that Ku70 and Ku80 are both important in regulating LPS-induced pro-inflammatory response in human macrophages and monocytes.LPS-TLR4 binding will recruit a number of adaptor proteins in TLR4 signaling pathway, activating the IκB kinase (IKK) complex , 22. TheOne important finding of the current study is that Ku70 and Ku80 are important regulators of LPS-induced NFκB activation. We demonstrated that Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in nuclei of LPS-treated THP-1 cells, required for full NFκB activation. In THP-1 human macrophages and primary human PBMCs, Ku70 plus Ku80 double knockdown or DKO potently inhibited LPS-induced NFκB activation. Yet, single shRNA or KO did not alter LPS-induced pro-inflammatory responses. On the contrary, forced overexpression of Ku70 plus Ku80 (D-OE) in THP-1 cells potentiated NFκB activation by LPS. Thus, Ku70 plus Ku80 are key regulators of NFκB activation by LPS.It has been shown that depleting Ku70/Ku80 could reduce cell viability and induce cytotoxicity in certain human cells , 24. HerKu80 mRNA and protein expression in THP-1 cells. Neither did Ku80 silencing inhibit Ku70 expression. Furthermore, ectopic overexpression of Ku70 did not alter Ku80 mRNA expression in THP-1 cells. Follow up studies will be needed to further explore the relationship between expression of Ku70 and Ku80 in human macrophages and monocytes.In murine cells the lack of Ku70 could lead to depleted expression of Ku80 . In humaTogether, we conclude that Ku70 and Ku80 promote LPS-induced NFκB activation and pro-inflammatory response possibly by enhancing DNA binding of p52-p65 in THP-1 macrophages and PBMCs.LPS (L4524), puromycin (MABE341), MTT -2, 5-diphenyl-2H-tetrazolium bromide,8 M212), trypan blue dye (T8154) were purchased from Sigma-Aldrich . Anti-Ku70 (#4588), Anti-Ku80 (#2180), anti-Toll-like Receptor 4  and other antibodies were purchased from Cell Signaling Technology . Fetal bovine serum , RPMI-1640 (SH30027), DMEM (SH30021) and other cell culture reagents were provided by Hyclone . mRNA primers, all sequences and vectors were provided by Shanghai Genechem Co. . TRIzol (15596026) and other reagents for the RNA assays were purchased from Thermo-Fisher Invitrogen . Enzyme-linked immunosorbent assay (ELISA) kits of human IL-1β (557966), IL-6 (550799) and TNF-α (550610) were purchased from BD Biosciences .From the Cell Bank of Shanghai Institute of Biological Science  THP-1 human macrophages were purchased. Cells were cultured in RPMI-1640 medium plus 10% FBS.As previously described , 29, by 5 cells/well THP-1 cells or the primary human PBMCs were seeded into 12-well plates. Following the applied LPS treatment , TRIzol reagent was utilized to extract total cellular RNA [−ΔΔCt method was utilized for the quantification of target mRNA [GAPDH. mRNA primers were listed in At a density of 1.5 ×10 ular RNA , 32. Forget mRNA , and nor5 cells/well THP-1 cells or the primary human PBMCs were seeded into six-well plates. Following the applied LPS treatment, 20-30 μg of protein lysates (from each treatment in each lane) were separated by a 10% SDS-PAGE gel, and transferred to a polyvinylidene difluoride (PVDF) blot. After blocking in 10% non-fat milk in PBST, the blot was incubated with applied primary and secondary antibodies. Enhanced chemiluminescence (ECL) reagents  were applied to visualize the immuno-reactive proteins through autoradiography [At a density of 3 ×10 iography . The qua4 cells/well THP-1 human macrophage or PBMCs were plated into 96-well plates. After the applied LPS treatment, MTT dye  was added. After incubation for another 2-3h, The optical density (OD) of MTT at 490 nm was recorded.Briefly, at a density of 1 ×10 5 cells/well THP-1 cells or the primary human PBMCs were seeded into the 12-well plates. After the applied LPS treatment, trypan blue staining was performed to quantify cell death. Trypan blue ratio was recorded [At a density of 1 ×10 recorded .4 cells/well THP-1 human macrophage or PBMCs were plated into 96-well plates. Following LPS treatment, supernatants were collected and cytokines were determined by using the commercial available ELISA kits.Briefly, at a density of 1 ×10 5 cells/well THP-1 cells or the primary human PBMCs were seeded into the six-well plates. The shRNA lentivirus was added to cultured cells (in polybrene-containing medium) for 24h. Afterwards, cells were cultured in fresh complete medium. Puromycin (5.0 μg/mL) was added to select resistant stable cells for four more passages. Expression of Ku70 and Ku80 was tested by qPCR and Western blotting. The non-sense control lentiviral shRNA  was transfected to the control cells.The verified Ku70 lentiviral shRNA (-a) and Ku80 lentiviral shRNA (-a) were provided by Dr. Xiang at Shanghai Jiao Tong University School of Medicine . At a deKu70 cDNA and Ku80 cDNA were provided by Dr. Xiang at Shanghai Jiao Tong University School of Medicine [5 cells/well THP-1 cells were seeded into six-well plates. The virus was enriched, filtered and added to THP-1 cells. Afterwards, cells were cultured in fresh complete medium. Puromycin (5.0 μg/mL) was added to select resistant stable cells for four more passages. Expression of Ku70 and Ku80 was verified by qPCR and Western blotting. Control cells were transfected with AAV with empty vector.The full-length human Medicine , individMedicine . At a dehuman Ku70 (targeted DNA sequence: CGAGGGCGATGAAGAAGCAG) or human Ku80 (targeted DNA sequence: GATACTGATCCCCACCAGAA) and a puromycin selection gene were provided by Shanghai Genechem Co. THP-1 cells or primary human PBMCs were seeded into six-well plates at a density of 3 ×10 5 cells/well. Cells were transfected with the lentiviral Ku70 and/or Ku80 knockout (KO) constructs, and selected by puromycin . To obtain monoclonal cells, GFP-positive cells were further sorted by fluorescence activated cell sorting (FACS), and cultured for another two weeks. In the stable cells Ku70 and Ku80 double knockout (DKO) was verified by qPCR and Western blotting. Control cells were transfected with CRISPR/Cas-9 PX458-GFP control plasmid (“Cas9-C”).The lentiviral CRISPR/Cas-9 PX458-green fluorescent protein (GFP)  plasmid 4 cells/well). After the colorimetric reaction, OD value was measured in an ELISA reader at the test wavelength of 450 nm.Following the applied treatment, nuclear proteins were extracted. By using a TransAM™ ELISA kit  the NFκB (p65) DNA-binding activity was tested. For each treatment, 1.0 μg of nuclear extracts were subjected to the binding of p65 to an immobilized consensus sequence in a 96-well plate . Statistics analyses were performed by using the SPSS software , with nificant . For comSupplementary Figures


Drosophila Yorkie, a transcriptional coactivator of the Hippo pathway. TAZ has been suggested to function as a regulator that modulates the expression of cell proliferation and anti-apoptotic genes in order to stimulate cell proliferation. TAZ has also been associated with a poor prognosis in several cancers, including breast cancer. However, the physiological role of TAZ in tumorigenesis remains unclear. We herein demonstrated that TAZ negatively regulated the activity of the tumor suppressor p53. The overexpression of TAZ down-regulated p53 transcriptional activity and its downstream gene expression. In contrast, TAZ knockdown up-regulated p21 expression induced by p53 activation. Regarding the underlying mechanism, TAZ inhibited the interaction between p53 and p300 and suppressed the p300-mediated acetylation of p53. Furthermore, TAZ knockdown induced cellular senescence in a p53-dependent manner. These results suggest that TAZ negatively regulates the tumor suppressor functions of p53 and attenuates p53-mediated cellular senescence.Transcriptional coactivator with a PDZ-binding motif (TAZ) is one of the mammalian orthologs of  Drosophila. TAZ has been shown to stimulate transcription by interacting with a number of transcription factors [Transcriptional coactivator with a PDZ-binding motif (TAZ), also called WW domain-containing transcriptional regulator 1, has been identified as a 14-3-3 binding phosphoprotein [ factors . TAZ pla factors . FurtherTP53 mutations or the inhibition of p53 activation by other factors [The tumor suppressor p53 plays an important role in regulating cell proliferation during various stimuli, including genotoxic stress and oncogenic activation. The majority of cancers show abnormalities in the p53 pathway due to  factors ,7,8,9. T factors ,11. In u factors . The ace factors . p53 ace factors .Acetylated p53 is deacetylated by multiple histone deacetylases (HDACs), including HDAC1/2 and SIRT1 . VariousAccumulating evidence suggests a complex and fine-tuning regulatory network connecting the p53 and Hippo pathways in a cellular context-dependent manner . Anotherv/v) fetal bovine serum (FBS) (Sigma), penicillin G (100 units/mL) , and streptomycin (100 μg/mL) (Meiji Seika Pharma) at 37 °C in a 5% CO2 incubator [2 incubator [H1299 (p53-null) cells were cultured in RPMI1640 medium  supplemented with 10% (ncubator . MCF7 (wncubator .TAZ siRNA (sense: 5′-AGACAUGAGAUCCAUCACUAA-3′) was purchased from FASMAC . siRNA oligo targeting human p53 mRNA was previously described [TM siRNA Luciferase Reporter Control (Invitrogen) was used as a control.Regarding DNA transfection, plasmids were transiently transfected with polyethylenimine (PEI)  or Lipofectamine 2000 . In short interfering RNA (siRNA) transfection, siRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen). Human escribed . Stealthp21 promoter-Luc (pGL4/p21) have been described previously [NOXA promoter-Luc (−198 to +45) was generated by ligating the human NOXA promoter region [TAZ Mission shRNA plasmid (TRCN0000319150) was obtained from Sigma. cDNA encoding TAZ was amplified by PCR and cloned into FLAG-pcDNA3, HA-pcDNA3, 6Myc-pcDNA3, or pGEX6P1 . YAP was amplified by PCR and cloned into FLAG-pcDNA3. The tetracycline-inducible lentiviral pCW57.1-FLAG-p53 vector was generated by subcloning FLAG-p53 from pcDNA3-FLAG-p53 [The original constructs encoding human p53, p300, SIRT1 and β-galactosidase  were described previously ,25. p53Reviously ,25. NOXAr region  with pGLr region . The TAZFLAG-p53  into pCWAn anti-p53 antibody (sc-126), horseradish peroxidase (HRP)-conjugated anti-p53 antibody (SC-126 HRP), anti-p21 antibody (sc-6246), anti-GST antibody (sc-138), and HRP-conjugated anti-HA antibody (SC-7392 HRP) were purchased from Santa Cruz Biotechnology . An anti-phospho-p53 (Ser15) antibody (9284), anti-acetyl-p53 (Lys382) antibody (2525), anti-PARP antibody (9542), and anti-TAZ antibody (4883) were purchased from Cell Signaling Technology . An anti-Mdm2 antibody (OP46) was purchased from Calbiochem . An anti-FLAG (M2) antibody (F3165), anti-β-actin antibody (A5441), actinomycin D (A9415), and Nutlin-3 (SML0580) were purchased from Sigma. An anti-phospho-p53 (Ser46) antibody (71-115) was obtained from BioAcademia .H1299 cells were transfected with the luciferase reporter plasmid, expression plasmids, pCMV/β-gal, and an empty vector. The total amount of transfected DNA was the same in each experiment. Twenty-four hours after transfection, cells were harvested and luciferase activity was measured. Luciferase activity was normalized against β-gal activity .Cells were lysed in TNTE buffer  supplemented with protease inhibitors and phosphatase inhibitors . After bEscherichia coli, and GST fusion proteins were purified as recommended by the instructions provided . H1299 cells were transfected with tagged-TAZ and TAZ deletion mutants. After 24 h, cells were lysed in TNTE buffer supplemented with protease inhibitors. Cell lysates were incubated with GST or GST-p53 at 4 °C for 2 h. Bound proteins were analyzed by immunoblotting.The recombinant p53 protein fused with GST was produced in p21 expression; 5′-TGTTGGTGCACAAAAAGACA-3′ and 5′-CACGCCAAACAAATCTCCTA-3′ for Mdm2 expression; 5′-GGCTGGGAGATGACCTTCAC-3′ and 5′-CTGAGTGGGGTGGTTCTGCT-3′ for TAZ expression; 5′-TTTCACCCTTCAGATCCGTGG-3′ and 5′-TTCCAAGGCCTCATTCAGCTC-3′ for p53 expression; 5′-TTTGCTTTCCTTGGTCAGGC-3′ and 5′-GCTTGCGACCTTGACCATCT-3′ for HPRT1 expression; 5′-TGGCACCCAGCACAATGAA-3′ and 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′ for β-actin expression.Total RNA from cells was extracted using RNAiso Plus  and reverse-transcribed with the PrimeScript first-strand cDNA Synthesis Kit (Takara Bio Inc.) following the manufacturer’s instructions. Quantitative PCR was performed using SYBR premix Ex Taq (Takara Bio Inc.) and the ABI Prism 7300 sequence detection system  with the following primers: 5′-GATTTCTACCACTCCAAACGCC-3′ and 5′-AGAAGATGTAGAGCGGGC-3′ for p21 promoter (-2283), 5′-CCAGGAAGGGCGAGGAAA-3′ and 5′-ACATCTCAGGCTGCTCAGAGTCT-3′ for the p21 promoter (-1391), 5′-TGTTTGGGCTATTTACTAGTTG-3′, and 5′-ATAAAATGACTTAAGCCCAGAG-3′ for the HPRT1 first intron primers.Cells were crosslinked with 1% formaldehyde, and then lysed in SDS lysis buffer . The ChIP procedure was performed as previously described . The priCells were washed with PBS. Crystal violet solution was added to stain cells, and stained cells were dissolved in 1% SDS and absorbance was measured at 595 nm. For apoptosis assay, both floating and attached cells were collected 48 h after Nutlin-3 treatment. Cells were then stained with Annexin V-FITC using the FITC Annexin V Apoptosis Detection Kit  . SA-β-gat-test. In multi-group analyses, significance was assessed using a one-way ANOVA with the post hoc Tukey-Kramer HSD test.The significance of differences between two groups was evaluated using two-tailed Student’s p21 promoter-Luc , the distal site at −2283 that binds p53 relatively strongly and the proximal site at −1391 that is more weakly bound by p53 [p21 promoter, but not to the region used as the control (HPRT1 first intron). However, as expected, TAZ knockdown increased the amount of p53 bound to DNA , which contributes to cancer invasion and metastasis . We here


Arabidopsis thaliana. Dark-triggered BR synthesis and the subsequent activation of BRASSINAZOLE RESISTANT 1 (BZR1), a BR signaling positive regulator, result in BZR1 binding to the AIF2 promoter in a dark-dependent manner, reducing AIF2 transcript levels and accelerating senescence. BR-induced down-regulation of AIF2 protein stability partly contributes to the progression of dark-induced leaf senescence. Furthermore, AIF2 interacts with INDUCER OF CBF EXPRESSION 1 (ICE1) via their C-termini. Formation of the AIF2–ICE1 complex and subsequent up-regulation of C-REPEAT BINDING FACTORs (CBFs) negatively regulates dark-triggered, BR-induced leaf senescence. This involves antagonistic down-regulation of PHYTOCHROME INTERACTING FACTOR 4 (PIF4), modulated through AIF2-dependent inhibition of ICE1’s binding to the promoter. PIF4-dependent activities respond to dark-induced early senescence and may promote BR synthesis and BZR1 activation to suppress AIF2 and accelerate dark-induced senescence. Taken together, these findings suggest a coordination of AIF2 and ICE1 functions in maintaining stay-green traits.ATBS1-INTERACTING FACTOR 2 (AIF2) is a non-DNA-binding basic helix–loop–helix (bHLH) transcription factor. We demonstrated that AIF2 retards dark-triggered and brassinosteroid (BR)-induced leaf senescence in  AIF2–ICE1 transcriptional complex is negatively involved in dark-triggered and brassinosteroid-induced leaf senescence, maintaining plant growth and enabling them to stay green longer. Aging or leaf senescence is the final stage of leaf development. Nutrients and energy from leaves are redirected to new and developing tissues or storage organs. Senescence involves substantial metabolic reprogramming including chlorophyll degradation, protein degradation, reallocation of nutrients, increase in reactive oxygen species (ROS), enhanced programmed cell death/necrosis, membrane ion leakage, and differential expression of numerous genes, including senescence-associated genes (SAGs) , jasmoniBrassinosteroids (BRs) are plant steroid hormones that play crucial roles in plant growth and development via extensive signal integration through direct interactions with numerous signaling pathways . Brassinde-etiolated 2 (det2), displays delayed leaf yellowing (bri1 (a BR receptor mutant) phenotype, and introduction of an RNAi construct of BAK7 in Arabidopsis resulted in severe growth retardation and early senescence  genes  encodes a nuclear-localized MYC-like bHLH transcription factor; its dominant negative mutant, Arabidopsis thaliana  and its BR-related mutants , AIF2 T-DNA knockout plant , AIF2-overexpressing p35S::AIF2-EGFP transgenic plants (AIF2ox), β-glucuronidase (GUS)-expressing pAIF2::AIF2-GUS  and its loss-of-function mutant of BR receptor BRI1 (bri1-5) . Seeds of a line carrying 35S::BIN2-myc were provided by Dr T.-W. Kim . Following 2 d of cold stratification, seeds were grown in pots containing Sunshine No. 5 soil (Polysciences) in an environmentally controlled growth room under a cycle of 16 h light (100–150 µmol m−2 s−1) and 8 h dark at 23–25 °C with 80–85% humidity.Wild-type pGBKT7 as the GAL4 DNA-binding domain vector and pGADT7 as the GAL4 DNA activation vector . Briefly, cDNAs encoding a full-length or truncated forms of AIF2 (At3g06590), ICE1 (At3g26744), and BIN2 (At4g18710) (depicted in JXB online) and subsequently inserted into either pGBKT7 or pGADT7 vector to generate constructs testing interactions between AIF2 and ICE1 in yeast cells (Saccharomyces cerevisiae AH109). These plasmids were co-transformed into yeast cells of equal densities. The transformed yeast cells were then quantitatively analysed for interactions between test proteins by measuring β-galactosidase activity, determined using chlorophenol red-β-D-galactopyranoside as a substrate.All procedures for yeast transformation and protein interaction were performed according to Clontech’s Yeast Protocols Handbook (PT3024-1) using in planta using co-immunoprecipitation assays, cDNAs encoding either full-length or truncated forms of AIF2, ICE1, and BIN2  was co-infiltrated through the underside of 2- to 4-week-old tobacco (Nicotiana benthamiana) leaves. Total proteins were then extracted from the infiltrated leaves, and immunoprecipitation was performed using anti-myc antibody -conjugated Protein G magnetic beads . Co-immunoprecipitated proteins were then separated using 12% SDS-PAGE, and the presence of AIF2FL–nYFP and BIN2–myc or ICE1–myc proteins was analysed by immunoblotting with anti-green fluorescent protein (GFP) antibodies or anti-myc antibodies , respectively. For an in planta interaction assay by bimolecular fluorescence complementation (BiFC), a combination of Agrobacterium containing full-length or part-length coding regions of AIF2, ICE1, and BIN2 in pPZP312-nYFP or pPZP312-cCFP, in fusion with the N-terminal half of YFP (nYFP) or the C-terminal half of CFP (cCFP), respectively  was co-infiltrated into the underside of 4-week-old tobacco leaves. After 36–48 h of incubation, epidermal cell layers were observed for fluorescence using the Zeiss LSM 710 confocal laser scanning microscope. 4′,6-Diamidino-2-phenylindole was used as the marker for indicating nuclear position.To test protein interactions and BIN2  were PCRand BIN2  and clonpGWB20  and diaminobenzidine (DAB) staining were performed as described previously . To measCBF2 promoter- or PIF4 promoter-driven luciferase reporter  in planta, a genomic DNA fragment covering 1292 or 1236 bp upstream of the CBF2 (AT4G25470) or PIF4 (AT2G43010) transcription start site, respectively, was PCR-amplified (see pBGWL7 in fusion with the full-length coding region of the luciferase gene (https://gateway.psb.ugent.be). Next, Agrobacterium tumefaciens GV301 carrying pCBF2::LuC or pPIF4::Luc together with Agrobacterium containing different combinations of AIF2 and ICE1 effectors was co-infiltrated, along with the transfection control (p35S::GUS), through the underside of 2- to 4-week-old tobacco leaves. After 36–48 h of incubation in the dark, luciferase activity in the transfected leaves was measured using a GloMax 20/20 luminometer . Data regarding luciferase activity were normalized to GUS activity of the same transfected samples. The GUS activity in leaves of transiently transformed tobacco and pAIF2::AIF2-GUS-expressing plants was measured according to a previously described method  and compared it with the genes differentially expressed in dark-induced Col-0 plants (ice1 mutant were identified using the R program (version 3.5.2), and the edgeR package was used for normalization and correction within arrays <−1 was applied to identify significantly regulated DEGs. A list of PIF4- or BZR1-targeted genes was obtained from chromatin-immunoprecipitation sequencing (ChIP-Seq) analysis . Gene ontology (GO) annotation and enrichment of GO terms were performed using resources from The Gene Ontology Consortium  produced dwarfed and greener plants, a phenotype typical of BR biosynthetic- or signaling-defective plants , less chlorophyll, and fewer ions from their leaves. TB staining visualizes cell death, while DAB staining reveals the presence of ROS such as hydrogen peroxide and superoxide. Differential outcomes of senescence phenotypes were reconfirmed by extensive leaf staining by TB and DAB in Col-0 and aif2-1 plants compared with that in AIF2ox plants  were highly up-regulated or acted as a positive regulator during senescence (SAG12 and AtNAP transcripts in Col-0 and aif2-1 plants than in AIF2ox plants (CHLOROPHYLL A/B BINDING PROTEIN 1 (CAB1) and RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL CHAIN 1A (RBCS1A), two genes involved in active photosynthesis, were relatively highly expressed (less repressed compared with the 0-day incubated plants) in AIF2ox compared with those in Col-0 and aif2-1 plants  stayed relatively green and had increased longevity compared with their respective wild-type counterparts, Col-0 and Ws-2. Overall, these results demonstrate that BR and its BZR1-mediated signaling pathways exhibit positive roles in dark-triggered leaf senescence.Hormones are major endogenous factors regulating onset and progression of leaf senescence  and an ienotypes , D. In cAIF2 in leaves have opposite effects on age- and dark-induced senescence. Interestingly, the AIF2 transcript level in the dark-induced leaves of Col-0 plants gradually declined in a time-dependent manner  during the initial period of dark induction (24 h), and the expression level of BR6ox continued to increase thereafter  point. Phosphorylation of proteins makes them more basic, causing the phosphorylated forms of proteins to migrate toward the more acidic area (toward pH 3) on an IEF strip. Interestingly, 1 or 2 d of dark incubation led BZR1 protein to migrate toward the basic area (toward pH 10) of the IEF strip  and/or the E-box (CANNTG) of target genes, function as both a transcriptional activator and a repressor, promoting transcription of growth-promoting genes, while repressing genes involved in BR biosynthesis such as EF strip . This imereafter . Accordiereafter . Similarrter see . Exogenorter see , and sucrter see . ATSK21/35S::AIF2FL-EGFP) or C-terminal-deleted AIF2  in Col-0 plants further facilitated dark-induced leaf senescence as determined by decreasing leaf autofluorescence (SAG15 (a marker of enhanced senescence) , and genes involved in ROS production and senescence, such as CCX1  in bzr1-1D plants greatly antagonized the BR-enhanced growth-promoting phenotype of bzr1-1D, and deletion of the AIF2 C-terminus negated this AIF2-antagonized effect  , and draynthesis . In contF2ox see . Moreoved effect . Similardeletion , F. ThesAIF2 is an atypical non-DNA-binding bHLH transcription factor and is a well-known member of the bHLH family that interacts with itself and with several other regulatory proteins. The bHLH transcription factors form complexes to activate or repress expression of genes that respond to diverse hormonal and developmental signals .6 yeast cells transformed by the Arabidopsis seedling cDNA library  and C-termini (23 amino acids) of AIF2 are necessary for interaction with ICE1  or C-terminus-deleted ICE1 (35S::ICE1dC-myc) in a Col-0 or aif2-1 background  shared the highly conserved helix–loop–helix domain and produced dwarfed plants when ectopically expressed in Arabidopsis  was drastically increased in the same AIF2ox plants. PIF4 promotes dark-induced ethylene biosynthesis to result in leaf senescence, and PIF4 directly binds to the promoter of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE C (PORC) to negatively regulate PORC transcription and chloroplast activity  and ICE1 (p35S::ICE1FL-EGFP) in tobacco leaves greatly enhanced pCBF2-Luc reporter expression , while deletion of the C-terminus nearly abolished this effect (PIF4 promotion was more evident in aif2-1 plants (>400-fold that of Col-0 plants). Thus, AIF2 may act as a negative regulator of ICE1 transcriptional activator for PIF4 gene expression. Supporting this idea, ectopic expression of AIF2 significantly decreased the dark-induced PIF4 gene expression compared with that of Col-0 plants  were significantly down-regulated both in Col-0 plants undergoing senescence and in ice1 plants. These results suggest that ICE1-induced retardation of leaf senescence involves suppression of diverse senescence-promoting hormones and stress responses, as well as promotion of auxin/cytokinin and other diverse cellular activities such as photosynthesis and cell expansion.ICE1 is a MYC-like bHLH transcription factor that binds to MYC-recognition motifs /dehydration-responsive element (DRE) present in the promoters of CBF-regulated genes  and PORC (At1g03630), key enzymes for chlorophyll biosynthesis, were significantly down-regulated in senescing leaves and in ice1 mutants , ANAC047, and ANAC087 were up-regulated both in senescing leaves and in ice1 plants  enhanced leaf senescence through transcriptional and post-translational suppression of AIF2 and up-regulation of PIF4 during senescence progression , while it functions as a transcriptional repressor of the senescence-promoting PIF4 gene  and RGA-LIKE 1 (RGL1) interact with WRKY6 and WRKY45, respectively, and negatively regulate up-regulation of senescence-related gene expression to retard both dark-induced  transcription, forming an amplification loop to accelerate age-dependent leaf senescence  found in promoters of test genes.pAIF2::AIF2-Luc plants.Fig. S4. Time-dependent AIF2–Luc expression activity in dark-triggered bzr1-1D-GFP-overexpressing p35S::bzr1-1D-GFP/Col-0 plants and the full-length AIF2 protein-expressing AIF2ox (p35S::AIF2FL-EGFP/Col-0) on dark-triggered leaf senescence.Fig. S5. Antagonistic effects of In vivo interaction test of AIF2 with ICE1 in tobacco.Fig. S6. Fig. S7. Functional analysis of AIF2–ICE1 interaction in the regulation of growth-related gene expression.Fig. S8. Functional analysis of AIF2–ICE1 interaction in tobacco, leading to retardation of dark-induced leaf senescence.CBF1 and ACS6 genes in the ICE1FL- or ICE1dC-overexpressing Col-0 or aif2-1 plants.Fig. S9. AIF2/ICE1-dependent expression of Fig. S10. Heatmap and gene ontology classification of PIF4- or non-PIF4-targeted genes in 542 ICE1- and senescence-regulated DEGs.AIF2, ICE1, BIN2, CBF2, and PIF4.Table S1. Primers used in cDNA or promoter amplification of Table S2. Primers used in quantitative real-time RT-PCR analysis.Table S3. Primers used in ChIP-qPCR analysis.ice1 mutant.Table S4. A list of differentially regulated genes grouped according to expression in Col-0 D0, D2, D5, and ice1 mutant.Table S5. Transcription factors differentially regulated both in leaves undergoing senescence and erz533_suppl_Supplementary_Figures_S1-S10Click here for additional data file.erz533_suppl_Supplementary_Table_S4Click here for additional data file.erz533_suppl_Supplementary_Table_S5Click here for additional data file.erz533_suppl_Supplementary_Tables_S1-S3Click here for additional data file.erz533_suppl_Supplementary_DataClick here for additional data file.


Small interfering RNA (siRNA) has been recognized as a powerful gene-silencing tool. For therapeutic application, chemical modification is often required to improve the properties of siRNA, including its nuclease resistance, activity, off-target effects, and tissue distribution. Careful siRNA guide strand selection in the RNA-induced silencing complex (RISC) is important to increase the RNA interference (RNAi) activity as well as to reduce off-target effects. The passenger strand-mediated off-target activity was previously reduced and on-target activity was enhanced by substitution with acyclic artificial nucleic acid, namely serinol nucleic acid (SNA). In the present study, the reduction of off-target activity caused by the passenger strand was investigated by modifying siRNAs with SNA. The interactions of SNA-substituted mononucleotides, dinucleotides, and oxyl (TEMPO)-labeled double-stranded RNA (dsRNA) with the MID domain of the Argonaute 2 (AGO2) protein, which plays a pivotal role in strand selection by accommodation of the 5’-terminus of siRNA, were comprehensively analyzed. The obtained nuclear magnetic resonance (NMR) data revealed that AGO2-MID selectively bound to the guide strand of siRNA due to the inhibitory effect of the SNA backbone located at the 5’ end of the passenger strand. Small interfering RNAs (siRNAs) are duplexes of approximately 23 base pairs composed of passenger strands and guide strands. They promote gene silencing of messenger RNA (mRNA) targets complementary to the guide strand via RNA interference (RNAi). Notably, synthetic siRNAs have shown therapeutic potential. The first siRNA drug, namely patisiran, was approved by the Food and Drug Administration (FDA) in 2018  NH4Cl ). The protein was purified using a Ni2+-NTA high-performance column . The hexahistidine-modified SUMO was cleaved by incubation with proTEV  and further purified employing a Ni2+-NTA high-performance column and a cation exchange column .DNA fragment encoding human AGO2-MID (432-578) was cloned between the BamHI and SalI sites of the pCold I plasmid , in which the SUMO-TEV coding region was inserted between the NdeI and BamHI sites. Hexahistidine-tagged SUMO-TEV-hAGO2-MID was expressed in the 2 in humidified air.The 293FT cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 80 µg/mL penicillin, and 90 µg/mL streptomycin. The cells were cultured at 37 °C with 5% COReporter plasmids pGL3-On and pGL3-Off or pGlo-On and pGlo-Off were used for the dual luciferase assay ,28. The The ITC experiments were performed at 25 °C using the iTC200 calorimeter . AGO2-MID was dialyzed against 25 mM MES (pH 6.5), 200 mM NaCl, and 1 mM tris(2-carboxyethyl)phosphine (TCEP). 20 μM AGO2-MID was first loaded into the sample cell and then 2 μL of adenosine (3 mM), AMP (3 mM), sA (3 mM), p-sA (3 mM), rArA (3 mM), p-rArA (0.3 mM), sArA (3 mM), p-sArA (2 mM), rAsA (3 mM), sAsA (3 mM), or p-sAsA (3 mM) was added every 150 s.v/v) D2O in the absence or presence of ascorbic acid (2 mM). The 1H-15N HSPQC spectra were acquired at 293 K using the Avance600 spectrometer equipped with a cryo-probe . The data were processed using the Topspin and Sparky software.AGO2-MID (0.06 mM) was mixed with TEMPO-labeled RNA duplex (0.4 mM) dissolved in 25 mM MES (pH 6.5), 100 mM NaCl, and 10% (