Figure 7Wild type , Traf3ip3 - / - and Mavs - / - mice ( three littermates per group ) were infected intravenously with VSV at a MOI of 2 × 108 . The spleens were collected 4 hours or 8 hours after infection and Ifna4 ( A ) , Ifnb ( B ) , Isg54 ( C ) and Il6 ( D ) inductions were measured respectively by qPCR . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . Wild type , Traf3ip3 - / - and Mavs - / - mice ( three littermates per group ) were infected intravenously with VSV at a MOI of 2 × 108 . The spleens were collected 4 hours or 8 hours after infection and VSV RNA levels were measured by qPCR ( E ) . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . Wild type , Traf3ip3 - / - and Mavs - / - mice ( three littermates per group ) were infected intravenously with VSV at a MOI of 2 × 108 . The sera were collected from the treated mice and used for the measurement of IFNβ production by ELISA ( F ) . All data are presented as the mean values based on three independent experiments , and error bars indicate s . d . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 . 05 and * * P < 0 . 01 . NS indicates no statistically significant difference . G Wild type , Traf3ip3 - / - and Mavs - / - mice ( n = 6 each ) were injected with VSV via tail vein injection at 5 × 107 pfu per mouse , and the survival rates were monitored for 15 days . P values were determined by unpaired two - tailed Student ' s t - test . * P < 0 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and Ifna4 (A), Ifnb (B), Isg54 (C) and Il6 (D) inductions were measured respectively by qPCR. All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The spleens were collected 4 hours or 8 hours after infection and VSV RNA levels were measured by qPCR (E). All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.Wild type, Traf3ip3-/- and Mavs-/- mice (three littermates per group) were infected intravenously with VSV at a MOI of 2×108. The sera were collected from the treated mice and used for the measurement of IFNβ production by ELISA (F). All data are presented as the mean values based on three independent experiments, and error bars indicate s.d. P values were determined by unpaired two-tailed Student's t-test. *P<0.05 and **P<0.01. NS indicates no statistically significant difference.G Wild type, Traf3ip3-/- and Mavs-/- mice (n=6 each) were injected with VSV via tail vein injection at 5×107 pfu per mouse, and the survival rates were monitored for 15 days. P values were determined by unpaired two-tailed Student's t-test. *P<0.05


Figure 1 | Representative analysis of B220 and IgM surface expression in bone marrow ( left ) and spleen cells ( right ) of mice from the indicated genotypes . Representative flow cytometric analysis of splenocytes from mice of the indicated genotypes ( pre - gated on B cells : B220 + / CD19 + ) for surface expression of IgM and the 3 - 83 idiotype ( 54 . 1 ) . Shown data are representative 11 - 35 individual mice per genotype . PCR fragments amplified with specific primers for Vκ and Jκ5 from genomic DNA of purified splenic B cells from Ptenf / f x 3 - 83ki and Ptenf / f x mb1 - cre x 3 - 83ki mice on the respective backgrounds . Genomic tail DNA from a 3 - 83ki mouse and DNA from purified splenic B cells of a control ( ctrl ) mouse were used as controls . PCR for the splicing factor Srp20 served as a loading control . Kb and Kd indicate the respective background of the mice , ( H2 - Kb : + Ag ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1| Representative analysis of B220 and IgM surface expression in bone marrow (left) and spleen cells (right) of mice from the indicated genotypes.Representative flow cytometric analysis of splenocytes from mice of the indicated genotypes (pre-gated on B cells: B220+/CD19+) for surface expression of IgM and the 3-83 idiotype (54.1). Shown data are representative 11 - 35 individual mice per genotype.PCR fragments amplified with specific primers for Vκ and Jκ5 from genomic DNA of purified splenic B cells from Ptenf/f x 3-83ki and Ptenf/f x mb1-cre x 3-83ki mice on the respective backgrounds. Genomic tail DNA from a 3-83ki mouse and DNA from purified splenic B cells of a control (ctrl) mouse were used as controls. PCR for the splicing factor Srp20 served as a loading control. Kb and Kd indicate the respective background of the mice, (H2-Kb: +Ag).


Figure 2A | Intracellular expression of IgM ( Ig - μHC ic ) in bone marrow ( left ) and spleen cells ( right ) was determined by flow cytometry and compared between the populations of B cells ( green , identified by B220 and CD19 expression ) and non - B cells ( gray ) . Numbers in the histograms indicate the percentages of the positive populations . Figures are representative of 11 - 35 individual mice per genotype . B | Intracellular Ca2 + influx was measured in CD90 . 2 / Thy1 . 2 - splenocytes derived from mice of the indicated genotypes following stimulation with 10 μg / ml α - κLC antibody . Figures are representative of at least 3 individual mice per genotype . C | Serum IgM concentrations measured in mice of the indicated genotypes . Mean ± SD , symbols represent IgM concentrations from individual mice ( n ) . Statistical significance was calculated by using the Kruskal - Wallis test ( see also Table S1 ) , n . s . = not significant ; * * * * p ≤ 0 . 0001 . D , E | Absolute numbers of B cells in bone marrow ( D ) and spleens ( E ) from mice of the respective genotypes , determined by B220 / CD19 surface expression . Mean ± SD , symbols indicate the numbers of B cells in individual mice ( n ) . Statistical significance was calculated by using the Kruskal - Wallis test ( see also Tables S2 - 3 ) , n . s . = not significant ; * p ≤ 0 . 05 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A | Intracellular expression of IgM (Ig-μHC ic) in bone marrow (left) and spleen cells (right) was determined by flow cytometry and compared between the populations of B cells (green, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Figures are representative of 11 - 35 individual mice per genotype.B | Intracellular Ca2+ influx was measured in CD90.2/Thy1.2- splenocytes derived from mice of the indicated genotypes following stimulation with 10 μg/ml α-κLC antibody. Figures are representative of at least 3 individual mice per genotype.C | Serum IgM concentrations measured in mice of the indicated genotypes. Mean ±SD, symbols represent IgM concentrations from individual mice (n). Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S1), n. s. = not significant; **** p ≤ 0.0001.D, E | Absolute numbers of B cells in bone marrow (D) and spleens (E) from mice of the respective genotypes, determined by B220/CD19 surface expression. Mean ±SD, symbols indicate the numbers of B cells in individual mice (n). Statistical significance was calculated by using the Kruskal-Wallis test (see also Tables S2-3), n. s. = not significant; * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001.


Figure 3Splenocytes from mice of the indicated genotypes , ( A ) Ptenf / f , Ptenf / f x mb1 - cre and Ptenf / f x mb1 - cre 3 - 83ki mice on the respective backgrounds were analyzed by flow cytometry . B cells were pre - gated by B220 and CD19 expression and the IgM / IgD surface expression ( dot plots , left ) was determined . Histograms ( right ) show a comparison of intracellular IgD expression ( Ig - δHC ic ) between the splenic populations of B cells ( orange , identified by B220 and CD19 expression ) and non - B cells ( gray ) . Numbers in the histograms indicate the percentages of the positive populations . Data are representative of 11 - 35 individual mice per genotype . ( B ) Ptenf / f x MD4tg and Ptenf / f x mb1 - cre x MD4tg mice in presence and absence of antigen ( ML5tg ) , were analyzed by flow cytometry . B cells were pre - gated by B220 and CD19 expression and the IgM / IgD surface expression ( dot plots , left ) was determined . Histograms ( right ) show a comparison of intracellular IgD expression ( Ig - δHC ic ) between the splenic populations of B cells ( orange , identified by B220 and CD19 expression ) and non - B cells ( gray ) . Numbers in the histograms indicate the percentages of the positive populations . Data are representative of 11 - 35 individual mice per genotype . C | Quantification of surface IgD mean fluorescence intensity ( MFI ) in Ptenf / f x MD4 - trangenic mice in presence ( mb1 - cre + / + ) and absence ( mb1 - cre + / ki ) of Pten . Single dots represent the average MFI of 4 independent measurements per mouse ( n ) , mean ± SD . Statistical significance was calculated by using an unpaired two - tailed t - test . D | Serum anti - HEL titers . Sera from MD4tg mice of the indicated genotypes were adjusted to an IgM concentration of 500 μg / ml and applied in triplicates to HEL - coated plates in dilution steps of 1 : 3 , mean ± SEM . Statistical significance was calculated by using the Kruskal - Wallis test ( see also Table S4 ) . Statements of significance in the figure refer to the comparison between Ptenf / f x MD4tg ( filled red squares ) and Ptenf / f x MD4tg x MD5tg sera ( filled orange triangles ) , * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 . E | Immunohistochemistry of sections from spleens of Ptenf / f , Ptenf / f x mb1 - cre , Ptenf / f x mb1 - cre x 3 - 83ki and Ptenf / f x mb1 - cre x MD4tg mice for CD169 ( green ) , CD5 ( red ) and IgM ( cyan ) at 10x magnification . Pictures in the second row show enlarged areas indicated by the white squares , respectively . Double - arrows indicate the distance between T cell ( red ) and marginal zone ( green ) . Shown pictures are representative of 2 - 3 mice per

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Splenocytes from mice of the indicated genotypes, (A) Ptenf/f, Ptenf/f x mb1-cre and Ptenf/f x mb1-cre 3-83ki mice on the respective backgrounds were analyzed by flow cytometry. B cells were pre-gated by B220 and CD19 expression and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig-δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11 - 35 individual mice per genotype.(B) Ptenf/f x MD4tg and Ptenf/f x mb1-cre x MD4tg mice in presence and absence of antigen (ML5tg), were analyzed by flow cytometry. B cells were pre-gated by B220 and CD19 expression and the IgM/IgD surface expression (dot plots, left) was determined. Histograms (right) show a comparison of intracellular IgD expression (Ig-δHC ic) between the splenic populations of B cells (orange, identified by B220 and CD19 expression) and non-B cells (gray). Numbers in the histograms indicate the percentages of the positive populations. Data are representative of 11 - 35 individual mice per genotype.C | Quantification of surface IgD mean fluorescence intensity (MFI) in Ptenf/f x MD4-trangenic mice in presence (mb1-cre +/+) and absence (mb1-cre +/ki) of Pten. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. Statistical significance was calculated by using an unpaired two-tailed t-test.D | Serum anti-HEL titers. Sera from MD4tg mice of the indicated genotypes were adjusted to an IgM concentration of 500 μg/ml and applied in triplicates to HEL-coated plates in dilution steps of 1 : 3, mean ±SEM. Statistical significance was calculated by using the Kruskal-Wallis test (see also Table S4). Statements of significance in the figure refer to the comparison between Ptenf/f x MD4tg (filled red squares) and Ptenf/f x MD4tg x MD5tg sera (filled orange triangles), ** p ≤ 0.01; *** p ≤ 0.001.E | Immunohistochemistry of sections from spleens of Ptenf/f, Ptenf/f x mb1-cre, Ptenf/f x mb1-cre x 3-83ki and Ptenf/f x mb1-cre x MD4tg mice for CD169 (green), CD5 (red) and IgM (cyan) at 10x magnification. Pictures in the second row show enlarged areas indicated by the white squares, respectively. Double-arrows indicate the distance between T cell (red) and marginal zone (green). Shown pictures are representative of 2 - 3 mice per genotype


Figure 4B | 3 days post transduction the surface expression of IgM / IgD ( dot plots , top ) , IgD and CD23 ( histograms , bottom ) was measured by flow cytometry and compared between EV - and FoxO1 - A3 - transduced cells . Fluorescence minus one ( FMO ) stainings , lacking α - IgD or α - CD23 antibodies respectively , served as controls ( ctrl ) . Bar diagrams below flow cytometry data display the percentages of IgD + cells ( n = 12 ; left ) and MFI of CD23 ( as relative units , RU ) , normalized to the MFI measured in the respective EV transduced cells ; n = 12 ; middle ) . Fcer2 transcript levels were measured by qRT - PCR ( right , n = 4 ) . Mean ± SD , symbols indicate data from individual mice . Statistical significance of IgD expression was determined by applying the Wilcoxon matched - pairs signed rank test for CD23 and by applying the one sample two - tailed t - test for Fcer2 expression . C | Cstf64 ( n = 8 ) , Ell2 ( n = 8 ) and Zfp318 ( n = 11 ) expression levels were measured at 3 days post transduction by qRT - PCR . FoxO1 - A3 - and EV - transduced splenic B cells were FACS - purified according to the gating strategy Mean ± SD , symbols indicate the expression in individual mice . Statistical significance was calculated by using the one - sample two - tailed t - test . E : IgD ( n = 6 ) , CD23 ( n = 4 ) and Fcer2 ( n = 4 ) . F : Cstf64 ( n = 4 ) , Ell2 ( n = 4 ) and Zfp318 ( n = 4 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 4B | 3 days post transduction the surface expression of IgM/IgD (dot plots, top), IgD and CD23 (histograms, bottom) was measured by flow cytometry and compared between EV- and FoxO1-A3-transduced cells. Fluorescence minus one (FMO) stainings, lacking α-IgD or α-CD23 antibodies respectively, served as controls (ctrl). Bar diagrams below flow cytometry data display the percentages of IgD+ cells (n = 12; left) and MFI of CD23 (as relative units, RU), normalized to the MFI measured in the respective EV transduced cells; n = 12; middle). Fcer2 transcript levels were measured by qRT-PCR (right, n = 4). Mean ±SD, symbols indicate data from individual mice. Statistical significance of IgD expression was determined by applying the Wilcoxon matched-pairs signed rank test for CD23 and by applying the one sample two-tailed t-test for Fcer2 expression.C | Cstf64 (n = 8), Ell2 (n = 8) and Zfp318 (n = 11) expression levels were measured at 3 days post transduction by qRT-PCR. FoxO1-A3- and EV-transduced splenic B cells were FACS-purified according to the gating strategy Mean ±SD, symbols indicate the expression in individual mice. Statistical significance was calculated by using the one-sample two-tailed t-test.E: IgD (n = 6), CD23 (n = 4) and Fcer2 (n = 4).F: Cstf64 (n = 4), Ell2 (n = 4) and Zfp318 (n = 4).


Figure 5B | Representative flow cytometric analysis of Ramos cells reconstituted with HH10 - mu IgM , HH10 - mu IgD . Cells were stained with α - murine μHC - , δHC - and κLC - antibodies , respectively . EV - transduced HL - KO cells , expressing GFP only , were used as negative control ( gray ) . C | Representative flow cytometric analysis of HEL - binding in reconstituted Ramos cells after staining with fluorescently - labeled HEL . EV - transduced HL - KO cells , expressing GFP only , were used as negative control ( gray ) . D | Representative intracellular Ca2 + influx in HH10 - mu IgM - and HH10 - mu IgD - expressing cells upon stimulation with multivalent HEL ( complex cHEL ) , monovalent ( soluble sHEL ) ( both at a concentration of 1 μg / ml ) , or 10 μg / ml α - mouse κLC antibody , respectively . E | Representative intracellular Ca2 + influx of HH10 - mu IgM - ( top ) and HH10 - mu IgD - ( bottom ) expressing cells upon stimulation with indicated ratios of 1 μg / ml cHEL and sHEL , and a 1 : 25 mixture of cHEL with bovine serum albumin ( BSA ) , where 1 = 1 μg / ml . F | Schematic overview of reconstitution of BCR - deficient Ramos ( HL - KO ) cells with HH10 - specific human IgM ( HH10 - hu IgM ) and IgD ( HH10 - hu IgD ) BCR isotype . G - J | using HH10 - hu IgM - and HH10 - hu IgD - expressing cells instead of HH10 - mu IgM - and HH10 - mu IgD - expressing cells , respectively . Data shown in G to J are representative of minimum three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B | Representative flow cytometric analysis of Ramos cells reconstituted with HH10-mu IgM, HH10-mu IgD. Cells were stained with α-murine μHC-, δHC- and κLC-antibodies, respectively. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray).C | Representative flow cytometric analysis of HEL-binding in reconstituted Ramos cells after staining with fluorescently-labeled HEL. EV-transduced HL-KO cells, expressing GFP only, were used as negative control (gray).D | Representative intracellular Ca2+ influx in HH10-mu IgM- and HH10-mu IgD-expressing cells upon stimulation with multivalent HEL (complex cHEL), monovalent (soluble sHEL) (both at a concentration of 1 μg/ml), or 10 μg/ml α-mouse κLC antibody, respectively.E | Representative intracellular Ca2+ influx of HH10-mu IgM- (top) and HH10-mu IgD- (bottom) expressing cells upon stimulation with indicated ratios of 1 μg/ml cHEL and sHEL, and a 1 : 25 mixture of cHEL with bovine serum albumin (BSA), where 1 = 1 μg/ml.F | Schematic overview of reconstitution of BCR-deficient Ramos (HL-KO) cells with HH10-specific human IgM (HH10-hu IgM) and IgD (HH10-hu IgD) BCR isotype. G - J | using HH10-hu IgM- and HH10-hu IgD-expressing cells instead of HH10-mu IgM- and HH10-mu IgD-expressing cells, respectively. Data shown in G to J are representative of minimum three independent experiments.


Figure 6A | Quantification of surface IgM ( top ) and IgD ( bottom ) MFI in B cells from mice of the indicated genotypes . Single dots represent the average MFI of 4 independent measurements per mouse ( n ) , mean ± SD . Statistical significance was calculated by using the Kruskal - Wallis test n . s . = not significant ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . | Representative intracellular Ca2 + influx measured in Thy1 . 2 - splenocytes , derived from mice of the indicated genotypes , upon stimulation with cHEL , monovalent sHEL ( both at a concentration of 1 μg / ml ) or 10 μg / ml α - mouse κLC antibody , respectively . Line diagrams below the dot plots show the overlayed MFI of the Ca2 + influx kinetics displayed in the plots above . C | Quantification of intracellular Ca2 + influx The median area under the curve of the Ca2 + influx kinetics was calculated , single dots represent data from individual mice , mean ± SD . Statistical significance was calculated by applying the Mann - Whitney - U test n . s . = not significant , * * p ≤ 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A | Quantification of surface IgM (top) and IgD (bottom) MFI in B cells from mice of the indicated genotypes. Single dots represent the average MFI of 4 independent measurements per mouse (n), mean ±SD. Statistical significance was calculated by using the Kruskal-Wallis test n. s. = not significant; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.| Representative intracellular Ca2+ influx measured in Thy1.2- splenocytes, derived from mice of the indicated genotypes, upon stimulation with cHEL, monovalent sHEL (both at a concentration of 1 μg/ml) or 10 μg/ml α-mouse κLC antibody, respectively. Line diagrams below the dot plots show the overlayed MFI of the Ca2+ influx kinetics displayed in the plots above.C | Quantification of intracellular Ca2+ influx The median area under the curve of the Ca2+ influx kinetics was calculated, single dots represent data from individual mice, mean ±SD. Statistical significance was calculated by applying the Mann-Whitney-U test n. s. = not significant, ** p ≤ 0.01.


Figure 7B | Analysis of GC B cells in splenic B220 + B cells from wild type ( WT , top ) and IgD - / - ( bottom ) animals after 4 , 7 and 10 days ( left to right ) post immunization . Percentages of follicular B cells ( Fo . B ; CD38 + / CD95 - ) and GC B cells ( CD38 - / CD95 + ) are depicted in the plots . GC B cells ( CD38 - / CD95 + ) were further analyzed for GL - 7 expression , compared to Fo . B cells and quantified . C | Quantification of Fo . B cells ( top , percentages ) and CD38 - / CD95 + / GL - 7 + GC cells ( percentages and absolute numbers , bottom panels ) in WT ( black ) and IgD - / - ( green ) animals after 4 , 7 , 10 and 14 days post immunization , mean ± SD ( numbers of animals indicated in the top plot ) . Statistical significance was analyzed by a two - tailed unpaired t - test . D | Analysis of GC in spleen sections from WT and IgD - / - animals after 4 , 7 and 10 days post immunization . Representative images of 2 x 2 mm sections stained with DAPI ( nuclei ) and peanut agglutinin ( PNA ) are shown for the indicated time points . Scale bar represents 500 μm . Dashed white or yellow lines mark distinct GC foci in the merged images . E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within the images of D are shown as enlarged single GCs . As no distinct GC foci were observed in IgD - / - samples from days 4 and 7 , an identical 800 x 800 μm region is shown in each case . F | Quantification of number of GCs per mm2 area of spleen sections from WT ( black ) and IgD - / - ( green ) animals after 4 , 7 and 10 days post immunization . Data represent mean ± SD of two sections ( from 3 animals per group and time point ) and were analyzed by two - tailed unpaired t - test . G | Total serum α - TNP IgG . Sera from PBS - or TNP - Ova - immunized WT ( left ) and IgD - / - ( middle ) mice , collected at day 7 following immunization , were adjusted to an IgG concentration of 1 μg / ml and applied in duplicates and dilution steps of 1 : 3 to TNP - coated plates , respectively , mean ± SEM . Right : Overlay of α - TNP IgG titers from TNP - Ova - immunized WT and IgD - / - mice . Statistical significance was calculated by using the Mann - Whitney - U test ( see also Table S7 ) , n . s . = not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B | Analysis of GC B cells in splenic B220+ B cells from wild type (WT, top) and IgD-/- (bottom) animals after 4, 7 and 10 days (left to right) post immunization. Percentages of follicular B cells (Fo.B; CD38+/CD95-) and GC B cells (CD38-/CD95+) are depicted in the plots. GC B cells (CD38-/CD95+) were further analyzed for GL-7 expression, compared to Fo.B cells and quantified.C | Quantification of Fo.B cells (top, percentages) and CD38-/CD95+/GL-7+ GC cells (percentages and absolute numbers, bottom panels) in WT (black) and IgD-/- (green) animals after 4, 7, 10 and 14 days post immunization, mean ±SD (numbers of animals indicated in the top plot). Statistical significance was analyzed by a two-tailed unpaired t-test.D | Analysis of GC in spleen sections from WT and IgD-/- animals after 4, 7 and 10 days post immunization. Representative images of 2 x 2 mm sections stained with DAPI (nuclei) and peanut agglutinin (PNA) are shown for the indicated time points. Scale bar represents 500 μm. Dashed white or yellow lines mark distinct GC foci in the merged images. E | Representative 800 x 800 μm regions of areas marked by yellow dashed lines within the images of D are shown as enlarged single GCs. As no distinct GC foci were observed in IgD-/- samples from days 4 and 7, an identical 800 x 800 μm region is shown in each case. F | Quantification of number of GCs per mm2 area of spleen sections from WT (black) and IgD-/- (green) animals after 4, 7 and 10 days post immunization. Data represent mean ±SD of two sections (from 3 animals per group and time point) and were analyzed by two-tailed unpaired t-test. G | Total serum α-TNP IgG. Sera from PBS- or TNP-Ova-immunized WT (left) and IgD-/- (middle) mice, collected at day 7 following immunization, were adjusted to an IgG concentration of 1 μg/ml and applied in duplicates and dilution steps of 1 : 3 to TNP-coated plates, respectively, mean ±SEM. Right: Overlay of α-TNP IgG titers from TNP-Ova-immunized WT and IgD-/- mice. Statistical significance was calculated by using the Mann-Whitney-U test (see also Table S7), n. s. = not significant, * p ≤ 0.05, ** p ≤ 0.01.


f1 ( a ) TbMCU - GFP co - localization with MitoTracker ( MT ) in mitochondria of PCF trypanosomes as detected by immunofluorescence analyses with antibodies against GFP ( Pearson ' s correlation coefficient ( PCC ) : 0 . 912 ) . ( b , c ) TbMCU co - localization with MitoTracker in the mitochondria of PCF ( b ) or BSF ( c ) trypanosomes , as detected by immunofluorescence analyses with antibodies against TbMCU . PCC : 0 . 930 and 0 . 920 , respectively . Merge images show the co - localization in yellow . DIC , differential interference contrast microscopy . Scale bar , 10 μm ( a - c ) . ( d , e ) Growth of BSF ( d ) and PCF ( e ) trypanosomes in the absence ( black lines , − Tet ) or presence ( red lines , + Tet ) of 1 μg ml − 1 tetracycline for the indicated number of days . Values are mean ± s . d . ( n = 3 ) . ( f , g ) Northern blot analyses of TbMCU RNAi of BSF ( f ) and PCF ( g ) trypanosomes grown in the absence ( 0 ) and presence ( 2 - 8 ; 2 - 10 ) of tetracycline ( top ) . Tubulin is shown as a loading control ( bottom ) . Markers are shown on the right . The transcript of TbMCU , including the 5 ′ - and 3 ′ - UTR was ~ 1 . 5 kb in length . ( h , i ) Western blot analyses of TbMCU RNAi of BSF ( h ) and PCF ( i ) trypanosomes grown in the absence ( 0 ) or presence ( 2 - 8 ; 2 - 10 ) of tetracycline . Total lysates ( 30 μg ) were subjected to 10 % SDS - polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against TbMCU ( top ) . One band of ~ 30 kDa was detected in T . brucei homogenates . Membranes were stripped and re - incubated with antibody against the voltage - dependent anion channel ( TbVDAC ) as a loading control ( bottom ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f1(a) TbMCU-GFP co-localization with MitoTracker (MT) in mitochondria of PCF trypanosomes as detected by immunofluorescence analyses with antibodies against GFP (Pearson's correlation coefficient (PCC): 0.912). (b,c) TbMCU co-localization with MitoTracker in the mitochondria of PCF (b) or BSF (c) trypanosomes, as detected by immunofluorescence analyses with antibodies against TbMCU. PCC: 0.930 and 0.920, respectively. Merge images show the co-localization in yellow. DIC, differential interference contrast microscopy. Scale bar, 10 μm (a-c).(d,e) Growth of BSF (d) and PCF (e) trypanosomes in the absence (black lines, −Tet) or presence (red lines, +Tet) of 1 μg ml−1 tetracycline for the indicated number of days. Values are mean±s.d. (n=3).(f,g) Northern blot analyses of TbMCU RNAi of BSF (f) and PCF (g) trypanosomes grown in the absence (0) and presence (2-8; 2-10) of tetracycline (top). Tubulin is shown as a loading control (bottom). Markers are shown on the right. The transcript of TbMCU, including the 5′- and 3′-UTR was ~1.5 kb in length.(h,i) Western blot analyses of TbMCU RNAi of BSF (h) and PCF (i) trypanosomes grown in the absence (0) or presence (2-8; 2-10) of tetracycline. Total lysates (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against TbMCU (top). One band of ~30 kDa was detected in T. brucei homogenates. Membranes were stripped and re-incubated with antibody against the voltage-dependent anion channel (TbVDAC) as a loading control (bottom).


f2 ( a ) The reaction buffer contained 2 mM succinate and 1 μM Calcium Green 5 - N . After several pulses of Ca2 + ( 8 μM final concentration ) cells ( 5 × 107 trypanosomes grown in the absence ( black tracing ) or presence ( green tracing ) of tetracycline ) were added to the reaction medium ( 2 . 45 ml ) and the reaction was started adding 50 μM digitonin . Ruthenium red ( RR , 40 μM , yellow and red tracing ) , and FCCP ( 5 μM ) were added where indicated . A decrease in fluorescence indicates decreasing medium Ca2 + or increasing vesicular Ca2 + . ( b ) Relative Ca2 + uptake at 700 s as compared with that of control trypanosomes grown in the absence of tetracycline considered as 1 . 0 ( − Tet ) ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( c ) Similar conditions as in a except that further CaCl2 additions ( 8 μM each time ) were added to − Tet trypanosomes to show the high mitochondrial capacity to take up Ca2 + . EGTA ( 8 μM ) and CaCl2 ( Ca2 + ) were added where indicated to show the presence of medium Ca2 + . ( d ) Relative initial rate of Ca2 + uptake by + Tet as compared with − Tet trypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( e ) Representative traces of Ca2 + uptake kinetics in digitonin - permeabilized TbMCU BSF trypanosomes cultured in the absence ( − Tet ) or presence ( + Tet ) of tetracycline . The reactions were started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate . Ca2 + uptake was monitored over time using 1 μM Calcium Green - 5N . Ruthenium red ( RR , 40 μM , yellow and red tracings ) , oligomycin ( Oligo , 2 . 5 μg ml − 1 ) , EGTA ( 0 . 4 μM ) and CaCl2 ( pulses of 0 . 4 μM ) were added where indicated . ( f ) Relative Ca2 + uptake at 200 s as compared with that of control BSF trypanosomes grown in the absence of tetracycline considered as 1 ( − Tet ) ( means ± s . d . , n = 3 , * ( + Tet ± RR ) , * * ( − Tet ± RR ) , * * * ( − Tet / + Tet ) , P0 . 001 , Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2(a) The reaction buffer contained 2 mM succinate and 1 μM Calcium Green 5-N. After several pulses of Ca2+ (8 μM final concentration) cells (5 × 107 trypanosomes grown in the absence (black tracing) or presence (green tracing) of tetracycline) were added to the reaction medium (2.45 ml) and the reaction was started adding 50 μM digitonin. Ruthenium red (RR, 40 μM, yellow and red tracing), and FCCP (5 μM) were added where indicated. A decrease in fluorescence indicates decreasing medium Ca2+ or increasing vesicular Ca2+. (b) Relative Ca2+ uptake at 700 s as compared with that of control trypanosomes grown in the absence of tetracycline considered as 1.0 (−Tet) (means±s.d., n=3, **P0.001, Student's t-test).(c) Similar conditions as in a except that further CaCl2 additions (8 μM each time) were added to −Tet trypanosomes to show the high mitochondrial capacity to take up Ca2+. EGTA (8 μM) and CaCl2 (Ca2+) were added where indicated to show the presence of medium Ca2+. (d) Relative initial rate of Ca2+ uptake by +Tet as compared with −Tet trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).(e) Representative traces of Ca2+ uptake kinetics in digitonin-permeabilized TbMCU BSF trypanosomes cultured in the absence (−Tet) or presence (+Tet) of tetracycline. The reactions were started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate. Ca2+ uptake was monitored over time using 1 μM Calcium Green-5N. Ruthenium red (RR, 40 μM, yellow and red tracings), oligomycin (Oligo, 2.5 μg ml−1), EGTA (0.4 μM) and CaCl2 (pulses of 0.4 μM) were added where indicated. (f) Relative Ca2+ uptake at 200 s as compared with that of control BSF trypanosomes grown in the absence of tetracycline considered as 1 (−Tet) (means±s.d., n=3, *(+Tet±RR), **(−Tet±RR), ***(−Tet/+Tet) , P0.001, Student's t-test).


f3 ( a ) PCFtrypanosomes ( 5 × 107 cells ) were added to the reaction buffer ( 2 . 45 ml ) containing 2 mM succinate , and 5 μM safranine , and the reaction was started with digitonin ( 50 μM ) . ADP ( 10 μM ) , oligomycin ( Oligo , 2 μg ml − 1 ) , CaCl2 ( 40 μM ) , EGTA ( 200 μM ) and FCCP ( 10 μM ) were added where indicated . ( b ) Changes in safranine fluorescence after addition of ADP or Ca2 + to − Tet and + Tet PCFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) . ( c ) BSFtrypanosomes ( 2 × 108 cells ) were added to the reaction buffer ( 2 . 45 ml ) containing EGTA ( 20 μM ) , ATP ( 1 mM ) , sodium orthovanadate ( 500 μM ) and 12 . 5 μM safranine . The reaction was started with digitonin ( 40 μM ) . CaCl2 ( 50 μM ) , EGTA ( 200 μM ) and FCCP ( 10 μM ) were added where indicated . ( d , e ) Changes in safranine fluorescence after addition of Ca2 + ( d ) or FCCP ( e ) to − Tet and + Tet BSFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3(a) PCFtrypanosomes (5 × 107 cells) were added to the reaction buffer (2.45 ml) containing 2 mM succinate, and 5 μM safranine, and the reaction was started with digitonin (50 μM). ADP (10 μM), oligomycin (Oligo, 2 μg ml−1), CaCl2 (40 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (b) Changes in safranine fluorescence after addition of ADP or Ca2+ to −Tet and +Tet PCFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).(c) BSFtrypanosomes (2 × 108 cells) were added to the reaction buffer (2.45 ml) containing EGTA (20 μM), ATP (1 mM), sodium orthovanadate (500 μM) and 12.5 μM safranine. The reaction was started with digitonin (40 μM). CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (d,e) Changes in safranine fluorescence after addition of Ca2+ (d) or FCCP (e) to −Tet and +Tet BSFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).


f4 ( a ) Comparison of AMP / ATP ratios between PCF trypanosomes grown in the absence ( − Tet ) or presence ( + Tet ) of tetracycline to induce RNAi , and with or without glucose ( Gl ) , expressed as fold increase ( means ± s . d . , n = 3 ; * P0 . 05 ; * * P0 . 01 , Student ' s t - test ) . ( b ) Comparison of AMP / ATP ratios between ( − Tet ) and ( + Tet ) PCF trypanosomes grown in a glucose - depleted medium ( SDM - 80 ) containing 5 . 2 mM L - proline for 2 days after 2 - day growth in SDM - 79 ± tetracycline , expressed as fold increase ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) . ( c ) Fluorescence microscopic images of TbATG8 . 2 in PCF trypanosomes cultured in SDM - 79 in the absence ( WT ) or presence ( RNAi ) of tetracycline to induce RNAi ; scale bars , 10 μm . ( d ) Quantification of autophagosome formation in RNAi PCF trypanosomes grown in SDM - 79 or SDM - 80 medium in the presence ( + Tet ) or absence ( − Tet ) of tetracycline . Over 500 cells from three experiments with 20 random fields / experiment were analysed ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( e ) Western blot analysis of ATG8 . 2 or tubulin in PCFtrypanosomes cultured in SDM - 79 or SDM - 80 in the absence ( − Tet ) or presence ( + Tet ) of tetracycline to induce RNAi . Cell lysates ( 30 μg ) were fractionated in 12 % urea - SDS - PAGE gel . Immunoblots were labelled with anti - ATG8 . 2 or anti - tubulin antibody . ( f ) Quantification of ATG8 . 2 - II / ( ATG8 . 2 - II + ATG8 . 2 - I ) presented as mean ratios ± s . d . ( n = 3 , * * P0 . 01 , Student ' s t - test ) . ( g ) Groups of five mice were infected with WT T . brucei ( black line ) or trypanosomes transfected with the construct for RNAi of TbMCU ( red line ) . Doxycycline ( green line ; 200 μg ml − 1 ) was given in the drinking water throughout a 30 - day period . ( h ) Parasitemia levels in surviving mice were monitored at 2 - day intervals after infection . The results are means ± s . d . ( n = 2 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4(a) Comparison of AMP/ATP ratios between PCF trypanosomes grown in the absence (−Tet) or presence (+Tet) of tetracycline to induce RNAi, and with or without glucose (Gl), expressed as fold increase (means±s.d., n=3; *P0.05; **P0.01, Student's t-test).(b) Comparison of AMP/ATP ratios between (−Tet) and (+Tet) PCF trypanosomes grown in a glucose-depleted medium (SDM-80) containing 5.2 mM L-proline for 2 days after 2-day growth in SDM-79±tetracycline, expressed as fold increase (means±s.d., n=3, **P0.01, Student's t-test).(c) Fluorescence microscopic images of TbATG8.2 in PCF trypanosomes cultured in SDM-79 in the absence (WT) or presence (RNAi) of tetracycline to induce RNAi; scale bars, 10 μm. (d) Quantification of autophagosome formation in RNAi PCF trypanosomes grown in SDM-79 or SDM-80 medium in the presence (+Tet) or absence (−Tet) of tetracycline. Over 500 cells from three experiments with 20 random fields/experiment were analysed (means±s.d., n=3, **P0.001, Student's t-test).(e) Western blot analysis of ATG8.2 or tubulin in PCFtrypanosomes cultured in SDM-79 or SDM-80 in the absence (−Tet) or presence (+ Tet) of tetracycline to induce RNAi. Cell lysates (30 μg) were fractionated in 12% urea-SDS-PAGE gel. Immunoblots were labelled with anti-ATG8.2 or anti-tubulin antibody. (f) Quantification of ATG8.2-II/(ATG8.2-II+ATG8.2-I) presented as mean ratios±s.d. (n=3, **P0.01, Student's t-test).(g) Groups of five mice were infected with WT T. brucei (black line) or trypanosomes transfected with the construct for RNAi of TbMCU (red line). Doxycycline (green line; 200 μg ml−1) was given in the drinking water throughout a 30-day period.(h) Parasitemia levels in surviving mice were monitored at 2-day intervals after infection. The results are means±s.d. (n=2).


f5 ( a ) Growth of TbMCU - KO BSF in the absence ( black line ) or presence ( red line ) of 1 μg ml − 1 tetracycline for the indicated number of days . Addition of 10 mM threonine to the medium ( green line ) partially rescued the mutant BSF trypanosomes but had no effect on + Tet cells ( yellow line ; means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( b ) Southern blot analysis of the BSF conditional KOs . Genomic DNA from the T . brucei SM parental strain ( + / + ) , single - ( + / − ) or double - allele ( − / − ) KO cells . ( c ) Northern blot analysis of wild - type ( WT ) and TbMCU - KO BSF cultured in the presence ( + Tet ) or absence ( − Tet ) of tetracycline for 2 days . Tubulin is shown as a loading control ( bottom panel ) . ( d ) Western blot analysis of total cell lysates . Proteins from BSF trypanosomes cultured in the presence ( + Tet ) or absence ( − Tet ) of tetracycline were detected using anti - HA antibody . TbVDAC was used as a loading control . ( e ) Immunofluorescence analysis of control ( upper panels ) and TbMCU - KO ( lower panels ) trypanosomes . TbMCU co - localized with MitoTracker ( MT ) in the control parasites ( + Tet ; PCC = 0 . 808 ) . Scale bar , 10 μm . ( f ) Conditional TbMCU - KO BSF trypanosomes were grown in the presence ( + Tet ) or absence ( − Tet ) of tetracycline for 2 days . BSF trypanosomes ( 2 × 108 cells ) were incubated as in Fig . 2e . Multiple pulses of CaCl2 ( arrowheads ) were added to a final concentration of 20 μM . A representative trace ( red or black ) of Ca2 + uptake from one of three independent experiments is shown . ( g ) Representative traces of mitochondrial membrane potential of digitonin - permeabilized BSF trypanosomes grown in the presence ( + Tet ) or absence ( − Tet ) of tetracycline . The reactions were incubated as in Fig . 3c . CaCl2 ( 50 μM ) , EGTA ( 200 μM ) and FCCP ( 10 μM ) were added where indicated . ( h , i ) Representative traces of mitochondrial membrane potential of digitonin - permeabilized BSFtrypanosomes grown in the presence ( + Tet ) or absence ( − Tet ) of tetracycline . The reactions were incubated as in Fig . 3c . CaCl2 25 , 50 and 100 μM CaCl2 ) or FCCP ( i ) to + Tet and − Tet BSFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5(a) Growth of TbMCU-KO BSF in the absence (black line) or presence (red line) of 1 μg ml−1 tetracycline for the indicated number of days. Addition of 10 mM threonine to the medium (green line) partially rescued the mutant BSF trypanosomes but had no effect on +Tet cells (yellow line; means±s.d., n=3, **P0.001, Student's t-test).(b) Southern blot analysis of the BSF conditional KOs. Genomic DNA from the T. brucei SM parental strain (+/+), single- (+/−) or double-allele (−/−) KO cells.(c) Northern blot analysis of wild-type (WT) and TbMCU-KO BSF cultured in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. Tubulin is shown as a loading control (bottom panel).(d) Western blot analysis of total cell lysates. Proteins from BSF trypanosomes cultured in the presence (+Tet) or absence (−Tet) of tetracycline were detected using anti-HA antibody. TbVDAC was used as a loading control.(e) Immunofluorescence analysis of control (upper panels) and TbMCU-KO (lower panels) trypanosomes. TbMCU co-localized with MitoTracker (MT) in the control parasites (+Tet; PCC=0.808). Scale bar, 10 μm.(f) Conditional TbMCU-KO BSF trypanosomes were grown in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. BSF trypanosomes (2 × 108 cells) were incubated as in Fig. 2e. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 20 μM. A representative trace (red or black) of Ca2+ uptake from one of three independent experiments is shown.(g) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSF trypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated.(h,i) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSFtrypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 25, 50 and 100 μM CaCl2) or FCCP (i) to +Tet and −Tet BSFtrypanosomes (means±s.d., n=3, **P0.001, Student's t-test).


f6 ( a ) Rhod - 2 fluorescence in control or overexpressing TbMCU ( TbMCU - OE ) PCF . Scale bar , 10 μm . ( b , c ) Rhod - 2 ( mitochondrial ) ( b ) and Fluo - 4 ( cytosolic ) ( c ) fluorescence in control ( − Tet ) and overexpressing TbMCU ( + Tet ) PCF ( means ± s . d . , n = 3 , * * P0 . 05 , Student ' s t - test ) ; NS , not significant . ( d ) Ca2 + uptake by digitonin - permeabilized PCF overexpressing TbMCU . Trypanosomes were incubated as in Fig . 2c . Multiple pulses of CaCl2 ( arrowheads ) were added to a final concentration of 8 μM . ( e ) Ca2 + uptake in TbMCU - OE PCF ( + Tet ) 200 s after addition of Ca2 + , as compared with uninduced trypanosomes considered as 1 ( − Tet ) ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) . ( f ) Mitochondrial membrane potential of digitonin - permeabilized control ( − Tet ) and TbMCU - OE ( + Tet ) PCF . The reactions were incubated as in Fig . 3a . ( g ) Changes in safranine fluorescence after addition of ADP , Ca2 + or FCCP to - Tet and + Tet PCF trypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) . ( h ) Mitochondrial oxidative stress as measured with MitoSOX Red in control ( C ) and TbMCU - overexpressing ( OE ) PCF permeabilized with 50 μM digitonin in the presence of 0 . 4 mM Ca2 + . Controls were treated with 2 μM antimycin A ( C + AA ) , or were trypanosomes exposed to 0 . 4 mM Ca2 + in the absence of digitonin ( − DIG + Ca2 + ) , and trypanosomes permeabilized with digitonin in the absence of Ca2 + ( C , yellow line ) . ( i ) Quantification of change at 900 s . Results are expressed as mean fluorescence ± s . d . ( n = 3 , * * P0 . 001 , Student ' s t - test ) . ( j ) Growth of trypanosomes in the absence ( control , black lines ) or presence ( TbMCU - OE , red lines ) of 1 μg ml − 1 tetracycline ( means ± s . d . , n = 3 ) . ( k ) Dead cells in representative fields of control ( − Tet ) and TbMCU - OE ( + Tet ) PCF are indicated ( white arrows ) . Scale bar , 10 μm . ( l ) Cell viability on apoptotic challenge of control ( − Tet ) or TbMCU - OE ( + Tet ) PCF trypanosomes with H2O2 and C2 - ceramide . The percentage of cell death was obtained from means ± s . d . by analysing > 60 fields ( including > 400 cells ) ( n = 3 ) . * * P0 . 01 , Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f6(a) Rhod-2 fluorescence in control or overexpressing TbMCU (TbMCU-OE) PCF. Scale bar, 10 μm. (b,c) Rhod-2 (mitochondrial) (b) and Fluo-4 (cytosolic) (c) fluorescence in control (−Tet) and overexpressing TbMCU (+Tet) PCF (means±s.d., n=3, **P0.05, Student's t-test); NS, not significant.(d) Ca2+ uptake by digitonin-permeabilized PCF overexpressing TbMCU. Trypanosomes were incubated as in Fig. 2c. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 8 μM. (e) Ca2+ uptake in TbMCU-OE PCF (+Tet) 200 s after addition of Ca2+, as compared with uninduced trypanosomes considered as 1 (−Tet) (means±s.d., n=3, **P0.01, Student's t-test).(f) Mitochondrial membrane potential of digitonin-permeabilized control (−Tet) and TbMCU-OE (+Tet) PCF. The reactions were incubated as in Fig. 3a.(g) Changes in safranine fluorescence after addition of ADP, Ca2+ or FCCP to -Tet and +Tet PCF trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).(h) Mitochondrial oxidative stress as measured with MitoSOX Red in control (C) and TbMCU-overexpressing (OE) PCF permeabilized with 50 μM digitonin in the presence of 0.4 mM Ca2+. Controls were treated with 2 μM antimycin A (C+AA), or were trypanosomes exposed to 0.4 mM Ca2+ in the absence of digitonin (−DIG+Ca2+), and trypanosomes permeabilized with digitonin in the absence of Ca2+ (C, yellow line). (i) Quantification of change at 900 s. Results are expressed as mean fluorescence±s.d. (n=3, **P0.001, Student's t-test).(j) Growth of trypanosomes in the absence (control, black lines) or presence (TbMCU-OE, red lines) of 1 μg ml−1 tetracycline (means±s.d., n=3). (k) Dead cells in representative fields of control (−Tet) and TbMCU-OE (+Tet) PCF are indicated (white arrows). Scale bar, 10 μm.(l) Cell viability on apoptotic challenge of control (−Tet) or TbMCU-OE (+Tet) PCF trypanosomes with H2O2 and C2-ceramide. The percentage of cell death was obtained from means±s.d. by analysing >60 fields (including >400 cells) (n=3). **P0.01, Student's t-test.


Figure 1Chl - HyPer2 response to chloroplast hydrogen peroxide elicited by BSMV infection in N . benthamiana epidermal cells . Ratiometric images ( F488 / 405 nm ) of fluorescence excitation at 488 and 405 nm show the oxidized state of chloroplast - targeted HyPer2 , respectively . The false - blue color indicates the chloroplasts . Scale bars , 30 μm . Data information : In ( A ) , data are representative of at least three independent experiments . Quantification of the BSMV - dependent changes in Chl - HyPer2 fluorescence at 0 to 6 dpi . The percentage of ROS - positive chloroplasts ( white ) among all chloroplasts ( blue and white ) per visual field . Data information : In ( B ) , error bars indicate mean ± SEM ( n = 5 ) . Western blot analysis of 2 - Cys Prx redox status in empty vector ( EV ) and BSMV inoculated leaves under non - reducing conditions at 3 dpi . Western blots were analyzed with anti - 2 - Cys Prx or anti - CP antibodies . The Rubisco large subunit ( RbcL ) served as a loading control . Data information : , representative data are shown , at least three independent replicates were performed with similar results . Relative mRNA levels of the sAPX , 2 - Cys Prx , GPXL , and PrxQ genes in response to BSMV infections at 1 , 3 and 5 dpi . The empty vector ( EV ) was used as a negative control , NbEF1α was used as an internal control . Data information : In ( D ) , error bars indicate mean ± SEM from three independent experiments ; * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * p - value < 0 . 001 , * * * * p - value < 0 . 0001 from Student ' s t test . BSMV accumulation in N . benthamiana leaves transiently expressing chloroplast ROS scavenging proteins . Agrobacterium harboring plasmids expressing various proteins are indicated above the panels . BSMV was agroinfiltrated into the same leaves at 1 . 5 dpi . Three days later , Western blot analyses were conducted with anti - TGB1 or anti - Flag antibodies . RbcL served as the loading control . 2 - CP , 2 - Cys Prx . Data information : representative data are shown , at least three independent replicates were performed with similar results . BSMV accumulation in 2 - Cys Prx or GPXL - silenced N . benthamiana plants using anti - TGB1 antibody . Empty vector TRV : 00 was used as negative control . Data information : representative data are shown , at least three independent replicates were performed with similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Chl-HyPer2 response to chloroplast hydrogen peroxide elicited by BSMV infection in N. benthamiana epidermal cells. Ratiometric images (F488/405 nm) of fluorescence excitation at 488 and 405 nm show the oxidized state of chloroplast-targeted HyPer2, respectively. The false-blue color indicates the chloroplasts. Scale bars, 30 μm. Data information: In (A), data are representative of at least three independent experiments.Quantification of the BSMV-dependent changes in Chl-HyPer2 fluorescence at 0 to 6 dpi. The percentage of ROS-positive chloroplasts (white) among all chloroplasts (blue and white) per visual field. Data information: In (B), error bars indicate mean ± SEM (n = 5).Western blot analysis of 2-Cys Prx redox status in empty vector (EV) and BSMV inoculated leaves under non-reducing conditions at 3 dpi. Western blots were analyzed with anti-2-Cys Prx or anti-CP antibodies. The Rubisco large subunit (RbcL) served as a loading control. Data information: , representative data are shown, at least three independent replicates were performed with similar results.Relative mRNA levels of the sAPX, 2-Cys Prx, GPXL, and PrxQ genes in response to BSMV infections at 1, 3 and 5 dpi. The empty vector (EV) was used as a negative control, NbEF1α was used as an internal control. Data information: In (D), error bars indicate mean ± SEM from three independent experiments; * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001 from Student's t test.BSMV accumulation in N. benthamiana leaves transiently expressing chloroplast ROS scavenging proteins. Agrobacterium harboring plasmids expressing various proteins are indicated above the panels. BSMV was agroinfiltrated into the same leaves at 1.5 dpi. Three days later, Western blot analyses were conducted with anti-TGB1 or anti-Flag antibodies. RbcL served as the loading control. 2-CP, 2-Cys Prx. Data information: representative data are shown, at least three independent replicates were performed with similar results.BSMV accumulation in 2-Cys Prx or GPXL-silenced N. benthamiana plants using anti-TGB1 antibody. Empty vector TRV: 00 was used as negative control. Data information: representative data are shown, at least three independent replicates were performed with similar results.


Figure 2Y2H analyses of interactions between γb and NbNTRC . Various Y2H combinations are indicated on the images . Self - interactions of γb served as a positive control , and combinations containing either empty AD or BD constructs served as negative controls . BiFC analyses of γb and NbNTRC interactions during BSMV infections . Various BiFC combinations are indicated on the left . YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false - green color and chloroplasts were visualized by chloroplast autofluorescence as a false - red color . Scale bars , 20 μm . Co - IP analyses of interactions between γb and NbNTRC in N . benthamiana . Leaf tissues were agroinfiltrated with various combinations indicated above the panels were harvested at 3 dpi . Total proteins were immunoprecipitated with anti - Flag beads . Total and immunoprecipitated ( IP ) proteins were analyzed by Western blotting with anti - GFP or anti - Flag antibodies . Data information : representative data are shown , and three replicates had similar results . GST pull - down assays to verify interactions between γb and NbNTRC in vitro . GST - tagged NTRC or GFP were incubated with γb - His or GFP - His . Input and pull - down products were analyzed by Western blot analyses with anti - GST or anti - His antibodies . GFP - His and GST - GFP served as negative controls . Data information : representative data are shown , and three replicates had similar results . Co - localization of γb and NbNTRC during BSMV infection . NbNTRC - mCherry was co - expressed with BSMVγb - GFP ( Jiang et al . , 2020 ) in N . benthamiana , and confocal analyses were conducted at 3 dpi , and chloroplast autofluorescence is displayed as a false - blue color . Scale bars , 20 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Y2H analyses of interactions between γb and NbNTRC. Various Y2H combinations are indicated on the images. Self-interactions of γb served as a positive control, and combinations containing either empty AD or BD constructs served as negative controls.BiFC analyses of γb and NbNTRC interactions during BSMV infections. Various BiFC combinations are indicated on the left. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color and chloroplasts were visualized by chloroplast autofluorescence as a false-red color. Scale bars, 20 μm.Co-IP analyses of interactions between γb and NbNTRC in N. benthamiana. Leaf tissues were agroinfiltrated with various combinations indicated above the panels were harvested at 3 dpi. Total proteins were immunoprecipitated with anti-Flag beads. Total and immunoprecipitated (IP) proteins were analyzed by Western blotting with anti-GFP or anti-Flag antibodies. Data information: representative data are shown, and three replicates had similar results.GST pull-down assays to verify interactions between γb and NbNTRC in vitro. GST-tagged NTRC or GFP were incubated with γb-His or GFP-His. Input and pull-down products were analyzed by Western blot analyses with anti-GST or anti-His antibodies. GFP-His and GST-GFP served as negative controls. Data information: representative data are shown, and three replicates had similar results.Co-localization of γb and NbNTRC during BSMV infection. NbNTRC-mCherry was co-expressed with BSMVγb-GFP (Jiang et al., 2020) in N. benthamiana, and confocal analyses were conducted at 3 dpi, and chloroplast autofluorescence is displayed as a false-blue color. Scale bars, 20 μm.


Figure 3Y2H assay to analyze interactions between NbNTRC and γb mutants . Various Y2H combinations are indicated on the images . Interactions of γb and NbNTRC were confirmed earlier and served as a positive control , combinations containing an empty BD construct served as a negative control . BiFC analysis of interactions between NbNTRC and γb mutants during BSMV infection of N . benthamiana . Various BiFC combinations are indicated at the top . YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false - green color and chloroplasts were visualized by chloroplast autofluorescence as a false - red color . RbcL - fused YFPc served as a negative control . Scale bars , 20 μm . Co - IP analysis of interactions between NbNTRC and γb mutants . NbNTRC - 3xFlag proteins together with γb - GFP , γbH85A - GFP or γbH85C - GFP were co - expressed in N . benthamiana leaves and harvested at 3 dpi . Total proteins were IP with anti - Flag beads . Total and IP products were detected in Western blots with anti - GFP or anti - Flag antibodies . Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information : , representative data were shown , and three independent experiments showed a similar result . GST pull - down assays to evaluate in vitro interactions between NbNTRC and γb mutants . Purified γb - His , γbH85A - His , γbH85C - His , or GFP - His proteins were incubated with GST - tagged NbNTRC . Input and pull - down products were analyzed by Western blotting with anti - GST or anti - His antibodies . GFP - His served as a negative control . Arrowhead indicates the specific band of the target protein . Data information : representative data were shown , and three independent experiments showed a similar result . Analysis of VSR activities of γb mutants . Infiltrated N . benthamiana leaves co - expressing sGFP plus γbH85A - 3xFlag , γbH85C - 3xFlag or the pGD empty vector ( EV ) were observed at 5 dpi under long - wave UV illumination for GFP fluorescence . The γb - 3xFlag protein served as a positive control . The white dashed circles indicate the infiltrated zone . Western blot analysis of the GFP protein in co - infiltrated leaves at 5 dpi with anti - GFP antibody . RbcL was used as loading control . Data information : representative data were shown , and three independent experiments showed a similar result .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Y2H assay to analyze interactions between NbNTRC and γb mutants. Various Y2H combinations are indicated on the images. Interactions of γb and NbNTRC were confirmed earlier and served as a positive control, combinations containing an empty BD construct served as a negative control.BiFC analysis of interactions between NbNTRC and γb mutants during BSMV infection of N. benthamiana. Various BiFC combinations are indicated at the top. YFP signals were visualized by confocal microscopy at 3 dpi and depicted as a false-green color and chloroplasts were visualized by chloroplast autofluorescence as a false-red color. RbcL-fused YFPc served as a negative control. Scale bars, 20 μm.Co-IP analysis of interactions between NbNTRC and γb mutants. NbNTRC-3xFlag proteins together with γb-GFP, γbH85A-GFP or γbH85C-GFP were co-expressed in N. benthamiana leaves and harvested at 3 dpi. Total proteins were IP with anti-Flag beads. Total and IP products were detected in Western blots with anti-GFP or anti-Flag antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information: , representative data were shown, and three independent experiments showed a similar result.GST pull-down assays to evaluate in vitro interactions between NbNTRC and γb mutants. Purified γb-His, γbH85A-His, γbH85C-His, or GFP-His proteins were incubated with GST-tagged NbNTRC. Input and pull-down products were analyzed by Western blotting with anti-GST or anti-His antibodies. GFP-His served as a negative control. Arrowhead indicates the specific band of the target protein. Data information: representative data were shown, and three independent experiments showed a similar result.Analysis of VSR activities of γb mutants. Infiltrated N. benthamiana leaves co-expressing sGFP plus γbH85A-3xFlag, γbH85C-3xFlag or the pGD empty vector (EV) were observed at 5 dpi under long-wave UV illumination for GFP fluorescence. The γb-3xFlag protein served as a positive control. The white dashed circles indicate the infiltrated zone.   Western blot analysis of the GFP protein in co-infiltrated leaves at 5 dpi with anti-GFP antibody. RbcL was used as loading control.  Data information:  representative data were shown, and three independent experiments showed a similar result.


Figure 4Effects of NbNTRC overexpression on BSMV protein accumulation . N . benthamiana leaves were first infiltrated with Agrobacterium containing various contructs indicated above the panel . After 1 . 5 dpi , BSMV was inoculated onto the leaves by agroinfiltration . Three days later , total protein was extracted for Western blotting with anti - TGB1 and anti - Flag antibodies . The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows RbcL was a loading control . Data information : representative data are shown and three eplicates similar results . Effects of NbNTRC overexpression on BSMV replication . Movement - deficient BSMV ( RNAα + RNAγ ) was infiltrated into leaves that had been agroinfiltrated previously with various constructs indicated above the panel . NbNTRC and viral γb proteins were detected by Western blotting with anti - Flag and anti - γb antibodies . Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows ( see Appendix Fig S4 ) . RT - qPCR analyses of viral RNA accumulation in 4B . NbPP2A was used as an internal control . Data information : representative data are shown and three eplicates similar results . In ( C ) , error bars indicate mean ± SEM from three independent experiments . The symptoms of infection after BSMV inoculation of a different N . benthamiana plant at 8 dpi . Magnified images of leaves ( in white boxes ) are shown in the lower panels . Scale bars , 2 cm . Statistical analysis of symptom appearance in different N . benthamiana plants after inoculation with BSMV . Western blot analysis of the virus accumulation in Figure 4D with anti - TGB1 antibody . RbcL served as loading control . Data information : , representative data are shown and three eplicates similar results . In ( E ) , error bars indicate mean ± SD from three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Effects of NbNTRC overexpression on BSMV protein accumulation. N. benthamiana leaves were first infiltrated with Agrobacterium containing various contructs indicated above the panel. After 1.5 dpi, BSMV was inoculated onto the leaves by agroinfiltration. Three days later, total protein was extracted for Western blotting with anti-TGB1 and anti-Flag antibodies. The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows RbcL was a loading control. Data information: representative data are shown and three eplicates similar results.Effects of NbNTRC overexpression on BSMV replication. Movement-deficient BSMV (RNAα + RNAγ) was infiltrated into leaves that had been agroinfiltrated previously with various constructs indicated above the panel. NbNTRC and viral γb proteins were detected by Western blotting with anti-Flag and anti-γb antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows (see Appendix Fig S4).   RT-qPCR analyses of viral RNA accumulation in 4B. NbPP2A was used as an internal control.   Data information: representative data are shown and three eplicates similar results. In (C), error bars indicate mean ± SEM from three independent experiments.The symptoms of infection after BSMV inoculation of a different N. benthamiana plant at 8 dpi. Magnified images of leaves (in white boxes) are shown in the lower panels. Scale bars, 2 cm.   Statistical analysis of symptom appearance in different N. benthamiana plants after inoculation with BSMV.   Western blot analysis of the virus accumulation in Figure 4D with anti-TGB1 antibody. RbcL served as loading control.   Data information: , representative data are shown and three eplicates similar results. In (E), error bars indicate mean ± SD from three independent experiments.


Figure 5Left panel : systemic symptoms of N . benthamiana plants inoculated with BSMV and BSMV γb mutants . Photographs were taken at 7 dpi . Right panel : schematic representation of inoculated ( ino . ) , older systemic leaves ( o - SL ) , and younger systemic leaves ( y - SL ) in N . benthamiana plants . Scale bars , 2 cm . Western blot analyses of TGB1 and CP accumulation in BSMV and BSMV γb mutant inoculated N . benthamiana leaves . Anti - TGB1 and anti - CP were used for protein detection . Data information : , representative data are shown , and three replicates had similar results . RbcL served as loading control . Western blot detection of BSMV with anti - CP antibodies in upper N . benthamiana leaves at 7 days ( o - SL ) or 28 days ( y - SL ) after infiltration . o - SL , older systemic leaves ; y - SL , younger systemic leaves . Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control . Effects of NbNTRC overexpression on infection of BSMV and BSMV γb mutants . A . tumefaciens harboring the indicated plasmids were infiltrated into N . benthamiana leaves . Note : Agrobacterium containing NbNTRC constructs were diluted 5 - fold compared to normal use ( OD600 = 0 . 3 ) . BSMV and the γb mutants were then agroinfiltrated into the same leaves at 1 . 5 dpi . Three days later , Western blots were conducted with anti - TGB1 and anti - Flag antibodies . Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control . Left panel : Western blot detection of BSMV CP accumulation in N . benthamiana leaves inoculated with BSMV and γb movement deficient mutants . Leaf samples were collected from different regions of the same leaf ( right panel ) Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control . Effects of γb and γb mutants on 2 - Cys Prx in N . benthamiana . Different combinantions of the pGD empty vector ( EV ) or γb and γb mutants are indicated above the panel . NbNTRC and γb proteins were detected by Western blotting with anti - Flag and anti - γb antibodies under reducing conditions , Western blot analyses of 2 - Cys Prx abundance with anti - 2 - Cys Prx antibody under non - reducing conditions . The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows , respectively . Data information : representative data are shown , and three replicates had similar results . RbcL served as loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Left panel: systemic symptoms of N. benthamiana plants inoculated with BSMV and BSMV γb mutants. Photographs were taken at 7 dpi. Right panel: schematic representation of inoculated (ino.), older systemic leaves (o-SL), and younger systemic leaves (y-SL) in N. benthamiana plants. Scale bars, 2 cm.Western blot analyses of TGB1 and CP accumulation in BSMV and BSMV γb mutant inoculated N. benthamiana leaves. Anti-TGB1 and anti-CP were used for protein detection. Data information: , representative data are shown, and three replicates had similar results. RbcL served as loading control.Western blot detection of BSMV with anti-CP antibodies in upper N. benthamiana leaves at 7 days (o-SL) or 28 days (y-SL) after infiltration. o-SL, older systemic leaves; y-SL, younger systemic leaves. Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.Effects of NbNTRC overexpression on infection of BSMV and BSMV γb mutants. A. tumefaciens harboring the indicated plasmids were infiltrated into N. benthamiana leaves. Note: Agrobacterium containing NbNTRC constructs were diluted 5-fold compared to normal use (OD600 = 0.3). BSMV and the γb mutants were then agroinfiltrated into the same leaves at 1.5 dpi. Three days later, Western blots were conducted with anti-TGB1 and anti-Flag antibodies. Uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.Left panel: Western blot detection of BSMV CP accumulation in N. benthamiana leaves inoculated with BSMV and γb movement deficient mutants. Leaf samples were collected from different regions of the same leaf (right panel) Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.Effects of γb and γb mutants on 2-Cys Prx in N. benthamiana. Different combinantions of the pGD empty vector (EV) or γb and γb mutants are indicated above the panel. NbNTRC and γb proteins were detected by Western blotting with anti-Flag and anti-γb antibodies under reducing conditions, Western blot analyses of 2-Cys Prx abundance with anti-2-Cys Prx antibody under non-reducing conditions. The uncleaved NTRC with cTP and cleaved NTRC without cTP are marked with black and gray arrows, respectively. Data information: representative data are shown, and three replicates had similar results. RbcL served as loading control.


Figure 6BiFC assays to identify interactions between NbNTRC and 2 - Cys Prx in N . benthamiana during BSMV infection . NbNTRC - YFPc and 2 - Cys Prx - YFPn were co - expressed with BSMV and γb mutant derivatives as indicated on the left . The EV was used as negative control . Scale bars , 30 μm . Quantificaiton of the BiFC intensities shown in Figure 6A with ImageJ software . The NbNTRC - 2 - Cys Prx interaction was normalized to that of the EV control , which was set to 1 . 0 . Data information : Representative data were shown in and three replicates had similar results . In B , error bars indicate the mean ± SEM , and a and b indicate statistically significant differences among different groups ( n = 5 , ANOVA , p - value < 0 . 05 ) . Split - LUC assays to determine NbNTRC - 2 - Cys Prx interactions in N . benthamiana during BSMV infection . NbNTRC - cLUC and 2 - Cys Prx - nLUC were co - expressed in N . benthamiana leaves infected with the indicated BSMV derivatives , Bioluminescence was observed at 3 dpi . The white dashed circles indicate the infiltrated regions . Quantification of the luciferase signals shown in Figure 6C as assessed by ImageJ software . Data information : Representative data were shown , and three replicates had similar results . In ( D ) , error bars indicate mean ± SEM ( n = 6 ) . c indicate statistically significant differences among different groups ( ANOVA , p - value < 0 . 05 ) . Competitive GST pull - down assays showing the effects of γb on interactions between NbNTRC and 2 - Cys Prx . GST - NbNTRC and 2 - Cys Prx - His were incubated with increasing amounts ( 5 , 10 , 20 , and 40 μg ) of γb and its mutant derivatives . Glutathione - Sepharose beads were washed and Western blot analysis was performed using anti - GST and anti - His antibodies . Data information : representative data are shown , and three independent experiments had similar results . Analysis of NbNTRC - 2 - Cys Prx interactions in the presence of the γb protein . NbNTRC - 3xFlag and 2 - Cys Prx - GFP were co - expressed in N . benthamiana leaves . Total proteins were extracted at 4 dpi and immunoprecipitated with anti - Flag beads , co - incubated with increasing amounts ( 20 , 40 , and 60 μg ) of γb - His and γb - His mutant derivatives . Input and IP proteins were analyzed by Western blotting with anti - His , anti - GFP or anti - Flag antibodies . The uncleaved 2 - Cys Prx with cTP and cleaved 2 - Cys Prx without cTP are marked with black and gray arrows , respectively . Data information : representative data are shown , and three independent experiments had similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6BiFC assays to identify interactions between NbNTRC and 2-Cys Prx in N. benthamiana during BSMV infection. NbNTRC-YFPc and 2-Cys Prx-YFPn were co-expressed with BSMV and γb mutant derivatives as indicated on the left. The EV was used as negative control. Scale bars, 30 μm.   Quantificaiton of the BiFC intensities shown in Figure 6A with ImageJ software. The NbNTRC-2-Cys Prx interaction was normalized to that of the EV control, which was set to 1.0.   Data information: Representative data were shown in and three replicates had similar results. In B, error bars indicate the mean ± SEM, and a and b indicate statistically significant differences among different groups (n = 5, ANOVA, p-value < 0.05).Split-LUC assays to determine NbNTRC-2-Cys Prx interactions in N. benthamiana during BSMV infection. NbNTRC-cLUC and 2-Cys Prx-nLUC were co-expressed in N. benthamiana leaves infected with the indicated BSMV derivatives, Bioluminescence was observed at 3 dpi. The white dashed circles indicate the infiltrated regions.   Quantification of the luciferase signals shown in Figure 6C as assessed by ImageJ software.   Data information: Representative data were shown , and three replicates had similar results. In (D), error bars indicate mean ± SEM (n = 6). c indicate statistically significant differences among different groups (ANOVA, p-value < 0.05).Competitive GST pull-down assays showing the effects of γb on interactions between NbNTRC and 2-Cys Prx. GST-NbNTRC and 2-Cys Prx-His were incubated with increasing amounts (5, 10, 20, and 40 μg) of γb and its mutant derivatives. Glutathione-Sepharose beads were washed and Western blot analysis was performed using anti-GST and anti-His antibodies. Data information: representative data are shown, and three independent experiments had similar results.Analysis of NbNTRC-2-Cys Prx interactions in the presence of the γb protein. NbNTRC-3xFlag and 2-Cys Prx-GFP were co-expressed in N. benthamiana leaves. Total proteins were extracted at 4 dpi and immunoprecipitated with anti-Flag beads, co-incubated with increasing amounts (20, 40, and 60 μg) of γb-His and γb-His mutant derivatives. Input and IP proteins were analyzed by Western blotting with anti-His, anti-GFP or anti-Flag antibodies. The uncleaved 2-Cys Prx with cTP and cleaved 2-Cys Prx without cTP are marked with black and gray arrows, respectively. Data information: representative data are shown, and three independent experiments had similar results.


Figure 7Chl - HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in N . benthamiana epidermal cells . The pCB301 empty vector ( EV ) was used as a negative control . Scale bars , 30 μm . The false - blue color indicates the chloroplasts . Quantification of LRSV - dependent changes in Chl - HyPer2 fluorescence at 3 dpi . Data information : , data are representative of at least three independent experiments . error bars represent mean ± SEM ( n = 5 ) ; * * p - value < 0 . 01 from Student ' s t test . Western blot analyses of 2 - Cys Prx abundance in EV and LRSV inoculated leaves under non - reducing conditions at 3 dpi . RbcL served as a loading control . Data information : representative data are shown , and three replicates had similar results . Western blot analyses to detect LRSV γb in NbNTRC - OE and NbNTRC - KO N . benthamiana plants challenged with LRSV at 8 dpi . RbcL was used as the loading control . Data information : representative data are shown , and three replicates had similar results . Chl - HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N . benthamiana epidermal cells . Scale bars , 30 μm . The false - blue color indicates the chloroplasts . Quantification of the TYMV - dependent changes in Chl - HyPer2 fluorescence at 5 dpi . Data information : data are representative of at least three independent experiments . error bars represent mean ± SEM ( n = 5 ) ; * * p - value < 0 . 01 from Student ' s t test . Western blot analysis of 2 - Cys Prx abundance in EV or TYMV inoculated leaves under non - reducing conditions at 5 dpi . RbcL was used as the loading control . Data information : representative data are shown , and three replicates had similar results . RT - qPCR analysis of TYMV RNA accumulation at 8 dpi . NbEF1α served as a internal control . Data information : representative data are shown , and three replicates had similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by LRSV infection at 3 dpi in N. benthamiana epidermal cells. The pCB301 empty vector (EV) was used as a negative control. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.   Quantification of LRSV-dependent changes in Chl-HyPer2 fluorescence at 3 dpi.   Data information: , data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.Western blot analyses of 2-Cys Prx abundance in EV and LRSV inoculated leaves under non-reducing conditions at 3 dpi. RbcL served as a loading control. Data information: representative data are shown, and three replicates had similar results.Western blot analyses to detect LRSV γb in NbNTRC-OE and NbNTRC-KO N. benthamiana plants challenged with LRSV at 8 dpi. RbcL was used as the loading control. Data information: representative data are shown, and three replicates had similar results.Chl-HyPer2 responds to chloroplast hydrogen peroxide caused by TYMV infection at 5 dpi in N. benthamiana epidermal cells. Scale bars, 30 μm. The false-blue color indicates the chloroplasts.   Quantification of the TYMV-dependent changes in Chl-HyPer2 fluorescence at 5 dpi.   Data information: data are representative of at least three independent experiments. error bars represent mean ± SEM (n = 5); ** p-value < 0.01 from Student's t test.Western blot analysis of 2-Cys Prx abundance in EV or TYMV inoculated leaves under non-reducing conditions at 5 dpi. RbcL was used as the loading control. Data information: representative data are shown, and three replicates had similar results.RT-qPCR analysis of TYMV RNA accumulation at 8 dpi. NbEF1α served as a internal control. Data information: representative data are shown, and three replicates had similar results.


Figure 1 . ( a , b ) Dual positive cells are more prevalent in epithelial organs and cells . ( a ) Proportion of ACE2 + TMPRSS2 + cells ( y axis ) per dataset ( dots ) from 21 tissues and organs ( rows ) . ( e - i ) In situ validation of dual positive cells in the lung , airways , and submucosal gland . ( e - g ) PLISH and immunostaining in human adult lung alveoli for ( e ) ACE2 ( white ) , TMPRSS2 ( green ) and CTSL ( red ) , ( f ) ACE2 ( white ) , TMPRSS2 ( green ) and HTII - 280 ( red ) , ( g ) ACE2 ( red ) and Pro - SFTPC ( green ) . ( h ) PLISH and immunostaining in human large airway submucosal glands . ACE2 ( red ) , ACTA2 ( green ) and DAPI ( blue ) . ( i ) PLISH in human adult large airway . ACE2 ( green ) and DAPI ( blue ) .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.(a,b) Dual positive cells are more prevalent in epithelial organs and cells. (a) Proportion of ACE2+TMPRSS2+ cells (y axis) per dataset (dots) from 21 tissues and organs (rows).(e-i) In situ validation of dual positive cells in the lung, airways, and submucosal gland. (e-g) PLISH and immunostaining in human adult lung alveoli for (e) ACE2 (white), TMPRSS2 (green) and CTSL (red), (f) ACE2 (white), TMPRSS2 (green) and HTII-280 (red), (g) ACE2 (red) and Pro-SFTPC (green). (h) PLISH and immunostaining in human large airway submucosal glands. ACE2 (red), ACTA2 (green) and DAPI (blue). (i) PLISH in human adult large airway. ACE2 (green) and DAPI (blue).


Figure 2 . ( a , c ) Single - cell ATAC - seq of lung samples from primary carina ( 1C ) and subpleural parenchyme ( RPL ) ( n = 1 patient , k = 3 samples , 3 , 366 cells from 1C , 8 , 340 cells from RPL ) . Uniform Manifold Approximation and Projection ( UMAP ) embedding of scATAC - seq profiles ( dots ) colored by ( a , left ) cell types , ( a , right ) cells with at least 1 fragment ( indicating accessibility , open chromatin ) mapping to the ACE2 gene locus ( defined as − 2kb upstream the Transcription Start Site to Transcription End Site ) , or by sample

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.(a,c) Single-cell ATAC-seq of lung samples from primary carina (1C) and subpleural parenchyme (RPL) (n=1 patient, k=3 samples, 3,366 cells from 1C, 8,340 cells from RPL). Uniform Manifold Approximation and Projection (UMAP) embedding of scATAC-seq profiles (dots) colored by (a, left) cell types, (a, right) cells with at least 1 fragment (indicating accessibility, open chromatin) mapping to the ACE2 gene locus (defined as −2kb upstream the Transcription Start Site to Transcription End Site), or by sample location