C ) Confocal imaging of dystrophic neurites found in a postmortem tissue of a patient with FTLD - TDP - C . Dystrophic neurites were identified using TDP - 43 ( cyan ) , and TDP - 43pS403 / 404 ( magenta ) . Neurite markers were used to visualize neuronal projections ( MAP2 for dendrites , PNF for axons ; yellow and orange , respectively ) . D ) 2D TEM ( first row ) of dystrophic neurites from FTLD - TDP - C , localized in postmortem human brain by correlative light and electron microscopy ( also shown in Fig . EV5D ) . 2D projections of the central slices of reconstructed 3D tomograms ( middle row ) taken at higher magnification from regions within each aggregate show a sparse arrangement of filaments which are interspersed with vesicles ( white asterisk ) . A similar arrangement of sparse filaments and vesicles is seen in a cross - section of a dystrophic neurite from FTLD - TDP - C ( cross - section , also shown in Fig . EV5G ) . The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram ( colored dots , last row ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Confocal imaging of dystrophic neurites found in a postmortem tissue of a patient with FTLD-TDP-C. Dystrophic neurites were identified using TDP-43 (cyan), and TDP-43pS403/404 (magenta). Neurite markers were used to visualize neuronal projections (MAP2 for dendrites, PNF for axons; yellow and orange, respectively). D) 2D TEM (first row) of dystrophic neurites from FTLD-TDP-C, localized in postmortem human brain by correlative light and electron microscopy (also shown in Fig. EV5D). 2D projections of the central slices of reconstructed 3D tomograms (middle row) taken at higher magnification from regions within each aggregate show a sparse arrangement of filaments which are interspersed with vesicles (white asterisk). A similar arrangement of sparse filaments and vesicles is seen in a cross-section of a dystrophic neurite from FTLD-TDP-C (cross-section, also shown in Fig. EV5G). The positions of filaments selected for subtomogram analysis are shown within a single orthoslice from the same tomogram (colored dots, last row).


a - c , Expression of CD45RO and CCR7 ( a ) , CD45RO and CD27 ( b ) , CD27 and CD28 ( c ) on peripheral blood CD8 T cells of three participants [ P1 ( heterozygous mutation ) , P2 ( homozygous mutation ) and P3 ( heterozygous ) for the c . 192G > C mutation in the DJ - 1 gene ] . d - g , Expression of CD57 ( d ) , PD - 1 ( e ) , EOMES ( f ) and T - bet ( g ) on peripheral blood CD8 T cells of three participants . The enlarged number in the corresponding gate represents the corresponding percentage out of the parent population .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

a-c, Expression of CD45RO and CCR7 (a), CD45RO and CD27 (b), CD27 and CD28 (c) on peripheral blood CD8 T cells of three participants [P1 (heterozygous mutation), P2 (homozygous mutation) and P3 (heterozygous) for the c.192G>C mutation in the DJ-1 gene]. d-g, Expression of CD57 (d), PD-1 (e), EOMES (f) and T-bet (g) on peripheral blood CD8 T cells of three participants. The enlarged number in the corresponding gate represents the corresponding percentage out of the parent population. h


b - c , Percentages of CD44low CD62Lhigh ( Tn ) ( b ) and CD44high CD62Llow ( Tem ) ( c ) cells among total CD8 T cells of spleen and pLNs from young and 45 - wk - old Dj - 1 KO and WT littermates ( young KO , n = 5 ; young WT , n = 5 ; 45 - wk - old KO , n = 8 ; 45 - wk - old WT , n = 6 ; for 45 - wk - old mice , data pooled from 2 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-c, Percentages of CD44low CD62Lhigh (Tn) (b) and CD44high CD62Llow (Tem) (c) cells among total CD8 T cells of spleen and pLNs from young and 45-wk-old Dj-1 KO and WT littermates (young KO, n=5; young WT, n=5; 45-wk-old KO, n=8; 45-wk-old WT, n=6; for 45-wk-old mice, data pooled from 2 independent experiments).


e , Percentages of Ki - 67 + cells among total CD8 T cells . f , Percentage of Ki - 67 + cells among splenic CD8 Tn , Tem and Tcm in 45 - wk - old Dj - 1 KO and age - and sex - matched WT mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e, Percentages of Ki-67+ cells among total CD8 T cells. f, Percentage of Ki-67+ cells among splenic CD8 Tn, Tem and Tcm in 45-wk-old Dj-1 KO and age- and sex-matched WT mice. g


j , Percentages of Tn ( top ) , Tvm ( middle ) , Tmem ( bottom ) among 60 - wk - old or young mice ( young KO , n = 4 ; young WT , n = 3 ; 60 - wk - old KO , n = 4 ; 60 - wk - old WT , n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

j, Percentages of Tn (top), Tvm (middle), Tmem (bottom) among 60-wk-old or young mice (young KO, n=4; young WT, n=3; 60-wk-old KO, n=4; 60-wk-old WT, n=5).


b , Percentages of KLRG1 + CD8 T cells derived from 45 - wk - old Dj - 1 KO and WT BM cells within young Dj - 1 KO ( n = 4 ) or WT recipients ( n = 5 ) . c , Percentages of KLRG1 + among CD8 Tem derived from 45 - wk - old Dj - 1 KO and WT BM cells within young Dj - 1 KO or WT recipients . d d , Percentages of PD - 1 + population among CD8 T cells derived from 45 - wk - old Dj - 1 KO and WT BM cells within young Dj - 1 KO ( n = 4 ) or WT recipients ( n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Percentages of KLRG1+ CD8 T cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO (n=4) or WT recipients (n=5). c, Percentages of KLRG1+ among CD8 Tem derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO or WT recipients. d d, Percentages of PD-1+ population among CD8 T cells derived from 45-wk-old Dj-1 KO and WT BM cells within young Dj-1 KO (n=4) or WT recipients (n=5).


j , Percentage of KLRG1 + population among total CD8 T cells ( 55 - wk - old KO , n = 4 ; 55 - wk - old WT , n = 5 ; young KO , n = 4 ; young WT , n = 4 ) . k , Percentage of KLRG1 + population among CD8 Tem cells . l , Percentage of KLRG1 + PD - 1 + population among total CD8 T cells ( 55 - wk - old KO , n = 4 ; 55 - wk - old WT , n = 5 ; young KO , n = 4 ; young WT , n = 4 ) . m , Percentage of KLRG1 + PD - 1 + population among CD8 Tem cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

j, Percentage of KLRG1+ population among total CD8 T cells (55-wk-old KO, n=4; 55-wk-old WT, n=5; young KO, n=4; young WT, n=4). k, Percentage of KLRG1+ population among CD8 Tem cells. l, Percentage of KLRG1+PD-1+ population among total CD8 T cells (55-wk-old KO, n=4; 55-wk-old WT, n=5; young KO, n=4; young WT, n=4). m, Percentage of KLRG1+PD-1+ population among CD8 Tem cells. Dat


b , the percentages of proliferating cells among living CD8 singlets ( CD8 Tn were from young mice ) .

b, the percentages of proliferating cells among living CD8 singlets (CD8 Tn were from young mice).


e , the percentages of proliferating cells among living CD8 singlets ( CD8 Tn were from 45 - wk - old mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O

e, the percentages of proliferating cells among living CD8 singlets (CD8 Tn were from 45-wk-old mice).


e - h , Oxygen consumption rate ( OCR ) of unstimulated ( e ) and stimulated CD8 Tn ( f ) by 2ug / ml of anti - CD3 and 1ug / ml of anti - CD28 for 24 hours . The CD8 Tn were isolated from 45 - wk - old mice . OCR of unstimulated ( g ) and stimulated CD8 Tn ( h ) by anti - CD3 and - CD28 for 24 hours . The CD8 Tn were isolated from young mice . Left of each OCR measurement panel , representation plots following Mito stress test . Right of each panel , quantitation of basal and maximum respiration under Mito stress test conditions The dashed line indicates the time when the corresponding modulator Oligomycin ( Oligo ) , Carbonyl cyanide - 4 ( trifluoromethoxy ) phenylhydrazone ( FCCP ) or Rotenone and Antimycin ( AA / Rot ) was added .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

e-h, Oxygen consumption rate (OCR) of unstimulated (e) and stimulated CD8 Tn (f) by 2ug/ml of anti-CD3 and 1ug/ml of anti-CD28 for 24 hours. The CD8 Tn were isolated from 45-wk-old mice. OCR of unstimulated (g) and stimulated CD8 Tn (h) by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each OCR measurement panel, representation plots following Mito stress test. Right of each panel, quantitation of basal and maximum respiration under Mito stress test conditions The dashed line indicates the time when the corresponding modulator Oligomycin (Oligo), Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) or Rotenone and Antimycin (AA/Rot) was added.


i - k , Extracellular acidification rate ( ECAR ) of unstimulated and stimulated CD8 Tn by anti - CD3 and - CD28 antibodies for 24 hours ( i ) . The CD8 Tn were isolated from 45 - wk - old mice . ECAR of unstimulated ( j ) and stimulated ( k ) CD8 Tn by anti - CD3 and - CD28 for 24 hours . The CD8 Tn were isolated from young mice . Left of each ECAR measurement panel , representation plots following Glycolysis stress test . Right of each panel , quantitation of glycolysis and glycolytic capacity under Glycolysis stress test conditions . The dashed line indicates the time when the corresponding modulator glucose , Oligomycin ( Oligo ) or 2 - deoxy - glucose ( 2 - DG ) was added .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

i-k, Extracellular acidification rate (ECAR) of unstimulated and stimulated CD8 Tn by anti-CD3 and -CD28 antibodies for 24 hours (i). The CD8 Tn were isolated from 45-wk-old mice. ECAR of unstimulated (j) and stimulated (k) CD8 Tn by anti-CD3 and -CD28 for 24 hours. The CD8 Tn were isolated from young mice. Left of each ECAR measurement panel, representation plots following Glycolysis stress test. Right of each panel, quantitation of glycolysis and glycolytic capacity under Glycolysis stress test conditions. The dashed line indicates the time when the corresponding modulator glucose, Oligomycin (Oligo) or 2-deoxy-glucose (2-DG) was added.


B , C Kinetic change of mRNA expression of Top1 , Btbd1 , Tdp1 , and Srsf1 in microglia following LPS / IFNγ stimulation ( n = 6 , technical replicates ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Statistical analyses were performed using two - way ANOVA with Dunnett ' s Multiple Comparison test for ( B and C ) ,

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C Kinetic change of mRNA expression of Top1, Btbd1, Tdp1, and Srsf1 in microglia following LPS/IFNγ stimulation (n = 6, technical replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using two-way ANOVA with Dunnett's Multiple Comparison test for (B and C),


D , E The protein expression of TOP1 was detected by Western blotting ( n = 3 , biological replicates ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E The protein expression of TOP1 was detected by Western blotting (n = 3, biological replicates). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


F - H Immunostaining of TOP1 ( red ) in stimulated microglia ( 4 h after LPS / IFNγ stimulation ) or resting microglia , co - stained with Hoechst ( blue ) ( bar = 20 μm ) ; for the lower - left panel in ( H ) , n = 3 technical replicates per group ( 2 to 5 random fields per well were analyzed ) ; for the lower right panel , 201 cells from unstimulated microglia and 256 cells from stimulated microglia were analyzed . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-H Immunostaining of TOP1 (red) in stimulated microglia (4 h after LPS/IFNγ stimulation) or resting microglia, co-stained with Hoechst (blue) (bar = 20 μm); for the lower-left panel in (H), n = 3 technical replicates per group (2 to 5 random fields per well were analyzed); for the lower right panel, 201 cells from unstimulated microglia and 256 cells from stimulated microglia were analyzed. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; Statistical analyses were performed using the student's unpaired two-tailed t-test.


G Immunofluorescent staining of spinal cord sections from mice at EAE peak and control mice with anti - F4 / 80 ( green ) , TOP1 ( magenta ) , and nuclei with Hoechst ( blue ) . For the left panels , bar = 400 μm ; for the magnified panels , bar = 100 μm . The yellow arrowhead indicates a representative F4 / 80 + cell with high TOP1 expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Immunofluorescent staining of spinal cord sections from mice at EAE peak and control mice with anti-F4/80 (green), TOP1 (magenta), and nuclei with Hoechst (blue). For the left panels, bar = 400 μm; for the magnified panels, bar = 100 μm. The yellow arrowhead indicates a representative F4/80+ cell with high TOP1 expression.


I A magnified image of the selected region ( white box ) in ( G ) ; bar = 50 F4 / 80 + cell with increased TOP1 expression in the nucleus in ( G ) . J The mean fluorescent intensity of TOP1 expression in the white matter in ( G ) was graphed ; n = 3 to 4 mice per group with 3 - 5 sections from each mouse . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I A magnified image of the selected region (white box) in (G); bar = 50 F4/80+ cell with increased TOP1 expression in the nucleus in (G). J The mean fluorescent intensity of TOP1 expression in the white matter in (G) was graphed; n = 3 to 4 mice per group with 3-5 sections from each mouse. Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


K TOP1 expression in the spinal cord homogenates of EAE mice dissected at disease peak ( scored 3 ) or control mice were examined by Western blotting . L The quantification of TOP1 protein expression of Western blot bands in ( K ) with normalization to GAPDH and β - actin ( n = 3 biological samples ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K TOP1 expression in the spinal cord homogenates of EAE mice dissected at disease peak (scored 3) or control mice were examined by Western blotting. L The quantification of TOP1 protein expression of Western blot bands in (K) with normalization to GAPDH and β-actin (n = 3 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


M , Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h ( n = 4 or 5 biological samples ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

M, Western blotting of TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples).


TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h ( n = 4 or 5 biological samples ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TOP1 expression from brain homogenates of mice receiving intracisternal LPS injection or PBS after 8 h (n = 4 or 5 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


O Microglia were sorted from LPS challenged mice ( i . p ) after 4 h or naive mice , and the mRNA expression of TOP1 was graphed ( n = 5 or 6 biological samples ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 Statistical analyses were performed using the student ' s unpaired two - tailed t - test .

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

O Microglia were sorted from LPS challenged mice (i.p) after 4 h or naive mice, and the mRNA expression of TOP1 was graphed (n = 5 or 6 biological samples). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001 Statistical analyses were performed using the student's unpaired two-tailed t-test.


P Brain sections from healthy control and MS brain tissues , including the active MS lesion , chronic active lesion , and the normal - appearing white matter ( NAWM ) , were stained with anti - HLA - DR , anti - TOP1 , and co - stained with Hoechst ( bar = 50 μm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

P Brain sections from healthy control and MS brain tissues, including the active MS lesion, chronic active lesion, and the normal-appearing white matter (NAWM), were stained with anti-HLA-DR, anti-TOP1, and co-stained with Hoechst (bar = 50 μm).


C , D Immunostaining of iNOS ( red ) and Phalloidin ( green ) in microglia ( n = 3 technical replicates with more than 5 random fields per well ) ( bar = 50 μm ) . Data information : Data shown are mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . = no statistical significance . Statistical analyses were performed using one - way ANOVA with Dunnett ' s Multiple Comparison Test for ( D )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, D Immunostaining of iNOS (red) and Phalloidin (green) in microglia (n = 3 technical replicates with more than 5 random fields per well) (bar = 50 μm). Data information: Data shown are mean ± s.e.m. *P<0.05; **P<0.01; ***P<0.001; n.s. = no statistical significance. Statistical analyses were performed using one-way ANOVA with Dunnett's Multiple Comparison Test for (D)


B Microglia in the hippocampus and thalamus were stained with Iba1 ( red ) ; bar = 50 μm ; microglia in the yellow boxes were presented in ( E ) .

O O O O O O O O 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 Microglia in the hippocampus and thalamus were stained with Iba1 (red); bar = 50 μm; microglia in the yellow boxes were presented in (E).


C , D Fluorescent intensity of Iba1 in hippocampus and thalamus in ( B ) ; n = 2 , 4 , 5 mice for naive , LPS , and LPS + TPT group , respectively ( 2 - 6 sections per mouse were analyzed ) . Data information : Data shown are mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; n . s . = no statistical significance . Statistical analyses comparing the LPS _ PBS group with the LPS _ TPT group were performed using the Student ' s unpaired two - tailed t -

O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

C, D Fluorescent intensity of Iba1 in hippocampus and thalamus in (B); n = 2, 4, 5 mice for naive, LPS, and LPS + TPT group, respectively (2-6 sections per mouse were analyzed). Data information: Data shown are mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; n.s. = no statistical significance. Statistical analyses comparing the LPS_PBS group with the LPS_TPT group were performed using the Student's unpaired two-tailed t-test


L , M Cytokine array on brain homogenates after 24 h of LPS challenge ( n = 2 mice for the naïve group ; n = 3 mice for the LPS and LPS + TPT groups ) .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O

L, M Cytokine array on brain homogenates after 24 h of LPS challenge (n = 2 mice for the naïve group; n = 3 mice for the LPS and LPS + TPT groups).


K Spinal cord sections dissected from Day 30 from the EAE batch in ( H ) were stained with CD11b ( red ) , Fluoromyelin ( green ) , and Hoechst ( blue ) ( bar = 200 μm ) . L The CD11b immunofluorescent intensity in ( K ) was graphed ( n = 2 mice for the control group ; n = 4 mice for the EAE - PBS and EAE - TPT groups ) . M The percentage of demyelinated area in the white matter of the spinal cord was graphed ( n = 2 mice for the control group , n = 4 mice for the EAE - PBS and EAE - TPT groups ) . Data information : Data are presented as the mean ± s . e . m . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; the Student ' s unpaired two - tailed t - test was used for comparing the EAE - PBS group with the EAE - TPT group in L , and M .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O

K Spinal cord sections dissected from Day 30 from the EAE batch in (H) were stained with CD11b (red), Fluoromyelin (green), and Hoechst (blue) (bar = 200 μm). L The CD11b immunofluorescent intensity in (K) was graphed (n = 2 mice for the control group; n = 4 mice for the EAE-PBS and EAE-TPT groups). M The percentage of demyelinated area in the white matter of the spinal cord was graphed (n = 2 mice for the control group, n = 4 mice for the EAE-PBS and EAE-TPT groups). Data information: Data are presented as the mean ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; the Student's unpaired two-tailed t-test was used for comparing the EAE-PBS group with the EAE-TPT group in L, and M.


A Bisulfite DNA methylation sequencing of the Athila6ATE transcriptional start site ( TSS ) in genotypes with silenced TEs ( left of the dashed line ) or transcriptionally active TEs ( right of the dashed line ) . Methylation in the CG ( red ) , CHG ( blue ) and CHH ( green ) sequence contexts are shown ( H = C , T , or A ) . Error bars represent Wilson score interval 95 % confidence limits . For multiple statistical tests , one ‐ way ANOVA was performed using Tukey ' s multiple comparison test . ns , not significant .

A Bisulfite DNA methylation sequencing of the Athila6ATE transcriptional start site (TSS) in genotypes with silenced TEs (left of the dashed line) or transcriptionally active TEs (right of the dashed line). Methylation in the CG (red), CHG (blue) and CHH (green) sequence contexts are shown (H = C, T, or A). Error bars represent Wilson score interval 95% confidence limits. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test. ns, not significant.


B The reads ‐ per ‐ million ‐ ( RPM ) ‐ normalized accumulation of Athila6ATE siRNAs from TE ‐ silenced epigenomes ( left ) and ddm1TE ‐ active epigenomes ( right ) . Inset line graphs reflecting the same values are shown for select genotypes . All line graphs have the same y ‐ axis scale and are shown for increased resolution in genotypes with low siRNA levels .

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B The reads‐per‐million‐(RPM)‐normalized accumulation of Athila6ATE siRNAs from TE‐silenced epigenomes (left) and ddm1TE‐active epigenomes (right). Inset line graphs reflecting the same values are shown for select genotypes. All line graphs have the same y‐axis scale and are shown for increased resolution in genotypes with low siRNA levels.


AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . A A representative microRNA , miR158B . The location of miR158B from this locus is shown in orange .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.A A representative microRNA, miR158B. The location of miR158B from this locus is shown in orange.


AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . B The SimpleHat2 non ‐ autonomous TE targeted by Pol IV ‐ RdDM . C The RDR6 ‐ RdDM target locus TAS3a .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.B The SimpleHat2 non‐autonomous TE targeted by Pol IV‐RdDM.C The RDR6‐RdDM target locus TAS3a.


AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . D The Athila6A consensus element in the wt Col epigenome . E The same Athila6A consensus element as in ( D ) upon transcriptional activation in the ddm1 epigenome .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.D The Athila6A consensus element in the wt Col epigenome.E The same Athila6A consensus element as in (D) upon transcriptional activation in the ddm1 epigenome.


AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . F The total small RNAs produced in the wt Col TE ‐ silenced epigenome . G The total small RNAs in ddm1 mutants .

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AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.F The total small RNAs produced in the wt Col TE‐silenced epigenome.G The total small RNAs in ddm1 mutants.


AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . H Bisulfite sequencing of different regions of the Athila6A element . Error bars represent Wilson score interval 95 % confidence limits . Differences in the methylation status were analyzed with a two ‐ tailed unpaired Student ' s t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.H Bisulfite sequencing of different regions of the Athila6A element. Error bars represent Wilson score interval 95% confidence limits. Differences in the methylation status were analyzed with a two‐tailed unpaired Student's t‐test.


AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . I A second biological replicate of the FLAG ‐ AGO6 ‐ IP and no ‐ antigen control followed by deep sequencing shown for the Athila6A consensus element in ddm1 mutants . The transcriptional start sites of the LTR TSS ‐ and IR ‐ driven transcripts are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.I A second biological replicate of the FLAG‐AGO6‐IP and no‐antigen control followed by deep sequencing shown for the Athila6A consensus element in ddm1 mutants. The transcriptional start sites of the LTR TSS‐ and IR‐driven transcripts are shown.


AGO6 relative enrichment of small RNAs ( solid lines ) and total small RNAs ( dashed lines ) are shown for each small RNA size class ( 21 , 22 and 24nt ) at four genomic loci . J Analysis of the first base of AGO6 ‐ enriched siRNAs from the wt Col TE ‐ silenced epigenome ( left ) and the ddm1 TE ‐ transcriptionally active epigenome ( right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AGO6 relative enrichment of small RNAs (solid lines) and total small RNAs (dashed lines) are shown for each small RNA size class (21, 22 and 24nt) at four genomic loci.J Analysis of the first base of AGO6‐enriched siRNAs from the wt Col TE‐silenced epigenome (left) and the ddm1 TE‐transcriptionally active epigenome (right).


B H3Ac control ChIP from the same chromatin used in the AGO6 ‐ IP for the At1g08200 constitutively expressed gene and the Athila6A TSS . Enrichment of H3Ac at the Athila6A TSS is increased upon transcriptional activation in ddm1 mutants , while enrichment of H3Ac at the gene At1g08200 is consistently high in both wt Col and ddm1 . Data are represented as mean ± SD of six biological replicates for each genotype .

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B H3Ac control ChIP from the same chromatin used in the AGO6‐IP for the At1g08200 constitutively expressed gene and the Athila6A TSS. Enrichment of H3Ac at the Athila6A TSS is increased upon transcriptional activation in ddm1 mutants, while enrichment of H3Ac at the gene At1g08200 is consistently high in both wt Col and ddm1. Data are represented as mean ± SD of six biological replicates for each genotype.


C - E AGO6 ‐ ChIP qPCR analysis of ( C ) the gene At1g08200 and the Pol IV ‐ RdDM target SimpleHat2 , ( D ) the Athila6A TSS in wt Col TE ‐ silenced epigenomes ( left ) and ddm1 TE ‐ active epigenomes ( right ) and ( E ) TAS3a . ChIP signals in ( D ) are normalized to the ago6 or ddm1 ago6 no ‐ antigen controls . ChIP signals in ( C ) and ( E ) are normalized to the ago6 no ‐ antigen control . In ( C - E ) , each biological replicate data point is shown as a gray dot . The data are represented as mean ± SD . For multiple statistical tests , one ‐ way ANOVA was performed using Tukey ' s multiple comparison test .

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C-E AGO6‐ChIP qPCR analysis of (C) the gene At1g08200 and the Pol IV‐RdDM target SimpleHat2, (D) the Athila6A TSS in wt Col TE‐silenced epigenomes (left) and ddm1 TE‐active epigenomes (right) and (E) TAS3a. ChIP signals in (D) are normalized to the ago6 or ddm1 ago6 no‐antigen controls. ChIP signals in (C) and (E) are normalized to the ago6 no‐antigen control. In (C-E), each biological replicate data point is shown as a gray dot. The data are represented as mean ± SD. For multiple statistical tests, one‐way ANOVA was performed using Tukey's multiple comparison test.


F ChIP qPCR analysis of the FLAG ‐ tagged POL V protein in wt Col and ddm1 . ChIP signals are normalized to the wt Col no ‐ antigen control .

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F ChIP qPCR analysis of the FLAG‐tagged POL V protein in wt Col and ddm1. ChIP signals are normalized to the wt Col no‐antigen control.


A AGO6 enrichment and depletion of 21 - 22nt siRNAs for the entire genome in 100 ‐ bp tiles for both wt Col and ddm1 . Tiles were categorized by annotation feature .

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A AGO6 enrichment and depletion of 21-22nt siRNAs for the entire genome in 100‐bp tiles for both wt Col and ddm1. Tiles were categorized by annotation feature.


B Of the tiles in ( A ) in both wt Col and ddm1 , we determined the number of tiles with ≥ 2 ‐ fold CHH methylation dependent on RDR6 .

B Of the tiles in (A) in both wt Col and ddm1, we determined the number of tiles with ≥ 2‐fold CHH methylation dependent on RDR6.


C Distribution of AGO6 enrichment across TEs ( solid lines ) and genes ( dashed lines ) for different sizes of small RNAs in either the wt Col TE ‐ silenced epigenome or the ddm1 TE ‐ transcriptionally active epigenome . Dashed lines have the same color codes as the solid lines . ' Start ' and ' Stop ' represent the edges of the TE and the transcriptional start and stop sites of the genes .

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C Distribution of AGO6 enrichment across TEs (solid lines) and genes (dashed lines) for different sizes of small RNAs in either the wt Col TE‐silenced epigenome or the ddm1 TE‐transcriptionally active epigenome. Dashed lines have the same color codes as the solid lines. 'Start' and 'Stop' represent the edges of the TE and the transcriptional start and stop sites of the genes.


D - G Heatmaps of AGO6 enrichment of TE siRNA size classes in wt Col and ddm1 epigenomes . Analyzed were ( D ) the position of the TE on a chromosome and TE size , ( E ) the copy number of the TE , ( F ) the TE type and ( G ) the TE family name . The color of the TE family name in ( G ) corresponds to the TE type in ( F ) .

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D-G Heatmaps of AGO6 enrichment of TE siRNA size classes in wt Col and ddm1 epigenomes. Analyzed were (D) the position of the TE on a chromosome and TE size, (E) the copy number of the TE, (F) the TE type and (G) the TE family name. The color of the TE family name in (G) corresponds to the TE type in (F).


H Bisulfite sequencing of TE targets . Error bars represent Wilson score interval 95 % confidence limits .

H Bisulfite sequencing of TE targets. Error bars represent Wilson score interval 95% confidence limits.


A Bisulfite sequencing of the Athila6A TSS in juvenile leaf tissue .

A Bisulfite sequencing of the Athila6A TSS in juvenile leaf tissue.


B qRT - PCR analysis of Athila6A gag / pol steady ‐ state mRNA transcript levels in inflorescence and leaf tissue .

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B qRT-PCR analysis of Athila6A gag/pol steady‐state mRNA transcript levels in inflorescence and leaf tissue.


C qRT - PCR analysis of AGO6 and AGO4 mRNA transcript levels in inflorescence and juvenile leaf tissue .

C qRT-PCR analysis of AGO6 and AGO4 mRNA transcript levels in inflorescence and juvenile leaf tissue.


D Western blot analysis of wt Col leaf and inflorescence . The arrow denotes the AGO6 protein ; the higher band is a previously observed cross ‐ reactive band ( see also Supplementary Fig S3A ) .

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D Western blot analysis of wt Col leaf and inflorescence. The arrow denotes the AGO6 protein; the higher band is a previously observed cross‐reactive band (see also Supplementary Fig S3A).


E , F Side view of an inflorescence with ( E ) or without ( F ) the AGO6 ‐ GFP transgene . G , H Top ‐ down view of an inflorescence with ( G ) or without ( H ) the AGO6 ‐ GFP transgene .

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E, F Side view of an inflorescence with (E) or without (F) the AGO6‐GFP transgene.G, H Top‐down view of an inflorescence with (G) or without (H) the AGO6‐GFP transgene.


J Nuclear and cytoplasmic fractionation of inflorescence protein . The cytoplasm ‐ enriched protein represents 2 . 5 % of the total nuclear ‐ enriched protein . H3Ac is a nuclear protein marker , while PEPC is a cytoplasmic protein .

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J Nuclear and cytoplasmic fractionation of inflorescence protein. The cytoplasm‐enriched protein represents 2.5% of the total nuclear‐enriched protein. H3Ac is a nuclear protein marker, while PEPC is a cytoplasmic protein.


K CHH methylation levels of the RDR6 ‐ RdDM targets TAS3a and Athila6A TSS in whole ‐ inflorescence ‐ tissue samples compared to dissected young buds corresponding to floral stages 6 - 8 .

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K CHH methylation levels of the RDR6‐RdDM targets TAS3a and Athila6A TSS in whole‐inflorescence‐tissue samples compared to dissected young buds corresponding to floral stages 6-8.


( a ) Beeswarm plot with Factor 1 values for each sample with colors corresponding to three clusters found by 3 - means clustering with low factor values ( LZ ) , intermediate factor values ( IZ ) and high factor values ( HZ )

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(a) Beeswarm plot with Factor 1 values for each sample with colors corresponding to three clusters found by 3-means clustering with low factor values (LZ), intermediate factor values (IZ) and high factor values (HZ)


( b ) Absolute loadings for the genes with the largest absolute weights in the mRNA data . Plus or minus symbols on the right indicate the sign of the loading . Genes highlighted in orange were previously described as prognostic markers in CLL and associated with IGHV status ( Maloum et al , 2009 ; Morabito et al , 2015 ; Plesingerova et al , 2017 ; Trojani et al , 2012 ; Vasconcelos et al , 2005 )

(b) Absolute loadings for the genes with the largest absolute weights in the mRNA data. Plus or minus symbols on the right indicate the sign of the loading. Genes highlighted in orange were previously described as prognostic markers in CLL and associated with IGHV status (Maloum et al, 2009; Morabito et al, 2015; Plesingerova et al, 2017; Trojani et al, 2012; Vasconcelos et al, 2005)


( c ) Heatmap of gene expression values for genes with the largest weights as in

(c) Heatmap of gene expression values for genes with the largest weights as in b


( d ) Absolute loadings of the drugs with the largest weights , annotated by target

(d) Absolute loadings of the drugs with the largest weights, annotated by target category


( e ) Drug response curves for two of the drugs , stratified by the clusters as in

(e) Drug response curves for two of the drugs, stratified by the clusters as in a


A Germination of dog1 - 3 ( knock - out ) , WT , and dog1 - 5 ( DOG1 upregulation ) seeds in media supplemented with NaCl for the indicated number of days after stratification ( DAS ) . Scale bar represents 5 mm . B Germination rate in 100 mM NaCl 7 days after stratification for seeds of different genotypes . Lines represent the fitted curves with a 95 % confidence interval ( grey area ) . ∗ p - value < 0 . 05 from two - tailed Student ' s t - test . Data for WT is the same as plotted in Fig 3C .

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A Germination of dog1-3 (knock-out), WT, and dog1-5 (DOG1 upregulation) seeds in media supplemented with NaCl for the indicated number of days after stratification (DAS). Scale bar represents 5 mm. B Germination rate in 100 mM NaCl 7 days after stratification for seeds of different genotypes. Lines represent the fitted curves with a 95% confidence interval (grey area). ∗ p-value < 0.05 from two-tailed Student's t-test. Data for WT is the same as plotted in Fig 3C. C


C shDOG1 expression levels normalized to UBC21 ( AT5G25760 ) in dog1 - 3 and dog1 - 5 relative to WT . D shDOG1 and lgDOG1 expression levels normalized to UBC21 in seeds treated with 100 mM NaCl relative to mock .

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C shDOG1 expression levels normalized to UBC21 (AT5G25760) in dog1-3 and dog1-5 relative to WT. D shDOG1 and lgDOG1 expression levels normalized to UBC21 in seeds treated with 100 mM NaCl relative to mock. E


A 3 ' RNA - seq read coverage on DOG1 locus . Black colour is used for coverage within the scale on the right - hand side ( 0 to 500 ) , and red is used for coverage within the scale on the left - hand side ( 0 to 50 ) . Above is a schematic representation of the annotated transcripts from the DOG1 locus and the chromosome coordinates .

A 3'RNA-seq read coverage on DOG1 locus. Black colour is used for coverage within the scale on the right-hand side (0 to 500), and red is used for coverage within the scale on the left-hand side (0 to 50). Above is a schematic representation of the annotated transcripts from the DOG1 locus and the chromosome coordinates.


Germination time - course in 100 mM NaCl after stratification for puppies - 1 ( C ) relative to WT . Lines represent fitted curves with a 95 % confidence interval ( grey area ) , dots represent data points , ∗ p - value < 0 . 05 from the two - tailed Student ' s t - test . ( C ) Data for WT is the same as

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Germination time-course in 100 mM NaCl after stratification for puppies-1 (C) relative to WT. Lines represent fitted curves with a 95% confidence interval (grey area), dots represent data points, ∗ p-value < 0.05 from the two-tailed Student's t-test. (C) Data for WT is the same as plotted


F RT - qPCR fold - change induction of PUPPIES - uns , PUPPIES - prom , PUPPIES - fusion , shDOG1 , and lgDOG1 in puppies - ox relative to WT ( blue dashed line ) , in dry ( light grey ) and imbibed seeds in the presence of 100 mM NaCl ( red ) . Significance from the two - tailed Student ' s t - test for comparing dry seeds of WT and puppies - ox is represented with light grey asterisks , and salt - imbibed seeds of WT and puppies - ox are represented with red asterisks . Black asterisks represent significance from two - way ANOVA with Tukey ' s multiple comparisons test for dry seeds vs imbibed seeds . ns p - value > 0 . 05 , ∗ p - value < 0 . 05 , ∗ ∗ p - value < 0 . 01 , ∗ ∗ ∗ p - value < 0 . 001 . Error bars represent the mean ± SD . n = 4 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F RT-qPCR fold-change induction of PUPPIES-uns, PUPPIES-prom, PUPPIES-fusion, shDOG1, and lgDOG1 in puppies-ox relative to WT (blue dashed line), in dry (light grey) and imbibed seeds in the presence of 100 mM NaCl (red). Significance from the two-tailed Student's t-test for comparing dry seeds of WT and puppies-ox is represented with light grey asterisks, and salt-imbibed seeds of WT and puppies-ox are represented with red asterisks. Black asterisks represent significance from two-way ANOVA with Tukey's multiple comparisons test for dry seeds vs imbibed seeds. ns p-value > 0.05, ∗ p-value < 0.05, ∗∗ p-value < 0.01, ∗∗∗ p-value < 0.001. Error bars represent the mean ± SD. n = 4 biological replicates.


G RT - qPCR with primers for shDOG1 and lgDOG1 on nascent RNA from seeds imbibed in 100 mM NaCl from puppies - ox relative to WT . Nascent RNA levels were normalized to UBC21 . Bars and error bars represent the mean ± SD . Points represent biological replicates . Statistical significance from two - tailed Student ' s t - test . ∗ p - value < 0 . 05 .

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G RT-qPCR with primers for shDOG1 and lgDOG1 on nascent RNA from seeds imbibed in 100 mM NaCl from puppies-ox relative to WT. Nascent RNA levels were normalized to UBC21. Bars and error bars represent the mean ± SD. Points represent biological replicates. Statistical significance from two-tailed Student's t-test. ∗ p-value < 0.05.


A z - stack max - projection image of smFISH for DOG1 RNA . The " Inferno " colour scale is used for the intensity of fluorescence from Quasar670 fluorophore ( DOG1 ) . The blue colour shows fluorescence from DAPI ( nuclei staining ) . Arrowheads point to foci corresponding to transcription sites ( TS ) . The scale bar is 20 μm . B Distribution of cytoplasmic DOG1 foci per cell in WT . The blue vertical dashed line indicates the average .

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A z-stack max-projection image of smFISH for DOG1 RNA. The "Inferno" colour scale is used for the intensity of fluorescence from Quasar670 fluorophore (DOG1). The blue colour shows fluorescence from DAPI (nuclei staining). Arrowheads point to foci corresponding to transcription sites (TS). The scale bar is 20 μm. B Distribution of cytoplasmic DOG1 foci per cell in WT. The blue vertical dashed line indicates the average. C


G z - stack max - projection images from seeds imbibed in 100 mM NaCl of WT ( left ) versus puppies - ox ( right ) . Arrowheads point to foci corresponding to TS .

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G z-stack max-projection images from seeds imbibed in 100 mM NaCl of WT (left) versus puppies-ox (right). Arrowheads point to foci corresponding to TS.


A - E The profile of Pol II during DOG1 transcription . Plots show the fraction of unique reads from targeted NET - Seq using rolling median ( 11 nt ) . Targeted libraries for sequencing of DOG1 locus were obtained by usage of 5 primers across the gene . Pol II dynamics is analyzed separately for each region sequenced in exon 1 ( A ) , exon 2 ( B ) , exon 3 ( C ) , intron 1 ( D ) , and intron 2 ( E ) . The x - axis shows the distance in bp from DOG1 . A light red colour fill highlights the regions where Pol II density is higher in puppies - ox . A light blue colour fill highlights the regions where Pol II density is higher in WT . For each plot , coloured tiles represent the fold - change ( puppies - ox / WT ) on a logarithmic scale . Greyscale tiles represent the result of a statistical test for the difference between genotypes from the two - tailed Student ' s t - test after Bonferroni correction . Tiles correspond to a 25 bp bin each . Vertical dashed lines connect the exon boundaries on the plots with the schematics of the DOG1 gene with grey boxes representing exons and black lines representing introns .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-E The profile of Pol II during DOG1 transcription. Plots show the fraction of unique reads from targeted NET-Seq using rolling median (11 nt). Targeted libraries for sequencing of DOG1 locus were obtained by usage of 5 primers across the gene. Pol II dynamics is analyzed separately for each region sequenced in exon 1 (A), exon 2 (B), exon 3 (C), intron 1 (D), and intron 2 (E). The x-axis shows the distance in bp from DOG1. A light red colour fill highlights the regions where Pol II density is higher in puppies-ox. A light blue colour fill highlights the regions where Pol II density is higher in WT. For each plot, coloured tiles represent the fold-change (puppies-ox / WT) on a logarithmic scale. Greyscale tiles represent the result of a statistical test for the difference between genotypes from the two-tailed Student's t-test after Bonferroni correction. Tiles correspond to a 25 bp bin each. Vertical dashed lines connect the exon boundaries on the plots with the schematics of the DOG1 gene with grey boxes representing exons and black lines representing introns.


F , G RT - qPCR measurement of alternative splicing of lgDOG1 alpha and beta isoforms and unspliced intron 1 DOG1 isoforms levels in puppies - ox , and puppies - 1 relative to WT , in seeds treated with 100 mM NaCl .

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F, G RT-qPCR measurement of alternative splicing of lgDOG1 alpha and beta isoforms and unspliced intron 1 DOG1 isoforms levels in puppies-ox, and puppies-1 relative to WT, in seeds treated with 100 mM NaCl.


Comparison of SARS - CoV - 2 and SARS - CoV replication kinetics in Vero E6 cells . ( a ) Growth curve showing the release of infectious viral progeny into the medium of infected Vero E6 cells ( m . o . i . 3 ) , as determined by plaque assay ( n = 4 ; mean ± sd is presented ) . ( b ) . Comparison of SARS - CoV - 2 Australia / VIC01 / 2020 and SARS - CoV Frankfurt - 1 plaque phenotype in Vero E6 cells .

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Comparison of SARS-CoV-2 and SARS-CoV replication kinetics in Vero E6 cells.(a) Growth curve showing the release of infectious viral progeny into the medium of infected Vero E6 cells (m.o.i. 3), as determined by plaque assay (n = 4; mean ± sd is presented). (b). Comparison of SARS-CoV-2 Australia/VIC01/2020 and SARS-CoV Frankfurt-1 plaque phenotype in Vero E6 cells.


Kinetics of SARS - CoV - 2 and SARS - CoV RNA synthesis in infected Vero E6 cells . ( a ) Hybridization analysis of viral mRNAs isolated from SARS - CoV - 2 - and SARS - CoV - infected Vero E6 cells , separated in an agarose gel and probed with a radiolabelled oligonucleotide recognizing the genome and subgenomic mRNAs of both viruses . Subsequently , the gel was re - hybridized to a probe specific for 18S ribosomal RNA , which was used as a loading control . ( b ) Analysis of the relative abundance of each of the SARS - CoV - 2 and SARS - CoV transcripts . Phosphorimager quantification was performed for the bands of the samples isolated at 12 , 14 and 24 h p . i . , which yielded essentially identical relative abundances . The table shows the average of these three measurements . SARS - CoV - 2 mRNA sizes were calculated on the basis of the position of the leader and body transcription - regulatory sequences ( ACGAAC ) in the viral genome ( Sawicki and Sawicki 1995 , Xu , Hu et al . 2003 ) .

O 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-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Kinetics of SARS-CoV-2 and SARS-CoV RNA synthesis in infected Vero E6 cells.(a) Hybridization analysis of viral mRNAs isolated from SARS-CoV-2- and SARS-CoV -infected Vero E6 cells, separated in an agarose gel and probed with a radiolabelled oligonucleotide recognizing the genome and subgenomic mRNAs of both viruses. Subsequently, the gel was re-hybridized to a probe specific for 18S ribosomal RNA, which was used as a loading control. (b) Analysis of the relative abundance of each of the SARS-CoV-2 and SARS-CoV transcripts. Phosphorimager quantification was performed for the bands of the samples isolated at 12, 14 and 24 h p.i., which yielded essentially identical relative abundances. The table shows the average of these three measurements. SARS-CoV-2 mRNA sizes were calculated on the basis of the position of the leader and body transcription-regulatory sequences (ACGAAC) in the viral genome (Sawicki and Sawicki 1995, Xu, Hu et al. 2003).


Cross - reactivity of antisera raised against SARS - CoV structural and non - structural proteins . Selected antisera previously raised against SARS - CoV nsps and structural proteins cross - react with corresponding SARS - CoV - 2 proteins . SARS - CoV - 2 - infected Vero E6 cells ( m . o . i . of 0 . 3 ) were fixed at 12 or 24 h p . i . For immunofluorescence microscopy , cells were ( double ) labelled with ( a ) a rabbit antiserum recognising nsp4 and a mouse mAb recognising dsRNA ; ( b ) anti - nsp4 rabbit serum and a mouse mAb directed against the N protein ; ( c - e ) rabbit antisera recognising against nsp3 , nsp13 and the M protein , respectively . Nuclear DNA was stained with Hoechst 33258 . Bar , 20 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cross-reactivity of antisera raised against SARS-CoV structural and non-structural proteins.Selected antisera previously raised against SARS-CoV nsps and structural proteins cross-react with corresponding SARS-CoV-2 proteins. SARS-CoV-2-infected Vero E6 cells (m.o.i. of 0.3) were fixed at 12 or 24 h p.i. For immunofluorescence microscopy, cells were (double)labelled with (a) a rabbit antiserum recognising nsp4 and a mouse mAb recognising dsRNA; (b) anti-nsp4 rabbit serum and a mouse mAb directed against the N protein; (c-e) rabbit antisera recognising against nsp3, nsp13 and the M protein, respectively. Nuclear DNA was stained with Hoechst 33258. Bar, 20 µm.


Assay to screen for compounds that inhibit SARS - CoV - 2 replication . Inhibition of SARS - CoV - 2 replication ( coloured bars ) was tested in Vero E6 cells by developing a CPE - reduction assay and evaluating several previously identified inhibitors of SARS - CoV , which was included for comparison ( grey bars ) . For each compound a two - fold serial dilution series in the low - micromolar range was tested ; ( a ) Remdesivir , ( b ) chloroquine , ( c ) Alisporivir and ( d ) pegylated interferon alpha - 2 . Cell viability was assayed using the CellTiter 96 ® Aqueous One Solution cell proliferation assay ( MTS assay ) . Compound toxicity ( solid line ) was evaluated in parallel using mock - infected , compound - treated cells . The graphs show the results of 3 independent experiments , each performed using quadruplicate samples ( mean ± SD are shown ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Assay to screen for compounds that inhibit SARS-CoV-2 replication.Inhibition of SARS-CoV-2 replication (coloured bars) was tested in Vero E6 cells by developing a CPE-reduction assay and evaluating several previously identified inhibitors of SARS-CoV, which was included for comparison (grey bars). For each compound a two-fold serial dilution series in the low-micromolar range was tested; (a) Remdesivir, (b) chloroquine, (c) Alisporivir and (d) pegylated interferon alpha-2. Cell viability was assayed using the CellTiter 96® Aqueous One Solution cell proliferation assay (MTS assay). Compound toxicity (solid line) was evaluated in parallel using mock-infected, compound-treated cells. The graphs show the results of 3 independent experiments, each performed using quadruplicate samples (mean ± SD are shown).


C Comparison of meiotic chromosome segregation in cells lacking H3K9me3 depending on Swi6 ' s affinity towards H3K9me2 . Exemplary IF microscopy images of the indicated fission yeast strains are shown . Homothallic strains were incubated on SPAS plates . DNA and tubulin were stained with DAPI and anti - TAT - 1 , respectively . Missegregating chromosome is indicated with a yellow arrow ; D Quantification of meiotic chromosome segregation phenotypes . The data for clr4 + and clr4F449Y is the same as in Fig 3B . P values were calculated using Fisher ' s exact test . Number of total cells counted is indicated in each bar .

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C Comparison of meiotic chromosome segregation in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary IF microscopy images of the indicated fission yeast strains are shown. Homothallic strains were incubated on SPAS plates. DNA and tubulin were stained with DAPI and anti-TAT-1, respectively. Missegregating chromosome is indicated with a yellow arrow; D Quantification of meiotic chromosome segregation phenotypes. The data for clr4+ and clr4F449Y is the same as in Fig 3B. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.


E Comparison of co - segregation of centromere I ( CenI ) during MI in cells lacking H3K9me3 depending on Swi6 ' s affinity towards H3K9me2 . Exemplary live cell microscopy images of the indicated fission yeast strains during MI and MII are shown . Heterothallic strains were crossed and dyads as well as tetrads were subjected to the analysis . Heterozygous CenI - GFP was used to follow the segregation pattern during meiosis . The GFP - signal is additionally displayed in greyscale for better visibility F Quantification of CenI - GFP segregation during meiosis . The data for clr4 + and clr4F449Y is the same as in Fig 3D . P values were calculated using Fisher ' s exact test . Number of total cells counted is indicated in each bar .

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E Comparison of co-segregation of centromere I (CenI) during MI in cells lacking H3K9me3 depending on Swi6's affinity towards H3K9me2. Exemplary live cell microscopy images of the indicated fission yeast strains during MI and MII are shown. Heterothallic strains were crossed and dyads as well as tetrads were subjected to the analysis. Heterozygous CenI-GFP was used to follow the segregation pattern during meiosis. The GFP-signal is additionally displayed in greyscale for better visibility F Quantification of CenI-GFP segregation during meiosis. The data for clr4+ and clr4F449Y is the same as in Fig 3D. P values were calculated using Fisher's exact test. Number of total cells counted is indicated in each bar.


G Tetrad dissection to asses spore viability in clr4F449Y swi6Chp1 - like - CD cells . Exemplary image ( left ) and quantification of the spore viability ( right ) are shown . Meiosis was induced in homothallic cells and the resulting spores were dissected . The data for clr4 + and clr4F449Y is the same as in Fig 3F . P values were calculated using Fisher ' s exact test . Number of dissected tetrads is indicated in each bar .

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G Tetrad dissection to asses spore viability in clr4F449Y swi6Chp1-like-CD cells. Exemplary image (left) and quantification of the spore viability (right) are shown. Meiosis was induced in homothallic cells and the resulting spores were dissected. The data for clr4+ and clr4F449Y is the same as in Fig 3F. P values were calculated using Fisher's exact test. Number of dissected tetrads is indicated in each bar.


A Western blot of whole - cell lysates ( WCL ) using a PhosTag gel for anti - FLAG ( top ) and a Bis - Tris gel for anti - Act1 detection ( bottom ) , respectively . WCL were prepared from mitotic and meiotic 3xFLAG - clr4 + / 3xFLAG - clr4 + cultures at the indicated time points .

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A Western blot of whole-cell lysates (WCL) using a PhosTag gel for anti-FLAG (top) and a Bis-Tris gel for anti-Act1 detection (bottom), respectively. WCL were prepared from mitotic and meiotic 3xFLAG-clr4+/3xFLAG-clr4+ cultures at the indicated time points.


C Western blot of FLAG IPs using a PhosTag gel to asses phosphorylation levels of Clr4 in the indicated homothallic strains .

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C Western blot of FLAG IPs using a PhosTag gel to asses phosphorylation levels of Clr4 in the indicated homothallic strains.


D Western blot of FLAG IPs in 3xFLAG - clr4 + cdc2 - as cells , treated with 2 µM 1 - NM - PP1 for the indicated amount of time . The blot was first probed with the phospho - specific anti - pS458 antibody , stripped , and reprobed with an anti - FLAG antibody .

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D Western blot of FLAG IPs in 3xFLAG-clr4+ cdc2-as cells, treated with 2 µM 1-NM-PP1 for the indicated amount of time. The blot was first probed with the phospho-specific anti-pS458 antibody, stripped, and reprobed with an anti-FLAG antibody.


[ A ] The same MVs from GB03 caused opposed impacts on plants grown in different medium . The petri dishes contain plastic partitions ( red dotted lines ) that separate different medium . 0 . 5 MS and 0 . 05 MS indicate 1 / 2 - strength ( nutrient - sufficient ) and 1 / 20th - strength ( nutrient - deficient ) Murashige & Skoog medium . Images were taken at 11 days after treatments ( DAT ) .

[A] The same MVs from GB03 caused opposed impacts on plants grown in different medium. The petri dishes contain plastic partitions (red dotted lines) that separate different medium. 0.5 MS and 0.05 MS indicate 1/2-strength (nutrient-sufficient) and 1/20th-strength (nutrient-deficient) Murashige & Skoog medium. Images were taken at 11 days after treatments (DAT).


[ B ] Quantification of total leaf area per plant ( square centimeter , cm2 ) . Values correspond to the means ± SE of three biological replicates . Different letters denote significant differences at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

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[B] Quantification of total leaf area per plant (square centimeter, cm2). Values correspond to the means ± SE of three biological replicates. Different letters denote significant differences at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ C ] Effective quantum yield of photosystem II in plants . n = 3 biological replicates , mean ± SE . Asterisks denote significant differences at * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 , Dunnett ' s multiple comparison test ( comparing with the control ) within each group of the same DAT

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[C] Effective quantum yield of photosystem II in plants. n=3 biological replicates, mean± SE. Asterisks denote significant differences at * p < 0.05, ** p < 0.01 and *** p < 0.001, Dunnett's multiple comparison test (comparing with the control) within each group of the same DAT


[ D ] Cell death visualization by trypan blue staining of 11 DAT leaves . Bar = 1mm

[D] Cell death visualization by trypan blue staining of 11 DAT leaves. Bar= 1mm


[ A ] Plants grown with 0 . 05 MS and GMVs ( G 0 . 05 ) showed hyper - induction of IPS1 and PS2 gene expression in both shoots and roots , compared to plants grown with 0 . 05 MS alone ( C 0 . 05 ) . Values are means ± SE of three biological replicates .

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[A] Plants grown with 0.05 MS and GMVs (G 0.05) showed hyper-induction of IPS1 and PS2 gene expression in both shoots and roots, compared to plants grown with 0.05 MS alone (C 0.05). Values are means ± SE of three biological replicates.


[ B ] Root acid phosphatase activity as detected by the blue color from the 5 - bromo - 4 - chloro - 3 - indolyl phosphate p - toluidine salt ( BCIP ) treatment . Image contrast enhanced for improved visualization of the blue color in roots

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[B] Root acid phosphatase activity as detected by the blue color from the 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) treatment. Image contrast enhanced for improved visualization of the blue color in roots


Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - accumulation of anthocyanin ( C ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-accumulation of anthocyanin (C) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


Supplementation of Pi to the nutrient - deficient plants ( 0 . 05 + Pi ) significantly reduced hyper - induction of IPS1 gene expression ( D ) triggered by GMVs . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Supplementation of Pi to the nutrient-deficient plants (0.05 + Pi) significantly reduced hyper-induction of IPS1 gene expression (D) triggered by GMVs. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ E ] Pi supplementation to the 0 . 05 MS medium partially restored GMV - induced plant vigor , as indicated by increases in total leaf area per plant . [ F ] A low Pi level ( LP ) , which was equal to 1 / 20 of that in the 0 . 5 MS medium , in plant growth medium blocked GMV - induced plant vigor . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

O O O B-CONTROLLED_VAR O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[E] Pi supplementation to the 0.05 MS medium partially restored GMV-induced plant vigor, as indicated by increases in total leaf area per plant. [F] A low Pi level (LP), which was equal to 1/20 of that in the 0.5 MS medium, in plant growth medium blocked GMV-induced plant vigor. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ G ] The Arabidopsis phr1phl1 mutant showed substantially decreased growth - promotion , compared to the wild type plants . Data information : The boxplots show representative data from three independent experiments ( n = 12 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group of the same DAT .

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[G] The Arabidopsis phr1phl1 mutant showed substantially decreased growth-promotion, compared to the wild type plants. Data information: The boxplots show representative data from three independent experiments (n=12). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group of the same DAT.


[ A ] Images of Arabidopsis plants exposed to four individual GMV components , including diacetyl ( DA ) , 2 , 3 - butanediol ( BTDL ) , 2 - methyl - 1 - propanol ( MP ) and acetoin ( ATN ) . The compounds were applied at dosages that , when the compounds totally evaporate from the agar - containing solid droplets , would yield in volatile concentrations of 9 . 7 µg ( DA ) , 32 . 5 µg ( BTDL ) , 7 . 9 µg ( MP ) and 28 . 5 µg ( ATN ) per mL free space in the petri dish , which resembled the 1 : 3 . 3 : 0 . 8 : 2 . 9 ratio among the four GMV components in natural GMVs22 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

[A] Images of Arabidopsis plants exposed to four individual GMV components, including diacetyl (DA), 2,3-butanediol (BTDL), 2-methyl-1-propanol (MP) and acetoin (ATN). The compounds were applied at dosages that, when the compounds totally evaporate from the agar-containing solid droplets, would yield in volatile concentrations of 9.7 µg (DA), 32.5 µg (BTDL), 7.9 µg (MP) and 28.5 µg (ATN) per mL free space in the petri dish, which resembled the 1 : 3.3 : 0.8 : 2.9 ratio among the four GMV components in natural GMVs22.


anthocyanin hyper accumulation ( B ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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anthocyanin hyper accumulation (B) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


PSR gene hyper induction ( C ) were observed in Pi - deficient plants exposed to DA but not to the other three GMV components . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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PSR gene hyper induction (C) were observed in Pi-deficient plants exposed to DA but not to the other three GMV components. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Compared to the wild type plants , phr1phl1 showed substantially decreased anthocyanin hyper accumulation ( D ) triggered by DA . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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Compared to the wild type plants, phr1phl1 showed substantially decreased anthocyanin hyper accumulation (D) triggered by DA. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Compared to the wild type plants , phr1phl1 showed substantially decreased PSR gene hyper induction ( E ) triggered by DA . Data information : qPCR results show values of means ± SE of two biological replicates . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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Compared to the wild type plants, phr1phl1 showed substantially decreased PSR gene hyper induction (E) triggered by DA. Data information: qPCR results show values of means ± SE of two biological replicates. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ A ] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency . Data information : qPCR results show values of means ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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[A] DA induces genes involved in SA biosynthesis and signaling in Arabidopsis grown with Pi deficiency. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ B ] DA elevates SA accumulation levels in Pi - deficient Arabidopsis . Means ± SE of three biological replicates . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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[B] DA elevates SA accumulation levels in Pi-deficient Arabidopsis. Means ± SE of three biological replicates. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ C ] Images of plants grown with different treatments using DA ( 9 . 7 µg per mL free space ) or SA ( 100 µM in plant growth medium ) .

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[C] Images of plants grown with different treatments using DA (9.7 µg per mL free space) or SA (100 µM in plant growth medium).


Exogenous application of SA mimics DA - induced anthocyanin accumulation ( D ) patterns in Pi - deficient plants . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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Exogenous application of SA mimics DA-induced anthocyanin accumulation (D) patterns in Pi-deficient plants. Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Exogenous application of SA mimics DA - induced IPS1 gene induction ( E ) patterns in Pi - deficient plants . Data information : qPCR results show values of means ± SE ( n = 3 ) , two biological replicates were analyzed with similar results . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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Exogenous application of SA mimics DA-induced IPS1 gene induction (E) patterns in Pi-deficient plants. Data information: qPCR results show values of means ± SE (n=3), two biological replicates were analyzed with similar results. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


Compared to the wild type plants , the NahG transgenic plants showed altered responses to DA under Pi - deficiency condition , as shown by plant images ( F )

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Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition, as shown by plant images (F)


Compared to the wild type plants , the NahG transgenic plants showed altered responses to DA under Pi - deficiency condition and quantification of anthocyanin accumulation levels ( G ) . Data information : The boxplots show representative data from three independent experiments ( n = 6 ) . Whiskers represent the min to max data range , the median is represented by the central horizontal line . The upper and lower limits of the box outline represent the first and third quartile . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test .

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Compared to the wild type plants, the NahG transgenic plants showed altered responses to DA under Pi-deficiency condition and quantification of anthocyanin accumulation levels (G). Data information: The boxplots show representative data from three independent experiments (n=6). Whiskers represent the min to max data range, the median is represented by the central horizontal line. The upper and lower limits of the box outline represent the first and third quartile. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test.


[ A ] Clustering of DA - regulated immunity - related genes in Arabidopsis with sufficient Pi , as revealed by RNAseq analysis .

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[A] Clustering of DA-regulated immunity-related genes in Arabidopsis with sufficient Pi, as revealed by RNAseq analysis.


[ B ] DA suppresses plant ROS burst induced by flg22 or elf18 . ROS accumulation was represented in relative luminescence units ( RLU ) . Data bar indicate mean ± SE ( n = 24 ) readings for 60 min . Three independent experiments were performed and similar results were observed . * * p < 0 . 01 , student t - test .

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[B] DA suppresses plant ROS burst induced by flg22 or elf18. ROS accumulation was represented in relative luminescence units (RLU). Data bar indicate mean ± SE (n=24) readings for 60 min. Three independent experiments were performed and similar results were observed. **p < 0.01, student t-test.


[ C ] DA induces gene expression of AZI1 in Arabidopsis . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group . Bar indicates mean ± SE ( n = 3 ) , two biological replicates were analyzed with similar results .

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[C] DA induces gene expression of AZI1 in Arabidopsis. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group. Bar indicates mean ± SE (n=3), two biological replicates were analyzed with similar results.


[ D ] flg22 - induced MPK3 / MPK6 phosphorylation in Arabidopsis is not decreased by DA . Kinase assays were performed at 5 min after flg22 treatment . Three independent experiments were performed and similar results were observed .

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[D] flg22-induced MPK3/MPK6 phosphorylation in Arabidopsis is not decreased by DA. Kinase assays were performed at 5 min after flg22 treatment. Three independent experiments were performed and similar results were observed.


[ E ] Plant resistance to the pathogen Pst DC3000 is not reduced by DA . The severity of pathogen infection was measured as colony forming units ( CFUs ) per mg FW plants at 0 and 3 days after inoculation . Data bar indicate mean ± SE ( n = 8 ) , two independent experiments were performed . Different letters denote significantly different means at p < 0 . 05 , Tukey ' s multiple comparison test within each group .

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[E] Plant resistance to the pathogen Pst DC3000 is not reduced by DA. The severity of pathogen infection was measured as colony forming units (CFUs) per mg FW plants at 0 and 3 days after inoculation. Data bar indicate mean ± SE (n=8), two independent experiments were performed. Different letters denote significantly different means at p < 0.05, Tukey's multiple comparison test within each group.


[ F ] Root colonization of GB03 in wild type Arabidopsis ( Col - 0 ) and the mutants / transgenic lines npr1 , NahG , and rbohd . * * * p < 0 . 001compared with Col - 0 without DA treatment , n = 9 biological replicates , student t - test .

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[F] Root colonization of GB03 in wild type Arabidopsis (Col-0) and the mutants/transgenic lines npr1, NahG, and rbohd. *** p < 0.001compared with Col-0 without DA treatment, n= 9 biological replicates, student t-test.


[ G ] Bacteria chemotaxis with different concentrations of DA . Data point indicates mean ± SE ( n = 6 ) . Two independent experiments were performed and similar results were observed . * * * p < 0 . 001 , student t - test .

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[G] Bacteria chemotaxis with different concentrations of DA. Data point indicates mean± SE (n=6). Two independent experiments were performed and similar results were observed. *** p < 0.001, student t-test.


A . Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons ( DIV16 ) expressing GFP - Sec61β . Zooms represent ( 1 ) an axonal structure with presynaptic boutons ( arrowheads ) , and ( 2 ) a dendritic structure . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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A. Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons (DIV16) expressing GFP-Sec61β. Zooms represent (1) an axonal structure with presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 µm (full size) and 5 µm (zoom).


C . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector or VAPA / B shRNAs . Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) .

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C. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).


D . Quantifications of fluorescence intensity of internalized endogenous Syt ( B , C ) at single presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector or VAPA / B shRNAs . N = 2 , n = 288 - 541 boutons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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D. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N=2, n=288-541 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


E . Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non - neuronal tissues . Cereb . , cerebellum . Hippoc . , hippocampus . Spin . , spinal .

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E. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal.


F . Pull down assay of HEK293T cells co - expressing Myc - VAPA with BioGFP or BioGFP - SCRN1 . G . Pull down assay of HEK293T cells co - expressing Myc - VAPB with BioGFP or BioGFP - SCRN1 . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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F. Pull down assay of HEK293T cells co-expressing Myc-VAPA with BioGFP or BioGFP-SCRN1. G. Pull down assay of HEK293T cells co-expressing Myc-VAPB with BioGFP or BioGFP-SCRN1.Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.


H . Scaled representation of SCRN1 - associated proteins identified with pull - down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP - SCRN1 from HEK293T cell lysates with adult rat brain extracts . All candidates showed > 10 enrichment of PSM compared to control .

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H. Scaled representation of SCRN1-associated proteins identified with pull-down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP-SCRN1 from HEK293T cell lysates with adult rat brain extracts. All candidates showed >10 enrichment of PSM compared to control.


I . Endogenous localization of SCRN1 and Syt in cortical neurons ( DIV18 ) expressing GFP . Zoom represents an axon structure with presynaptic boutons ( arrowheads ) . Scale bars : 10 µm ( full size ) and 5 µm ( zoom ) .

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I. Endogenous localization of SCRN1 and Syt in cortical neurons (DIV18) expressing GFP. Zoom represents an axon structure with presynaptic boutons (arrowheads). Scale bars: 10 µm (full size) and 5 µm (zoom).


J . Representative image of Syt antibody uptake at axons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA or SCRN1 shRNA with GFP - SCRN1 . Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt , respectively . Zooms represent typical boutons . Scale bars : 5 µm ( full size ) and 2 µm ( zoom ) .

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J. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA or SCRN1 shRNA with GFP-SCRN1. Yellow and grey arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 µm (full size) and 2 µm (zoom).


K . Quantifications of fluorescence intensity of internalized endogenous Syt ( B , C ) at single presynaptic boutons of hippocampal neurons ( DIV18 ) co - expressing RFP and pSuper empty vector , SCRN1 shRNA alone or SCRN1 shRNA with GFP - SCRN1 . N = 2 , n = 201 - 300 boutons . Data information : Data represent Mean ± SEM ; NS : not significant ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by Mann - Whitney U test .

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K. Quantifications of fluorescence intensity of internalized endogenous Syt (B, C) at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA alone or SCRN1 shRNA with GFP-SCRN1. N=2, n=201-300 boutons. Data information: Data represent Mean ± SEM; NS: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann-Whitney U test.