HIV - 1 - VSV - ZsGreen at various dilutions ( corresponding to multiplicities of infection ( m . o . i ) 0 . 06 , 0 . 03 and 0 . 02 ) ( F ) infection is reduced in differentiated THP - 1 depleted of CLIP170 determined by luciferase assays or FACS analysis , respectively .

B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O 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-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O

HIV-1-VSV-ZsGreen at various dilutions (corresponding to multiplicities of infection (m.o.i) 0.06, 0.03 and 0.02) (F) infection is reduced in differentiated THP-1 depleted of CLIP170 determined by luciferase assays or FACS analysis, respectively.


( G ) HIV - 1 - WT - luc infection is decreased in CLIP170 depleted CHME3 cells .

O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE O O

(G) HIV-1-WT-luc infection is decreased in CLIP170 depleted CHME3 cells.


Effects of CLIP170 on fusion of viral particles by endocytosis . CHME3 cells either treated with control or CLIP170 siRNAs ( A ) were infected with Mock or HIV - 1 - VSV - BlaM - Vpr . Representative FACS analysis of cells ( n = 3 ) showing ∼ 9 % ( Control siRNA ) and 3 % ( CLIP170 siRNA ) shift ( A ) from green ( uncleaved CCF2 ) to blue ( cleaved CCF2 ) . % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot .

O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O 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-EXP_ASSAY O O

Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells either treated with control or CLIP170 siRNAs (A) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing ∼ 9% (Control siRNA) and 3% (CLIP170 siRNA) shift (A) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot.


HIV - 1 - VSV - luc infection is enhanced in Flag - CLIP170 , but not in Flag control , expressing CHME3 cells .

B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O

HIV-1-VSV-luc infection is enhanced in Flag-CLIP170, but not in Flag control, expressing CHME3 cells.


Molecular weight markers ( in kDa ) are shown to the right of WBs in C .

O O O O O O O O O O O O O B-EXP_ASSAY O O O

Molecular weight markers (in kDa) are shown to the right of WBs in C.


Effects of CLIP170 on fusion of viral particles by endocytosis . CHME3 cells expressing Flag control or Flag - CLIP170 ( D ) were infected with Mock or HIV - 1 - VSV - BlaM - Vpr . Representative FACS analysis of cells ( n = 3 ) showing no difference ( D ) from green ( uncleaved CCF2 ) to blue ( cleaved CCF2 ) . % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot .

O O B-GENEPROD O O O O O O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O 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-EXP_ASSAY O O

Effects of CLIP170 on fusion of viral particles by endocytosis. CHME3 cells expressing Flag control or Flag-CLIP170 (D) were infected with Mock or HIV-1-VSV-BlaM-Vpr. Representative FACS analysis of cells (n = 3) showing no difference (D) from green (uncleaved CCF2) to blue (cleaved CCF2). % infected cells is indicated at the top left corner of each plot and quantification from independent repeats is presented as bar graphs at the right side of each FACS plot.


( E ) The percentage fused double - labelled ( GFP - Vpr + / S15 - tomato - ) HIV - 1 - DL - WT are not affected in CHME3 cells depleted of CLIP170 . Data represent mean of ≥ 895 virus particles analyzed in ≥ 11 fields of view ( ≥ 17 cells ) for each condition . One - way ANOVA was used to calculate statistical significance ( mean ± SEM ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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 percentage fused double-labelled (GFP-Vpr+/S15-tomato-) HIV-1-DL-WT are not affected in CHME3 cells depleted of CLIP170. Data represent mean of ≥895 virus particles analyzed in ≥11 fields of view (≥17 cells) for each condition. One-way ANOVA was used to calculate statistical significance (mean ±SEM).


( F ) Percentage HIV - 1 - WT - GFP - Vpr particles within 2 μm of the nucleus is reduced in CHME3 depleted of CLIP170 at 2 and 4 h . p . i , scatterplot with mean of ≥ 354 virus particles quantified in ≥ 13 cells per sample . Statistical significance was determined by one - way ANOVA * = p < 0 . 05 , * * * = p < 0 . 001 .

O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentage HIV-1-WT-GFP-Vpr particles within 2 μm of the nucleus is reduced in CHME3 depleted of CLIP170 at 2 and 4 h.p.i, scatterplot with mean of ≥354 virus particles quantified in ≥13 cells per sample. Statistical significance was determined by one-way ANOVA * = p<0.05, *** = p<0.001.


Infected cells in F analyzed by live cell imaging . Scatterplots representing displacement over time ( velocity ) Each dot represents one viral particle tracked , red line indicates the mean . 188 trajectories were analyzed from 25 movies . Statistical significance was determined by t test . * * * = p < 0 . 001 .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Infected cells in F analyzed by live cell imaging. Scatterplots representing displacement over time (velocity) Each dot represents one viral particle tracked, red line indicates the mean. 188 trajectories were analyzed from 25 movies. Statistical significance was determined by t test. *** = p<0.001.


Infected cells in F analyzed by live cell imaging . cumulative distance travelled over time ( speed ) ( H ) . Each dot represents one viral particle tracked , red line indicates the mean . 188 trajectories were analyzed from 25 movies . Statistical significance was determined by t test . * * * = p < 0 . 001 .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Infected cells in F analyzed by live cell imaging. cumulative distance travelled over time (speed) (H). Each dot represents one viral particle tracked, red line indicates the mean. 188 trajectories were analyzed from 25 movies. Statistical significance was determined by t test. *** = p<0.001.


( I ) The induction of Ac - MTs is blocked in HIV - 1 - VSV - luc ( Virus ) infected , but not in uninfected ( NI ) or Mock infected CHME3 depleted of EB1 , but not CLIP170 at 6 h . p . i . Tyrosinated MTs ( Tyr - MTs ) are not affected . Representative fields are shown . Similar results were obtained in three independent experiments . Scale bar , 50 μm .

O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O 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) The induction of Ac-MTs is blocked in HIV-1-VSV-luc (Virus) infected, but not in uninfected (NI) or Mock infected CHME3 depleted of EB1, but not CLIP170 at 6 h.p.i. Tyrosinated MTs (Tyr-MTs) are not affected. Representative fields are shown. Similar results were obtained in three independent experiments. Scale bar, 50 μm.


CHME3 treated with negative control , EB1 or CLIP170 siRNAs were either lysed and analyzed by WB Molecular weight markers ( in kDa ) are shown to the right of WBs in A .

B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY O O O

CHME3 treated with negative control, EB1 or CLIP170 siRNAs were either lysed and analyzed by WB Molecular weight markers (in kDa) are shown to the right of WBs in A.


CHME3 treated with negative control , EB1 or CLIP170 siRNAs were infected with GFP - Vpr / S15 - tomato - labeled VSV - G pseudotyped HIV - 1 ( B ) . Scatterplot of maximum p24 intensity of fused viral particles normalized to the control mean .

B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O

CHME3 treated with negative control, EB1 or CLIP170 siRNAs were infected with GFP-Vpr/S15-tomato-labeled VSV-G pseudotyped HIV-1 (B). Scatterplot of maximum p24 intensity of fused viral particles normalized to the control mean.


( C - D ) Amounts of pelleted p24 CA normalized to the input CA at 3 h . p . i . with HIV - 1 - VSV - luc in 293A ( C ) or CHME3 ( D ) treated with control , EB1 or CLIP170 siRNAs . Mean + / - SEM from three independent experiments is shown . Statistical significance was determined by one - way ANOVA . Error bars = standard error of the mean ( * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 ) .

O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Amounts of pelleted p24 CA normalized to the input CA at 3 h.p.i. with HIV-1-VSV-luc in 293A (C) or CHME3 (D) treated with control, EB1 or CLIP170 siRNAs. Mean +/- SEM from three independent experiments is shown. Statistical significance was determined by one-way ANOVA. Error bars = standard error of the mean (* = p-value ≤0.05, ** = p-value ≤0.01, *** = p-value ≤0.001).


Representative ( n = 3 ) WB analysis demonstrating that while GAPDH - HA control do not bind ( A FEZ1 - Flag control pelleted with in vitro assembled HIV - 1 CA - NC complexes .

O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O

Representative (n=3) WB analysis demonstrating that while GAPDH-HA control do not bind (A FEZ1-Flag control pelleted with in vitro assembled HIV-1 CA-NC complexes.


Representative ( n = 3 ) WB analysis demonstrating that while EB1 - Flag do not bind pelleted with in vitro assembled HIV - 1 CA - NC complexes .

O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O

Representative (n=3) WB analysis demonstrating that while EB1-Flag do not bind pelleted with in vitro assembled HIV-1 CA-NC complexes.


Representative ( n = 3 ) WB analysis demonstrating that while Flag - CLIP170 pelleted with in vitro assembled HIV - 1 CA - NC complexes .

O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O

Representative (n=3) WB analysis demonstrating that while Flag-CLIP170 pelleted with in vitro assembled HIV-1 CA-NC complexes.


( D ) Quantification of the levels of bound CLIP170 as a ratio of total protein across experimental replicates ( n = 3 ) from C . Statistical significance was determined by t test ( mean ± SEM ) * * * = p < 0 . 001 .

O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O 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) Quantification of the levels of bound CLIP170 as a ratio of total protein across experimental replicates (n=3) from C. Statistical significance was determined by t test (mean ±SEM) *** = p<0.001.


( E ) Representative WB analysis ( n = 3 ) showing the levels of HIV - 1 p24 CA in mock and virus fractions separated by sucrose gradient centrifugation . Fractions 1 - 3 contain free p24 not associated with the intact cores , while fractions # 8 and # 9 harbouring intact HIV - 1 cores .

O O O O B-EXP_ASSAY O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O

(E) Representative WB analysis (n=3) showing the levels of HIV-1 p24 CA in mock and virus fractions separated by sucrose gradient centrifugation. Fractions 1-3 contain free p24 not associated with the intact cores, while fractions #8 and # 9 harbouring intact HIV-1 cores.


( F ) Flag - CLIP170 , but not Flag control , also binds to fractions containing intact HIV - 1 cores ( Virus # 8 and # 9 ) but not with Mock fractions .

O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O

(F) Flag-CLIP170, but not Flag control, also binds to fractions containing intact HIV-1 cores (Virus #8 and #9) but not with Mock fractions.


( G ) HIV - 1 - WT - luc infection is increased in CHME3 cells stably expressing Flag - CLIP170 , but not control Flag or Flag - CLIP170ΔCAPgly . Molecular weight markers ( in kDa ) are shown to the right of WBs .

O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O

(G) HIV-1-WT-luc infection is increased in CHME3 cells stably expressing Flag-CLIP170, but not control Flag or Flag-CLIP170ΔCAPgly. Molecular weight markers (in kDa) are shown to the right of WBs.


( H ) Representative images ( n = 3 ) of CHME3 expressing Flag - CLIP170 or Flag - CLIP170ΔCAPgly stained for Flag and Tyr - MTs . Scale bar , 10 μm .

O O O O O O O O O O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O

(H) Representative images (n=3) of CHME3 expressing Flag-CLIP170 or Flag-CLIP170ΔCAPgly stained for Flag and Tyr-MTs. Scale bar, 10 μm.


( A ) Super resolution microscopy ( SIM ) imaging of Flag - tagged proteins , cyclophilin A ( CypA - Flag ) , EB1 - Flag , or Flag - CLIP170 bound to in vitro assembled CA - NC complexes , stained with anti - p24 AG3 . 0 ( Red ) , anti - Flag or anti - CLIP170 ( Green ) . Scale bar , 2 μm . Representative images from different fields of view are shown .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

(A) Super resolution microscopy (SIM) imaging of Flag-tagged proteins, cyclophilin A (CypA-Flag), EB1-Flag, or Flag-CLIP170 bound to in vitro assembled CA-NC complexes, stained with anti-p24 AG3.0 (Red), anti-Flag or anti-CLIP170 (Green). Scale bar, 2 μm. Representative images from different fields of view are shown.


( B ) WB analysis confirming protein expression in samples from A and C . Molecular weight markers ( in kDa ) are shown to the right of WBs .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

(B) WB analysis confirming protein expression in samples from A and C. Molecular weight markers (in kDa) are shown to the right of WBs.


( C ) Quantification ( n = 3 ) of the association of each protein with 97 to 137 CA - NC complexes , using untransfected lysates ( Control ) and EB1 - Flag as controls for non - specific background staining .

O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O

(C) Quantification (n=3) of the association of each protein with 97 to 137 CA-NC complexes, using untransfected lysates (Control) and EB1-Flag as controls for non-specific background staining.


( D ) Lysates from untransfected , CypA - Flag - or Flag - CLIP170 - transfected cells were subjected to CA - NC binding assays and WB analysis .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O

(D) Lysates from untransfected, CypA-Flag- or Flag-CLIP170-transfected cells were subjected to CA-NC binding assays and WB analysis.


( E ) Levels of CypA or CLIP170 bound to WT or R18L CA - NC assemblies in samples from D were quantified using Fiji ( n = 4 experimental replicates ; data is presented as a ratio of CypA or CLIP170 in bound versus input for each CA - NC assembly , normalized to WT CA - NC arbitrarily set to 1 ) . Statistical significance was determined by t test ( mean ± SEM ) . * * * = p < 0 . 001 .

O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O 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) Levels of CypA or CLIP170 bound to WT or R18L CA-NC assemblies in samples from D were quantified using Fiji (n = 4 experimental replicates; data is presented as a ratio of CypA or CLIP170 in bound versus input for each CA-NC assembly, normalized to WT CA-NC arbitrarily set to 1). Statistical significance was determined by t test (mean ±SEM). *** = p<0.001.


Anti - HA co - IP showing that GAPDH fused to the HIV - 1 MHR ( GAPDH - MHRhiv ) , but not HA - tagged GAPDH ( GAPDH ) binds to Flag - CLIP170 ( C ) in 293T cells . Results are representative of three experimental replicates .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O

Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv), but not HA-tagged GAPDH (GAPDH) binds to Flag-CLIP170 (C) in 293T cells. Results are representative of three experimental replicates.


Anti - HA co - IP showing that GAPDH fused to the HIV - 1 MHR ( GAPDH - MHRhiv ) endogenous forms of various human SxIP - containing + TIPs , but not vimentin ( D ) in 293T cells . Results are representative of three experimental replicates .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O

Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv) endogenous forms of various human SxIP-containing +TIPs, but not vimentin (D) in 293T cells. Results are representative of three experimental replicates.


Anti - HA co - IP showing that GAPDH fused to the HIV - 1 MHR ( GAPDH - MHRhiv ) or to Flag - tagged SxIP fragment ( Flag - Clasp2M ) ( E ) in 293T cells . Results are representative of three experimental replicates .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O

Anti-HA co-IP showing that GAPDH fused to the HIV-1 MHR (GAPDH-MHRhiv) or to Flag-tagged SxIP fragment (Flag-Clasp2M) (E) in 293T cells. Results are representative of three experimental replicates.


HIV - 1 - VSV - luc ( Virus ) , but not uninfected ( NI ) or Mock , infection induces the formation of Ac - MTs in CHME3 expressing GAPDH - MHRhiv , but not GAPDH , as shown by WB analysis ( F )

B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O

HIV-1-VSV-luc (Virus), but not uninfected (NI) or Mock, infection induces the formation of Ac-MTs in CHME3 expressing GAPDH-MHRhiv, but not GAPDH, as shown by WB analysis (F)


HIV - 1 - VSV - luc ( Virus ) , but not uninfected ( NI ) or Mock , infection induces the formation of Ac - MTs in CHME3 expressing GAPDH - MHRhiv , but not GAPDH or imaging at 6 h . p . i ( G ) . Scale bar , 15 μm . Representative fields are shown . Results are representative of three experimental replicates .

B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O B-CELL_LINE O B-GENEPROD O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HIV-1-VSV-luc (Virus), but not uninfected (NI) or Mock, infection induces the formation of Ac-MTs in CHME3 expressing GAPDH-MHRhiv, but not GAPDH or imaging at 6 h.p.i (G). Scale bar, 15 μm. Representative fields are shown. Results are representative of three experimental replicates.


( H ) GAPDH - MHRhiv expression in CHME3 reduces infection with HIV - 1 - WT - luc . Statistical significance was determined by t test . * = p < 0 . 05 . Data are mean values from three independent experiments + / - SEM . Molecular weight markers ( in kDa ) are shown to the right of WBs .

O O O B-GENEPROD O O O O B-CELL_LINE O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O

(H) GAPDH-MHRhiv expression in CHME3 reduces infection with HIV-1-WT-luc. Statistical significance was determined by t test. * = p<0.05. Data are mean values from three independent experiments +/- SEM. Molecular weight markers (in kDa) are shown to the right of WBs.


( C ) Anti - HA co - IP in CHME3 expressing GAPDH - MHRhiv , GAPDH - MHRsiv or GAPDH - MHRmulv showing binding to endogenous CLIP170 . Results are representative of three experimental replicates .

O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O

(C) Anti-HA co-IP in CHME3 expressing GAPDH-MHRhiv, GAPDH-MHRsiv or GAPDH-MHRmulv showing binding to endogenous CLIP170. Results are representative of three experimental replicates.


Anti - HA co - IP in Rhesus macaque FRhK4 ( D ) cells expressing GAPDH alongside GAPDH -

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM I-ORGANISM B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O

Anti-HA co-IP in Rhesus macaque FRhK4 (D) cells expressing GAPDH alongside GAPDH-MHRsiv


Anti - HA co - IP in rat fibroblast Rat2 ( E ) cells expressing GAPDH alongside GAPDH - MHRmulv Anti - EB1 co - IP was included as positive control to demonstrate CLIP170 binding by EB1 in Rat2 cells . Results are representative of three experimental replicates .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM B-CELL_TYPE B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O O O O O O O

Anti-HA co-IP in rat fibroblast Rat2 (E) cells expressing GAPDH alongside GAPDH-MHRmulv Anti-EB1 co-IP was included as positive control to demonstrate CLIP170 binding by EB1 in Rat2 cells. Results are representative of three experimental replicates.


FRhK4 cells infected with SIV - VSV - luc ( F ) stained for Ac - MTs , Tyr - MTs , and the nucleus ( Hoechst ) at 6 h . p .

B-CELL_LINE O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O

FRhK4 cells infected with SIV-VSV-luc (F) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i


Rat2 cells infected with MuLV - VSV - luc ( G ) stained for Ac - MTs , Tyr - MTs , and the nucleus ( Hoechst ) at 6 h . p . i ,

B-CELL_LINE O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O

Rat2 cells infected with MuLV-VSV-luc (G) stained for Ac-MTs, Tyr-MTs, and the nucleus (Hoechst) at 6 h.p.i,


FRhK4 cells infected with SIV - VSV - luc analyzed by

B-CELL_LINE O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O

FRhK4 cells infected with SIV-VSV-luc analyzed by WB


Rat2 cells infected with MuLV - VSV - luc analyzed by

Rat2 cells infected with MuLV-VSV-luc analyzed by WB


Measurements of SIV - VSV - luc infectivity in FRhK4 ( J ) cells depleted of CLIP170 . Statistical significance was determined by t test . * * * = p < 0 . 001 . Data are mean values from two independent experiments + / - SEM .

O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Measurements of SIV-VSV-luc infectivity in FRhK4 (J) cells depleted of CLIP170. Statistical significance was determined by t test. *** = p<0.001. Data are mean values from two independent experiments +/- SEM.


Measurements of MuLV - VSV - luc infectivity in Rat2 ( K ) cells depleted of CLIP170 .

O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O

Measurements of MuLV-VSV-luc infectivity in Rat2 (K) cells depleted of CLIP170.


( L ) 293T transfected with GAPDH - MHRhiv or R162Q mutant ( GAPDH - MHRhivQ ) were subjected to anti - HA co - IP . Representative WB analysis is shown ( n = 2 ) . Molecular weight markers ( in kDa ) are shown to the right of WBs .

O O O B-CELL_LINE O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

(L) 293T transfected with GAPDH-MHRhiv or R162Q mutant (GAPDH-MHRhivQ) were subjected to anti-HA co-IP. Representative WB analysis is shown (n = 2). Molecular weight markers (in kDa) are shown to the right of WBs.


( A ) Phenotypes of wild type ( Col ) , kup9 mutants ( kup9 - 1 and kup9 - 2 ) and the kup9 - 1 / ProKUP9 : KUP9 complementation lines ( COM1 and COM2 ) . Seeds were germinated and grown on low - K + ( LK , 50 μM ) or high - K + ( HK , 5 mM ) medium for 7 d . Scale bar , 1 cm .

O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(A) Phenotypes of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on low-K+ (LK, 50 μM) or high-K+ (HK, 5 mM) medium for 7 d. Scale bar, 1 cm.


( B ) Primary root length of the plants tested Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents the control .

O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.


( D ) RT - qPCR analysis of KUP9 transcript levels in various materials . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 120 individual plants ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O 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) RT-qPCR analysis of KUP9 transcript levels in various materials. Data are means ± SE (n = 3, biological replicates; each replicate contains 120 individual plants).


( E ) and ( F ) K + content of the various plant materials tested in ( A ) . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 120 - 150 individual plants ) .

O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O 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) and (F) K+ content of the various plant materials tested in (A). Data are means ± SE (n = 3, biological replicates; each replicate contains 120-150 individual plants).


( A ) Root meristem zones of wild type ( Col ) , kup9 mutants ( kup9 - 1 and kup9 - 2 ) and the kup9 - 1 / ProKUP9 : KUP9 complementation lines ( COM1 and COM2 ) . Seeds were germinated and grown on LK or HK medium for 7 d . The meristem zone lengths are marked with red lines . Scale bars , 50 μm .

O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O O O O O O O O O

(A) Root meristem zones of wild type (Col), kup9 mutants (kup9-1 and kup9-2) and the kup9-1/ProKUP9:KUP9 complementation lines (COM1 and COM2). Seeds were germinated and grown on LK or HK medium for 7 d. The meristem zone lengths are marked with red lines. Scale bars, 50 μm.


( B ) , ( C ) and ( D ) represent different zone lengths , different zone cell numbers , and cell length of the indicated seedlings germinated and grown on LK or HK medium for 7 d . MEZ , meristem zone ; EZ , elongation zone ; MAZ , maturation zone . Data are means ± SE ( n = 10 - 15 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents the control .

O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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) and (D) represent different zone lengths, different zone cell numbers, and cell length of the indicated seedlings germinated and grown on LK or HK medium for 7 d. MEZ, meristem zone; EZ, elongation zone; MAZ, maturation zone. Data are means ± SE (n = 10-15, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents the control.


( A ) to ( F ) Expression of KUP9 as determined in the ProKUP9 : GUS line . GUS staining of 5 - day - old seedling ( A ) , root maturation zone ( B ) , transverse section of maturation zone ( C ) and ( D ) , primary root tip ( E ) , and root apex ( F ) . Ep , epidermis ; Co , cortex ; En , endodermis ; Pe , pericyle ; Xy , xylem ; Ph , phloem . Scale bars , 5 mm in ( A ) , 1 mm in ( B ) , 10 μm in ( C ) , 5 μm in ( D ) , 1 mm in ( E ) , and 20 μm in ( F ) .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD B-EXP_ASSAY O O O O O O B-ORGANISM O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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) to (F) Expression of KUP9 as determined in the ProKUP9:GUS line. GUS staining of 5-day-old seedling (A), root maturation zone (B), transverse section of maturation zone (C) and (D), primary root tip (E), and root apex (F). Ep, epidermis; Co, cortex; En, endodermis; Pe, pericyle; Xy, xylem; Ph, phloem. Scale bars, 5 mm in (A), 1 mm in (B), 10 μm in (C), 5 μm in (D), 1 mm in (E), and 20 μm in (F).


( G ) and ( H ) Expression of KUP9 protein as determined in the kup9 - 1 / ProKUP9 : KUP9 - GFP transgenic plants . Green fluorescence shows the subcellular localization of KUP9 - GFP . PI ( propidium iodide ) and ER tracker were used to indicate the positions of the plasma membrane ( PM ) and endoplasmic reticulum ( ER ) and are shown as red and cyan fluorescence , respectively . Scale bars for root tip , 10 μm in ( G ) and 5 μm in ( H ) . Scale bars for EZ and MAZ , 20 μm .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O

(G) and (H) Expression of KUP9 protein as determined in the kup9-1/ProKUP9:KUP9-GFP transgenic plants. Green fluorescence shows the subcellular localization of KUP9-GFP. PI (propidium iodide) and ER tracker were used to indicate the positions of the plasma membrane (PM) and endoplasmic reticulum (ER) and are shown as red and cyan fluorescence, respectively. Scale bars for root tip, 10 μm in (G) and 5 μm in (H). Scale bars for EZ and MAZ, 20 μm.


( I ) Co - localization analyses of KUP9 - GFP with ER marker NIP1 ; 1 - mCherry in their crossing line . Scale bars , 20 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O

(I) Co-localization analyses of KUP9-GFP with ER marker NIP1;1-mCherry in their crossing line. Scale bars, 20 μm.


( J ) Co - localization analyses of KUP9 - GFP with the ER marker BIP2 using immunological staining . Scale bars , 5 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(J) Co-localization analyses of KUP9-GFP with the ER marker BIP2 using immunological staining. Scale bars, 5 μm.


( A ) QC cell observation in wild type and the kup9 mutants using the QC marker lines ProWOX5 : GFP and ProQC25 : GUS . Seeds were germinated on HK medium for 4 d , then the seedlings were transferred to LK or HK medium for the indicated times . Scale bars , 10 μm .

O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD O B-TISSUE O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

(A) QC cell observation in wild type and the kup9 mutants using the QC marker lines ProWOX5:GFP and ProQC25:GUS. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bars, 10 μm.


( B ) QC division rates of wild type and the kup9 mutants after LK treatment for 96 h . The statistical method is described in Materials and Methods . Data are means ± SE ( n = 150 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) QC division rates of wild type and the kup9 mutants after LK treatment for 96 h. The statistical method is described in Materials and Methods. Data are means ± SE (n = 150, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


( A ) Auxin levels in root tips , as indicated by the ProDR5 : GFP reporter line . Seeds were germinated on HK medium for 4 d , then the seedlings were transferred to LK or HK medium for the indicated times . Scale bar , 10 μm . ( B ) Quantification of GFP fluorescence in the QC cells shown in ( A ) . Data are means ± SE ( n = 20 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance .

O O O B-SMALL_MOLECULE O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O B-TISSUE O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O 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) Auxin levels in root tips, as indicated by the ProDR5:GFP reporter line. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium for the indicated times. Scale bar, 10 μm. (B) Quantification of GFP fluorescence in the QC cells shown in (A). Data are means ± SE (n = 20, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance.


( A ) Phenotypes of various plants . Seeds were germinated and grown on LK or HK medium with or without NAA for 7 d . Scale bar , 1 cm .

O O O O O O O O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(A) Phenotypes of various plants. Seeds were germinated and grown on LK or HK medium with or without NAA for 7 d. Scale bar, 1 cm.


( C ) QC cell observation using the QC marker line ProWOX5 : GFP . Seeds were germinated on HK medium for 4 d , then the seedlings were transferred to LK or HK medium with or without NAA ( 100 nM ) for 96 h . Scale bar , 10 μm .

O O O B-TISSUE I-TISSUE O O O B-TISSUE O O O O B-GENEPROD O B-TISSUE O B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(C) QC cell observation using the QC marker line ProWOX5:GFP. Seeds were germinated on HK medium for 4 d, then the seedlings were transferred to LK or HK medium with or without NAA (100 nM) for 96 h. Scale bar, 10 μm.


( D ) QC division rates of the plants tested The statistical method is described in Materials and Methods . Data are means ± SE ( n = 50 - 60 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) QC division rates of the plants tested The statistical method is described in Materials and Methods. Data are means ± SE (n = 50-60, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


Phenotypes of the kup9 - 1 / ProWOX5 : AMI1 ( A ) transgenic lines . Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d . Scale bars , 1 cm .

O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

Phenotypes of the kup9-1/ProWOX5:AMI1 (A) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.


Primary root length of the plants tested Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


Phenotypes of the kup9 - 1 / ProKUP9 : AMI1 ( C ) transgenic lines . Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d . Scale bars , 1 cm .

Phenotypes of the kup9-1/ProKUP9:AMI1 (C) transgenic lines. Seeds were germinated and grown on LK or HK medium with or without IAM for 7 d. Scale bars, 1 cm.


( A ) Expression of KUP9 - GFP in BY - 2 tobacco cells . HDEL - mCherry was used as an ER marker . Scale bar , 10 μm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O

(A) Expression of KUP9-GFP in BY-2 tobacco cells. HDEL-mCherry was used as an ER marker. Scale bar, 10 μm.


( B ) 3H - IAA accumulation in the BY - 2 cells expressing the indicated proteins . PIN4 - GFP and KUP10 - GFP were used as positive control and negative control , respectively . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 3 - 4 mL suspension cells ) . Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represent control .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 3H-IAA accumulation in the BY-2 cells expressing the indicated proteins. PIN4-GFP and KUP10-GFP were used as positive control and negative control, respectively. Data are means ± SE (n = 3, biological replicates; each replicate contains 3-4 mL suspension cells). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.


( C ) Expression of KUP9 - GFP and OST4 - GFP - KUP9 in Xenopus oocytes . Scale bar , 100 μm .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM B-CELL_TYPE O O O O O O O

(C) Expression of KUP9-GFP and OST4-GFP-KUP9 in Xenopus oocytes. Scale bar, 100 μm.


( D ) 3H - IAA accumulation in the oocytes expressing KUP9 - GFP and PIN4 at the indicated times . PIN4 was used as a positive control . The oocytes were incubated in the bath solution containing 3H - IAA for the indicated times . Data are means ± SE ( n = 6 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O

(D) 3H-IAA accumulation in the oocytes expressing KUP9-GFP and PIN4 at the indicated times. PIN4 was used as a positive control. The oocytes were incubated in the bath solution containing 3H-IAA for the indicated times. Data are means ± SE (n = 6, biological replicates; each replicate contains 6 oocytes).


( E ) IAA content in the oocytes expressing the indicated proteins . The oocytes were injected with the same amount of IAA and incubated in IAA - free bath solution for 6 h . IAA content was measured using UPLC - MS . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O

(E) IAA content in the oocytes expressing the indicated proteins. The oocytes were injected with the same amount of IAA and incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( F ) K + efflux activity of the oocytes expressing KUP9 and NRT1 . 5 . The oocytes were not injected with IAA . The oocytes were incubated in K + - free bath solution for 6 h . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O 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-CELL_TYPE O O

(F) K+ efflux activity of the oocytes expressing KUP9 and NRT1.5. The oocytes were not injected with IAA. The oocytes were incubated in K+-free bath solution for 6 h. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( G ) K + efflux activity of the oocytes expressing the indicated proteins . The oocytes were injected with H2O , IAA , or BA . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O

(G) K+ efflux activity of the oocytes expressing the indicated proteins. The oocytes were injected with H2O, IAA, or BA. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes).


( H ) K + efflux activity of the oocytes expressing the indicated proteins . IAA was injected into the oocytes . Then the oocytes were incubated in K + - free bath solutions at different pH for 6 h . Data are means ± SE ( n = 3 - 6 , biological replicates ; each replicate contains 6 oocytes ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O B-CELL_TYPE O O O B-CELL_TYPE O O O O O O O O O O O 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-CELL_TYPE O O

(H) K+ efflux activity of the oocytes expressing the indicated proteins. IAA was injected into the oocytes. Then the oocytes were incubated in K+-free bath solutions at different pH for 6 h. Data are means ± SE (n = 3-6, biological replicates; each replicate contains 6 oocytes).


( I ) IAA efflux activity of oocytes expressing the indicated proteins under different external K + conditions ( - K , 0 mM ; + K , 50 mM ) . The oocytes were injected with the same amount of IAA , and then incubated in IAA - free bath solution for 6 h . IAA content was measured using UPLC - MS . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 6 oocytes ) . Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represent control .

O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O 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) IAA efflux activity of oocytes expressing the indicated proteins under different external K+ conditions (-K, 0 mM; +K, 50 mM). The oocytes were injected with the same amount of IAA, and then incubated in IAA-free bath solution for 6 h. IAA content was measured using UPLC-MS. Data are means ± SE (n = 3, biological replicates; each replicate contains 6 oocytes). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represent control.


( A ) K + levels of various materials using the fluorescent dye Asante Potassium Green - 2 ( AM ) . Scale bar , 50 μm . ( B ) Quantification of K + concentration under LK and HK conditions . Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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) K+ levels of various materials using the fluorescent dye Asante Potassium Green-2 (AM). Scale bar, 50 μm. (B) Quantification of K+ concentration under LK and HK conditions. Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance, and "#" represents control.


( C ) to ( E ) The contents of free IAA ( C ) , IA - Asp ( D ) , and IA - Glu ( E ) in the root tips of wild type and the kup9 - 1 mutant . Data are means ± SE ( n = 3 , biological replicates ; each replicate contains 1000 - 1200 individual plants ) . Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 ) was used to analyze statistical significance , and " # " represents control .

O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) to (E) The contents of free IAA (C), IA-Asp (D), and IA-Glu (E) in the root tips of wild type and the kup9-1 mutant. Data are means ± SE (n = 3, biological replicates; each replicate contains 1000-1200 individual plants). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represents control.


( F ) Phenotype of the kup9 - 1 / ProKUP9 : PIN8 transgenic lines . Seeds were germinated and grown on LK or HK medium for 7 d . Scale bar , 1 cm .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O O B-TISSUE O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O

(F) Phenotype of the kup9-1/ProKUP9:PIN8 transgenic lines. Seeds were germinated and grown on LK or HK medium for 7 d. Scale bar, 1 cm.


( G ) Primary root length of the plants tested Data are means ± SE ( n = 25 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical significance .

O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O 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) Primary root length of the plants tested Data are means ± SE (n = 25, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance.


( H ) QC division rates of the indicated plants . The statistical method is described in Materials and Methods . Data are means ± SE ( n = 50 - 60 , individual plants ) . Student ' s t - test ( * * P < 0 . 01 ) was used to analyze statistical

O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) QC division rates of the indicated plants. The statistical method is described in Materials and Methods. Data are means ± SE (n = 50-60, individual plants). Student's t-test (**P < 0.01) was used to analyze statistical significance


a , TEM analysis of P7 wt retinae ( N = 4 eyes ) showing a ( photoreceptor - Multivesicular Body ) PR - MVB in close proximity to ( Muller glia ) MG ( red dashed box ) . PR - photoreceptor cell , MG ( blue dashed lines ) - muller glia cell , MVB ( yellow and yellow arrow ) - multivesicular body , m ( green ) - mitochondria , cc ( pink ) - connecting cilium , RPE ( purple and purple dashed line ) - retina pigment epithelium , RPE melanin - black arrows . Scale bar = 2µm left , 1µm right ;

O O B-EXP_ASSAY O O O O B-TISSUE O O O O B-TISSUE O O O O B-CELL_TYPE O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_TYPE O B-SUBCELLULAR O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE O O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O

a, TEM analysis of P7 wt retinae (N = 4 eyes) showing a (photoreceptor- Multivesicular Body) PR-MVB in close proximity to (Muller glia) MG (red dashed box). PR-photoreceptor cell, MG (blue dashed lines) -muller glia cell, MVB (yellow and yellow arrow)- multivesicular body, m (green)-mitochondria, cc (pink)-connecting cilium, RPE (purple and purple dashed line) -retina pigment epithelium, RPE melanin-black arrows. Scale bar = 2µm left, 1µm right;


f , Representative Dot Blot of 100K EV pellet ( each dot represents 3 pooled EV isolations , derived from 60 * 106 cells ; N = 8 experiments ) vs cell lysate ( CL ) from P8 Nrl . Gfp + / + photoreceptors . EV markers = LAMP1 , CD8 , CD9 ; Golgi marker = GM130 ; phototransduction markers = Recoverin , Rhodopsin , Rod α - Transducin ;

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

f, Representative Dot Blot of 100K EV pellet (each dot represents 3 pooled EV isolations, derived from 60*106 cells; N = 8 experiments) vs cell lysate (CL) from P8 Nrl.Gfp+/+ photoreceptors. EV markers = LAMP1, CD8, CD9; Golgi marker = GM130; phototransduction markers = Recoverin, Rhodopsin, Rod α-Transducin;


g , RTqPCR analysis of 100K P8 photoreceptor pellets for Gnat1 , Rec , Rho , Crx , Cre and Gfp , relative to β - actin , comparing EVs ( N = 8 ) against the appropriate ( Nrl . Gfp + / + or Nrl . Cre + / - ) photoreceptor cell lysate ( N = 3 ) . Gnat1 , Cre and Gfp mRNA were present in all relevant samples ;

O O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O

g, RTqPCR analysis of 100K P8 photoreceptor pellets for Gnat1, Rec, Rho, Crx, Cre and Gfp, relative to β-actin, comparing EVs (N = 8) against the appropriate (Nrl.Gfp+/+ or Nrl.Cre+/-) photoreceptor cell lysate (N = 3). Gnat1, Cre and Gfp mRNA were present in all relevant samples;


i , representative flow cytometry analysis of Nrl . Cre + / - & mTmGfloxed co - cultures . Samples were analyzed for expression of myrGFP ( recombination ) and CD73 ( photoreceptor identity ) versus CD73 - ve fraction ( other retinal cells ) . N > 3 independent cultures for each condition with technical triplicate for each sample ; One way ANOVA , non - parametric , Kruskal - Wallis with Dunns ' multiple comparisons

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O 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, representative flow cytometry analysis of Nrl.Cre+/- & mTmGfloxed co-cultures. Samples were analyzed for expression of myrGFP (recombination) and CD73 (photoreceptor identity) versus CD73-ve fraction (other retinal cells). N > 3 independent cultures for each condition with technical triplicate for each sample; One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test


g , Quantification of no . of TdTomato + ve host photoreceptor cells seen following transplantation of live ( 224 ± 181 ) versus UV - treated ( dead ) ( 3 ± 4 ) Nrl . Cre + / - P8 photoreceptors ( N = 4 eyes per condition ) . Graph shows mean ± S . D .

O O O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O B-TISSUE O O O O O O O O O O O O

g, Quantification of no. of TdTomato+ve host photoreceptor cells seen following transplantation of live (224 ± 181) versus UV-treated (dead) (3 ± 4) Nrl.Cre+/- P8 photoreceptors (N = 4 eyes per condition). Graph shows mean ± S.D.


i & j , Representative images of depth colour coding ( ImageJ ) of x , y , z live imaging of Nrl . Gfp + / + photoreceptor cultures showing ( i ) neurites ( blue arrows ) growing along the substrate ( asterisks denote secondary branching of long GFP + neurites ) , and in ( j ) , a free - floating PhNT ( magenta arrow ) . Scale bar = 10 µm ;

O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

i & j, Representative images of depth colour coding (ImageJ) of x,y,z live imaging of Nrl.Gfp+/+ photoreceptor cultures showing (i) neurites (blue arrows) growing along the substrate (asterisks denote secondary branching of long GFP+ neurites), and in (j), a free-floating PhNT (magenta arrow). Scale bar = 10 µm;


k & l , Quantification of the localization of Nrl . Gfp + / + processes in the z - axis ( depth ) ( N = 4 independent cultures , n = 1096 cells , nneurites = 440 , nnanotubes = 31 where ( k ) neurites . P values = * * * * p < 0 . 0001 ( bottom vs top ) ; * * p = 0 . 002 ( bottom vs middle ) ; * * p = 0 . 003 ( middle vs top ) and ( l ) PhNTs ; P - values = * * * * p < 0 . 0001 ( bottom vs middle & middle vs top ) . One way ANOVA , non - parametric , Kruskal - Wallis with Dunns ' multiple comparisons test shows means vary significantly .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O 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 & l, Quantification of the localization of Nrl.Gfp+/+ processes in the z-axis (depth) (N = 4 independent cultures, n = 1096 cells, nneurites = 440, nnanotubes = 31 where (k) neurites. P values = **** p < 0.0001 (bottom vs top); **p = 0.002 (bottom vs middle); **p = 0.003 (middle vs top) and (l) PhNTs; P-values = **** p < 0.0001 (bottom vs middle & middle vs top) . One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test shows means vary significantly.


d , representative time lapse images of cGFP mobility between two PhNT - connected Nrl . Gfp + / + photoreceptors ( dashed ellipse = bleached area ) versus an isolated cell ( solid ellipse = bleached area ) and clustered cells ( dot ellipse = bleached area ) . Images show pre - bleach ( t = - 5 ' ' ) , bleach ( t = 0 ' ' ) , and post - bleach ( t = 35 ' ' and 215 ' ' ) . Unbleached reference cells also indicated ( arrows ) ; Green = cytoplasm ; Scale bar = 5µm .

O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O

d, representative time lapse images of cGFP mobility between two PhNT-connected Nrl.Gfp+/+ photoreceptors (dashed ellipse = bleached area) versus an isolated cell (solid ellipse = bleached area) and clustered cells (dot ellipse = bleached area). Images show pre-bleach (t = -5''), bleach (t = 0''), and post-bleach (t = 35'' and 215''). Unbleached reference cells also indicated (arrows); Green = cytoplasm; Scale bar = 5µm.


d , Lysosomes can be exchanged , rarely , between PhNT - connected cells . Lysosomes were labelled with SiR - Lyso and their movements were analysed by TrackMate software . Images show deconvolution of 3D reconstructions of lysosomes ( surface ; red ) and cGFP ( volume ; green ) from time - lapse live imaging series of Nrl . Gfp + / + photoreceptor cultures ( green ) ; the position of a transferred lysosome is marked as1 ( t = 0 ) , 2 ( t = 3 ' ) , 3 ( t = 6 ' ) , 4 ( t = 9 ' ) ; N . B . Deconvolution shows a segment - like process extending from cell " A " and a PhNT connecting cells " A " and " B " ; Scale bar = 2 µm ;

O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O

d, Lysosomes can be exchanged, rarely, between PhNT-connected cells. Lysosomes were labelled with SiR-Lyso and their movements were analysed by TrackMate software. Images show deconvolution of 3D reconstructions of lysosomes (surface; red) and cGFP (volume; green) from time-lapse live imaging series of Nrl.Gfp+/+ photoreceptor cultures (green); the position of a transferred lysosome is marked as1 (t = 0), 2 (t = 3'), 3 (t = 6'), 4 (t = 9'); N.B. Deconvolution shows a segment-like process extending from cell "A" and a PhNT connecting cells "A" and "B"; Scale bar = 2 µm;


e , Mitochondria can be exchanged , rarely , between PhNT - connected cells . Mitochondria movements determined by TrackMate software . Images show deconvolution of 3D reconstructions of mitochondria ( surface ; red ) and cGFP ( volume ; green ) from time - lapse live imaging series ; position of transferred mitochondrion is marked as 1 ( t = 0 ) , 2 ( t = 6 ' ) , 3 ( t = 12 ' ) , 4 ( t = 18 ' ) ; Mitochondria labelled with mito - Tracker - Orange ; Scale bar = 5 µm .

O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O 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-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

e, Mitochondria can be exchanged, rarely, between PhNT-connected cells. Mitochondria movements determined by TrackMate software. Images show deconvolution of 3D reconstructions of mitochondria (surface; red) and cGFP(volume; green) from time-lapse live imaging series; position of transferred mitochondrion is marked as 1 (t = 0), 2 (t = 6'), 3 (t = 12'), 4 (t = 18'); Mitochondria labelled with mito-Tracker-Orange; Scale bar = 5 µm.


a , MIP image of wildtype ( wt ) retina transplanted with P8 Nrl . Gfp + / + CD73 + MACS - enriched photoreceptors ; dashed box indicates ROI subjected to 3D deconvolution and volume reconstruction ( right ) and shows PhNT - like connection between donor and host photoreceptor at the level of host inner segment . Additional images show 3D segmentation of z = 0 and z = 38 focal planes , and reveal the PhNT ; green = GFP , blue = nuclei ; Scale bar = 10 µm in MIP and 5 µm volume reconstruction ; b & c , assessment of properties of PhNT - like processes between donor and host photoreceptors ( n = 19 PhNT - like processes ; N = 10 eyes ) showing ( b ) diameter and ( c ) length ;

O O O O O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O B-TISSUE O O O O O O O O O O O O

a, MIP image of wildtype (wt) retina transplanted with P8 Nrl.Gfp+/+ CD73+ MACS-enriched photoreceptors; dashed box indicates ROI subjected to 3D deconvolution and volume reconstruction (right) and shows PhNT-like connection between donor and host photoreceptor at the level of host inner segment. Additional images show 3D segmentation of z = 0 and z = 38 focal planes, and reveal the PhNT; green = GFP, blue = nuclei; Scale bar = 10 µm in MIP and 5 µm volume reconstruction; b & c, assessment of properties of PhNT-like processes between donor and host photoreceptors (n = 19 PhNT-like processes; N = 10 eyes) showing (b) diameter and (c) length;


d , MIP image of wt retina transplanted with P8 Nrl . Gfp + / + x myrRFP / + photoreceptors ; showing wt host ONL photoreceptor labeled with cGFP ( green ) but not with myrRFP ( red ) ; Scale bar = 50µm ; e , quantification and statistical analysis of material transfer events following subretinal transplantation of live ( cGFP = 3775 ± 804 , myrRFP = 436 ± 145 ) or UV - treated ( cGFP = 1 . 3 ± 2 . 5 , myrRFP = 4 . 3 ± 5 . 7 ) Nrl . Gfp + / + x myr - Rfp + / + photoreceptors into wt hosts ( N = 8 and N = 4 retinae , respectively ) ; One - way ANOVA , parametric , two tail , Tukey ' s multiple comparisons ; Graph shows mean ± S . D .

O O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O B-TISSUE B-CELL_TYPE O O B-GENEPROD O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O B-TISSUE O O O O 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, MIP image of wt retina transplanted with P8 Nrl.Gfp+/+ x myrRFP/+ photoreceptors; showing wt host ONL photoreceptor labeled with cGFP (green) but not with myrRFP (red); Scale bar = 50µm; e, quantification and statistical analysis of material transfer events following subretinal transplantation of live (cGFP = 3775 ± 804, myrRFP = 436 ± 145) or UV-treated (cGFP = 1.3 ± 2.5, myrRFP = 4.3 ± 5.7) Nrl.Gfp+/+x myr-Rfp+/+ photoreceptors into wt hosts (N = 8 and N = 4 retinae, respectively); One-way ANOVA, parametric, two tail, Tukey's multiple comparisons; Graph shows mean ± S.D.


f - h , Assessment of potential for transfer of donor Gfp and Gnat1 mRNA during transplantation using RNAscopeTM . f , Representative MIP images of Gnat1 - / - eyes transplanted with Nrl . Gfp + / + donor cells and processed in situ for Gfp mRNA ( red ) and Gnat1 mRNA ( magenta ) ; blue = nuclei ; ROIs indicated in ( f ) show GFP + ve donor cell mass ( CM ) and GFP + ve cells within Gnat1 - / - host outer nuclear layer ( ONL ) ; g & h , quantification of Gfp and Gnat1 mRNA ( mean fluorescence intensity per cell ) , respectively ( N = 3 retinae ) ; One - way ANOVA , non - parametric , two tailed analysis showed no significant differences in signal intensity for either Gnat1 or Gfp mRNA between GFP + ve and GFP - ve host photoreceptors ;

O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O

f-h, Assessment of potential for transfer of donor Gfp and Gnat1 mRNA during transplantation using RNAscopeTM. f, Representative MIP images of Gnat1-/- eyes transplanted with Nrl.Gfp+/+ donor cells and processed in situ for Gfp mRNA (red) and Gnat1 mRNA (magenta); blue = nuclei; ROIs indicated in (f) show GFP+ve donor cell mass (CM) and GFP+ve cells within Gnat1-/- host outer nuclear layer (ONL); g & h, quantification of Gfp and Gnat1 mRNA (mean fluorescence intensity per cell), respectively (N = 3 retinae); One-way ANOVA, non-parametric, two tailed analysis showed no significant differences in signal intensity for either Gnat1 or Gfp mRNA between GFP+ve and GFP-ve host photoreceptors;


i , Manipulation of actin signalling alters material transfer in vivo : ( far left ) MIP image showing GFP + ve host photoreceptor ( green ) expressing Rod α - transducin ( red ) after transplantation of retinal cells transduced with lenti - CAG - P2A . Gfp ( transduction control ) into Gnat1 - / - recipient ; Scale bar = 10 µm ; adjacent images show Gnat1 - / - eyes transplanted with P2 retinal cells transduced with ( left ) plenti - CAG - P2A . GFP ( green ) , or ( middle ) plenti - CAG - RhoA - P2A . GFP ( green ) , or ( right ) plenti - CAG - ΔΝRac1 - P2A . GFP ( green ) ; green = GFP , blue = nuclei ; Scale bar = 20 µm ; j , quantification of the number of GFP + cells in Gnat1 - / - host ONL , ( N = 5 retinae per condition ) ; plenti - CAG - P2A . GFP ( 2328 ± 234 ) , plenti - CAG - RhoA - P2A . GFP ( 668 ± 481 ) , plenti - CAG - ΔΝRac1 - P2A . GFP ( 547 ± 306 ) ; One - way ANOVA , non - parametric , Dunn ' s multiple comparisons test . Graph shows mean ± SD .

O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O 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, Manipulation of actin signalling alters material transfer in vivo: (far left) MIP image showing GFP+ve host photoreceptor (green) expressing Rod α-transducin (red) after transplantation of retinal cells transduced with lenti-CAG-P2A.Gfp (transduction control) into Gnat1-/- recipient; Scale bar = 10 µm; adjacent images show Gnat1-/- eyes transplanted with P2 retinal cells transduced with (left) plenti-CAG-P2A.GFP (green), or (middle) plenti-CAG-RhoA-P2A.GFP (green), or (right) plenti-CAG-ΔΝRac1-P2A.GFP (green); green = GFP, blue = nuclei; Scale bar = 20 µm; j, quantification of the number of GFP+ cells in Gnat1-/- host ONL, (N = 5 retinae per condition); plenti-CAG-P2A.GFP (2328 ± 234), plenti-CAG-RhoA-P2A.GFP (668 ± 481), plenti-CAG-ΔΝRac1-P2A.GFP (547 ± 306); One-way ANOVA, non-parametric, Dunn's multiple comparisons test. Graph shows mean ± SD.


b , Representative tile scan of un - injected Nrl . Cre + / - / TdTomato + / - chimera showing endogenous recombination ( white ) ; ROIs , as indicated with dashed or dot boxes ; Scale bars = 50 µm ;

O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O 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, Representative tile scan of un-injected Nrl.Cre +/-/TdTomato+/- chimera showing endogenous recombination (white); ROIs, as indicated with dashed or dot boxes; Scale bars = 50 µm;


c & d , ROI from un - injected Nrl . Cre + / - / TdTomato + / - chimera showing spontaneous recombination presenting in mosaic stripe patterning ; Scale bar = 50 µm in c and 10 µm in d ;

O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O 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, ROI from un-injected Nrl.Cre +/-/TdTomato+/- chimera showing spontaneous recombination presenting in mosaic stripe patterning; Scale bar = 50 µm in c and 10 µm in d;


e & f , Representative images of Nrl . Cre + / - / TdTomato + / - chimera after receiving a subretinal injection of AAV - Nrl . Cre . The injected eyes show widespread recombination , which also shows the anticipated mosaic stripe patterning ; Scale bar = 50 µm in ( e ) and 10 µm in ( f ) ;

O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O O 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 & f, Representative images of Nrl.Cre+/-/TdTomato+/- chimera after receiving a subretinal injection of AAV-Nrl.Cre. The injected eyes show widespread recombination, which also shows the anticipated mosaic stripe patterning; Scale bar = 50 µm in (e) and 10 µm in (f);


A , B . Quantification of oryzalin root growth assays in single ( A ) and double ( B ) mutants . DAG = days after germination . Graphs show mean ± SD of three biological replicates with at least 10 plants per genotype per replicate . Asterisks indicate a significant difference in root length in a two - way ANOVA followed by Tukey ' s multiple comparisons test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 and * * * * P < 0 . 0001 ) .

O O O O O O B-SMALL_MOLECULE B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-TISSUE O O 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-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B. Quantification of oryzalin root growth assays in single (A) and double (B) mutants. DAG = days after germination. Graphs show mean ± SD of three biological replicates with at least 10 plants per genotype per replicate. Asterisks indicate a significant difference in root length in a two-way ANOVA followed by Tukey's multiple comparisons test (* P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001).


E . Silique pictures of cycb1 double mutant combinations . White arrowheads indicate aborted ovules and seeds . Scale bars : 500 μm . F , G . Quantification of aborted seeds in single ( F ) and double ( G ) mutants . Graphs represents the average seed abortion rate per plant ± SD of three biological replicates , n = 550 - 1029 seeds analyzed per genotype . Asterisks indicate significant differences in seed abortion rate in an ordinary one - way ANOVA test , followed by a Dunnett ' s multiple comparisons test ( * * * * P < 0 . 0001 ) .

O O B-TISSUE O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O B-TISSUE O O O O O O O O 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. Silique pictures of cycb1 double mutant combinations. White arrowheads indicate aborted ovules and seeds. Scale bars: 500 μm. F, G. Quantification of aborted seeds in single (F) and double (G) mutants. Graphs represents the average seed abortion rate per plant ± SD of three biological replicates, n = 550-1029 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).


C , D . Quantification of endosperm nuclei in cycb1 single ( C ) and double ( D ) mutants . Boxes and whiskers represent min to max values with the median indicated as a central horizontal line , n = 26 - 30 seeds per genotype . Asterisks show significant differences in the number of endosperm nuclei per seed in a Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ( * * * * P < 0 . 0001 ) .

O O O O O O B-TISSUE B-SUBCELLULAR O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O B-TISSUE B-SUBCELLULAR O B-TISSUE O O 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. Quantification of endosperm nuclei in cycb1 single (C) and double (D) mutants. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 26-30 seeds per genotype. Asterisks show significant differences in the number of endosperm nuclei per seed in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (**** P < 0.0001).


A . DIC images of abnormal embryo sacs in cycb1 mutant combinations . Red arrowheads indicate the visible nuclei in Col - 0 embryo sacs ( central and egg cells ) and the corresponding structures in the quadruple cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cyb1 ; 4 - mutant . Scale bars : 20 μm . B . Quantification of the different abnormal embryo sac structures in cycb1 mutant combinations ( n = 202 - 459 embryo sacs per genotype ) . Different letters indicate significant differences in the proportion of abnormal embryo sacs in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons . WT = wildtype .

O O B-EXP_ASSAY O O O B-TISSUE I-TISSUE O O O O O O O O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O

A. DIC images of abnormal embryo sacs in cycb1 mutant combinations. Red arrowheads indicate the visible nuclei in Col-0 embryo sacs (central and egg cells) and the corresponding structures in the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant. Scale bars: 20 μm. B. Quantification of the different abnormal embryo sac structures in cycb1 mutant combinations (n = 202-459 embryo sacs per genotype). Different letters indicate significant differences in the proportion of abnormal embryo sacs in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. WT = wildtype.


C . DIC images of embryo sacs 3 DAP with wildtype pollen ( female x male ) . Red arrowheads indicate the visible embryo sac nuclei in the crosses with the quadruple cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cyb1 ; 4 - mutant as a female donor , while the control Col - 0 x Col - 0 cross exhibits a developing embryo . Scale bars : 20 μm . D . Quantification of seed abortion in different cycb1 mutant combinations . Graph represents the average seed abortion rate per plant ± SD of three biological replicates , n = 463 - 579 seeds analyzed per genotype . Asterisks indicate significant differences in seed abortion rate in an ordinary one - way ANOVA test , followed by a Dunnett ' s multiple comparisons test ( * * * * P < 0 . 0001 ) .

O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O B-TISSUE O O O O O O O O O O O B-TISSUE I-TISSUE B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O 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-TISSUE O O O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY 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-TISSUE O O O O O O O O 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. DIC images of embryo sacs 3 DAP with wildtype pollen (female x male). Red arrowheads indicate the visible embryo sac nuclei in the crosses with the quadruple cycb1;1-/- cycb1;2+/- cycb1;3+/- cyb1;4- mutant as a female donor, while the control Col-0 x Col-0 cross exhibits a developing embryo. Scale bars: 20 μm. D. Quantification of seed abortion in different cycb1 mutant combinations. Graph represents the average seed abortion rate per plant ± SD of three biological replicates, n = 463-579 seeds analyzed per genotype. Asterisks indicate significant differences in seed abortion rate in an ordinary one-way ANOVA test, followed by a Dunnett's multiple comparisons test (**** P < 0.0001).


A , B . DAPI staining of pollen in cycb1 mutants , including pollen configurations found in cycb1 ; 1 - / - cycb1 ; 2 + / - cycb1 ; 3 + / - cycb1 ; 4 - / - mutants ( B ) . Scale bars : 5 μm . C . Quantification of DAPI - stained pollen configurations in different cycb1 mutant combinations , n = 420 - 616 pollen grains per genotype . Different letters indicate significant differences in the proportion of abnormal pollen ( uni - and bicellular ) in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons .

O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O O O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-TISSUE O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O

A, B. DAPI staining of pollen in cycb1 mutants, including pollen configurations found in cycb1;1-/- cycb1;2+/- cycb1;3+/- cycb1;4-/- mutants (B). Scale bars: 5 μm. C. Quantification of DAPI-stained pollen configurations in different cycb1 mutant combinations, n = 420-616 pollen grains per genotype. Different letters indicate significant differences in the proportion of abnormal pollen (uni- and bicellular) in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.


D . Alexander staining of mature pollen indicating pollen viability . Scale bars : 5 μm . E . Quantification of Alexander - stained pollen viability , n = 403 - 498 pollen grains per genotype . Different letters indicate significant differences in the proportion of dead pollen in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons .

O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-TISSUE O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-TISSUE B-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O

D. Alexander staining of mature pollen indicating pollen viability. Scale bars: 5 μm. E. Quantification of Alexander-stained pollen viability, n = 403-498 pollen grains per genotype. Different letters indicate significant differences in the proportion of dead pollen in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons.


A , B , C . Co - immunolocalization against tubulin ( magenta ) and KNOLLE ( green ) in root meristematic cells . Nuclei were counterstained with DAPI for the DNA ( cyan ) . White arrowheads indicate laggards in the metaphase stage . Scale bars : 5 μm .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O

A, B, C. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). White arrowheads indicate laggards in the metaphase stage. Scale bars: 5 μm.


D . Quantification of wildtype ( WT ) , double and misplaced PPBs . Different letters indicate significant differences in the proportions of the different arrays per category in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype . E . Quantification of PPBs with prominent perinuclear microtubules ( MTs ) . Boxes and whiskers represent min to max values with the median indicated as a central horizontal line , n = 10 roots per genotype . Asterisks show significant differences in the percentage of PPBs with prominent perinuclear microtubules per root in an ANOVA test followed by Dunnett ' s multiple comparisons test ( * * P < 0 . 01 and * * * * P < 0 . 0001 ) . F . Quantification of spindles with lagging chromosomes . Boxes and whiskers represent min to max values with the median indicated as a central horizontal line , n = 10 roots per genotype . Asterisks show significant differences in the percentage of spindles with lagging chromosomes per root in a Kruskal - Wallis test followed by Dunn ' s multiple comparisons test ( * * P < 0 . 01 and ns = non - significant ) .

O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O 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-TISSUE O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-TISSUE O O O O O O O O O O O O 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-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O 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-SUBCELLULAR O O B-SUBCELLULAR O B-TISSUE O O O O O O 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. Quantification of wildtype (WT), double and misplaced PPBs. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. E. Quantification of PPBs with prominent perinuclear microtubules (MTs). Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of PPBs with prominent perinuclear microtubules per root in an ANOVA test followed by Dunnett's multiple comparisons test (** P < 0.01 and **** P < 0.0001). F. Quantification of spindles with lagging chromosomes. Boxes and whiskers represent min to max values with the median indicated as a central horizontal line, n = 10 roots per genotype. Asterisks show significant differences in the percentage of spindles with lagging chromosomes per root in a Kruskal-Wallis test followed by Dunn's multiple comparisons test (** P < 0.01 and ns = non-significant).


A . Co - immunolocalization against tubulin ( magenta ) and KNOLLE ( green ) in root meristematic cells . Nuclei were counterstained with DAPI for the DNA ( cyan ) . Scale bars : 5 μm . B . Quantification of wildtype ( WT ) and abnormal phragmoplasts . Different letters indicate significant differences in the proportions of the different arrays per category in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype . C . Quantification of the different mitotic stages in roots of the different genotypes . Different letters indicate significant differences in the proportions of the different arrays per category in a Chi - squared test followed by the Marascuilo procedure to identify significant pairwise comparisons . Ten roots were analyzed per genotype .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O 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-TISSUE O O O O O

A. Co-immunolocalization against tubulin (magenta) and KNOLLE (green) in root meristematic cells. Nuclei were counterstained with DAPI for the DNA (cyan). Scale bars: 5 μm. B. Quantification of wildtype (WT) and abnormal phragmoplasts. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype. C. Quantification of the different mitotic stages in roots of the different genotypes. Different letters indicate significant differences in the proportions of the different arrays per category in a Chi-squared test followed by the Marascuilo procedure to identify significant pairwise comparisons. Ten roots were analyzed per genotype.


A . Kinase assays against Histone H1 . Top and middle panels indicate shorter and longer exposures respectively of the same kinase assays . Bottom panel is a CBB staining of Histone H1 showing equal loading of the protein . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . B . Kinase assays against GIP1 . Top and and middle panels indicate shorter and longer exposures respectively of the same kinase assays . Bottom panel is a CBB staining of GIP1 showing equal loading of the protein . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . C . Western blot against StrepIII - tagged proteins to show loaded amounts of the CDKs . A : CDKA ; 1 , B1 : CDKB1 ; 1 , B2 : CDKB2 ; 2 . Double CDKB2 ; 2 bands are likely due to a truncation of the expressed protein .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O

A. Kinase assays against Histone H1. Top and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of Histone H1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. B. Kinase assays against GIP1. Top and and middle panels indicate shorter and longer exposures respectively of the same kinase assays. Bottom panel is a CBB staining of GIP1 showing equal loading of the protein. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. C. Western blot against StrepIII-tagged proteins to show loaded amounts of the CDKs. A: CDKA;1, B1: CDKB1;1, B2: CDKB2;2. Double CDKB2;2 bands are likely due to a truncation of the expressed protein.


D . Time - lapse of confocal microscope pictures of root meristematic cells tagged with GFP - GIP1 in Col - 0 ( top panel ) and cycb1 ; 1 cycb1 ; 2 ( bottom panel ) . GIP1 localizes at the nuclear polar caps , followed by co - localization with microtubules at the spindle and phragmoplast arrays . In cycb1 ; 1 cycb1 ; 2 double mutants , GIP1 exhibited an abnormal localization , being found at the spindle ( magenta arrowheads ) and phragmoplast ( white arrowheads ) midzones , which are normally devoid of the protein . Scale bars : 5 μm .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

D. Time-lapse of confocal microscope pictures of root meristematic cells tagged with GFP-GIP1 in Col-0 (top panel) and cycb1;1 cycb1;2 (bottom panel). GIP1 localizes at the nuclear polar caps, followed by co-localization with microtubules at the spindle and phragmoplast arrays. In cycb1;1 cycb1;2 double mutants, GIP1 exhibited an abnormal localization, being found at the spindle (magenta arrowheads) and phragmoplast (white arrowheads) midzones, which are normally devoid of the protein. Scale bars: 5 μm.


C Wild - type and tel1 ∆ strains expressing a fully functional Top1 - HA tagged protein were arrested in G1 with α - factor ( αf ) and released into YEPD supplemented with CPT ( 50 μM ) . Western blot with anti - HA antibodies ( top ) and Coomassie staining ( bottom ) of protein extracts prepared at the indicated time points

O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

C Wild-type and tel1∆ strains expressing a fully functional Top1-HA tagged protein were arrested in G1 with α-factor (αf) and released into YEPD supplemented with CPT (50 μM). Western blot with anti-HA antibodies (top) and Coomassie staining (bottom) of protein extracts prepared at the indicated time points


A - C Exponentially growing cell cultures ( exp ) were arrested in G1 with α - factor ( αf ) and released into YEPD with or without CPT ( 50 μM ) . Cell samples were harvested at the indicated time points to evaluat Rad53 phosphorylation by western blot with anti - Rad53 antibodies ( C )

O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O

A-C Exponentially growing cell cultures (exp) were arrested in G1 with α-factor (αf) and released into YEPD with or without CPT (50 μM). Cell samples were harvested at the indicated time points to evaluat Rad53 phosphorylation by western blot with anti-Rad53 antibodies (C)


E , F G1 - arrested cell cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) ( E ) or in YEPD supplemented with CPT ( 10 μM ) with ( + noc ) or without ( - noc ) nocodazole ( F ) . Western blot analysis with anti - Rad53 antibodies

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O

E, F G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM) (E) or in YEPD supplemented with CPT (10 μM) with (+noc) or without (-noc) nocodazole (F). Western blot analysis with anti-Rad53 antibodies


A - C Exponentially growing cell cultures were arrested in G1 with α - factor ( αf ) and released in YEPD supplemented with CPT ( 50 μM ) . ( A ) Western blot analysis with anti - Rad53 antibodies

O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O

A-C Exponentially growing cell cultures were arrested in G1 with α-factor (αf) and released in YEPD supplemented with CPT (50 μM). (A) Western blot analysis with anti-Rad53 antibodies


( B - E ) G1 - arrested cell cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) . Sample collected at the indicated time points were subjected to western blot analysis with anti - Rad53 antibodie

O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O

(B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodie


( B - E ) G1 - arrested cell cultures ( αf ) were released in YEPD supplemented with CPT ( 50 μM ) . Sample collected at the indicated time points were subjected to western blot analysis with anti - Rad53 antibodies ( B , D

O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O

(B-E) G1-arrested cell cultures (αf) were released in YEPD supplemented with CPT (50 μM). Sample collected at the indicated time points were subjected to western blot analysis with anti-Rad53 antibodies (B, D


F , G G1 - arrested cell cultures ( αf ) were released into YEPD supplemented with CPT ( 50 μM ) . Western blot analysis with anti - Rad53 antibodies

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O

F, G G1-arrested cell cultures (αf) were released into YEPD supplemented with CPT (50 μM). Western blot analysis with anti-Rad53 antibodies


A Agarose gel image illustrating PCR amplicons of ltaS ( LBA0447 ) , slpB , and slpX deletions in NCK2187 .

O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O

A Agarose gel image illustrating PCR amplicons of ltaS (LBA0447), slpB, and slpX deletions in NCK2187.


B SDS - PAGE gel of 5 M LiCl ‐ purified S ‐ layer fractions from the parental strains , NCK56 and NCK1909 , NCK2030 ( LTA + , SlpA + , SlpB − , SlpX − ) , and NCK2187 ( LTA − , SlpA + , SlpB − , SlpX − ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O

B SDS-PAGE gel of 5 M LiCl‐purified S‐layer fractions from the parental strains, NCK56 and NCK1909, NCK2030 (LTA+, SlpA+, SlpB−, SlpX−), and NCK2187 (LTA−, SlpA+, SlpB−, SlpX−).


C B6 mice were orally gavaged with 109 CFU erythromycin ‐ resistant NCK56 or NCK2187 . Fecal pellets were collected daily and tested for the presence of erythromycin ‐ resistant strains . n = 3 mice / group . Data are representative of five independent experiments and are shown as mean ± SEM .

O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O B-ORGANISM O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

C B6 mice were orally gavaged with 109 CFU erythromycin‐resistant NCK56 or NCK2187. Fecal pellets were collected daily and tested for the presence of erythromycin‐resistant strains. n = 3 mice/group. Data are representative of five independent experiments and are shown as mean ± SEM.


D ColonicLPL cells were co ‐ cultured with NCK56 or NCK2187 ( 1 : 1 ) , and secreted cytokines were measured in the supernatants . Data are shown as mean ± SEM and are representative of two experiments performed in triplicate . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

O B-TISSUE B-CELL_TYPE O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ColonicLPL cells were co‐cultured with NCK56 or NCK2187 (1:1), and secreted cytokines were measured in the supernatants. Data are shown as mean ± SEM and are representative of two experiments performed in triplicate. *P 0.05, **P 0.01, ***P 0.001.


A B6 mice were orally gavaged with 109CFU NCK56 ( blue ) or NCK2187 ( green ) on days 0 , 3 , 6 , and 9 , or left untreated , and immune responses in the colon analyzed at day 14 by flow cytometry .

O O B-ORGANISM O O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O

A B6 mice were orally gavaged with 109CFU NCK56 (blue) or NCK2187 (green) on days 0, 3, 6, and 9, or left untreated, and immune responses in the colon analyzed at day 14 by flow cytometry.