V . cholerae ΔvrrA ΔmicV strains carrying pBAD - micV , pBAD - vrrA or an empty vector control ( pCtr ) were cultivated to early stationary phase ( OD600 of 1 . 5 ) in LB . Cells were treated with L - arabinose ( 0 . 2 % final conc . ) and RNA samples were collected at the indicated time points after induction . Northern blot analysis was performed to determine VrrA , MicV and ompT levels . 5S rRNA served as loading control . For comparison , RNA samples of a wild - type strain carrying pCtr were collected during various growth phases , which indicated ~ 18 - fold and ~ 7 - fold higher levels of VrrA and MicV expressed from the pBAD plasmids , respectively ( see Source data for quantifications ) .

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

V. cholerae ΔvrrA ΔmicV strains carrying pBAD-micV, pBAD-vrrA or an empty vector control (pCtr) were cultivated to early stationary phase (OD600 of 1.5) in LB. Cells were treated with L-arabinose (0.2% final conc.) and RNA samples were collected at the indicated time points after induction. Northern blot analysis was performed to determine VrrA, MicV and ompT levels. 5S rRNA served as loading control. For comparison, RNA samples of a wild-type strain carrying pCtr were collected during various growth phases, which indicated ~18-fold and ~7-fold higher levels of VrrA and MicV expressed from the pBAD plasmids, respectively (see Source data for quantifications).


V . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to ( B ) : regulated by both

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

V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to (B): regulated by both sRNAs


V . cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids ( x - axis ) and either an empty vector control ( pCtr ) , the pMicV , or the pVrrA plasmid , were cultivated in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 . The target genes were classified according to regulated only by

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

V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by MicV


V. cholerae ΔvrrA ΔmicV strains carrying the indicated reporter plasmids (x-axis) and either an empty vector control (pCtr), the pMicV, or the pVrrA plasmid, were cultivated in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1. The target genes were classified according to regulated only by VrrA


V . cholerae ΔvrrA ΔmicV strains carrying the ompT : : gfp or ompT M1 * : : gfp fusions ( D ) , ushA : : gfp or ushA M1 * : : gfp fusions ( E ) , or lpp : : gfp or lpp M2 * : : gfp fusions ( F ) and an empty vector control ( pCtr ) , the micV expression plasmids ( pMicV , pMicV M1 ) or the vrrA expression plasmids ( pVrrA , pVrrA M2 ) were grown in M9 and GFP fluorescence was measured . M1 and M2 denote the mutations indicated in ( A , B , C ) . Fluorescence of the control strains was set to 1 .

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

V. cholerae ΔvrrA ΔmicV strains carrying the ompT::gfp or ompT M1*::gfp fusions (D), ushA::gfp or ushA M1*::gfp fusions (E), or lpp::gfp or lpp M2*::gfp fusions (F) and an empty vector control (pCtr), the micV expression plasmids (pMicV, pMicV M1) or the vrrA expression plasmids (pVrrA, pVrrA M2) were grown in M9 and GFP fluorescence was measured. M1 and M2 denote the mutations indicated in (A, B, C). Fluorescence of the control strains was set to 1.


V . cholerae ΔrpoE , ΔrpoE ΔvrrA , ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD - rpoE or an empty vector control ( pCtr ) were grown to OD600 of 1 . 5 and L - arabinose ( 0 . 2 % final conc . ) was added . RNA samples were collected at the indicated time points and monitored for ompT ( G ) , ushA ( H ) , or lpp ( I ) levels using qRT - PCR .

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

V. cholerae ΔrpoE, ΔrpoE ΔvrrA, ΔrpoE ΔmicV or ΔrpoE ΔvrrA ΔmicV strains carrying pBAD-rpoE or an empty vector control (pCtr) were grown to OD600 of 1.5 and L-arabinose (0.2% final conc.) was added. RNA samples were collected at the indicated time points and monitored for ompT (G), ushA (H), or lpp (I) levels using qRT-PCR.


V . cholerae ΔvrrA ΔmicV strains carrying the ompT : : 3XFLAG gene and pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and analyzed for MicV , VrrA and RybB expression on Northern blots . OmpT : : 3xFLAG production was tested on Western blots . RNAPα and 5S rRNA served as loading controls for Western and Northern blots , respectively .

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

V. cholerae ΔvrrA ΔmicV strains carrying the ompT::3XFLAG gene and pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to an OD600 of 2.0. RNA and protein samples were collected and analyzed for MicV, VrrA and RybB expression on Northern blots. OmpT::3xFLAG production was tested on Western blots. RNAPα and 5S rRNA served as loading controls for Western and Northern blots, respectively.


E . coli wild - type strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were grown in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and investigated on Northern blots and SDS - PAGE , respectively . For comparison , we included the E . coli insertional mutant strains ompA : : kanR and ompC : : kanR for specific assignment of OmpA and OmpC bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. coli wild-type strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and investigated on Northern blots and SDS-PAGE, respectively. For comparison, we included the E. coli insertional mutant strains ompA::kanR and ompC::kanR for specific assignment of OmpA and OmpC bands.


V . cholerae wild - type and ΔrpoE strains carrying pMicV , pVrrA , pRybB , or an empty vector control ( pCtr ) were cultivated in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

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

V. cholerae wild-type and ΔrpoE strains carrying pMicV, pVrrA, pRybB, or an empty vector control (pCtr) were cultivated in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


V . cholerae wild - type and ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or the sRNA library after consecutive selection experiments ( Sel1 , Sel2 , Sel3 ) were grown in LB to OD600 of 0 . 2 . Cells were treated with ethanol ( 3 . 5 % final conc . ) for 6 hours . Serial dilutions were prepared and spotted onto agar plates . R1 and R2 indicate two independent biological replicates .

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

V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library after consecutive selection experiments (Sel1, Sel2, Sel3) were grown in LB to OD600 of 0.2. Cells were treated with ethanol (3.5% final conc.) for 6 hours. Serial dilutions were prepared and spotted onto agar plates. R1 and R2 indicate two independent biological replicates.


V . cholerae wild - type and ΔrpoE strains carrying an empty vector control ( pCtr ) , pRybB , or the sRNA library before ( input ) or after consecutive ethanol selection experiments ( Sel1 , Sel2 , Sel3 ) were cultivated in LB to OD600 of 2 . 0 . Membrane fractions were analyzed by SDS - PAGE . The indicated bands were analyzed by mass spectrometry .

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

V. cholerae wild-type and ΔrpoE strains carrying an empty vector control (pCtr), pRybB, or the sRNA library before (input) or after consecutive ethanol selection experiments (Sel1, Sel2, Sel3) were cultivated in LB to OD600 of 2.0. Membrane fractions were analyzed by SDS-PAGE. The indicated bands were analyzed by mass spectrometry.


V . cholerae ΔvrrA ΔmicV cells expressing the ompA : : 3xFLAG gene and carrying an empty vector control ( pCtr ) , or plasmids producing the 15 most highly enriched sRNA variants ( sRNA variants 1 - 15 ) were grown in LB to an OD600 of 2 . 0 . RNA and protein samples were collected and tested for sRNA and OmpA : : 3xFLAG expression on Northern and Western blots , respectively ( with RNAPα and 5S rRNA as loading controls ) .

V. cholerae ΔvrrA ΔmicV cells expressing the ompA::3xFLAG gene and carrying an empty vector control (pCtr), or plasmids producing the 15 most highly enriched sRNA variants (sRNA variants 1-15) were grown in LB to an OD600 of 2.0. RNA and protein samples were collected and tested for sRNA and OmpA::3xFLAG expression on Northern and Western blots, respectively (with RNAPα and 5S rRNA as loading controls).


V . cholerae ΔvrrA ΔmicV strains carrying the ompA : : gfp fusion and an empty vector control ( pCtr ) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured . Fluorescence of the control strains was set to 1 .

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

V. cholerae ΔvrrA ΔmicV strains carrying the ompA::gfp fusion and an empty vector control (pCtr) or the enriched sRNA expression plasmids were grown in M9 and GFP fluorescence was measured. Fluorescence of the control strains was set to 1.


V . cholerae wild - type , ΔrpoE , ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

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

V. cholerae wild-type, ΔrpoE, ΔompA or ΔrpoE ΔompA strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.


Macrophages produce little to no inflammatory cytokines when incubated with either ( a ) whole iRBC for 24 h . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( a ) n = 4 ; Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared with

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

Macrophages produce little to no inflammatory cytokines when incubated with either (a) whole iRBC for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC


Macrophages produce little to no inflammatory cytokines when incubated with either ( b ) fresh iRBCL for 24 h . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( b ) n = 5 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared with

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

Macrophages produce little to no inflammatory cytokines when incubated with either (b) fresh iRBCL for 24 h. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (b) n=5 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC


( c ) Macrophages were pre - incubated with IFNγ ( 1000U / ml ) for 18 h before iRBCL stimulation . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( c ) n = 3 Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared with

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Macrophages were pre-incubated with IFNγ (1000U/ml) for 18 h before iRBCL stimulation. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=3 Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared with RBC


( d ) Phase - contrast microscopy of macrophages 24 h after the addition of RBC , iRBC or LPS . Dark hemozoin crystals can be observed within macrophages . Bar is 30 µm .

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

(d) Phase-contrast microscopy of macrophages 24 h after the addition of RBC, iRBC or LPS. Dark hemozoin crystals can be observed within macrophages. Bar is 30 µm.


( e ) Macrophages were incubated with iRBCL 24 h before addition of LPS . Paired t - test was performed to determine statistical significance . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( e ) n = 3 . Asterisks indicate significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) when values were compared RBCL ( e ) .

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

(e) Macrophages were incubated with iRBCL 24 h before addition of LPS. Paired t-test was performed to determine statistical significance. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (e) n=3. Asterisks indicate significance (*p<0.05, **p<0.01 and ***p<0.001) when values were compared RBCL (e).


Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . RBCL , iRBCL , XO were added to macrophages for 24 h . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with RBCL ( a ) n = 7 One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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

Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. RBCL, iRBCL, XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL (a) n=7 One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . , XO , Hypoxanthine ( Hyx ) were added to macrophages for 24 h . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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

Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO, Hypoxanthine (Hyx) were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . XO added to macrophages for 24 h . Febuxostat ( Feb ) was pre - incubated for 30 minutes with XO before addition to macrophages . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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

Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. XO added to macrophages for 24 h. Febuxostat (Feb) was pre-incubated for 30 minutes with XO before addition to macrophages. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h . , XO the anti - oxidants N - acetyl - L - cysteine ( NAC ) and 1 - Thioglycerol ( 1 - TG ) , and heat inactivated ( HI ) XO were added to macrophages for 24 h . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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

Cytokine secretion of macrophages incubated with the indicated stimuli for 24 h. , XO the anti-oxidants N-acetyl-L-cysteine (NAC) and 1-Thioglycerol (1-TG), and heat inactivated (HI) XO were added to macrophages for 24 h. Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with XO Grey asterisks mark comparison with Control n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


Cytokine secretion of PBMC ( e ) incubated with the indicated stimuli for 24 h . RBC , iRBC , RBCL , iRBCL , XO Each symbol represents the value obtained for cells from an independent donor in an independent experiment . Insets show paired samples by donor in two different conditions . Blue asterisks indicate significance when values were compared with RBCL n = 3 . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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

Cytokine secretion of PBMC (e) incubated with the indicated stimuli for 24 h. RBC, iRBC, RBCL, iRBCL, XO Each symbol represents the value obtained for cells from an independent donor in an independent experiment. Insets show paired samples by donor in two different conditions. Blue asterisks indicate significance when values were compared with RBCL n=3. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance (*p<0.05, **p<0.01 and ***p<0.001).


( a ) XO was incubated with macrophages for the indicated times , when wells were washed twice and media was replaced . Supernatants were collected for cytokine determinations 24 h later ( n = 3 ) . Kruskal - Wallis statistical method was used for statistical analysis . Blue asterisks indicate significance when values were compared with time 0 ( * p < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) XO was incubated with macrophages for the indicated times, when wells were washed twice and media was replaced. Supernatants were collected for cytokine determinations 24 h later (n=3). Kruskal-Wallis statistical method was used for statistical analysis. Blue asterisks indicate significance when values were compared with time 0 (*p<0.05).


( b ) Diagram shows experimental set up with macrophages in the bottom of the well and XO added either in the dialysis cassette ( DC , blue ) or the well ( black ) . XO activity panels show that the enzyme did not leak through the DC membrane . Secretion of IL6 and TNF was measured 24 h after addition of XO in each compartment . A representative result of two independent experiments is shown .

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

(b) Diagram shows experimental set up with macrophages in the bottom of the well and XO added either in the dialysis cassette (DC, blue) or the well (black). XO activity panels show that the enzyme did not leak through the DC membrane. Secretion of IL6 and TNF was measured 24 h after addition of XO in each compartment. A representative result of two independent experiments is shown.


( a ) Plasma levels of TNF and IL8 from patients with uncomplicated ( black circles , n = 14 ) or cerebral ( red circles , n = 9 ) malaria correlate with levels of XO - produced ROS detected in each sample . ROS is expressed as fold change over plasma from healthy controls . Uncomplicated malaria , gray circles , cerebral malaria , red circles . Linear regression ( a ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

(a) Plasma levels of TNF and IL8 from patients with uncomplicated (black circles, n=14) or cerebral (red circles, n=9) malaria correlate with levels of XO-produced ROS detected in each sample. ROS is expressed as fold change over plasma from healthy controls. Uncomplicated malaria, gray circles, cerebral malaria, red circles. Linear regression (a) were performed to determine statistical significance (*p<0.05, **p<0.01).


( b ) Levels of XO activity in malaria patients correlate with disease severity UM ( uncomplicated malaria ) , CM ( cerebral malaria ) . Mann - Whitney test ( b ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Levels of XO activity in malaria patients correlate with disease severity UM (uncomplicated malaria), CM (cerebral malaria). Mann-Whitney test (b) were performed to determine statistical significance (*p<0.05, **p<0.01).


( c ) Plasma from malaria patients induce macrophages to secrete cytokines , which are inhibited by a XO specific inhibitor . Plasma from a healthy control ( HP ) and from three patients : P1 ( with cerebral malaria ) , P2 and P3 ( both with uncomplicated malaria ) was pre - incubated for 30 min with febuxostat ( Feb ) or alone at 37ºC before addition to macrophages at 1 : 2 dilution in media for 30 min . Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation . unpaired t - tests ( c ) were performed to determine statistical significance ( * p < 0 . 05 , * * p < 0 . 01 ) .

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(c) Plasma from malaria patients induce macrophages to secrete cytokines, which are inhibited by a XO specific inhibitor. Plasma from a healthy control (HP) and from three patients: P1 (with cerebral malaria), P2 and P3 (both with uncomplicated malaria) was pre-incubated for 30 min with febuxostat (Feb) or alone at 37ºC before addition to macrophages at 1:2 dilution in media for 30 min. Cells were washed and cytokine secretion measured in triplicates after 24 h of incubation. unpaired t-tests (c) were performed to determine statistical significance (*p<0.05, **p<0.01).


Cytokine secretion by macrophages after 24 h incubation with the indicated stimuli . ( b ) IL1β levels after addition of increasing concentration of RBCL or iRBCL ( 1 : 2 , 1 : 4 and 1 : 8 macrophage : iRBCL ) . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance . Asterisks indicate significance when values are compared with XO + RBCL ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( a ) n = 4 ; ( b ) n = 2 Insets show paired samples by donor in two different conditions .

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Cytokine secretion by macrophages after 24 h incubation with the indicated stimuli. (b) IL1β levels after addition of increasing concentration of RBCL or iRBCL (1:2, 1:4 and 1:8 macrophage:iRBCL). One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (a) n=4; (b) n=2 Insets show paired samples by donor in two different conditions.


chemokine secretion by macrophages after 24 h incubation with the indicated stimuli . One - way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance . Asterisks indicate significance when values are compared with XO + RBCL ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) . Each symbol represents the value obtained for cells from an independent donor in an independent experiment . ( c ) n = 4 . Insets show paired samples by donor in two different conditions .

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chemokine secretion by macrophages after 24 h incubation with the indicated stimuli. One-way ANOVA with Tukey test for multiple comparisons was performed to determine statistical significance. Asterisks indicate significance when values are compared with XO+RBCL (*p<0.05, **p<0.01 and ***p<0.001). Each symbol represents the value obtained for cells from an independent donor in an independent experiment. (c) n=4. Insets show paired samples by donor in two different conditions.


( a ) Gene expression analysis of macrophages incubated with the indicated stimuli expressed as fold change over RBCL . Dotted red line indicates 2 - fold increase in RNA expression .

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(a) Gene expression analysis of macrophages incubated with the indicated stimuli expressed as fold change over RBCL. Dotted red line indicates 2-fold increase in RNA expression.


( b ) Western Blot analysis of proIL1β . Relative abundance is the average of 3 independent experiments with different donors .

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(b) Western Blot analysis of proIL1β. Relative abundance is the average of 3 independent experiments with different donors.


( c ) FLICA analysis of activated caspase - 1 . Results are representative of 2 independent experiments with different donors .

(c) FLICA analysis of activated caspase-1. Results are representative of 2 independent experiments with different donors.


( d ) Two distinct siRNAs against NLRP3 and an irrelevant control siRNA were utilized to knock down RNA levels by lipofectamine transfection in macrophages that were later incubated with iRBCL , or RBCL as control , with or without XO , before quantification of secreted IL1β . Results are representative of 2 independent experiments with different donors .

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(d) Two distinct siRNAs against NLRP3 and an irrelevant control siRNA were utilized to knock down RNA levels by lipofectamine transfection in macrophages that were later incubated with iRBCL, or RBCL as control, with or without XO, before quantification of secreted IL1β. Results are representative of 2 independent experiments with different donors.


B . Images of rosettes and seedlings ( 15 DAG ) of Col - 0 , arf7 / arf19 , slr , and gLBD16 - SRDX as well as the inducible lateral - root - less lines pGATA23 : : shy2 - 2 - GR and pGATA23 : : slr1 - GR grown on DMSO control medium or 30 µM Dexamethasone ( DEX ) , Scale bar 1 cm . The rosettes were stained with a Lugol ' s Iodine solution for starch accumulation at the end of the dark period ( representative images of n = 3 biological replicates ) . C . Box plots of starch quantification in rosette tissues at 15 DAG ( n = 4 biological replicates ) . Comparison between samples was performed by one - way ANOVA and post - hoc Tukey HSD Test ( α = 0 . 05 ) ; different letters indicate significant differences .

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

B. Images of rosettes and seedlings (15 DAG) of Col-0, arf7/arf19, slr, and gLBD16-SRDX as well as the inducible lateral-root-less lines pGATA23::shy2-2-GR and pGATA23::slr1-GR grown on DMSO control medium or 30 µM Dexamethasone (DEX), Scale bar 1 cm. The rosettes were stained with a Lugol's Iodine solution for starch accumulation at the end of the dark period (representative images of n=3 biological replicates). C. Box plots of starch quantification in rosette tissues at 15 DAG (n=4 biological replicates). Comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences.


E . Representative confocal sections of calcofluor counterstained ( E , n = 5 biological replicates ) and differential interference contrast ( DIC ) images of GUS - stained root bends ( F , n = 16 biological replicates ) in DR5 : GUS seedlings treated as indicated 48h after gravistimulation . In E , orange arrows indicate the pericycle ( marked in yellow ) . F . Fraction of root bends forming an LRP and showing DR5 GUS staining after treatment with either control or 2D containing agar blocks ( n = 16 biological replicates , ± SE ) .

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E. Representative confocal sections of calcofluor counterstained (E, n=5 biological replicates) and differential interference contrast (DIC) images of GUS-stained root bends (F, n=16 biological replicates) in DR5:GUS seedlings treated as indicated 48h after gravistimulation. In E, orange arrows indicate the pericycle (marked in yellow). F. Fraction of root bends forming an LRP and showing DR5 GUS staining after treatment with either control or 2D containing agar blocks (n=16 biological replicates, ± SE).


A . Representative western blot of root tissues of pUB10 : S6K1 - 3xHA treated by the indicated combination of auxin ( IAA , 10µM ) , sucrose ( Suc , 110mM ) , 2 - deoxy - d - glucose ( 2D , 20mM ) and AZD8055 ( AZD , 10µM ) and probed with anti - S6K1 / 2 , anti - HA or anti - S6K1 - T449P . Blot is one of three biological replicates . B . Quantification of the relative S6K activation . Box plots show three biological replicates , and comparison between samples was performed by one - way ANOVA and post - hoc Tukey HSD Test ( α = 0 . 05 ) ; different letters indicate significant differences .

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

A. Representative western blot of root tissues of pUB10:S6K1-3xHA treated by the indicated combination of auxin (IAA, 10µM), sucrose (Suc, 110mM), 2-deoxy-d-glucose (2D, 20mM) and AZD8055 (AZD, 10µM) and probed with anti-S6K1/2, anti-HA or anti-S6K1-T449P. Blot is one of three biological replicates. B. Quantification of the relative S6K activation. Box plots show three biological replicates, and comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences.


C . Representative DIC images showing RPT1B promoter expression at different stages of LR development in 10 DAG seedlings . Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells . Scale bars : 50 µm . D . Representative confocal section showing S6K1 expression in different stages of LR development in 10 DAG pS6K1 : gS6K1 - CFP seedlings . Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells . Scale bars : 50 µm .

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C. Representative DIC images showing RPT1B promoter expression at different stages of LR development in 10 DAG seedlings. Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells. Scale bars: 50 µm. D. Representative confocal section showing S6K1 expression in different stages of LR development in 10 DAG pS6K1:gS6K1-CFP seedlings. Orange arrowheads indicate the accumulation of reporter signals in the dividing lateral root founder cells. Scale bars: 50 µm.


A - C . Box plots of emerged LR in TOR - oe ( A ) , rpt1b ( B ) , and lst8 ( C ) at 14 DAG . Comparison between samples was performed by one - way ANOVA and post - hoc Tukey HSD Test ( α = 0 . 05 ) ; different letters indicate significant differences . The number of roots scored is indicated .

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A-C. Box plots of emerged LR in TOR-oe (A), rpt1b (B), and lst8 (C) at 14 DAG. Comparison between samples was performed by one-way ANOVA and post-hoc Tukey HSD Test (α = 0.05); different letters indicate significant differences. The number of roots scored is indicated.


H . Fraction of bends developing lateral root primordia and stained for GUS activity in primary root vasculature of UB10pro > > amiR - TOR / DR5 : GUS , n = 18 bends .

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H. Fraction of bends developing lateral root primordia and stained for GUS activity in primary root vasculature of UB10pro>>amiR-TOR/DR5:GUS, n=18 bends.


E , F . Abundance of ARF19 ( E ) and LBD16 ( F ) transcripts in RNAseq samples . mRNA accumulation in response to auxin is comparable for both , whether TOR is knocked down . n = 3 biological replicates .

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E, F. Abundance of ARF19 (E) and LBD16 (F) transcripts in RNAseq samples. mRNA accumulation in response to auxin is comparable for both, whether TOR is knocked down. n=3 biological replicates.


G , H . Relative expression levels ( normalized to ACTIN ) of ARF19 ( G ) and LBD16 ( H ) measured by RT - qPCR upon TOR activity inhibition with AZD8055 . Comparison between samples was performed by one - way ANOVA . Different letters indicate significant differences based on a post - hoc Tukey HSD Test ( n = 5 biological replicates , α = 0 . 05 ) .

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G, H. Relative expression levels (normalized to ACTIN) of ARF19 (G) and LBD16 (H) measured by RT-qPCR upon TOR activity inhibition with AZD8055. Comparison between samples was performed by one-way ANOVA. Different letters indicate significant differences based on a post-hoc Tukey HSD Test (n=5 biological replicates, α = 0.05).


L , M . Total lysates prepared from lateral roots treated or not with IAA and AZD were fractionated through sucrose gradients , and the relative redistribution ( percentage of total ) of ACTIN , ARF7 , ARF19 , and LARP1 mRNAs in every 8 fractions were studied by RT - qPCR analysis . ( L ) Polysome profiles . 40S , small ribosomal subunit ; 60S , large ribosomal subunit ; 80S , mono - ribosome ; polysomes , polyribosomes . AU is arbitrary units of RNA absorbance at A260 nanometers . ( M ) RT - qPCR analysis of mRNA redistribution through a sucrose gradient ( 8 fractions collected ) . Translation efficiency was computed as a percentage of mRNA in non - polysome fractions ( 40 / 60 / 80S ; fractions 1 - 3 ) against both lights ( fractions 4 - 5 ) and heavy polysomes ( fractions 6 - 8 ) . The plot is representative of three independently performed experiments with similar results .

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L, M. Total lysates prepared from lateral roots treated or not with IAA and AZD were fractionated through sucrose gradients, and the relative redistribution (percentage of total) of ACTIN, ARF7, ARF19, and LARP1 mRNAs in every 8 fractions were studied by RT-qPCR analysis. (L) Polysome profiles. 40S, small ribosomal subunit; 60S, large ribosomal subunit; 80S, mono-ribosome; polysomes, polyribosomes. AU is arbitrary units of RNA absorbance at A260 nanometers. (M) RT-qPCR analysis of mRNA redistribution through a sucrose gradient (8 fractions collected). Translation efficiency was computed as a percentage of mRNA in non-polysome fractions (40/60/80S; fractions 1-3) against both lights (fractions 4-5) and heavy polysomes (fractions 6-8). The plot is representative of three independently performed experiments with similar results.


A . Abundance of WOX11 transcripts in RNAseq samples . mRNA accumulation in response to auxin is elevated when TOR is knocked down . n = 3 biological replicates .

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A. Abundance of WOX11 transcripts in RNAseq samples. mRNA accumulation in response to auxin is elevated when TOR is knocked down. n=3 biological replicates.


B . Distribution of GUS - staining in UB10pro > > amiR - TOR / WOX11pro : : GUS seedlings 6 days after transfer to 10µM Est . The proportion of root bends with the depicted phenotype is indicated .

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B. Distribution of GUS-staining in UB10pro>>amiR-TOR/WOX11pro::GUS seedlings 6 days after transfer to 10µM Est. The proportion of root bends with the depicted phenotype is indicated.


C . The arf7 / arf19 mutant shows defective LR initiation . D . Formation of roots ( arrows ) close to the wound site of arf7 / arf19 primary roots 6 days after excision ( DAE ) . E . Blocking TOR - activity via exposure to 10µM AZD8055 ( AZD ) resulted in the loss of rooting capability on the wound site of arf7 / arf19 primary roots at 6 DAE .

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C. The arf7/arf19 mutant shows defective LR initiation. D. Formation of roots (arrows) close to the wound site of arf7/arf19 primary roots 6 days after excision (DAE). E. Blocking TOR-activity via exposure to 10µM AZD8055 (AZD) resulted in the loss of rooting capability on the wound site of arf7/arf19 primary roots at 6 DAE. S


F , G . Close - ups indicate root formation location or absence of root formation on ctrl media and AZD - containing media , respectively , in arf7 / arf19 primary roots at 6 DAE . The proportion of root wound sites with the depicted phenotype is indicated .

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F, G. Close-ups indicate root formation location or absence of root formation on ctrl media and AZD-containing media, respectively, in arf7/arf19 primary roots at 6 DAE. The proportion of root wound sites with the depicted phenotype is indicated.


Growth analysis of wild type Xcc8004 , EPS deficient mutants ( B5 and B12 ) , ndvB and ndvB ( pHMJ ) grown in PS medium ( A ) ; PS medium containing 50 µm extracellularion chelator , diethylenetriaminepentaacetic acid ( DETAPAC ) ( B ) ; or supplemented with either with ( C ) secreted glucan ( 0 . 5 mg / ml ) ; ( D ) xanthan ( 0 . 5 mg / ml ) ; ( E ) FeSO4 ( 40µM ) ; and ( F ) MnCl2 ( 40µM ) . Data shown are mean ± SD ( n = 3 ) . From here onward , until not mentioned , the ' n ' indicates three biological replicates .

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Growth analysis of wild type Xcc8004, EPS deficient mutants (B5 and B12), ndvB and ndvB (pHMJ) grown in PS medium (A); PS medium containing 50 µm extracellularion chelator, diethylenetriaminepentaacetic acid (DETAPAC) (B); or supplemented with either with (C) secreted glucan (0.5 mg/ml); (D) xanthan (0.5 mg/ml); (E) FeSO4 (40µM); and (F) MnCl2 (40µM). Data shown are mean ± SD (n=3). From here onward, until not mentioned, the 'n' indicates three biological replicates.


( A ) MALDI - MS analysis on the 2 . 1 RT eluted fraction from the wild type Xcc8004 EPS . Shown one quasimolecular ion at m / z ( mass - to - charge ratio ) 2632 . 8 which matched with the calculated mass for an [ M + Na ] + ion based on an unsubstituted 16 - member cyclic glucan ( inset ) of Xanthomonas campestris . Peaks with m / z of below 1100 were matrix derived peaks after comparing the MALDI - MS of matrix only control ( See also Fig . EV3A - B ; Fig . EV4D ) .

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(A) MALDI-MS analysis on the 2.1 RT eluted fraction from the wild type Xcc8004 EPS. Shown one quasimolecular ion at m/z (mass-to-charge ratio) 2632.8 which matched with the calculated mass for an [M+Na] + ion based on an unsubstituted 16-member cyclic glucan (inset) of Xanthomonas campestris. Peaks with m/z of below 1100 were matrix derived peaks after comparing the MALDI-MS of matrix only control (See also Fig. EV3A-B; Fig. EV4D).


( B ) Top : GC - MS analysis of acid hydrolyzed and BSTFA - derivatized , 2 . 1 RT eluted fraction from the EPS of wild type Xcc strain . Bottom : matching spectrum from NIST database , corresponding to glucopyranose pentakis - O - trimethyl silyl ( See also Fig . EV3C ) .

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(B) Top: GC-MS analysis of acid hydrolyzed and BSTFA-derivatized, 2.1 RT eluted fraction from the EPS of wild type Xcc strain. Bottom: matching spectrum from NIST database, corresponding to glucopyranose pentakis-O-trimethyl silyl (See also Fig. EV3C).


( C ) Quantification of total EPS , secreted ( S ) and periplasmic ( P ) glucan by phenol - sulfuric acid colorimetric assay from 2 - day - old cultures . Secreted xanthan and glucan was isolated from the cell - free culture supernatant by addition of KCl [ 1 % ( w / v ) ] and 2 volumes of ethanol , to precipitate xanthan . Supernatant containing cyclic - β - ( 1 , 2 ) was purified by BioGel - P4 size - exclusion chromatography . To isolate periplasmic glucan , cell pellet was treated with 1 % trichloroacetic acid ( TCA ) to release periplasmic glucans , purified by BioGel - P4 size - exclusion chromatography . Data shown are mean ± SD ( n = 3 ) .

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(C) Quantification of total EPS, secreted (S) and periplasmic (P) glucan by phenol-sulfuric acid colorimetric assay from 2-day-old cultures. Secreted xanthan and glucan was isolated from the cell-free culture supernatant by addition of KCl [1% (w/v)] and 2 volumes of ethanol, to precipitate xanthan. Supernatant containing cyclic-β-(1, 2) was purified by BioGel-P4 size-exclusion chromatography. To isolate periplasmic glucan, cell pellet was treated with 1% trichloroacetic acid (TCA) to release periplasmic glucans, purified by BioGel-P4 size-exclusion chromatography. Data shown are mean ± SD (n=3).


( A ) Iron ( Fe2 + ) - binding potential of different polysaccharide [ used at 5 . 56 micromole ( glucose equivalent ) ] using the Fe2 + - ferrozine colorimetric assay . Lipopolysacchrides ( LPS ; E . coli 0111 : B4 ) , mannan ( Saccharomyces cerevisiae ) , isolated and commercial ( purchased from Sigma Aldrich ) xanthan , extracellular glucan from Xcc . Data shown are mean ± SD ( n = 3 ) .

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(A) Iron (Fe2+)-binding potential of different polysaccharide [used at 5.56 micromole (glucose equivalent)] using the Fe2+-ferrozine colorimetric assay. Lipopolysacchrides (LPS; E. coli 0111:B4), mannan (Saccharomyces cerevisiae), isolated and commercial (purchased from Sigma Aldrich) xanthan, extracellular glucan from Xcc. Data shown are mean ± SD (n=3).


( B ) Colorimetric iron - binding assay . Glucan from diverse source binds Fe2 + iron . Extracellular ( ECG ) or periplasmic ( PPG ) glucan isolated from the wild type Xcc8004 or the ndvB ( pHM1J ) ; commercial glucan from yeast , barley and Euglena purchased from Sigma Aldrich . Glucans from various sources were incubated with FeSO4 at room temperature for 15 min , amount of free iron was determined using the standard concentration of ferrozine - Fe2 + complex .

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(B) Colorimetric iron-binding assay. Glucan from diverse source binds Fe2+ iron. Extracellular (ECG) or periplasmic (PPG) glucan isolated from the wild type Xcc8004 or the ndvB (pHM1J); commercial glucan from yeast, barley and Euglena purchased from Sigma Aldrich. Glucans from various sources were incubated with FeSO4 at room temperature for 15 min, amount of free iron was determined using the standard concentration of ferrozine-Fe2+ complex.


( C ) Iron - binding measured by Inductively Coupled Plasma - Optical Emission Spectrometry ( ICP - OES ) . Xanthan and various glucans were mixed with 2 mM FeSO4 , dialysed against deionized water before determination of Fe2 + content by ICP - OES . Data shown are mean ± SD ( n ≥ 3 ) . * * indicates p - value < 0 . 01 and * * * indicates p - value < 0 . 001 by paired student t - test .

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

(C) Iron-binding measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Xanthan and various glucans were mixed with 2 mM FeSO4, dialysed against deionized water before determination of Fe2+ content by ICP-OES. Data shown are mean ± SD (n≥3). ** indicates p-value < 0.01 and *** indicates p-value < 0.001 by paired student t-test.


( D ) Specificity of glucan - Fe2 + interaction . Purified Xcc β - ( 1 , 2 ) - glucan was incubated with 100 µM FeSO4 and increasing concentrations of Ca2 + , Co2 + , Ni2 + , Mn2 + , Mg2 + or Zn2 + , and free or unbound iron was measured by Fe2 + - ferrozine colorimetric assay . Data shown are mean ± SD ( n = 3 ) .

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

(D) Specificity of glucan-Fe2+ interaction. Purified Xcc β-(1, 2)-glucan was incubated with 100 µM FeSO4 and increasing concentrations of Ca2+, Co2+, Ni2+, Mn2+, Mg2+ or Zn2+, and free or unbound iron was measured by Fe2+-ferrozine colorimetric assay. Data shown are mean ± SD (n=3).


( A ) Analysis of total iron levels measured by Inductively Coupled Plasma - Optical Emission Spectrometry ( ICP - OES ) . Cells were grown under either rich - media ( PS ) , iron - restricted ( PS + 150 μM DP ) or glucan supplemented iron - restricted media ( PS + 150 μM DP + 0 . 5 mg / ml glucan ) till the late exponential growth phase . Bacterial cells were harvested , washed and then iron content was determined using ICP - OES . Data shown are mean ± SD ( n = 3 ) . * * indicates p value < 0 . 01 determined by paired student t - test ( two tailed with equal variance ) .

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

(A) Analysis of total iron levels measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Cells were grown under either rich-media (PS), iron-restricted (PS + 150 μM DP) or glucan supplemented iron-restricted media (PS + 150 μM DP + 0.5 mg/ml glucan) till the late exponential growth phase. Bacterial cells were harvested, washed and then iron content was determined using ICP-OES. Data shown are mean ± SD (n=3). ** indicates p value < 0.01 determined by paired student t-test (two tailed with equal variance).


( B ) Co - elution of periplasmic glucan and radiolabellediron - glucan complex . Periplasmic glucan was isolated from the 55Fe2 + - loaded cells , and analyzed by BioGel - P4 size - exclusion chromatography . Fractions ( 1 . 5 ml ) were collected at a flow rate of 20 ml / h and assayed for glucan using the phenol - sulphuric acid colorimetric assay , and radioactivity was measured to detect the presence of 55Fe .

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

(B) Co-elution of periplasmic glucan and radiolabellediron-glucan complex. Periplasmic glucan was isolated from the 55Fe2+-loaded cells, and analyzed by BioGel-P4 size-exclusion chromatography. Fractions (1.5 ml) were collected at a flow rate of 20 ml/h and assayed for glucan using the phenol-sulphuric acid colorimetric assay, and radioactivity was measured to detect the presence of 55Fe.


( C ) Iron localization assay using atomic absorption spectroscopy . Two sets of bacterial cells were grown in iron - restricted media ( PS + 150 μM DP ) till late exponential phase . Cold iron ( 50 μM FeSO4 ) was added to the one set of bacterial cultures and incubated for one hour at 28 ° C . Bacterial cells were harvested , washed from the both sets of bacterial cultures . Periplasmic and cytoplasmic fractions were collected , freeze dried , and then iron content in each fraction was determined by Inductively Coupled Plasma - Optical Emission Spectrometry ( ICP - OES ) . Iron content is represented on Y - axis as microgram of iron present per milligram of proteins in respective fractions . Data shown are mean ± S . E . ( n = 3 ) . * indicates p - value < 0 . 05 and * * indicates p - value < 0 . 01 by paired student t - test .

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

(C) Iron localization assay using atomic absorption spectroscopy. Two sets of bacterial cells were grown in iron-restricted media (PS + 150 μM DP) till late exponential phase. Cold iron (50 μM FeSO4) was added to the one set of bacterial cultures and incubated for one hour at 28°C. Bacterial cells were harvested, washed from the both sets of bacterial cultures. Periplasmic and cytoplasmic fractions were collected, freeze dried, and then iron content in each fraction was determined by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Iron content is represented on Y-axis as microgram of iron present per milligram of proteins in respective fractions. Data shown are mean ± S.E. (n = 3). * indicates p-value < 0.05 and ** indicates p-value < 0.01 by paired student t-test.


Xanthomonas campestris : ( A ) wild type Xcc 8004 ; ( B ) B5 ( EPS deficient ) ( C ) B12EPS deficient ) ; ( D ) ndvB ( glucan deficient ) ; and ( E ) ndvB ( pHM1J ) + ( complementing strain ) ; cells were treated in the presence or absence of either FeSO4 , H2O2 or FeSO4 + Dp ( dipyridyl ) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry .

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

Xanthomonas campestris: (A) wild type Xcc 8004; (B) B5 (EPS deficient) (C) B12EPS deficient); (D) ndvB (glucan deficient); and (E) ndvB(pHM1J)+ (complementing strain); cells were treated in the presence or absence of either FeSO4, H2O2 or FeSO4+Dp (dipyridyl) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry.


( A ) Xanthomonas campestris cells treated with FeSO4 , H2O2 and FeSO4 + Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry . Data shown are mean ± S . E . ( n = 3 ) .

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(A) Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry. Data shown are mean ± S.E. (n = 3).


( B ) The percent of ROS positive Xanthomonas campestris cells treated with FeSO4 , H2O2 and FeSO4 + Dp determined by flow cytometry at 3 h after treatment . Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99 % of untreated cells . Data shown are mean ± SE ( n = 3 ) . * * * indicates p - value < 0 . 001 compared with data obtained from iron treated wild type and mutant by paired student t - test .

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

(B) The percent of ROS positive Xanthomonas campestris cells treated with FeSO4, H2O2 and FeSO4+Dp determined by flow cytometry at 3 h after treatment. Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99% of untreated cells. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.


( C ) Iron sensitivity assay . Cells were grown overnight in PS medium and cultures were diluted in fresh media at an density of 1 x 106 cells / ml , treated in the absence or presence of 500 µM FeSO4 . Viability of cultures was measured at the indicated time point by dilution plating . Percent survival was determined as the number of viable cells at each time divided by the number of viable cells before exposure to FeSO4 . Data shown are mean ± SE ( n = 3 ) . * indicates p - value < 0 . 05 and * * indicates p - value < 0 . 01 compared with data obtained from iron treated wild type and mutant by paired student t - test .

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

(C) Iron sensitivity assay. Cells were grown overnight in PS medium and cultures were diluted in fresh media at an density of 1 x 106 cells/ml, treated in the absence or presence of 500 µM FeSO4. Viability of cultures was measured at the indicated time point by dilution plating. Percent survival was determined as the number of viable cells at each time divided by the number of viable cells before exposure to FeSO4. Data shown are mean ± SE (n=3). * indicates p-value < 0.05 and ** indicates p-value < 0.01 compared with data obtained from iron treated wild type and mutant by paired student t-test.


( A ) Wild type E . coli ( MG1655 ) ; ΔopgG ( ΔmdoG ; JW1035 ) and ΔopgH ( ΔmdoH ; JW1037 ) , mutants blocked in periplasmic glucan synthesis were grown in LOS medium , and cells were treated in the presence or absence of either FeSO4 , H2O2 or FeSO4 + DP ( dipyridyl ) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry .

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

(A) Wild type E. coli (MG1655); ΔopgG (ΔmdoG; JW1035) and ΔopgH (ΔmdoH; JW1037), mutants blocked in periplasmic glucan synthesis were grown in LOS medium, and cells were treated in the presence or absence of either FeSO4, H2O2 or FeSO4+DP (dipyridyl) were analyzed for ROS by monitoring DCF fluorescence with flow cytometry.


( B ) The percent of ROS positive E . coli cells treated with FeSO4 , H2O2 and FeSO4 + DP determined by flow cytometry at 3 h after treatment . Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99 % of untreated cells . Data shown are mean ± SE ( n = 3 ) . * * * indicates p - value < 0 . 001 compared with data obtained from iron treated wild type and mutant by paired student t - test .

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

(B) The percent of ROS positive E. coli cells treated with FeSO4, H2O2 and FeSO4+DP determined by flow cytometry at 3 h after treatment. Shown are the mean percentages of ROS positive cells exceeding the fluorescence of 99% of untreated cells. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.


( C ) E . coli cells treated with FeSO4 , H2O2 and FeSO4 + Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry . Data shown are mean ± SE ( n = 3 ) . * * * indicates p - value < 0 . 001 compared with data obtained from iron treated wild type and mutant by paired student t - test .

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

(C) E. coli cells treated with FeSO4, H2O2 and FeSO4+ Dp were analyzed for ROS by monitoring DCF fluorescence with fluorometry. Data shown are mean ± SE (n=3). *** indicates p-value < 0.001 compared with data obtained from iron treated wild type and mutant by paired student t-test.


( D ) Stretching of the PDMS sandwich concomitantly stretches the wing disc inside the microchannel ( top - down view ) ; wing disc resting on a stretching device ( anchor ) and 10 min after stretch ; M = microchannel .

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(D) Stretching of the PDMS sandwich concomitantly stretches the wing disc inside the microchannel (top-down view); wing disc resting on a stretching device (anchor) and 10 min after stretch; M=microchannel.


( E ) zipper RNAi clones ( zip iR , in red ) in Arm : : GFP expressing anchored and stretched third instar wing disc ; lower panel shows color coded cell aspect ratio in anchored and stretched discs ( bright colors indicate high elongation ) ; zip RNAi clone is outlined in white .

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(E) zipper RNAi clones (zip iR, in red) in Arm::GFP expressing anchored and stretched third instar wing disc; lower panel shows color coded cell aspect ratio in anchored and stretched discs (bright colors indicate high elongation); zip RNAi clone is outlined in white.


( F ) Box plot showing distribution of cell aspect ratio for control ( wild - type ) and zipper RNAi cells in anchored and stretched ( 20 min ) wing disc ; median represented by horizontal line , 75th and 25th centiles are represented by top and bottom of the boxes respectively ; n = 8 wing discs .

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(F) Box plot showing distribution of cell aspect ratio for control (wild-type) and zipper RNAi cells in anchored and stretched (20 min) wing disc; median represented by horizontal line, 75th and 25th centiles are represented by top and bottom of the boxes respectively; n=8 wing discs.


( G ) Color - coded aspect ratio in control and zip RNAi cells ( zip RNAi clone outlined in white ) subjected to anchoring , stretching ( 20 min ) and relaxation ( 10 min , post stretch ) .

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(G) Color-coded aspect ratio in control and zip RNAi cells (zip RNAi clone outlined in white) subjected to anchoring, stretching (20 min) and relaxation (10 min, post stretch).


( H ) Box plot showing distribution of cell aspect ratio in anchored , stretched ( 20 min ) and relaxed ( 10 min ) wing disc for control ( wild - type ) and zipper RNAi cells ; median represented by horizontal line ; 75th and 25th centiles are represented by top and bottom of the boxes respectively , n = 3 wing discs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Box plot showing distribution of cell aspect ratio in anchored, stretched (20 min) and relaxed (10 min) wing disc for control (wild-type) and zipper RNAi cells; median represented by horizontal line; 75th and 25th centiles are represented by top and bottom of the boxes respectively, n=3 wing discs.


( I ) zip - RNAi clone in Arm : : GFP wing disc at the beginning ( 5 min ) and at the end ( 40 min ) of stretching experiment . Yellow and red arrows indicate the control and zip RNAi cells respectively .

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(I) zip-RNAi clone in Arm::GFP wing disc at the beginning (5 min) and at the end (40 min) of stretching experiment. Yellow and red arrows indicate the control and zip RNAi cells respectively.


( J ) Change of zip - RNAi aspect ratio ( a . r . ) relative to change in control cells aspect ratio ( a . r . ) in the course of stretch , blue line shows fitted regression ; dotted line represents 95 % confidence bounds of the best fit line ; n = 3 wing discs .

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(J) Change of zip-RNAi aspect ratio (a.r.) relative to change in control cells aspect ratio (a.r.) in the course of stretch, blue line shows fitted regression; dotted line represents 95% confidence bounds of the best fit line; n=3 wing discs.


( A ) Wing disc expressing E - cad : : GFP and Sqh : : mCherry prior to stretch ( anchor ) and after stretch ( 15 min ) . ( A " ) , Insets of discs shown in ( A ) . ( B ) Schematics demonstrating change in Sqh concentration before and after stretch on vertical ( V ) and horizontal ( H )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wing disc expressing E-cad::GFP and Sqh::mCherry prior to stretch (anchor) and after stretch (15 min).
(A"), Insets of discs shown in (A).
(B) Schematics demonstrating change in Sqh concentration before and after stretch on vertical (V) and horizontal (H) junctions


( C ) Quantification of mean fluorescent intensity of E - cad : : GFP and Sqh : : mCherry on horizontal ( H ) junctions relative to the intensity on vertical ( V ) junctions ( referred to as " polarity " ) prior to and after 15 min stretch . The detailed rationale of intensity determination is described in Fig . S3 and Experimental Procedures .

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(C) Quantification of mean fluorescent intensity of E-cad::GFP and Sqh::mCherry on horizontal (H) junctions relative to the intensity on vertical (V) junctions (referred to as "polarity") prior to and after 15 min stretch. The detailed rationale of intensity determination is described in Fig. S3 and Experimental Procedures.


( D - E ) , Quantification of E - cad : : GFP ( D ) and Sqh : : mCherry ( E ) concentration ( i . e . mean intensity per junctional unit area ) prior and after stretch on both horizontal and vertical junctions normalized to starting concentration on unstretched ( anchored ) vertical junctions .

(D-E), Quantification of E-cad::GFP (D) and Sqh::mCherry (E) concentration (i.e. mean intensity per junctional unit area) prior and after stretch on both horizontal and vertical junctions normalized to starting concentration on unstretched (anchored) vertical junctions.


( F ) Quantification of total amount of Sqh : : mCherry and E - cad : : GFP fluorescence on vertical and horizontal junctions in anchored and stretched discs .

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(F) Quantification of total amount of Sqh::mCherry and E-cad::GFP fluorescence on vertical and horizontal junctions in anchored and stretched discs.


( G ) Tissue expressing E - cad : : GFP and Sqh : : mCherry subjected to increasing levels of stretch .

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(G) Tissue expressing E-cad::GFP and Sqh::mCherry subjected to increasing levels of stretch.


( H ) Quantification of Sqh : : mCherry polarity ( as in c ) of experiment described in ( G ) . The level of stretch is measured as a change in tissue strain ( deformation relative to unstretched tissue ) .

(H) Quantification of Sqh::mCherry polarity (as in c) of experiment described in (G). The level of stretch is measured as a change in tissue strain (deformation relative to unstretched tissue).


( I - K ) Sqh : : mCherry polarity emergence as a function of cell aspect ratio change ( I ) , stress ( J ) , stress applied on average cell area of 10 μm2 ( K ) . Yellow asterisks indicate equivalent regions in the tissue . N = 8 wing discs ( A - F ) , N = 3 wing discs ( G - K ) ;

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

(I-K) Sqh::mCherry polarity emergence as a function of cell aspect ratio change (I), stress (J), stress applied on average cell area of 10 μm2 (K). Yellow asterisks indicate equivalent regions in the tissue. N=8 wing discs (A- F), N=3 wing discs (G-K);


( A ) Short ( 15 min ) stretch and relaxation ( 10 min ) of E - cad : : GFP and Sqh : : mCherry expressing discs .

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(A) Short (15 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs.


( B ) Quantification of Sqh : : mCherry polarity , E - cad : : GFP polarity and aspect ratio change of experiment described in ( A ) ; schematics below the graph illustrates quantified changes in cell aspect ratio ( yellow ) and Sqh polarity ( red ) . Statistical test compares Sqh polarity between anchored and stretched or stretched and relaxed disc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (A); schematics below the graph illustrates quantified changes in cell aspect ratio (yellow) and Sqh polarity (red). Statistical test compares Sqh polarity between anchored and stretched or stretched and relaxed disc.


( C ) Long ( 200 min ) stretch and relaxation ( 10 min ) of E - cad : : GFP and Sqh : : mCherry expressing discs .

(C) Long (200 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs.


( D ) Quantification of Sqh : : mCherry polarity , E - cad : : GFP polarity and aspect ratio change of experiment described in ( C ) ; schematics below the graph illustrates quantified changes in aspect ratio ( yellow ) and Sqh polarity ( red ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (C); schematics below the graph illustrates quantified changes in aspect ratio (yellow) and Sqh polarity (red).


( A ) Overview of the E - cad : : GFP disc prior to ( anchor ) and after compression .

(A) Overview of the E-cad::GFP disc prior to (anchor) and after compression.


( B ) E - cad : : GFP and Sqh : : mCherry localization in anchored and compressed wing disc .

(B) E-cad::GFP and Sqh::mCherry localization in anchored and compressed wing disc.


( C ) Laser cuts ( perpendicular to the line of stretch ) across circa 10 cells in E - cad : : GFP expressing wing disc tissue subjected to anchoring , short ( < 20 min ) stretching and short ( < 20 min ) compression . Plot shows increase in distance between recoiling short axis [ μm ] of the fitted ellipse ( see Fig . S5A for details ) against time [ sec . ] ; data is plotted as mean ± S . E . M , n = 7 - 10 discs / condition .

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(C) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) stretching and short (<20 min) compression. Plot shows increase in distance between recoiling short axis [μm] of the fitted ellipse (see Fig. S5A for details) against time [sec.]; data is plotted as mean ± S.E.M, n= 7-10 discs/condition.


( D ) Activated Myosin ( anti - phosphorylated - Sqh ; in red ) and anti - E - cad ( green ) staining in compressed discs . ( D " ) Close up of compression experiment described in ( D ) ; yellow arrow points towards an example of polarized phosphorylated - Sqh cable in compressed discs .

(D) Activated Myosin (anti-phosphorylated-Sqh; in red) and anti-E-cad (green) staining in compressed discs. (D") Close up of compression experiment described in (D); yellow arrow points towards an example of polarized phosphorylated-Sqh cable in compressed discs.


( A ) Laser cuts ( perpendicular to the line of stretch ) across circa 10 cells in E - cad : : GFP expressing wing disc tissue subjected to anchoring , short ( < 20 min ) and long ( > 120 min ) stretching and stretch retraction ( tissue forced back to the original , pre - stretch shape .

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(A) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) and long (>120 min) stretching and stretch retraction (tissue forced back to the original, pre-stretch shape.


( C ) An overlay of a junction prior to ( red ) and 15 . 44 sec . after ablation ( green ) .

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(C) An overlay of a junction prior to (red) and 15.44 sec. after ablation (green).


( E ) Laser cuts ( perpendicular or parallel to the line of stretch ) across circa 10 cells in E - cad : : GFP expressing wing disc tissue subjected to anchoring or short stretching ( < 30 min ) . .

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(E) Laser cuts (perpendicular or parallel to the line of stretch) across circa 10 cells in E-cad::GFP expressing wing disc tissue subjected to anchoring or short stretching (< 30 min)..


( H ) An overlay of anchored and stretched discs prior to ablation ( red ) and 15 . 44 seconds post ablation ( green ) . Inset zooms in on the area next to ablation site ( marked with yellow asterisk ) .

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(H) An overlay of anchored and stretched discs prior to ablation (red) and 15.44 seconds post ablation (green). Inset zooms in on the area next to ablation site (marked with yellow asterisk).


( A ) RokK116a : : Venus localization in anchored , stretched , relaxed third instar imaginal discs .

(A) RokK116a::Venus localization in anchored, stretched, relaxed third instar imaginal discs.


discs . ( B ) Drok2 mitotic clones ( RFP - ) in Sqh : : GFP third instar imaginal disc . Inset demonstrates Sqh : : GFP polarity inside Drok2 clone .

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discs.
(B) Drok2 mitotic clones (RFP-) in Sqh::GFP third instar imaginal disc. Inset demonstrates Sqh::GFP polarity inside Drok2 clone.


( C ) Sqh : : GFP , enGal4 > UAS - rok RNAi , UAS - Act5CRFP discs subjected to stretching ; inset demonstrates Sqh : : GFP polarity in rok RNAi cells ( marked by red .

(C) Sqh::GFP, enGal4>UAS-rok RNAi, UAS-Act5CRFP discs subjected to stretching; inset demonstrates Sqh::GFP polarity in rok RNAi cells (marked by red.


( D ) zip RNAi clones ( labeled as zip iR , indicated in red ) in stretched third instar RokK116a : : Venus discs .

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(D) zip RNAi clones (labeled as zip iR, indicated in red) in stretched third instar RokK116a::Venus discs.


( A ) Discs expressing Sqh : : GFP and enGal4 > UAS - dia RNAi , UAS - Act5c - RFP that were stretched ; UAS - Act5C : : RFP marks dia iR ( posterior side ) in red . ( A " ) Insets comparing Sqh : : GFP polarity in stretchedenGal4 > dia iR , UAS - Act5c - RFP discs ; panel 1 refers to anterior ( ctrl ) side ; panel 2 refers to posterior ( dia iR ) side .

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(A) Discs expressing Sqh::GFP and enGal4>UAS-dia RNAi, UAS-Act5c-RFP that were stretched; UAS-Act5C::RFP marks dia iR (posterior side) in red. (A") Insets comparing Sqh::GFP polarity in stretchedenGal4>dia iR, UAS- Act5c-RFP discs; panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side.


( B ) Tissue marked with E - cad : : GFP and Sqh : : mCherry , expressing dia - RNAi ( dia iR ) with hhGal4 driver and subjected to stretching . Dotted line indicates anterior - posterior ( A - P ) compartment boundary . ( B " ) Inset demonstrates lack of Sqh : : GFP polarity in dia - RNAi cells .

(B) Tissue marked with E-cad::GFP and Sqh::mCherry, expressing dia-RNAi (dia iR) with hhGal4 driver and subjected to stretching. Dotted line indicates anterior-posterior (A-P) compartment boundary. (B") Inset demonstrates lack of Sqh::GFP polarity in dia-RNAi cells.


( C - D ) , Quantification of E - cad : : GFP polarity ( C ) and Sqh : : mCherry polarity ( D ) in experiment described in ( B ) ; polarity is measured as mean fluorescent intensity on horizontal junctions relative to vertical junctions ( see Experimental Procedures for details ) ; n = 4 wing discs .

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(C-D), Quantification of E-cad::GFP polarity (C) and Sqh::mCherry polarity (D) in experiment described in (B); polarity is measured as mean fluorescent intensity on horizontal junctions relative to vertical junctions (see Experimental Procedures for details); n=4 wing discs.


( E ) Stretched discs expressing Sqh : : GFP and enGal4 > UAS - cofilin ( upper panel ) or UAS - AIP1 ( lower panel ) RNAi and UAS - Act5c - RFP ( marks posterior side in red ) . ( E " ) , Insets comparing Sqh : : GFP polarity in stretched discs described in ( E ) ; panel 1 refers to anterior ( ctrl ) side ; panel 2 refers to posterior ( dia iR ) side .

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(E) Stretched discs expressing Sqh::GFP and enGal4>UAS-cofilin (upper panel) or UAS-AIP1 (lower panel) RNAi and UAS-Act5c-RFP (marks posterior side in red).
(E"), Insets comparing Sqh::GFP polarity in stretched discs described in (E); panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side.


( F ) Sqh : : GFP polarity in enGal4 > dia iR , UAS - Act5C : : RFP discs labeled with Phalloidin - 647 ; Inset ( Phall - 647 ) zooms in on cell shape in anterior and posterior disc compartments .

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(F) Sqh::GFP polarity in enGal4> dia iR, UAS-Act5C::RFP discs labeled with Phalloidin-647; Inset (Phall-647) zooms in on cell shape in anterior and posterior disc compartments.


( G ) Discs expressing nubGal4 and Cd8 : : GFP ( control ) or dia iR ( KK ) labeled with DAPI ; ellipse is fitted into the pouch region ( red dotted line ) and aspect ratio determined as long ellipse axis ( b ) divided by short ellipse axis ( a ) ( white lines ) .

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(G) Discs expressing nubGal4 and Cd8::GFP (control) or dia iR (KK) labeled with DAPI; ellipse is fitted into the pouch region (red dotted line) and aspect ratio determined as long ellipse axis (b) divided by short ellipse axis (a) (white lines).


( H ) Box plot showing distribution of wing disc pouch aspect ratios in conditions described in ( G ) , n = 19 ( ctrl ) and 16 ( dia iR ) wing pouches ; horizontal line indicates median .

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(H) Box plot showing distribution of wing disc pouch aspect ratios in conditions described in (G), n=19 (ctrl) and 16 (dia iR) wing pouches; horizontal line indicates median.


Immunofluorescence images of U2OS cells expressing FLAG - HA - FAM134 after 24hrs doxycycline induction stained for HA ( green ) and endogenous calnexin ( CANX ; red ) . Scale bar : 20μm .

Immunofluorescence images of U2OS cells expressing FLAG-HA-FAM134 after 24hrs doxycycline induction stained for HA (green) and endogenous calnexin (CANX; red). Scale bar: 20μm.


Lysates of HEK293T cells transiently transfected with HA - tagged wild - type FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) purified GST , wild type GST - LC3s , GST - GABARAPs , GST - Ubiquitin ( Ub ) , GST - Tetra - Ubiquitin , A representative Ponceau staining is shown .

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Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) purified GST, wild type GST-LC3s, GST-GABARAPs, GST-Ubiquitin (Ub), GST-Tetra-Ubiquitin, A representative Ponceau staining is shown.


Lysates of HEK293T cells transiently transfected with HA - tagged wild - type and wild - type or ∆ LIR mutant FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) A representative Ponceau staining is shown .

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Lysates of HEK293T cells transiently transfected with HA-tagged wild-type and wild-type or ∆LIR mutant FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) A representative Ponceau staining is shown.


Lysates of HEK293T cells transiently transfected with HA - tagged wild - type FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) using GST - LC3B and GST - LC3B F52A - V53A as baits . A representative Ponceau staining is shown .

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Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) using GST-LC3B and GST- LC3B F52A-V53A as baits. A representative Ponceau staining is shown.


Immunofluorescence images of U2OS expressing wild - type or ∆ LIR mutant FLAG - HA - FAM134 proteins after 24hrs doxycycline induction under different conditions ( basal = DMSO , Baf . A1 = 2h 200nM Bafilomycin A1 , EBSS = 2h starvation in EBSS ) Scale bar : 10μm ; staining against FAM134 ( HA ; green ) and endogenous LC3B ( red ) .

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Immunofluorescence images of U2OS expressing wild-type or ∆LIR mutant FLAG-HA-FAM134 proteins after 24hrs doxycycline induction under different conditions (basal=DMSO, Baf.A1=2h 200nM Bafilomycin A1, EBSS=2h starvation in EBSS) Scale bar: 10μm; staining against FAM134 (HA; green) and endogenous LC3B (red).


Small sections of sample pictures of U2OS cells stably expressing the ER - phagy reporter ssRFP - GFP - KDEL . Pictures were obtained by the IncuCyte ® S3 ( 10x ) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time . Scale bar 25µm . Time lapse of representative total image views can be found in Movie EV7 and Movie EV8 .

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Small sections of sample pictures of U2OS cells stably expressing the ER-phagy reporter ssRFP-GFP-KDEL. Pictures were obtained by the IncuCyte® S3 (10x) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time. Scale bar 25µm. Time lapse of representative total image views can be found in Movie EV7 and Movie EV8.


Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction . Autophagy flux was induced and blocked by treatment with Torin1 ( 8hrs ) and Bafilomycin A1 ( Baf . A1 , 8hrs ) , respectively . n =

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Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction. Autophagy flux was induced and blocked by treatment with Torin1 (8hrs) and Bafilomycin A1 (Baf.A1, 8hrs), respectively. n=3


Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts ( MEFs ) .

Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts (MEFs).


Western blot for Fam134 proteins in indicated MEFs , expressing the ER - phagy reporter ssRFP - GFP - KDEL and being reconstituted with the respective wild - type or ∆ LIR mutant Fam134 protein .

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Western blot for Fam134 proteins in indicated MEFs, expressing the ER-phagy reporter ssRFP-GFP-KDEL and being reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein.


Profile plots showing the Log2 LFQ intensity ( G ) and representative immunofluorescence ( H ) of Climp63 ( green ) in wild - type and Fam134 knockout MEFs .

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Profile plots showing the Log2 LFQ intensity (G) and representative immunofluorescence (H) of Climp63 (green) in wild-type and Fam134 knockout MEFs.


Profile plots showing the Log2 LFQ intensity ( I ) and representative immunofluorescence ( J ) for collagen I ( red ) expression in wild - type and Fam134 knockout MEFs . Scale bar : 10μm . Inset scale bar : 5μm . Nuclei were stained with Hoechst 33342 .

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Profile plots showing the Log2 LFQ intensity (I) and representative immunofluorescence (J) for collagen I (red) expression in wild-type and Fam134 knockout MEFs. Scale bar: 10μm. Inset scale bar: 5μm. Nuclei were stained with Hoechst 33342.


Electron microscopy of wild - type and Fam134 single knockout MEFs ( ER = endoplasmic reticulum ) . Scale bar : 500nm .

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Electron microscopy of wild-type and Fam134 single knockout MEFs (ER=endoplasmic reticulum). Scale bar: 500nm.


Immunofluorescence images stained for endogenous Calnexin ( Canx ) or Climp63 in wild - type ( WT ) , Fam134 knockout MEFs , and Fam134 knockout MEFs reconstituted with the respective wild - type or ∆ LIR mutant Fam134 protein . Scale bar : 10μm . Inset scale bar : 5μm .

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Immunofluorescence images stained for endogenous Calnexin (Canx) or Climp63 in wild-type (WT), Fam134 knockout MEFs, and Fam134 knockout MEFs reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. Scale bar: 10μm. Inset scale bar: 5μm.


Representative Western blots of total cell lysate derived from MEFs used in ( B ) and detected for endogenous Fam134 and Actin , respectively .

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Representative Western blots of total cell lysate derived from MEFs used in (B) and detected for endogenous Fam134 and Actin, respectively.


Representative Western Blot of Collagen I in wild - type MEFs and Fam134 knockout MEFs reconstituted for the respective wild - type ( WT ) or ∆ LIR mutant Fam134 protein . Vinculin has been used as reference for ratio calculation . The statistical significance was estimated by unpaired Student ' s t test . All data are represented as mean ± s . e . m . of n = 3 biological experiments and the statistical significance is defined as * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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Representative Western Blot of Collagen I in wild-type MEFs and Fam134 knockout MEFs reconstituted for the respective wild-type (WT) or ∆LIR mutant Fam134 protein. Vinculin has been used as reference for ratio calculation. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.


Representative Western Blot ( D ) and the relative bar plot ( E ) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆ LIR Fam134a protein .

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Representative Western Blot (D) and the relative bar plot (E) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆LIR Fam134a protein.


Representative Western Blot ( F ) and the relative bar plot ( G ) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c . Representative Western Blot ( H ) and the relative bar plot ( I ) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b .

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Representative Western Blot (F) and the relative bar plot (G) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c. Representative Western Blot (H) and the relative bar plot (I) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b.


Representative Western Blot ( J ) and the relative bar plot ( K ) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b . Representative Western Blot ( L ) and the relative bar plot ( M ) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c .

Representative Western Blot (J) and the relative bar plot (K) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134b. Representative Western Blot (L) and the relative bar plot (M) showing collagen expression in Fam134a knockout MEFs overexpressing wild type Fam134c.


( A ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 12 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 16 ) mice ( age 9 - 12 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles . Mice were monitored for up to 200 days post - treatment with tamoxifen . Data are presented as % survival post - treatment with tamoxifen and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test ; * * * * p < 0 . 0001 .

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(A) Bcl-xfl/fl;RosaCreERT2+/Ki (n=12) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=16) mice (age 9-12 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. Mice were monitored for up to 200 days post-treatment with tamoxifen. Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.