E Representative sections of brains from vehicle DEBIO - 0932 treated mice in ( B - D ) . dotted lines surround metastases ( GFP + ) . Representative field of view of metastasis stained with GFP caspase 3 . Scale bars : slices , 1mm ; caspase 3 , 50 µm . F of established metastases found in vehicle DEBIO - 0932 treated brains from panel ( E ) . Values are shown in box - - whisker plots where every dot represents a different brain in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( vehicle : n = 10 brains ; DEBIO - 0932 : n = 14 brains ) . P value was calculated using two - t - . G of number of caspase 3 ( CC3 + ) in cancer found in vehicle DEBIO - 0932 treated brains from panel ( E ) . Values are shown in box - - whisker plots where every dot is a metastatic lesion in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( n = 8 metastatic lesions from 4 brains per condition ) . P value was calculated using two - t - .

O O O O O O 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 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 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 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

E Representative sections of brains from vehicle and DEBIO-0932 treated mice in (B-D). The dotted lines surround the metastases (GFP+). Representative field of view of metastasis stained with GFP and cleaved caspase 3. Scale bars: slices,1mm; cleaved caspase 3, 50 µm. F Quantification of established metastases found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot represents a different brain and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (vehicle: n=10 brains; DEBIO-0932: n=14 brains). P value was calculated using two-tailed t-test. G Quantification of number of cleaved caspase 3 (CC3+) in cancer cells found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 metastatic lesions from 4 brains per condition). P value was calculated using two-tailed t-test.


I Representative BrM - PDOC stained with proliferation markers ( BrdU ) markers of microenvironment ( GFAP for astrocytes , Iba1 for microglia / macrophages ) . Scale bar : 50 µm .

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I Representative BrM-PDOC stained with proliferation markers (BrdU) and markers of the microenvironment (GFAP for astrocytes, Iba1 for microglia/ macrophages). Scale bar: 50 µm.


F qRT - PCR of H2030 - BrM brain metastases obtained from during neurosurgery compared to relapsed metastases from corresponding . A panel of five genes related to HSP90 pathway is evaluated . Values are shown in box - - whisker plots where every dot represents a different in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( n = 5 mice per experimental condition ) . P value was calculated using two - t - .

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

F qRT-PCR of H2030-BrM brain metastases obtained from animals during neurosurgery compared to relapsed metastases from the corresponding animals. A panel of five genes related to HSP90 pathway is evaluated. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=5 mice per experimental condition). P value was calculated using two-tailed t-test.


H Representative of vehicle DEBIO - 0932 treated mice after neurosurgery until experimental endpoint at 6 8 for vehicle DEBIO - 0932 treated mice , respectively .

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H Representative images of vehicle and DEBIO-0932 treated mice after neurosurgery until experimental endpoint at 6 and 8 weeks for vehicle and DEBIO-0932 treated mice, respectively.


C Volcano plot with deregulated ( red : upregulated ; green : downregulated ) found in brain metastases treated with DEBIO - 0932 compared to DMSO ( n = 3 biological replicates ( mice ) per condition , n ≥ 12 brain metastases per mouse were pooled together ) . with a P value < 0 . 05 a log2 > 1 or < - 1 were defined as deregulated . Gray dotted lines indicate P value log2 cut offs . names of top deregulated are shown .

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C Volcano plot with deregulated proteins (red: upregulated; green: downregulated) found in brain metastases treated with DEBIO-0932 compared to DMSO (n=3 biological replicates (mice) per condition, n≥12 brain metastases per mouse were pooled together). Proteins with a P value <0.05 and a log2 ratio >1 or <-1 were defined as deregulated. Gray dotted lines indicate P value and log2 ratio cut offs. The names of the top deregulated proteins are shown.


G Kaplan - Meier curves showing significant correlation between worse survival post - brain metastasis high expression of HSP90 signature ( AHR , DDA1 , UBE4B , GPATCH8 ) in a cohort of 45 breast cancer brain metastasis .

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G Kaplan-Meier curves showing significant correlation between worse survival post-brain metastasis and high expression levels of the HSP90 signature (AHR, DDA1, UBE4B, GPATCH8) in a cohort of 45 breast cancer brain metastasis patients.


H Representative ( selected cases obtained from Fig EV6M ) histological score of AHR , DDA1 UBE4B in human brain metastases ( n = 16 ) according to signal of corresponding in cancer .

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H Representative images (selected cases obtained from Fig EV6M) and histological score of AHR, DDA1 and UBE4B in human brain metastases (n=16) according to the signal intensity of the corresponding protein in cancer cells.


K of ex vivo BLI of brains thoracic regions at endpoint of experiment . Values are shown in box - - whisker plots where every dot represents a different in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( n = 9 shControl mice n = 10 shAHR # 1 mice ) . P value was calculated using two - t - .

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

K Quantification of ex vivo BLI of brains and thoracic regions at the endpoint of the experiment. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=9 shControl mice and n=10 shAHR#1 mice). P value was calculated using two-tailed t-test.


N Representative of tumors with high ( red dot ) or low ( grey dot ) UBE4B . A few cases of matched - metastases allowed to evaluate HSP90 - dependent . Scale bar : : 100 µm .

N Representative images of primary tumors with high (red dot) or low (grey dot) UBE4B levels. A few cases of matched primary-metastases allowed to evaluate the HSP90-dependent protein. Scale bar: : 100 µm.


P Kaplan - Meier curve comparing relapse - free survival of tumors with high low values of UBE4B . P value was calculated using log - rank ( Mantel - Cox ) .

P Kaplan-Meier curve comparing relapse-free survival of primary tumors with high and low values of UBE4B. P value was calculated using log-rank (Mantel-Cox) test.


D Representative organotypic cultures from experiment in panel ( C ) . RFP is an internal control probe labelling cancer independent of autophagy flux GFP indicate GFP - LC3 + . dotted in upper panel delimits a high magnification shown in lower panel respect to GFP signal derived from GFP - LC3 accumulation . Dotted lines in lower panel surround cancer . Asterisk labels in magnified in high magnification panel showing GFP - LC3 + . Scale bar : low magnificantion , 25 µm ; high magnification ( ) , 10 µm ; high magnification ( ) , 2 . 5 µm . E of GFP - LC3 + vesicles per of experiment in panel ( C ) . Values are shown in box - - whisker plots where every dot represents a field of view of an organotypic culture in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( DMSO : n = 15 fields of view , 2232 cancer from 3 organotypic cultures ; DEBIO - 0932 : n = 20 fields of view , 3260 cancer from 4 organotypic cultures ) . P value was calculated using two - t - .

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

D Representative organotypic cultures from the experiment in panel (C). RFP is an internal control probe labelling cancer cells independent of autophagy flux and GFP indicate GFP-LC3+ puncta. The dotted line in the upper panel delimits a high magnification area shown in the lower panel respect to the GFP signal derived from GFP-LC3 accumulation. Dotted lines in lower panel surround individual cancer cells. Asterisk labels the area in the cell magnified in the high magnification panel showing the GFP-LC3+ puncta. Scale bar: low magnificantion, 25 µm; high magnification (cells), 10 µm; high magnification (puncta), 2.5 µm. E Quantification of GFP-LC3+ vesicles per cell of the experiment in panel (C). Values are shown in box-and-whisker plots where every dot represents a field of view of an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (DMSO: n=15 fields of view, 2232 cancer cells from 3 organotypic cultures; DEBIO-0932: n=20 fields of view, 3260 cancer cells from 4 organotypic cultures). P value was calculated using two-tailed t-test.


Growth of wild type Xcc8004 , EPS deficient mutants ( B5 B12 ) , ndvB 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 ) ; ( F ) MnCl2 ( 40µM ) . Data shown are mean ± SD ( n = 3 ) . From here onward , until not mentioned , ' 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 on 2 . 1 RT eluted fraction from wild type Xcc8004 EPS . Shown one quasimolecular ion at m / z ( mass - to - charge ) 2632 . 8 which matched with calculated mass for an [ M + Na ] + ion on an unsubstituted 16 - member cyclic glucan ( inset ) of Xanthomonas campestris . Peaks with m / z of below 1100 were matrix derived peaks after comparing 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 of acid hydrolyzed BSTFA - derivatized , 2 . 1 RT eluted fraction from EPS of wild type Xcc . Bottom : matching spectrum from NIST database , corresponding to glucopyranose pentakis - O - trimethyl silyl ( See also Fig . EV3C ) .

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

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

O O O O O B-MEASURED_VAR 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 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 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 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

(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 Fe2 + - ferrozine colorimetric . Lipopolysacchrides ( LPS ; E . coli 0111 : B4 ) , mannan ( Saccharomyces cerevisiae ) , isolated commercial ( purchased from Sigma Aldrich ) xanthan , extracellular glucan from Xcc . Data shown are mean ± SD ( n = 3 ) .

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

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

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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 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 * * * indicates p - value < 0 . 001 by paired student t - .

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(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 increasing concentrations of Ca2 + , Co2 + , Ni2 + , Mn2 + , Mg2 + or Zn2 + , free or unbound iron was measured by Fe2 + - ferrozine colorimetric . 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 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 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

(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 ) of total iron measured by Inductively Coupled Plasma - Optical Emission Spectrometry ( ICP - OES ) . 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 late exponential growth phase . were harvested , washed 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 - ( two with equal variance ) .

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 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 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

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

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(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 using atomic absorption spectroscopy . Two sets of were grown in iron - restricted media ( PS + 150 μM DP ) till late exponential phase . Cold iron ( 50 μM FeSO4 ) was added to one set of cultures incubated for one hour at 28 ° C . were harvested , washed from both sets of cultures . Periplasmic fractions were collected , freeze dried , 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 in respective fractions . Data shown are mean ± S . E . ( n = 3 ) . * indicates p - value < 0 . 05 * * indicates p - value < 0 . 01 by paired student t - .

O O O B-MEASURED_VAR 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 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

(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 ) ; ( E ) ndvB ( pHM1J ) + ( complementing ) ; were treated in presence or absence of either FeSO4 , H2O2 or FeSO4 + Dp ( dipyridyl ) were analyzed for ROS by monitoring DCF with flow cytometry .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-CONTROLLED_VAR 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 ) ΔopgH ( ΔmdoH ; JW1037 ) , mutants blocked in periplasmic glucan synthesis were grown in LOS medium , were treated in presence or absence of either FeSO4 , H2O2 or FeSO4 + DP ( dipyridyl ) were analyzed for ROS by monitoring DCF 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 B-CONTROLLED_VAR 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

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

O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 treated with FeSO4 , H2O2 FeSO4 + Dp were analyzed for ROS by monitoring DCF with fluorometry . Data shown are mean ± SE ( n = 3 ) . * * * indicates p - value < 0 . 001 compared with data obtained from iron treated wild type mutant by paired student t - .

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


200 µg of yeast extract in different buffer compositions was processed by R2 - P2 followed by DDA - MS peptide . Sample preparation was performed in triplicate . Mean of unique phosphopeptides are shown for different conditions ( n = 3 ) . Fraction of phosphopeptides with 0 , 1 or 2 missed cleavage sites . mean is displayed ( n = 3 ) . Boxplot depicting phosphopeptide MS1 signal distributions . Boxplot depicting coefficients of variation ( CVs ) distributions for phosphopeptide MS1 signal intensities ( n = 3 ) .

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 B-MEASURED_VAR 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 B-MEASURED_VAR 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

200 µg of yeast protein extract in different buffer compositions was processed by R2-P2 followed by DDA-MS peptide analysis. Sample preparation was performed in triplicate. Mean count of unique phosphopeptides are shown for the different conditions (n=3).   Fraction of phosphopeptides with 0, 1 or 2 missed cleavage sites. The mean is displayed (n=3).   Boxplot depicting phosphopeptide MS1 signal intensity distributions.   Boxplot depicting coefficients of variation (CVs) distributions for phosphopeptide MS1 signal intensities (n=3).


Different amounts ( 25 µg , 50 µg , 100 µg , 200 µg 400 µg ) of yeast extract were processed by R2 - P2 or by in - solution digestion reversed phase C18 SPE desalting , phospho - enriched by robotic Fe3 + - IMAC followed by peptide with DDA - MS . Sample preparation was performed in triplicate . Boxplot depicting MS1 signal distributions for phosphopeptides detected in samples .

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

Different amounts (25 µg, 50 µg, 100 µg, 200 µg and 400 µg) of yeast protein extract were processed by R2-P2 or by in-solution digestion and reversed phase C18 SPE desalting, phospho-enriched by robotic Fe3+-IMAC followed by peptide analysis with DDA-MS. Sample preparation was performed in triplicate. Boxplot depicting MS1 signal intensity distributions for phosphopeptides detected in all samples.


plot of log2 phosphopeptide versus input amounts with each representing an phosphopeptide . For every phosphopeptide a linear regression was performed lines are colored according to slope of linear regression . Black represents linear regression of median phosphopeptide intensities corresponding function is shown on top . Boxplot depicting distribution of slope values resulting from linear regressions of phosphopeptides shown in B . Boxplot depicting distribution of r squared values resulting from linear regressions of phosphopeptides shown in B .

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

Line plot of log2 phosphopeptide intensity versus protein input amounts with each line representing an individual phosphopeptide. For every phosphopeptide a linear regression was performed and lines are colored according to the slope of the linear regression. Black line represents the linear regression of the median phosphopeptide intensities and the corresponding function is shown on top.   Boxplot depicting the distribution of slope values resulting from linear regressions of individual phosphopeptides shown in B.  Boxplot depicting the distribution of r squared values resulting from linear regressions of individual phosphopeptides shown in B.


Average number of unique peptides identified for R2 - P1 performed on 5 different days ( n = 5 ) . Average number of unique phosphopeptides identified for R2 - P2 performed on 5 different days ( n = 5 ) . Boxplot depicting CVs of peptide MS1 signal intensities for reproducibility of R2 - P1 within same batch ( n = 5 ) between different batches conducted on different days ( n = 5 ) . For each peptide , one replicate per batch was randomly chosen for inter - batch comparisons . Boxplot depicting CVs of phosphopeptide MS1 signal intensities for reproducibility of R2 - P2 within same batch ( n = 5 ) between different batches conducted on different days ( n = 5 ) . For each peptide , one replicate per batch was randomly chosen for inter - batch comparisons .

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

Average number of unique peptides identified for R2-P1 performed on 5 different days (n=5).   Average number of unique phosphopeptides identified for R2-P2 performed on 5 different days (n=5).   Boxplot depicting CVs of peptide MS1 signal intensities for reproducibility analysis of R2-P1 within the same batch (n=5) and between different batches conducted on different days (n=5). For each peptide, one replicate per batch was randomly chosen for inter-batch comparisons.   Boxplot depicting CVs of phosphopeptide MS1 signal intensities for reproducibility analysis of R2-P2 within the same batch (n=5) and between different batches conducted on different days (n=5). For each peptide, one replicate per batch was randomly chosen for inter-batch comparisons.


250 µg of yeast extract was processed by R2 - P2 using either Fe3 + - IMAC , Ti4 + - IMAC , Zr4 + - IMAC or TiO2 magnetic beads followed by peptide with DDA - MS . Phosphopeptide enrichment was performed in triplicate . Average number of unique phosphopeptides identified by different enrichments ( n = 3 ) . Phosphopeptide enrichment efficiency shown as fraction of phosphorylated peptides over total peptides ( n = 3 ) . Fraction of phosphopeptides with 1 , 2 or 3 phosphorylation sites ( n = 3 ) .

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

250 µg of yeast protein extract was processed by R2-P2 using either Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic beads and followed by peptide analysis with DDA-MS. Phosphopeptide enrichment was performed in triplicate.   Average number of unique phosphopeptides identified by the different enrichments (n=3).   Phosphopeptide enrichment efficiency shown as the fraction of phosphorylated peptides over total peptides (n=3).   Fraction of phosphopeptides with 1, 2 or 3 phosphorylation sites (n=3).


250 µg of yeast extract was processed by R2 - P2 using either Fe3 + - IMAC , Ti4 + - IMAC , Zr4 + - IMAC or TiO2 magnetic beads followed by peptide with DDA - MS . Phosphopeptide enrichment was performed in triplicate . Boxplot depicting distributions of phosphopeptide MS1 signals ( n = 3 ) . Boxplot depicting distributions of phosphopeptide MS1 signal CVs ( n = 3 ) .

O O O O O O O O O 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 B-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

250 µg of yeast protein extract was processed by R2-P2 using either Fe3+-IMAC, Ti4+-IMAC, Zr4+-IMAC or TiO2 magnetic beads and followed by peptide analysis with DDA-MS. Phosphopeptide enrichment was performed in triplicate. Boxplot depicting distributions of phosphopeptide MS1 signals (n=3).   Boxplot depicting distributions of phosphopeptide MS1 signal CVs (n=3).


Unique phosphopeptides present in at least two ( out of three ) replicates identified in DIA - MS experiment . Boxplot depicting distributions of Pearson correlations for biological replicates different treatments for different time points . P - values are calculated by Wilcoxon rank - sum . Significantly regulated phosphopeptide isoforms for different stimulation time points compared to corresponding untreated control as determined by a two sample t - ( permutation - FDR < 0 . 05 , fold change > 1 . 5 ) . Only phosphopeptide isoforms with phosphosites localized were considered .

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 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 B-MEASURED_VAR O O O O O O O

Unique phosphopeptides present in at least two (out of three) replicates identified in the DIA-MS experiment.   Boxplot depicting distributions of Pearson correlations for biological replicates and different treatments for different time points. P-values are calculated by Wilcoxon rank-sum test.   Significantly regulated phosphopeptide isoforms for the different stimulation and time points compared to the corresponding untreated control as determined by a two sample t-test (permutation-based FDR < 0.05, and fold change > 1.5). Only phosphopeptide isoforms with all phosphosites localized were considered.


Principal component ( PCA ) of DIA - MS phosphoproteomic data for different stimuli separated by time points . Hierarchical clustering of z - scored values of significantly regulated ( ANOVA , permutation - FDR < 0 . 05 ) phosphopeptides of different time points . Column clustering hierarchy is indicated at top . Row clusters for term enrichment are color coded indicated on right .

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

Principal component analysis (PCA) of the DIA-MS phosphoproteomic data for the different stimuli separated by time points. Hierarchical clustering analysis of z-scored intensity values of significantly regulated (ANOVA, permutation-based FDR <0.05) phosphopeptides of the different time points. Column clustering hierarchy is indicated at the top. Row clusters for term enrichment analysis are color coded and indicated on the right.


A ) Summary of computationally identified circuit using a constraint of maximum three inputs . Shown are input miRNAs predicted circuit output . Expression of identified miRNAs inputs are given as fold change over pre - set input abundance threshold ( t ) of total miRNA pool ( where t = 1 % ) ( left ) . Calculated circuit output are given as mol / ( middle ) logic connectivity of identified miRNA is depicted ( right ) . EB : embryoid bodies , MPC : mesenchymal progenitor , NPC : neural progenitor , R - NSC : neural rosettes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Summary of computationally identified circuit using a constraint of maximum three inputs. Shown are input miRNAs and predicted circuit output levels. Expression levels of identified miRNAs inputs are given as fold change over the pre-set input abundance threshold (t) of the total miRNA pool (where t = 1%) (left). Calculated circuit output levels are given as mol / cell (middle) and logic connectivity of the identified miRNA is depicted (right). EB: embryoid bodies, MPC: mesenchymal progenitor cells, NPC: neural progenitor cells, R-NSC: neural rosettes.


C ) Bar chart showing relative DsRed expression of sensors containing indicated miRNA target sites as four fully complementary repeats ( discrete miRNA sensing ) . control vector ( n . t . ) does not contain any target sites . Two - unpaired t - were performed to compare definitive endoderm derived from H1 ( H1 DE ) to H1 control vectors . For comparison to H1 , data were normalized with control vector ( nt ) before performing t - . * Pvalue = 0 . 011 * * P - value = 0 . 006 are both considered very significant . Representative scatterplots of HES - 2 are shown on top . Data information : Each bar in C corresponds to mean + / - s . d . from at least three 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar chart showing relative DsRed expression of sensors containing the indicated miRNA target sites as four fully complementary repeats (discrete miRNA sensing). The control vector (n.t.) does not contain any target sites. Two-tailed unpaired t-tests were performed to compare the definitive endoderm derived from H1 (H1 DE) to H1 and the control vectors. For comparison to H1, data were normalized with the control vector (nt) before performing the t-test. *Pvalue = 0.011 and ** P- value = 0.006 are both considered very significant. Representative scatterplots of HES-2 are shown on top. Data information: Each bar in C corresponds to mean +/- s.d. from at least three biological replicates.


D ) Bar chart showing relative DsRed expression of sensors containing different mutated target of miR - 17 Additionally , a sensor containing no target ( n . t . ) two wildtype ( WT ) T17 target sites with 1x 4x repeats were tested . Two - unpaired t - were performed to compare H1 HES - 2 for each sensor . * P - values < 0 . 01 , were considered very significant ( * * ) , < 0 . 02 significant ( * ) > 0 . 5 not significant . . Representative scatterplots of HES - 2 are shown on top . Data information : Each bar in D corresponds to mean + / - s . d . from at least three 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 O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bar chart showing relative DsRed expression of sensors containing different mutated target sequence of miR-17 Additionally, a sensor containing no target (n.t.) and two wildtype (WT) T17 target sites with 1x and 4x repeats were tested. Two-tailed unpaired t-tests were performed to compare H1 and HES-2 for each sensor. *P-values < 0.01, were considered very significant (**), < 0.02 significant (*) and > 0.5 not significant.. Representative scatterplots of HES-2 are shown on top. Data information: Each bar in D corresponds to mean +/- s.d. from at least three biological replicates.


C ) Combinatorial screen . Computational combinatorial screening of Act1MAX Act2MAX using model depicted in A ( left ) . Experimental combinatorial screening in H1 ( middle ) with circuit schematic on top . Two different Act1 - Act2 circuit configurations are depicted : PIT2 - tTA ( left ) rtTA - PIT2 ( right ) . Calculated FF4 Output are shown for On , Off Off / On ( FF4 ) or On / Off ( Output ) . Color plots show normalized mCitrine ( left ) mCherry ( right ) expression for each Act1 - Act2 combination . Color bars show lowest highest expression range on a linear scale with median as midpoint D ) Correlation plots with calculated values on X axis ( Model ) experimental data on Y axis ( Data ) . A linear regression model ( y = a + b * x ) was used to compare experimental data with model predictions . Linear fit coefficient of determination ( R2 ) as indicated . PIT2 - tTA circuit ( black ) , rtTA - PIT2 circuit ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Combinatorial screen. Computational combinatorial screening of Act1MAX and Act2MAX using the model depicted in A (left). Experimental combinatorial screening in H1 (middle) with circuit schematic on top. Two different Act1-Act2 circuit configurations are depicted: PIT2-tTA (left) and rtTA-PIT2 (right). Calculated FF4 and Output levels are shown for On, Off and Off/On (FF4) or On/Off (Output). Color plots show normalized mCitrine (left) and mCherry (right) expression levels for each Act1-Act2 combination. Color bars show lowest and highest expression range on a linear scale with median as the midpoint D) Correlation plots with calculated values on X axis (Model) and experimental data on Y axis (Data). A linear regression model (y = a + b*x) was used to compare experimental data with model predictions. Linear fit and coefficient of determination (R2) as indicated. PIT2-tTA circuit (black), rtTA-PIT2 circuit (red).


C ) Bar chart showing performance of circuit 1 - 3 in H1 , HES - 2 HEK293 . mCitrine ( top ) mCherry ( bottom ) expression was normalized to internal Ef1a - iRFP control ( rel . U . ) ( left ) further normalized to maximal mCitrine ( circuit 2 ) ( top ) or maximal mCherry ( bottom ) ( rel . U . / Ctrl ) ( right ) . Samples below detection limit are labelled with b . d . Data information : Each bar show mean ± s . d . of at least three 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 O O O O O O O O O O O O O O O O O O O O O O

C) Bar chart showing the performance of circuit 1 - 3 in H1, HES-2 and HEK293. mCitrine (top) and mCherry (bottom) expression was normalized to internal Ef1a-iRFP control (rel.U.) (left) and further normalized to maximal mCitrine (circuit 2) (top) or maximal mCherry (bottom) (rel.U./Ctrl) (right). Samples below detection limit are labelled with b.d. Data information: Each bar show mean ± s.d. of at least three biological replicates.


D ) Bar chart showing performance of circuit 1 in response to administration of miRNA mimics in H1 ( left ) HEK293 ( right ) . First sample ( with profile + + - - - for H1 - - - - - for HEK292 ) has no miRNA administered . For other samples , miRNA dose with highest change in response has been selected from miRNA titration experiments Maximal mCitrine mCherry ( as calculated in B ) are depicted as yellow red dashed lines , respectively . Chart is shown in a base 10 logarithmic scale . Data information : Each bar show mean ± s . d . of at least three 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 O O O O O O O O O O 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) Bar chart showing performance of circuit 1 in response to administration of miRNA mimics in H1 (left) and HEK293 (right). First sample (with profile + + - - - for H1 and - - - - - for HEK292) has no miRNA administered. For the other samples, miRNA dose with highest change in response has been selected from miRNA titration experiments Maximal mCitrine and mCherry levels (as calculated in B) are depicted as yellow and red dashed lines, respectively. Chart is shown in a base 10 logarithmic scale. Data information: Each bar show mean ± s.d. of at least three biological replicates.


E ) Bar chart showing fine tuning of output using miSFITs library . Shown are FF4 / mCitrine ( top ) Output / mCherry ( bottom ) of circuit 1 3 for different T17 variants . Data were normalized to control circuits . Data information : Each bar show mean ± s . d . of at least three 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

E) Bar chart showing fine tuning of output using miSFITs library. Shown are FF4 / mCitrine (top) and Output / mCherry (bottom) of circuit 1 and 3 for different T17 variants. Data were normalized to control circuits. Data information: Each bar show mean ± s.d. of at least three biological replicates.


A ) Circuit schematics ( left ) . Chart shows normalized Output production in response to changing amounts of plasmids encoding Act2 , tTA ( left ) PIT2 ( right ) . Two transactivator constructs have been tested , onecontains miR302a / b target sites ( Off ) , other by mock miRFF3 / FF6 target sites ( ON ) . Data information : Charts show mean ± s . d . of at least three 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

A) Circuit schematics (left). Chart shows normalized Output production in response to changing amounts of plasmids encoding Act2, tTA (left) and PIT2 (right). Two transactivator constructs have been tested, onecontains miR302a/b target sites (Off), the other by mock miRFF3/FF6 target sites (ON). Data information: Charts show mean ± s.d. of at least three biological replicates.


B ) Discrete circuit - mediated control of BMP4 by endogenous miRNAs ( On ) . B ) Micropatterned RUES2 have been transfected with On Offcircuits . Bar chart shows BMP4 as determined by ELISA average iRFP expression as measured by Flow Cytometry ( left ) . Corresponding bar chart showing average fraction of expressing a given germ - layer marker ( right ) . Data information : Each bar show mean ± s . d . from at least three biological replicates for BMP4 iRFP data from at least 20 colonies for germ - layer data . Scale bar represents 200 μm .

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

B) Discrete circuit-mediated control of BMP4 by endogenous miRNAs (On).B) Micropatterned RUES2 have been transfected with On and Offcircuits. Bar chart shows BMP4 concentration as determined by ELISA and average iRFP expression as measured by Flow Cytometry (left). Corresponding bar chart showing the average fraction of cells expressing a given germ-layer marker (right). Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and iRFP data and from at least 20 colonies for germ-layer data. Scale bar represents 200 μm.


D , Circuit - mediated tuning of BMP4 = using miSFITs library . D ) . Bar chart on left shows BMP4 for each circuit variant determined from culture media by ELISA . Bar chart on right shows same secreted BMP4 along internal iRFP expression signal , measured by Flow Cytometry in comparison to untransfected controls in absence ( - BMP4 ) presence ( + BMP4 ) of BMP4 ( right ) . E Data information : Each bar show mean ± s . d . from at least three biological replicates for BMP4 iRFP data from at least 20 colonies for germ - layer data . Scale bar represents 200 μm .

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

D, Circuit-mediated tuning of BMP4 =using miSFITs library. D). Bar chart on the left shows BMP4 concentration for each circuit variant determined from culture media by ELISA. Bar chart on the right shows the same secreted BMP4 concentration along cell internal iRFP expression signal, measured by Flow Cytometry in comparison to untransfected controls in the absence (-BMP4) and presence (+ BMP4) of BMP4 (right).E Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and iRFP data and from at least 20 colonies for germ-layer data. Scale bar represents 200 μm.


E , Circuit - mediated tuning of BMP4 = using miSFITs library . ( right ) . E ) Bar chart showing relative fraction of expressing a given germ - layer marker as analyzed from confocal microscopy ( right ) . Dose response chart of marker fraction in response to measured BMP4 ( left ) . Data information : Each bar show mean ± s . d . from at least three biological replicates for BMP4 from at least 20 colonies for germ - layer data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Circuit-mediated tuning of BMP4 =using miSFITs library. (right).E) Bar chart showing the relative fraction of cells expressing a given germ-layer marker as analyzed from confocal microscopy images (right). Dose response chart of the marker fraction in response to measured BMP4 concentration (left). Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and from at least 20 colonies for germ-layer data.


F ) Circuit - mediated tuning of BMP4 = using miSFITs library . EV5C . F ) Radial marker profiles of each germ - layer marker analyzed from confocal microscopy . Charts on left show normalized of marker expression at a given colony position , where x = 0 is center of colony x = 1 is edge of colony . Representative microscopy image of a single colony ( top ) showing composite of Sox2 / mCitrine ( yellow ) , Sox17 / mCherry ( red ) TBXT / AF647 ( blue ) . Data information : Each bar show mean ± s . d . from at least three biological replicates for BMP4 from at least 20 colonies for germ - layer data . Scale bar represents 200 μm .

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

F) Circuit-mediated tuning of BMP4 =using miSFITs library. EV5C.F) Radial marker profiles of each germ-layer marker analyzed from confocal microscopy images. Charts on the left show normalized intensity of marker expression at a given colony position, where x=0 is the center of the colony and x=1 is the edge of the colony.Representative microscopy image of a single colony (top) showing composite of Sox2/mCitrine (yellow), Sox17/mCherry (red) and TBXT/AF647 (blue). Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and from at least 20 colonies for germ-layer data. Scale bar represents 200 μm.


A 3 - D reconstructions of propidium iodide ( PI ) - stained walls ( red ) BCECF - stained vacuoles ( green ) of epidermal atrichoblasts in early late meristem in early late elongation zone . Scale bars : 5 µm .

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

A 3-D reconstructions of propidium iodide (PI)-stained cell walls (red) and BCECF-stained vacuoles (green) of epidermal atrichoblasts in the early and late meristem and in the early and late elongation zone. Scale bars: 5 µm.


B Boxplots showing vacuolar occupancy of in defined zones ( n = 7 - 11 ) . Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

O O O O O O O O O O O O O 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 Boxplots showing vacuolar occupancy of cells in the defined zones (n=7-11). Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


c ) Graph depicts absolute values of volume vacuolar volume in depicted root zones ( data points display mean , error bars depict s . e . m . , n = 7 - 11 ) . " cytosol calc . " is approximated as difference between volume vacuolar volume .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph depicts absolute values of cell volume and vacuolar volume in the depicted root zones (data points display mean, error bars depict s.e.m., n=7-11). "cytosol calc." is approximated as the difference between cell volume and vacuolar volume.


A Representative of vacuolar of late meristematic . PI ( green ) pUBQ10 : : VAMP711 ( yellow ) depict wall tonoplast , respectively . Seedlings were treated with DMSO ( solvent control ) or 5 µM FC ( Fusicoccin ) for 2 . 5 h in liquid medium ( n = 24 ) . Mann Whitney U ( * * * p < 0 . 001 ) . Data information : Scale bars : 5 µm .

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

A Representative images and quantification of vacuolar morphology of late meristematic cells. PI (green) and pUBQ10::VAMP711 (yellow) depict cell wall and tonoplast, respectively. Seedlings were treated with DMSO (solvent control) or 5 µM FC (Fusicoccin) for 2.5 h in liquid medium (n=24). Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


B Left : 3 - D reconstructions of PI - stained wall ( red ) BCECF - stained vacuole ( green ) of late meristematic . Right : Boxplot depicts vacuolar occupancy of treated with solvent DMSO or 5 µM FC for 2 . 5 h in liquid medium ( n = 11 ) . Student ´ s t - ( * p < 0 . 05 ) . Data information : Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

B Left: 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Right: Boxplot depicts vacuolar occupancy of the cell treated with the solvent DMSO or 5 µM FC for 2.5 h in liquid medium (n = 11). Student´s t-test (*p < 0.05). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


C wall vacuolar was visualized with PI ( green ) MDY - 64 ( yellow ) . pER8 : : GFP - SAUR19 seedlings were treated with DMSO ( n = 60 ) or 10 µM β - estradiol ( n = 56 ) for 6 h in liquid medium . Mann Whitney U ( * * * p < 0 . 001 ) . Data information : Scale bars : 5 µm .

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

C Cell wall and vacuolar membrane was visualized with PI (green) and MDY-64 (yellow). pER8::GFP-SAUR19 seedlings were treated with DMSO (n=60) or 10 µM β-estradiol (n=56) for 6 h in liquid medium. Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


D Left : 3 - D reconstructions of PI - stained wall ( red ) BCECF - stained vacuole ( green ) of late meristematic in pER8 : : GFP - SAUR19 lines . Right : Boxplot depicts vacuolar occupancy of . Seedlings were treated with solvent control DMSO ( n = 11 ) or 10 µM β - estradiol ( n = 8 ) for 6 h in liquid medium . Student ´ s t - ( * * p < 0 . 01 ) . Data information : Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

D Left: 3-D cell reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells in pER8::GFP-SAUR19 lines. Right: Boxplot depicts vacuolar occupancy of the cell. Seedlings were treated with the solvent control DMSO (n=11) or 10 µM β-estradiol (n=8) for 6 h in liquid medium. Student´s t-test (**p < 0.01). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


E wall vacuolar was visualized with PI ( green ) pUBQ10 : : VAMP711 ( E ) ( yellow ) . Col - 0 wild type seedlings were treated for 3 h in liquid medium adjusted to pH 5 . 7 ( n = 44 ) or pH 4 . 5 ( n = 40 ) . Mann Whitney U ( * * * p < 0 . 001 ) . Data information : Scale bars : 5 µm .

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

E Cell wall and vacuolar membrane was visualized with PI (green) and pUBQ10::VAMP711 (E) (yellow). Col-0 wild type seedlings were treated for 3 h in liquid medium adjusted to pH 5.7 (n=44) or pH 4.5 (n=40). Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


F Left : 3 - D reconstructions of PI - stained wall ( red ) BCECF - stained vacuole ( green ) of late meristematic . Right : Boxplot depicts vacuolar occupancy of . Seedlings were treated for 3 h in liquid medium adjusted to pH 5 . 7 or pH 4 . 5 ( n = 11 ) . Student ´ s t - ( * * * p < 0 . 001 ) . Data information : Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

F Left: 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Right: Boxplot depicts vacuolar occupancy of cell. Seedlings were treated for 3 h in liquid medium adjusted to pH 5.7 or pH 4.5 (n=11). Student´s t-test (***p < 0.001). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


A Representative of vacuolar of late meristematic . PI ( green ) MDY - 64 ( yellow ) depict wall vacuolar , respectively . Seedlings were treated with DMSO solvent control or 50 µM EGCG for 22 h on solid medium ( n = 40 ) . Mann Whitney U ( * * * p < 0 . 001 ) . Data information : Scale bars : 5 µm .

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

A Representative images and quantification of vacuolar morphology of late meristematic cells. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Seedlings were treated with DMSO solvent control or 50 µM EGCG for 22 h on solid medium (n=40). Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


B 3 - D reconstructions of PI - stained wall ( red ) BCECF - stained vacuole ( green ) of late meristematic . Boxplot depicts vacuolar occupancy of . Seedlings were treated with DMSO ( n = 16 ) or 50 µM EGCG ( n = 15 ) for 22 h on solid medium . Student ´ s t - ( * * p < 0 . 01 ) . Data information : Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

B 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of the cell. Seedlings were treated with DMSO (n=16) or 50 µM EGCG (n=15) for 22 h on solid medium. Student´s t-test (**p < 0.01). Data information: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


C Representative of vacuolar of late meristematic . PI ( green ) MDY - 64 ( yellow ) depict wall vacuolar , respectively . Seedling roots were grown on surface ( n = 28 ) or into matrix ( n = 28 ) of 2 % agar - containing solid medium . Mann Whitney U ( * * * p < 0 . 001 ) . Data information : Scale bars : 5 µm .

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

C Representative images and quantification of vacuolar morphology of late meristematic cells. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Seedling roots were grown on the surface (n=28) or into the matrix (n=28) of 2 % agar-containing solid medium. Mann Whitney U test (***p < 0.001). Data information: Scale bars: 5 µm.


D 3 - D reconstructions of PI - stained wall ( red ) BCECF - stained vacuole ( green ) of late meristematic . Boxplot depicts vacuolar occupancy of surface ( n = 10 ) into medium ( n = 11 ) grown seedling roots . Student ´ s t - ( * * p < 0 . 01 ) . Data information : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of surface (n=10) and into the medium (n=11) grown seedling roots. Student´s t-test (**p < 0.01). Data information: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( A ) Vacuolar of Col - 0 ( n = 64 ) fer - 4 ( n = 60 ) . Mann Whitney U ( * * * p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (A) Vacuolar morphology of Col-0 (n=64) and fer-4 (n=60). Mann Whitney U test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . ( B ) 3 - D reconstructions of PI - stained wall ( red ) BCECF - stained vacuole ( green ) . Boxplot depicts vacuolar occupancy of Col - 0 control ( n = 12 ) fer - 4 ( n = 10 ) mutant . Mann Whitney U ( * * p < 0 . 01 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. (B) 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green). Boxplot depicts vacuolar occupancy of Col-0 control (n=12) and fer-4 (n=10) mutant cells. Mann Whitney U test (**p < 0.01). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( C ) Col - 0 ( n = 52 ) fer - 4 ( n = 44 ) seedlings were treated with solvent DMSO or 50 µM EGCG for 22 h on solid medium . Kruskal Wallis followed by Dunn ´ s multiple comparison ( b : p < 0 . 05 , c : p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (C) Col-0 (n=52) and fer-4 (n=44) seedlings were treated with solvent DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( D ) Col - 0 ( n = 28 ) fer - 4 ( n = 28 ) mutant seedling roots were grown on surface or into matrix of 2 % agar - containing solid medium . Kruskal Wallis followed by Dunn ´ s multiple comparison ( b : p < 0 . 05 , c : p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (D) Col-0 (n=28) and fer-4 (n=28) mutant seedling roots were grown on the surface or into the matrix of 2 % agar-containing solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( A ) Vacuolar of Col - 0 control ( n = 52 ) lrx3 / 4 / 5 triple mutants ( n = 48 ) . Mann Whitney U ( * * * p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (A) Vacuolar morphology of Col-0 control (n=52) and lrx3/4/5 triple mutants (n=48). Mann Whitney U test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . ( B ) 3 - D reconstructions of PI - stained wall ( red ) BCECF - stained vacuole ( green ) of late meristematic . Boxplot depicts vacuolar occupancy of in Col - 0 control ( n = 11 ) lrx3 / 4 / 5 ( n = 10 ) . Student ´ s t - ( * * * p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. (B) 3-D reconstructions of PI-stained cell wall (red) and BCECF-stained vacuole (green) of late meristematic cells. Boxplot depicts vacuolar occupancy of the cell in Col-0 control (n=11) and lrx3/4/5 (n=10). Student´s t-test (***p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( C ) Col - 0 ( n = 40 - 44 ) lrx3 / 4 / 5 ( n = 36 ) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium . Kruskal Wallis followed by Dunn ´ s multiple comparison ( b : p < 0 . 01 , c : p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (C) Col-0 (n=40-44) and lrx3/4/5 (n=36) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.01, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Representative of vacuolar of late meristematic atrichoblast . PI ( green ) MDY - 64 ( yellow ) depicts wall vacuolar , respectively . ( D ) Col - 0 ( n = 40 - 48 ) fer - 4 ( n = 28 - 32 ) seedlings were grown on surface or into matrix of 2 % agar - containing solid medium . Kruskal Wallis followed by Dunn ´ s multiple comparison ( b : p < 0 . 05 , c : p < 0 . 001 ) . scale bars : 5 µm . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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

Representative images and quantification of vacuolar morphology of late meristematic atrichoblast cells. PI (green) and MDY-64 (yellow) staining depicts cell wall and vacuolar membrane, respectively. (D) Col-0 (n=40-48) and fer-4 (n=28-32) seedlings were grown on the surface or into the matrix of 2 % agar-containing solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). scale bars: 5 µm. Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


A Rosette phenotype of 3 - - old Col - 0 , fer - 4 , lrx3 / 4 / 5 fer - 4 / lrx3 / 4 / 5 ( upper panel ) . Vacuolar ( MDY - 64 - stained ) of late meristematic atrichoblast of Col - 0 , fer - 4 , lrx3 / 4 / 5 fer - 4 / lrx3 / 4 / 5 ( lower panel ) . Scale bars : 1 cm ( upper row ) 5 µm ( lower row ) .

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

A Rosette phenotype of 3-weeks-old Col-0, fer-4, lrx3/4/5 and fer-4/lrx3/4/5 (upper panel). Vacuolar morphology (MDY-64-stained) of late meristematic atrichoblast cells of Col-0, fer-4, lrx3/4/5 and fer-4/lrx3/4/5 (lower panel). Scale bars: 1 cm (upper row) and 5 µm (lower row).


B LRR4 - HA RALF1 - FLAG were transiently expressed ( as indicated by + - ) in Nicotiana benthamiana Immunoprecipitation subsequent detection of by western blotting was done as labelled .

O O O O O O O O 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 LRR4-HA and RALF1-FLAG were transiently expressed (as indicated by + and -) in Nicotiana benthamiana Immunoprecipitation and subsequent detection of the proteins by western blotting was done as labelled.


C Relative root [ % of control ] of Col - 0 ( n = 11 - 13 ) , lrx3 / 4 / 5 ( n = 9 - 13 ) fer - 4 ( n = 11 - 12 ) after 3 days of RALF1 treatment . Statistical analyses were performed for each using one - way ANOVA followed by Bonferroni post ( 1 µM 1 . 25 µM b : p < 0 . 001 ; 1 . 5 µM b : p < 0 . 05 , c : p < 0 . 001 ) . Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values . Representative experiment is shown .

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C Relative root length [% of control] of Col-0 (n=11-13 ), lrx3/4/5 (n=9-13 ) and fer-4 (n=11-12) after 3 days of RALF1 treatment. Statistical analyses were performed for each concentration using one-way ANOVA followed by Bonferroni post test (1 µM and 1.25 µM b: p < 0.001; 1.5 µM b: p < 0.05, c: p < 0.001). Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values. Representative experiment is shown.


D Nicotiana benthamiana was transiently transformed with LRR4 - HA NtermFER - FLAG . Immunoprecipitation subsequent detection of by western blotting was done as labelled .

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D Nicotiana benthamiana was transiently transformed with LRR4-HA and NtermFER-FLAG. Immunoprecipitation and subsequent detection of the proteins by western blotting was done as labelled.


E Yeast was transformed with either empty plasmids ( e / e ) , NtermFER - FLAG or LRR4 - HA with empty plasmid , or both NtermFER - FLAG LRR4 - HA . Growth of yeast on quadruple drop - out medium was only observed in transformed with NtermFER - FLAG LRR4 - HA .

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E Yeast was transformed with either empty plasmids (e / e), NtermFER-FLAG or LRR4-HA with the empty plasmid, or both NtermFER-FLAG and LRR4-HA. Growth of yeast on quadruple drop-out medium was only observed in cells transformed with NtermFER-FLAG and LRR4-HA.


A Rosette phenotype of 3 - - old Col - 0 , lrx3 / 4 / 5 35S : : LRR4 - cit . Data information : Scale bars : 1

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A Rosette phenotype of 3-weeks-old Col-0, lrx3/4/5 and 35S::LRR4-cit. Data information: Scale bars: 1 cm


B Representative of vacuolar of late meristematic of Col - 0 35S : : LRR4 - cit . PI ( green ) MDY - 64 ( yellow ) depict wall vacuolar , respectively . Col - 0 ( n = 36 ) 35S : : LRR4 - cit ( n = 32 - 36 ) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium . Kruskal Wallis followed by Dunn ´ s multiple comparison ( b : p < 0 . 05 , c : p < 0 . 001 ) . Data information : 5 µm ( B ) . Boxplots : Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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B Representative images and quantification of vacuolar morphology of late meristematic cells of Col-0 and 35S::LRR4-cit. PI (green) and MDY-64 (yellow) staining depict cell wall and vacuolar membrane, respectively. Col-0 (n=36) and 35S::LRR4-cit (n=32-36) seedlings were treated with DMSO or 50 µM EGCG for 22 h on solid medium. Kruskal Wallis test followed by Dunn´s multiple comparison (b: p < 0.05, c: p < 0.001). Data information: 5 µm (B). Boxplots: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


C Relative root of Col - 0 ( n = 11 - 13 ) 35S : : LRR4 - cit ( n = 11 - 14 ) after 3 days of RALF1 treatment . Statistical analyses were performed for each using Student ´ s t - ( * * * p < 0 . 001 ) . Boxplots : Boxplots : Box limits represent 25th 75th percentile , horizontal represents median . Whiskers display min . to max . values .

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C Relative root length of Col-0 (n=11-13) and 35S::LRR4-cit (n=11-14) after 3 days of RALF1 treatment. Statistical analyses were performed for each concentration using Student´s t-test (***p < 0.001). Boxplots: Boxplots: Box limits represent 25th and 75th percentile, horizontal line represents median. Whiskers display min. to max. values.


Heat map showing absolute of unique peptides for S . indica apoplastic present at three time points in culture filtrate . S1 / S2 / S3 = biologically independent sample 1 , 2 3 ; d = deglycosylated .

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Heat map showing absolute counts of unique peptides for S. indica apoplastic proteins present at all three time points and in culture filtrate. S1/S2/S3 = biologically independent sample 1, 2 and 3; d = deglycosylated.


eATP in APF from S . indica inoculated mock - treated barley root samples collected at predominantly biotrophic phase ( 3 5 dpi ) as well as predominantly associated phase ( 7 10 dpi ) . Error bars show standard error of mean obtained from three independent biological replicates at p < 0 . 05 ( * ) , 0 . 01 ( * * ) analyzed by Student ' s t - .

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eATP levels in APF from S. indica inoculated and mock-treated barley root samples collected at the predominantly biotrophic phase (3 and 5 dpi) as well as the predominantly cell death associated phase (7 and 10 dpi). Error bars show the standard error of the mean obtained from three independent biological replicates at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.


eATP measured from culture medium collected from mock treated S . indica colonized Arabidopsis seedlings at 3 , 5 , 7 10 dpi . Error bars represent ± SE of mean from three independent biological replicates . RLU : relative units . Asterisks indicate significance at p < 0 . 05 ( * ) , 0 . 01 ( * * ) analyzed by Student ' s t - .

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eATP levels measured from culture medium collected from mock treated and S. indica colonized Arabidopsis seedlings at 3, 5, 7 and 10 dpi. Error bars represent ±SE of the mean from three independent biological replicates. RLU: relative light units. Asterisks indicate significance at p< 0.05 (*), 0.01 (**) analyzed by Student's t-test.


S . indica colonization of Arabidopsis dorn1 - 3 mutant parental Col - 0aq lines quantified by qPCR at 3 , 5 , 7 10 dpi . of fungal ( SiTEF ) to ( AtUBI ) amplicons representing fungal colonization in root tissue was calculated using gDNA as template 2 - ΔCT method . Error bars represent standard error of mean of three technical replicates . experiment was repeated 3 times for 3 , 5 7 dpi with similar outcomes .

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S. indica colonization of Arabidopsis dorn1-3 mutant and the parental Col-0aq lines quantified by qPCR at 3, 5, 7 and 10 dpi. The ratio of fungal (SiTEF) to plant (AtUBI) amplicons representing fungal colonization levels in plant root tissue was calculated using gDNA as template and the 2-ΔCT method. Error bars represent standard error of the mean of three technical replicates. The experiment was repeated 3 times for 3, 5 and 7 dpi with similar outcomes.


Transcript of S . indica E5 ′ NT during colonization of barley Arabidopsis at different symbiotic stages in axenic culture . Error bars represent standard error of mean of three independent biological replicates . CM = medium .

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Transcript levels of S. indica E5′NT during colonization of barley and Arabidopsis at different symbiotic stages and in axenic culture. Error bars represent standard error of the mean of three independent biological replicates. CM = complex medium.


Ecto - 5 ′ - nucleotidase activity measured in preparations of Arabidopsis expressing Pro35S : : E5 ′ NT ( # 303 ) , Pro35S : : SPE5 ′ NT : mCherry : E5 ′ NTwoSP ( # 304 ) or Pro35S : : mCherry ( # 305 ) . E5 ′ NT activity was measured after incubation with 100 µM of either ATP , ADP or AMP . In preparations from Pro35S : : E5 ′ NT ( # 303 ) lines phosphate release was specifically increased upon incubation with purines . Error bars represent standard error of mean from three technical repetitions . coomassie stained SDS - PAGE shows pattern of fractions for transgenic lines . Equal volumes were loaded . experiment was repeated two times with similar results .

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Ecto-5′-nucleotidase activity measured in membrane protein preparations of Arabidopsis plants expressing Pro35S::E5′NT (#303), Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) or Pro35S::mCherry (#305). E5′NT activity was measured after incubation with 100 µM of either ATP, ADP or AMP. In the membrane protein preparations from Pro35S::E5′NT (#303) lines phosphate release was specifically increased upon incubation with purines. Error bars represent the standard error of the mean from three technical repetitions. The coomassie stained SDS-PAGE shows the protein pattern of the membrane fractions for the individual transgenic lines. Equal volumes were loaded. The experiment was repeated two times with similar results.


Confocal microscopy of Arabidopsis roots expressing either mCherry ( # 305 ) or Pro35S : : SPE5 ′ NT : mCherry : E5 ′ NTwoSP ( # 304 ) showing secretion of E5 ′ NT fusion . mCherry show z - stacks of 14 image planes of 1 µm each . Scale bar = 20 µm .

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Confocal microscopy images of Arabidopsis roots expressing either cytosolic mCherry (#305) or Pro35S::SPE5′NT:mCherry:E5′NTwoSP (#304) showing secretion of the E5′NT fusion protein. mCherry images show z-stacks of 14 image planes of 1 µm each. Scale bar=20 µm.


transgenic Arabidopsis Pro35S : : E5 ′ NT ( # 303 ) expressing untagged full SiE5 ′ NT was better colonized by S . indica . Error bars of qPCR data represent ± SE of mean from three independent biological replicates . Asterisks indicate significance ( Student ' s t - , * p < 0 . 05 ) .

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The transgenic Arabidopsis line Pro35S::E5′NT (#303) expressing untagged full length SiE5′NT was better colonized by S. indica. Error bars of the qPCR data represent ±SE of the mean from three independent biological replicates. Asterisks indicate significance (Student's t-test, * p< 0.05).


S . indica induced eATP release in different Arabidopsis transgenic lines . Culture medium was collected from mock treated or S . indica inoculated seedlings growing in liquid medium at 5 dpi released eATP was measured . RLU : relative units . Error bars represent ± SE of mean from three biological replicates . Asterisks indicate significance to other samples within same treatment group ( ANOVA , p < 0 . 05 ) .

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S. indica induced eATP release in different Arabidopsis transgenic lines. Culture medium was collected from mock treated or S. indica inoculated seedlings growing in liquid medium at 5 dpi and released eATP was measured. RLU: relative light units. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).


S . indica colonization of transgenic lines at 5 dpi . Error bars represent ± SE of mean from three biological replicates . Asterisks indicate significance to other samples within same treatment group ( ANOVA , p < 0 . 05 ) .

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S. indica colonization of transgenic lines at 5 dpi. Error bars represent ±SE of the mean from three biological replicates. Asterisks indicate significance to all other samples within the same treatment group (ANOVA, p<0.05).


Expression of eATP responsive gene At1g58420 measured by qRT - PCR . Error bars represent ± SE of mean from three independent biological replicates ( independent from those shown in Fig . 2C ) . Letters indicate significant groups ( ANOVA , p < 0 . 01 , for 305 p < 0 . 05 ) .

Expression analysis of the eATP responsive gene At1g58420 measured by qRT-PCR. Error bars represent ±SE of the mean from three independent biological replicates (independent from those shown in Fig. 2C). Letters indicate significant groups (ANOVA, p<0.01, for the line 305 p<0.05).


Relative fluxes of phosphates ( Pi ) sugars ( C ) across fungal plasma . Without additional Pi - source in apoplast ( value 0 at x - axis , black dots ) there is a constant flux of phosphate via proton - coupled phosphate ( H / P ) transporter from fungus to apoplast a constant flux of sugar from apoplast to fungus . For better comparison these fluxes were normalized to 1 - 1 , respectively , other fluxes were calculated relative to these control values . With increasing ATP - release Pi - production Pi - efflux gets smaller is zero at a relative Pi - production rate of 1 ( grey triangles ) . At higher ATP - release Pi - production rates fungus imports Pi , i . e . transport direction of phosphate has been inverted in comparison to control condition ( square ) . C - flux via proton - coupled sugar ( H / C ) transporter is not affected by additional Pi - source ( horizontal grey ) .

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Relative fluxes of phosphates (Pi) and sugars (C) across the fungal plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of phosphate via the proton-coupled phosphate (H/P) transporter from the fungus to the apoplast and a constant flux of sugar from the apoplast to the fungus. For better comparison these fluxes were normalized to 1 and -1, respectively, and all other fluxes were calculated relative to these control values. With increasing ATP-release and Pi-production the Pi-efflux gets smaller and is zero at a relative Pi-production rate of 1 (grey triangles). At higher ATP-release and Pi-production rates the fungus imports Pi, i.e. the transport direction of phosphate has been inverted in comparison to the control condition (white square). The C-flux via the proton-coupled sugar (H/C) transporter is not affected by the additional Pi-source (horizontal grey line).


Relative fluxes of Pi C across plasma . Without additional Pi - source in apoplast ( value 0 at x - axis , black dots ) there is a constant flux of sugar via H / C transporter from to apoplast ( thereafter to fungus ) a constant flux of phosphate ( coming from fungus ) via H / P transporter from apoplast to . With increasing ATP - release Pi - production Pi - influx via H / P transporter increases while C - flux via H / C transporter is unaffected by additional Pi - source ( horizontal grey ) .

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Relative fluxes of Pi and C across the plant plasma membrane. Without additional Pi-source in the apoplast (value 0 at the x-axis, black dots) there is a constant flux of sugar via the H/C transporter from the plant to the apoplast (and thereafter to the fungus) and a constant flux of phosphate (coming from the fungus) via the H/P transporter from the apoplast to the plant. With increasing ATP-release and Pi-production the Pi-influx via the H/P transporter increases while the C-flux via the H/C transporter is unaffected by the additional Pi-source (horizontal grey line).


Phosphate fluxes across plasma . Besides Pi - uptake via H / P transporter ( B , black , for clarity not shown in C ) , loses Pi due to ATP - release ( grey ) . difference between Pi - uptake Pi - loss is net Pi - balance of ( grey ) . Without ATP - release ( value 0 at x - axis , black dots ) gains Pi . At a relative ATP - release Pi - production of 1 ( grey triangles ) release as much phosphate as it takes up , while at higher ATP - release values , loses Pi .

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Phosphate fluxes across the plant plasma membrane. Besides the Pi-uptake via the H/P transporter (B, black line, for clarity not shown in C), the plant loses Pi due to the ATP-release (light grey line). The difference between Pi-uptake and Pi-loss is the net Pi-balance of the plant (dark grey line). Without ATP-release (value 0 at the x-axis, black dots) the plant gains Pi. At a relative ATP-release and Pi-production of 1 (grey triangles) the plant release as much phosphate as it takes up, while at higher ATP-release values, the plant loses Pi.


( a ) Immunoblot of caspase - 1 IL - 1β in lysates ( Lys ) supernatants ( Sup ) of peritoneal macrophages obtained from Map1lc3b - / - mice Becn1 + / - mice corresponding wild - type littermate mice , then left unstimulated ( - ) or stimulated with LPS alone ( LPS ) or LPS followed by 30 min of ATP treatment ( L + A ) . p10 , mature form of caspase - 1 . Right margin , molecular in kilodaltons ( kDa ) .

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(a) Immunoblot analysis of caspase-1 and IL-1β in lysates (Lys) and supernatants (Sup) of peritoneal macrophages obtained from Map1lc3b-/- mice and Becn1+/- mice and the corresponding wild-type littermate mice, then left unstimulated (-) or stimulated with LPS alone (LPS) or LPS followed by 30 min of ATP treatment (L + A). p10, mature form of caspase-1. Right margin, molecular size in kilodaltons (kDa).


( b ) Enzyme - linked immunosorbent ( ELISA ) of cytokine secretion by peritoneal macrophages stimulated with LPS , followed by treatment for 1 h with ATP .

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(b) Enzyme-linked immunosorbent assay (ELISA) of cytokine secretion by peritoneal macrophages stimulated with LPS, followed by treatment for 1 h with ATP.