( B ) Immunoblot of netrin - 1 , DCC , UNC5B , active caspase - 3 , alpha synuclein FL , alpha synuclein pS129 , and Tyrosine Hydroxylase ( TH ) levels in age - matched controls ( n = 3 ) vs Parkinson ' s disease patients brain samples ( n = 3 ) ( left panel ) . Band quantification bar graph ( right panel ) . Bars and error bars represent the mean ± SEM . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey test for multiple group comparison . P * * < 0 . 01 . N = 3

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(B) Immunoblot of netrin-1, DCC, UNC5B, active caspase-3, alpha synuclein FL, alpha synuclein pS129, and Tyrosine Hydroxylase (TH) levels in age-matched controls (n=3) vs Parkinson's disease patients brain samples (n=3) (left panel). Band quantification bar graph (right panel). Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. P**< 0.01. N=3 replicates


F Representative BLI and histology of organotypic cultures with H2030 - BrM cancer cells treated with DMSO or decreasing concentrations of DEBIO - 0932 . Scale bar : 100 µm ; high magnification : 50 µm . G Quantification of the bioluminescence signal emitted by each condition shown in ( F ) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with DMSO . Day 0 is considered 12 - 16h after the addition of cancer cells and treatment or DMSO . Values are shown in box - and - whisker plots where each dot is 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 ( n = 8 DMSO , n = 8 BKM120 and n = 7 per concentration of DEBIO - 0932 - treated organotypic cultures , 2 independent experiments ) . P value was calculated using two - tailed t - test .

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F Representative BLI and histology of organotypic cultures with H2030-BrM cancer cells treated with DMSO or decreasing concentrations of DEBIO-0932. Scale bar: 100 µm; high magnification: 50 µm. G Quantification of the bioluminescence signal emitted by each condition shown in (F) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with DMSO. Day 0 is considered 12-16h after the addition of cancer cells and treatment or DMSO. Values are shown in box-and-whisker plots where each dot is 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 (n=8 DMSO, n=8 BKM120 and n=7 per concentration of DEBIO-0932-treated organotypic cultures, 2 independent experiments). P value was calculated using two-tailed t-test.


B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled . Scale bar : 75 µm . Each individual condition was evaluated in several organotypic cultures ( 3 - 6 slices ) .

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B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled. Scale bar: 75 µm. Each individual condition was evaluated in several organotypic cultures (3-6 slices).


D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation . Values are shown as mean + s . e . m . ( n = 3 organotypic cultures per experimental condition , 1 independent experiment ) .

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D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation. Values are shown as mean + s.e.m. (n=3 organotypic cultures per experimental condition, 1 independent experiment).


A - C Immunofluorescence against HSP90 in mouse brains with established metastases . ( A ) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula , where neurons co - localize with the chaperone . Scale bars : 100 µm ( low magnification ) , 50 µm ( medial habenula nucleus ) , 12 µm ( high magnification neurons ) . ( B ) Established metastases from different primary origins and oncogenomic profiles stained with HSP90 . Dotted lines delineate the metastasis ( cc : cancer cells ) . Scale bars : 75 µm . ( C ) Iba1 colocalizes with HSP90 within areas affected by metastases . BB : bisbenzamide . Scale bar : 75 µm ( low magnification ) , 12 µm ( high magnification ) .

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A-C Immunofluorescence against HSP90 in mouse brains with established metastases. (A) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula, where neurons co-localize with the chaperone. Scale bars: 100 µm (low magnification), 50 µm (medial habenula nucleus), 12 µm (high magnification neurons). (B) Established metastases from different primary origins and oncogenomic profiles stained with HSP90. Dotted lines delineate the metastasis (cc: cancer cells). Scale bars: 75 µm. (C) Iba1 colocalizes with HSP90 within areas affected by metastases. BB: bisbenzamide. Scale bar: 75 µm (low magnification), 12 µm (high magnification).


E Representative human brain metastases showing different intensities or scores for HSP90 . Scale bar : 50 µm . F Quantification of HSP90 in human brain metastases . 59 out of 60 ( 98 % ) showed positive staining of HSP90 in the tumor , 15 ( 25 % ) scored with 3 ( strong ) , 36 ( 60 % ) with 2 ( moderate ) and 8 ( 13 % ) with 1 ( weak ) according to the signal intensity of HSP90 in the cytoplasm of cancer cells .

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E Representative human brain metastases showing different intensities or scores for HSP90. Scale bar: 50 µm. F Quantification of HSP90 in human brain metastases. 59 out of 60 (98%) showed positive staining of HSP90 in the tumor, 15 (25%) scored with 3 (strong), 36 (60%) with 2 (moderate) and 8 (13%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells.


Representative human brain metastases showing different percentages of nuclear HSP90 . Scale bars : ( J ) 50 µm Black arrows point to cancer cells positive for HSP90 in the nucleus .

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Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (J) 50 µm Black arrows point to cancer cells positive for HSP90 in the nucleus.


Representative human brain metastases showing different percentages of nuclear HSP90 . Scale bars : ( K ) low magnification : 100 µm ; high magnification : 10 µm .

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Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (K) low magnification: 100 µm; high magnification: 10 µm.


L Quantification of nuclear HSP90 in human brain metastases . 54 out of 60 samples ( 90 % ) showed positive nuclear HSP90 in the tumor . 27 ( 45 % ) showed 1 - 5 % ( moderate ) and 27 ( 45 % ) showed > 5 % ( high ) of nuclear HSP90 . M Quantification of nuclear HSP90 in human primary tumors . 19 out of 30 ( 63 % ) showed positive nuclear HSP90 in the tumor . 9 ( 30 % ) showed 1 - 5 % ( moderate ) and 10 ( 33 % ) showed > 5 % ( high ) of nuclear HSP90 .

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L Quantification of nuclear HSP90 in human brain metastases. 54 out of 60 samples (90%) showed positive nuclear HSP90 in the tumor. 27 (45%) showed 1-5% (moderate) and 27 (45%) showed >5% (high) of nuclear HSP90. M Quantification of nuclear HSP90 in human primary tumors. 19 out of 30 (63%) showed positive nuclear HSP90 in the tumor. 9 (30%) showed 1-5% (moderate) and 10 (33%) showed >5% (high) of nuclear HSP90.


B Representative in vivo and ex vivo images of vehicle and DEBIO - 0932 treated mice 5 weeks ( experimental endpoint ) after intracardiac inoculation of H2030 - BrM cells .

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B Representative in vivo and ex vivo images of vehicle and DEBIO-0932 treated mice 5 weeks (experimental endpoint) after intracardiac inoculation of H2030-BrM cells.


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 .

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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 ) and markers of the 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 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 .

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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 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 .

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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 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 .

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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 and high expression levels of the HSP90 signature ( AHR , DDA1 , UBE4B , GPATCH8 ) in a cohort of 45 breast cancer brain metastasis patients .

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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 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 .

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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 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 .

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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 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 .

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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 primary tumors with high and low values of UBE4B . P value was calculated using log - rank ( Mantel - Cox ) test .

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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 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 .

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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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 ) .

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 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 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 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 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 .

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

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.


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 .

O O O O B-MEASURED_VAR O O O O 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 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 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

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 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 .

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 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 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 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 ) .

O O O O O O O O O O O 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 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 B-MEASURED_VAR O 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 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 ) .

O O O O O O O O O O O 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 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 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 .

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

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

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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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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 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 .

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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 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 .

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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 ) .

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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 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 .

O O O O O O O O O O O O O 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 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 .

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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 and NtermFER - FLAG . Immunoprecipitation and subsequent detection of the proteins 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 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 .

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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 - weeks - old Col - 0 , lrx3 / 4 / 5 and 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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.