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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV230692	2/4	Xanthomonas oryzae	X. oryzae pv. oryzae PXO99	(d)(U)C DG Filtration	Bahar O	2016	33%
Study summary Full title Bacterial Outer Membrane Vesicles Induce Plant Immune Responses. All authors Bahar O, Mordukhovich G, Luu DD, Schwessinger B, Daudi A, Jehle AK, Felix G, Ronald PC Journal Mol Plant Microbe Interact Abstract Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane v (show more...)Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions. (hide) EV-METRIC 33% (74th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: EF-TU non-EV: None Proteomics no EV density (g/ml) not specified Show all info Study aim Function Sample Species Xanthomonas oryzae Sample Type Cell culture supernatant EV-producing cells X. oryzae pv. oryzae PXO99 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: speed (g) 170000 Density gradient Type Discontinuous Number of initial discontinuous layers not specified Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) not specified Sample volume (mL) not spec Orientation Top-down Speed (g) 246000 Duration (min) 360 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 120 Pelleting: rotor type not specified Pelleting: speed (g) 170000 Filtration steps 0.22µm or 0.2µmBetween 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins EF-TU Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-200

	EV230692	1/4	Xanthomonas campestris	X. campestris pv. campestris 33913	(d)(U)C DG Filtration	Bahar O	2016	29%
Study summary Full title Bacterial Outer Membrane Vesicles Induce Plant Immune Responses. All authors Bahar O, Mordukhovich G, Luu DD, Schwessinger B, Daudi A, Jehle AK, Felix G, Ronald PC Journal Mol Plant Microbe Interact Abstract Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane v (show more...)Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions. (hide) EV-METRIC 29% (67th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: PXO_03968 (Ax21) non-EV: None Proteomics no EV density (g/ml) not specified Show all info Study aim Function Sample Species Xanthomonas campestris Sample Type Cell culture supernatant EV-producing cells X. campestris pv. campestris 33913 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: speed (g) 170000 Density gradient Type Discontinuous Number of initial discontinuous layers not specified Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) not specified Sample volume (mL) not spec Orientation Top-down Speed (g) 246000 Duration (min) 360 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 120 Pelleting: rotor type not specified Pelleting: speed (g) 170000 Filtration steps 0.22µm or 0.2µmBetween 0.22 and 0.45 µm Characterization: Protein analysis Protein Concentration Method Bradford Western Blot Detected EV-associated proteins PXO_03968 (Ax21) Characterization: Lipid analysis No Characterization: Particle analysis EM EM-type Transmission-EM Image type Wide-field Report size (nm) 20-200

	EV230692	3/4	Pseudomonas syringae group genomosp. 3	P. syringae pv. tomato DC3000	(d)(U)C DG Filtration	Bahar O	2016	29%
Study summary Full title Bacterial Outer Membrane Vesicles Induce Plant Immune Responses. All authors Bahar O, Mordukhovich G, Luu DD, Schwessinger B, Daudi A, Jehle AK, Felix G, Ronald PC Journal Mol Plant Microbe Interact Abstract Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane v (show more...)Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions. (hide) EV-METRIC 29% (67th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) not specified Show all info Study aim Function Sample Species Pseudomonas syringae group genomosp. 3 Sample Type Cell culture supernatant EV-producing cells P. syringae pv. tomato DC3000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: speed (g) 170000 Density gradient Type Discontinuous Number of initial discontinuous layers not specified Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) not specified Sample volume (mL) not spec Orientation Top-down Speed (g) 246000 Duration (min) 360 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 120 Pelleting: rotor type not specified Pelleting: speed (g) 170000 Filtration steps 0.22µm or 0.2µmBetween 0.22 and 0.45 µm Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis None

	EV230692	4/4	Adivorax citrulli	Adivorax citrulli M6	(d)(U)C DG Filtration	Bahar O	2016	29%
Study summary Full title Bacterial Outer Membrane Vesicles Induce Plant Immune Responses. All authors Bahar O, Mordukhovich G, Luu DD, Schwessinger B, Daudi A, Jehle AK, Felix G, Ronald PC Journal Mol Plant Microbe Interact Abstract Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane v (show more...)Gram-negative bacteria continuously pinch off portions of their outer membrane, releasing membrane vesicles. These outer membrane vesicles (OMVs) are involved in multiple processes including cell-to-cell communication, biofilm formation, stress tolerance, horizontal gene transfer, and virulence. OMVs are also known modulators of the mammalian immune response. Despite the well-documented role of OMVs in mammalian-bacterial communication, their interaction with plants is not well studied. To examine whether OMVs of plant pathogens modulate the plant immune response, we purified OMVs from four different plant pathogens and used them to treat Arabidopsis thaliana. OMVs rapidly induced a reactive oxygen species burst, medium alkalinization, and defense gene expression in A. thaliana leaf discs, cell cultures, and seedlings, respectively. Western blot analysis revealed that EF-Tu is present in OMVs and that it serves as an elicitor of the plant immune response in this form. Our results further show that the immune coreceptors BAK1 and SOBIR1 mediate OMV perception and response. Taken together, our results demonstrate that plants can detect and respond to OMV-associated molecules by activation of their immune system, revealing a new facet of plant-bacterial interactions. (hide) EV-METRIC 29% (67th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles outer membrane vesicles Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Filtration Protein markers EV: None non-EV: None Proteomics no EV density (g/ml) not specified Show all info Study aim Function Sample Species Adivorax citrulli Sample Type Cell culture supernatant EV-producing cells Adivorax citrulli M6 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 120 Pelleting: speed (g) 170000 Density gradient Type Discontinuous Number of initial discontinuous layers not specified Lowest density fraction 20% Highest density fraction 45% Total gradient volume, incl. sample (mL) not specified Sample volume (mL) not spec Orientation Top-down Speed (g) 246000 Duration (min) 360 Fraction volume (mL) 1 Fraction processing Centrifugation Pelleting: volume per fraction 12 Pelleting: duration (min) 120 Pelleting: rotor type not specified Pelleting: speed (g) 170000 Filtration steps 0.22µm or 0.2µmBetween 0.22 and 0.45 µm Characterization: Protein analysis None Protein Concentration Method Bradford Characterization: Lipid analysis No Characterization: Particle analysis None

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EV-TRACK ID	EV230692
species	Xanthomonas oryzae	Xanthomonas campestris	Pseudomonas syringae group genomosp. 3	Adivorax citrulli
sample type	Cell culture	Cell culture	Cell culture	Cell culture
cell type	X. oryzae pv. oryzae PXO99	X. campestris pv. campestris 33913	P. syringae pv. tomato DC3000	Adivorax citrulli M6
condition	Control condition	Control condition	Control condition	Control condition
separation protocol	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration	dUC/ Density gradient/ Filtration
Exp. nr.	2	1	3	4
EV-METRIC %	33	29	29	29