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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV240106	1/2	Sinorhizobium fredii	HH103	(d)(U)C DG Filtration UF	Li D	2022	67%
Study summary Full title Analysis of Outer Membrane Vesicles Indicates That Glycerophospholipid Metabolism Contributes to Early Symbiosis Between HH103 and Soybean. All authors Li D, Li Z, Wu J, Tang Z, Xie F, Chen D, Lin H, Li Y Journal Mol Plant Microbe Interact Abstract Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OM (show more...)Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OMVs have been focused on mammalian-bacterial interactions. However, research on the OMVs of rhizobia is still limited. In this work, we isolated and purified OMVs from HH103 under free-living conditions that were set as control (C-OMVs) and symbiosis-mimicking conditions that were induced by genistein (G-OMVs). The soybean roots treated with G-OMVs displayed significant deformation of root hairs. G-OMVs significantly induced the expression of nodulation genes related to early symbiosis, while they inhibited that of the defense genes of soybean. Proteomics analysis identified a total of 93 differential proteins between C-OMVs and G-OMVs, which are mainly associated with ribosome synthesis, flagellar assembly, two-component system, ABC transporters, oxidative phosphorylation, nitrogen metabolism, quorum sensing, glycerophospholipid metabolism, and peptidoglycan biosynthesis. A total of 45 differential lipids were identified through lipidomics analysis. Correlation analysis of OMV proteome and lipidome data revealed that glycerophospholipid metabolism is the enriched Kyoto Encyclopedia of Genes and Genomes metabolic pathway, and the expression of phosphatidylserine decarboxylase was significantly up-regulated in G-OMVs. The changes in three lipids related to symbiosis in the glycerophospholipid metabolism pathway were verified by enzyme-linked immunosorbent assay. Our results indicate that glycerophospholipid metabolism contributes to rhizobia-soybean symbiosis via OMVs.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license. (hide) EV-METRIC 67% (94th 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 Vesicle 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 Ultrafiltration Adj. k-factor 0 (washing) Protein markers EV: phosphatidylcholine/ phosphatidylserine/ phosphatidylinositol/ None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Sinorhizobium fredii Sample Type Cell culture supernatant EV-producing cells HH103 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 265000 Wash: adjusted k-factor TDB Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 25% Highest density fraction 55% Total gradient volume, incl. sample (mL) 38 Orientation Bottom-up Speed (g) 265000 Duration (min) 960 Fraction processing Centrifugation Pelleting: speed (g) 265000 Pelleting: adjusted k-factor TDB Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BSA method Protein Yield (µg) per liter per OD600 unit ELISA Detected EV-associated proteins phosphatidylcholine/ phosphatidylserine/ phosphatidylinositol Not detected EV-associated proteins Detected contaminants None Not detected contaminants None Proteomics database No Detected EV-associated proteins None Not detected EV-associated proteins None Detected contaminants None Not detected contaminants None Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean Reported size (nm) 82 NTA EV concentration Yes Particle yield as number of particles per liter per OD unit: 1.5*10^8 EM EM-type Transmission-EM Image type Wide-field Extra information - filtration step of separation protocol: The pore size of the filter was 0.45 µm, I reported this as ‘between 0.22 and 0.45 µm’.

	EV240106	2/2	Sinorhizobium fredii	HH103	(d)(U)C DG Filtration UF	Li D	2022	67%
Study summary Full title Analysis of Outer Membrane Vesicles Indicates That Glycerophospholipid Metabolism Contributes to Early Symbiosis Between HH103 and Soybean. All authors Li D, Li Z, Wu J, Tang Z, Xie F, Chen D, Lin H, Li Y Journal Mol Plant Microbe Interact Abstract Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OM (show more...)Gram-negative bacteria can produce outer membrane vesicles (OMVs), and most functional studies of OMVs have been focused on mammalian-bacterial interactions. However, research on the OMVs of rhizobia is still limited. In this work, we isolated and purified OMVs from HH103 under free-living conditions that were set as control (C-OMVs) and symbiosis-mimicking conditions that were induced by genistein (G-OMVs). The soybean roots treated with G-OMVs displayed significant deformation of root hairs. G-OMVs significantly induced the expression of nodulation genes related to early symbiosis, while they inhibited that of the defense genes of soybean. Proteomics analysis identified a total of 93 differential proteins between C-OMVs and G-OMVs, which are mainly associated with ribosome synthesis, flagellar assembly, two-component system, ABC transporters, oxidative phosphorylation, nitrogen metabolism, quorum sensing, glycerophospholipid metabolism, and peptidoglycan biosynthesis. A total of 45 differential lipids were identified through lipidomics analysis. Correlation analysis of OMV proteome and lipidome data revealed that glycerophospholipid metabolism is the enriched Kyoto Encyclopedia of Genes and Genomes metabolic pathway, and the expression of phosphatidylserine decarboxylase was significantly up-regulated in G-OMVs. The changes in three lipids related to symbiosis in the glycerophospholipid metabolism pathway were verified by enzyme-linked immunosorbent assay. Our results indicate that glycerophospholipid metabolism contributes to rhizobia-soybean symbiosis via OMVs.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license. (hide) EV-METRIC 67% (94th 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 genistein treated condition Focus vesicles Outer Membrane Vesicle 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 Ultrafiltration Adj. k-factor 0 (washing) Protein markers EV: phosphatidylcholine/ phosphatidylserine/ phosphatidylinositol/ None non-EV: None Proteomics yes Show all info Study aim Function/Identification of content (omics approaches) Sample Species Sinorhizobium fredii Sample Type Cell culture supernatant EV-producing cells HH103 EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: rotor type SW 32 Ti Pelleting: speed (g) 265000 Wash: adjusted k-factor TDB Density gradient Type Discontinuous Number of initial discontinuous layers 7 Lowest density fraction 25% Highest density fraction 55% Total gradient volume, incl. sample (mL) 38 Orientation Bottom-up Speed (g) 265000 Duration (min) 960 Fraction processing Centrifugation Pelleting: speed (g) 265000 Pelleting: adjusted k-factor TDB Filtration steps 0.45µm > x > 0.22µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type NS Characterization: Protein analysis Protein Concentration Method BSA method Protein Yield (µg) per liter per OD600 unit ELISA Detected EV-associated proteins phosphatidylcholine/ phosphatidylserine/ phosphatidylinositol Not detected EV-associated proteins Detected contaminants None Not detected contaminants None Proteomics database No Detected EV-associated proteins None Not detected EV-associated proteins None Detected contaminants None Not detected contaminants None Characterization: Lipid analysis Yes Characterization: Particle analysis DLS Report type Mean Reported size (nm) 56 NTA EV concentration Yes Particle yield as number of particles per liter per OD unit: 11*10^8 EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV240106
species	Sinorhizobium fredii
sample type	Cell culture
cell type	HH103
condition	Control condition	genistein treated condition
separation protocol	dUC/ Density gradient/ Filtration/ Ultrafiltration	dUC/ Density gradient/ Filtration/ Ultrafiltration
Exp. nr.	1	2
EV-METRIC %	67	67