diff --git a/assays/ANOVAForDifferentialEnrichmentOfMetabolites/README.md b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/README.md new file mode 100644 index 0000000000000000000000000000000000000000..f730cd57340035f270819ee8fa4e97a8a219b2a1 --- /dev/null +++ b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/README.md @@ -0,0 +1,89 @@ +<img src=./dataset/gr2.jpg width=60%> + +**Figure 2. Diversity and region-specificity of metabolites along the root axis** + +(A) Principal-component analysis (PCA) of metabolites extracted from the full-length root, and segments 0–2, 2–4, and 4–6 cm from the root tip. The colors of clusters correspond to regions of the root depicted in the plant cartoon in Figure 1A. Centroids are indicated for each cluster of root region. +(B) Heatmap depicting the enrichment or depletion of indicated metabolites in respective root segments. Clustering performed based on average linkage Pearson hierarchical clustering of metabolites. Clusters are indicated in red roman numbers. Colors on the scale bar indicate the level of enrichment or depletion of each metabolite across the root segments. + +<img src=./dataset/gr4.jpg width=60%> + +**Figure 4. Metabolite profiles of root segments from *sweet2, sweet4*, and *swee11;12* are distinct from WT** + +(A) Principal-component analysis (PCA) of total metabolites extracted from the whole root and root segments 2, 4, and 6 cm above the root tip of *sweet2, sweet4, sweet11;12*, and WT plants. WT: N = 16, *sweet2*: N = 19, *sweet4*: N = 20, *sweet11;12*: N = 21. +(B) Relative abundance of metabolite superclasses significantly (p value < 0.05) enriched and depleted in each root segment of sweet mutants compared with corresponding segments of WT. +(C) Heatmap depicting the enrichment (or depletion) of indicated metabolites in respective root segments for WT and sweet plants (abbreviated as *swt*). Heatmap generated based on average linkage Pearson hierarchical clustering of metabolites and root segments. Clusters are indicated in red roman numbers. Colors indicate the level of enrichment or depletion of each metabolite across the plant genotypes and root segments. + +**Table 2. Contribution of spatial distribution and/or plant genotype to the abundance of root metabolites** + +| **Metabolite** | **Superclass** | **sweet2** | **sweet4** | **sweet11;12** | +|--------------------|-------------------------|-------------------|-------------------|-------------------| +| Sterol 23.74 | sterol lipid | N/A | N/A | N/A | +| Sterol 23.16 | sterol lipid | genotype | genotype | genotype | +| Fructose | carbohydrate | genotype | genotype | genotype | +| Glucose | carbohydrate | genotype | genotype | genotype | +| Disaccharide 14.98 | carbohydrate | genotype | genotype | genotype | +| Sorbitol | carbohydrate | genotype | genotype | genotype | +| Sucrose | carbohydrate | genotype | genotype | genotype | +| Hexose 13.45 | carbohydrate | genotype | genotype | genotype | +| Disaccharide 15.98 | carbohydrate | genotype | genotype | genotype | +| Mannitol | carbohydrate | genotype | N/A | genotype | +| Disaccharide 14.35 | carbohydrate | N/A | genotype | N/A | +| Gluconate | carbohydrate | genotype, spatial | genotype | N/A | +| Hexose-P | carbohydrate | spatial | genotype | N/A | +| Glycerol | carbohydrate | genotype, spatial | genotype | spatial | +| Carb 11.60 | carbohydrate | spatial | spatial | spatial | +| Disaccharide 21.9 | carbohydrate | spatial | genotype | genotype, spatial | +| Glycerate | carbohydrate | spatial | genotype, spatial | genotype, spatial | +| 5-oxo-Proline | carbohydrate | genotype, spatial | genotype, spatial | genotype, spatial | +| Disaccharide 21.5 | carbohydrate | genotype, spatial | genotype, spatial | genotype, spatial | +| Raffinose | carbohydrate | genotype, spatial | genotype, spatial | N/A | +| Disaccharide 14.71 | carbohydrate | N/A | N/A | N/A | +| Disaccharide 20.5 | carbohydrate | N/A | N/A | N/A | +| Myoinositol | carbohydrate | N/A | N/A | N/A | +| Shikimate | organic oxygen compound | N/A | N/A | N/A | +| γ-Aminobutyrate | organic acid | N/A | N/A | N/A | +| Malate | organic acid | spatial | spatial | spatial | +| Ketoglutarate | organic acid | spatial | spatial | spatial | +| Fumarate | organic acid | spatial | spatial | spatial | +| Aspartate | organic acid | genotype, spatial | genotype, spatial | genotype, spatial | +| Isocitrate | organic acid | genotype, spatial | genotype, spatial | genotype, spatial | +| Glutamine | organic acid | genotype, spatial | genotype, spatial | genotype, spatial | +| Alanine | organic acid | genotype | genotype, spatial | genotype, spatial | +| Glycine | organic acid | genotype, spatial | genotype | genotype | +| Succinate | organic acid | genotype | genotype | genotype, spatial | +| Isoleucine | organic acid | genotype | genotype | genotype | +| Leucine | organic acid | genotype | genotype | genotype | +| Lysine | organic acid | genotype | genotype | genotype | +| Asparagine | organic acid | genotype | genotype | genotype | +| Methionine | organic acid | genotype | genotype | genotype | +| Phenylalanine | organic acid | genotype | genotype | genotype | +| Proline | organic acid | genotype | genotype | genotype | +| Pyruvate | organic acid | genotype | genotype | genotype | +| Serine | organic acid | genotype | genotype | genotype | +| Threonine | organic acid | genotype | genotype | genotype | +| Valine | organic acid | genotype | genotype | genotype | +| Glutamate | organic acid | genotype | genotype | genotype | + +**N/A: not applicable. The abundance of indicated metabolites is not significantly different from WT plants (two-way ANOVA, p > 0.05).** + +## Key Resources Table + +| **REAGENT or RESOURCE** | **SOURCE** | **IDENTIFIER** | +|-----------------------------------------------|------------------------------------------------------------------|-----------------------------------------------------------------------| +| **Deposited Data** | | | +| 16S sequencing data | This paper | https://www.ebi.ac.uk/ena/browser/home (Accession number: PRJEB63568) | +| Metabolomics data | This paper | https://www.metabolomicsworkbench.org/ (Study ID: ST002779) | +| Arabidopsis root spatial microarray | [https://doi.org/10.1126/science.1146265] | GEO dataset: GSE8934 ID: 200008934 | +| NIST14 Mass Spectral Library | https://www.nist.gov/srd/nist-standard-reference-database-1a-v14 | N/A | +| Arabidopsis thaliana Col-0 metabolic pathways | https://pmn.plantcyc.org/organism-summary?object=ARA | N/A | +| **Software and Algorithms** | | | +| iGraph | https://CRAN.R-project.org/package=igraph. | RRID:SCR_019225 | +| COBRApy | http://opencobra.sourceforge.net | RRID:SCR_012096 | +| GUROBI | https://www.gurobi.com/ | N/A | +| statsmodels | https://www.statsmodels.org/stable/index.html | RRID:SCR_016074 | +| Vegan | https://cran.r-project.org/web/packages/vegan/index.html | RRID:SCR_011950 | +| Qiime2 | https://qiime2.org/ | RRID:SCR_008249 | +| Flash2 | https://github.com/dstreett/FLASH2 | RRID:SCR_005531 | +| Dada2 | https://benjjneb.github.io/dada2 | RRID:SCR_008205 | +| **Others** | | | +| 16S data analyses | This work | GitHub https://github.com/duranpa/sweet_collaboration | \ No newline at end of file diff --git a/assays/ANOVAForDifferentialEnrichmentOfMetabolites/dataset/.gitkeep b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/ANOVAForDifferentialEnrichmentOfMetabolites/dataset/gr2.jpg b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/dataset/gr2.jpg new file mode 100644 index 0000000000000000000000000000000000000000..e5ef95b8cb4266095078b7c41e8a0ce33dcb52f3 --- /dev/null +++ b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/dataset/gr2.jpg @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:b15bdb9906a602f26a6ec87b116e6884804546fd5839e8f0b3d7bf2a0c85ebc3 +size 52917 diff --git a/assays/ANOVAForDifferentialEnrichmentOfMetabolites/dataset/gr4.jpg b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/dataset/gr4.jpg new file mode 100644 index 0000000000000000000000000000000000000000..aebf05a3a56716746c828335dba58582f5ad75d2 --- /dev/null +++ b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/dataset/gr4.jpg @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:0cd28b653835bb0a158b2da5dcdcb841e645286319519b729f8dedc490b31cd3 +size 102333 diff --git a/assays/ANOVAForDifferentialEnrichmentOfMetabolites/isa.assay.xlsx b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..494ab1bc2ff88fedd3f1e5794f1a89a3561c3b7c Binary files /dev/null and b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/isa.assay.xlsx differ diff --git a/assays/ANOVAForDifferentialEnrichmentOfMetabolites/protocols/.gitkeep b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/ANOVAForDifferentialEnrichmentOfMetabolites/protocols/ANOVAForDifferentialEnrichmentOfMetabolitesProtocol.md b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/protocols/ANOVAForDifferentialEnrichmentOfMetabolitesProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..941712ccb91229a3a43a4fba1216fe1b009d7ff4 --- /dev/null +++ b/assays/ANOVAForDifferentialEnrichmentOfMetabolites/protocols/ANOVAForDifferentialEnrichmentOfMetabolitesProtocol.md @@ -0,0 +1,3 @@ +## ANOVA for differential enrichment of metabolites + +We applied the python package *statsmodels* (Seabold, S. and Perktold, J., 2010) to perform two-way analysis of variance (ANOVA) (Girden, E., 1992). For a particular metabolite, we collected its mass spectrometry relative response in the WT or in sweet knockouts (line: WT vs. SWEET), and at different segments of the root (region: 2cm vs. 4cm vs. 6cm vs. whole). We fit a model that decomposes the relative response of the metabolite as a combination of line-specific and region-specific factors; this model is described by the following string in the code: *‘relativeResponse ∼ Line + Region'* and calculates a *P*-value for *Line* and one for *Region*. Using the *P*-value cutoff of 0.05 for *Line* (and *Region*), we determined the relative response of the metabolite to change significantly with *Line* (*Region*) if *P* <0.05. \ No newline at end of file diff --git a/assays/AmpliconSequencingDataAnalysis/README.md b/assays/AmpliconSequencingDataAnalysis/README.md new file mode 100644 index 0000000000000000000000000000000000000000..c3a56c53564599bba82ea6fec75f1e7eba02f118 --- /dev/null +++ b/assays/AmpliconSequencingDataAnalysis/README.md @@ -0,0 +1,28 @@ +<img src=./dataset/gr1.jpg width=60%> + +**Figure 1. Differential spatial colonization by root microbiota along the longitudinal root axis** + +(A) Phenotype of wild-type *A. thaliana* Col-0 grown on CD-Rhizotron, with root segments marked. CPCoA of the Bray-Curtis dissimilarities of endospheric (n = 63) or rhizosphere samples (n = 65), constrained by the distance to the tip from which they were harvested. Different colors represent respective longitudinal segments or whole root samples, with colors of clusters corresponding to the root segments depicted in the plant cartoon. Centroids are indicated for each cluster of root region. +(B) Phenotype of wild-type *A. thaliana* Col-0 grown on ArtSoil. CPCoA of the Bray-Curtis dissimilarities of root endosphere (n = 39) or rhizosphere samples (n = 40), constrained by the distance to the tip from which they were harvested. Colors of clusters correspond to segments of the root depicted in the plant cartoon. Centroids are indicated for each cluster of root region. + +## Key Resources Table + +| **REAGENT or RESOURCE** | **SOURCE** | **IDENTIFIER** | +|-----------------------------------------------|------------------------------------------------------------------|-----------------------------------------------------------------------| +| **Deposited Data** | | | +| 16S sequencing data | This paper | https://www.ebi.ac.uk/ena/browser/home (Accession number: PRJEB63568) | +| Metabolomics data | This paper | https://www.metabolomicsworkbench.org/ (Study ID: ST002779) | +| Arabidopsis root spatial microarray | [https://doi.org/10.1126/science.1146265] | GEO dataset: GSE8934 ID: 200008934 | +| NIST14 Mass Spectral Library | https://www.nist.gov/srd/nist-standard-reference-database-1a-v14 | N/A | +| Arabidopsis thaliana Col-0 metabolic pathways | https://pmn.plantcyc.org/organism-summary?object=ARA | N/A | +| **Software and Algorithms** | | | +| iGraph | https://CRAN.R-project.org/package=igraph. | RRID:SCR_019225 | +| COBRApy | http://opencobra.sourceforge.net | RRID:SCR_012096 | +| GUROBI | https://www.gurobi.com/ | N/A | +| statsmodels | https://www.statsmodels.org/stable/index.html | RRID:SCR_016074 | +| Vegan | https://cran.r-project.org/web/packages/vegan/index.html | RRID:SCR_011950 | +| Qiime2 | https://qiime2.org/ | RRID:SCR_008249 | +| Flash2 | https://github.com/dstreett/FLASH2 | RRID:SCR_005531 | +| Dada2 | https://benjjneb.github.io/dada2 | RRID:SCR_008205 | +| **Others** | | | +| 16S data analyses | This work | GitHub https://github.com/duranpa/sweet_collaboration | \ No newline at end of file diff --git a/assays/AmpliconSequencingDataAnalysis/dataset/.gitkeep b/assays/AmpliconSequencingDataAnalysis/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/AmpliconSequencingDataAnalysis/dataset/gr1.jpg b/assays/AmpliconSequencingDataAnalysis/dataset/gr1.jpg new file mode 100644 index 0000000000000000000000000000000000000000..c9524cc64426660003d0e32ee1e2bb3c35c564a4 --- /dev/null +++ b/assays/AmpliconSequencingDataAnalysis/dataset/gr1.jpg @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:e3a52b436d674bf4ed37fa19bfeee97f38209537f77736138344de5874257395 +size 86727 diff --git a/assays/AmpliconSequencingDataAnalysis/isa.assay.xlsx b/assays/AmpliconSequencingDataAnalysis/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..419398f43d7d6f9ede46238daf8a729f41599c39 Binary files /dev/null and b/assays/AmpliconSequencingDataAnalysis/isa.assay.xlsx differ diff --git a/assays/AmpliconSequencingDataAnalysis/protocols/.gitkeep b/assays/AmpliconSequencingDataAnalysis/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/AmpliconSequencingDataAnalysis/protocols/AmpliconSequencingDataAnalysisProtocol.md b/assays/AmpliconSequencingDataAnalysis/protocols/AmpliconSequencingDataAnalysisProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..057ea25347d00bffb5aee95e6b3ce34222f10d4e --- /dev/null +++ b/assays/AmpliconSequencingDataAnalysis/protocols/AmpliconSequencingDataAnalysisProtocol.md @@ -0,0 +1,3 @@ +## Amplicon sequencing data analysis + +Sequencing reads were demultiplexed using Qiime2 (qiime demux emp_paired) (Bolyen, E. et al., 2019), and merged using Flash2 (MagoÄ, T. and Salzberg, S.L., 2011). Reads were denoised and dereplicated using Dada2 (Callahan, B.J. et al., 2016) and remaining individual reads were denoted as ASVs. Chimeras were removed using Qiime2 (vsearch uchime-denovo). Taxonomic classification was done via the Qiime feature classifier using the silva_138 and sequences classified as mitochondrial or chloroplast were removed from the dataset. Remaining ASVs were included in count tables. Bray-Curtis dissimilarities between samples were calculated normalized ASV tables and performed PCAs using the cmdscale function (*vegan* R package). To quantify the effects of genotype and root region we used a constrained PCoA using the *capscale* function (*vegan* R package). To quantify the contribution of different variables and their interactions to the variance in pairwise Bray-Curtis dissimilarities, we analysed the Bray-Curtis distance matrix between pairs of samples with 999 iterations of a permutation-based test (permutational multivariate analysis of variance (PERMANOVA), adonis function, vegan R package), and removed technical and batch effects, using the formula as follows: *Bray-curtis ∼ VariableX + Condition(Technical_replicates + Biological_replicates)*. We further inspected the effects of the variables using a constrained PCoA using the capscale function (vegan R package). Sequencing data from the SynCom experiment was pre-processed similarly as natural community 16S rRNA data. Quality-filtered merged paired-end reads were aligned to a reference set of sequences extracted from the whole-genome assemblies of the 60 strains included in the SynCom using Rbec79 (v1.0.0). A count table that was employed for downstream analyses of diversity was generated in R (v4.0.3) with the R package vegan (v2.5–6). Scripts for microbiota analysis and data visualization can be found at https://github.com/duranpa/sweet_collaboration \ No newline at end of file diff --git a/assays/ConstructionOfGUSReporterLines/README.md b/assays/ConstructionOfGUSReporterLines/README.md new file mode 100644 index 0000000000000000000000000000000000000000..5b273388d5431ed557450987fc8f0a6109d7150e --- /dev/null +++ b/assays/ConstructionOfGUSReporterLines/README.md @@ -0,0 +1,38 @@ +**Table 1. SWEETs accumulation along the longitudinal region of roots from A. thaliana grown on ½ Murashige-Skoog (½ MS) media supplemented with or without sucrose, and ArtSoil** + +| **SWEET** | **½ MS − sucrose** | **½ MS + sucrose** | **ArtSoil** | +|:---------:|:------------------------------------:|--------------------------------------|------------------------| +| 1 | regions below 2 cm | regions below 2 cm | regions below 2 cm | +| 2 | regions below 0.05 cm and above 2 cm | regions below 0.05 cm and above 2 cm | entire root | +| 3 | not tested | not tested | not tested | +| 4 | not detected | regions above 2 cm (patchy) | not detected | +| 5 | not tested | not tested | not tested | +| 6 | not tested | not tested | not tested | +| 7 | not detected | not detected | not detected | +| 8 | not detected | not detected | not detected | +| 9 | not tested | not tested | not tested | +| 10 | not tested | not tested | not tested | +| 11 | regions below 2 cm | at 2.5–3 cm region | regions below 2 cm | +| 12 | regions below 2 cm | regions 2 cm and above | regions 2 cm and above | +| 13 | regions below 2.5 cm | regions below 2.5 cm | regions below 2.5 cm | +| 14 | not tested | not tested | not tested | +| 15 | not tested | not tested | not tested | +| 16 | not tested | not tested | not tested | +| 17 | entire root | entire root | entire root | + +## Key Resources Table + +| **REAGENT or RESOURCE** | **SOURCE** | **IDENTIFIER** | +|-----------------------------|-------------------------------------------------------------|-------------------------------------------| +| **Oligonucleotides** | | | +| AtSWEET4-XbaI-F | IDT | GCAGGTCGACTCTAGAAGTGGTTCCACG GAGATGACG | +| AtSWEET4-BamHI-R | IDT | CGGTACCCGGGGATCCAGCTGAAACTCG TTTAGCTTGTCC | +| AtSWEET5-XbaI-F | IDT | GCAGGTCGACTCTAGATTAGGACTGACAC CAGCGATGC | +| AtSWEET5-BamHI-R | IDT | CGGTACCCGGGGATCCAGCCTGGCCAAG TTCGATTC | +| AtSWEET7-XbaI-F | IDT | GCAGGTCGACTCTAGAATTCAGGCTTGGC GTAACTTG | +| AtSWEET7-BamHI-R | IDT | CGGTACCCGGGGATCCAACATTGTTAGGT TCTTGGTTGG | +| AtSWEET8-XbaI-F | IDT | GCAGGTCGACTCTAGAACCATGACAATTT GGCTCCGAG | +| AtSWEET8-BamHI-R | IDT | CGGTACCCGGGGATCCAACCCTCTCCGT AGCAGAAATC | +| **Recombinant DNA** | | | +| pMDC163 vector | Arabidopsis Biological Resource Center https://abrc.osu.edu | TAIR accession Vector:1009003758 | +| promoterless GUSplus vector | Yang et al.81 | N/A | \ No newline at end of file diff --git a/assays/ConstructionOfGUSReporterLines/dataset/.gitkeep b/assays/ConstructionOfGUSReporterLines/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/ConstructionOfGUSReporterLines/isa.assay.xlsx b/assays/ConstructionOfGUSReporterLines/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..451d99bd725ca119119860e062d7ee70debdc103 Binary files /dev/null and b/assays/ConstructionOfGUSReporterLines/isa.assay.xlsx differ diff --git a/assays/ConstructionOfGUSReporterLines/protocols/.gitkeep b/assays/ConstructionOfGUSReporterLines/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/ConstructionOfGUSReporterLines/protocols/ConstructionOfTranslationalGUSReporterLines.md b/assays/ConstructionOfGUSReporterLines/protocols/ConstructionOfTranslationalGUSReporterLines.md new file mode 100644 index 0000000000000000000000000000000000000000..a5b632e2ab24395393c97dadf55157d7d14e8ea3 --- /dev/null +++ b/assays/ConstructionOfGUSReporterLines/protocols/ConstructionOfTranslationalGUSReporterLines.md @@ -0,0 +1,3 @@ +## Construction of translational GUS reporter lines + +Genomic fragments comprising the 5′-upstream region and the entire coding region of *SWEET1, SWEET4, SWEET5, SWEET7, SWEET8, SWEET10,* and *SWEET13* were amplified by PCR using wild-type *A. thaliana* Col-0 genomic DNA as a template (primers for each gene are listed in Key Resource Table). The amplified products of *SWEET4, SWEET5, SWEET7, SWEET8*, and *SWEET10* were cloned into promoterless GUSplus vector (Yang, J. et al., 2018) while *SWEET1* and *SWEET13* were cloned into pMDC163 using the Gateway cloning system. All constructs were confirmed by sequencing. Wild-type *A. thaliana* Col-0 were transformed by *Agrobacterium tumefaciens* harboring SWEET-GUS constructs using the floral dip method. Transgenic seedlings were selected on ½ MS media supplemented with 1% sucrose and 25 μg/mL hygromycin. Transgenic *SWEET2-GUS, SWEET11-GUS, SWEET12-GUS* and *SWEET17-GUS* were previously reported in Chen et al. (2012), Chen et al. (2015), and Guo et al. (2014). \ No newline at end of file diff --git a/assays/CorrelationBetweenMicrobesAndMetabolites/README.md b/assays/CorrelationBetweenMicrobesAndMetabolites/README.md new file mode 100644 index 0000000000000000000000000000000000000000..3916c9db06ba78b6a3183986eaf7554e2b6d2eaa --- /dev/null +++ b/assays/CorrelationBetweenMicrobesAndMetabolites/README.md @@ -0,0 +1,29 @@ +<img stc=./dataset/gr5.jpg width=60%> + +**Figure 5 Loss of spatial microbiota colonization in roots of *sweet2, sweet4*, and *sweet11;12*** + +(A) CPCoA of the Bray-Curtis dissimilarities of endospheric bacteria derived from full-length roots of WT, *sweet2, sweet4*, and *sweet11;12* plants, constrained by plant genotype. Different colors represent plant genotypes with centroids indicated for each cluster of genotype. WT: N = 60, *sweet2*: N = 40, *sweet4*: N = 41, *sweet11;12* and N = 42. +(B) Bray-Curtis dissimilarities distances of endospheric bacteria derived from the 0–2, 2–4, and 4–6 cm segments from the root tip of WT, *sweet2, sweet4*, and *sweet11;12* plants (sweet abbreviated as “swtâ€) compared with each other. The colors and sizes of the dots correspond to the mean of the Bray-Curtis distance. +(C) Spearman correlation for metabolite-microbe (class level) pairs. Metabolites ranked based on Spearman average hierarchical clustering. The color of each point indicates the degree of correlation. Highlights indicate the class of metabolite: blue, sugar/carbohydrate; red, organic acid; and yellow, lipid. Spearman correlations significance, p < 0.05. + +## Key Resources Table + +| **REAGENT or RESOURCE** | **SOURCE** | **IDENTIFIER** | +|-----------------------------------------------|------------------------------------------------------------------|-----------------------------------------------------------------------| +| **Deposited Data** | | | +| 16S sequencing data | This paper | https://www.ebi.ac.uk/ena/browser/home (Accession number: PRJEB63568) | +| Metabolomics data | This paper | https://www.metabolomicsworkbench.org/ (Study ID: ST002779) | +| Arabidopsis root spatial microarray | [https://doi.org/10.1126/science.1146265] | GEO dataset: GSE8934 ID: 200008934 | +| NIST14 Mass Spectral Library | https://www.nist.gov/srd/nist-standard-reference-database-1a-v14 | N/A | +| Arabidopsis thaliana Col-0 metabolic pathways | https://pmn.plantcyc.org/organism-summary?object=ARA | N/A | +| **Software and Algorithms** | | | +| iGraph | https://CRAN.R-project.org/package=igraph. | RRID:SCR_019225 | +| COBRApy | http://opencobra.sourceforge.net | RRID:SCR_012096 | +| GUROBI | https://www.gurobi.com/ | N/A | +| statsmodels | https://www.statsmodels.org/stable/index.html | RRID:SCR_016074 | +| Vegan | https://cran.r-project.org/web/packages/vegan/index.html | RRID:SCR_011950 | +| Qiime2 | https://qiime2.org/ | RRID:SCR_008249 | +| Flash2 | https://github.com/dstreett/FLASH2 | RRID:SCR_005531 | +| Dada2 | https://benjjneb.github.io/dada2 | RRID:SCR_008205 | +| **Others** | | | +| 16S data analyses | This work | GitHub https://github.com/duranpa/sweet_collaboration | \ No newline at end of file diff --git a/assays/CorrelationBetweenMicrobesAndMetabolites/dataset/.gitkeep b/assays/CorrelationBetweenMicrobesAndMetabolites/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/CorrelationBetweenMicrobesAndMetabolites/dataset/gr5.jpg b/assays/CorrelationBetweenMicrobesAndMetabolites/dataset/gr5.jpg new file mode 100644 index 0000000000000000000000000000000000000000..ad6ed7fb7cad7f2cc0245deb59ef4fec37781018 --- /dev/null +++ b/assays/CorrelationBetweenMicrobesAndMetabolites/dataset/gr5.jpg @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:19d713ddc403f0a0b78434f3fe1f2d8cd92baeee6ca56369d24d84a6eff77ea2 +size 146839 diff --git a/assays/CorrelationBetweenMicrobesAndMetabolites/isa.assay.xlsx b/assays/CorrelationBetweenMicrobesAndMetabolites/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..26c80915aaa6b89dfd9230a9eaa195eb409534c3 Binary files /dev/null and b/assays/CorrelationBetweenMicrobesAndMetabolites/isa.assay.xlsx differ diff --git a/assays/CorrelationBetweenMicrobesAndMetabolites/protocols/.gitkeep b/assays/CorrelationBetweenMicrobesAndMetabolites/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/CorrelationBetweenMicrobesAndMetabolites/protocols/CorrelationBetweenMicrobesAndMetabolitesProtocol.md b/assays/CorrelationBetweenMicrobesAndMetabolites/protocols/CorrelationBetweenMicrobesAndMetabolitesProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..e1e95280fb934ffe41e60e8aaffa0d383ab5bc80 --- /dev/null +++ b/assays/CorrelationBetweenMicrobesAndMetabolites/protocols/CorrelationBetweenMicrobesAndMetabolitesProtocol.md @@ -0,0 +1,7 @@ +## Correlation between microbes and metabolites + +We calculated the correlation between every pair of microbes and metabolites to reveal the potential cross feeding between the microbe species. At each combination of plant lineages (WT / SWEET2 / SWEET4 / SWEET11,22) and root segments (2cm / 4cm / 6cm / whole root), we performed 16s RNA sequencing to quantify the abundance of different microbial classes and also mass spectrometry to measure the abundance of different metabolites. + +For each pair of microbe class and metabolite, we calculated their spearman correlation across different plant lineages and root locations to construct the correlation matrix; matrix elements that correspond to p-value >0.05 are deemed insignificant and replaced with 0 (i.e., not connected in a complex network). We used the iGraph package of R to display this bipartite network (Csárdi, G. et al., 2023). Communities within this network are detected using the label propagation algorithm (Raghavan, U.N., 2007). + +Reference ID: KRT64abe6afd69ae \ No newline at end of file diff --git a/assays/DNAExtractionAndLibraryPreparation/README.md b/assays/DNAExtractionAndLibraryPreparation/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/DNAExtractionAndLibraryPreparation/dataset/.gitkeep b/assays/DNAExtractionAndLibraryPreparation/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/DNAExtractionAndLibraryPreparation/isa.assay.xlsx b/assays/DNAExtractionAndLibraryPreparation/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..427dbbe1b5abae77a96120ea6c569e1a5922d27a Binary files /dev/null and b/assays/DNAExtractionAndLibraryPreparation/isa.assay.xlsx differ diff --git a/assays/DNAExtractionAndLibraryPreparation/protocols/.gitkeep b/assays/DNAExtractionAndLibraryPreparation/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/DNAExtractionAndLibraryPreparation/protocols/DNAExtractionAndLibraryPreparationProtocol.md b/assays/DNAExtractionAndLibraryPreparation/protocols/DNAExtractionAndLibraryPreparationProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..8d0264565ce49225fabf96a4836c85aedf6040b4 --- /dev/null +++ b/assays/DNAExtractionAndLibraryPreparation/protocols/DNAExtractionAndLibraryPreparationProtocol.md @@ -0,0 +1,3 @@ +## DNA extraction and library preparation + +Total DNA was extracted from the aforementioned samples using the FastDNAâ„¢ SPIN Kit for Soil (MP Biomedicals, Solon, USA) or NuceloSpin Soil Mini kit for DNA from soil (Macherey-Nagel GmbH & Co. KG, Düren, Germany) following instructions from the manufacturers. DNA samples were eluted in 50 μL nuclease-free water and used for microbial community profiling. DNA samples were used in a two-step PCR amplification protocol. In the first step, V4-V7 (799F: AACMGGATTAGATACCCKG; 1192R: ACGTCATCCCCACCTTCC) of the bacterial 16S rRNA, was amplified. Under a sterile hood, each sample was amplified in triplicate in a 25 μL reaction volume containing 2 U DFS-Taq DNA polymerase, 1× incomplete buffer (Bioron GmbH, Ludwigshafen, Germany), 2 mM MgCl2, 0.3% BSA, 0.2 mM dNTPs (Life technologies GmbH, Darmstadt, Germany) and 0.3 μM forward and reverse primers. PCR was performed using the following parameters: 94 °C/2 min, 94 °C/30 s, 55 °C/30 s, 72 °C/30 s, 72 °C/10 min for 25 cycles. Afterwards, single-stranded DNA and proteins were digested by adding 1 μL of Antarctic phosphatase, 1 μL Exonuclease I, and 2.44 μL Antarctic phosphatase buffer (New England BioLabs GmbH, Frankfurt, Germany) to 20 μl of the pooled PCR product. Samples were incubated at 37 °C for 30 min and enzymes were deactivated at 85 °C for 15 min. Samples were centrifuged for 10 min at 3,000 × g and 3 μL of this reaction were used for a second PCR, prepared in the same way as described above using the same protocol but with cycles reduced to 10 and with primers including barcodes and Illumina adapters (Thiergart, T. et al., 2020). PCR quality was controlled by loading 5 μL of each reaction on a 1% agarose gel and affirming that no band was detected within the negative control. Amplicon concentration was determined fluorescently (Quant-iTâ„¢ PicoGreenâ„¢, Invitrogen), and equivalent DNA amounts of each of the barcoded amplicons were pooled in one library. Then, 80 μL of the pooled library was loaded in a 1.5% agarose gel and run for 2 h at 80 V. Subsequently, bands with a size of ∼500 bp were cut out and purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany). The final library concentration was estimated fluorescently (Quantusâ„¢ Fluorometer, Promega). Paired-end Illumina sequencing was performed in-house using the MiSeq sequencer and custom sequencing primers at the Max Planck Institute for Plant Breeding Research. \ No newline at end of file diff --git a/assays/FluxBalanceAnalysisToDefinePathways/README.md b/assays/FluxBalanceAnalysisToDefinePathways/README.md new file mode 100644 index 0000000000000000000000000000000000000000..8d56edfaa6d82ee0abdde738ec47492803801e47 --- /dev/null +++ b/assays/FluxBalanceAnalysisToDefinePathways/README.md @@ -0,0 +1,21 @@ +## Key Resources Table + +| **REAGENT or RESOURCE** | **SOURCE** | **IDENTIFIER** | +|-----------------------------------------------|------------------------------------------------------------------|-----------------------------------------------------------------------| +| **Deposited Data** | | | +| 16S sequencing data | This paper | https://www.ebi.ac.uk/ena/browser/home (Accession number: PRJEB63568) | +| Metabolomics data | This paper | https://www.metabolomicsworkbench.org/ (Study ID: ST002779) | +| Arabidopsis root spatial microarray | [https://doi.org/10.1126/science.1146265] | GEO dataset: GSE8934 ID: 200008934 | +| NIST14 Mass Spectral Library | https://www.nist.gov/srd/nist-standard-reference-database-1a-v14 | N/A | +| Arabidopsis thaliana Col-0 metabolic pathways | https://pmn.plantcyc.org/organism-summary?object=ARA | N/A | +| **Software and Algorithms** | | | +| iGraph | https://CRAN.R-project.org/package=igraph. | RRID:SCR_019225 | +| COBRApy | http://opencobra.sourceforge.net | RRID:SCR_012096 | +| GUROBI | https://www.gurobi.com/ | N/A | +| statsmodels | https://www.statsmodels.org/stable/index.html | RRID:SCR_016074 | +| Vegan | https://cran.r-project.org/web/packages/vegan/index.html | RRID:SCR_011950 | +| Qiime2 | https://qiime2.org/ | RRID:SCR_008249 | +| Flash2 | https://github.com/dstreett/FLASH2 | RRID:SCR_005531 | +| Dada2 | https://benjjneb.github.io/dada2 | RRID:SCR_008205 | +| **Others** | | | +| 16S data analyses | This work | GitHub https://github.com/duranpa/sweet_collaboration | \ No newline at end of file diff --git a/assays/FluxBalanceAnalysisToDefinePathways/dataset/.gitkeep b/assays/FluxBalanceAnalysisToDefinePathways/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/FluxBalanceAnalysisToDefinePathways/isa.assay.xlsx b/assays/FluxBalanceAnalysisToDefinePathways/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..c45891c2d7d1db9e35c8b3828c28ce5aaabebe52 Binary files /dev/null and b/assays/FluxBalanceAnalysisToDefinePathways/isa.assay.xlsx differ diff --git a/assays/FluxBalanceAnalysisToDefinePathways/protocols/.gitkeep b/assays/FluxBalanceAnalysisToDefinePathways/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/FluxBalanceAnalysisToDefinePathways/protocols/FluxBalanceAnalysisToDefinePathwaysProtocol.md b/assays/FluxBalanceAnalysisToDefinePathways/protocols/FluxBalanceAnalysisToDefinePathwaysProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..d9d3d1c35c2cd54256cbf165792d15448f9def04 --- /dev/null +++ b/assays/FluxBalanceAnalysisToDefinePathways/protocols/FluxBalanceAnalysisToDefinePathwaysProtocol.md @@ -0,0 +1,9 @@ +## Flux balance analysis (FBA) to define pathways + +As we suspect that the root may convert sucrose into other sugars before secretion, we would like to find out the genes involved in these conversion pathways for further analysis. To assign genes in the metabolic network to these pathways, we applied FBA (Orth, J.D. et al., 2010), which searches for the combination of genes and reactions that results in the highest yield for such conversions, assuming sucrose to be the sole input carbon source of the metabolic network. + +We used the AraGEM (de Oliveira Dal’Molin, C.G. et al., 2009) a curated FBA model of *Arabidopsis*, to perform our simulations and define the genes associated with each conversion pathway. The AraGEM model is published with 3 different sets of parameters that correspond to their own case of metabolism: (a) photosynthesis, (b) photorespiration, and (c) non-photosynthetic cell. Case (c) is relevant to the root condition, as it considers the conversion of sucrose into other biomass metabolites. Hence, our simulations are developed from case (c), which uses sucrose as the input carbon source. + +We switched off the default objective function of AraGEM, which includes a set of metabolites with weights found in real plants. To investigate the conversion of sucrose into different sugars, we set different monosaccharides, disaccharides and combinations of monosaccharides as the objective; these include all possible monosaccharides and disaccharides in AraGEM. In total, we analyzed 11 different objective functions: (1) maltose, (2) glucose, (3) beta-D-fructose, (4) D-galactose, (5) D-xylose, (6) glucose + beta-D-fructose, (7) glucose + D-galactose, (8) glucose + D-xylose, (9) beta-D-fructose + D-galactose, (10) beta-D-fructose + D-xylose, (11) D-galactose + D-xylose. Note that the molecules within objective functions have equal weights, as some of them have multiple sugar molecules. + +We used the python package COBRApy (Ebrahim, A. et al., 2013) along with the GUROBI solver (Gurobi Optimizer Version 3.0. Houston, Texas: Gurobi Optimization, Inc., April 2010. (software program)) to perform FBA simulation. For each of the 11 objective functions, we performed parsimony-FBA simulation (Holzhütter, H.-G., 2004) to find out the most efficient reactions that perform the conversion of sucrose into the objective sugars. We also calculated the shadow price (Reznik, E., 2013) of each metabolite involved in these reactions; a metabolite with negative shadow price is deemed critical, as an extra supply of this metabolite leads to a higher flux of the objective function. Reactions that have critical metabolites both at their input and output are also considered critical. We collected all the critical reactions of an objective function, and the genes associated with any critical reactions are assigned to the pathway (refer to Table S6 for the genes in these pathways). \ No newline at end of file diff --git a/assays/GUSHistochemicalStaining/README.md b/assays/GUSHistochemicalStaining/README.md new file mode 100644 index 0000000000000000000000000000000000000000..81a7b249834cdc2c25ba7d01a82f86f8e9bae95a --- /dev/null +++ b/assays/GUSHistochemicalStaining/README.md @@ -0,0 +1,5 @@ +<img src=./dataset/gr3.jpg width=60%> + +**Figure 3. Accumulation of SWEETs along the root in the absence or presence of sugar and soil microbes** + +Accumulation of respective SWEETs fused with a translational GUS reporter gene in 14-days-old A. thaliana seedlings grown on ½ MS-salt media supplemented with or without 1% sucrose (½ MS-salt −suc: ½ MS media without sucrose supplementation, ½ MS-salt +suc: ½ MS media with sucrose supplementation), on microbiome-inoculated or microbiome-killed ArtSoil (ArtSoil −microbes: ArtSoil inoculated with heat-killed soil microbes, ArtSoil +microbes: ArtSoil inoculated with soil microbes). The root segment depicted for SWEET2-GUS is <0.2 cm, and the root segments depicted for SWEET12-GUS and SWEET4-GUS are >2 cm. Scale bars, 500 μm. The yellow arrow indicates the accumulation of SWEET2-GUS in the root zone of interest. Green arrows indicate the sporadic accumulation of SWEET4-GUS. Representative image for each genotype from N > 10. \ No newline at end of file diff --git a/assays/GUSHistochemicalStaining/dataset/.gitkeep b/assays/GUSHistochemicalStaining/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/GUSHistochemicalStaining/dataset/gr3.jpg b/assays/GUSHistochemicalStaining/dataset/gr3.jpg new file mode 100644 index 0000000000000000000000000000000000000000..b169b63ed9c1b64a3bf052e20dc7e3e3d96bba0d --- /dev/null +++ b/assays/GUSHistochemicalStaining/dataset/gr3.jpg @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:4a0dfcce1170dcd48b8e5fd06f5bf2006398d122c5ebee0f7c02c469c78123f9 +size 49348 diff --git a/assays/GUSHistochemicalStaining/isa.assay.xlsx b/assays/GUSHistochemicalStaining/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..e1070256789273cff09c47b2acb18eb0a5a6ee89 Binary files /dev/null and b/assays/GUSHistochemicalStaining/isa.assay.xlsx differ diff --git a/assays/GUSHistochemicalStaining/protocols/.gitkeep b/assays/GUSHistochemicalStaining/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/GUSHistochemicalStaining/protocols/GUSHistochemicalStainingProtocol.md b/assays/GUSHistochemicalStaining/protocols/GUSHistochemicalStainingProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..e41f48d05196e728047d4375e4ca2e29da850da9 --- /dev/null +++ b/assays/GUSHistochemicalStaining/protocols/GUSHistochemicalStainingProtocol.md @@ -0,0 +1,3 @@ +## GUS histochemical staining + +Three-week-old plants were carefully removed from ArtSoil and rinsed with 70% ethanol. Each plant was inserted into one well in a 12-well plate before 1 mL of GUS staining solution (10 mM EDTA, 50 mM phosphate buffer, 0.1% Triton X-100, 1 mM potassium ferrocyanide, 1 mM potassium ferricyanide, 2 mM X-Gluc, 20% methanol) was added into each well. The plate was subsequently incubated in the dark at 37 °C for 1 h (SWEET1, 2, 17) or 4 h (SWEET4, 7, 8, 11, 12, 13). Prior to imaging, plants were removed from GUS staining buffer and rinsed with 70% ethanol. \ No newline at end of file diff --git a/assays/MicrobiotaProfiling/README.md b/assays/MicrobiotaProfiling/README.md new file mode 100644 index 0000000000000000000000000000000000000000..8d56edfaa6d82ee0abdde738ec47492803801e47 --- /dev/null +++ b/assays/MicrobiotaProfiling/README.md @@ -0,0 +1,21 @@ +## Key Resources Table + +| **REAGENT or RESOURCE** | **SOURCE** | **IDENTIFIER** | +|-----------------------------------------------|------------------------------------------------------------------|-----------------------------------------------------------------------| +| **Deposited Data** | | | +| 16S sequencing data | This paper | https://www.ebi.ac.uk/ena/browser/home (Accession number: PRJEB63568) | +| Metabolomics data | This paper | https://www.metabolomicsworkbench.org/ (Study ID: ST002779) | +| Arabidopsis root spatial microarray | [https://doi.org/10.1126/science.1146265] | GEO dataset: GSE8934 ID: 200008934 | +| NIST14 Mass Spectral Library | https://www.nist.gov/srd/nist-standard-reference-database-1a-v14 | N/A | +| Arabidopsis thaliana Col-0 metabolic pathways | https://pmn.plantcyc.org/organism-summary?object=ARA | N/A | +| **Software and Algorithms** | | | +| iGraph | https://CRAN.R-project.org/package=igraph. | RRID:SCR_019225 | +| COBRApy | http://opencobra.sourceforge.net | RRID:SCR_012096 | +| GUROBI | https://www.gurobi.com/ | N/A | +| statsmodels | https://www.statsmodels.org/stable/index.html | RRID:SCR_016074 | +| Vegan | https://cran.r-project.org/web/packages/vegan/index.html | RRID:SCR_011950 | +| Qiime2 | https://qiime2.org/ | RRID:SCR_008249 | +| Flash2 | https://github.com/dstreett/FLASH2 | RRID:SCR_005531 | +| Dada2 | https://benjjneb.github.io/dada2 | RRID:SCR_008205 | +| **Others** | | | +| 16S data analyses | This work | GitHub https://github.com/duranpa/sweet_collaboration | \ No newline at end of file diff --git a/assays/MicrobiotaProfiling/dataset/.gitkeep b/assays/MicrobiotaProfiling/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/MicrobiotaProfiling/isa.assay.xlsx b/assays/MicrobiotaProfiling/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..bb8b0147b40fc1c4676a77fee60739b402f00df5 Binary files /dev/null and b/assays/MicrobiotaProfiling/isa.assay.xlsx differ diff --git a/assays/MicrobiotaProfiling/protocols/.gitkeep b/assays/MicrobiotaProfiling/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/MicrobiotaProfiling/protocols/MicrobiotaProfilingProtocol.md b/assays/MicrobiotaProfiling/protocols/MicrobiotaProfilingProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..02284f831a4cab50e95573fd9ad6d9ecc601e805 --- /dev/null +++ b/assays/MicrobiotaProfiling/protocols/MicrobiotaProfilingProtocol.md @@ -0,0 +1,3 @@ +## Microbiota profiling + +Roots of 3-weeks-old plants grown via the CD-Rhizotrons were cut into 2-cm segments as described in “Root metabolite profiling†(Figures S1E and S1F). Each root segment was manually separated from the surrounding soil leaving only tightly adhered soil particles. The roots were then placed in 15 mL centrifuge tubes filled with 10 mL of deionized sterile water and inverted ten times to further displace residual soil from the roots. To harvest the root (endospheric) fraction, the roots were transferred to clean centrifuge tubes filled with 10 mL of detergent (0.1% Triton X-100 diluted in 1x TE) and shaken for two minutes. The roots were then transferred into clean centrifuge tubes filled with 10 mL of 80% ethanol and shaken for 30 seconds. The same step was repeated but replacing 80% ethanol with 2% bleach. Finally, roots were rinsed three times with deionized sterile water before they were dried on sterile filter papers and flash-frozen in liquid nitrogen and stored at -80 °C until further processing. To harvest the rhizospheric fraction, the soil wash-offs were centrifuged at 3000 × g for 10 min. The supernatants were discarded and the pellets were resuspended and transferred to clean 2 mL screwcap tubes. The tubes were centrifuged at 3000 × g for 10 min, after which the supernatant was discarded, and the pellets were flash-frozen in liquid nitrogen and stored in -80 °C for further processing. The similar protocol was applied for harvesting the root fraction of plants grown on ArtSoil. For rhizospheric fraction of plants grown on ArtSoil, a clean scalpel was used to scrape approximately 0.5 cm (wide) of agar along the surface where the roots grew. The agar scraps in clean microcentrifuge tubes were flash-frozen and stored at -80 °C for further processing. \ No newline at end of file diff --git a/assays/QuantifyMetabolicActivitiesUsingPAS/README.md b/assays/QuantifyMetabolicActivitiesUsingPAS/README.md new file mode 100644 index 0000000000000000000000000000000000000000..8d56edfaa6d82ee0abdde738ec47492803801e47 --- /dev/null +++ b/assays/QuantifyMetabolicActivitiesUsingPAS/README.md @@ -0,0 +1,21 @@ +## Key Resources Table + +| **REAGENT or RESOURCE** | **SOURCE** | **IDENTIFIER** | +|-----------------------------------------------|------------------------------------------------------------------|-----------------------------------------------------------------------| +| **Deposited Data** | | | +| 16S sequencing data | This paper | https://www.ebi.ac.uk/ena/browser/home (Accession number: PRJEB63568) | +| Metabolomics data | This paper | https://www.metabolomicsworkbench.org/ (Study ID: ST002779) | +| Arabidopsis root spatial microarray | [https://doi.org/10.1126/science.1146265] | GEO dataset: GSE8934 ID: 200008934 | +| NIST14 Mass Spectral Library | https://www.nist.gov/srd/nist-standard-reference-database-1a-v14 | N/A | +| Arabidopsis thaliana Col-0 metabolic pathways | https://pmn.plantcyc.org/organism-summary?object=ARA | N/A | +| **Software and Algorithms** | | | +| iGraph | https://CRAN.R-project.org/package=igraph. | RRID:SCR_019225 | +| COBRApy | http://opencobra.sourceforge.net | RRID:SCR_012096 | +| GUROBI | https://www.gurobi.com/ | N/A | +| statsmodels | https://www.statsmodels.org/stable/index.html | RRID:SCR_016074 | +| Vegan | https://cran.r-project.org/web/packages/vegan/index.html | RRID:SCR_011950 | +| Qiime2 | https://qiime2.org/ | RRID:SCR_008249 | +| Flash2 | https://github.com/dstreett/FLASH2 | RRID:SCR_005531 | +| Dada2 | https://benjjneb.github.io/dada2 | RRID:SCR_008205 | +| **Others** | | | +| 16S data analyses | This work | GitHub https://github.com/duranpa/sweet_collaboration | \ No newline at end of file diff --git a/assays/QuantifyMetabolicActivitiesUsingPAS/dataset/.gitkeep b/assays/QuantifyMetabolicActivitiesUsingPAS/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/QuantifyMetabolicActivitiesUsingPAS/isa.assay.xlsx b/assays/QuantifyMetabolicActivitiesUsingPAS/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..d066c9beec6c35237190e3931542c2434cc6e963 Binary files /dev/null and b/assays/QuantifyMetabolicActivitiesUsingPAS/isa.assay.xlsx differ diff --git a/assays/QuantifyMetabolicActivitiesUsingPAS/protocols/.gitkeep b/assays/QuantifyMetabolicActivitiesUsingPAS/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/QuantifyMetabolicActivitiesUsingPAS/protocols/QuantifyMetabolicActivitiesUsingPASProtocol.md b/assays/QuantifyMetabolicActivitiesUsingPAS/protocols/QuantifyMetabolicActivitiesUsingPASProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..856ea3e406629d48263ffd920b457aa75299b0b1 --- /dev/null +++ b/assays/QuantifyMetabolicActivitiesUsingPAS/protocols/QuantifyMetabolicActivitiesUsingPASProtocol.md @@ -0,0 +1,13 @@ +## Quantify metabolic activities using PAS + +Pathway activity score (PAS) was introduced to quantify the activity of different metabolic pathways in single-cell transcriptomes (Xiao, Z. et al., 2019) (Kim, J.-Y. et al., 2021). It is designed with a permutation test along with a P-value to examine whether the gene expression of a pathway at a particular cell cluster is significantly higher or lower than the sample average. Since we are working with bulk RNA-seq and not scRNA-seq in the current study, this algorithm is modified, and the permutation test is no longer suitable and discarded. + +There are 3 sources for pathways used in our analysis. (A) we obtained the pathway data of Arabidopsis from PlantCyc (Hawkins, C. et al., 2021) (link to the tables https://pmn.plantcyc.org/organism-summary?object=ARA, downloaded on 18-NOV-2022). Pathways with patterns ‘glucos’, ‘galactos’, ‘fructos’, ‘xylos’, ‘sucros’, or ‘maltos’ in their name are considered relevant to sugar metabolism. (B) we are also interested in the potential to convert sucrose coming from the phloem into different sugars for secretion at different root segments, and these pathways are not clearly defined in PlantCyc. Thus, we applied flux balanced analysis to define the pathways of relevant metabolic reactions. See section ‘Flux balance analysis (FBA) to define pathways’ for the details of the algorithm. Table S6 shows the genes involved in each pathway. (C) we also assign the SWEET and SUC genes to their own single-gene-pathway to facilitate our analysis. This means the gene SWEET1 is assigned to a new pathway ‘SWEET1’, and so on. + +Brady et al. performed bulk transcriptomics on different segments of Arabidopsis root (Brady, S.M. et al., 2007). The experiment involved two replicate roots. In the published dataset, the 13 segments of root 1 are labeled as: *LCOLUMELLASB, L1SB, L2SB, …, L11SB, L12SB*; the 12 segments of root 2 are labeled as: *Slice1JW, Slice2JW, …, Slice11JW, Slice12JW*. We found that the two roots are in different developmental stages, therefore we dropped root 2 and used only root 1 in our analysis. + +Given the matrix of gene expression across different samples, we normalized the data using trimmed mean of M values (TMM) normalization (Robinson, M.D. and Oshlack, A., 2010). In practice, this is implemented by the function *calcNormFactors* within the R package *edgeR* (Robinson, M.D., 2010). We used the function argument *method="TMM"* to call TMM and set *logratioTrim=0.3*. + +Let us denote gi,j to be the normalized read count of gene i in sample j. The read count of a gene is normalized to give the relative transcript level, which is 1 when averaged over different samples. Mathematically, the relative transcript level of gene i at sample j is denoted as ri,j, and is defined as ð‘Ÿð‘–,ð‘—=ð‘”ð‘–,ð‘—/(1/ð‘*â¢âˆ‘ð‘˜ð‘”ð‘–,ð‘˜), where N is the total number of samples, and the label k goes over all samples. The PAS of pathway t at sample j is denoted as pt,j, which is a weighted average of the relative transcript levels across the genes of the pathway: ð‘ð‘¡,ð‘—=∑ð‘šð‘¡ +ð‘–=1ð‘¤ð‘–â¢ð‘Ÿð‘–,ð‘—/∑ð‘šð‘¡ +ð‘–=1ð‘¤ð‘–. Here mt is the number of genes in pathway t, and wi is the weight of gene i, defined as the reciprocal of the number of pathways that gene i is involved in. Because ri,j is centered around 1, so do pt,j. Thus, if pt,j>1, the expression of genes associated with pathway t in sample j is higher than the average over all samples, and vice versa. \ No newline at end of file diff --git a/assays/RootMetaboliteProfiling/README.md b/assays/RootMetaboliteProfiling/README.md index e69de29bb2d1d6434b8b29ae775ad8c2e48c5391..8d56edfaa6d82ee0abdde738ec47492803801e47 100644 --- a/assays/RootMetaboliteProfiling/README.md +++ b/assays/RootMetaboliteProfiling/README.md @@ -0,0 +1,21 @@ +## Key Resources Table + +| **REAGENT or RESOURCE** | **SOURCE** | **IDENTIFIER** | +|-----------------------------------------------|------------------------------------------------------------------|-----------------------------------------------------------------------| +| **Deposited Data** | | | +| 16S sequencing data | This paper | https://www.ebi.ac.uk/ena/browser/home (Accession number: PRJEB63568) | +| Metabolomics data | This paper | https://www.metabolomicsworkbench.org/ (Study ID: ST002779) | +| Arabidopsis root spatial microarray | [https://doi.org/10.1126/science.1146265] | GEO dataset: GSE8934 ID: 200008934 | +| NIST14 Mass Spectral Library | https://www.nist.gov/srd/nist-standard-reference-database-1a-v14 | N/A | +| Arabidopsis thaliana Col-0 metabolic pathways | https://pmn.plantcyc.org/organism-summary?object=ARA | N/A | +| **Software and Algorithms** | | | +| iGraph | https://CRAN.R-project.org/package=igraph. | RRID:SCR_019225 | +| COBRApy | http://opencobra.sourceforge.net | RRID:SCR_012096 | +| GUROBI | https://www.gurobi.com/ | N/A | +| statsmodels | https://www.statsmodels.org/stable/index.html | RRID:SCR_016074 | +| Vegan | https://cran.r-project.org/web/packages/vegan/index.html | RRID:SCR_011950 | +| Qiime2 | https://qiime2.org/ | RRID:SCR_008249 | +| Flash2 | https://github.com/dstreett/FLASH2 | RRID:SCR_005531 | +| Dada2 | https://benjjneb.github.io/dada2 | RRID:SCR_008205 | +| **Others** | | | +| 16S data analyses | This work | GitHub https://github.com/duranpa/sweet_collaboration | \ No newline at end of file diff --git a/assays/RootMetaboliteProfiling/isa.assay.xlsx b/assays/RootMetaboliteProfiling/isa.assay.xlsx index 123c5a7c2206f7a8e9a54523265ff0c499b76398..5b13c1b7c1cdbaf8c3598fc95bc32c862d4eac7d 100644 Binary files a/assays/RootMetaboliteProfiling/isa.assay.xlsx and b/assays/RootMetaboliteProfiling/isa.assay.xlsx differ diff --git a/assays/RootMetaboliteProfiling/protocols/RootMetaboliteProfilingProtocol.md b/assays/RootMetaboliteProfiling/protocols/RootMetaboliteProfilingProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..2f500053972554a32f2524db96cf10977cf3cb5d --- /dev/null +++ b/assays/RootMetaboliteProfiling/protocols/RootMetaboliteProfilingProtocol.md @@ -0,0 +1,3 @@ +## Root metabolite profiling + +Approximately 25-35 sterilized *A. thaliana* Col-0 seeds were sown in a row on ArtSoil and grown for 3 weeks. After 3 weeks, both primary and lateral roots longer than 8 cm were harvested by slicing with a scalpel. The roots were segmented into 2-cm segments measuring 0-2, 2-4, and 4-6 cm from the root tip. Lateral roots that emerged from primary roots were removed, if present. The roots were rinsed in distilled water, and blotted dry on Whatman filter papers before they were weighed and kept in 1.5-ml microcentrifuge tubes, each containing 2 metal beads. The samples were flash-frozen in liquid nitrogen and stored at -80 °C until the extraction process. For metabolite extraction, 0.5 mL of chilled extraction buffer (2:5:2 ratio of ddH2O: methanol: chloroform containing 5 μM ribitol as internal standard) was added into each root sample and mixed by vortex for 20 sec. Metal beads in the tubes were removed. Sample tubes were shaken on a rotary shaker for 30 mins at 4 °C. The samples were then centrifuged at 20, 000 × g for 5 min at 4 °C. After centrifugation, 0.5 ml of supernatant was carefully aspirated and transferred to a clean 1.5-ml microcentrifuge tube. Samples were stored at -80 °C until they were subjected to GC-MS. For GC-MS analysis 30 μl of sample were dried by vacuum centrifugation in glass inlet tubes. Dried samples were derivatized according to Gu (2012) and Shim et al.(2019). Raw data files were converted to the mzXML format using ProteoWizard (Chambers, M.C. et al., 2012) and to the NetCDF format via MetAlign (Lommen, A. and Kools, H.J., 2012) using default parameters. Deconvolution of mass spectra was conducted using the free deconvolution software AMDIS (Automated Mass Spectral Deconvolution and Identification System from NIST). Deconvoluted mass spectra were matched against the NIST14 Mass Spectral Library (https://www.nist.gov/srd/nist-standard-reference-database-1a-v14). Database matches with more than 70% were further compared with an in-house chemical standard library for compound annotation. Compounds, that could not be verified by the in-house library are named according to the matched compound class and the retention time. Extracted ion peaks were integrated using MassHunter Quantitative (v b08.00, Agilent Technologies). For relative quantification, all metabolite peak areas were normalized to fresh weight and the peak area of the internal standard ribitol (Sigma-Aldrich) to correct for technical error. Here, a GC-based system was employed for metabolomics, which may detect only the fraction of metabolites that can be vaporized. It may be useful for a more in-depth analysis to use LC-based systems to identify more metabolites and improve coverage. \ No newline at end of file diff --git a/studies/ArtSoilPreparation/README.md b/studies/ArtSoilPreparation/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/ArtSoilPreparation/isa.study.xlsx b/studies/ArtSoilPreparation/isa.study.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..033f4ea69f716ba0977037986625eb9fd467b7db Binary files /dev/null and b/studies/ArtSoilPreparation/isa.study.xlsx differ diff --git a/studies/ArtSoilPreparation/protocols/.gitkeep b/studies/ArtSoilPreparation/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/ArtSoilPreparation/protocols/ArtSoilPreparationProtocol.md b/studies/ArtSoilPreparation/protocols/ArtSoilPreparationProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..388d10d19bbba169f794f2cea1fb726dad6c668b --- /dev/null +++ b/studies/ArtSoilPreparation/protocols/ArtSoilPreparationProtocol.md @@ -0,0 +1,3 @@ +## ArtSoil preparation + +For the preparation of 10% autoclaved soil suspension, sieved CAS (to remove pebbles) was dissolved in deionized water and autoclaved three times before use. Live bacterial suspension was prepared by inoculating fresh CAS into 10% autoclaved soil suspension to achieve desired bacterial dilution (Korenblum, E. et al., 2020). Autoclaved half-strength Murashige-Skoog (½ MS) media including MES (Duchefa Biochemie) and supplemented with 1% agar was cooled to 40–45°C before it was inoculated with live bacterial suspension. Approximately 50 mL of ArtSoil media was used for each 12 cm x 12 cm culture plate (Figure S2B). Sterilized *A. thaliana Col-0* seeds were sown and germinated on ArtSoil, and incubated in a plant growth chamber for 4 weeks under standard growth conditions. \ No newline at end of file diff --git a/studies/ArtSoilPreparation/resources/.gitkeep b/studies/ArtSoilPreparation/resources/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/CD-Rhizotron/README.md b/studies/CD-Rhizotron/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/CD-Rhizotron/isa.study.xlsx b/studies/CD-Rhizotron/isa.study.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..64423defd65535ec3408a9874d75662f1babb0d7 Binary files /dev/null and b/studies/CD-Rhizotron/isa.study.xlsx differ diff --git a/studies/CD-Rhizotron/protocols/.gitkeep b/studies/CD-Rhizotron/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/CD-Rhizotron/protocols/CD-RhizotronProtocol.md b/studies/CD-Rhizotron/protocols/CD-RhizotronProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..09014bb8117fe103e05efa56cc15c8f308bb74b3 --- /dev/null +++ b/studies/CD-Rhizotron/protocols/CD-RhizotronProtocol.md @@ -0,0 +1,3 @@ +## CD-Rhizotron + +To assemble a CD-Rhizotron, a small opening, measuring approximately 1.5-2 cm, was cut on the top of a slimline jewel CD case (14.3 cm x 12.4 cm x 0.52 cm; Figure S1A). The teeth of the tray were snipped off and sanded down to produce a smooth surface. The resulting hole was sealed on both sides with packing tape. CD-Rhizotrons were filled with soil (Cologne Agricultural Soil, CAS (Bulgarelli, D. et al., 2012)) and wrapped with aluminum foil to provide a dark environment for proper root growth (Figures S1A–S1D). Up to four sterilized *A. thaliana* Col-0 seeds were sown directly onto each CD-Rhizotron from the top aperture of the CD-Rhizotron. CD-Rhizotrons were incubated under standard growth conditions. After 5-7 days, germinated seedlings were removed to leave each CD-Rhizotron with a single seedling. Plants were grown for another 3 weeks before they were harvested for microbiota profiling. Preparation of SynCom was adapted from (Durán et al., 2018). The 60-member SynCom was curated based on differences in 16S rRNA sequence with a 97% threshold. Briefly, bacterial strains (Table S5) were cultivated in 50% Tryptic Soy Broth (Sigma) for one week at 28°C with shaking at 200 rpm. The bacterial cultures were pooled in equal ratio and centrifuged at 4000x g for 10 mins. The bacterial pellet was re-suspended in 10 mM MgCl2 to remove residual media and bacteria-derived metabolites. The washing step was repeated three times. The washed bacterial suspension was adjusted to an OD600 of 0.02 (107 cells/mL) prior to inoculation onto sterile peat soil. \ No newline at end of file diff --git a/studies/CD-Rhizotron/resources/.gitkeep b/studies/CD-Rhizotron/resources/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/PlantMaterialAndGrowthConditions/README.md b/studies/PlantMaterialAndGrowthConditions/README.md new file mode 100644 index 0000000000000000000000000000000000000000..b88d689899dc45a5779d980cf0ad79356ea56f38 --- /dev/null +++ b/studies/PlantMaterialAndGrowthConditions/README.md @@ -0,0 +1,30 @@ +## Key Resources Table + +| **REAGENT or RESOURCE** | **SOURCE** | **IDENTIFIER** | +|---------------------------------------------------|-------------------------------------------------------------|-------------------------------------| +| **Biological Samples** | | | +| Arabidopsis thaliana Col-0 PSWEET17:SWEET17 -GUS | Guo et al.80 | N/A | +| Arabidopsis thaliana Col-0 PSWEET2:SWEET2-GUS | Chen et al.34 | N/A | +| Arabidopsis thaliana Col-0 PSWEET12:SWEET12-GUS | Chen et al.28 | N/A | +| Arabidopsis thaliana Col-0 PSWEET11:SWEET11-GUS | Chen et al.28 | N/A | +| Arabidopsis thaliana Col-0 sweet2c | SIGnAL http://signal.salk.edu/ | SALK_048430.36.85.x | +| Arabidopsis thaliana Col -0 sweet11;12 | Arabidopsis Biological Resource Center https://abrc.osu.edu | TAIR Germplasm: CS68845 | +| Arabidopsis thaliana Col-0 sweet4a | SIGnAL http://signal.salk.edu/ | SALK_072225.23.65.x | +| **Chemicals, Peptides, and Recombinant Proteins** | | | +| DFS-Taq DNA polymerase | Bioron | Cat # 101005 | +| Antarctic phosphatase | New England BioLabs | Cat # M0289 | +| Exonuclease I | New England BioLabs | Cat # M0293 | +| Ribitol | Sigma-Aldrich | Cat # PHR3526 | +| Murashige&Skoog media including MES buffer | Duchefa Biochemie | Cat # M0254 | +| Tritonâ„¢ X-100,for molecular biology | Sigma-Aldrich | Cat # T8787 | +| Agar | Sigma-Aldrich | Cat # 05040 | +| Hygromycin B | Carl Roth | Cat # CP12.2 | +| Thermo Scientific X Gluc | Thermo Fisher Scientific | Cat # R0851 | +| **Critical Commercial Assays** | | | +| FastDNAâ„¢ SPIN Kit for Soil | MP Biomedicals | Cat # SKU116560200-CF | +| NuceloSpin Soil Mini kit for DNA from soil | Macherey-Nagel | Cat # 740780 | +| QIAquick gel extraction kit | Qiagen | Cat # 28706X4 | +| Quant-iTâ„¢ PicoGreenâ„¢ | Invitrogen | Cat # P7581 | +| Gatewayâ„¢ LR Clonaseâ„¢ Enzyme mix | Invitrogen | Cat # 11791019 | +| **Experimental Models: Organisms/Strains** | | | +| Arabidopsis thaliana Col-0 | TAIR | TAIR accession Germplasm:1008804532 | \ No newline at end of file diff --git a/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx b/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..214ceb7e958bb928660f60a6de2bf4536196cab1 Binary files /dev/null and b/studies/PlantMaterialAndGrowthConditions/isa.study.xlsx differ diff --git a/studies/PlantMaterialAndGrowthConditions/protocols/.gitkeep b/studies/PlantMaterialAndGrowthConditions/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/studies/PlantMaterialAndGrowthConditions/protocols/ExperimentalModel.md b/studies/PlantMaterialAndGrowthConditions/protocols/ExperimentalModel.md new file mode 100644 index 0000000000000000000000000000000000000000..e984a92a5e39ea926112a49cb24d1f6b95ae3618 --- /dev/null +++ b/studies/PlantMaterialAndGrowthConditions/protocols/ExperimentalModel.md @@ -0,0 +1,3 @@ +## Experimental Model + +*Arabidopsis thaliana* Col-0 seeds were sterilized in a 1.5-mL microcentrifuge tube with 1 mL of 30% chlorine + 0.1% Triton X-100 for 15-20 min at room temperature with agitation. The seeds were rinsed once with 1 mL of 80% ethanol, followed by five rinses with autoclaved ddH2O. The microcentrifuge tube was wrapped in aluminum foil and cold-treated at 4 °C for 2-5 days before sowing. *A. thaliana* Col-0 seeds were incubated under standard growth conditions, i.e., 22 °C, 10 h light/ 14 h dark, 60% humidity, 180-230 μmol/s/m2 light intensity. \ No newline at end of file diff --git a/studies/PlantMaterialAndGrowthConditions/resources/.gitkeep b/studies/PlantMaterialAndGrowthConditions/resources/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391