Supplementary Files
Creators: name: Kudjordjie, Enoch Narh
name: Hooshmand, Kourosh
name: Sapkota, Rumakanta
name: Darbani, Behrooz
name: Fomsgaard, Inge S.
name: Nicolaisen, Mogens
Publisher: name: figshare
Year: 2022
Subjects: subject: 60504 Microbial Ecology
subject: FOS: Biological sciences
subject: Microbiology
subject: Plant Biology
subject: 60704 Plant Pathology
subject: 60202 Community Ecology (excl. Invasive Species Ecology)

description: Supplementary files (Tables and Figures) for<br> Kudjordjie et al., Fusarium oxysporum disrupts microbiome-metabolome networks in Arabidopsis thaliana roots <br> <br> <strong>Supplementary files</strong> <strong>Supplementary Figures</strong> <strong>Figure S1</strong>. Structure and pathways of quantified metabolites and hormones. A) Chemical structure of glucosinolates and their degradation products quantified in this study B) A schematic view of the biosynthetic pathways of phenylpropanoids and representative intermediates and chemical structures. For simplicity, only the enzymes involved in the formation of 4-coumaryl CoA from phenylalanine are shown. Solid arrows represent single enzymatic steps. Dashed arrows indicate multiple consecutive enzymatic steps. The colors are representative of following chemical classes: coumarins (green), cinnamic acid derivatives (black), benzoic acids (dark red), lignin precursors (monolignols) (blue), and lignans (light red). <strong>Figure S2. </strong>(<strong>A</strong>) FOM quantities in shoots of inoculated and non-inoculated <em>Arabidopsis</em>. Distribution of (<strong>B</strong>) bacterial and (<strong>C</strong>) fungal sequence reads and OTUs in samples used in this study. Rarefaction curves showing the coverage of (<strong>D</strong>) bacterial and (<strong>E</strong>) fungal OTU richness (species richness in number of OTUs) as a function of sequencing depth (sample size in number of reads). <strong>Figure S3. </strong>Bacterial and fungal community analysis. A) Bacterial and B) fungal communities in inoculated and non-inoculated Col-0 and Ler-0. C) Relative abundance of fOTUs assigned to the <em>Fusarium</em> genus in Col-0 control and inoculated samples. D) Relative abundance of fOTUs assigned to the <em>Fusarium</em> genus in Ler-0 non-inoculated and inoculated samples at different DAI. Red colour shades represented inoculated samples while green colour shades represent non-inoculated samples at different DAI. Bacterial alpha diversity E) and F) fungal beta diversity visualized using Principal coordinates analysis (PCoA) in inoculated and control Col-0 and Ler-0. ANOVA was used to determine statistically significant differences between sample means, followed by the Tukey post hoc test using R statistical package “<em>agricolae</em>”. Significance of test indicated as *** for p &lt; 0.001, ** p &gt; 0.01, *p &lt; 0.05. <strong>Figure S4. </strong>Analysis of differential OTUs in inoculated and non-inoculated samples. Heatmap showing top50 bacterial (A) and B) fungal OTU relative abundances in inoculated and non-inoculated samples across different 5 DAI. (C) Global analysis revealed two bacterial OTUs; bOTU6 (<em>Rhizobium</em>) and bOTU10 (<em>Pseudomonas protegens</em>) to be significantly abundant in inoculated and non-inoculated samples of Col-0 and Ler-0. (D) bOTU4 (<em>Streptomyces</em>) abundance in inoculated and non-inoculated samples of Col-0 and Ler-0. One-way ANOVA was used to determine statistically significant differences between sample means, followed by the Tukey post hoc test using R statistical package. Significance of test indicated as *** for p &lt; 0.001, ** p &lt; 0.01, *p &lt; 0.05. <strong>Figure S5. </strong>Differentially abundant bacterial (A) and fungal (B) taxa between samples of Col-0 and Ler-0. Log2fold changes greater than and less than zero indicate enrichment OTUs in Ler-0 and Col-0, respectively. C) Heatmap showing differentially abundant bacteria taxa in inoculated and non-inoculated samples of Col-0 and Ler-0. D) Heatmap showing differentially abundant fungal taxa in inoculated and non-inoculated samples of Col-0 and Ler-0. OTU1 (FOM) was significantly enriched in the inoculated samples. In C and D, taxa enrichment in non-inoculated are indicated in blue while enrichment in inoculated samples are indicated in red. Log2fold changes greater than and less than zero indicated an enrichment of the relevant genera in Ler-0 and Col-0 respectively. The analysis was performed using DESeq2 with phyloseq. Globally enriched OTUS; bOTU6 and bOTU10 are indicated in astericks. Differences were considered significant with the p-value (adjusted for false discovery rate using Benjamini–Hochberg correction) cut-off of 0.01, except for fOTUs differential abundance estimation in Col-0 and Ler-0 comparison where a cut-off of 0.05 was used. <strong>Figure S6</strong>. Abundance of indicator (A) bacterial and (B) fungal taxa identified in inoculated and non-inoculated root samples of Col-0 and Ler-0. Bars correspond to summed abundances for taxonomic divisions at phylum level, with individual classes (and lower-level taxa) denoted by stacked boxes. C) Fungal indicator OTU enrichment in non-inoculated and inoculated samples of Col-0 and Ler-0 at different days after inoculation (DAI). Number of indicator microbial OTUs identified in inoculated and non-inoculated Col-0 and Ler-0 samples are shown in the table. Highly significant OTUs (p <strong>Figure S7</strong>: The missense variants in Ler-0 <em>Fusarium</em> resistance proteins as compared with Col-0. <strong>Supplementary tables </strong> <strong>Table S1</strong>: MRM transitions and compound-dependent parameters for LC-MS/MS analysis of the target analytes in both negative and positive ESI modes. Each analyte has two MRM transitions. Q1= quadrupole 1, protonated or deprotonated molecule (m/z ratio); Q3 (1) = quadrupole 3 (quantifier); Q3 (2) = quadrupole 3 (qualifier); DP= declustering potential; EP= entrance potential; CE = collision energy. Aliphatic glucosinolates (AG); Indole glucosinolates (IG); Hydroxybenzyl glucosinolates (OH-benzyl-G); Benzoic acid (BZ); Cinnamic acids (CA); ISTD (internal standard). <strong>Table S2</strong>: Sequence read statistics for bacterial and fungal reads. <strong>Table S3</strong>: Permutational analysis of variance (PERMANOVA) using ‘adonis’ test on Bray-Curtis distance matrices for bacterial and fungal community dissimilarity assessment using 1000 permutations. <strong>Table S4</strong>: Differentially affected taxa in genotypes and inoculated and non-inoculated samples. Differences were considered significant with the p-value (adjusted for false discovery rate using Benjamini–Hochberg correction) cut-off of 0.01, except for fOTUs differential abundance estimation in Col-0 and Ler-0 comparison where a cut-off of 0.05 was used. <strong>Table S5</strong>: Bacterial and fungal indicator OTUs identified in inoculated and non-inoculated Col-0 and Ler-0 lines. Indicator value and relative abundance of each indicator taxa is provided. <strong>Table S6</strong>: Summary of co-occurrence network properties including keystone OTUs, and their relative abundances identified in inoculated and non-inoculated <em>Arabidopsis</em> lines. <strong>Table S7</strong>: Summary of bOTU_fOTU1 correlations identified in inoculated and non-inoculated <em>Arabidopsis</em> lines. <strong>Table S8</strong>: Absolute concentrations (μg/g) of glucosinolates, glucosinolates hydrolysis products, camalexin, C) coumarins, lignin precursors, lignans, phenolic acids, and hormones in the root of <em>Arabidopsis</em> accessions after root was challenged with the FOM spores (inoculated) or water (non-inoculated). Values represent the mean of five biological replicates + standard error. At each time‐point, significant differences between the root of non­-inoculated samples and those exposed to FOM are indicated by asterisks (*: p&lt;0.05, **: p&lt;0.01, ***: p&lt;0.001). <strong>Table S9: </strong>Pathway enrichment analysis highlights different pathogen response mechanisms in which the co-expressed genes are involved <strong>Table S10</strong>: Summary of OTU-metabolite correlation analysis using all dataset obtained and partitions of inoculated and non-inoculated <em>Arabidopsis</em> samples. <strong>Table S11</strong>: Summary of metabolite-fOTU1 correlations identified in inoculated and non-inoculated <em>Arabidopsis</em> lines. <strong>Table S12</strong>: Summary of indicator hubOTU metabolite-correlations identified in inoculated and non-inoculated <em>Arabidopsis</em> lines.