Additional file 2 of Dicer-like proteins influence Arabidopsis root microbiota independent of RNA-directed DNA methylation

Additional file 1: Figure S1. The largest separation between bacterial communities is spatial proximity to the root as revealed by PCoA plotted from wUniFrac metrics (Related to Fig. 1). [A] PCo 1 vs PCo 2. [B] PCo 2 vs PCo 3. [C] PCo 1 vs PCo 3. Figure S2. Microbe richness in different compartments reflects the selectivity of plants on root-associated microbes (Related to Fig. 1). [A] Numbers of observed OTUs in the different compartments. [B] Numbers of estimated OTUs based on the Chao1 estimator. [C] Shannon index of the microbe richness. Samples were rarefied to 23000 reads prior to the analysis. Soil1 is the initial bulk soil and Soil2 is the final bulk soil. Letters denote statistical significance (p ≤ 0.05, Wilcox test) compared to Soil 1. Figure S3. Taxonomic structure of Abundant Community members (ACM) is affected by compartments and plant genotype (Related to Fig. 1). [A] Relative abundance (RA) of the bacteria within the initial bulk soil (Soil 1) and the final bulk soil (Soil 2) as classified at the phylum level. [B] Relative abundance of the bacteria phyla that were identified within the rhizosphere samples. [C] Relative abundance of the bacteria phyla that were identified within the root samples. Figure S4. The dcl234 triple mutation alters Arabidopsis root microbiota (Related to Fig. 1). [A] Relative abundance of the top 5 abundant phyla in roots of the wild type Arabidopsis (Col-0) and the RdDM pathway mutants. Mean ± SE, n ≥ 3. Asterisks indicate significant difference (FDR ≤ 0.05) between the mutant and the wild type. Taxa with RA > 5% in at least one sample were included in the statistical analysis. [B] A heatmap showing the levels of OTUs with significantly different enrichment (FDR ≤ 0.05) in dcl234 compared to Col-0. Phyla are annotated on the left side of the heatmap; on the right side, OTUs are annotated to different levels, F, family, G, genus, O, order; NR, New Reference. Figure S5. Read counts of the metagenomic sequencing. Stacked bars are shown to indicate the read counts of both plant DNA and microbial DNA sequences in each sample. Mean ±SE, n = 4. * indicates p ≤ 0.05; NS, non-significant, student t-test. Figure S6. The dcl234 triple mutation causes alterations in Arabidopsis defense-related processes (Related to Fig. 2). [A] A heatmap of DEGs involved in phenylpropanoid production. [B] Root anthocyanin visualization and measurements in plants at 18 days after germination. Mean ± SE, n = 6 biological replicates. Each biological replicate consisted of roots from 3 plants. **, p ≤ 0.01, student t-test. [C] A heatmap of DEGs involved in sulfur metabolism. DEGs were identified in the mRNAseq transcriptome analysis. The two heatmaps use the same Z-Score color scale that is shown in panel C. Figure S7. The dcl234 mutant shows both decreased and increased accumulation of different sRNAs (Related to Fig. 3). [A] The MA plot of the sRNA population identified in Col-0 and dcl234. Y-axis displays fold changes (dcl234 vs Col-0) of sRNA abundance; X-axis displays average signal intensities of the sRNA in all samples. The red, purple, and black colors indicate sRNAs that were increased, decreased, or not changed in dcl234 compared to Col-0 based on FDR p ≤ 0.05. [B] A heatmap of miRNAs identified as increased or decreased in dcl234 compared to Col-0. [C] Examples of genetic loci where sRNA abundance was affected by the dcl234 triple mutation. Snapshot images were obtained from whole-genome sRNA sequencing results. Vertical gray bars indicate the sRNA sequencing coverage normalized to the same scale in Col-0 and dcl234. Figure S8. The dcl234 mutant shows altered expression of cell wall-associated genes (Related to Fig. 4). [A] A heatmap of DEGs related to cellulose synthesis. [B] Expression levels of DEGs related to callose synthesis and pectin metabolism. Snapshot images were obtained from whole-genome sRNA sequencing results. Vertical gray bars indicate the mRNA sequencing coverage normalized to the same scale in Col-0 and dcl234. [C] Quantitative RT-PCR measurements of two pectin-related DEGs. Mean ± SE, n=3 technical replicates. Two biological replicates were analyzed with similar results. * indicates p ≤ 0.05, student t-test. Figure S9. The dcl234 mutant shows altered transcription regulation of metabolism that potentially connects to alterations in root exudates (Related to Table 2). [A] Gas Chromatography-Mass Spectrometry (GC-MS) analysis of root exudates from Col-0 and dcl234. Representative profiles were shown. n =3 biological replicates. [B] A heatmap of gibberellin-related DEGs. [C] Expression levels of miR827 and its target gene BAH1. Snapshot images were obtained from whole-genome sRNA and mRNA sequencing results. Vertical gray bars indicate the sequencing coverage normalized to the same scale in Col-0 and dcl234.