Additional file 2 of RNA silencing is a key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions
Creators: name: Piombo, Edoardo
name: Vetukuri, Ramesh Raju
name: Konakalla, Naga Charan
name: Kalyandurg, Pruthvi B.
name: Sundararajan, Poorva
name: Jensen, Dan Funck
name: Karlsson, Magnus
name: Dubey, Mukesh
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: Plant Biology

description: Additional file 2: Fig. S1: Gene ontology terms referring to biological processes enriched in wheat genes or C. rosea genes differentially expressed during the interaction between the two organisms. The analysis was done with BLAST2GO, using a Fisher test corrected with the FDR method. The adjusted pvalue threshold was set at 0.05, and enriched biological processes were visualized using Python seaborn v. 0.12.2 and Scientific Inkscape ( https://github.com/burghoff/Scientific-Inkscape ). The heatmap shows the negative LOG10 of the FDR-corrected p-value obtained in a Fisher test to calculate gene ontology enrichment. Fig. S2: The heatmap shows the Spearman correlation between the module eigengenes of co-expression modules generated with WGCNA and the conditions examined in this study. Wheat roots (Wheat Control), C. rosea WT interacting with wheat roots (Cr-Wr), C. rosea Δdcl1 interacting with wheat roots (Δdcl1-Wr), C. rosea Δdcl2 interacting with wheat roots (Δdcl2-Wr). The modules were generated using the normalized expression values of differentially expressed wheat genes. Asterisks indicate significant correlation or anticorrelation. Fig. S3: The heatmap shows the Spearman correlation between the module eigengenes of co-expression modules generated with WGCNA and the conditions examined in this study. C. rosea WT growing in PDB media (Cr Control), C. rosea WT interacting with wheat roots (Cr-Wr), C. rosea Δdcl1 interacting with wheat roots (Δdcl1-Wr), C. rosea Δdcl2 interacting with wheat roots (Δdcl2-Wr). The modules were generated using the normalized expression values of differentially expressed C. rosea genes. Asterisks indicate significant correlation or anticorrelation. Fig. S4: The figure contains information regarding the wheat miRNAs detected in this study. A: length distribution. B: differential expression. C: number of putative gene targets showing inverse expression pattern with the miRNAs. Fig. S5: The figure contains information regarding the C. rosea milRNAs detected in this study. A: length distribution. B: differential expression. C: number of putative gene targets showing an inverse expression pattern with the milRNAs. Fig. S6: Gene expression validation by RT-qPCR. Expression profiles of selected C. rosea (A) and wheat (B) genes were analyzed during interactions. Relative expression levels in C. rosea and wheat were normalized by respective C. rosea and wheat β-tubulin (TUB) expression and presented in relation to non-interaction control. Error bars represent standard deviation based on three biological replicates. Different letters indicate statistically significant differences (p &lt; 0.05) based on Fisher’s exact test. The table highlighted in green indicates gene expression patterns from RNAseq. *Indicates endogenous gene targets (C. rosea genes targeted by C. rosea milRNAs, see Table 4), # indicates cross-kingdom gene targets (C. rosea gene targeted by three wheat miRNAs mir_17532_x1, mir_16010_x2, mir_12061_x13 (see Table 4). Fig. S7: Trafficking of Cy3 labeled wheat mir_12061_x13 mimics (miR17532) (A) and Phytophthora infestans miR8788 (B) from wheat roots to C. rosea-GFP conidia and hyphae A. Representative confocal images showing the co-localization of Cy3-miR17532 (Magenta), C. rosea-GFP (Green) and merge (right panel). B. Representative confocal images showing the co-localization of Cy3-miR8788 (Magenta), C. rosea-GFP (Green) and merge (right panel). Twenty-four hours post incubation (hpi) with the mimics, wheat roots were washed with 0.M KCl and 0.01 M Triton X100 to remove surface-bound miRNA oligos. Conidia from C. rosea-GFP were applied to the roots, and Cy3 fluorescence was determined 72 hpi.