RNAseq analysis identifies a core set of UPR regulated genes.

(A) ER stress assay of U. maydis strain JB1 (WT), JB1Δcib1 and UVO151 (Pcrg:clp1). Serial 10-fold dilutions were spotted on YNB solid medium supplemented with 1% arabinose (YNBA) and indicated concentrations of tunicamycin (TM). Plates were incubated for 48 h at 28°C. (B) Venn diagram of differentially expressed genes identified by RNAseq analysis. Numbers depict differentially regulated genes (log2FC≥2) in WT +TM compared to the untreated control (blue circle) and in WT +TM compared to the Δcib1 +TM control (yellow circle). Both sets share 115 genes (overlap), of which 65 were upregulated and 50 downregulated in a cib1-dependent manner. Upward arrows indicate upregulation, downward arrows indicate downregulation. (C) Enriched functional categories of the 65 UPR core genes identified by RNAseq analysis. FunCat analysis was performed using the “Functional Catalogue” (FunCat) of the MIPS U. maydis database (http://mips.helmholtz-muenchen.de/funcatDB/). Bars reflect the percentage of core genes belonging to the respective functional category. Please note that individual genes can be assigned to more than one category. Only overrepresented categories are shown. *P value ≤ 0.05, **P value ≤ 0.01 and ***P value ≤ 0.001. (D) Heat map showing expression levels of 65 upregulated genes as determined by RNAseq analysis. Expression values were log2-transformed, mean-centered and SD-scaled reads per million (RPM) (Row z-Score) were calculated and visualized using the ClustVis tool (https://biit.cs.ut.ee/clustvis/). Blue and red color intensity of the row z-Score indicates downregulation and upregulation, respectively. Hierarchical clustering on the y-axis groups genes with similar expression profiles. Euclidean distance and complete linkage for genes were used as clustering method. Sample types (WT, Pcrg:clp1, Δcib1) and treatment conditions (without tunicamycin [-TM], with tunicamycin [+TM]) are indicated at the bottom. Regulation of the 65 UPR core genes in planta [22] is depicted for 2, 4 and 6 days post inoculation (dpi). Green bars indicate upregulation, grey indicate the absence of differential gene expression and red bars indicate downregulation.