Fig 1.
Dual-culture interaction plate setup.
Fusarium graminearum DAOMC 180378 hyphae (dark grey) was grown from an agar plug placed at the centre of the plate on a cellulose membrane. Colonies of Bacillus velezensis E68 (light grey) were grown from aliquots of cell suspension spotted 2.5 cm from the centre of the plate. Striped areas were collected with a sterile loop or spatula for RNA extraction. Diagram is not to scale.
Fig 2.
Schematic of experimental workflow of RNA-seq analysis of Bacillus velezensis and Fusarium graminearum.
Cultures of B. velezensis E68 and F. graminearum DAOMC 180378 were grown in single and dual culture. The striped areas were collected with a sterile spatula before RNA extraction. Total RNA was sent for library preparation and RNA sequencing. Bacterial RNA was depleted for ribosomal RNA, while fungal RNA was enriched with poly-A selection. Samples were sequenced on an Illumina HiSeq using a paired-end 150 bp protocol. Raw sequencing reads were pre-processed by removing adaptors and poor quality bases and reads. Filtered reads were aligned to their respective genomes and counted for each gene. Gene counts were normalized by the TMM method, low expression genes were removed from analysis and differential expression was calculated. In order to functionally annotate the genes, each gene sequence was used in a BLASTx search to compare to the UniprotKB protein database, annotating each gene with a description and gene ontology terms. The resulting GO terms were used in GO enrichment analysis. Known biosynthetic gene clusters were used in rotation gene set testing to determine their overall regulation pattern.
Table 1.
List of primers used in this study.
Fig 3.
B. velezensis E68 inhibits the growth of F. graminearum DAOMC 180378 in dual culture.
(A) F. graminearum DAOMC 180378 grown in single culture on PDA, (B) Dual culture condition for B. velezensis E68 and F. graminearum DAOMC 180378 on PDA. (C) Hyphal morphology of F. graminearum in single culture. (D) and (E) Hyphal morphology of F. graminearum in dual culture with B. velezensis. Microscopy performed at 40x magnification.
Table 2.
Number of sequencing reads from each sample after trimming.
Table 3.
Genes of B. velezensis E68 and F. graminearum DAOMC 180378.
Table 4.
Significantly over-represented Gene Ontology (GO) terms following enrichment analysis on up- and down-regulated genes from B. velezensis E68 and F. graminearum DAOMC 180378 in dual culture.
Table 5.
Rotation gene set testing of biosynthetic gene clusters encoding secondary metabolites in B. velezensis E68.
Table 6.
Normalized expression of core biosynthetic genes from biosynthetic gene clusters encoding secondary metabolites in B. velezensis E68.
Table 7.
Fold change of expression of trichothecene pathway genes in F. graminearum DAOMC 180378 in dual culture with B. velezensis E68.
Fig 4.
Log2 fold change of selected genes between single and dual culture based on RNA-seq and qPCR for (A) B. velezensis E68 and (B) F. graminearum DAOMC 180378. Asterisks (*) indicate significantly differentially expressed genes for RNA-seq and indicate p < 0.05 for qPCR.