Supporting Information Figs S1-S7 & S9
Figure S1. The spilt-marker deletion strategies for FgCYP51 gene deletion in F. graminearum
isolate PH-1. A. FgCYP51A gene deletion and confirmation strategy. FgCYP51A deletion constructs
were created by two fusion PCR products, 5’ flanks of FgCYP51A gene plus upstream of hygromycin
phosphotransferase cassette (hph) and 3’ flanks of FgCYP51A gene plus downstream of hph, to
replace the FgCYP51A gene. After selection with hygromycin, transformants were confirmed by PCR
by using the primers F2-5outF with GC1, and GC2 with F2-3outR. The agarose gel photo shows the
products size of the confirming PCR. Lane 1 is the 1kb DNA ladder, lane 2 is the confirming PCR with
genomic DNA of the ΔFgCYP51A2 mutant as template, Lane3 is the confirming PCR with the genomic
DNA of the ΔFgCYP51A3 mutant as template. B. FgCYP51B gene deletion and confirmation strategy.
FgCYP51B deletion constructs were created by two fusion PCR products, 5’ flanks of FgCYP51B gene
plus upstream of hph and 3’ flanks of FgCYP51A gene plus downstream of hph, to replace the
FgCYP51B gene. After selection with hygromycin, the transformants were confirmed by PCR by using
the primers F1-5outF with GC1, and GC2 with F1-3outR. The photo of 1% agarose gel shows the
products size of confirming PCR. Lane 1 is the 1kb DNA ladder, lane 2 is the confirming PCR with
genomic DNA of the ΔFgCYP51B3 mutant as template, Lane3 is the confirming PCR with the genomic
DNA of the ΔFgCYP51B4 mutant as template. C. FgCYP51C gene deletion and confirmation strategy.
FgCYP51C deletion construct were created by two fusion PCR products, 5’ flanks of FgCYP51C gene
plus upstream of hph and 3’ flanks of FgCYP51A gene plus downstream of hph, to replace the
FgCYP51C gene. After selection with hygromycin, the transformants were confirmed by PCR by using
the primers F3-5outF with GC1, and GC2 with F3-3outR. The photo of 1% agarose gel shows the
products size of confirming PCR. Lane 1 is the 1kb DNA ladder, lane 2 is the conforming PCR with
genomic DNA of the ΔFgCYP51C2 mutant as template, Lane3 is the conforming PCR with the
genomic DNA of the ΔFgCYP51C3 mutant as template. D. FgCYP51C gene deletion and confirmation
strategy. FgCYP51C deletion construct was created by two fusion PCR products, 5’ flanks of
FgCYP51C gene plus upstream of neomycine expression gene (NEO) and 3’ flanks of FgCYP51C gene
plus downstream of NEO, to replace the FgCYP51C gene. After selection with neomycin,
transformants were confirmed by PCR by using the primers F3-5outF with U151, and U150 with F33outR (the middle). The photo of 1% agarose gel shows the products size of confirming PCR. Lane 1
is the 1kb DNA ladder, lane 2 is the conforming PCR with genomic DNA of the ΔFgCYP51C1-NEO
mutant as a template, Lane3 is the conforming PCR with the genomic DNA of the ΔFgCYP51C2-NEO
mutant as a template. E. ΔFgCYP51AC mutants confirmation. Double deleted FgCYP51A and
FgCYP51C is based on the ΔFgCYP51C1-NEO mutant to delete the FgCYP51A gene with hph marker
(see Figure S1-A). The photo of 1% agarose gel shows the products size of confirming PCR. F.
ΔFgCYP51BC mutants confirmation. Double deleted FgCYP51B and FgCYP51C is based on the
ΔFgCYP51C1-NEO mutant to delete the FgCYP51B gene with hph marker (see Figure S1-B). The
photo of 1% agarose gel shows the products size of confirming PCR.
Figure S2. In vitro transcript levels of ERG genes. Mycelia were collected after 48 hours incubation
in PDB media. Relative quantification of ERG genes was analysed with the 2-ΔΔCt method with βtubulin as the reference gene and wild type PH-1 as a calibrator. There were three biological
replicates for statistical analyses. Greek letters show the statistically significant differences. Error
bars show the standard error between biological replicates.
Figure S3. Relative quantification of Actin and β-tubulin expression in vitro. Relative quantification
(RQ) of Actin was analysed with the β-tubulin as the reference gene and wild type PH-1 as a
calibrator. RQ of β-tubulin was calculated with Actin gene as a reference gene and PH-1 as a
calibrator. Greek letters show statistically significant differences from three biological replicates.
Error bars show the standard error between biological replicates.
Figure S4. Relative quantification of gene expression in infected wheat ears. Twenty two spikelets
of each ear were inoculated. The inoculated spikelets were collected at 7 dpi. Relative gene
expression was analysed with 2-ΔΔCt method with Actin gene as the reference gene and PH-1 as a
calibrator. Greek letters show the statistically significant differences from three biological replicates.
Error bars show the standard error between biological replicates.
Figure S5. Infection of Arabidopsis floral tissue with PH-1 and FgCYP51 gene deletion mutants.
Each plant was spray inoculated with approximately 5 x 104 spores in sterile water. Infected floral
tissue at a) 8 dpi. and b) 16 dpi. Scale bar = 5mm
Figure S6. Pathogenicity of PH-1 and FgCYP51 gene deletion mutants on apple sections. . Apple
sections were inoculated with a 5µl droplet of a spore suspension containing 5 × 105 ml-1 conidia.
Each fruit section was inoculated with wild type PH-1, an FgCYP51 deletion mutant, and water as a
negative control. Photos were taken at 6 dpi. Bottom right is a schematic image of the inoculation.
Figure S7. Infection of tomato fruits with PH-1 and FgCYP51 gene deletion mutants. Tomato fruits
were inoculated with 3 times 5µl droplet of 5 × 105 ml-1 spore suspensions applied to wounds. The
top lane shows the surface growth on tomato fruits. The bottom lane shows a longitudinal section of
the infected area. Photos were taken at 6 dpi.
Figure. S9. Alignment of predicted FgCYP51 amino acid sequences. FgCYP51 sequences were
aligned by ClustalW. Residues identical in all FgCYP51 paralogues are highlighted in black. Residues
identical in all sequenced eukaryotic CYP51s are marked with black triangles and residues identical in
more than 99% of sequences are marked with grey triangles (Lepesheva and Waterman, 2011).