Supporting information for the manuscript:
The global regulator FfSge1 is required for expression of secondary
metabolite gene clusters, but not for pathogenicity in Fusarium fujikuroi
C.B. Michielse1,5#, L. Studt1#, S. Janevska1, C.M.K. Sieber2, B. Arndt3,4, J.J. Espino1, H-U.
Humpf3,4, U. Güldener2 and B. Tudzynski1
1
Institute of Biology and Biotechnology of Plants, Westfälische Wilhelms-University,
Schlossplatz 8, 48143 Münster, Germany
2
Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Germany
Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, 85764 Neuherberg,
Germany
NRW Graduate School of Chemistry, Westfälische Wilhelms-University, Wilhelm-Klemm-
3
Strasse 10, 48149 Münster, Germany
4
Institute of Food Chemistry, Westfälische Wilhelms-University, Corrensstr. 45, 48149 Münster,
Germany
5
Present address: Dyadic Netherlands, Nieuwe Kanaal 7-S, 6709 PA Wageningen, The
Netherlands
# both authors contributed equally
Running title: FfSge1, a regulator of secondary metabolism
Corresponding author: B. Tudzynski, Institute of Biology and Biotechnology of Plants,
Westfälische Wilhelms-University, Schlossplatz 8, 48143 Münster, Germany.
E-mail: tudzynsb@uni-muenster.de, phone: +49 251 8324801, fax: +49 251 8321601
Tab. S1. Primer used during this study.
Fig. S1. Domain structure of FfSge1. FfSge1 start (1) and stop (333) codon are highlighted by
bars. The protein (grey bar) contains all typical conserved domains, the TOS9 motif (COG5037)
present at aa 5 to 209 (green bar), the Gti/Pac2 superfamily domain (Pfam 09729) located at aa
10 to 183 (blue bar), a putative protein kinase A phosphorylation site (KRWTDS) at aa residues
65 to 70 (yellow bar) and a nuclear localization signal (PPGEKKR) present at aa residues 93 to
99 (red bar).
Fig. S2. Analysis of ffsge1 deletion and FfSGE1C complementation mutants. A) Schematic
representation of the FfSGE1 knock-out strategy drawn to scale. The arrow heads represent the
primers used to check homologous recombination (a and b; e and f) and the absence of the open
reading frame (c and d). BglII (B) and XbaI (X) restriction sites are depicted by short vertical
bars. B) Schematic representation of the complementation strategy drawn to scale. The arrow
heads indicate the primers used to check homologous recombination (a and b; e and f) and the
presence of the open reading frame (c and d). BglII (B) restriction sites are depicted by short
vertical bars. C) Verification of correct homologous recombination at the FfSGE1 locus in the
deletion mutant by PCR. D) Verification of correct homologous recombination at the FfSGE1
locus in the complementation mutant by PCR. E) Verification of correct homologous
recombination at the FfSGE1 locus in the deletion and complemented strain by Southern
analysis. To this end, genomic DNA of the mutants was digested with BglII, size separated,
blotted, followed by hybridization with a 947 bp probe corresponding to the FfSGE1 promoter
region. The FfSGE1 locus of the wild-type strain is visible as a 1.9 kb fragment. In the ∆ffsge1
deletion mutant, introduction of the gene replacement cassette by homologous recombination led
to the replacement of the 1.9 kb fragment with a fragment > 10 kb. Introduction of the FfSGE1
complementation cassette by homologous recombination led to the expected replacement of the
> 10 kb fragment with the 1.9 kb fragment. orf, open reading frame. lf, upstream (left) flanking
region. rf, downstream (right) flanking region.
Fig. S3. Analysis of FfSGE1 overexpression and FfSGE1:GFP mutants. A) A diagnostic
PCR verifies the presence of the FfSGE1 overexpression construct in the wild type using primers
gpd-dia-fwd/sge-yOE-r2. B) A diagnostic PCR verifies the presence of the FfSGE1::GFP fusion
construct in the wild type using primers sge-f1/gpd-dia-rev. C) Ectopic integration of the FfSGE1
overexpression construct was verified by Southern analysis. Genomic DNA was digested with
XbaI, size separated and blotted followed by hybridization with a probe corresponding to the
FfSGE1 open reading frame. The FfSGE1 locus of the wild-type strain is visible as a 2.7 kb
fragment. Ectopic introduction of the FfSGE1 overexpression cassette led to the expected 2.7 kb
wild-type FfSGE1 fragment and an additional fragment corresponding to the additional FfSGE1
gene copy. D) FfSGE1 overexpression in the OE:FfSGE1 mutant was verified via Northern
analysis. The wild-type and the mutant strain were grown for three days at 28 C in liquid ICI
medium supplemented with 6 and 60 mM glutamine. As loading control, rRNA was visualised
by UV light.
Fig. S4. FfSGE1 is not required for conidiogenesis, but is involved in vegetative growth. A)
Growth phenotype of the wild type, the ∆ffsge1 deletion and FfSGE1c mutants grown for seven
days at 28 C on various solidified complex and minimal media (V8, vegetable fruit juice. CM,
complete medium. PDA, potato dextrose agar. CDA, Czapek Dox agar. ICI, Imperial Chemical
Industries (synthetic) medium). B) Fungal growth rate (± SD) of the wild type, the ∆ffsge1
deletion and FfSGE1c mutants grown in liquid ICI media for seven days at 28 C containing
either low (6 mM gln) or high (60 mM gln) nitrogen levels. Fungal dry mass of the wild type was
set to 1. C) Average number of microconidia per cm2 (± SD) of the wild type, the ∆ffsge1
deletion and FfSGE1c mutant.
Fig. S5. Biosynthesis of fusaric acid is decreased after only three days of incubation in the
OE:FfSGE1 mutant. FU production levels (± SD) in the wild type (set to 100%) and FfSGE1
overexpression mutant (in a wild-type background). The strains were grown in liquid ICI
medium supplemented with 60 mM glutamine for three days, followed by harvest of the culture
filtrate. FU production was quantified using HPLC-UV/DAD and a student’s t-test was
performed to determine significant differences (n = 3). *, significantly different at a confidence
interval of 95%.
Fig. S6. Diagnostic PCR analysis of double mutants generated in order to gain insight into
the hierarchical gene regulatory network. In each case, primer combinations were chosen both
for testing the presence of the corresponding OE-vector, as well as for testing whether an in
locus integration occured. Double mutants of Δffsge1/OE:APF2 (A) were analysed with primer
pairs POliC-seqF2/00012_apf2_R (presence, 0.73 kb) and POliC-seqF2/OE_apf2_diag (in locus
integration, 2.20 kb). The transformant T3 carries a homologous integration of pOE:APF2:GFP
that was also used as a positive control (P) in the PCR reaction. Double mutants of
Δffvel1/OE:FfSGE1 (B) as well as ΔareA/OE:FfSGE1 (C) were analysed with primer pairs gpd-
dia-fwd/Sge-yOE-r2 (presence, 1.24 kb) and gpd-dia-fwd/sge1-r9 (in locus integration, 1.24 kb).
Δffvel1/OE:FfSGE1 T2, T3 (B) as well as ΔareA/OE:FfSGE1 T1-4, T8 (C) carry a homologous
integration of pOE:SGE1 that was also used as a positive control (P) in the PCR reaction. In each
case, genomic DNA of the WT was applied as a negative control. As size markers (M),
GeneRuler DNA Ladder Mix (A), as well as λ/HindIII (B, C) were used.
Fig. S7 Induced FfVEL1 expression in OE:FfSGE1 compared to the wild type. FfVEL1
expression was analysed in the OE:FfSGE1 mutant compared to the wild type (WT) under low (6
mM gln) and high (60 mM gln) nitrogen concentrations. The wild-type and the mutant strain
were grown for three days at 28 C in liquid ICI medium supplemented with 6 and 60 mM
glutamine (gln). As loading control, rRNA was visualised by UV light.
Tab. S1. Primers used in this study.
Name
sequence (5’-3’)
sge-KOf4
ccggaattcaagtgcaactagcctttagcc
sge-KOr7
cccaagcttggagaaggcagggatagtg
sge-KOf5
acgcgtcgacacgccgatcctgaatgac
sge-KOr8
cggggtacctgggtcggactactcttcg
sgecom-f1-ApaI
aagggcccgtccatccatctatgccatg
sgecom-r1-HindIII
cccaagcttgagggatacgagactcgttg
Sge-yOE-f
taccccgcttgagcagacataacatggctggaactatgccacttc
Sge-yOE-r2
gcggataacaatttcacacaggaaacagcctaccaccacgttgaagaag
Sge-yGFP-f
agctctgtacagtgaccggtgaccgaccacatgaccgtgagc
Sge-yGFP-r
ggagcgcgtggcgatggagcgccaccacgttgaagaaggcgcgt
sge-KOf1
gtaacgccagggttttcccagtcacgacggaatgacacgcgtggatatg
pAN-T
cccagaatgcacaggtacac
sge-KOr5
aacttataccgcccgcttg
pOli-R2
agccatggatccattgtgatgtga
sge-f1
atggctggaactatgccacttc
sge1-r8
ctaccaccacgttgaagaag
gpd-dia-fwd
catcttcccatccaagaacc
gpd-dia-rev
cattaatgcagctggcacgac
POliC-seqF2
gggagacgtatttaggtgctaggg
00012_apf2_R
ccacggctctgtgccgc
OE_apf2_diag
caccagcgatgtgtgccagg
sge1-r9
gtcattcaggatcggcgtc
sge-KOr1
atccacttaacgttactgaaatctccaacgggtggtttgagtgtgtgtg
bik2-F
cttgagtctgatagaggcgc
bik2-R
acggcgcagcagaaagtgcc
cps/ks-RT-for
gtgtagctggatcatagcgacactcctg
cps/ks-RT-rev
ccattggccctggctaagtttccc
FUM2_WT_F
tatgtacaacatcttcctcc
FUM2_WT_R
agcgatcatgaagatgtccg
FUB3_WT_F
tcgagataattcctgggtggc
FUB3_WT_R
taccactgtctatcaggctcc
00005_apf9_F
cctcattggaggagtggtcacg
00005_apf9_R
ggacatgtcgccagcacagg
WT-Diag-78-5'
ctcgcaacctttctgaagc
WT-Diag-78-3'
ctaaagcctctctaatcgg
sge-r
cggtcgaagtggcatagttc
sge-f
tccaagggtttaagatcactca
FFUJ_02115-f
agttcgttcgctacgacgtt
FFUJ_02115-r
agactctggagaccgacgaa
FFUJ_05795-f
cgctactatgtcaccgtcattgacgc
FFUJ_05795-r
caatgatgagaatggcgcaatcggcc
CPSKSRTPCRFW2
gacgacgaggctgaagattacctgag
CPSKSRTPCRRV2
cgcgcaagccatcaccatcgatttg
FUBRTPCRFW
ccaaccctgacgatcctcttgtgc
FUBRTPCRRV
tactttcgagtccactcccgagctg
FP450-1RTPCRFW
gatcacatgggcttcgggtacgg
FP450-1RTPCRRV
tacttcctcggctccatgctcgac
FGMTRTPCRFW
atgctgtgatggcaacaatg
FGMTRTPCRRV
cttgttgagagcaccaaccatggagta
FRAC-RT-PCR-FW
gagaacgagcgtgtcttgattgagcc
FRAC-RT-PCR-RV
tttcctccgcagaatgaagaaggactc
FvSGE1-r3
gttcctgaagtcgtacgag
FfSGE1-f2
catctcaaaagcccgacaac
Fig. S1
Fig. S2
Fig. S3
Fig. S4
Fig. S5
Fig. S6
Fig. S7