Supplementary Material
Characterization of the fusaric acid biosynthetic gene cluster and
its regulation in F. fujikuroi
Eva-Maria Niehaus[a]#, Katharina W. von Bargen[b]#, José J. Espino[a], Andreas Pfannmüller[a],
Hans-Ulrich Humpf[b]* and Bettina Tudzynski[a]*
a
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität
Münster, Schlossplatz 8, D-48143 Münster
b
Institut für Lebensmittelchemie, Westfälische Wilhelms-Universität Münster, Corrensstr. 45,
D-48149 Münster
*Prof. Dr. B. Tudzynski, fon: +49 251 83 24801, fax: +49 251 83 21601, e-mail:
tudzynsb@uni-muenster.de
*Prof. Dr. H.-U. Humpf, fon: +49 251 83 33391, fax: +49 251 83 33396, e-mail: humpf@unimuenster.de
#
authors contributed equally
Keywords: Fusarium fujikuroi, fusaric acid, polyketide synthase, mycotoxin, HPLC-HRMS,
nitrogen regulation
1
Figures
Fig. S1: Deletion strategy and southern blot of three independent FUB1 deletion
mutants. (a) Southern strategy with the wild type (WT) and deletion mutant (∆FUB1) which
has a hygromycin resistance gene: hygromycin phosphotransferase (hph). The WT and the
three mutants were digested with SspI. The shaded area depict the 5’ and the 3’ flank,
respectively. (b) Southern blot of the WT and ∆FUB1 (T5, T11, T17). The WT allele (~ 2.9
kb) is absent in the three deletion mutants. The 5’ flank was used as probe
Fig. S2: Plate assay with the wild type F. fujikuroi IMI58289 and two independent FUB1
deletion mutants on CM agar. The plates were inoculated with 5-mm-diameter mycelial disks
of the fungal strains and incubated for three days at 28 °C
2
8.54
8.54
8.17 residual formic acid
7.97
7.97
7.95
7.95
7.80
7.80
7.78
7.78
3000
-0.02 TMS
1.09
1.07
2.75
2.69
2.67
2.65
2.50 DMSO
2.50 DMSO
2.50 DMSO
2.50 DMSO
2.49 DMSO
1.66
1.64
1.62
1.62
1.60
1.58
3.62
3.60
3.59
3.57
3.56
3.54
3.41 impurity
4.28
2800
2600
2400
2200
2000
1800
(dd)
7.79
(d)
8.54
1600
(dd)
7.96
(h)
3.58
(m)
2.72
(m)
1.63
(d)
1.08
1400
1200
1000
800
600
400
200
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
3.00
2.34
2.00
0.98
1.14
1.04
1.14
0
1.0
-200
0.5
0.0
40.1
40.0 DMSO
39.9 DMSO
39.7 DMSO
39.5 DMSO
39.3 DMSO
39.1 DMSO
38.9 DMSO
31.9
28.6 Impurity
27.0 Impurity
23.6
60.4 Impurity
65.1
124.4
136.9
141.7
146.1
149.4
166.2
Fig. S3: 1H-NMR spectrum (d6-DMSO, 400 MHz) of fusarinolic acid. Impurities were not
completely removed by the used chromatographic separation
40000
35000
30000
25000
20000
15000
10000
5000
0
170
160
150
140
130
120
110
100
90
80
f1 (ppm)
70
60
50
40
30
20
10
0
Fig. S4: 13C-NMR spectrum (d6-DMSO, 100 MHz) of fusarinolic acid. Impurities were not
completely removed by the used chromatographic separation
3
Fig. S5: COSY spectrum (d6-DMSO, 400 MHz) of fusarinolic acid
Fig. S6: HMQC spectrum (d6-DMSO) of fusarinolic acid
4
250000
-0.02 TMS
-0.02 TMS
-0.02 TMS
-0.03 TMS
240000
2.86
2.84
2.84
2.82
2.45
2.43
2.41
2.39
3.47 residual MeOH
3.74 Impurity
7.26 CDCl3
5.85
5.84
5.83
5.82
5.82
5.81
5.80
5.80
5.79
5.78
5.78
5.77
5.76
5.76
5.74
5.74
5.04
5.04
5.03
5.03
5.02
5.02
4.99
4.99
4.98
4.88 Impurity
8.19
8.17
8.17
7.80
7.79
7.78
7.77
8.63
Fig. S7: HMBC spectrum (d6-DMSO) of fusarinolic acid
230000
220000
210000
200000
190000
180000
170000
160000
150000
140000
130000
(m)
5.02
(s)
8.63
(d)
8.18
(dd)
7.78
(m)
5.79
(m)
4.99
(t)
2.84
120000
(q)
2.42
110000
100000
90000
80000
70000
60000
50000
40000
30000
20000
10000
0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
f1 (ppm)
3.5
3.0
1.98
1.84
0.88
0.90
0.99
0.98
0.85
0.82
-10000
2.5
-20000
2.0
1.5
1.0
0.5
0.0
Fig. S8: 1H-NMR spectrum (CDCl3, 400 MHz) of dehydrofusaric acid
5
34.7
32.4
77.5 CDCl3
77.4 CDCl3
77.2 CDCl3
76.8 CDCl3
116.5
124.4
148.0
144.9
142.3
138.8
136.4
165.1
7000
6500
6000
5500
5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0
-500
170
160
150
140
130
120
110
100
90
80
f1 (ppm)
70
60
50
40
30
20
10
0
Fig. S9: 13C-NMR spectrum (CDCl3, 100 MHz) of dehydrofusaric acid
Fig. S10: COSY spectrum (CDCl3, 400 MHz) of dehydrofusaric acid
6
Fig. S11: HMQC spectrum (CDCl3) of dehydrofusaric acid
Fig. S12: HMBC spectrum (CDCl3) of dehydrofusaric acid
7
Tables
Table S1: List of all primers and nucleotide sequence that were used in this study.
Primer
Sequence
Amplification of the gene flanks
fub1-5F
GTAACGCCAGGGTTTTCCCAGTCACGACGGAACAGAAGAATGGAGCGCAGG
fub1-5R
ATCCACTTAACGTTACTGAAATCTCCAACGATTGTGTGATGGGCGCTGG
fub1-3F
CTCCTTCAATATCATCTTCTGTCTCCGACGGCACTGGTGCTGTCGCTGG
fub1-3R
GCGGATAACAATTTCACACAGGAAACAGCCCTAATGCTGGCCGGACTATC
fub2-5F
GTAACGCCAGGGTTTTCCCAGTCACGACGTCTCAACATCATCAAGGCCGC
fub2-5R
ATCCACTTAACGTTACTGAAATCTCCAACCTGTTCTGATGTGCCAATGGC
fub2-3F
CTCCTTCAATATCATCTTCTGTCTCCGACAAGTTGATCATGGGAGACGCG
fub2-3R
GCGGATAACAATTTCACACAGGAAACAGCGTAGCTTGAGATGGTGCGAGG
fub3-5F
GTAACGCCAGGGTTTTCCCAGTCACGACGATTACTGACAGCTGAGCACCG
fub3-5R
ATCCACTTAACGTTACTGAAATCTCCAACTGGCAAGCTTCGCAAGATACG
fub3-3F
CTCCTTCAATATCATCTTCTGTCTCCGACTCCGTAAACAACCAAGCACGC
fub3-3R
GCGGATAACAATTTCACACAGGAAACAGCACAATGGTGTGGTCAGCTGGG
fub4-5F
GTAACGCCAGGGTTTTCCCAGTCACGACGATACTTAGCGGGTGACTGTGG
fub4-5R
ATCCACTTAACGTTACTGAAATCTCCAACGAAGGTCAAAGGATGCTTGGG
fub4-3F
CTCCTTCAATATCATCTTCTGTCTCCGACCTGGATGGCTGATTGCAAACG
fub4-3R
GCGGATAACAATTTCACACAGGAAACAGCCAAGGTCTTCACCATTCTGGC
fub5-5F
GTAACGCCAGGGTTTTCCCAGTCACGACGTCCGTGAATACAGTGGAGACG
fub5-5R
ATCCACTTAACGTTACTGAAATCTCCAACTCAAAATACACTGGGGACGGG
fub5-3F
CTCCTTCAATATCATCTTCTGTCTCCGACCCGAGAGAAGATAAGAGTGCC
fub5-3R
GCGGATAACAATTTCACACAGGAAACAGCTACCTCGTATCTCTAGGTGCG
Diagnostic primers
fub1-5F-diag
CAGGCGAACTGGCTGGCG
fub1-3R-diag
GGGCAGTGGTGGAGATGTTGG
fub1-WT-F
CCAGGTTGTCGAGCTTCTGTCC
fub1-WT-R
GCGAGTCGGCAAACCAGTAGGG
fub2-5F-diag
TTCCAGCTTCTCAAGGATGGC
fub2-3R-diag
ATGGCTACAATGCCTCTCAGC
fub2-WT-F
CCGAGCTCAAAGAATACCTCG
fub2-WT-R
ATCTCCTTGATCTCCTCCTCG
fub3-5F-diag
GATAGTCCGGCCAGCATTAGG
fub3-3R-diag
ATGGCCATCTTCACATCAGCC
8
fub3-WT-F
TCGAGATAATTCCTGGGTGGC
fub3-WT-R
TACCACTGTCTATCAGGCTCC
fub4-5F-diag
CTAGGACCAACTTGTGAAGCC
fub4-3R-diag
AAACAGTCCATGTCTCACGGC
fub4-WT-F
TTCTTTGTCTCCACGGGTACG
fub4-WT-R
TCTGCAAAGTGCACCAACTCC
fub5-5F-diag
TTGTACCAGCTGTAGTTGGGC
fub5-3R-diag
GAGGACGATTCCAGGAATTGC
fub5-WT-F
TCACGATGGCCTTGGTAATGG
fub5-WT-R
ACAATGCTGTCATACGAGCCG
Diagnostic primers for the resistance cassette
pCSN44-hph-trpC-T
GGAATAGAGTAGATGCCGACCGG
pCSN44-trpC-P2
GTGATCCGCCTGGACGACTAAACC
Primers for the overexpression mutants
gpd-prom-out-F1
CCATACTCCATCCTTCCCATCC
OE-fub2-3R
GCGGATAACAATTTCACACAGGAAACAGCCGATGTCAGCGC
OE-fub2-5F
TACCCCGCTTGAGCAGACATGTCATGGCTACCGAG
OE-fub3-3R
GCGGATAACAATTTCACACAGGAAACAGCTAGATGTCAGTGTCG
OE-fub3-5F
TACCCCGCTTGAGCAGACATAGCATGCGTAGT
OE-fub4-3R
GCGGATAACAATTTCACACAGGAAACAGCGGTGATTAGAGG
OE-fub4-5F
TACCCCGCTTGAGCAGACATAAGATGAGATTTCTT
OE-fub5-3R
GCGGATAACAATTTCACACAGGAAACAGCCAAGACACTCGC
OE-fub5-5F
TACCCCGCTTGAGCAGACATAGAATGACGACG
Wild type primers of the border genes of the fusaric acid cluster
FFUJ_02104-F
CCTGTTACCATTCTTCTCGC
FFUJ_02104-R
GCCTGGGTGGTCCATTCTG
FFUJ_02110-F
GGTAGAGATGGTACTGTCTTCTGG
FFUJ_02110-R
GCCGCAATATCCAACGCCG
Amplification of the resistance cassette
hphF
GTCGGAGACAGAAGATGATATTGAAGGAGC
hphR
GTTGGAGATTTCAGTAACGTTAAGTGGAT
Real Time PCR primers
FRACRTPCRFW
GAGAACGAGCGTGTCTTGATTGAGCC
FRACRTPCRRV
TTTCCTCCGCAGAATGAAGAAGGACTC
FUJGMTRTPCRFW CGGGCCATTCTCTATTCTTTC
FUJGMTRTPCRRV
ATGCTGTGATGGCAACAATG
FUBRTPCRFW
CCAACCCTGACGATCCTCTTGTGC
9
FUBRTPCRRV
TACTTTCGAGTCCACTCCCGAGCTG
FFUB1RTPCRFW
TGGATCGTCAAGGCACTGGTGC
FFUB1RTPCRRV
TCCACATCAGCCTTGATGATCTTGGAAAC
FFUB5RTPCRFW
CAGCAACTGCTACATCGCCCTCAC
FFUB5RTPCRRV
GCAAGCGTATACAGCCCATCAC
10