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Fig. S1a-i: MS2 spectra of infection markersThe putative identity of infection markers obtained by metabolite fingerprinting was confirmed by MS2 fragmentation experiment by LC 1290 Infinity coupled with a 6540 UHD Accurate-Mass Q-TOF LC MS instrument with Agilent Jet Stream Technology as ESI source.
O
O-Glc
O-CH3
OH
O-CH3
OH
m/z 359.1474
4x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
-ESI Product Ion (2.799 min) Frag=160.0V [email protected] (567.2078[z=1] -> **) KF_Steffi_MSMSI_CE10_polar_neg_110923_01.d
419.1327
521.1997
329.1385
223.0604
133.0145 385.1472
Counts vs. Mass-to-Charge (m/z)120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560
Lariciresinol glucoside (M+FA-H+)-
[M-H+]-
[M-H++FA]-
[M-Glc-H+]-[M-Glc-CH2O-H+]-
[M-Glc-C9H12O-H+]- 359.1474
- 162.0528, C6H10O5
- 136.088, C9H12O
- 30.0196, CH2O
- 164.0825, C10H12O2
195.0649
m/z 195.065
[C10H11O4]-
Supporting Information Figure S1a
4x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
-ESI Product Ion (2.284 min) Frag=150.0V [email protected] (729.2606[z=1] -> **) KF_Steffi_vl21_MSMSI_CE20_polar_neg_110919_01.d
521.2001
359.1491
329.1383
445.1137
Counts vs. Mass-to-Charge (m/z)125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725
[M-Glc-Glc-H+]-
[M-Glc-Glc-CH2O-H+]-
[M-H+]-
[M-H++FA]-
Lariciresinol diglucoside (M+FA-H+)-
[M-Glc-H+]-
- 162.0528 Da, C6H10O5
- 2 x 162.0528 Da, C12H20O10
- 30.0196 Da, CH2O
Supporting Information Figure S1b
O
O-Glc
O-CH3
Glc-O
O-CH3
OH
m/z 359.1491
m/z 521.2001
4x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
-ESI Product Ion (3.169 min) Frag=160.0V [email protected] (519.1872[z=1] -> **) KF_Steffi_MSMSIV_CE10_polar_neg_110926_01.d
357.1343
419.1349
498.1601 193.0497
Counts vs. Mass-to-Charge (m/z)140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520
Pinoresinol glucoside [M+FA-H+]-
- 162.0528, C6H10O5
[M-H+]-
[M-Glc-H+]-
Supporting Information Figure S1c
O
O
OH
CH3-O
O-Glc
O-CH3
m/z 357.1343
- 2 x 162.0528, 2 x C6H10O5
- 206.0942, C12H14O3 [X]
Pinoresinol diglucoside [M+FA-H+]-
4x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
-ESI Product Ion (2.480 min) Frag=160.0V [email protected] (681.2400[z=1] -> **) KF_Steffi_MSMSIV_CE40_polar_neg_110926_01.d
151.0398
357.1338
112.9845
Counts vs. Mass-to-Charge (m/z)75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675
[M-Glc-Glc-H+]-
-ESI Product Ion (2.462 min) Frag=160.0V [email protected] (681.2400[z=1] -> **) KF_Steffi_MSMSIV_CE10_polar_neg_110926_01.d
519.1859
357.1342
681.2383
Counts vs. Mass-to-Charge (m/z)350 400 450 500 550 600 650 700
[M-H+]-
[M-Glc-H+]-
[M-Glc-Glc-H+]-
Supporting Information Figure S1d
O
O
O
CH3-O
O
O-CH3
Glc
Glc
x
m/z 357.1338
[C8H7O3]-
[X]-
m/z 151.0398 (X-)
m/z 519.1859
Supporting Information Figure S1e
3x10
0
1
2
3
4
5
6
7
8
-ESI Product Ion (3.194 min) Frag=160.0V [email protected] (579.2083[z=1] -> **) KF_Steffi_21vl_MSMSV_CE10_polar_neg_110926_02.d
417.1555
223.0591 577.1522 181.0504 361.0535 533.1669
Counts vs. Mass-to-Charge (m/z)140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580
Syringaresinol glucoside [M-H+]-
[M-Glc-H+]-
[M-H+]-
- 162.0528, C6H10O5
- 236.1051, C13H16O4
[C9H9O4]-
[Y]-
O
O
OH
O-CH3
CH3-O
O-Glc
O-CH3
O-CH3
m/z 417.1555
Ym/z 181.0504 [Y] -
m/z 181.0504 [Y] -
Supporting Information Figure S1f
4x10
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
-ESI Product Ion (3.553 min) Frag=100.0V [email protected] (551.1770[z=1] -> **) KF28_Steffi_35vl_24_MSMSV_CE10_meoh_neg_120921_01.d
389.1239
551.1760 193.0504 341.1023
492.1027 165.0566
Counts vs. Mass-to-Charge (m/z)140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560
Guaiacylglycerol ferulic acid ether glucoside [M-H+]-
- 48.0216, -H2O - CH2O- 162.0528, - C6H10O5
- 196.0735, - C10H12O4
[M-Glc-H+]-
[M-H+]-
- 196.0735 -27.9938,- CO - C10H12O4
Ferulic acid[C10H9O4]-
O-CH3
O-Glc
O
OHOH
OHO-CH3
O
m/z 193.0504
m/z 389.1239
m/z 165.0566
[C9H9O3]-
Supporting Information Figure S1g
3x10
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
-ESI Product Ion (3.207 min) Frag=160.0V [email protected] (533.1642[z=1] -> **) KF_Steffi_MSMSII_CE10_polar_neg_110923_01.d
371.1113
353.1017
533.1635
387.1414
498.1571
223.0581 419.1305
Counts vs. Mass-to-Charge (m/z)180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
[M-H+]-
[M-Glc-H+]-
[M-Glc-CH2O-H+]-
[M-Glc-H2O-H+]-
- 162.0528, C6H10O5
- 30.0103, CH2O
- 18.0096, H2O
Sesamolinol glucoside [M-H+]-
341.1010
O
O
O-CH3
O
O
O-Glc
O-CH3
m/z 371.1105
m/z 341.1010
Supporting Information Figure S1h
4x10
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
-ESI Product Ion (3.757 min) Frag=160.0V [email protected] (487.1246[z=1] -> **) KF_Steffi_MSMSII_CE10_polar_neg_110923_01.d
353.1017
371.1105
133.0134 487.1219
316.9401
Counts vs. Mass-to-Charge (m/z)140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
341.1009
[M-H+]-
[M-Malyl-H+]-
[M-Malyl-CH2O-H+]-
[M-Malyl-H2O-H+]-
- 116.0114, C4H4O4
- 30.0103, CH2O
- 18.0096, H2O
Malyl sesamolinol [M-H+]-
O
O
O-CH3
O
O
O
O-CH3
O
OH
OH
O
m/z 371.1105
m/z 341.1009
Supporting Information Figure S1i
3x10
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
3.25
3.5
3.75
-ESI Product Ion (2.727 min) Frag=100.0V [email protected] (399.0928[z=1] -> **) KF28_Steffi_35vl_24_MSMSV_CE10_meoh_neg_120921_01.d
191.0347
310.9121 119.0351 176.0120 243.8791 354.8957
Counts vs. Mass-to-Charge (m/z)120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
Scopolin [M+FA-H+]-
[M-H++FA]-
[M-Glc-H+]-
[M-Glc-CH3-H+]-
- 162.0528 -44.9898, C6H10O5 -FA
- 15.0227, -CH3
O-CH3
O-GlcOO
m/z 176.0120
m/z 191.0347
(a)
(b)
Supporting Information Figure S2in
tens
ityin
tens
ity
valine (cl.11) glutamine (cl.12)
arginine (cl. 14)
asparagine (cl. 11)
35dpi21 dpi10 dpi
control VL control VL control VL control VL control VL control VL
10 dpi 21 dpi 35dpi
control VL control VL control VL
10 dpi 21 dpi 35dpi
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
pyrrolidone-5-carboxylic acid (cl. 12)leucine (cl. 10)
Fig. S2: UPLC-TOF-MS based metabolite fingerprinting of V. longisporum infected Arabidopsis leaves.Arabidopsis plants of different time points after inoculation with V. longisporum (10, 21 and 35 dpi) and the corresponding control plants were harvested and the aerial parts were extracted by two phase partitioning. The fingerprint of metabolites of the polar extraction phase was generated by UPLC-TOF-MS analysis. A subset of high-quality 862 metabolite marker candidates (FDR <10-4) derived from the positive as well as the negative ionization mode were used for clustering and visualization by means of one-dimensional self-organizing map (1D-SOM). (a) 1D-SOM heat map after metabolite-based clustering with 20 prototypes. Horizontal and vertical dimensions correspond to prototypes and experimental conditions, respectively. The heat map colors represent average intensity values according to the color map on the right hand side. The width of each prototype column is proportional to the number of marker candidates assigned to this prototype. (b) Box plots of prominent markers from different clusters (cl.) which accumulate in V. longisporum inoculated plants (VL). Four samples per treatment from two independent experiments were analyzed. Boxes indicate the 25th (q1) and 75th (q3) intensity percentiles and median of the data. The whiskers indicate the range between q1-1.5(q3-q1) and q3+1.5(q3-q1), respectively.
control VL43
hypocotyl
petiole
Supporting Information Figure S3
Fig. S3: Lignin accumulation in V. longisporum infected vascular tissue of Arabidopsis.Cross-sections of hypocotyls and petioles of V. longisporum inoculated (VL) and control plants at 21 dpi. Material was paraffin embedded, hand sectioned and stained with phloroglucin/HCl for lignified tissue. Scale bar: 200 µm.
Col 0
C4H:F
5H-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
rela
tive
units
Col 0
C4H:F
5H
0
10
20
30
40
50
60
70
pg m
g-1
FW
A B Crelative leaf area VL-DNAcontrol
Col 0
C4H:F5H
VL
Supporting Information Figure S4
Fig. S4: Infection of the C4H:F5H mutant with V. longisporum.Col 0 and C4H:F5H plants were infected by root dip infection and harvested at 21 dpi. (a) Pictures of infected and control plants at 21 dpi. (b) The relative leaf area was determined from pictures taken of the rosettes of the plants: The leaf area of infected plants was divided by the leaf area of control plants and the results of the mutant plants were set in relation to the value of the wild type. The data represent the mean values of five independent experiments ±SD. (c) Determination of VL-DNA in infected leaves. The mean values of ten samples of three independent experiments are shown ±SE.
Supporting Information Figure S5
Fig. S5: Sinapoyl glucose, coniferin and syringin in glycosyltransferase overexpressor plants and the fah1-2 mutant. Quantification of sinapoyl glucose and monolignols by HPLC-DAD of V. longisporum-inoculated (black bars) and control (white bars) plants. Syringin could not be quantified in the UGT72E2-OE because of high coniferin peaks. Each data point represents the mean value of eight samples from two independent experiments + SD. (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 )
0
0.5
1
1.5
2
2.5
3
0
5
10
15
µm
ol g
-1 F
W
syringinconiferin
UGT72E3-
OE
UGT72E2-
OE
UGT72E1-
OECol
0
Col 0
fah1
-20
20
40
60
80
100
nmol
g-1
FW
-1.66533453693773E-16
0.2
0.4
0.6
0.8
1
1.2sinapoyl glucose
0
0.2
0.4
0.6
0.8
1
1.2
1.4
µm
ol g
-1 F
W
Col 0
fah1
-2
controlVL
UGT72E3-
OE
UGT72E2-
OE
UGT72E1-
OECol
0
UGT72E3-
OE
UGT72E2-
OE
UGT72E1-
OECol
0
coniferin sinapoyl glucose
n.d.
Fig S6: Influence of ferulic and sinapic acid on V. longisporum growth.PDB plates were supplemented with ferulic acid and sinapic acid in the indicated concentrations. After inoculation the fungus was grown for 18 days on the plates. Pictures of colonies were taken under a binocular and the colony area was determined. The data represent the mean values ±SD of seven different plates of two independent experiments. Asterisks indicate significant differences between control and supplemented plates according to student’s t-test (*, P ≤ 0.05; ***, P ≤ 0.001).
Supporting Information Figure S6
cont
rol
1 µM
10 µ
M
100µ
M1
µM
10 µ
M
100
µM
0
200colony area
mm
2
ferulic acid sinapic acid
****
Supporting Information Table S1
Table S1: Primers used for the quantitative RT-PCR.
QuantPrime: Samuel Arvidsson, Miroslaw Kwasniewski, Diego M. Riano-Pachon and Bernd Mueller-Roeber: QuantPrime - a flexible tool for reliable high-throughput primer design for quantitative PCR. BMC Bioinformatics 2008, 9:465
name ATG Primer referencePhenylalanine ammonia lyase 1 (PAL1)
At2g37040PAL1neuF: AGAAGTGGAAGCAGCAAGAGCAG
PAL1neuR: ATCCTGTTCGGGATAGCCGATG
QuantPrime
Phenylalanine ammonia lyase 2 (PAL2)
At3g53260PAL2R: TGCTACTTCTCACCGGAGA
PAL2F: TATTCCGGCGTTCAAAAATC
Huang et al. 2010
Ferulate-5-hydroxylase (F5H)
At4g36220F5H_F: ATGATGGGGATGTTGTCGAT
F5H_R: CGTCCATGATGATTGCTTTG
Vanholme et al. 2010
Cinnamyl alcohol dehydrogenase 5 (CAD5)
At4g34230CAD5F: CCTGCGAGAGAGATCTGGAA
CAD5R:CAAAGTGGCTCAGTGGACTGT
Tronchet et al. 2010
Cinnamyl alcohol dehydrogenase 8 (CAD8)
At4g37990CAD8F: GCTGCACCGAAGGTATGGAA
CAD8R: CGTCCAAGGGGAGATTGTCT
Tronchet et al. 2010
UGT72E1 At3g5074072E1F: AAAGAGACCGCTGCCGAAT
72E1R: CCTGACACGCTCCAAAAGATG
Lanot et al. 2006
UGT72E2 At5g6669072E2neuF: TGGTGGTTGTCTATCGGCGAAAC
72E2neuR: ATACGAACCGTTGCTGGCTCTG
QuantPrime
UGT72E3 At5g2631072E3neuF: AGGGTTCGTGACTCGTACTTGC
72E3neuR: TGGGCTAGGATTTCAGCTTGCG
QuantPrime
UGT84A3 At4g1549084A3F: AGCCAACTTGAAGCAAGAGCAG
84A3R: ACCACCCATAAGACCGACAAGC
QuantPrime
Dirigent protein 6 (DIR6)
At4g23690DIR6F: ACCCTGCAAACACTTCTCGTTC
DIR6R: CGGAGGTTGCGTTTGCTACATTG
QuantPrime
Supporting Information Table S2
Table S2: Data matrix of 826 high quality marker candidates (false discovery rate<10 -4) obtained by metabolite fingerprinting (UPLC-ESI-TOF-MS analysis) of mock treated (control) or V. longisporum infected (VL) leaves of Arabidopsis (10, 21, 35 dpi) (see separate file).
Supporting Information Table S3
Table S3: Infection markers identified by metabolite fingerprinting (UPLC-ESI-TOF-MS analysis) and verified by UHPLC-ESI-QTOF-MS2 analysis or co-elution (see separate file).
Linie Atg plant line literature effected gene susceptibility
fah1-2 At4g36220 CS6172 (Chapple et al. 1992) ferulate-5-hydroxylase defect
++
ref 1_SALK At3g24503 SALK_027911 aldehyde-dehydrogenase defect
(+)
sng1-1 At3g24503 N3737 (Lorenzen et al. 1996) sinapoylglucose-malate O-sinapoyltransferase defect
(+)
UGT72E1-OE At3g50740 (Lanot et al. 2008) glycosyltransferase overexpressed
+/-
UGT72E2 -OE At5g66690 (Lanot et al. 2006) glycosyltransferase overexpressed
--
UGT72E3 -OE At5g26310 (Lanot et al. 2008) glycosyltransferase overexpressed
+/-
C4H::F5H At4g36220 (Meyer et al. 1998) ferulate-5-hydroxylase overexpressed
(- )
Atprr1-1atprr2 At1g32100At4g13660
SALK_058467SALK_123621
(Nakatsobu et al. 2008)
pinoresinol-reductase defect
+/-
nac42 At2g43000 N536474 transcription factor defect
+/-
NAC42_20-OE At2g43000 transkription factor overexpressed
+/-
NAC42_6 -OE At2g43000 transcription factor overexpressed
+/-
cad4 At4g39330 SAIL_1265_A06 cinnamyl-alcohol-dehydrogenase defect
+/-
cad5 At4g34230 SALK_040062 cinnamyl-alcohol-dehydrogenase defect
+/-
Supporting Information Table S4
Table S4: Mutants and transgenic lines tested for their susceptibility against V. longisporum.
