Methods S1. Supplementary information to materials and methods.
Metabolome Analysis
Samples were frozen, homogenized and extracted in methanol (Acros Organics,
http://www.acros.com, 12,5 µL/mg of sample) at room temperature for 15 min. Samples
were centrifuged and 450 µL of the supernatant was lyophilised and resuspended in 50
µL of Milli-Q water. The solution was phase partitioned with 50 µL of cyclohexane
(Acros Organics), and 45 µL of the water phase collected for liquid-chromatographymass spectrometry (LC-MS) analysis. LC-MS was performed on a Waters Acquity
UPLC system (Waters, Milford, MA, USA, http://www.waters.com), equipped with an
Acquity BEH C18 column (2.1 mm x 150 mm, 1.7 µm) (Waters Corp, Milford, MA,
USA) connected to a Synapt HDMS Q-TOF mass spectrometer (MS) equipped with an
electrospray ionization source and lockspray interface (Waters Corp, Milford, MA,
USA). Mobile phases were (A) water containing 1% ACN and 0.1% formic acid and
(B) ACN containing 1% water and 0.1% formic acid. Column temperature 40°C,
autosampler temperature 10°C, flow rate 350 µL/min, gradient elution increasing from
5 to 50% B (0 to 30 min). MS source parameters were as following: capillary voltage
2.7 kV, sampling cone 38 V, extraction cone 2 V, source temperature 120°C,
desolvation temperature 350°C, cone gas flow 50 L/h, desolvation gas 550 L/h. The
collision energy for the trap and transfer cell were set at 6 and 4 V, respectively. For
data acquisition, the dynamic range enhancement mode was activated. Full scan data
was recorded in negative centroid V-mode, the mass range was set between m/z 1001000 with a scan speed of 0.2 s/scan using Masslynx software (Waters Corp, Milford,
MA, USA). Leucin-enkephalin (400 pg/µL dissolved in water/ACN (1:1, v/v) acidified
with 0.1% formic acid) was used for lock mass calibration by scanning every 10
seconds with a scan time of 0.5 seconds; 3 scans were averaged. For MS/MS purposes,
the same settings were applied, except the trap collision energy was ramped from 10 to
45 V. All solvents used were ULC/MS grade (Biosolve, Valkenswaard, The
Netherlands, http://www.biosolve-chemicals.com), water was produced by a DirectQUV
water
purification
system
(Millipore
SAS,
Molsheim,
France,
http://www.millipore.com). Data processing was carried out by using Markerlynx (an
add-in to Masslynx, Waters, Milford, MA, USA) for peak detection and alignment. For
peak detection, the m/z range was set from 100 to 1000, with an extraction window of
0.05 Da, and marker intensity threshold was set at 30 counts. For peak alignment, a
mass window of 0.05 Da and a retention time window of 0.2 minutes were applied.
Detected peaks were finally deisotoped. The aligned dataset was subsequently subjected
to PCA and multivariate OPLS-DA analysis.
Protoplast transactivation assay
Accession numbers for reporters (phenylpropanoid biosynthesis genes):
AtPAL1, phenylalanine ammonia-lyase (At2g37040); AtC4H, cinnamate 4-hydroxylase
(At2g30490);
At4CL1,
coumaroylshikimic
4-coumarate:CoA
acid/quinic
acid
AtCCoAOMT1,
(At1g51680);
3-hydroxylase
hydroxycinnamoyl-CoA:shikimate/quinate
(At5g48930);
ligase
caffeoyl-CoA
(At2g40890);
AtC3H1,
p-
AtHCT,
p-
p-hydroxycinnamoyltransferase
O-methyltransferase
(At4g34050);
AtCCR1, cinnamoyl-CoA reductase (At1g15950); AtCOMT, caffeic acid Omethyltransferase (At5g54160); AtF5H1, ferulic acid 5-hydroxylase (At4g36220);
AtCAD6, cinnamyl alcohol dehydrogenase (At4g34230); AtCHS, chalcone synthase
(At5g13930).
Accession numbers for effectors (transcription factors):
AtMYB20 (At1g66230), AtMYB63 (At1g79180), AtMYB69 (At4g33450), AtMYB103
(At1g63910), AtNST1 (At2g46770), AtSND1 (At1g32770), AtSND2 (At4g28500),
AtSND3 (At1g28470).
Primers used for cloning Medicago truncatula F5H promoter:
 pro_MtF5H_FW_GW:
GGGGACAACTTTGTATAGAAAAGTTGCTGATTCTCACATTTGTTTTTA
 pro_MtF5H_RV_GW:
GGGGACTGCTTTTTTGTACAAACTTGCGTTAACTTGTGTGTTTCTAT